WO2003048345A1

WO2003048345A1 - Phenotypic screen of chimeric proteins

Info

Publication number: WO2003048345A1
Application number: PCT/KR2002/002309
Authority: WO
Inventors: Jin-Soo Kim; Kyung-Soon Park; Dong-Ki Lee; Wongi Seol; Horim Lee; Seong-Il Lee; Hyo-Young Yang; Yangsoon Lee; Young-Soon Jang
Original assignee: Toolgen, Inc.
Priority date: 2001-12-07
Filing date: 2002-12-07
Publication date: 2003-06-12
Also published as: JP2005511049A; CN1610737A; AU2002365796A1; KR20040065235A; EP1451297A1; CN100463962C; US20040209277A1; CA2469477A1; US20030194727A1; EP1451297A4; US7514257B2

Abstract

In one aspect, a library of nucleic acids that encode different artificial, chimeric proteins is screened to identify a chimeric protein that alters a phenotypic trait of a cell or organism. The chimeric protein can be identified without a priori knowledge of a particular target gene or pathway. Some chimeric proteins include multiple zinc finger domains and can induce, for example, thermotolerance, solvent-tolerance, altered cellular growth, insulin production, differentiation, and drug resistance.

Description

PHENOTYPIC SCREEN OF CHIMERIC PROTEINS

BACKGROUND

Most genes are regulated at the transcriptional level by polypeptide transcription factors that bind to specific DNA sites within the gene, typically in promoter or enhancer regions. These proteins activate or repress transcriptional initiation by RNA polymerase at the promoter, thereby regulating expression of the target gene. Many transcription factors, both activators and repressors, include structurally distinct domains that have specific functions, such as DNA binding, dimerization, or interaction with the transcriptional machinery. The DNA binding portion of the transcription factor itself can be composed of independent structural domains that contact DNA. The three- dimensional stmctures of many DNA-binding domains, including zinc finger domains, homeodomains, and helix-turn-helix domains, have been determined from NMR and X- ray crystallographic data. Effector domains such as activation domains or repression domains retain their function when transferred to DNA-binding domains of heterologous transcription factors (Brent and Ptashne, (1985) Cell 43:729-36; Dawson et al, (1995) Mol Cell Biol. 15:6923-31).

Artificial transcription factors can be produced that are chimeras of zinc finger domains. For example, WO 01/60970 (Kim et al.) describes methods for determining the specificity of zinc finger domains and for constructing artificial transcription factors that recognize particular target sites. One application for artificial transcription factors is to alter the expression of a particular target gene. Target sites are identified in the regulatory region of the target gene, and artificial transcription factors are engineered to recognize one or more of the target sites. When such artificial transcription factors are introduced into cells, they may bind to the corresponding target sites and modulate transcription. This strategy for controlling the expression of a target gene is sometimes referred to as the "target-driven" approach for identifying transcription factors.

SUMMARY

In one aspect, the invention features a method that includes: (1) providing a library of cells, the library comprising a plurality of cells that each have a heterologous nucleic acid that expresses an artificial, chimeric polypeptide comprising a first and a second binding domain, wherein the first and second binding domain are heterologous to each other, and the first and second binding domain of each member of the plurality differ from those of the other members of the plurality; and (2) identifying, from the library, a cell that has a trait that is altered relative to a reference cell. The binding domains can be, for example, independently folded modules, e.g., zinc finger domains. In many embodiments, the binding domains are DNA binding domain. Typically, the reference cell is a cell which does not include a library nucleic acid or which includes a control nucleic acid. The reference cell may be a parental cell from which the library of cells was made, or a derivative thereof. The trait can be any detectable phenotype, e.g., a phenotype that can be observed, selected, inferred, and/or quantitated. As used herein, a chimeric protein includes at least two binding domains that are heterologous to each other. The two binding domains can be from different naturally occurring proteins. The two regions can also be from the same naturally occurring protein, but are positioned in a different configuration in the chimeric protein relative to the corresponding naturally occurring protein.

In many embodiments, the cell does not include a reporter gene. In other words, the cells can be screened without having, a priori, infonnation about a target gene whose regulation is altered by expression of the chimeric polypeptide. In addition, the cell may include a reporter gene as an additional indicator of a marker that is related or unrelated to the trait. Likewise, one or more target genes may be known prior to the screening. In another example, the trait is production of a compound (e.g., a natural or artificial compound. The compound can be an antibiotic, an anti-proliferative drug, an analgesic, a protein and so on.

In yet another example, the trait is resistance to an environmental condition, e.g., heavy metals, salinity, environmental toxins, biological toxins, pathogens, parasites, other environmental extremes (e.g., desiccation, heat, cold), and so forth. In a related example, the trait is stress resistance (e.g., to heat, cold, extreme pH, chemicals, such as ammonia, drags, osmolarity, and ionizing radiation). In yet another example, the trait is drug resistance. The change in the trait can be in either direction, e.g., towards sensitivity or further resistance.

In yet another example, the cell is a plant, animal (e.g., mammalian), fungal, or bacterial cell. For mammals, the trait can be cell proliferation, production of a cytokine, hormone, or signaling molecule, activation of a cell signaling pathway, activation of a physiological pathway (e.g., glucose homeostasis, metabolism, obesity).

The DNA binding domains can be, for example, zinc finger domains. Typically, the first zinc finger domain varies among nucleic acids of the library, and the second zinc finger domain also varies among nucleic acids of the plurality. The nucleic acid can also express at least a third DNA binding domain, e.g., a third zinc finger domain.

The zinc finger domains of each expressed polypeptide can be identical to zinc finger domains from different naturally occurring proteins, or can be variants of naturally- occurring proteins, e.g., mutants at the DNA contacting positions. The naturally- occurring protein can be any eukaryotic zinc finger protein: for example, a fungal (e.g., yeast), plant, or animal protein (e.g., a mammalian protein, such as a human or murine protein). Each polypeptide can further include a third, fourth, fifth, and/or sixth zinc finger domain. Each zinc finger domain can be a mammalian, e.g., human zinc fmger domain. Optionally, the nucleic acids of the plurality of cells encode a sufficient number of different zinc finger domains to recognize at least 10, 20, 30, 40, or 50 different 3-base pair DNA sites. In one embodiment, the nucleic acids the nucleic acids of the plurality encode a sufficient number of different zinc fmger domains to recognize no more than 30, 20, 10, or 5 different 3-base pair DNA sites. The polypeptide expressed from each nucleic acid of the library of cells can also include a functional transcriptional regulatory domain, e.g., a transcription activation, repression domain methylation domain, acetylation domain, or deacetylation domain. Also many chimeric polypeptides are functional without fusion to a particular transcriptional regulatory domain. The nucleic acid encoding the polypeptide can be operably linked to a constitutive or inducible promoter.

The method can further include isolating the nucleic acid from the identified cell. The nucleic acid can be sequenced. The polypeptide encoded by the nucleic acid can be isolated. The method can further include identifying a nucleic acid binding site specifically recognized by the polypeptide. The binding site can be identified, e.g., by a computer string or profile search of a sequence database, particularly, a database of regulatory sequences or by selecting in vitro nucleic acids that bind to the polypeptide (e.g., SELEX). A computer database of nucleic acid sequences can be analyzed to identify occurrences of the identified nucleic acid binding site or sites similar to the identified binding site.

The method can include analyzing the expression of one or more endogenous genes or the level/activity of one or more endogenously expressed polypeptides in the identified cell, e.g., using mRNA profiling (e.g., using microarray analysis), 2-D gel electrophoresis, an array of protein ligands (e.g., antibodies), and/or mass spectroscopy.

Also, a single or small number of genes or proteins can also be profiled. In one embodiment, the profile is compared to a database of reference profiles. In another embodiment, regulatory regions of genes whose expression is altered by expression of the identified chimeric polypeptide are compared to identify candidate sites that determine coordinate regulation that results directly or indirectly from expression of the chimeric polypeptide.

The method can also include cultivating the cell to exploit the altered trait. For example, if the altered trait is increased production of a metabolite, the method can include cultivating the cell to produce the metabolite. The cell can be the cell isolated from the libraiy, or a cell into which the nucleic acid encoding the chimeric polypeptide has been re-introduced. Expression of the chimeric polypeptide can be tuned, e.g., using an inducible promoter, in order to finely vary the trait, or another conditional promoter

(e.g., a cell type specific promoter). A cell containing the nucleic acid encoding the chimeric polypeptide can be introduced into an organism (e.g., ex vivo treatment), or used to make a transgenic organism.

In one embodiment, at least some library members encode proteins having different regulatory domains. For example, some libraiy members can include an activation domain, and other members, an inhibitory domain. A particular combination of DNA binding domains, for example, may be represented in the libraiy in one instance as a fusion to an activation domain, and in another instance as a fusion to a repression domain.

In another example, some library members include an activation domain, whereas others do not have a regulatory domain.

The following are some exemplary phenotypes: expansion of stem cells populations (e.g., hematopoietic stem cells, neuronal stem cells, epidermal stem cells, or cord blood stem cells) and other cells having a limited ability to expand in vitro; inhibition of stem cell differentiation s not differentiate; increased pluripotency of a cell (e.g., a differentiated cell or a stem cell); altered stress resistance (e.g., increased or decreased resistance to a condition such as heat, cold, extreme pH, chemicals such as ammonia which can be produced during cell culture, drugs, salt (osmality), ionizing irradiation, etc.); sensitivity to ionizing irradiation or a toxic agent (e.g., an anticancer drag cells), e.g., increased sensitivity in cancer cells; ability to support viral infection/replication (e.g., hepatitis C replication); ability to resist a pathogen, e.g., a virus, bacteria, or protist; enhancement of RNAi efficiency in cells; enhancing transfection efficiency; retardation or delay of the aging process in cells or organisms; growth in a serum-free, chemically-defined media, in the absence of added growth factors; reduction or elimination of inclusion body formation; and enhanced protein secretion in a cell.

Exemplary applications of these methods include: identifying essential genes in a pathogen (e.g., a pathogenic microbe), identifying genes (of the host or pathogen) required for pathogenesis of microbes, identifying targets of drag candidates, gene discovery in signal transduction pathways, microbial engineering and industrial biotechnology, increasing yield of metabolites of commercial interests, and modulating growth behavior (e.g. improving growth of a microorganism, or reducing growth of a cancer cell).

In another aspect, the invention features a cultured cell that includes (a) a gene (endogenous or exogenous) encoding a protein, and (b) an artificial transcription factor, wherein the cell produces the protein at a higher level than does an identical cell that comprises the gene but not the transcription factor, and wherein the transcription factor exerts its effect on production of the protein in a manner other than by binding to a regulatory region operably linked to the gene. The term "artificial" means not naturally- occurring. The term "gene" means a "coding sequence", either cDNA or genomic (i.e., with introns), and either endogenous or exogenous (transiently or stably fransfected).

The artificial transcription factor can include a chimeric DNA binding domain that includes at least two, three, or four zinc finger domains. The artificial transcription factor can further include a regulatory domain (list of activation and repression domain in summary). At least one or two of the individual zinc finger domains may be naturally- occurring (e.g., mammalian, plant, or human). In one embodiment, all the zinc finger domains present are naturally occurring. The artificial transcription factor can be encoded by a heterologous gene of the cell. The heterologous gene encoding the heterologous transcription factor can be regulated by an inducible promoter. The cell can further include at least a second artificial transcription factor. For example, (i) the artificial transcription factor causes the cell or a culture cell lacking the first gene, but otherwise identical to produce a second protein encoded by a second gene operably linked to a regulatory region other than the regulatory region operably linked to the first gene at a higher level than does an identical cell including the second gene but not the transcription factor, and (ii) the transcription factor exerts its effect on production of the second protein in a manner other than by binding to a regulatory region operably linked to the second gene. In another example, (i) the cell further includes a second gene encoding a second protein, (ii) the cell produces the second protein at a higher level than does an identical cell including the second gene but not the transcription factor, and (iii) the transcription factor exerts its effect on production of the second protein in a manner other than by binding to a regulatory region operably linked to the second gene. The regulatory region operably linked to the first gene can be different from the regulatory region operably linked to the second gene.

The first and second transcription factors are selected from the group consisting of: a) a polypeptide including the amino acid sequence of SEQ ID NO:21

(FECKDCGKAFIQKSNLIRHQRTHTGEKPYACPVESCDRRFSDSSNLTRHIRIHTGE KPYACPVESCDRRFSDSSNLTRHIRIH); b) a polypeptide including the amino acid sequence of SEQ ID NO:22 (SCGICGKSFSDSSAKRRHCILHTGEKPYNCDNEGCTWKFARSDI LNRHKKRHTG EKPYNCDNEGCTWKFARSDELNRHKKRHTGEKPYECHDCGKSFRQSTHLTRHR RIH) c) a polypeptide including the amino acid sequence of SEQ ID NO:23 (YECDHCGKSFSQSSHLNNHKRTHTGEKPYRCEECGKAFRWPSNLTRHKRIHTGE KPYRCEECGKAFRWPSNLTRHI RIHTGEKPFACPECPKRFMRSDNLTQHIKTH). In another aspect, the invention features a method of producing a protein. The method includes providing a cell described herein (e.g., above); culturing the cell under conditions that permit production of the protein at a level higher (e.g., at least two, three, five, ten, or a hundred fold) than the level produced by an identical cell that includes the gene but not the transcription factor; detecting the protein produced by the cell and/or purifying the expressed protein from the cell and/or from a medium that surrounds the cell. The gene can be an endogenous or exogenous gene. An endogenous gene can be a naturally occurring gene or a gene that is genetically altered relative to a naturally- occurring gene (e.g., by insertion or modification of regulatory sequences). Examples of endogenous genes include genes encoding a hormone, a cell surface receptor, an antibody, a growth factor, an adhesion protein, a neurotransmitter, and an enzyme. An exogenous gene can be operably linked to a viral promoter, e.g., a CMN or adenovirus promoter. The cell can be a mammalian cell.

The method can further include introducing the cell into a subject. The method can further include formulating the purified protein with a pharmaceutically acceptable carrier.

The method can include one or more of the following features: the transcription factor is present in the cell in an amount effective to increase production of a luciferase marker protein encoded by a gene operably linked to a CMV promoter at least 1.1 , or 2 fold; the transcription factor is present in the cell in an amount effective to increase production of a secreted alkaline phosphatase marker protein encoded by a gene operably linked to an SN40 promoter at least 2, 5, 7, 10 fold; the transcription factor directly alters the expression of a plurality of endogenous genes; the transcription factor alters the rate of division of the cell; the transcription factor competes for binding to a naturally- occurring DΝA binding site specifically recognized by PB08, K_F02, or KJD10, and has a dissociation constant for a DΝA site of less than 50 nM; the transcription factor specifically recognizes a DΝA site that partially overlaps the DΝA binding site specifically recognized by PB08, K_F02, or K_D10; the transcription factor includes two consecutive zinc finger domains selected from the group of QSΝR-DSΝR; DSΝR-DSΝR; DSAR-RDKR; RDKR-RDER; RDER-QTHR; QSHN-WSΝR; WSΝR-WSΝR; WSΝR- RDΝQ, QSΝR1-QSΝK; QSΝK-CSΝR1 wherein each four letter identifier identifies the amino acid present at the -1, +2, +3, and +6 DΝA contacting residues of a zinc finger domain; the transcription factor includes the amino acid sequence of SEQ ID ΝO:21, 22, or 23. The transcription factor can include an amino acid sequence that differs by 1 to 8 amino acid substitutions, insertions, or deletions of SEQ ID NO:21, 22, or 23. The substitution may be at a position other than a DNA. contacting residue, e.g., between a metal coordinating cysteine and position — 1. The substitutions can be conservative substitutions. The transcription factor includes an amino acid encoded by a nucleic acid sequence in FIGs. 17, 18, and 19.

In another example, the transcription factor includes the following three zinc finger domains: a) Cys-X₂.₅-Cys-X₃-X_a-X-Gln-X-Ser-Asn-Xb-X-Arg-His-X₃.5-His (SEQ ID NO:24); b) Cys-X₂.₅-Cys-X₃-X_a-X-Asp-X-Ser-Asn-X_b-X-Arg-His-X₃.₅-His (SEQ ID NO:25); and c) Cys-X₂.₅-Cys-X₃-X_a-X-Asp-X-Ser-Asn-X_b-X-Arg-His-X₃.₅-His (SEQ ID NO:26), e.g., with between one and three substitutions at the invariant positions. In another example, the transcription factor includes at least three of the following four zinc fmger domains (in this order): a) Cys-X₂.₅-Cys-X₃-X_a-X-Asp-X-Ser-Ala-X_b-X-Arg-His-X₃.5-His (SEQ ID NO:27); b) Cys-X₂.5-Cys-X₃-X_a-X-Arg-X-Asp-Lys-Xb-X-Arg-His-X₃.₅-His (SEQ ID NO:28); c) Cys-X₂.₅-Cys-X₃-X_a-X-Arg-X-Asp-Glu-X_b-X-Arg-His-X₃.₅-His (SEQ ID NO:29); and d) Cys-X₂.₅-Cys-X₃-X_a-X-Gln-X-Tlii--His-Xb-X-Arg-His-X₃.₅-His (SEQ ID NO: 30), e.g., with between one and three substitutions at the invariant positions. In another example, the transcription factor includes at least three of the following four zinc finger domains (in this order): a) Cys-X₂.₅-Cys-X₃-X_a-X-Gln-X-Ser-His-Xb-X-Nal-His-X₃_₅-His (SEQ ID ΝO:31); b) Cys-X₂.₅-Cys-X₃-X_a-X-Tιp-X-Ser-Asn-X_b-X-Arg-His-X₃.₅-His (SEQ ID NO:32); c) Cys-X₂-₅-Cys-X₃-X_a-X-T -X-Ser-Asn-X_b-X-Arg-His-X₃.₅-His (SEQ ID NO:33); and d) Cys-X₂.₅-Cys-X₃-X_a-X-Arg-X-Asp-Asn-Xb-X-Lys-His-X₃.₅-His (SEQ ID NO:34), e.g., with between one and three substitutions at the invariant positions. In the above listings, X_a is any amino acid, or optionally phenylalanine or tyrosine; and X is any amino acid, or optionally, a hydrophobic amino acid. In another aspect, the invention features a method of producing a target protein that includes expressing a nucleic acid encoding the target protein in a cell (e.g., in vitro or in vivo), wherein the cell includes a heterologous, artificial transcription factor that increase the amount of protein produced relative to a cell that does not include the heterologous transcription factor. In one embodiment, the heterologous transcription factor causes the increase by a mechanism other than by directly regulating transcription of the gene encoding the target protein. For example, the heterologous transcription factor does not bind to a transcriptional regulatory region that directly regulates the nucleic acid encoding the target gene. The transcription factor can be introduced into the cell as a protein or by introduction and transcription of a nucleic acid encoding it. The method can include other features described herein.

In still another aspect, the invention features a method of identifying a transcription factor. The method includes: providing a nucleic acid library that includes a plurality of nucleic acids, each encoding a different artificial transcription factor, each transcription factor including at least two zinc finger domains that are chimeric with respect to each other; introducing a member of the library into each of a plurality of cells; identifying a cell from the plurality that has enhanced production of a first target protein, wherein the first target protein is encoded by a gene operably linked to a first transcriptional regulatory sequence; and evaluating the ability of the member of the library to enhance production of a second target protein encoded by a gene operably linked to a second transcriptional regulatory sequence that is different from the first transcriptional regulatory sequence.

The method can include one or more of the following features: the first and second target proteins are the same; the cells into which the library members are introduced each includes the gene encoding the second target protein operably linked to the second transcriptional regulatory sequence; the cell is a eukaryotic cell; the first and/or second transcriptional regulatory sequences includes a viral regulatory sequence; the method further includes preparing a host cell that includes a gene encoding transcription factor encoded by a member of the library that enhances production of the first and the second target protein; the method further includes producing a third target protein, different from the first and second target protein from the host cell; and the evaluating includes evaluating the identified cell. The second or third target protein can be, for example, a secreted protein, e.g., erythropoietin, thrombopoietin, a growth factor, an interleukin, or a chemokine. In another example, the target protein is an enzyme, e.g., that catalyzes a reaction in a metabolite producing pathway. Other features described herein can also be included.

In a related aspect, the invention features a method of identifying a protein chimera. The method includes: providing a nucleic acid libraiy that comprises a plurality of nucleic acids, each encoding a different artificial protein chimera that comprises at least two zinc finger domains; providing a test cell that produces a given level of a first target protein under a specific condition; wherein the first target protein is encoded by a gene operably linked to a first transcriptional regulatory sequence; introducing each member of the plurality into a replicate of the test cell to provide a plurality of transformed cells; identifying, from the plurality of transformed cells or progeny cells thereof, a cell that produces, under the specific condition, a level of the first target protein that differs from the given level; and evaluating the ability of the member of the libraiy in the identified cell to enhance production of a second target protein, encoded by a gene operably linked to a second transcriptional regulatory sequence, different from the first transcriptional regulatory sequence. Other features described herein can also be included.

In another aspect, the invention features a host cell, whose genetic material includes: a heterologous gene that encodes an artificial transcription factor that increases production of a target protein encoded by a target gene at least 30% relative to an otherwise identical control host cell lacking the heterologous gene. In one embodiment, the transcription factor does not directly regulate transcription of the target protein. In another embodiment, the transcription factor directly regulates transcription of the target protein. The host cell can include a sequence or a reporter construct such as a sequence encoding lacZ, secreted alkaline phόsphatase (SEAP), GFP, luciferase etc) operably linked to a promoter (e.g., a viral promoter). The cell can include other features described herein. In another aspect, the invention features a host cell, whose genetic material includes a heterologous gene that encodes a polypeptide that includes at least two, three, or four zinc finger domains, binds to an naturally-occurring DNA site, e.g., with an equilibrium dissociation constant of less than 50 nM, and competes with PB08, K_F02, or K_D10 for binding to the naturally-occurring DNA binding site. The cell can include other features described herein.

In another aspect, the invention features a host cell, whose genetic material includes a first heterologous gene that encodes a target protein, and a second heterologous gene that encodes an artificial transcription factor that increases production of the target protein at least 30% relative to an otherwise identical control host cell lacking the second heterologous gene, wherein the transcription factor does not directly regulate transcription of the target protein. The cell can include other features described herein.

In still another aspect, the invention features an isolated polypeptide that includes: the sequence: X_a-X-Cys-X₂.5-Cys-X₃-X_a-X₅-X_b-X-Arg-His-X₃.₅-His-Xι.₆-X_a-X-Cys-X₂.₅- Cys-X₃-X_a-X₅-Xb-X-Arg-His-X₃.5-His-Xι.₆-X_a-X-Cys-X₂.₅-Cys-X₃-X_a-X₅-X_b-X-Arg-His- X₃.₅-His (SEQ ID NO: 35), and that has one or more of the following effects when present at an effective concentration in a human 293 cell: a) increases expression of a gene encoding a luciferase operably linked to a CMV promoter at least 2 fold; b) increases expression of a gene encoding SEAP operably linked to a SV40 promoter at least 2 fold and c) increases or decreases the rate of cell division at least 50%.

In another aspect, the invention features a method of altering the differentiated state of a metazoan cell, the method including: expressing an artificial transcription factor in the cell in an amount effective to alter the differentiated state of the cell. In one embodiment, the differentiated state can be characterized by a neuronal phenotype (e.g., neurite extension, synapse formation, or neuronal marker expression) or an osteoblasts phenotype (e.g., osteoblasts marker expression). In one embodiment, the differentiated state is altered so as to increase the pluripotency of the cell, e.g., to make it less differentiated, e.g., functional as a stem cell or a precursor cell. In one embodiment, the differentiated state is altered from one differentiated state to another (e.g., from a myogenic cell state to an osteoblast state, from a neuronal state to a glial state, and so forth). In one embodiment, the artificial transcription factor induces neurite extension. The cell can be, for example, a stem cell, a neuronal cell, a neural crest cell, or a neuronal progenitor cell. The artificial transcription factor can be a transcription factor described herein, e.g., Neurol-p65 or a Neurol related molecule (see below). The artificial transcription factor can compete for binding to a natural DNA binding site with Neurol - p65. The artificial transcription factor may bind to the same site as Neurol -p65 or a site that overlaps a DNA site bound by Neurol -p65.

In another embodiment, the artificial transcription factor induces an osteoblasts- specific marker, e.g., in an initially non-osteoblast cell, e.g., a myoblast.

In another aspect, the invention features a method of identifying a transcription factor. The method includes: providing a nucleic acid library that includes a plurality of nucleic acids, each encoding a different artificial transcription factor, each transcription factor including at least two zinc finger domains; introducing a member of the library into each of a plurality of cells; and identifying a cell from the plurality that has an altered differentiated state. The method can include one or more of the following features: the cell is a stem cell; the cell is a neuronal cell, a neural crest cell, or a neuronal progenitor cell; and the differentiated state includes neuronal outgrowth or neurite formation. Other features can also be included.

In still another aspect, the invention features a method of identifying a plurality of transcription factors. The method includes: providing a nucleic acid libraiy that includes a plurality of nucleic acids, each encoding a different artificial transcription factor, each transcription factor including at least two zinc finger domains; identifying a first member of the library which alters a given trait of a cell; and screening cells to identify a cell in which the given trait is further altered, wherein each screened cell expresses the transcription factor encoded by the first member of the libraiy and a transcription factor encoded by second member of the same nucleic acid libraiy or another nucleic acid library of artificial transcription factors. The method can include additional rounds of screening in the presence of the first and second transcription factors. In some cases, the method includes identifying a second member of the library that reverses the change in phenotypic state effected by the first member. The method can include other features described herein. In another aspect, the invention features a method of preparing a modified cell, the method including providing a nucleic acid libraiy that includes a plurality of nucleic acids, each encoding a different artificial transcription factor, each transcription factor including at least two zinc finger domains; identifying a first and a second member of the library which alters a given trait of a cell; and preparing a cell that can express first and second polypeptides, the first and second polypeptides being encoded respectively by the first and second identified library members. The method can also be extended to additional member, e.g., a third member. The method can further include evaluating the given trait for the prepared cell.

The method can further include one or more of the following features: the preparing includes introducing a first gene encoding the first polypeptide and a second gene encoding the second polypeptide into the cell; the first and second gene are components of the same nucleic acid; wherein the preparing includes fusing a first cell that includes a first gene encoding the first polypeptide to a second cell that includes a second gene encoding the second polypeptide; the given trait is production of a metabolite; the given trait is production of a target polypeptide; the production includes secretion. Other features described herein can also be used. The following method relates to using virases to screening organismal properties of a ZFP libraiy. A method of identifying a transcription factor can include: providing a nucleic acid library that includes a plurality of nucleic acids, each encoding a different artificial transcription factor, each transcription factor including at least two zinc finger domains; packaging each member of the libraiy into a virus or viral particle that infects mammalian cells to form a plurality of virases or viral particles; introducing the plurality of virases or viral particles into a plurality of non-human mammalian subjects; and identifying a subject from the plurality that has an altered phenotype. For example, each subject has a detectable disorder; the plurality of viruses or viral particles is divided into pools, and each pool is introduced into one of the subjects from the plurality of subjects; the plurality of viruses or viral particles includes separate samples, each sample having packaged within a single nucleic acid of the nucleic acid library, and a single sample is introduced into each subject.

In one aspect, the invention features a modified cell that includes a heterologous nucleic acid encoding an artificial transcription factor that confers stress resistance to the modified cell relative to a reference cell that is substantially identical to the modified cell and that lacks the heterologous nucleic acid and the artificial transcription factor. For example, the artificial transcription factor includes at least two' zinc finger domains. One or more of the zinc finger domains can be naturally occurring, e.g., a naturally occurring domain in Table 1. Exemplary artificial transcription factors include transcription factors that have one or more consecutive motifs as described herein, e.g., a thermotolerant or solvent tolerant protein described herein. The modified cell can be a prokaryotic or eukaryotic cell. The stress resistance can include one or more the following traits: heat resistance, solvent resistance, heavy metal resistance, osmolarity resistance, resistance to extreme pH, chemical resistance, cold resistance, and resistance to a genotoxic agent, resistance to radioactivity. Stress resistance enables a resistant cell to survive or grow in a condition in which a non- resistant cell would die or fail to grow. For example, the modified cell can express the artificial transcription factor and resist stress to a greater extent than a substantially identical culture cell that lacks the artificial transcription factor. The invention also provides such artificial proteins, and proteins that alter sensitivity of the cell to a toxic agent relative to an identical cell that does not contain the nucleic acid. In another aspect, the method includes a method of producing a cellular product.

The method includes providing a modified cell that includes a heterologous nucleic acid encoding an artificial transcription factor; maintaining the modified cell under conditions in which the artificial transcription factor is produced; and recovering a product produced by the cultured cell, wherein the product is other than the artificial transcription factor. For example, the artificial transcription factor can confer stress resistance, or another property described herein, e.g., altered protein production, altered metabolite production, and so forth. For example, the artificial transcription factor includes at least two zinc fmger domains. One or more of the zinc finger domains can be naturally occurring, e.g., a naturally occurring domain in Table 1. Exemplary artificial transcription factors include transcription factors that have one or more consecutive motifs (e.g., at least two, three or four consecutive motifs, or at least three motifs in the same pattern, including non- consecutive patterns) as described herein.

Exemplary products include a metabolite or a protein (e.g., an endogenous or heterologous protein. For example, the modified cell includes a nucleic acid encoding a heterologous protein, other than the artificial transcription factor, and the product is the heterologous protein. In another example, the modified cell further includes a second nucleic acid encoding a heterologous protein, and the heterologous protein participates in production of the metabolite. The modified cell can be maintained at a temperature between 20°C and 40°C or greater than 37°C. In one embodiment, the modified cell is maintained under conditions which would inhibit the growth of a substantially identical cell that lacks the artificial transcription factor. In one embodiment, the zinc finger domain includes a set of DNA contacting residues that correspond to DNA contacting residues of a zinc finger domain listed in Table 15. In a related embodiment, the artificial transcription factor includes an array of at least three zinc finger domains, wherein the DNA contacting residues of each of the zinc finger domains of the array correspond respectively to DNA contacting residues of any three consecutive zinc finger domains listed in a row of Table 15. In still another embodiment, the artificial transcription factor competes with a protein that includes the array of zinc finger domains listed in a row of Table 15.

In another aspect, the invention features a cell that contains a gene (e.g., an endogenous or heterologous gene) encoding a target protein and a heterologous nucleic acid that includes a sequence encoding an artificial protein chimera that (1) increases the amount of the protein produced by the cell relative to a cell that does not include the heterologous nucleic acid, and (2) does not bind to a transcriptional regulatory region that directly regulates transcription of the gene encoding the target protein. For example, the artificial transcription factor includes at least two zinc finger domains, and, e.g., binds DNA. One or more of the zinc finger domains can be naturally occurring, e.g., a naturally occurring domain in Table 1. Exemplary artificial transcription factors include transcription factors that have one or more consecutive motifs as described herein, In one embodiment, the cell is a eukaryotic cell, and the artificial protein chimera competes with PB08, KJF02, or K_D10 for binding to a genomic DNA site. In one embodiment, the artificial protein chimera alters (e.g., increases or decrease) the rate of cell cycle progression by the cell. The invention also provides such artificial transcription factors.

The cell can be used in a method of producing a protein, the method including: providing the cell, and maintaining the cell under conditions in which the artificial protein chimera increases the amount of the target protein produced by the cell relative to a cell that does not include the heterologous nucleic acid. For example, the protein is a secreted protein, a cytoplasmic protein, or a nuclear protein. In another aspect, the invention features a cell that contains an endogenous gene encoding a secreted protein and a heterologous nucleic acid that includes a sequence encoding an artificial transcription factor that increases the amount of the secreted protein produced by the cell relative to a cell that does not include the heterologous nucleic acid. In one embodiment, the cell is a eukaryotic cell, and the secreted protein is insulin. In one embodiment, the artificial transcription factor specifically binds to an endogenous DNA site that is also specifically bound by 08_D04_p65. In another embodiment, the artificial transcription factor specifically binds to an endogenous DNA site and the artificial transcription factor competes with 08_D04_p65 for binding to the endogenous DNA site. For example, the artificial transcription factor includes an amino acid sequence as follows:

CX₍₂-5₎CXXXBXRXSHJXRHX(₃.₅)HX(i.₆)BXCX₍₂.5)CXXXBXRXDHJXTHX₍₃. ₅₎H (SEQ ID O:45); or

CX₍₂.5)CXXXBXRXDHJXTHX(₃.₅)HX(i.₆₎BXCX(₂.5)CXXXBXNXSSJXRHX₍₃. ₅₎H (SEQ ID ΝO:46) where B is phenylalanine or tyrosine; and J is a hydrophobic amino acid. The cell can be used in a method of producing a secreted protein (e.g., insulin). For example, the cell is maintained under conditions in which the artificial transcription factor increases the amount of insulin produced by the cell relative to a cell that does not include the heterologous nucleic acid. Similarly, the invention provides an artificial transcription factor including at least two zinc finger domains, wherein the artificial transcription factor induces expression of an endogenous insulin gene in a mammalian cell that does not express an endogenous insulin gene in the absence of the artificial transcription factor. In another aspect, the invention features an artificial transcription factor that alters sensitivity of the cell to a toxic agent (e.g., a drug, e.g., an anti-fungal drug, e.g., ketoconazole) relative to an identical cell that does not contain the nucleic acid. The sensitivity can be increased or decreased. In one embodiment, the cell is a fungal cell. For example, the artificial transcription factor includes at least two zinc finger domains, e.g., at least three. One or more of the zinc finger domains can be naturally occumng, e.g., a naturally occumng domain in Table 1. Exemplary artificial transcription factors include transcription factors that have one or more consecutive motifs as described herein. For example, the artificial transcription factor binds to an endogenous DNA site and the artificial transcription factor competes with a zinc finger protein listed in Table 5 for binding to the endogenous DNA site.

In another aspect, the invention features a method of altering the drug resistance of a fungal cell. The method includes altering the expression or activity of a protein that is at least 70% identical to AQY1, YJR147W, YLL052C, YLL053C, or YPL091W in the cell. The expression can be altered, e.g., using a transcription factor.

In still another aspect, the invention features a method of identifying an artificial chimeric protein that alters the sensitivity of a cell to a toxic agent, the method including: providing a nucleic acid library that includes a plurality of nucleic acids, each nucleic acid of the plurality encoding a chimeric protein that includes an array of at least three zinc finger domains wherein at least two adjacent zinc finger domains do not occur adjacent to each other in a naturally occurring protein; introducing members of the library into replicates of a test cell to yield transformed cells; cultivating the transformed cells in the presence of a toxic agent; and identifying, from the transformed cells, a cell whose sensitivity to the toxic agent is altered relative to the test cell. For example, the test cell is a fungal cell, and the toxic agent is an anti-fungal agent. In another example, the test cell is a cancer cell, and the toxic agent is an anti-mitotic agent. The chimeric protein encoded by each nucleic acid of the plurality can include a transcriptional regulatoiy domain.

The method can further include constructing a nucleic acid that encodes a second chimeric protein that includes the array of zinc finger domains of the chimeric protein encoded by the libraiy member in the selected cell, but does not include the transcriptional regulatoiy domain of the chimeric protein encoded by the libraiy member in the identified cell. The method can further include constructing a nucleic acid that encodes a second chimeric protein that includes (i) the array of zinc finger domains of the chimeric protein encoded by the library member in the selected cell, and (ii) a transcriptional regulatoiy domain, other than the transcriptional regulatoiy domain of the chimeric protein encoded by the libraiy member in the identified cell. In another aspect, the invention features a nucleic acid that includes a sequence encoding an artificial transcription factor including three zinc finger domains, wherein expression of the artificial transcription factor induces a neuronal phenotype in at least one vertebrate cell. In one example, at least one of the zinc fii ger domains has the sequence:

Cys-X₂.₅-Cys-X₃-X_a-X-Gln-Xb-X-Ser-Asn-His-X₃.₅-His (SEQ ID NO:250) Cys-X₂.₅-Cys-X₃-X_a-X-Gln-Xb-X-Ser-Asn-His-X₃.₅-His (SEQ ID NO:251); or Cys-X₂.5-Cys-X₃-X_a-X-Cys-X_b-X-Ser-Asn-His-X₃.₅-His (SEQ ID NO:252), wherein X_a is phenylalanine or tyrosine; and Xb is a hydrophobic amino acid. E.g., the artificial transcription factor includes the sequence: Cys-X₂.₅-Cys-X₃-X_a-X-Gln-X_b-X- Ser-Asn-His-X₃.5-His-Xι.6-Cys-X₂.5-Cys-X₃-X_a-X-Gln-X_b-X-Ser-Asn-His-X₃.₅-His-X₁.6- Cys-X₂-₅-Cys-X3-X_a-X-Cys-Xb-X-Ser-Asn-His-X₃.₅-His (SEQ ID NO:253), wherein X_a is phenylalanine or tyrosine; and Xb is a hydrophobic amino acid.

One method includes providing a vertebrate cell (e.g., a mammalian cell, a human) that contains the nucleic acid; and maintaining the vertebrate cell under conditions in which the artificial transcription factor is produced and neurite formation is induced. In one example, the vertebrate cell is a stem cell prior to production of the artificial transcription factor.

In another aspect, the invention features a nucleic acid that includes a sequence encoding an artificial transcription factor included of three zinc finger domains, wherein expression of the artificial transcription factor induces osteogenesis in at least one vertebrate cell. For example, at least one of the zinc finger domains has the sequence:

Cys-X₂-₅-Cys-X₃-Xa-X-Arg-Xb-X-Asp-Lys-His-X₃.₅-His (SEQ ID NO:254);

Cys-X₂.₅-Cys-X₃-Xa-X-Gln-X_b-X-Thr-His-His-X₃.₅-His (SEQ ID NO:255);

Cys-X₂.₅-Cys-X₃-Xa-X-Val-X_b-X-Ser-Thr-His-X₃.₅-His (SEQ ID NO:256) or;

Cys-X₂.₅-Cys-X₃-Xa-X-Arg-X_b-X-Asp-Lys-His-X₃.₅-His (SEQ ID NO:257), wherein X_a is phenylalanine or tyrosine; and Xb is a hydrophobic amino acid; or the artificial transcription factor includes the amino acid sequence:

Cys-X₂-₅-Cys-X₃-X_a-X-Arg-X_b-X-Asp-Lys-His-X₃.₅-His-Xi.₆-Cys-X₂.₅-Cys-X₃- X_a-X-Gln-X_b-X-Tlιr-His-His-X₃.₅-His-Xι.₆-Cys-X₂.5-Cys-X₃-X_a-X-Nal-Xb-X-Ser-Thr- His-X₃.₅-His-Xi.₆-Cys-X₂.₅-Cys-X₃-X_a-X-Arg-Xb-X-Asp-Lys-His-X₃-₅-His (SEQ ID ΝO:258), wherein X_a is phenylalanine or tyrosine; and X_ is a hydrophobic amino acid..

One method includes providing a vertebrate cell that contains the nucleic acid; and maintaining the vertebrate cell under conditions in which the artificial transcription factor is produced and osteogenesis is induced.

In another aspect, the invention features a method of altering the differentiative capacity of a stem cell. The method includes: providing a stem cell and a nucleic acid that includes a sequence encoding an artificial transcription factor includes of a plurality of zinc finger domains, wherein the artificial transcription factor alters the differentiative capacity of the stem cell; introducing the nucleic into the stem cell; and maintaining the stem cell under conditions in which the artificial transcription factor is produced thereby altering the differentiative capacity of the stem cell. For example, the artificial transcription factor induces differentiation of the stem cell. In another example, the artificial transcription factor enhances self-renewal potential of the stem cell. The stem cell can be an embryonic stem cell, a vertebrate stem cell, a plant stem cell, a hematopoietic stem cell, a neuronal progenitor cell or muscular progenitor cell.

In still another aspect, the invention features a method that includes: providing a nucleic acid library that includes a plurality of nucleic acids, each encoding a different artificial transcription factor, each artificial transcription including an array of at least two zinc finger domains and a regulatory domain that activates or represses transcription; providing cell that have a given trait; introducing members of the nucleic acid library into the cells; identifying a member of the library that alters the given trait; and preparing a coding nucleic acid that includes a sequence encoding a DNA binding polypeptide that includes the array of zinc finger domains from the identified member, but does not include a regulatoiy domain identical to the regulatory domain of the identified member. For example, the DNA binding polypeptide is lacks the regulatory domain of the identified member. In another example, the DNA binding polypeptide includes a regulatoiy domain that is mutated relative to the regulatoiy domain of the identified member. In one embodiment, the DNA binding polypeptide includes a regulatory domain of opposite functionality relative to the regulatoiy domain of the identified member.

The method can further include introducing the coding nucleic acid into a cell and assessing the given trait of the cell. The step of identifying can include identifying cells having a property selected from the group consisting of: resistance to a given environmental condition; differentiation; dedifferentiation; proliferation; apoptosis; serum-independence; pathogen resistance; and pathogen sensitivity. In another aspect, the invention features an isolated nucleic acid encoding an artificial transcription factor that improves solubility of a heterologous, over-expressed protein in a cell that produces the artificial transcription factor. For example, the artificial transcription factor includes a plurality of zinc finger domains. The cell can be a bacterial cell or a eukaryotic cell. In one example, the protein is a mammalian protein, e.g., AKT protein. In one embodiment, the plurality of zinc finger domains include domains: QSTR-DSAR-RDHT-WSNR or VSTR-DGNV-QSNR-QSNK. The invention also features a modified cell that includes the heterologous nucleic acid of any one of claims 99 to 104. The invention also provides a method of producing a heterologous target protein. The method includes providing the modified cell wherein the modified cell includes a second nucleic acid that includes a sequence encoding a heterologous target protein; and maintaining the modified cell under conditions wherein the artificial transcription factor and the heterologous target protein are produced. The modified cell can be a cultured cell or an in vivo cell, e.g., in a subject. In another aspect, the invention features a method of transferring an altered trait from a first cell to a second cell. The method includes: providing a nucleic acid library that includes a plurality of nucleic acids, each encoding a different artificial protein chimera that includes at least two zinc fmger domains; introducing members of the nucleic acid library into first cells that have a given trait to provide transfoπned cells; identifying an altered cell from the transfomied cells in which a member of the libraiy alters the given trait; recovering a nucleic acid library member from the identified, altered cell; introducing the nucleic acid library member into second cells, wherein the second cells differ from the first cells by a phenotypic trait other than the given trait; and evaluating a second cell that includes the nucleic acid libraiy member and expresses the artificial protein chimera that the nucleic acid library member encodes. For example, the first and second cells are eukaryotic, e.g., yeast cells or mammalian cells. The first and second cells can differ by the proliferative properties or differentiative properties. For example, the first cells are cancer cells, and the second cells are non-cancerous, or vice versa. The evaluated second cell can be evaluated for an alteration to the given trait. In other aspects, the invention features a nucleic acid including a sequence encoding an artificial transcription factor included of three zinc finger domains. For example, expression of the artificial transcription factor alters a property of at least one eukaryotic cell, wherein the property is selected from the group consisting of viral replication, virus production, and viral infectivity; expression of the artificial transcription factor alters an ability of a eukaryotic cell to regulate a stem cell that is co-cultured with the eukaryotic cell or that is cultured in media conditioned by the eukaryotic cell; expression of the artificial transcription factor alters an ability of a mammalian culture cell (e.g., a CHO cell) to glycosylate a secreted protein (e.g., an antibody); and expression of the artificial transcription factor alters the ability of a cell to take up exogenous nucleic acid.

In still another aspect, the invention features a method of identifying a transcription factor. The method includes: providing a nucleic acid library that includes a plurality of nucleic acids, each encoding a different artificial transcription factor, each transcription factor including at least two zinc finger domains; introducing members of the nucleic acid libraiy into cells that have a given trait; identifying a member of the library which alters the given trait; and preparing a second library that includes a plurality of nucleic acids, each encoding (1) a variant that differs from the artificial protein chimera corresponding to the identified member by between one and six amino acid substitutions, insertions, or deletions, (2) a protein chimera that comprises the zinc finger domains of the artificial protein chimera corresponding to the identified member and an additional zinc finger domain, wherein the additional zinc finger domain varies among the members of the second library, (3) a variant of the artificial protein chimera corresponding to the identified member, wherein the variant has a subset of the zinc finger domain positions replaced with other zinc finger domains and at least one invariant zinc finger domain that is identical to the zinc finger domain at a corresponding position in the artificial protein chimera corresponding to the identified member, and/or (4) a variant of the artificial protein chimera corresponding to the identified member, wherein, among the members of the second library, one or more zinc finger domain positions are varied such that the particular domain in the artificial protein chimera corresponding to the identified member at that position occurs at a frequency greater than other zinc finger domains at that position. The method can include other features described herein. In another aspect, the invention features a method of identifying a transcription factor, the method includes: providing a nucleic acid libraiy that includes a plurality of nucleic acids, each encoding a different artificial transcription factor, each transcription factor including a DNA binding component having at least two zinc finger domains and a first regulatory domain that activates or represses transcription; introducing members of the nucleic acid library into cells that have a given trait; identifying a member of the library which alters the given trait; and preparing a nucleic acid that encodes the DNA binding component of the identified member and a second regulatory domain that differs from the first regulatory domain. A related method includes identifying a transcription factor that lacks a regulatory domain, and adding one; or removing a regulatory domain from a transcription factor that includes one. Some transcription factors can function without a transcriptional regulatory domain. The invention also features an artificial transcription factor that comprises a plurality of zinc finger domains and a first regulatory domain. The artificial transcription factor produces a first trait when produced in a cell, but produces a second trait if the first regulatory domain is inactivated and a second regulatory domain, of opposite functionality from the first, is included.

In one example, the first regulatory domain activates transcription and the second regulatory domain represses transcription. In another example, the first regulatoiy domain represses transcription and the second regulatory domain activates transcription. In still another example, the first regulatoiy domain activates transcription to a first extent and the second regulatory domain activates transcription to an extent at least 50% less than the first extent. The method can further include introducing the prepared nucleic acid into a test cell and assessing the test cell, e.g., the given trait of the test cell. In one embodiment, the identified member increases the rate of cell division, and the prepared nucleic acid encodes a transcription factor that decreases the rate of cell division. In another embodiment, the identified member causes resistance to a compound, and the prepared nucleic acid encodes a transcription factor that causes sensitivity to the compound.

In still another aspect, the invention features a method of identifying a transcription factors, the method including: providing a nucleic acid libraiy that includes first and second pluralities of nucleic acids, wherein each nucleic acid of the first plurality encodes a different artificial transcription factor that includes at least two zinc finger domains and a regulatoiy domain that activates transcription, and each nucleic acid of the second plurality encodes a different artificial transcription factor that includes at least two zinc finger domains and a regulatory domain that represses transcription; introducing members of the nucleic acid library into cells that have a given trait; and identifying a member of the library which alters the given trait.

The invention also features a method that includes evaluating the phenotype of a chimeric artificial zinc fmger protein in a first cell, expressing the protein in a second cell, and evaluating a phenotype of the second cell. For example, the method can include identifying a member of a transcription factor library that alters a given phenotype in a first cell (a yeast strain or a human cell line such as 293 cells, for example) and then expressing the member in a different cell (a different yeast strain or HeLa cells, for example). The first cell may be more amenable for screening than other strains or cell lines.

In another aspect, the invention features a method of altering the sensitivity of a fungal strain to an anti-fungal agent. The method includes: introducing a nucleic acid encoding an artificial chimeric protein that comprises at least three zinc finger domains into a fungal cell; and maintaining the fungal cell under conditions that allow for expression of the introduced nucleic acid in the cell. For example, the fungal cell is a yeast cell, e.g., Candida, Pichia, Hanseula, Histoplasma, or Cryptococcus. In one embodiment, the artificial chimeric protein comprises the amino acid sequence of the zinc finger array of a protein selected from Kl through Kl 1 (see Example 3).

The method can include one or more of the following features: the artificial chimeric protein comprises a transcriptional regulatoiy domain (e.g., an activation or repression domain); expression of the artificial chimeric protein in the cell alters the transcript level of a water transporter; expression of the artificial chimeric protein in a S. cerevisiae cell alters the transcript level of the YLL053 gene or the PDR5 gene; the artificial chimeric protein competes a polypeptide selected from Kl through Kl 1 for binding to a specific DNA site.

In another aspect, the invention features a method of altering the sensitivity of a fungal strain to an anti-fungal agent. The method includes altering the activity or expression of a protein that comprises an amino acid sequence of at least 50 amino acids that is at least 30, 50, 60, 70, 80, 90, or 95% identical to YLL053, AQY1, YJR147W, YLL052C, or YPL091W. For example, the fungal cell is a yeast cell, e.g., Candida, Pichia, Hanseula, Histoplasma, or Ciγptococcus. In one embodiment, the activity or expression is increased to reduce sensitivity, i.e., increase resistance. In another embodiment, the activity or expression is reduced to increase sensitivity. Altering the activity or expression can include expressing an artificial transcription factor, contacting the cell with a double-stranded RNA (dsRNA) that includes a sequence of at least 20 nucleotides complementary to YLL053, AQY1, YJR147W, YLL052C, or YPL091 W gene, or contacting the cell with a chemical compound.

A related method includes screening a test compound (e.g., a small organic compound) for interaction with such a protein, e.g., to identify an inhibitor of a YLL053/AQY1 -related protein or screening a test compound for ability to alter the activity or expression of AQY1, YJR147W, YLL052C, YLL053C or YPL091W. In another aspect, the invention features a method of identifying an artificial chimeric protein that alters the sensitivity of a fungal strain to an anti-fungal agent. The method includes: providing a nucleic acid library that comprises a plurality of nucleic acids, each nucleic acid of the plurality comprising a coding sequence that encodes an artificial, chimeric protein that comprises an array of at least three zinc finger domains wherein at least two adjacent zinc finger domains do not occur adjacent to each other in a naturally occumng protein; introducing each nucleic acid of the plurality into a fungal cell to yield transfomied fungal cells; maintaining (e.g., culturing) the transfomied fungal cells in the presence of an anti-fungal agent; and selecting a cell from the transfomied fungal cells whose sensitivity to the anti-fungal agent is altered relative to a control fungal cell.

The method can include one or more of the following features: the control fungal cell is not transfomied or is transfomied with a reference nucleic acid; the chimeric protein encoded by each nucleic acid of the plurality comprises a transcriptional regulatory domain; the fungal cell is a pathogenic fungal cell, e.g., a Candida, Histoplasma, or Cryptococcus.

The method can further include, for example, constructing a nucleic acid that encodes a second chimeric protein that comprises (i) the array of zinc fmger domains of the chimeric protein encoded by the libraiy member in the selected cell, and (ii) a transcriptional regulatoiy domain, other than the transcriptional regulatory domain of the chimeric protein encoded by the library member in the selected cell. The method can include other features described herein. In a further aspect, the invention features a method of identifying an agent that counters resistance of a fungal cell to an anti-fungal agent. The method includes: contacting a test compound to a polypeptide that includes YJR147W, YLL052C, YLL053C or YPL091W; and evaluating interaction between the test compound and the polypeptide, wherein an interaction indicates that the test compound may be useful as an agent to counter resistance to an anti-fungal agent. The method can further include contacting a fungal cell with the test compound and the anti-fungal agent, e.g., and evaluating viability or growth of the cell. The cell can be a cell that is resistant to the anti-fungal agent (e.g., ketoconazole). In another aspect, the invention features a method of identifying a target gene that is regulated by artificial transcription factors. The method includes: providing a nucleic acid libraiy that comprises a plurality of nucleic acids, each encoding a different artificial transcription factor, each transcription factor comprising at least two zinc finger domains; introducing each member of the plurality into a replicate of a test cell to provide a plurality of transfomied cells; identifying a plurality of phenotypically altered cells from the plurality of transfomied cells, wherein each phenotypically altered cell has an altered phenotype relative to the test cell; and identifying one or more transcripts or proteins whose abundance is similarly altered in at least two phenotypically altered cells of the plurality relative to the test cell. In one embodiment, the method further includes, in a test cell, altering the activity of a transcript or protein whose abundance is similarly altered in at least two phenotypically altered cells, e.g., by genetic alteration (e.g., mutation or overexpression) or otherwise (e.g., RNA interference, anti-sense, or antibody binding). In some cases, the activity of a plurality of transcripts or proteins is altered. In one embodiment, the similarly altered transcripts or proteins are identified by profiling transcripts or protein abundance in each phenotypically altered cell of the plurality to provide a profile for each phenotypically altered cell; and comparing the profiles to each other. Profiles can be obtained using, for example, a nucleic acid or protein array, SAGE tags, differential display, or subtractive hybridization. In one embodiment, the cell is a cancer cell, e.g., a human cancer cell. One or more of the identified transcripts can be a transcript that is absent from the cell in the absence of any artificial transcription factors. A polypeptide encoded by one of the identified transcripts can be used as a target polypeptide for screening to find test compounds that interact with the target. The test compound can be evaluated to determine if they enhance or inhibit activity of the target polypeptide. In one embodiment, the test compound is a small molecule having a molecular weight of less than 10, 5, or 2 kDa.

With respect to all methods described herein, a library of nucleic acids that encode chimeric zinc fmger proteins can be used. The term "library" refers to a physical collection of similar, but non-identical biomolecules. The collection can be, for example, together in one vessel or physically separated (into groups or individually) in separate vessels or on separate locations on a solid support. Duplicates of individual members of the library may be present in the collection. A library can include at least 10, IO², 10³, IO⁵, IO⁷, or IO⁹ different members, or fewer than 10¹³, IO¹², 10¹⁰, IO⁹, IO⁷, IO⁵, or 10³ different members.

A first exemplary library includes a plurality of nucleic acids, each nucleic acid encoding a polypeptide comprising at least a first, second, and third zinc fmger domains. As used herein, "first, second and third" denotes three separate domains that can occur in any order in the polypeptide: e.g., each domain can occur N-terminal or C-terminal to either or both of the others. The first zinc finger domain varies among nucleic acids of the plurality. The second zinc finger domain varies among nucleic acids of the plurality. At least 10 different first zinc finger domains are represented in the libraiy. In one implementation, at least 0.5, 1, 2, 5%, 10%, or 25% of the members of the libraiy have one or both of the following properties: (1) each represses transcription of at least one plG reporter plasmid at least 1.25 fold in vivo; and (2) each binds at least one target site with a dissociation constant of no more than 7, 5, 3, 2, 1, 0.5, or 0.05 nM. The first and second zinc finger domains can be from different naturally-occurring proteins or are positioned in a configuration that differs from their relative positions in a naturally- occurring protein. For example, the first and second zinc finger domains may be adjacent in the polypeptide, but may be separated by one or more intervening zinc finger domains in a naturally occurring protein. A second exemplary library includes a plurality of nucleic acids, each nucleic acid encoding a polypeptide that includes at least first and second zinc finger domains. The first and second zinc finger domains of each polypeptide (1) are identical to zinc fmger domains of different naturally occumng proteins (and generally do not occur in the same naturally occurring protein or are positioned in a configuration that differs from their relative positions in a naturally-occurring protein), (2) differ by no more than four, three, two, or one amino acid residues from domains of naturally occumng proteins, or (3) are non-adjacent zinc fmger domains from a naturally occumng protein. Identical zinc finger domains refer to zinc finger domains that are identical at each amino acid from the first metal coordinating residue (typically cysteine) to the last metal coordinating residue (typically histidine). The first zinc finger domain varies among nucleic acids of the plurality, and the second zinc finger domain varies among nucleic acids of the plurality. The naturally occurring protein can be any eukaryotic zinc finger protein: for example, a fungal (e.g., yeast), plant, or animal protein (e.g., a mammalian protein, such as a human or murine protein). Each polypeptide can further include a third, fourth, fifth, and/or sixth zinc finger domain. Each zinc fmger domain can be a mammalian, e.g., human, zinc finger domain. Other types of libraries can also be used, e.g., including mutated zinc fmger domains.

In some embodiments, a libraiy of nucleic acids encoding zinc finger proteins or a library of such proteins themselves can include members with different regulatory domains. For example, the library can include at least 10% of members with an activation domain, and at least another 10%> of members with a repression domain. In another example, at least 10%> have an activation domain or repression domain; another at least 10% has no regulatory domain. In still another example, some include an activation domain; others, a repression domain; still others, no regulatory domain at all. Other percentages, e.g., at least 20, 25, 30, 40, 50, 60% can also be used.

1. Neurolp65 and Related Molecules

In still another aspect, the invention features an artificial polypeptide that includes: the sequence:

CX₍₂.₅₎CXXXBXQXSNJXRHX₍₃.₅)HX₍₁.₆)BXCX(2.₅)CXXXBXQXSNJXKH X₍₃.₅₎HX₍i.₆₎BXCX₍₂.₅₎CXXXBXCXSNJXRHX₍₃.₅)H (SEQ ID NO:36), where B is any amino acid, or optionally phenylalanine or tyrosine; and J is any amino acid, or optionally, a hydrophobic amino acid. This array is also abbreviated as: QSNR-QSNK-CSNR. Other exemplary artificial polypeptides include:

CX₍2.₅)CXXXBXQXSNJXRHX₍₃.5)HX₍₁.₆₎BXCX(2.₅)CXXXBXQXSNJXKHX(₃. ₅₎H (SEQ ID NO:37), and

CX(2-₅)CXXXBXQXSNJXI^«αHX₍₃_₅)HX(i.6)BXCX₍₂.₅)CXXXBXCXSNJXRHX₍₃. ₅₎H (SEQ ID NO:38).

The polypeptide can, for example, induce neurites when present at an effective concentration in a mouse Neuro2a cell. For example, the isolated polypeptide can include an amino acid sequence that is identical to the zinc fmger array within SEQ ID NO:2 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:2. The substitutions can be conservative substitutions. The isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:2. In one embodiment, the polypeptide can specifically bind to a target DNA site. For example, the polypeptide can compete with the Neurol p chimeric ZFP (SEQ ID NO:2) for binding to a target DNA site, e.g., a site bound with a Jζ_ of less than 10 nM. The polypeptide can further include a transcriptional regulatoiy domain, e.g., an activation or repression domain. The polypeptide can include one, two, or three or more additional zinc finger domains. Also featured are nucleic acids encoding the above polypeptides. For example, the isolated polypeptide that includes the amino acid of SEQ ID NO:2 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO: 1. Featured nucleic acids can include operably linked regulatoiy sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth. In one embodiment, the coding nucleic acid is operably linlced to a conditional promoter such as an inducible promoter or a cell-type specific promoter. The nucleic acid can be included in a vector or integrated into a chromosome.

In addition, the invention features host cells (e.g., mammalian host cells) that include a polypeptide described above. The host cell can include a nucleic acid described above and express the nucleic acid. For example, the host cell can be a neuronal cell that extends neurites (e.g., at least in part as a consequence of the polypeptide). 2. Osteolp65 and Related Molecules

CX₍2.₅)CXXXBXRXDKJXRHX₍₃.₅)HX(i.₆₎BXCX(₂_₅)CXXXBXQXTHJXRHX(₃. 5)HX(₁.6)BXCX(₂.5)CXXXBXVXSTJXRHX(₃.5)HX₍i.₆₎BXCX₍₂. ₅₎CXXXBXRXDKJXRHX(₃.₅₎H (SEQ ID NO:39), where B is any amino acid, or optionally phenylalanine or tyrosine; and J is any amino acid, or optionally, a hydrophobic amino acid. This array is also abbreviated as: RDKR-QTHR-NSTR-RDKR. Other exemplary artificial polypeptides include: CX(2-₅)CXXXBXRXDKJXRHX(₃.₅)HX(_I.6)BXCX(₂.₅)CXXXBXQXTHJXRHX(₃.

₅₎HX₍i.₆₎BXCX₍₂.₅₎CXXXBXVXSTJXRHX₍₃.₅₎H (SEQ ID ΝO:40);

CX(2-₅)CXXXBXQXTHJXRHX₍₃.5)HX₍₁.₆)BXCX₍2.₅)CXXXBXNXSTJXRHX₍₃. ₅₎HX₍i.₆₎BXCX₍₂.₅₎CXXXBXRXDKJXRHX₍₃.₅₎H (SEQ ID ΝO:41);

CX(2-₅)CXXXBXRXDKJXRHX(₃.5)HX₍₁.₆₎BXCX(2.₅)CXXXBXQXTHJXRHX₍₃. ₅)H (SEQ ID NO:42); and

CX₍2-₅)CXXXBXNXSTJXRHX₍₃.5)HX₍₁.₆)BXCX₍₂.₅)CXXXBXRXDKJXRHX₍₃. ₅)H (SEQ ID ΝO:43).

The polypeptide can, for example, induce alkaline phosphatase or other indicators of osteoblasts differentiation, when present at an effective concentration in a C2C12 myoblast cell line cell. For example, the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO:4 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:4. The substitutions can be conservative substitutions. The isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:4. In one embodiment, the polypeptide can specifically bind to a target DNA site. For example, the polypeptide can compete with the Osteolp chimeric ZFP (SEQ ID NO:4) for binding to a target DNA site, e.g., a site bound with a K_<- of less than 10 nM. The polypeptide can further include a transcriptional regulatoiy domain, e.g., an activation or repression domain. The polypeptide can include one, two, or three or more additional zinc finger domains.

Also featured are nucleic acids encoding the above polypeptides. For example, the isolated polypeptide that includes the amino acid of SEQ ID NO:4 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:3. Featured nucleic acids can include operably linked regulatoiy sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth. In one embodiment, the coding nucleic acid is operably linked to a conditional promoter such as an inducible promoter or a cell-type specific promoter. The nucleic acid can be included in a vector or integrated into a chromosome.

In addition, the invention features host cells (e.g., mammalian host cells) that include a polypeptide described above. The host cell can include a nucleic acid described above and express the nucleic acid. For example, the host cell can be a stem cell that has the phenotype of an osteoblasts, e.g., at least partially as a consequence of the artificial chimeric polypeptide.

3. 08 P04 6 and Related Molecules

CX₍₂.₅)CXXXBXRXSHJXRHX₍₃.5)HX₍₁.₆₎BXCX₍₂.₅)CXXXBXRXDHJXTHX₍₃_ ₅₎HX₍₁-₆₎BXCX₍₂.₅₎CXXXBXVXSSJXRHX₍3-₅)H (SEQ ID NO:44), where B is any amino acid, or optionally phenylalanine or tyrosine; and J is any amino acid, or optionally, a hydrophobic amino acid. This array is also abbreviated as: RSHR-RDHT-VSSR. Other exemplary artificial polypeptides include:

CX(₂.₅)CXXXBXRXSHJXRHX₍₃.₅₎HX₍₁.₆₎BXCX(₂.₅)CXXXBXRXDHJXTHX₍₃. ₅₎H (SEQ ID NO:45); and

CX₍₂-₅₎CXXXBXRXDHJXTHX(₃.₅)HX_(I.₆₎BXCX₍₂.₅)CXXXBXVXSSJXRHX₍₃. ₅₎H (SEQ ID NO:46) The polypeptide can, for example, induce insulin gene expression, e.g., when present at an effective concentration in a human 293 cell. For example, the isolated polypeptide can include an amino acid sequence that is identical to the zinc fmger array within SEQ ID NO:6 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO: 6. The substitutions can be conservative substitutions. The isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:6. In one embodiment, the polypeptide can specifically bind to a target DNA site. For example, the polypeptide can compete with the 08_D04_6 chimeric ZFP (SEQ ID NO:6) for binding to a target DNA site, e.g., a site bound with a K of less than 10 nM. The polypeptide can further include a transcriptional regulatoiy domain, e.g., an activation or repression domain. The polypeptide can include one, two, or three or more additional zinc finger domains.

Also featured are nucleic acids encoding the above polypeptides. For example, the isolated polypeptide that includes the amino acid of SEQ ID NO:6 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:5. Featured nucleic acids can include operably linked regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth. In one embodiment, the coding nucleic acid is operably linlced to a conditional promoter such as an inducible promoter or a cell-type specific promoter. The nucleic acid can be included in a vector or integrated into a chromosome.

In addition, the invention features host cells (e.g., mammalian host cells) that include a polypeptide described above. The host cell can include a nucleic acid described above and express the nucleic acid. For example, the host cell can be a human cell that expresses the insulin gene, e.g., at least partially as a consequence of the artificial chimeric polypeptide. The invention also features a method of producing insulin that includes culturing a cell described herein in vitro, or of producing insulin in a subject by introducing a polypeptide described above or a nucleic acid encoding the polypeptide into a cell of the subject.

4. P B08 and Related Molecules

CX₍₂.₅₎CXXXBXQXSNJXRHX(₃.₅)HX(i.₆)BXCX₍₂.₅₎CXXXBXDXSNJXRHX₍₃_ ₅₎HX₍i.₆₎BXCX₍₂.₅₎CXXXBXDXSNJXRHX₍₃_₅)H (SEQ ID NO:47), where B is any amino acid, or optionally phenylalanine or tyrosine; and J is any amino acid, or optionally, a hydrophobic amino acid. This array is also abbreviated as: QSNR-DSNR-DSNR. Other exemplary artificial polypeptides include: CX₍2.5)CXXXBXQXSNJXRHX₍3-₅)HX(₁-6)BXCX(2.₅)CXXXBXDXSNJXRHX(₃. ₅₎H (SEQ ID NO:48); and

CX₍2.₅)CXXXBXDXSNJXRHX₍₃.₅)HX₍₁.6)BXCX₍₂.₅₎CXXXBXDXSNJXRHX₍₃. ₅₎H (SEQ ID NO:49). The polypeptide can, for example, increase expression of a heterologous polypeptide, e.g., a reporter polypeptide encoded by a SV40-SEAP reporter construct in a 293 cell when present at an effective concentration. For example, the isolated polypeptide can include an amino acid sequence that is identical to the zinc fmger array within SEQ ID NO: 8 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:8. The substitutions can be conservative substitutions. The isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 91% identical to the zinc finger array within SEQ ID NO: 8. In one embodiment, the polypeptide can specifically bind to a target DNA site. For example, the polypeptide can compete with the P_B08 chimeric ZFP (SEQ ID NO: 8) for binding to a target DNA site, e.g., a site bound with a K_d of less than 10 nM. The polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain. The polypeptide can include one, two, or three or more additional zinc finger domains.

Also featured are nucleic acids encoding the above polypeptides. For example, the isolated polypeptide that includes the amino acid of SEQ ID NO: 8 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:7. Featured nucleic acids can include operably linked regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a poIyA addition site, and so forth. In one embodiment, the coding nucleic acid is operably linlced to a conditional promoter such as an inducible promoter or a cell-type specific promoter. The nucleic acid can be included in a vector or integrated into a chromosome.

In addition, the invention features host cells (e.g., mammalian host cells) that include a polypeptide described above. The host cell can include a nucleic acid described above and express the nucleic acid. For example, the host cell can be a mammalian cell that has increased heterologous polypeptide production. 5. K DIP and Related Molecules

CX₍2.5)CXXXBXQXSHJXVHX₍₃.5)HX(_I.₆)BXCX₍2-₅)CXXXBXWXSNJXRHX(3. ₅)HX(₁_₆)BXCX₍2-₅)CXXXBXWXSNJXRHX₍₃.₅)HX₍i.6)BXCX₍₂. ₅₎CXXXBXRXDNJXQHX₍₃.₅₎H (SEQ ID NO:50), where B is any amino acid, or optionally phenylalanine or tyrosine; and J is any amino acid, or optionally, a hydrophobic amino acid. This array is also abbreviated as: QSHV-WSNR-WSNR-RDNQ. Other exemplary artificial polypeptides include: CX₍₂.₅₎CXXXBXWXSNJXRHX₍₃.5)HX₍ι.₆)BXCX₍2-5)CXXXBXWXSNJXRHX₍₃.

₅₎HX₍i.₆₎BXCX₍₂.₅₎CXXXBXRXDNJXQHX₍₃.₅₎H (SEQ ID NO:51); and

CX₍₂.5)CXXXBXQXSHJXVHX(₃.₅)HX(i.₆₎BXCX(2-₅)CXXXBXWXSNJXRHX(₃. ₅₎HX₍i.₆₎BXCX(₂.₅₎CXXXBXWXSNJXRHX₍₃.₅₎H (SEQ ID NO:52).

The polypeptide can decrease the cell proliferation when present at an effective concentration in a mammalian cell, e.g., by at least 30, 40, 50, or 60%. Cell proliferation may be assessed by cell number after a predefined incubation time. For example, the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO: 10 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO: 10. The substitutions can be conservative substitutions. The isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc fmger array within SEQ ID NO: 10. In one embodiment, the polypeptide can specifically bind to a target DNA site. For example, the polypeptide can compete with the K_D10 chimeric ZFP (SEQ ID NO: 10) for binding to a target DNA site, e.g., a site bound with a Kd of less than 10 nM. The polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain. The polypeptide can include one, two, or three or more additional zinc finger domains.

Also featured are nucleic acids encoding the above polypeptides. For example, the isolated polypeptide that includes the amino acid of SEQ ID NO: 10 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:9. Featured nucleic acids can include operably linked regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatoiy regions, a polyA addition site, and so forth. In one embodiment, the coding nucleic acid is operably linlced to a conditional promoter such as an inducible promoter or a cell-type specific promoter. The nucleic acid can be included in a vector or integrated into a chromosome.

In addition, the invention features host cells (e.g., eukaryotic, e.g., mammalian host cells) that include a nucleic acid described herein, e.g., a nucleic acid encoding polypeptide described herein. The host cell can also express the nucleic acid. For example, the host cell can be a mammalian cell that has a reduced proliferation rate relative to an otherwise identical cell that does not include the polypeptide.

6. K F02 and Related Molecules In still another aspect, the invention features an artificial polypeptide that includes: the sequence:

CX₍2.₅)CXXXBXDXSAJXRHX₍₃.₅)HX₍i-₆)BXCX(2.₅)CXXXBXRXDKJXRHX₍₃. ₅)HX(,.₆)BXCX(2.₅)CXXXBXRXDEJXRHX₍₃.₅)HX₍₁.₆₎BXCX(2. ₅₎CXXXBXQXTHJXRHX₍₃_₅₎H (SEQ ID NO:53), where B is any amino acid, or optionally phenylalanine or tyrosine; and J is any amino acid, or optionally, a hydrophobic amino acid. This array is also abbreviated as: DSAR-RDKR-RDER-QTHR. Other exemplary artificial polypeptides include:

CX(2-₅)CXXXBXRXDKJXRHX₍₃.₅)HX(i_₆)BXCX₍₂.₅)CXXXBXRXDEJXRHX₍₃. ₅₎HX₍,.₆₎BXCX₍₂-₅₎CXXXBXQXTHJXRHX₍₃_₅₎ (SEQ ID NO:54); and CX₍₂.₅₎CXXXBXDXSAJXRHX₍3.₅)HX₍i.₆)BXCX₍2.₅)CXXXBXRXDKJXRHX₍₃.

₅₎HX₍i.₆₎BXCX₍₂.₅₎CXXXBXRXDEJXRHX₍₃.₅₎H (SEQ ID NO:55).

The polypeptide can increase cell proliferation, e.g., by at least 50, 100, or 120%, in a mammalian cell, e.g., a 293 cell. For example, the isolated polypeptide can include an amino acid sequence that is identical to the zinc fmger array within SEQ ID NO: 12 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc fmger domains present in SEQ ID NO: 12. The substitutions can be conservative substitutions. The isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc fmger array within SEQ ID NO: 12. In one embodiment, the polypeptide can specifically bind to a target DNA site. For example, the polypeptide can compete with the K_F02 chimeric ZFP (SEQ ID NO: 12) for binding to a target DNA site, e.g., a site bound with a K of less than 10 nM. The polypeptide can further include a transcriptional regulatoiy domain, e.g., an activation or repression domain. The polypeptide can include one, two, or three or more additional zinc finger domains.

Also featured are nucleic acids encoding the above polypeptides. For example, the isolated polypeptide that includes the amino acid of SEQ ID NO: 12 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO: 11. Featured nucleic acids can include operably linked regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth. In one embodiment, the coding nucleic acid is operably linked to a conditional promoter such as an inducible promoter or a cell-type specific promoter. The nucleic acid can be included in a vector or integrated into a chromosome.

In addition, the invention features host cells (e.g., mammalian host cells) that include a nucleic acid described above. The host cell can include a nucleic acid described above and express the nucleic acid. For example, the host cell can be a mammalian cell that has an increased rate of cell proliferation relative to an otherwise identical cell that does not include the polypeptide.

7. K12 All and Related Molecules

In still another aspect, the invention features an artificial polypeptide that includes: the sequence: CX₍2.5)CXXXBXRXDHJXTHX(3-5)HX₍ι.₆₎BXCX₍2.5)CXXXBXQXSNJXVHX₍₃_

₅)_HX₍i-₆₎BXCX₍₂-₅₎CXXXBXQXTHJXRHX(3-₅₎HX₍ι.₆₎BXCX₍₂. ₅₎CXXXBXQXFNJXRHX₍₃.₅)H (SEQ ID NO:56), where B is any amino acid, or optionally phenylalanine or tyrosine; and J is any amino acid, or optionally, a hydrophobic amino acid. This array is also abbreviated as: RDHT-QSNV-QTHR-QFNR. Other exemplary polypeptides include:

CX(₂.₅)CXXXBXRXDHJXTHX(₃-₅)HX(i.₆₎BXCX₍₂.₅)CXXXBXQXSNJXNHX₍₃. ₅₎HX₍ι.₆₎BXCX₍₂-₅₎CXXXBXQXTHJXRHX₍₃.₅₎H (SEQ ID ΝO:57); and

CX(2-5)CXXXBXQXSNJXVHX(3-₅)HX₍₁.₆₎BXCX(2.₅)CXXXBXQXTHJXRHX₍₃. ₅₎HX₍i_₆₎BXCX₍₂-₅₎CXXXBXQXFNJXRHX₍₃-₅₎H (SEQ ID NO:58) The polypeptide can increase heterologous gene expression, e.g., a gene operable linked to a strong promoter such as a viral promoter, in a mammalian cell. For example, the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO:260 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:260. The substitutions can be conservative substitutions. The isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:260. In one embodiment, the polypeptide can specifically bind to a target DNA site. For example, the polypeptide can compete with the K12_A_11 chimeric ZFP (SEQ ID NO:260) for binding to a target DNA site, e.g., a site bound with a K_d of less than 10 nM. The polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain. The polypeptide can include one, two, or three or more additional zinc finger domains. Since the K12_A_11 chimeric ZFP includes a repression domain it is unlikely to increase heterologous gene expression by directly binding to the promoter of the heterologous gene.

Also featured are nucleic acids encoding the above polypeptides. For example, the isolated polypeptide that includes the amino acid of SEQ ID NO:260 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:259. Featured nucleic acids can include operably linked regulatoiy sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth. In one embodiment, the coding nucleic acid is operably linked to a conditional promoter such as an inducible promoter or a cell-type specific promoter. The nucleic acid can be included in a vector or integrated into a chromosome.

In addition, the invention features host cells (e.g., mammalian host cells) that include a nucleic acid described above. The host cell can include a nucleic acid described above and express the nucleic acid.

8. K44-16-E12 and Related Molecules

CX(2-₅)CXXXBXQXSHJXVHX(₃.₅)HX₍ι.₆)BXCX₍2.₅)CXXXBXQXSSJXRHX₍₃. ₅₎HX₍i.₆₎BXCX₍₂-₅₎CXXXBXQXTHJXRHX₍₃.₅₎H (SEQ ID NO:59), where B is any amino acid, or optionally phenylalanine or tyrosine; and J is any amino acid, or optionally, a hydrophobic amino acid. This array is also abbreviated as: QSHV-QSSR-QTHR. Other exemplary polypeptides include:

CX(₂-₅)CXXXBXQXSSJXRHX₍₃.5)HX(i.₆₎BXCX(₂.₅)CXXXBXQXTHJXRHX₍₃. ₅)H (SEQ ID NO:60); and

CX(₂.₅)CXXXBXQXSHJXNHX(₃.₅₎HX₍ι.₆₎BXCX(₂.₅)CXXXBXQXSSJXRHX₍₃. ₅₎H (SEQ ID ΝO:61).

The polypeptide can increase heterologous gene expression, e.g., a gene operable linlced to a strong promoter such as a viral promoter, in a mammalian cell. For example, the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO:262 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:262. The substitutions can be conservative substitutions. The isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:262. In one embodiment, the polypeptide can specifically bind to a target DNA site. For example, the polypeptide can compete with the K44-16-E12 chimeric ZFP (SEQ ID NO:262) for binding to a target DNA site, e.g., a site bound with a K_d of less than 10 nM. The polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain. The polypeptide can include one, two, or three or more additional zinc finger domains. Since the K44-16-E12 chimeric ZFP includes a repression domain it is unlikely to increase heterologous gene expression by directly binding to the promoter of the heterologous gene.

Also featured are nucleic acids encoding the above polypeptides. For example, the isolated polypeptide that includes the amino acid of SEQ ID NO:262 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:261. Featured nucleic acids can include operably linlced regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatoiy regions, a polyA addition site, and so forth. In one embodiment, the coding nucleic acid is operably linked to a conditional promoter such as an inducible promoter or a cell-type specific promoter. The nucleic acid can be included in a vector or integrated into a chromosome.

In addition, the invention features host cells (e.g., mammalian host cells) that include a nucleic acid described above. The host cell can include a nucleic acid described above and express the nucleic acid. For example, the host cell can be a mammalian cell (e.g., a 293 cell that has the phenotype of increased heterologous protein production (e.g., both a secreted and intracellular reporter protein encoded by genes operably linked to the CMN promoter).

9. K13 B08 and Related Molecules

CX(2-₅)CXXXBXQXSΝJXRHX₍₃_5)HX₍₁.₆₎BXCX₍2.₅)CXXXBXQXSSJXRHX₍₃. ₅)HX₍i.₆₎BXCX(2-₅)CXXXBXQXTHJXRHX(₃.₅)HX₍i.₆₎BXCX(2- ₅)CXXXBXRXDKJXRHX₍₃.₅₎H (SEQ ID NO:62), where B is any amino acid, or optionally phenylalanine or tyrosine; and J is any amino acid, or optionally, a hydrophobic amino acid. This array is also abbreviated as: QSNR-QSSR-QTHR-RDKR. Other exemplary artificial polypeptides include: CX(2-₅)CXXXBXQXSSJXRHX₍3.5)HX₍ι.₆₎BXCX(2-₅)CXXXBXQXTHJXRHX(3-

₅₎HX_(I.₆₎BXCX₍₂-₅₎CXXXBXRXDKJXRΗX₍₃.₅₎H (SEQ ID NO:63); and

CX(2-₅)CXXXBXQXSNJXRHX(3.₅)HX₍i.₆₎BXCX(2-₅)CXXXBXQXSSJXRHX₍₃. ₅₎HX₍i.₆₎BXCX₍₂-₅₎CXXXBXQXTHJXRHX₍₃.₅₎H (SEQ ID NO:64).

The polypeptide can cause increased SEAP production for an SEAP protein encoded by a heterologous gene encoded operably linked to the CMV promoter. For example, the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO:264 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:264. The substitutions can be conservative substitutions. The isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:264. In one embodiment, the polypeptide can specifically bind to a target DNA site. For example, the polypeptide can compete with the K13_B08 chimeric ZFP (SEQ ID NO:264) for binding to a target DNA site, e.g., a site bound with a K_d of less than 10 nM. The polypeptide can further include a transcriptional regulatoiy domain, e.g., an activation or repression domain. The polypeptide can include one, two, or three or more additional zinc finger domains. Also featured are nucleic acids encoding the above polypeptides. For example, the isolated polypeptide that includes the amino acid of SEQ ID NO:264 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:263. Featured nucleic acids can include operably linked regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth. In one embodiment, the coding nucleic acid is operably linlced to a conditional promoter such as an inducible promoter or a cell-type specific promoter. The nucleic acid can be included in a vector or integrated into a chromosome.

10. F104 p65 and Related Molecules

CX(2-5)CXXXBXRXDHJXTHX(3-5)HX(i.₆₎BXCX(2-₅)CXXXBXRXSHJXRHX(3. ₅₎HX₍i.₆₎BXCX₍₂-₅₎CXXXBXQXSHJXRHX₍₃.5)H (SEQ ID NO:65), where B is any amino acid, or optionally phenylalanine or tyrosine; and J is any amino acid, or optionally, a hydrophobic amino acid. This array is also abbreviated as: RDHT-RSHR-QSHR. Other exemplary artificial polypeptides include:

CX₍₂.₅)CXXXBXRXSHJXRHX(3-₅)HX(i.₆)BXCX₍₂.₅)CXXXBXQXSHJXRHX(3- ₅₎H (SEQ ID NO:66); and

CX₍₂-₅)CXXXBXRXDHJXTHX(3-5)HX(μ₆)BXCX(2-₅)CXXXBXRXSHJXRHX₍3. ₅)H (SEQ ID NO:67). For example, the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO: 18 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO: 18. The substitutions can be conservative substitutions. The isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 91% identical to the zinc fmger array within SEQ ID NO: 18. In one embodiment, the polypeptide can specifically bind to a target DNA site. For example, the polypeptide can compete with the F104_p65 chimeric ZFP (SEQ ID NO: 18) for binding to a target DNA site, e.g., a site bound with a K of less than 10 nM. The polypeptide can further include a transcriptional regulatoiy domain, e.g., an activation or repression domain. The polypeptide can include one, two, or three or more additional zinc finger domains. 5 Also featured are nucleic acids encoding the above polypeptides. For example, the isolated polypeptide that includes the amino acid of SEQ ID NO: 18 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO: 17. Featured nucleic acids can include operably linlced regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA o addition site, and so forth. In one embodiment, the coding nucleic acid is operably linlced to a conditional promoter such as an inducible promoter or a cell-type specific promoter. The nucleic acid can be included in a vector or integrated into a chromosome.

In addition, the invention features host cells (e.g., mammalian host cells) that include a nucleic acid described above. The host cell can include a nucleic acid described 5 above and express the nucleic acid.

11. F121 p65 and Related Molecules

In still another aspect, the invention features an artificial polypeptide that includes: the sequence: 0 CX₍₂.5)CXXXBXQXSHJXTHX₍₃.₅₎HX₍ι.₆₎BXCX₍₂.5)CXXXBXRXSHJXRHX₍₃.

₅₎HX₍₁.₆₎BXCX₍₂.₅₎CXXXBXRXDHJXTHX₍₃.₅₎H (SEQ ID NO:68), where B is any amino acid, or optionally phenylalanine or tyrosine; and J is any amino acid, or optionally, a hydrophobic amino acid. This array is also abbreviated as: QSHT-RSHR-RDHT. Other exemplary polypeptides include: 5 CX(₂.₅₎CXXXBXRXSHJXRHX(₃.₅)HX₍₁.₆)BXCX(2.₅)CXXXBXRXDHJXTHX₍₃.

₅₎H (SEQ ID NO:69); and

CX₍₂-₅₎CXXXBXQXSHJXTHX₍₃.₅)HX₍₁.₆₎BXCX₍₂.₅₎CXXXBXRXSHJXRHX₍₃. ₅₎H (SEQ ID NO:70).

For example, the isolated polypeptide can include an amino acid sequence that is 0 identical to the zinc finger array within SEQ ID NO:20 or that differs by no more than 8,

6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:20. The substitutions can be conservative substitutions. The isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:20. In one embodiment, the polypeptide can specifically bind to a target DNA site. For example, the polypeptide can compete with the F121_p65 chimeric ZFP (SEQ ID NO:20) for binding to a target DNA site, e.g., a site bound with a K_d of less than 10 nM. The polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain. The polypeptide can include one, two, or three or more additional zinc finger domains. The polypeptide can be used, for example, to regulate expression of insulin-like growth factor 2. Also featured are nucleic acids encoding the above polypeptides. For example, the isolated polypeptide that includes the amino acid of SEQ ID NO:20 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO: 19. Featured nucleic acids can include operably linlced regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth. In one embodiment, the coding nucleic acid is operably linked to a conditional promoter such as an inducible promoter or a cell-type specific promoter. The nucleic acid can be included in a vector or integrated into a chromosome.

12. K44-11-D01 and K44-11-G12 and Related Molecules

CX₍₂-₅₎CXXXBXQXSHJXVHX(₃-₅)HX(i-₆)BXCX₍2-₅₎CXXXBXQXSNJXIHX(₃. ₅₎HX(i.₆₎BXCX₍2.₅)CXXXBXQXTHJXRHX₍₃_5)HX₍₁.₆)BXCX(2. ₅₎CXXXBXCXSNJXRHX(₃.₅₎H (SEQ ID NO:265),

CX₍₂.₅)CXXXBXQXSHJXVHX(₃.₅)HX₍₁.₆₎BXCX(₂.₅)CXXXBXVXSTJXRHX₍₃. ₅₎HX₍₁_₆₎BXCX(2.₅)CXXXBXRXDNJXQHX₍₃.₅)HX₍₁.₆)BXCX(₂. ₅₎CXXXBXQXTHJXRHX(₃_₅₎H (SEQ ID NO:266), where B is any amino acid, or optionally phenylalanine or tyrosine; and J is any amino acid, or optionally, a hydrophobic amino acid. These arrays are also abbreviated as: QSHN-SQNI-QTHR-CSNR and QSHV-VSTR-RDNQ-QTHR.

For example, the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO: 14 or 16 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:20. The substitutions can be conservative substitutions. The isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO: 14 or 16. In one embodiment, the polypeptide can specifically bind to a target DNA site. For example, the polypeptide can compete with the F121_p65 chimeric ZFP (SEQ ID NO:14 or 16) for binding to a target DNA site, e.g., a site bound with a K_d of less than 10 nM. The polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain. The polypeptide can include one, two, or three or more additional zinc fmger domains. The polypeptide can be used, for example, to regulate protein production.

Also featured are nucleic acids encoding the above polypeptides. For example, the isolated polypeptide that includes the amino acid of SEQ ID NO: 14 or 16 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO.T3 or 15. Featured nucleic acids can include operably linlced regulatoiy sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth. In one embodiment, the coding nucleic acid is operably linlced to a conditional promoter such as an inducible promoter or a cell-type specific promoter. The nucleic acid can be included in a vector or integrated into a chromosome.

In addition, the invention features host cells (e.g., mammalian host cells) that include a nucleic acid described above. The host cell can include a nucleic acid described above and express the nucleic acid. * * *

As used herein, the "dissociation constant" refers to the equilibrium dissociation constant of a polypeptide for binding to a 28-basepair double-stranded DNA that includes one 9-basepair target site. The dissociation constant is determined by gel shift analysis using a purified protein that is bound in 20 mM Tris pH 7.7, 120 mM NaCl, 5 mM MgCl₂, 20 μM ZnSO₄, 10% glycerol, 0.1% Nonidet P-40, 5 mM DTT, and 0.10 mg/mL BSA

(bovine serum albumin) at room temperature. Additional details are provided in Example 10 and Rebar and Pabo (1994) Science 263:671-673. As used herein, the term "screen" refers to a process for evaluating members of a library to find one or more particular members that have a given property. In a direct screen, each member of the library is evaluated. For example, each cell is evaluated to detemiine if it is extending neurites. In another type of screen, temied a "selection," each member is not directly evaluated. Rather the evaluation is made by subjecting the members of the library to conditions in which only members having a particular property are retained. Selections may be mediated by survival (e.g., drag resistance) or binding to a surface (e.g., adhesion to a substrate). Such selective processes are encompassed by the term "screening." The term "base contacting positions," "DNA contacting positions," or "nucleic acid contacting positions" refers to the four amino acid positions of zinc finger domains that structurally correspond to the positions of amino acids arginine 73, aspartic acid 75, glutamic acid 76, and arginine 79 of ZIF268.

Glu Arg Pro Tyr Ala Cys Pro Val Glu Ser Cys Asp Arg Arg Phe Ser 1 5 10 15

Arg Ser Asp Glu Leu Thr Arg His lie Arg lie His Thr Gly Gin Lys

20 25 30

Pro Phe Gin Cys Arg lie Cys Met Arg Asn Phe Ser Arg Ser Asp His 35 40 45

Leu Thr Thr His lie Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys

50 55 60

Asp lie Cys Gly Arg Lys Phe Ala Arg Ser Asp Glu Arg Lys Arg His 65 70 75 80 Thr Lys lie His Leu Arg Gin Lys Asp (SEQ ID NO: 71)

85

These positions are also referred to as positions -1, 2, 3, and 6, respectively. To identify positions in a query sequence that correspond to the base contacting positions, the query sequence is aligned to the zinc finger domain of interest such that the cysteine and histidine residues of the queiy sequence are aligned with those of finger 3 of Zif268. The ClustalW WWW Service at the European Bioinformatics Institute (Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680) provides one convenient method of aligning sequences. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; a group of amino acids having acidic side chains is aspartic acid and glutamic acid; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Depending on circumstances, amino acids within the same group may be interchangeable. Some additional conservative amino acids substitution groups are: valine-leucine-isoleucine; phenylalanine-tyrosine; lysine-arginine; alanine-valine; aspartic acid-glutamic acid; and asparagine-glutamine. The term "heterologous polypeptide" refers either to a polypeptide with a non- naturally occurring sequence (e.g., a hybrid polypeptide) or a polypeptide with a sequence identical to a naturally occurring polypeptide but present in a milieu in which it does not naturally occur.

The terms "hybrid" and "chimera" refer to a non-naturally occurring polypeptide that comprises amino acid sequences derived from either (i) at least two different naturally occurring sequences, or non-contiguous regions of the same naturally occumng sequence, wherein the non-contiguous regions are made contiguous in the hybrid; (ii) at least one artificial sequence (i.e., a sequence that does not occur naturally) and at least one naturally occurring sequence; or (iii) at least two artificial sequences (same or different). Examples of artificial sequences include mutants of a naturally occurring sequence and de novo designed sequences. An "artificial sequence" is not present among naturally occumng sequences. With respect to any artificial sequence (e.g., protein or nucleic acid) described herein, the invention also refers to a sequence with the same elements, but which is not presenting each of the following organisms whose genomes are sequenced: Homo sapiens, Mus musculus, Arabidopsis thaliana, Drosophila melanogaster, Escherichia coli, Saccharomyces cerevisiae, and Oiγza sativa. A molecule with such a sequence can be expressed as a heterologous molecule in a cell of one of the aforementioned organisms.

The invention also includes sequences (not necessarily termed "artificial") which are made by a method described herein, e.g., a method of joining nucleic acid sequences encoding different zinc finger domains or a method of phenotypic screening. The invention also features a cell that includes such a sequence. As used herein, the term "hybridizes under stringent conditions" refers to conditions for hybridization in 6X sodium chloride/sodium citrate (SSC) at 45°C, followed by two washes in 0.2 X SSC, 0.1% SDS at 65°C.

The term "binding preference" refers to the discriminative property of a polypeptide for selecting one nucleic acid binding site relative to another. For example, when the polypeptide is limiting in quantity relative to two different nucleic acid binding sites, a greater amount of the polypeptide will bind the preferred site relative to the other site in an in vivo or in vitro assay described herein.

A "reference cell" refers to any standard cell. In one example, the reference cell is a parental cell for a cell that expresses a zinc finger protein, e.g., a cell that is substantially identical to the zinc finger protein expressing cell, but which does not produce the zinc finger protein.

Among other advantages, many of the methods and compositions relate to the identification and use of new and useful chimeric proteins, e.g., chimeric transcription factors. Some embodiments may include one or more of the following advantages: i) Endogenous genes can be either up- or down-regulated. Any given artificial chimeric transcription factor can be converted to transcriptional activator by fusing to appropriate transcriptional activation domains or to transcriptional repressor by fusing to repression domains. Moreover, even without a transcriptional regulatoiy domain, chimeric transcription factors can be potent repressors, e.g., when they bind to sites near the TATA box and the initiator element. Moreover, it is possible to screen a library that includes both activators and repressors with different DNA binding specificity, ii) Gene expression can be finely regulated. Depending on the DNA-binding affinity, chimeric transcription factors can cause a range of effects, e.g., moderate to strong activation and repression. This may lead to diverse phenotypes that are not necessarily obtained by completely inactivation or high level over-expressed of a particular target gene. For example, in some cases, broad specificity may be an advantage where the coordinated action of several genes is required to drive a desired phenotype. In other cases, narrow specificity is desired. One method for flexibly controlling specificity is by adding or removing zinc finger domains. 3-finger ZFPs theoretically regulate more genes than 6- finger ZFPs. iii) ZFP library approach is universally applicable. Since transcriptional regulatoiy mechanisms are highly conserved and because all known organism use DNA and transcription for life, chimeric proteins that bind to DNA can be used to regulate any desired cell. Further, as described herein, many methods do not require a priori infonnation (e.g., genome sequence) of the cell in order to identify useful chimeric proteins, iv) Artificial chimeric proteins can be used as a tool to dissect pathways within a cell. For example, target genes responsible for the phenotypic changes in selected clones can be identified, e.g., as described herein, v) A ZFP-TF may mimic the function of a master regulatoiy protein, such as a master regulatory transcription factor. For example, the ZFP-TF may bind to the same site as the master regulatory, or to an overlapping site, vi) The level of gene expression change, thus the extent of the phenotype generated by ZFP-TF, can be precisely controlled by altering the expression level of ZFP-TF in cells.

All patents, patent applications, and references cited herein are incorporated by reference in their entirety. The following patent applications: WO 01/60970 (Kim et al); U.S. Serial No. 60/338,441, filed December 7, 2001; U.S. Serial No. 60/313,402, filed August 17, 2001; U.S. Serial No. 60/374,355, filed April 22, 2002; U.S. Serial

No.60/376,053, filed April 26, 2002; U.S. Serial No. 60/400,904, filed August 2, 2002; U.S. Serial No. 60/401,089, filed August 5, 2002; and U.S. Serial No. 10/223,765, filed August 19, 2002, are expressly incorporated by reference in their entirety for all purposes. The details of one or more embodiments of the invention are set forth in the accompa- nying drawings and the description below. Any feature described herein can be used in combination with another compatible feature also described herein. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a region of the yeast plasmid pYTC-Lib. FIG. 2 is a schematic of an exemplary target-driven approach. FIG. 3 A is a schematic of a method for preparing an exemplary zinc finger protein library. FIG. 3B is a schematic of an exemplary phenotype-driven approach. FIG. 4 depicts an expression profile of transiently fransfected ZFPs. In the left panel, a cell expressing p65 activation domain alone is compared to a control. In the right panel, a cell expressing the zinc finger protein F121 jp65 is compared to a control. Each dot displaced from the diagonal represents a gene whose expression is substantially altered.

FIG. 5 depicts expression profiles of stably fransfected ZFPs.

FIG. 6 A depicts the effects of DNA-binding domain mutants and effector domain alterations to the K5 ZFP on drug resistance.

FIG. 6B depicts the effects of K5 and YLL053C overexpression. FIG. 7 depicts one method of constructing a diverse three finger libraiy.

FIG. 8 is a photograph of control and ZFP-expressing cells in which neurite formation is induced. FIG. 8A. pcDNA3(empty vector)-transfected Neuro2A cells without retinoic acid (RA) treatment. FIG. 8B. Neurol -p65 (also called 08_D01-65) expression without RA treatment. FIG. 8C. pcDNA3 (empty vector)-transfected Neuro2A cells with 10 μM RA. FIG. 8D. Neurol -p65 expression with 10 μM RA.

FIG. 9 depicts comparative microarray data for cells in which the insulin gene is over-expressed. The chimeric ZFP is 08_D04-p65. The sequence of which is listed in FIG. 16.

FIG. 10A is a truncated table listing ZFP DNA binding specificities. FIG. 10B is a schematic illustrating the correspondence between zinc finger domains and the recognition site sequence.

FIG. 11 is a flowchart for a method of identifying targets of artificial ZFPs.

FIG. 12 is a schematic of a method of identifying ZFPs that induce differentiation.

FIG. 13 depicts comparative microarray data at various time points for cells in which the F104-p65 ZFP is present.

FIG. 14 lists the coding nucleic acid (SEQ ID NO:l) and amino acid (SEQ ID NO:2) sequences for Neurol-p65, a protein that can induce neurites. Neurol-p65 includes the zinc finger domains QSNRl-QSNK-CSNRl and the p65 activation domain. Other artificial proteins with at least two or three consecutive zinc fmger domains that have the same pattern of DNA contacting residues as domains in QSNR1-QSNK-CSNR1- p65 may also induce neurites. FIG. 15 lists the coding nucleic acid (SEQ ID NO: 3) and amino acid (SEQ ID NO:4) sequences for Osteol-p65, a protein that can induce osteocytes. Osteol-p65 includes the zinc finger domains RDKR-QTHR1-VSTR-RDKR and the p65 activation domain. Other artificial proteins with at least two or three consecutive zinc finger domains that have the same pattern of DNA contacting residues as domains in RDKR- QTHRl-NSTR-RDKR-p65 may also induce osteocytes.

FIG. 16 lists the coding nucleic acid (SEQ ID ΝO:5) and amino acid (SEQ ID NO:6) sequences for 08_D04_p65, a protein that can enhance insulin production (See FIG. 9). 08_D04jp65 includes the zinc fmger domains RSHR-RDHT-VSSR and the p65 activation domain. Other artificial proteins with at least two or three consecutive zinc finger domains that have the same pattern of DNA contacting residues as domains in RSHR-RDHT-NSSR may also enhance insulin production.

FIG. 17 lists the coding nucleic acid (SEQ ID ΝO:7) and amino acid (SEQ ID NO:8) sequences for P_B08, a protein that can enhances SV40-SEAP. P_B08 includes the zinc finger domains QSNRl-DSNR-DSNR and the p65 activation domain. Other artificial proteins with at least two or three consecutive zinc finger domains that have the same pattern of DNA contacting residues as domains in QSNRl-DSNR-DSNR may also enhances SV40-SEAP.

FIG. 18 lists the coding nucleic acid (SEQ ID NO:9) and amino acid (SEQ ID NO: 10) sequences for K_D10, a protein that can decrease cell proliferation. K_D10 includes the zinc fmger domains QSHV-WSNR-WSNR-RDNQ and the kid repression domain. Other artificial proteins with at least two or three consecutive zinc finger domains that have the same pattem of DNA contacting residues as domains in QSHV- WSNR-WSNR-RDNQ may also decrease cell proliferation. FIG. 19 lists the coding nucleic acid (SEQ ID NO: 11) and amino acid (SEQ ID

NO: 12) sequences for K_F02, a protein that can enhance cell proliferation. K_F02 includes the zinc finger domains DSAR2-RDKR-RDER1-QHR1 and the kid repression domain. Other artificial proteins with at least two or three consecutive zinc fmger domains that have the same pattern of DNA contacting residues as domains in DSAR2- RDKR-RDERl-QHRl-kid may also enhance cell proliferation.

FIG. 20 lists the coding nucleic acid (SEQ ID NO: 13) and amino acid (SEQ ID NO: 14) sequences for K44_ 11_D01, a protein that can enhance protein expression. K44_11_D01 includes the zinc finger domains QSHN-QSNI-QTHRl-CSNRl and the kid repression domain. Other artificial proteins with at least two or three consecutive zinc finger domains that have the same pattern of DNA contacting residues as domains in QSHV-QSNI-QTHR1-CSNR1 may also enhance protein expression. FIG. 21 lists the coding nucleic acid (SEQ ID NO: 15) and amino acid (SEQ ID

NO: 16) sequences for K44-11-G12, a protein that can enhance protein expression. K44- 11-G12 includes the zinc finger domains QSHV-VSTR-RDNQ-QTHR1 and the kid repression domain. Other artificial proteins with at least two or three consecutive zinc finger domains that have the same pattern of DNA contacting residues as domains in QSHV-NSTR-RDNQ-QTHRl may also enhance protein expression.

FIG. 22 lists the coding nucleic acid (SEQ ID NO: 17) and amino acid (SEQ ID NO: 18) sequences for F104_p65. F104_p65 includes the zinc finger domains RDHT- RSHR-QSHR2-p65.

FIG. 23 lists the coding nucleic acid (SEQ ID NO: 19) and amino acid (SEQ ID NO:20) sequences for F121 _p65. F121__p65 includes the zinc finger domains QSHT- RSHR-RDHT-p65. Other artificial proteins with at least two or three consecutive zinc fmger domains that have the same pattem of DNA contacting residues as domains in QSHT-RSHR-RDHT may also increase transcription of insulin-like growth factor 2

FIG. 24 lists the coding nucleic acid (SEQ ID NO:259) and amino acid (SEQ ID NO: 260) sequences for K12_A11, which includes the zinc finger domains RDHT- QSNV2-QTHR1 -QFNR-kid.

FIG. 25 lists the coding nucleic acid (SEQ ID NO:261) and amino acid (SEQ ID NO:262) sequences for K44-16-E12, which includes the zinc fmger domains QSHV- QSSRl-QTHRl-kid. FIG. 26 lists the coding nucleic acid (SEQ ID NO:263) and amino acid (SEQ ID

NO:264) sequences for K13_B08, which includes the zinc finger domains QSNR1- QSSR1-QTHR1-RDKR-KID.

DNA sequences in the FIGs. 14 to 26 can include a sequence encoding a HA tag (gray underlined), and a sequence encoding a nuclear localization signal (boxed). The initiation and stop codons are underlined. The shaded letters indicate sequences encoding a regulatory domain, e.g., a p65 or kid domain. The small letters indicated the linker sequences. With respect to the amino acid sequences, italicized and shaded letters 1 VI ' "-re, U f \) CJ \J V

indicate zinc finger domains and shaded letters, not italicized indicate a regulatoiy domain, e.g., a Kid or p65 domain.

DNA sequences in the FIGs. 14 to 24 include a sequence encoding a HA tag (gray underlined), and a sequence encoding a nuclear localization signal (boxed). The initiation and stop codons are underlined. The shaded letters indicate sequences encoding a regulatory domain, e.g., a p65 or kid domain. The small letters indicated the linker sequences. With respect to the amino acid sequences, italicized and shaded letters indicate zinc finger domains and shaded letters, not italicized indicate a regulatoiy domain, e.g., a Kid or p65 domain.

DETAILED DESCRIPTION

In one aspect of the invention, a library of nucleic acids that encode different artificial, chimeric proteins is screened to identify a chimeric protein that alters a phenotypic trait of a cell or organism. The chimeric protein can be identified without a priori knowledge of a particular target gene or pathway.

In one example, each nucleic acid of the library encodes an artificial polypeptide that includes a plurality of zinc finger domains. A nucleic acid library that encodes different chimeric proteins can be prepared, for example, as described in the sections below. Members of the libraiy are introduced into cells in culture or cells in an organism. After an interval to allow for expression of the encoded polypeptides, cells or organisms which have an altered phenotypic trait are identified. The libraiy nucleic acid in at least one of these phenotypically altered cells is recovered, thus identifying an artificial chimeric polypeptide that produces the phenotypic effect.

Although this method is generally described below in the context of chimeras of zinc fmger domains, it is easily adapted to other stractural domains, including other DNA binding domains and cell signaling domains.

Library Construction: 1. Exemplary Structural Domains

The nucleic acid library is constructed so that it includes nucleic acids that each encode and can express an artificial protein that is a chimera of one or more stractural domains. In some aspects, the stractural domains are nucleic acid binding domains that vary in specificity such that the libraiy encodes a population of proteins with different binding specificities.

A variety of structural domains are known to bind nucleic acids with high affinity and high specificity. For reviews of stractural motifs which recognize double stranded DNA, see, e.g., Pabo and Sauer (1992) Annu. Rev. Biochem. 61:1053-95; Patikoglou and Burley (1997) Annu. Rev. Biophys. Biomol. Struct. 26:289-325; Nelson (1995) Curr Opin Genet Dev. 5:180-9. A few non-limiting examples of nucleic acid binding domains include:

Zinc fingers. Zinc fingers are small polypeptide domains of approximately 30 amino acid residues in which there are four amino acids, either cysteine or histidine, appropriately spaced such that they can coordinate a zinc ion (For reviews, see, e.g., Klug and Rhodes, (1987) Trends Biochem. ScU2:464-469(1987); Evans and Hollenberg, (1988) Cell 52:1-3; Payre and Vincent, (1988) FEBSLett. 234:245-250; Miller et al, (1985) EMBOJ. 4: 1609-1614; Berg, (1988) Proc. Natl. Acad. Sci. U.S.A. 85:99-102; Rosenfeld and Margalit, (1993) J. Biomol. Struct. Dyn. 11:557-570). Hence, zinc finger domains can be categorized according to the identity of the residues that coordinate the zinc ion, e.g., as the Cys2-His2 class, the Cys2-Cys₂ class, the Cys₂-CysHis class, and so forth. The zinc coordinating residues of Cys₂-His2 zinc fingers are typically spaced as follows: X_a-X-C-X₂-₅-C-X₃-X_a-X₅-ψ-X₂-H-X₃.₅-H (SEQ ID NO:72), where ψ (psi) is a hydrophobic residue (Wolfe et al, (1999) Annu. Rev. Biophys. Biomol. Struct. 3:183-212), wherein "X" represents any amino acid, wherein X_a is phenylalanine or tyrosine, the subscript indicates the number of amino acids, and a subscript with two hyphenated numbers indicates a typical range of intervening amino acids. Typically, the intervening amino acids fold to form an anti-parallel β-sheet that packs against an α-helix, although the anti-parallel β-sheets can be short, non-ideal, or non-existent. The fold positions the zinc-coordinating side chains so they are in a tetrahedral conformation appropriate for coordinating the zinc ion. The base contacting residues are at the N-terminus of the finger and in the preceding loop region.

For convenience, the primary DNA contacting residues of a zinc finger domain are numbered: -1, 2, 3, and 6 based on the following example: - 1 1 2 3 4 5 6 X_a-X- C -X₂-₅ - C-X₃ -X_a-X- C-X- S -N-X_b-X-R-H-X₃-₅ -H ( SEQ ID NO : 73 ) , where X_a is typically phenylalanine or tyrosine, and X_b is typically a hydrophobic residue. As noted in the example above, the DNA contacting residues are Cys (C), Ser (S), Asn (N), and Arg (R). The above motif can be abbreviated CSNR. As used herein, such abbreviation refers to a class of sequences which include a domain corresponding to the motif. The class also includes a species whose sequence includes a particular polypeptide sequence, typically a sequence listed in Table 1 or 2 which corresponds to the motif. Where two sequences have the same motif, a number may be used to indicate the sequence.

A zinc finger protein typically consists of a tandem array of three or more zinc finger domains. For example, zinc finger domains whose motifs are listed consecutively are not interspersed with other folded domains, but may include a linker, e.g., a flexible linker described herein between domains.

The zinc finger domain (or "ZFD") is one of the most common eukaryotic DNA- binding motifs, found in species from yeast to higher plants and to humans. By one estimate, there are at least several thousand zinc finger domains in the human genome alone, possibly at least 4,500. Zinc finger domains can be isolated from zinc fmger proteins. Non-limiting examples of zinc finger proteins include CF2-II, Krappel, WTl, basonuclin, BCL-6/LAZ-3, erythroid Kruppel-like transcription factor, Spl, Sp2, Sp3, Sp4, transcriptional repressor YY1, EGRl/Krox24, EGR2/Krox20, EGR3/Pilot, EGR4/AT133, Evi-1, GLI1, GLI2, GLI3, HIV-EP1/ZNF40, HIV-EP2, KR1, ZfX, ZfY, and ZNF7. Computational methods described below can be used to identify all zinc fmger domains encoded in a sequenced genome or in a nucleic acid database. Any such zinc finger domain can be utilized. In addition, artificial zinc finger domains have been designed, e.g., using computational methods (e.g., Dahiyat and Mayo, (1997) Science 278:82-7). It is also noteworthy that at least some zinc finger domains bind to ligands other than DNA, e.g., RNA or protein. Thus, a chimera of zinc finger domains or of a zinc finger domain and another type of domain can be used to recognize a variety of target compounds, not just DNA.

WO 01/60970, U.S. Serial No. 60/374,355, filed April 22, 2002, and U.S. Serial No. 10/223,765, filed August 19, 2002, describe exemplary zinc finger domains which can be used to construct an artificial zinc finger protein. See also the Table 1, below. Homeodomains. Homeodomams are eukaryotic domains that consist of a

N-tem inal ami that contacts the DNA minor groove, followed by three α-helices that contact the major groove (for a review, see, e.g., Laughon, (1991) Biochemistiy 30:11357- 67). The third α-helix is positioned in the major groove and contains critical DNA- contacting side chains. Homeodomains have a characteristic highly-conserved motif present at the rum leading into the third α-helix. The motif includes an invariant tryptophan that packs into the hydrophobic core of the domain. This motif is represented in the Prosite database as PDOC00027 ([L/I/V/M/F/Y/G]-[A/S/L/V/R]-X(2)- [L/I/V/M/S/T/A/C/N]-X-[L/I/V/M]-X(4)-[L/I/V]- [R/K/N/Q/E/S/T/A/I/Y]- [L/I/V/F/S/T/N/K/H]-W-[F/Y/V/C]-X-[N/D/Q/T/A H]-X(5)- [R/K/N/A/I/M/W]. Homeodomains are commonly found in transcription factors that detennine cell identity and provide positional infonnation during organismal development. Such classical homeodomains can be found in the genome in clusters such that the order of the homeodomains in the cluster approximately corresponds to their expression pattem along a body axis. Homeodomains can be identified by alignment with a homeodomain, e.g., Hox-1, or by alignment with a homeodomain profile or a homeodomain hidden Markov Model (HMM; see below), e.g., PF00046 of the Pfam database or "HOX" of the SMART database, or by the Prosite motif PDOC00027 as mentioned above.

Helix-turn-helix proteins. This DNA binding motif is common among many prokaryotic transcription factors. There are many subfamilies, e.g., the Lad family, the AraC family, to name but a few. The two helices in the name refer to a first α-helix that packs against and positions a second α-helix in the major groove of DNA. These domains can be identified by alignment with a HMM, e.g., HTH_ARAC, HTH_ARSR, HTH_ASNC, HTH_CRP, HTH_DEOR, HTH_DTXR, HTH_GNTR, HTH CLR, HTH LACI, HTH LUXR, HTH_MARR, HTH_MERR, and HTH_XRE profiles available in the SMART database. Library Construction: 2. Identification of Structural Domains

A variety of methods can be used to identify stractural domains. Nucleic acids encoding identified domains are used to construct the nucleic acid library. Further, nucleic acid encoding these domains can also be varied (e.g., mutated) to provide additional domains that are encoded by the libraiy.

Computational Methods. To identify additional naturally-occurring stractural domains, the amino acid sequence of a known stractural domain can be compared to a database of known sequences, e.g., an annotated database of protein or nucleic acid sequences. In another implementation, databases of uncharacterized sequences, e.g., unannotated genomic, EST or full-length cDNA sequence; of characterized sequences, e.g., SwissProt or PDB; and of domains, e.g., Pfam, ProDom (Corpet et al. (2000) Nucleic Acids Res. 28:267-269), and SMART (Simple Modular Architecture Research Tool, Letunic et al. (2002) Nucleic Acids Res 30, 242-244) can provide a source of stractural domain sequences. Nucleic acid sequence databases can be translated in all six reading frames for the purpose of comparison to a queiy amino acid sequence. Nucleic acid sequences that are flagged as encoding candidate nucleic acid binding domains can be amplified from an appropriate nucleic acid source, e.g., genomic DNA or cellular RNA. Such nucleic acid sequences can be cloned into an expression vector. The procedures for computer-based domain identification can be interfaced with an oligonucleotide synthesizer and robotic systems to produce nucleic acids encoding the domains in a high- throughput platform. Cloned nucleic acids encoding the candidate domains can also be stored in a host expression vector and shuttled easily into an expression vector, e.g., into a translational fusion vector with other domains (of a similar or different type), either by restriction enzyme mediated subcloning or by site-specific, recombinase mediated subcloning (see U.S. Patent No. 5,888,732). The high-throughput platfomi can be used to generate multiple microtitre plates containing nucleic acids encoding different candidate chimeras.

Detailed methods for the identification of domains from a starting sequence or a profile are well known in the art. See, for example, Prosite (Hofmann et al, (1999) Nucleic Acids Res. 27:215-219), FASTA, BLAST (Altschul et al, (1990) J. Mol. Biol. 215:403-10.), etc. A simple string search can be done to find amino acid sequences with identity to a queiy sequence or a query profile, e.g., using Perl to scan text files. Sequences so identified can be about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater identical to an initial input sequence.

Domains similar to a query domain can be identified from a public database, e.g., using the XBLAST programs (version 2.0) of Altschul et al, (1990) J. Mol. Biol 215 :403- 10. For example, BLAST protein searches can be perfomied with the XBLAST parameters as follows: score = 50, wordlength = 3. Gaps can be introduced into the query or searched sequence as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402. Default parameters for XBLAST and Gapped BLAST programs are available at National Center for Biotechnology Infonnation (NCBI), National Institutes of Health, Bethesda MD.

The Prosite profiles PS00028 and PS50157 can be used to identify zinc finger domains. In a SWISSPROT release of 80,000 protein sequences, these profiles detected 3189 and 2316 zinc fmger domains, respectively. Profiles can be constructed from a multiple sequence alignment of related proteins by a variety of different techniques. Gribslcov and co-workers (Gribskov et al, (1990) Meth. Enzymol 183:146-159) utilized a symbol comparison table to convert a multiple sequence alignment supplied with residue frequency distributions into weights for each position. See, for example, the PROSITE database and the work of Luethy et al, (1994) Protein Sci. 3:139-1465.

Hidden Markov Models (HMM's) representing a DNA binding domain of interest can be generated or obtained from a database of such models, e.g., the Pfam database, release 2.1. A database can be searched, e.g., using the default parameters, with the HMM in order to find additional domains (see, e.g., Bateman et al. (2002) Nucleic Acids Research 30:276-280). Alternatively, the user can optimize the parameters. A threshold score can be selected to filter the database of sequences such that sequences that score above the threshold are displayed as candidate domains. A description of the Pfam database can be found in Sonhammer et al, (1997) Proteins 28(3):405-420, and a detailed description of HMMs can be found, for example, in Gribskov et al, (1990) Meth. Enzymol 183:146-159; Gribskov et al, (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al, (1994) J. Mol. Biol. 235:1501-1531; and Stultz et al, (1993) Protein Sci. 2:305-314.

The SMART database of HMM's (Simple Modular Architecture Research Tool, Schultz et al, (1998) Proc. Natl. Acad. Sci. USA 95:5857 and Schultz et al, (2000) Nucl Acids Res 28:231) provides a catalog of zinc finger domains (ZnF_C2H2; ZnF_C2C2; ZnF_C2HC; ZnF_ C3Hl; ZnF_C4; ZnF_CHCC; ZnF_GATA; and ZnF_NFX) identified by profiling with the hidden Markov models of the HMMer2 search program (Durbin et al, (1998) Biological sequence analysis: probabilistic models of proteins and nucleic acids. Cambridge University Press).

Hybridization-based Methods. A collection of nucleic acids encoding various forms of a structural domain can be analyzed to profile sequences encoding conserved amino- and carboxy-tenninal boundary sequences. Degenerate oligonucleotides can be designed to hybridize to sequences encoding such conserved boundary sequences. Moreover, the efficacy of such degenerate oligonucleotides can be estimated by comparing their composition to the frequency of possible annealing sites in known genomic sequences. If desired, multiple rounds of design can be used to optimize the degenerate oligonucleotides.

Comparison of known Cys₂-His2 zinc fingers, for example, revealed a common sequence in the linker region between adjacent fingers in natural sequence (Agata et al, (1998) Gene 213:55-64). Degenerate oligonucleotides that anneal to nucleic acid encoding the conserved linker region were used to amplify a plurality of zinc finger domains. The amplified nucleic acid encoding the domains can be used to construct nucleic acids that encode a chimeric array of zinc fingers.

Library Construction: 3. Nucleic Acids Encoding Structural Domains

Nucleic acids that are used to assemble the library can be obtained by a variety of methods. Some component nucleic acids of the library can encode naturally occumng domains. In addition, some component nucleic acids are variants that are obtained by mutation or other randomization methods. The component nucleic acids, typically encoding just a single domain, can be joined to each other to produce nucleic acids encoding a fusion of the different domains.

Isolation of a natural repertoire of domains. A library of domains can be constracted by isolation of nucleic acid sequences encoding domains from genomic DNA or cDNA of eukaiyotic organisms such as humans. Multiple methods are available for doing this. For example, a computer search of available amino acid sequences can be used to identify the domains, as described above. A nucleic acid encoding each domain can be isolated and inserted into a vector appropriate for the expression in cells, e.g., a vector containing a promoter, an activation domain, and a selectable marker. In another example, degenerate oligonucleotides that hybridize to a conserved motif are used to amplify, e.g., by PCR, a large number of related domains containing the motif. For example, Kruppel-like Cys2His₂ zinc fingers can be amplified by the method of Agata et al., (1998) Gene 213:55-64. This method .also maintains the naturally occurring zinc fmger domain linker peptide sequences, e.g., sequences with the pattern: Thr-Gly- (Glu/Gln)-(Lys/Arg)-Pro-(Tyr/Phe) (SEQ ID NO:74). Moreover, screening a collection limited to domains of interest, unlike screening a library of unselected genomic or cDNA sequences, significantly decreases library complexity and reduces the likelihood of missing a desirable sequence due to the inherent difficulty of completely screening large libraries.

The human genome contains numerous zinc finger domains, many of which are uncharacterized and unidentified. It is estimated that there are thousands of genes encoding proteins with zinc fmger domains (Pellegrino and Berg, (1991) Proc. Natl. Acad. Sci. USA 88:671-675). These human zinc finger domains represent an extensive collection of diverse domains from which novel DNA-binding proteins can be constracted. Many exemplary human zinc finger domains are described in WO 01/60970, U.S. Serial No. 60/374,355, filed April 22, 2002, and U.S. Serial No. 10/223,765, filed August 19, 2002. See also Table 1 below.

Table 1.

ZFD Amino acid sequence SEQ Target

ID subsite (s)

NO:

CSNR1 YKCKQCGKAFGCPSNLRRHGRTH 75 GAA>GAC>GAG

CSNR2 YQCNICGKCFSCNSNLHRHQRTH 76 GAA>GAC>GAG

DSAR YSCGICGKSFSDSSAKRRHCILH 77 GTC

DSCR YTCSDCGKAFRDKSCLNRHRRTH 78 GCC

HSNK YKCKECGKAFNHSSNFNKHHRIH 79 GAC

HSSR FKCPVCGKAFRHSSSLVRHQRTH 80 GTT

ISNR YRCKYCDRSFSISSNLQRHVRNIH 81 GAA>GAT>GAC

ISNV YECDHCGKAFSIGSNLNVHRRIH 82 AAT SNR YGCHLCGKAFSKSSNLRRHEMIH 83 GAG

QAHR YKCKECGQAFRQRAHLIRHHK H 84 GGA

QFNR YKCHQCGKAFIQSFNLRRHERTH 85 GAG

QGNR FQCNQCGASFTQKGNLLRHIKLH 86 GAA QSHR1 YACHLCGKAFTQSSHLRRHEKTH 87 GGA>GAA>AGA

QSHR2 YKCGQCGKFYSQVSHLTRHQKIH 88 GGA

QSHR3 YACHLCGKAFTQCSHLRRHEKTH 89 GGA>GAA

QSHR4 YACHLCAKAFIQCSHLRRHEKTH 90 GGA>GAA

QSHR5 YVCRECGRGFRQHSHLVRHKRTH 91 GGA>AGA>GAA>CGA

QSHT YKCEECGKAFRQSSHLTTHKIIH 92 AGA, CGA>TGA>GGA

QSHV YECDHCGKSFSQSSHLNVHKRTH 93 CGA>AGA>TGA

QSNI YMCSECGRGFSQKSNLIIHQRTH 94 AAA, CAA

QSNK YKCEECGKAFTQSSNLTKHKKIH 95 GAA>TAA>AAA

QSNR1 FECKDCGKAFIQKSNLIRHQRTH 96 GAA

QSNR2 YVCRECRRGFSQKSNLIRHQRTH 97 GAA

QSNR3 YECEKCGKAFNQSSNLTRHKKSH 98 GAA

QSNV1 YECNTCRKTFSQKSNLIVHQRTH 99 AAA>CAA

QSNV2 YVCSKCGKAFTQSSNLTVHQKIH 100 AAA>CAA

QSNV3 YKCDECGKNFTQSSNLIVHKRIH 101 AAA

QS V4 YECDVCGKTFTQKSNLGVHQRTH 102 AAA

QSNT YECVQCGKGFTQSSNLI HQRVH 103 AAA

QSSR1 YKCPDCGKSFSQSSSLIRHQRTH 104 GTA>GCA

QSSR2 YECQDCGRAFNQNSSLGRHKRTH 105 GTA

QSSR3 YECNECGKFFSQSSSLIRHRRSH 106 GTA>GCA

QSTR YKCEECGKAFNQSSTLTRHKIVH 107 GTA>GCA

QSTV YECNECGKAFAQNSTLRVHQRIH 108 ACA

QTHQ YECHDCGKSFRQSTHLTQHRRIH 109 AGA>CGA, TGA

QTHR1 YECHDCGKSFRQSTHLTRHRRIH 110 GGA>AGA, GAA

QTHR2 H CLECGKCFSQNTHLTRHQRT 111 GGA

RDER1 YVCDVEGCTWKFARSDELNRHKKRH 112 GCG>GTG,GAC

RDER2 YHCDWDGCGWKFARSDELTRHYRKH 113 GCG>GTG

RDER3 YRCSWEGCEWRFARSDELTRHFRKH 114 GCG>GTG

RDER4 FSCSWKGCERRFARSDELSRHRRTH 115 GCG>GTG

RDER5 FACSWQDCNKKFARSDELARHYRTH 116 GCG

RDER6 YHCNWDGCGWKFARSDELTRHYRKH 117 GCG>GTG

RDHR1 FLCQYCAQRFGRKDHLTRHMKKSH 118 GAG,GGG

RDHT FQCKTCQRKFSRSDHLKTHTRTH 119 AGG,CGG,GGG,TGG

RDKI FACEVCGVRFTRNDKLKIHMRKH 120 GGG

RDKR YVCDVEGCTWKFARSDKLNRHKKRH 121 GGG>AGG

RSHR YKCMECGKAFNRRSHLTRHQRIH 122 GGG

RSNR YICRKCGRGFSRKSNLIRHQRTH 123 GAG>GTG

RTNR YLCSECDKCFSRSTNLIRHRRTH 124 GAG

SSNR YECKECGKAFSSGSNFTRHQRIH 125 GAG>GAC

VSNV YECDHCGKAFSVSSNL VHRRIH 126 AAT>CAT>TAT

VSSR YTCKQCGKAFSVSSSLRRHETTH 127 GTT>GTG>GTA

VSTR YECNYCGKTFSVSSTLIRHQRIH 128 GCT>GCG

WSNR YRCEECGKAFRWPSNLTRHKRIH 129 GGT>GGA

If each zinc finger domain recognizes a unique 3- to 4-bp sequence, the total number of domains required to bind eveiy possible 3- to 4-bp sequence is only 64 to 256 (4³ to 4⁴). It is possible that the natural repertoire of the human genome contains a sufficient number of unique zinc fmger domains to span all possible recognition sites. These zinc finger domains are a valuable resource for constructing artificial chimeric DNA-binding proteins. A nucleic acid library can include nucleic acids encoding proteins that include naturally occurring zinc finger domains, artificial mutants of such domains, and combinations thereof.

Mutated Domains. In one implementation, the library includes nucleic acids encoding at least one structural domain that is an artificial variant of a naturally-occunϊng sequence. In one embodiment, such variant domains are assembled from a degenerate patterned library. In the case of a nucleic acid binding domains, positions in close proximity to the nucleic acid binding interface or adjacent to a position so located can be targeted for mutagenesis. A mutated test zinc fmger domain, for example, can be constrained at any mutated position to a subset of possible amino acids by using a patterned degenerate libraiy. Degenerate codon sets can be used to encode the profile at each position. For example, codon sets are available that encode only hydrophobic residues, aliphatic residues, or hydrophilic residues. The library can be selected for full- length clones that encode folded polypeptides. Cho et al. ((2000) J. Mol. Biol. 297(2):309-19) provides a method for producing such degenerate libraries using degenerate oligonucleotides, and also provides a method of selecting library nucleic acids that encode full-length polypeptides. Such nucleic acids can be easily inserted into an expression plasmid, e.g., using convenient restriction enzyme cleavage sites.

Selection of the appropriate codons and the relative proportions of each nucleotide at a given position can be determined by simple examination of a table representing the genetic code, or by computational algorithms. For example, Cho et al, supra, describe a computer program that accepts a desired profile of protein sequence and outputs a prefen-ed oligonucleotide design that encodes the sequence.

See also Zhang et al, (2000) J Biol. Chem. 275:33850-33860; Rebar and Pabo (1994) Science 263:671-673; Segal (1999) Proc. Natl. Acad. Sci. USA 96:2758; Gogus et al, (1996) Proc. Natl. Acad. Sci. USA. 93:2159-2164; Drier et al, (2001) J. Biol. Chem. 276: 29466-29478; Liu et al. (2001) J. Biol. Chem. 276(14):11323-11334; and Hsu et α/., (1992) Science 257:1946-50 for some available zinc finger domains. Library Construction: 4. A Library of Chimeric Zinc Finger Proteins

A libraiy of nucleic acids encoding diverse chimeric zinc finger proteins can be formed by serial ligation, e.g., as described in Example 1. The library can be constracted such that each nucleic acid encodes a protein that has at least three, four, or five zinc finger domains. In some implementations, particularly for large libraries, each zinc finger coding segment can be designed to randomly encode any one of a set of zinc finger domains. The set of zinc finger domains can be selected to represent domains with a range of specificities, e.g., covering 30, 40, 50 or more of the 64 possible 3-basepair subsites. The set can include at least about 12, 15, 20, 25, 30, 40 or 50 different zinc finger domains. Some or all of these domains can be domains isolated from naturally occunϊng proteins. Moreover, because there may be little or no need for more than one zinc fmger domain for a given 3-basepair subsite, it may be possible to generate a libraiy using a small number of component domains, e.g., less than 500, 200, 100, or even less than 64 total component domains. One exemplary libraiy includes nucleic acids that encode a chimeric zinc finger protein having three fingers and 30 possible domains at each finger position. In its fully represented form, this libraiy includes 27,000 sequences (i.e., the result of 30³). The library can be constructed by serial ligation in which a nucleic acid from a pool of nucleic acids encoding all 30 possible domains is added at each step. In one embodiment, the libraiy can be stored as a random collection. In another embodiment, individual members can be isolated, stored at an addressable location (e.g., arrayed), and sequenced. After high throughput sequencing of 40 to 50 thousand constracted libraiy members, missing chimeric combinations can be individually assembled in order to obtain complete coverage. Once arcayed, e.g., in microtitre plates, each individual member can be recovered later for further analysis, e.g., for a phenotypic screen. For example, equal amounts of each arrayed member can be pooled and then transfomied into a cell. Cells with a desired phenotype are selected and characterized. In another example, each member is individually transfomied into a cell, and the cell is characterized, e.g., using a nucleic acid microarcay to detennine if the transcription of endogenous genes is altered (see "Profiling Regulatory Properties of a Chimeric Zinc Finger Protein," below). Introducing Nucleic Acid Libraries into Cells

Library nucleic acids can be introduced into cells by a variety of methods. In one example, the library is stored as a random pool including multiple replicates of each library nucleic acid. An aliquot of the pool is transfomied into cells. In another embodiment, individual library members are stored separately (e.g., in separate wells of a microtitre plate or at separate addresses of an array) and are individually introduced into cells.

In still another embodiment, the libraiy members are stored in pools that have a reduced complexity relative to the library as a whole. For example, each pool can include 10³ different library members from a libraiy of IO⁵ or 10° different members. When a pool is identified as having a member that causes a particular effect, the pool is deconvolved to identify the individual library member that mediates the phenotypic effect. This approach is useful when recovery of the altered cell is difficult, e.g., in a screen for chimeric proteins that cause apoptosis. Library nucleic acids can be introduced into cells by a variety of methods.

Exemplary methods include electroporation (see, e.g., U.S. 5,384,253); microprojectile bombardment techniques (see, e.g., U.S. 5,550,318; 5,538,880; and 5,610,042; and WO 94/09699); liposome-mediated transfection (e.g., using LIPOFECTAMINE™ (Invitrogen) or SUPERFECT™ (QIAGEN GmbH); see, e.g., Nicolau et al, Methods Enzymol, 149: 157-176, 1987.); calcium phosphate or DEAE-Dextran mediated transformation (see, e.g., Rippe et al, (1990) Mol. Cell Biol, 10:689-695); direct microinjection or sonication loading; receptor mediated transfection (see, e.g., EP 273 085); and Agrobacteriw7i-mediated transfoπnation (see, e.g., U.S. 5,563,055 and 5,591,616). The term "transform," as used herein, encompasses any method that introduces an exogenous nucleic acid into a cell.

It is also possible to use a viral particle to deliver a libraiy nucleic acid into a cell in vitro or in vivo. In one embodiment, viral packaging is used to deliver the libraiy nucleic acids to cells within an organism. In another embodiment, the library nucleic acids are introduced into cells in vitro, after which the cells are transferred into an organism.

After introduction of the libraiy nucleic acids, the library nucleic acids are expressed so that the chimeric proteins encoded by the library are produced by the cells. Constant regions of the libraiy nucleic acid can provide necessary regulatoiy and supporting sequences to enable expression. Such sequences can include transcriptional promoters, transcription terminators, splice site donors and acceptors, untranslated regulatoiy regions (such as polyA addition sites), bacterial origins of replication, markers for indicating the presence of the library nucleic acid or for selection of the library nucleic acid.

Screening Nucleic Acid Libraries Encoding Chimeric Proteins

In a screen, the cells or organisms are evaluated to identify ones that have an altered phenotype. This process can be adapted to the phenotype of interest. As the number of possible pheno types is vast, so too are the possibilities for screening. Numerous genetic screens and selections have been conducted to identify mutants or overexpressed naturally occurring genes that result in particular phenotypes. Any of these methods can be adapted to identify useful members of a nucleic acid library encoding chimeric proteins. A screen can include evaluating each cell or organism that includes a library nucleic acid or a selection, e.g., evaluating cells or organisms that survive or otherwise withstand a particular treatment.

Exemplary methods for evaluating cells include microscopy (e.g., light, confocal, fluorescence, scanning electron, and transmission electron), fluorescence based cell sorting, differential centrifugation, differential binding, immunoassays, enzymatic assays, growth assays, and in vivo assays.

Some screens involve particular environmental conditions. Cells that are sensitive or resistant to the condition are identified.

Some screens require detection of a particular behavior of a cell (e.g., chemotaxis, morphological changes, or apoptosis), or a particular behavior of an organism (e.g., phototaxis by a plant, mating behavior by a Drosophila, and so forth). In one embodiment, the cells or organisms can be evaluated directly, e.g., by visual inspection, e.g., using a microscope and optionally computer software to automatically detect altered cells. In another embodiment, the cells or organisms can be evaluated using an assay or other indicator associated with the desired phenotype. Some screens relate to cell proliferation. Cells that proliferate at a different rate relative to a reference cell (e.g., a normal cell) are identified. In addition, cells that have an altered response to a proliferative signal (e.g., a growth factors or other mitogen) can be identified. The cells may be more or less sensitive to the signal. Screens that relate to cell differentiation can also be used. The screening and use of chimeric zinc finger proteins can be used to modulate the differentiative and proliferative capacity of a variety of cells, including stem cells, such as ES cells and somatic stem cells, both human and otherwise. Zinc finger proteins can be found that direct ES cells to differentiate into a restricted lineage, such as neuronal progenitor cells or hematopoietic stem cells. It should be also possible to screen for zinc finger proteins that can direct differentiation of stem cells toward a defined post-mitotic cell subtype, for example, directing differentiation of ES cells and/or neural stem cells to dopaminergic or cholinergic neurons.

Among other phenotypes to evaluate differentiation, it is possible to look at expression of marker genes and marker proteins. Examples of such markers include:

^■ FLK1 for endothelial cells (Cho et al, (2001) Blood 98:3635-42; Nishikawa et al, Development 125: 1747-1757),

^■ vascular smooth muscle cell-specific myosin heavy chain for smooth muscle (Drab et al, (1997) FASEB J 11 :905-15) ^■ Bone-specific alkaline phosphatase (BAP) and osteocalci for osteoblasts,

(Demers et al, (2000) Cancer 88:2919-26)

^■ CD4, CD8, and CD45 for white blood cells (Ody et al, (2000) Blood 96:3988- 90, Martin et al, (2000) Blood 96:2511-9)

^■ Fllc-2 and CD34 for hematopoietic stem cells, (Julie et al, Proc. Natl. Acad. Sci. USA, 2001,Vol. 98, Issue 25, 14541-14546, Woodward & Jenkinson. Eur

J Immunol 2001 Nov;31(11):3329-38, George AA et al, Blood 2001 Jun 15;97(12):3925-30)

^■ CFU for hematopoietic stem cells, MSC progenitors (Frimberger et al, Exp Hematol 2001 May;29(5):643-52) ^■ Muc- 18 (CD 146) for bone marrow fibroblasts, (Filshie et al , ( 1998) Leukemia

12:414-21) ^■ collagen type II, collagen type IV and chondrocyte expressed protein-68 for chondrocytes (Carlberg et al, (2001) Differentiation 67:128-38, Steck et al, (2001) Biochem J 353 : 169-74)

^■ adipocyte lipid-binding protein (ALBP) and fatty acid transporter for adipocytes (Amri, et al, (1995) J Biol. Chem. 270:2367-2371, Bastie et al,

(1999) JBiol Chem 274:21920-5, Frohnert et al, (1999) J. Biol. Chem. 274, 3970-3977, Teboul et al, (2001) Biochem. J. 360:305-312) - CD133 for neural stem cells (Uchida N et al, (2000) Proc. Natl Acad. Sci. USA 97:14720-5) ■ GF AP for astrocytes (Dai et al , (2001 ) Genes Dev 15:1913-25)

■ microrubule-associated protein-2 for neurons (Roy et al, (2000) Nat Med 6:271-7)

It is also possible to screen mammalian cells for other properties, such as anti- tumorigenesis, altered apoptosis, and anti-viral phenotypes. For example, by selecting for cells that are resistant to viral infection or virus production, it is possible to identify artificial chimeric proteins that can be used as anti-viral agents.

Similarly changes in cell signaling pathways can be detected by the use of probes coιτelated with activity or inactivity of the pathway or by observable indications con-elated with activity or inactivity of the pathway. Some screens relate to production of a compound of interest, e.g., a metabolite, a secreted protein, and a post-translationally modified protein. For example, cells can be identified that produce an increased amount of a compound. In another example, cells can be identified that produce a reduced amount of a compound, e.g., an undesired byproduct. Cells of interest can be identified by a variety of means, including the use of a responder cell, microaιτays, chemical detection assays, and immunoassays. More examples of particular embodiments include:

1) Protein solubility: InE. coli, many heterologous proteins are expressed as inclusion bodies. We identified chimeric zinc finger proteins that increase the soluble fraction of a human protein expressed in E. coli. See Example 12. Accordingly, the invention features an artificial transcription factor or a chimeric zinc fmger protein that alters (e.g., increases) the solubility of a heterologous protein expressed (e.g., overexpressed in a cell). 2) Glycosylation: Therapeutic proteins including antibodies are often produced in CHO cells. However, such proteins do not have optimal glycosylation pattern. In one embodiment, a library encoding chimeric proteins is screened to identify a CHO cell that is modified so that a secreted protein, e.g., an antibody, includes one or more (e.g., all) glycosylations that characterize an antibody produced by a B cell. Accordingly, the invention features an artificial transcription factor or a chimeric zinc finger protein that alters glycosylation of a secreted protein, e.g., a protein secreted by a CHO cell, e.g., an antibody secreted by a CHO cell.

3) Viral titer: In one embodiment, a library encoding chimeric proteins is screened to identify a chimeric protein that increases or decreases viral titer in a cell culture. Virases can be used as a delivery vehicle, e.g., a gene delivery vehicle. For example, therapeutic viruses are now being developed to cure certain type of cancer (e.g. an adenovirus). Increasing the viral titer is useful for preparing virases for therapy. On the other hand suppressing viral production in a cell culture and in vivo is useful for treating viral disease. Accordingly, the invention features an artificial transcription factor or a chimeric zinc finger protein that alters (e.g., increases or decreases) virus production in a cell, e.g., a eukaryotic or mammalian cell.

4) Transformation efficiency: Genetic engineering in many eukaryotic cell lines or prokaryotic organisms is limited by low transfection or transformation efficiency. Artificial transcription factors can be selected that modify cells so that transfection or transformation efficiency is improved. A selection for such a factor can be performed by a transformation with a reporter or marker at limiting concentrations, followed by selection of those cells that take up the reporter. Accordingly, the invention features an artificial transcription factor or a chimeric zinc finger protein that alters (e.g., increases) the DNA uptake efficiency or tolerance of DNA uptake procedures of a cell.

5) Feeder cells. It is possible to identify artificial transcription factors and other chimeras that modify the properties of a culture cell so that the culture cell can support proliferation or differentiation of a stem cells, e.g., thereby producing a feeder cell. The culture cell can be a human or mammalian cell. The cells can be screened (e.g., by pooling library members) to identify a cell which causes a stem cell cultivated in the same milieu (e.g., the same well) to proliferate or differentiation. The artificial transcription factor may activate key cytokines and growth factors, which are secreted to the media. The media can be used to induce differentiation or proliferate (e.g., by supporting self- renewal) the stem cell. Accordingly, the invention features an artificial transcription factor or a chimeric zinc finger protein that alters (e.g., increases) the ability of a mammalian cell to condition media or otherwise alter the behavior of a stem cell, e.g., regulate proliferation or differentiation of a stem cell.

It is also possible to screen for an artificial transcription factor using one type of cell and then to express the artificial transcription factor in a second type of cell. This process an be used to generally transfer the phenotypic change induced by the transcription factoring a first cell into a second cell. For example, we determined the expression profiles of a particular zinc fmger protein in two widely different cell lines; human embiyonic kidney 293 cell, which is non-cancerous, and human cervical cancer cell HeLa. We found that the profiles were strikingly similarity. Similarly, in yeast we demonstrated that a phenotype induced by a ZFP in one strain can be transferred to a different strain.

Stem Cells

The approaches described here, while generally applicable to any cell, are also particularly useful for regulating the behavior of stem cells from any metazoan organism. Stem cells are cells with the capacity self-renewal and also the potential for differentiation. The self-renewal can be prolonged, and even indefinite. Stem cells can produce highly differentiated descendants (Watt and Hogan (2000) Science 287:1427-1430). Recent success in culturing human embiyonic stem cells has provided a potential source of cells for cell-based therapies. However, maintaining, replicating, and differentiating stem cells can, at least in some cases, be difficult. ES cells, for example, have a tendency to differentiate randomly in vitro. A libraiy of chimeric transcription factors can be used to identify proteins that can control cell differentiation, e.g., stem cell differentiation. For example, one may identify chimeric transcription factors that direct the differentiation of stem cells towards a defined post-mitotic cell subtype (for example, a dopaminergic or cholinergic neuron). Proteins can be identified that increase the potential for self-renewal, that prevent differentiation, or that direct the extent and character of differentiation. These proteins are generally identified by introducing nucleic acids encoding artificial zinc finger proteins into stem cells or stem cell progenitors, and evaluating the phenotype of the cell.

A means to control the differentiative and proliferative potential of stem cells would enable, among other things, the provision of a large supply of undifferentiated cells, and the regulated differentiation toward a specific cell types. Such control can be tailored to a therapeutic use or other applications (e.g., development of transgenic animals, in vitro cell culture, and so forth).

In other examples, one can identify chimeric transcription factors that direct embryonic stem (ES) cells to differentiate into a restricted lineage. Thus, one may produce neuronal progenitor cells or hematopoietic stem cells from ES cells. It is also possible to identify chimeric proteins, e.g., chimeric ZFPs, that cause 1) a differentiated cell to adopt a different differentiated state or 2) a differentiated cell to a adopt a non- differentiated state, e.g., thereby generating a stem cell or a pluripotent progenitor cell.

The identification method does not require information about a particular target gene. Target genes can be identified after screening, e.g., by transcript or protein profiling to identify genes whose expression or activity is altered by an identified chimeric transcription factor. The identification of genes regulated by ZFP-TFs will advance understanding of cell differentiation.

Production of cellular products.

The invention features artificial transcription factors (e.g., chimeric zinc finger proteins) that alter the ability of a cell to produce a cellular product, e.g., a protein or metabolite. A cellular product can be an endogenous or heterologous molecule. For example, it is possible to identify an artificial transcription factor that increases the ability of a cell to produce proteins, e.g., particular proteins (e.g., particular endogenous proteins), overexpressed proteins, heterologous proteins, or mis-folded proteins. In one embodiment, cells are screened for their ability to produce a reporter protein, e.g., a protein that can be enzymatically or fluorescently detected. In one example, the reporter protein is insoluble when overexpressed in a reference cell. For example, bacterial cells can be screened for artificial transcription factors that reduce inclusion bodies. In another example, the reporter protein is secreted, e.g., by a prokaryotic or eukaiyotic cell. Cells can be screened for higher secretoiy through-put, or improved post-translational modification, e.g., glycosylation, phosphorylation, or proteolytic processing.

In one embodiment, cells are screened for their ability to alter (e.g., increase or decrease) the activity of two different reporter proteins. The reporter proteins may differ, e.g., by activity, localization (e.g., secreted/cytoplasmic/nuclear), size, solubility, isoelectric point, oligomeric state, post-translational regulation, translational regulation, and transcriptional regulation (e.g., the gene encoding them may be regulated by different regulatory sequences). The invention includes artificial transcription factors (e.g., zinc finger proteins) that alter at least two different reporter genes that differ by these properties, and zinc finger proteins that selectively regulate a reporter gene, or a class of reporter genes defined by one of these properties.

Because the phenotypic screening method can be used to isolate the artificial transcription factor, it is not necessary to know a priori how the zinc finger protein mediates increased protein production. Possible mechanisms, which can be verified, include alteration of one or more of the following: translation machinery, transcript processing, transcription, secretion, protein degradation, stress resistance, catalytic activity, e.g., metabolite production. In one example, an artificial transcription factor may modulate expression of one or more enzymes in a metabolic pathway and thereby enhance production of a cellular product such as a metabolite or a protein.

Iterative Design

Once a chimeric DNA binding protein is identified, its ability to alter a phenotypic trait of a cell can be further improved by a variety of strategies. Small libraries, e.g., having about 6 to 200 or 50 to 2000 members, or large libraries can be used to optimize the properties of a particular identified chimeric protein. In a first exemplary implementation of an iterative design, mutagenesis techniques are used to alter the original chimeric DNA binding protein. The techniques are applied to construct a second libraiy whose members include members that are variants of an original protein, for example, a protein identified from a first libraiy. Examples of these techniques include: error-prone PCR (Leung et al. (1989) Technique 1:11-15), recombination, DNA shuffling using random cleavage (Stemmer (1994) Nature 389-391), Coco et al. (2001) Nature Biotech. 19:354, site-directed mutagenesis (Zollner et al. (1987) Nucl Acids Res 10:6487-6504), cassette mutagenesis (Reidhaar-Olson (1991) Methods Enzymol 208:564-586) incorporation of degenerate oligonucleotides (Griffiths et al. (1994) EMBO J 13:3245); serial ligation, pooling specific library members from a prefabricated and arrayed library, recombination (e.g., sexual PCR and "DNA Shuffling™" (Maxygen, Inc., CA)), or by combinations of these methods.

In one embodiment, a library is constracted that mutates a set of amino acid positions. For example, for a chimeric zinc finger protein, the set of amino acid positions may be positions in the vicinity of the DNA contacting residues, but not the DNA contacting residues themselves. In another embodiment, the libraiy varies each encoded domain in a chimeric protein, but to a more limited extent than the initial library from which the chimeric DNA binding protein was identified. For a chimeric zinc finger protein, the nucleic acids that encode a particular domain can be varied among other zinc finger domains whose recognition specificity is known to be similar to that of the domain present in the original chimeric protein. Some techniques include generating new chimeric DNA binding proteins from nucleic acids encoding domains of at least two chimeric DNA binding proteins that are known to have a particular functional property. These techniques, which include DNA shuffling and standard domain swapping, create new combinations of domains. See, e.g., U.S. Patent No. 6,291,242. DNA shuffling can also introduce point mutations in addition to merely exchanging domains. The shuffling reaction is seeded with nucleic acid sequences encoding chimeric proteins that induces a desired phenotype. The nucleic acids are shuffled. A secondary library is produced from the shuffling products and screened for members that induce the desired phenotype, e.g., under similar or more stringent conditions. If the initial libraiy is comprehensive such that chimeras of all possible domain combinations are screened, DNA shuffling of domains isolated from the same initial libraiy may be of no avail. DNA shuffling may be useful in instances where coverage is comprehensive and also in instances where comprehensive screening may not be practical.

In a second exemplary implementation of an iterative design, a chimeric DNA binding protein that produces a desired phenotype is altered by varying each domain. Domains can be varied sequentially, e.g., one-by-one, or greater than one at a time. The following example refers to an original chimeric protein that includes three zinc finger domains: fingers I, II, and III and that produces a desired phenotype. A second library is constructed such that each nucleic acid member of the second libraiy encodes the same fmger II and fmger III as the initially identified protein. However, the libraiy includes nucleic acid members whose finger I differs from finger I of the original protein. The difference may be a single nucleotide that alters the amino acid sequence of the encoded chimeric protein or may be more substantial. The second library can be constructed, e.g., such that the base-contacting residues of fmger I are varied, or that the base-contacting residues of fmger I are maintained but that adjacent residues are varied. The second library can also to include a large enough set of zinc fmger domains to recognize at least 20, 30, 40, or 60 different trinucleotide sites.

The second library is screened to identify members that alter a phenotype of a cell or organism. The extent of alteration can be similar to that produced by the original protein or greater than that produced by the original protein. Concuπently, or subsequently, a third library can be constracted that varies finger

II, and a fourth library can be constracted that varies fmger III. It may not be necessaiy to further improve a chimeric protein by varying all domains, if the chimeric protein or already identified variants are sufficient. In other cases, it is desirable to re-optimize each domain. If other domains are varied concuπently, improved variants from each particular library can be recombined with each other to generate still another libraiy. This library is similarly screened.

In a third exemplary implementation of an iterative design, the method includes adding, substituting, or deleting a domain, e.g., a zinc fmger domain or a regulatory domain. An additional zinc fmger domain may increase the specificity of a chimeric protein and may increase its binding affinity. In some cases, increased binding affinity may enhance the phenotype that the chimeric protein produces. An additional regulatoiy domain, e.g., a second activation domain or a domain that recruits an accessoiy factor, may also enhance the phenotype that the chimeric protein produces. A deletion may improve or broaden the specificity of the activity of the chimeric protein, depending on the contribution of the domain that is deleted, and so forth. In a fourth exemplary implementation of an iterative design, the method includes co-expressing the original chimeric protein and a second chimeric DNA binding protein in a cell. The second chimeric protein can be also identified by screening a nucleic acid library that encodes different chimeras. In one embodiment, the second chimeric protein is identified by screening the libraiy in a cell that expresses the original chimeric protein. In another embodiment, the second chimeric protein is identified independently.

Profiling Regulatory Properties of a Chimeric Zinc Finger Protein A chimeric transcription factor that alters a phenotype of a cell can be further characterized to identify the endogenous genes that it directly or indirectly regulates. Typically, the chimeric transcription factor is produced within the cell. At an appropriate time, e.g., before, during, or after the phenotypic change occurs, the cell is analyzed to detennine the levels of transcripts or proteins present in the cell or in the medium sunounding the cell. For example, mRNA can be harvested from the cell and analyzed using a nucleic acid microarray.

Nucleic acid microarrays can be fabricated by a variety of methods, e.g., photolithographic methods (see, e.g., U.S. Patent No. 5,510,270), mechanical methods (e.g., directed-flow methods as described in U.S. Patent No. 5,384,261), and pin based methods (e.g., as described in U.S. 5,288,514). The array is synthesized with a unique capture probe at each address, each capture probe being appropriate to detect a nucleic acid for a particular expressed gene.

The mRNA can be isolated by routine methods, e.g., including DNase treatment to remove genomic DNA and hybridization to an oligo-dT coupled solid substrate (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y). The substrate is washed, and the mRNA is eluted. The isolated mRNA is then reversed transcribed and optionally amplified, e.g., by rtPCR, e.g., as described in (U.S. Patent No. 4,683,202). The nucleic acid can be labeled during amplification or reverse transcription, e.g., by the incorporation of a labeled nucleotide. Examples of prefeixed labels include fluorescent labels, e.g., red-fluorescent dye Cy5 (Amersham) or green-fluorescent dye Cy3 (Amersham). Alternatively, the nucleic acid can be labeled with biotin, and detected after hybridization with labeled streptavidin, e.g., streptavidin-phycoerythrin (Molecular Probes). The labeled nucleic acid is then contacted to the aπay. In addition, a control nucleic acid or a reference nucleic acid can be contacted to the same an-ay. The control nucleic acid or reference nucleic acid can be labeled with a label other than the sample nucleic acid, e.g., one with a different emission maximum. Labeled nucleic acids are contacted to an array under hybridization conditions. The array is washed, and then imaged to detect fluorescence at each address of the an-ay.

A general scheme for producing and evaluating profiles includes detecting hybridization at each address of the aπay. The extent of hybridization at an address is represented by a numerical value and stored, e.g., in a vector, a one-dimensional matrix, or one-dimensional array. The vector x has a value for each address of the aιτay. For example, a numerical value for the extent of hybridization at a particular address is stored in variable x_a. The numerical value can be adjusted, e.g., for local background levels, sample amount, and other variations. Nucleic acid is also prepared from a reference sample and hybridized to the same or a different array. The vector y is construct identically to vector x. The sample expression profile and the reference profile can be compared, e.g., using a mathematical equation that is a function of the two vectors. The comparison can be evaluated as a scalar value, e.g., a score representing similarity of the two profiles. Either or both vectors can be transformed by a matrix in order to add weighting values to different genes detected by the aιτay. The expression data can be stored in a database, e.g., a relational database such as a SQL database (e.g., Oracle or Sybase database environments). The database can have multiple tables. For example, raw expression data can be stored in one table, wherein each column coιτesponds to a gene being assayed, e.g., an address or an aπay, and each row corresponds to a sample. A separate table can store identifiers and sample infonnation, e.g., the batch number of the array used, date, and other quality control infonnation.

Genes that are similarly regulated can be identified by clustering expression data to identify coregulated genes. Such cluster may be indicative of a set of genes coordinately regulated by the chimeric zinc finger protein. Genes can be clustered using hierarchical clustering (see, e.g., Sokal and Michener (1958) Univ. Kans. Sci. Bull 38: 1409), Bayesian clustering, k-means clustering, and self-organizing maps (see, Tamayo et al. (1999) Proc. Natl. Acad. Sci. USA 96:2907). The similarity of a sample expression profile to a reference expression profile (e.g., a control cell) can also be determined, e.g., by comparing the log of the expression level of the sample to the log of the predictor or reference expression value and adjusting the comparison by the weighting factor for all genes of predictive value in the profile. Proteins can also be profiled in a cell that has an active chimeric protein with in it.

One exemplary method for profiling proteins includes 2-D gel electrophoresis and mass spectroscopy to characterize individual protein species. Individual "spots" on the 2-D gel are proteolyzed and then analyzed on the mass spectrometer. This method can identify both the protein component and, in many cases, translational modifications. The protein and nucleic acid profiling methods can not only provide information about the properties of the chimeric protein, but also information about natural mechanisms operating within the cell. For example, the proteins or nucleic acids upregulated by expression of the chimeric protein may be the natural effectors of the phenotypic change caused by expression of the chimeric protein. In addition, other methods can be used to identify target genes and proteins that are directly or indirectly regulated by the artificial chimeric protein. In one example, alterations that compensate (e.g., suppress) the phenotypic effect of the artificial chimeric protein are characterized. These alterations include genetic alterations such as mutations in chromosomal genes and overexpression of a particular gene, as well as other alterations, such as RNA interference (e.g., by double-stranded RNA).

In a particular example, a chimeric ZFP is isolated that causes a growth defect or lethality when conditionally expressed in a cell, e.g., a pathogenic bacteria or fungi. Such a ZFP can be identified by transforming the cell with the ZFP libraries that include nucleic acids encoding ZFPs, expression of the nucleic acids being controlled by an inducible promoter. Transfomiants are cultured on non-inducible media and then replica- plated on both inducible and non-inducible plates. Colonies that grow noπnally on non- inducible plate, but show defective growth on inducible plate are identified as "conditional lethal" or "conditional growth defective" colonies.

(a) Identification of target genes using a cDNA library A cDNA expression libraiy is then transfomied into the "conditional lethal" or

"conditional growth defective" strains described above. Transfomiants are plated on inducible plates. Colonies that survive, despite the presence and expression of the ZFP that causes the defect, are isolated. The nucleic acid sequences of cDNAs that complement the defect are characterized. These cDNA can be transcripts of direct or indirect target genes that are regulated by chimeric ZFP that mediates the defect.

(b) Identification of target genes using a secondary ZFP libraiy

A second chimeric protein that suppresses the effect of the first chimeric protein is identified. The targets of the second chimeric protein (in the presence or absence of the first chimeric protein) are identified.

For example, a ZFP library is transfomied into "conditional lethal" or "conditional growth defective" colonies (which include a first chimeric ZFP that causes the defect). Transfomiants are plated on inducible plates. Colonies that can survive by the expression of introduced ZFP are identified as "suppressed strains". Target genes of the second ZFPs can be characterized by DNA microan-ay analysis. The comparative analysis can be done between four strains: 1) no ZFP; 2) the first ZFP alone; 3) the second ZFP alone; and 4) the first and second ZFP. For example, genes that are regulated in opposing directions by the first and second chimeric ZFPs are candidates for targets that mediate the growth-defective phenotype. This method can be applied to any phenotype, not just a growth defect.

(c) Co-regulated genes identified by expression profiling analysis A candidate target of a chimeric ZFP can be identified by expression profiling.

Subsequently, to detemiine if the candidate target mediates the phenotype of the chimeric ZFP, the candidate target can be independently over-expressed or inhibited (e.g., by genetic deletion or RNA interference). In addition, it may be possible to apply this analysis to multiple candidate targets since in at least some cases more than one candidate may need to be perturbed to cause the phenotype. An example of this approach is provided in Example 3 (Ketoconazole resistance).

(d) Time-Course Analysis

The targets of a chimeric ZFP can be identified by a characterizing changes in gene expression with respect to time after a cell is exposed to the chimeric ZFP. For example, a gene encoding the chimeric ZFP can be attached to an inducible promoter. An exemplary inducible promoter is regulated by a small molecule such as doxycycline. The gene encoding the chimeric ZFP is introduced into cells. mRNA samples are obtained from cells at various times after induction of the inducible promoter. See, e.g., FIG. 13, which depicts genes that are activated and repressed in the course of induction of the ZFP F104-p65.

(e) Identification ofprimaiy target genes ofZFP-TFsfrom mammalian cells using protein transduction and cDNA microarray technologies.

It is also possible to introduce a chimeric protein into a cell by transduction. The protein is provided to the extracellular milieu and the cell transduces the protein into itself. Thus, the cell does not have to include a gene encoding the chimeric protein. This approach may obviate concerns about exogenous nucleic acid integration, propagation, and so forth. Levels of the protein can be precisely controlled. In one embodiment, the chimeric ZFP is fused to protein transduction domain of Tat or VP22.

To analyze the effects of a transduced chimeric protein in culture cells, the chimeric protein is added to the culture media, e.g., as a fusion to a protein transduction domain. Detection of regulated target genes can be enhanced by addition of an inhibitor of protein synthesis such as cycloheximide. Thus, translation of the primary target genes is blocked, and genes that would be regulated by proteins encoded by the primary target genes would be detected. The identity of primary target genes can be found by DNA microarray analysis.

The use of cyclohexamide to identify primary target genes can also be used when the chimeric protein is encoded by a heterologous nucleic acid in the cell. For example, expression of the heterologous nucleic acid can be induced for less than 30, 20, 15, 10, or 5 minutes, and then cyclohexamide can be added.

(f) Activity Analysis

The function of a potential target gene can be evaluated by inhibiting activity of the target gene, e.g., by RNA interference (RNAi) with double-stranded RNAs (dsRNA), anti-sense technology, ribozymes, or targeted genetic mutation. A cell or organism in which activity of the target gene is reduced can be evaluated and compared to a control cell or organism that is not treated with RNAi. In another example, a cell or organism that expresses the artificial zinc finger protein believed to regulate the target gene is treated with RNAi. The ability of the artificial zinc fmger protein to induce a phenotype is evaluated in the presence and absence of RNAi. In some cases, if the potential target gene is indeed a critical target, the RNAi treatment inactivating the potential target may attenuate the phenotype induced by the artificial zinc finger protein. dsRNA can be produced by transcribing a cassette in both directions, for example, by including a T7 promoter on either side of the cassette. The insert in the cassette is selected so that it includes a sequence complementary to the potential target gene. See also, the HiScribe™ RNAi Transcription Kit (New England Biolabs, MA) and Fire, A. (1999) Trends Genet. 15, 358-363. dsRN A can be digested into smaller fragments. See, e.g., US Patent Application 2002-0086356. dsRNAs can be used to silence gene expression in mammalian cells. See, e.g., Clemens, et al. (2000) Proc. Natl. Sci. USA 97, 6499-6503; Billy, E. et al. (2001) Proc. Natl Sci. USA 98, 14428-14433; Elbashir et al. (2001) Nature. 411(6836):494-8; Yang, D. et al. (2002) Proc. Natl Acad. Sci. USA 99, 9942-9947.

Target DNA Site Identification

With respect to chimeric DNA binding proteins, a variety of methods can be used to determine the target site of a chimeric DNA binding protein that produces a phenotype of interest. Such methods can be used, alone or in combination, to find such a target site. In one embodiment, infonnation from expression profile is used to identify the target site recognized by a chimeric zinc fmger protein. The regulatoiy regions of genes that are co-regulated by the chimeric zinc finger protein are compared to identify a motif that is common to all or many of the regulatoiy regions. In another embodiment, biochemical means are used to detemiine what DNA site is bound by the chimeric zinc fmger protein. For example, chromatin immuno- precipitation experiments can be used to isolate nucleic acid to which the chimeric zinc finger protein is bound. The isolated nucleic acid is PCR amplified and sequence. See, e.g., Gogus et al. (1996) Proc. Natl. Acad. Sci. USA. 93:2159-2164. The SELEX method is another exemplary method that can be used. Further, infonnation about the binding specificity of individual zinc fmger domains in the chimeric zinc finger protein can be used to predict the target site. The prediction can be validated or can be used to guide interpretation of other results (e.g., from chromatin immunoprecipitation, in silico analysis of co-regulated genes, and SELEX).

In still another embodiment, a potential target site is inferred based on infonnation about the binding specificity of each component zinc fmger. For example, as shown in FIG. 10A and 10B, a chimera that includes the zinc finger domains, from N- to C- tenninus, : CSNR, RSNR, and QSNR is expected to recognize the target site 5'-GAAGAGGACC-3' (SEQ ID NO: 130). The domains CSNR, RSNR, and QSNR have the following respective DNA binding specificities GAC, GAG, and GAA. The expected target site is fomied by considering the domains in C temiinal to N-temiinal order and concatenating their recognition specificities to obtain one strand of the target site in 5' to 3' order.

Although in most cases, chimeric zinc finger proteins are likely to function as transcriptional regulators, it is possible that in some cases the chimeric zinc finger proteins mediate their phenotypic effect by binding to an RNA or protein target. Some naturally-occunϊng zinc finger proteins in fact bind to these macromolecules.

Additional Features for Chimeric Transcription Factors

With respect to a libraiy encoding chimeric nucleic acid binding domains, the encoded polypeptides can also include one or more of the following features. These features may be constant among all members of the library or may also vaiy. In one example, some nucleic acids encode polypeptides that include an activation domain, whereas others include a repression domain, or no transcriptional regulatoiy domain.

Activation domains. Transcriptional activation domains that may be used in the present invention include but are not limited to the Gal4 activation domain from yeast and the VP16 domain from herpes simplex viras. The ability of a domain to activate transcription can be validated by fusing the domain to a known DNA binding domain and then detennining if a reporter gene operably linked to sites recognized by the known DNA-binding domain is activated by the fusion protein.

An exemplary activation domain is the following domain from p65: YLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKP APQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVS ALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLG NSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTAQRPP DPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQ (SEQ ID NO:131)

The sequence of an exemplaiy Gal4 activation domain is as follows: NFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSNVHDNFMNNEITAS KIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDDEDTPPNPK KEISMAYPYDVPDYAS (SEQ ID NO: 132)

In bacteria, activation domain function can be emulated by a domain that recruits a wild-type RNA polymerase alpha subunit C-tenninal domain or a mutant alpha subunit C-terminal domain, e.g., a C-tenninal domain fused to a protein interaction domain.

Repression domains. If desired, a repression domain instead of an activation domain can be fused to the DNA binding domain. Examples of eukaiyotic repression domains include repression domains from Kid, UME6, ORANGE, groucho, and WRPW (see, e.g., Dawson et α/., (1995) Mol. Cell Biol. 15:6923-31). The ability of a domain to repress transcription can be validated by fusing the domain to a known DNA binding domain and then detennining if a reporter gene operably linlced to sites recognized by the known DNA-binding domain is repressed by the fusion protein.

An exemplary repression domain is the following domain from UME6 protein:

NSASSSTKLDDDLGTAAAVLSNMRSSPYRTHDKPISNVNDMNNTNALGVPASRPH SSSFPSKGVLRPILLRIHNSEQQPIFESNNSTACI (SEQ ID NO: 133)

Another exemplary repression domain is from the Kid protein: VSVTFEDVAVLFTRDEWKKLDLSQRSLYREVMLENYSNLASMAGFLFTKPKVIS LLQQGEDPW (SEQ ID NO: 134)

Still other chimeric transcription factors include neither an activation or repression domain. Rather, such transcription factors may alter transcription by displacing or otherwise competing with a bound endogenous transcription factor (e.g., an activator or repressor).

Peptide Linkers. DNA binding domains can be connected by a variety of linkers. The utility and design of linkers are well known in the art. A particularly useful linker is a peptide linker that is encoded by nucleic acid. Thus, one can construct a synthetic gene that encodes a first DNA binding domain, the peptide linker, and a second DNA binding domain. This design can be repeated in order to construct large, synthetic, multi-domain DNA binding proteins. PCT WO 99/45132 and Kim and Pabo ((1998) Proc. Natl Acad. Sci. USA 95:2812-7) describe the design of peptide linkers suitable for joining zinc fmger domains.

Additional peptide linkers are available that form random coil, α-helical or β- pleated tertiary structures. Polypeptides that fomi suitable flexible linkers are well known in the art (see, e.g., Robinson and Sauer (1998) Proc Natl Acad Sci USA. 95:5929-34). Flexible linkers typically include glycine, because this amino acid, which lacks a side chain, is unique in its rotational freedom. Serine or threonine can be interspersed in the linker to increase hydrophilicity. In additional, amino acids capable of interacting with the phosphate backbone of DNA can be utilized in order to increase binding affinity. Judicious use of such amino acids allows for balancing increases in affinity with loss of sequence specificity. If a rigid extension is desirable as a linker, α-helical linkers, such as the helical linker described in Pantoliano et al. (1991) Biochem. 30:10117-10125, can be used. Linkers can also be designed by computer modeling (see, e.g., U.S. 4,946,778). Software for molecular modeling is commercially available (e.g., from Molecular Simulations, Inc., San Diego, CA). The linker is optionally optimized, e.g., to reduce antigenicity and/or to increase stability, using standard mutagenesis techniques and appropriate biophysical tests as practiced in the art of protein engineering, and functional assays as described herein.

For implementations utilizing zinc finger domains, the peptide that occurs naturally between zinc fingers can be used as a linker to join zinc fingers together. An example of a naturally occumng linker is: Thr-Gly-(Glu or Gln)-(Lys or Arg)-Pro-(Tyr or Phe) (SEQ ID NO:74) (Agata et al, supra). Generally, linkers can be selected or based on the sequences that join zinc fingers in naturally occumng proteins.

Dimerization Domains. An alternative method of linking DNA binding domains is the use of dimerization domains, especially heteiOdimerization domains (see, e.g.,

Pomerantz et al (1998) Biochemistiy 37:965-970). In this implementation, DNA binding domains are present in separate polypeptide chains. For example, a first polypeptide encodes DNA binding domain A, linker, and domain B, while a second polypeptide encodes domain C, linker, and domain D. An artisan can select a dimerization domain from the many well-characterized dimerization domains. Domains that favor heterodimerization can be used if homodimers are not desired. A particularly adaptable dimerization domain is the coiled-coil motif, e.g., a dimeric parallel or anti-parallel coiled-coil. Coiled-coil sequences that preferentially form heterodimers are also available (Lumb and Kim, (1995) Biochemistry 34:8642-8648). Another species of dimerization domain is one in which dimerization is triggered by a small molecule or by a signaling event. For example, a dimeric fonn of FK506 can be used to dimerize two FK506 binding protein (FKBP) domains. Such dimerization domains can be utilized to provide additional levels of regulation.

Chimeric Proteins for Non-DNA Applications

It is also possible to modify the examples herein to make libraries of nucleic acids that encode chimeric proteins that include different combinations of non-DNA binding domains, e.g., intracellular signal transduction domains (e.g., SH2, SH3, PDZ, Che domains, or kinase domains). The chimeric proteins encoded by the library can be expressed in cells, and cells having an altered phenotypic trait are identified. For example, chimeric proteins fomied of different combinations of signaling domains can be identified that decrease or increase the rate of cell proliferation.

Expression of Zinc Finger Proteins

Method described herein can include use of routine techniques in the field of molecular biology, biochemistry, classical genetics, and recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al, Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al, eds., 1994)).

In addition to other methods described herein, nucleic acids encoding zinc proteins can be constructed using synthetic oligonucleotides as linkers to construct a synthetic gene. In another example, synthetic oligonucleotides are used and/or primers to amplify sequences encoding one or more zinc finger domains, e.g., from an RNA or DNA template, artificial or synthetic. See U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al, eds, 1990)). Methods such as polymerase chain reaction (PCR) can be used to amplify nucleic acid sequences directly from mRNA, from cDNA, from genomic, cDNA, or zinc fmger protein libraries. Degenerate oligonucleotides can be designed to amplify homologs using the sequences provided herein. Restriction endonuclease sites can be incorporated into the primers. Gene expression of zinc finger proteins can also be analyzed by techniques known in the art, e.g., reverse transcription and amplification of mRNA, isolation of total RNA or polyA⁺ RNA, northern blotting, dot blotting, in situ hybridization, RNase protection, nucleic acid aιτay technology, e.g., and the like. The polynucleotide encoding an artificial zinc finger protein can be cloned into vectors before transfonnation into prokaryotic or eukaiyotic cells for replication and/or expression. These vectors are typically prokaiyote vectors, e.g., plasmids, phage or shuttle vectors, or eukaiyotic vectors.

Protein Expression. To obtain recombinant expression (e.g., high level) expression of a polynucleotide encoding an artificial zinc fmger protein, one can subclone the relevant coding nucleic acids into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator, and a ribosome binding site for translational initiation. Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook et al, and Ausubel et al, supra. Bacterial expression systems for expression are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al, (1983) Gene 22:229-235; Mosbach et al, (1983) Nature 302:543-545. Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast (e.g., S. cerevisiae, S. pombe, Pichia, and Hanseula), and insect cells are well known in the art and are also commercially available. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function. In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for expression in host cells. A typical expression cassette thus contains a promoter operably linked to the coding nucleic acid sequence and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation teπnination. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the stractural gene, introns with functional splice donor and acceptor sites. In addition to a promoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes. The particular expression vector used to transport the genetic infonnation into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST, and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc-, or a hexa-histidine tag. Expression vectors can contain regulatory elements from eukaiyotic virases, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculoviras pDSVE, and any other vector allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaiyotic cells.

Expression of proteins from eukaryotic vectors can be also be regulated using inducible promoters. With inducible promoters, expression levels are tied to the concentration of inducing agents, such as tetracycline or ecdysone, by the incorporation of response elements for these agents into the promoter. Generally, high level expression is obtained from inducible promoters only in the presence of the inducing agent; basal expression levels are minimal. Inducible expression vectors are often chosen if expression of the protein of interest is detrimental to eukaryotic cells.

Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculoviras vector in insect cells, with mitochondrial respiratory chain protein encoding sequences and glycolysis protein encoding sequence under the direction of the polyhedrin promoter or other strong baculoviras promoters The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to pem it selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences. The prokaryotic sequences can be chosen such that they do not interfere with the replication of the DNA in eukaryotic cells.

Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of zinc finger proteins, which are then purified using standard techniques (see, e.g., Colley et al, J. Biol. Chem. 264:17619- 17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaiyotic and prokaryotic cells are perfomied according to standard techniques (see, e.g., Morrison, J. Bad. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al, eds, 1983). Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al, supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell.

After the expression vector is introduced into the cells, the fransfected cells are cultured under conditions favoring expression or activating expression. The protein can then be isolated from a cell extract, cell membrane component or vesicle, or media. Expression vectors with appropriate regulatory sequences can also be used to express a heterologous gene encoding an artificial zinc finger in a model organism, e.g., a Drosophila, nematode, zebrafish, Xenopus, or mouse. See, e.g., Riddle et al, eds., C. elegans ll. Plainview (NY): Cold Spring Harbor Laboratory Press; 1997.

Protein Purification. Zinc fmger protein can be purified from materials generated by any suitable expression system, e.g., those described above.

Zinc finger proteins may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, affinity purification, immunopurification methods, and others (see, e.g. , Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4,673,641; Ausubel et al, supra; and Sambrook et al, supra). For example, zinc fmger proteins can include an affinity tag that can be used for purification, e.g., in combination with other steps.

Recombinant proteins are expressed by transformed bacteria in large amounts, typically after promoter induction; but expression can be constitutive. Promoter induction with IPTG is one example of an inducible promoter system. Bacteria are grown according to standard procedures in the art. Fresh or frozen bacteria cells are used for isolation of protein. Proteins expressed in bacteria may form insoluble aggregates

("inclusion bodies"). Several protocols are suitable for purifying proteins from inclusion bodies. See, e.g., Sambrook et al, supra; Ausubel et al, supra). If the proteins are soluble or exported to the periplasm, they can be obtained from cell lysates or periplasmic preparations. Differential Precipitation. Salting-in or out can be used to selectively precipitate a zinc finger protein or a contaminating protein. An exemplary salt is ammonium sulfate. Ammonium sulfate precipitates proteins on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol includes adding saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This concentration precipitates many of the more hydrophobic proteins. The precipitate is analyzed to determine if the protein of interest is precipitated or in the supernatant. Ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, either through dialysis or diafiltration.

Column chromatography. A zinc finger protein can be separated from other proteins on the basis of its size, net surface charge, hydrophobicity, and affinity for ligands. In addition, antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well known in the art. Chromatographic techniques can be perfomied at any scale and using equipment from many different manufacturers (e.g., Phaπnacia Biotech). See, generally, Scopes, Protein Purification: Principles and Practice (1982). Similarly general protein purification procedures can be used to recover a protein whose production is altered (e.g., enhanced) by expression of an artificial zinc finger protein in a producing cell.

Gene and Cell-based Therapeutics

One aspect of the invention, described above, provides for isolated DNA molecules which encode an artificial zinc fmger protein. These isolated DNA molecules can be inserted into a variety of DNA constracts and vectors for the purposes of gene therapy. As used herein, a "vector" is a nucleic acid molecule competent to transport another nucleic acid molecule to which it has been covalently linked. Vectors include plasmids, cosmids, artificial chromosomes, and viral elements. The vector can be competent to replicate in a host cell or to integrate into a host DNA. Viral vectors include, e.g., replication defective retrovirases, adenovirases and adeno-associated viruses. A gene therapy vector is a vector designed for administration to a subject, e.g., a mammal, such that a cell of the subject is able to express a therapeutic gene contained in the vector. The gene therapy vector can contain regulatory elements, e.g., a 5' regulatory element, an enhancer, a promoter, a 5' untranslated region, a signal sequence, a 3' untranslated region, a polyadenylation site, and a 3' regulatory region. For example, the 5' regulatory element, enhancer or promoter can regulate transcription of the DNA encoding the therapeutic polypeptide. The regulation can be tissue specific. For example, the regulation can restrict transcription of the desired gene to brain cells, e.g., cortical neurons or glial cells; hematopoietic cells; or endothelial cells. Alternatively, regulatoiy elements can be included that respond to an exogenous drag, e.g., a steroid, tetracycline, or the like. Thus, the level and timing of expression of the therapeutic zinc finger polypeptide (e.g., a polypeptide that regulates VEGF) can be controlled.

Gene therapy vectors can be prepared for delivery as naked nucleic acid, as a component of a virus, or of an inactivated virus, or as the contents of a liposome or other delivery vehicle. Alternatively, where the gene delivery agent, e.g., a viral vector, can be produced from recombinant cells which produce the gene delivery system. Appropriate viral vectors include retrovirases, e.g., Moloney retrovirus, adenovirases, adeno- associated viruses, and lentiviruses, e.g., Herpes simplex virases (HSV). HSV is potentially useful for infecting nervous system cells.

A gene therapy vector can be administered to a subject, for example, by intravenous injection, by local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054- 3057). The gene therapy agent can be further fonnulated, for example, to delay or prolong the release of the agent by means of a slow release matrix. One method of providing a recombinant therapeutic tri-domain polypeptide, is by inserting a gene therapy vector into bone man-ow cells harvested from a subject. The cells are infected, for example, with a retroviral gene therapy vector, and grown in culture. Meanwhile, the subject is iιτadiated to deplete the subject of bone man-ow cells. The bone marrow of the subject is then replenished with the infected culture cells. The subject is monitored for recovery and for production of the therapeutic polypeptide.

Cell based-therapeutic methods include introducing a nucleic acid that encoding the artificial zinc finger protein operably linlced to a promoter into a cell in culture. The artificial zinc finger protein can be selected to regulate an endogenous gene in the culture cell or to produce a desired phenotype in the cultured cell. Further, it is also possible to modify cells, e.g., stem cells, using nucleic acid recombination, e.g., to insert a transgene, e.g., a transgene encoding an artificial zinc finger protein that regulates an endogenous gene. The modified stem cell can be administered to a subject. Methods for cultivating stem cells in vitro are described, e.g., in US Application 2002-0081724. In some examples, the stem cells can be induced to differentiate in the subject and express the transgene. For example, the stem cells can be differentiated into liver, adipose, or skeletal muscle cells. The stem cells can be derived from a lineage that produces cells of the desired tissue type, e.g., liver, adipose, or skeletal muscle cells. In another embodiment, recombinant cells that express or can express an artificial zinc fmger protein, e.g., as described herein, can be used for replacement therapy in a subject. For example, a nucleic acid encoding the artificial zinc finger protein operably linked to a promoter (e.g., an inducible promoter, e.g., a steroid hormone receptor- regulated promoter) is introduced into a human or nonhuman, e.g., mammalian, e.g., porcine recombinant cell. The cell is cultivated and encapsulated in a biocompatible material, such as poly-lysine alginate, and subsequently implanted into the subject. See, e.g., Lanza (1996) Nat. Biotechnol. 14:1107; Jolci et al. (2001) Nat. Biotechnol. 19:35; and U.S. Patent No. 5,876,742. In implementations where the artificial zinc finger protein regulates an endogenous gene that encodes a secreted protein, production of the secreted polypeptide can be regulated in the subject by administering an agent (e.g., a steroid hormone) to the subject. In still another embodiment, the recombinant cells that express or can express an artificial zinc finger protein are cultivated in vitro. A protein produced by the recombinant ceils can be recovered (e.g., purified) from the ceils or from media surrounding the cells.

Target for Altered Protein Production

In one embodiment, a nucleic acid libraiy is screened to identify an artificial zinc fmger protein that alters production, synthesis or activity of one or more particular target proteins. The alteration can increase or decrease activity or abundance of the target protein. The phenotype screened for can be associated with altered production or activity of one or more target proteins or can be the level of production or activity itself. For example, it is possible to screen a nucleic acid library for artificial transcription factors that activate or suppress expression of a reporter gene (such as those encoding luciferase, LacZ, or GFP) under the control of a regulatory sequence (e.g., the promoter) of an endogenous target gene. Some exemplary target proteins include: cell surface proteins (e.g., glycosylated surface proteins), cancer-associated proteins, cytokines, chemokines, peptide hormones, neurotransmitters, cell surface receptors (e.g., cell surface receptor kinases, seven transmembrane receptors, virus receptors and co-receptors, extracellular matrix binding proteins, cell-binding proteins, antigens of pathogens (e.g., bacterial antigens, malarial antigens, and so forth). Additional protein targets include enzymes such as enolases, cytochrome P450s, acyltransferases, methylases, TIM banel enzymes, isomerases, acyl transferases, and so forth.

More specific examples include: integrins, cell attachment molecules or "CAMs" such as cadherins, selections, N-CAM, E-CAM, U-CAM, I-CAM and so forth); proteases (e.g., subtilisin, tiypsin, chymotiypsin; a plasminogen activator, such as urokinase or human tissue-type plasminogen activator); bombesin; factor IX, thrombin; CD-4; platelet- derived growth factor; insulin-like growth factor-I and -II; nerve growth factor; fibroblast growth factor (e.g., aFGF and bFGF); epidem al growth factor (EGF); VEGFa; transfom ing growth factor (TGF, e.g., TGF-α and TGF-β; insulin-like growth factor binding proteins; eiytliropoietin; thrombopoietin; mucins; human serum albumin; growth hormone (e.g., human growth homione); proinsulin, insulin A-chain insulin B-chain; parathyroid hormone; thyroid stimulating hormone; thyroxine; follicle stimulating homione; calcitonin; atrial natriuretic peptides A, B or C; leutinizing hormone; glucagon; factor VIII; hemopoietic growth factor; tumor necrosis factor (e.g., TNF-α and TNF-β); enlcephalinase; Mullerian-inhibiting substance; gonadotropin-associated peptide; tissue factor protein; inhibin; activin; vascular endothehal growth factor; receptors for homiones or growth factors; rheumatoid factors; osteoinductive factors; an interferon, e.g., interferon-α,β,γ; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1, IL-2, IL-3, IL-4, etc.; decay accelerating factor; and immunoglobulins. In some embodiments, the target is associated with a disease, e.g., cancer.

The present invention will be described in more detail through the following examples. However, it should be noted that these examples are not intended to limit the scope of the present invention.

EXAMPLE 1 : CONSTRUCTION OF ZFP LIBRARIES

In one example, various phenotypes of Saccharomyces cerevisiae are altered by regulating gene expression using zinc fmger protein (ZFP) expression libraries. The zinc finger proteins in these exemplary libraries consist of three or four zinc f ger domains (ZFDs) and recognize 9- to 12-bp DNA sequences respectively. The chimeric zinc finger protein is identified without a priori knowledge of the target genes. Three different class of transcription factors are produced from the ZFPs in the libraries: isolated ZFPs themselves function as efficient transcriptional repressors when they bind to a site near the promoter region; ZFPs are also expressed as fusion proteins to a transcriptional activation domain or to a repression domain to yield transcriptional activators or repressors, respectively. We used 40 different zinc fmger domains as modular building blocks to construct 3-finger or 4-fmger zinc finger protein. At their upper limits, a 3-finger ZFP library consists of 64,000 (= 40³) sequences and a 4-fmger libraiy consists of 2.6 million (= 40⁴) sequences. These libraries of ZFP expression plasmids were then transfomied into yeast cells.

In each transfomied cell, a different ZFP transcription factor would be expressed and regulate unspecified target genes in the genome. This alteration of gene expression pattern can lead to phenotypic changes. By screening a large number of transformed cells, one can isolate clones with desired phenotypes. In addition, the regulated target genes can be identified by genome-wide analyses of gene expression profile (e.g., via DNA microarray analysis) or in silico prediction of target DNA sequences after identifying zinc finger proteins introduced to the transfomiants.

(1) Yeast Strains The S. cerevisiae strain used for this experiment was YPH499a (MATa, ade2-101, ura -52, lys2-801, trpl- 63, his3- 200, leu2- 1, GAL+). Transfonnation of yeast cells was earned out by using the lithium acetate transformation method (see, e.g., Gietz et al, (1992) Nucl. Acids Res 20:1245).

(2) Construction of plasmid p3

The parental vector that we used to construct libraries of zinc fmger proteins is the plasmid p3. p3 was constracted by modifying the pcDNA3 vector (Invitrogen, San Diego CA) as follows. The pcDNA3 vector was digested with Hindlll and Xhol. A synthetic oligonucleotide duplex with compatible overhangs was ligated into the digested pcDNA3. The duplex contains nucleic acid that encodes the hemagglutinin (HA) tag and a nuclear localization signal. The duplex also includes: restriction sites for BamHI, EcoRI, Notl, and Bglll; and a stop codon. The Xmal site in SV40 origin of the vector was destroyed by digestion with Xmal, filling in the overhanging ends of the digested Xmal restriction site, and religation of the ends.

(3) Construction of plasmid pYCT-Lib

We used pYCT-Lib as the parental vector for conditional expression of zinc finger proteins in yeast. pYCT-Lib is a yeast shuttle vector that includes the inducible GAL1 promoter (FIG. 1). Other features can include (i) a sequence encoding a nuclear localization signal (NLS) and a hemagglutinin tag (HA) and (ii) a sequence including the TRP1 gene for selection of plasmid containing cells on synthetic minimal media lacking tryptophan.

The polylinker from the T7 primer site to the Sphl site region can include:

TAATACGACTCACTATAGGGAATATTAAGCTAAGCTCACCATGGGTAAGCCT ATCCCTAACCCTCTCCTCGGTCTCGATTCTACACAAGCTATGGGTGCTCCTCC AAAAAAGAAGAGAAAGGTAGCTGGATCCACTAGTAACGGCCGCCAGTGTGCT GGAATTCTGCAGATATCCATCACACTGGCGGCGCTCGAGGCATGCATCTA (SEQ ID NO: 135) p-YCT-Lib was constracted as follows. The yeast expression plasmid pYESTrp2 (Invitrogen, San Diego CA) was digested with NgoM4 and then was partially digested with Pstl to remove the 2μ ori fragment from the vector. The 5.0 kb DNA fragment from the NgoM4-PstI digested vector was purified after gel electrophoresis and ligated with a CEN-ARS fragment that was amplified from pRS313 (forward primer: 5'-CGATCTGCAGGG TCCTTTTCATCACGTGCT-3 ' (SEQ ID NO:136), reverse primer: 5 '-CGATCGATGCCG GCGGACGGATCGCTTGCCT (SEQ ID NO: 137)). The DNA segment that encodes the B42 activation domain was removed by digestion with Ncol and BamHI and replaced with a DNA fragment encoding the V5 epitope tag and the nuclear localization signal. The latter DNA fragment was PCR- amplified from pYESTrp2 (forward primer: 5'-AATTCCATGGGTAAGCCTATCCCTAACC-3' (SEQ ID NO:138), reverse primer: 5'-AATTGGATCCAGCTACCTTTCTCTTCTT-3'(SEQ ID NO: 139)) and ligated into the Ncol and BamHI sites. The resulting plasmid was named as pYTC-Lib (FIG. 1).

(4) Construction of plasmid pYCT-Lib-Ga 14 To generate pYCT-Lib-Gal4, the Gal4 activation domain was PCR-amplified from yeast genomic DNA (forward primer: 5'-AAGGAAGGAAGGAAGCGGC CGCAGCCAATTTTAATCAAAGTGG-3' (SEQ ID NO: 140), reverse primer: 5'-ACATACATGCATGCGCCGTTACTAGTGGATCC-3' sequence (SEQ ID NO:141)) and inserted between the Notl and Sphl recognition sites of pYTC-Lib to generate pYTC-Lib-Gal4. An exemplary sequence encoding the Gal4 activation domain and linking sequences includes:

GGCCGCCAGTGTGCTGGAATTCTGCAGATATCCATCACACTGGCGGCCGCAG CCAATTTTAATCAAAGTGGGAATATTGCTGATAGCTCATTGTCCTTCACTTTC ACTAACAGTAGCAACGGTCCGAACCTCATAACAACTCAAACAAATTCTCAAG CGCTTTCACAACCAATTGCCTCCTCTAACGTTCATGATAACTTCATGAATAAT GAAATCACGGCTAGTAAAATTGATGATGGTAATAATTCAAAACCACTGTCAC CTGGTTGGACGGACCAAACTGCGTATAACGCGTTTGGAATCACTACAGGGAT GTTTAATACCACTACAATGGATGATGTATATAACTATCTATTCGATGATGAAG ATACCCCACCAAACCCAAAAAAAGAGATCTCTATGGCTTACCCATACGATGT TCCAGATTACGCTAGCTAAGGATCCACTAGTAACGGCGCATGCATCTAGAGG GCC (SEQ ID NO: 142)

(5) Construction of plasmid pYCT-Lib-Ume6 To generate pYCT-Lib-Ume6, a DNA fragment encoding amino acids 508 to 594 of S. cerevisiae Ume6 was amplified from yeast genomic DNA (forward primer: 5 '-AAGGAAGGAAGGAAGCGGCCGCAAATTCTGCATCTTCATCTACC-3 ' (SEQ ID NO: 143), reverse primer: 5'-ACATACATGCATGCTGTAGAATTGTTGCTTTCG- 3' (SEQ ID NO: 144)) and inserted between the Notl and Sphl recognition sites of pYTC- Lib. This 87-amino acid region functions as a transcriptional repression domain (Kadosh and Strahl (1997) Cell 89:365-371). An exemplary sequence encoding the Ume6 repression domain and linking sequences includes:

GGCCGCCAGTGTGCTGGAATTCTGCAGATATCCATCACACTGGCGGCCGCAA ATTCTGCATCTTCATCTACCAAACTAGACGACGACTTGGGTACAGCAGCAGCA GTGCTATCAAACATGAGATCATCCCCATATAGAACTCATGATAAACCCATTTC CAATGTCAATGACATGAATAACACAAATGCGCTCGGTGTGCCGGCTAGTAGG CCTCATTCGTCATCTTTTCCATCAAAGGGTGTCTTAAGACCAATTCTGTTACGT ATCCATAATTCCGAACAACAACCCATTTTCGAAAGCAACAATTCTACAGCATG CATCTAGAGGGCC (SEQ ID NO: 145)

(6) Libraiy construction

A three-fingered protein library (the "3-F library"), encoding zinc finger proteins that have an array of three ZFDs, was constructed from nucleic acids encoding 40 different ZFDs or "fingers." A four-fingered protein library (the "4-F library") was constracted from nucleic acids encoding 27 different ZFDs (Table 2, below). Table 2: Zinc finger domains for constraction of 3-finger or 4-finger ZFP libraries.

Domain Source Target Sites Amino acid sequences SEQ Library Name ID

NO:

DSAR Mutated¹ GTC FMCTWSYCGKRFTDRSALARHKRTH 146 3F

DSHR Mutated' GGC HICHIQGCGKVYGDRSHLTRHLRWH 147 3F

DSKR Mutated' GGT FACPECPKRFMDSSKLSRHIKTH 148 3F

DSNR Mutated² GAC YACPVESCDRRFSDSSNLTRHIRIH 149 3F

DSSR Mutated³ GCC HICHIQGCGKVYGDRSSLTRHLRWH 150 3F

HSNK Human GAC YKCKECGKAFNHSSNFNKHHRIH 151 3F

HSSR Human GTT FKCPVCGKAFRHSSSLVRHQRTH 152 3F

ISNR Human GAA>GAT>GAC YRCKYCDRSFSISSNLQRHVRNIH 153 3F

KSNR Human GAG YGCHLCGKAFSKSSNLRRHEMIH 154 3F

QAHR Human GGA YKCKECGQAFRQRAHLIRHHKLH 155 3F

QFNR Human GAG YKCHQCGKAFIQSFNLRRHERTH 156 3F

QGNR Human GAA FQCNQCGASFTQKGNLLRHIKLH 157 3F

QNTQ Drosophila⁴ ATA YTCSYCGKSFTQSNTLKQHTRIH 158 3F

QSHR5 Human GGA>AGA>GAA>CGA YVCRECGRGFRQHSHLVRHKRTH 159 3F-

QSHV Human CGA>AGA>TGA YECDHCGKSFSQSSHLNVHKRTH 160 3F

QSNI Human AAA, CAA YMCSECGRGFSQKSNLIIHQRTH 161 3F

QSNK Human GAA>TAA>AAA YKCEECGKAFTQSSNLTKHKKIH 162 3F

QSTR Human GTA>GCA YKCEECGKAFNQSSTLTRHKIVH 163 3F

QTHR1 Human GGA>AGA,GAA>TGA,CGA YECHDCGKSFRQSTHLTRHRRIH 164 3F

RDHR1 Human GAG, GGG FLCQYCAQRFGRKDHLTRHMKKSH 165 3F

RDKR Human GGG>AGG YVCDVEGCTWKFARSDKLNRHKKRH 166 3F

RDNQ Mutated⁶ AAG FACPECPKRFMRSDNLTQHIKTH 167 3F

SADR Mutated³ AGA FQCRICMRNFSSPADLTRHIRTH 168 3F

SSNR Human GAG>GAC YECKECGKAFSSGSNFTRHQRIH 169 3F

TIDR Mutated⁵ ACT FQCRICMRNFSTHIDLIRHIRTH 170 3F

VSNV Human AAT>CAT>TAT YECDHCGKAFSVSSNLNVHRRIH 171 3F

VSTR Human GCT>GCG YECNYCGKTFSVSSTLIRHQRIH 172 3F

CSNR1 Human GAA>GAOGAG YKCKQCGKAFGCPSNLRRHGRTH 173 3F-.4F-

DGNV Mutated⁵ AAC FQCRICMRNFSDSGNLRVHIRTH 174 3F-.4F-

QSHR3 Human GGA>GAA YACHLCGKAFTQCSHLRRHEKTH 175 3F-

QSHT Human AGA,CGA>TGA>GGA YKCEECGKAFRQSSHLTTHKIIH 176 3F-.4F-

QSNR1 Human GAA FECKDCGKAFIQKSNLIRHQRTH 177 3F-.4F-

QSNV2 Human AAA.CAA YVCSKCGKAFTQSSNLTVHQKIH 178 3F-.4F-

QSSR1 Human GTA>GCA YKCPDCGKSFSQSSSLIRHQRTH 179 3F-.4F-

QTHQ Human CGA>TGA,AGA YECHDCGKSFRQSTHLTQHRRIH 180 3F-,4F-

RDER1 Human GCG>GTG, GAG YVCDVEGCTWKFARSDELNRHK RH 181 3F-.4F-

RDHT Human TGG,AGG, CGG.GGG FQCKTCQRKFSRSDHLKTHTRTH 182 3F-,4F-

RSHR Human GGG YKCMECGKAFNRRSHLTRHQRIH 183 3F-,4F-

RSNR Human GAG>GTG YICRKCGRGFSRKSNLIRHQRTH 184 3F-.4F-

VDYK Drosophila⁷ TAT, GAT FHCGYCEKSFSVKDYLTKIRTH 185 3F-.4F-

VSSR Human GTT>GCT>GTG>GTA YTCKQCGKAFSVSSSLRRHETTH 186 3F-.4F-

DGAR Mutated³ GTC FQCRICMRNFSDPGA VRHIRTH 187 4F-

DGHR Mutated³ GGC FQCRICMRNFSDPGHLVRHIRTH 188 4F-

DGNR Mutated³ GAC FQCRICMRNFSDPGNLKRHIRTH 189 4F-

DRDR Mutated³ GCC FQCRICMRNFSDCRDLARHIRTH 190 4F-

MHHE Mutated⁸ TGT YACPVESCDRRFSMSHHLKEHIRTH 191 4F-

QASA Mutated⁸ ATA FQCRICMRNFSQQASLNAHIRTH 192 4F-

QGDR Mutated³ GCA, GCC FQCRICMRNFSQSGDLRRHIRTH 193 4F-

QSDR Mutated⁹ GCT FQCRICMRNFSQSSDLVRHIRTH 194 4F-

QGTR Mutated⁸ ACA FQCRICMRNFSQRGTLTRHIRTH 195 4F-

RDTN Mutated⁵ AAG FQCRICMRNFSRSDTLSNHIRTH 196 4F-

TDKR Mutated³ GGG, GGT FQCRICMRNFSTADKLSRHIRTH 197 4F-

TGNR Mutated³ GAT>GAA FQCRICMRNFSTSGNLVRHIRTH 198 4F-

TIDR Mutated⁵ ACT FQCRICMRNFSTHIDLIRHIRTH 199 4F-

Superscripts in column 2 of Table 2 refer to 1) Zhang et al, (2000) J. Biol. Chem. 275:33850-33860; 2) Rebar and Pabo (1994) Science 263:671-673; 3) Segal (1999) Proc. Natl. Acad. Sci. USA 96:2758 ; 4) Gogus et al, (1996) Proc. Natl. Acad. Sci. USA. 93:2159-2164; 5) Drier et al, (2001) J. Biol. Chem. 276: 29466-29478; 6) Liu et al. (2001) J Biol. Chem. 276(14):11323-11334; 7) Hsu et α/., (1992) Science 257:1946-50.

FIG. 7 depicts a method of constructing a diverse three fmger library. Nucleic acid fragments encoding each ZFD were individually cloned into the p3 vector to form "single fingered" vectors. Equal amounts of each "single fingered" vector were combined to form a pool. One aliquot of the pool was digested with Agel and Xhol to obtain digested vector fragments. These vector fragments were treated with phosphatase for 30 minutes. Another aliquot of the pool was digested with Xmal and Xhol to obtain segments encoding single fingers. The digested vector nucleic acids from the Agel and Xhol digested pool were ligated to the nucleic acid segments released from the vector by the Xmal and Xhol digestion. The ligation generated vectors that each encodes two zinc finger domains. After transformation into E. coli, approximately 1.4 x IO⁴ independent transfomiants were obtained, thereby forming a two-fingered libraiy. The size of the insert region of the two-fingered libraiy was verified by PCR analysis of 40 colonies. The con-ect size insert was present in 95% of the library members.

To prepare a three-fingered library, DNA segments encoding one finger were inserted into plasmids encoding two fingers. The 2-fingered libraiy was digested with Agel and Xhol. The digested plasmids, which retain nucleic acid sequences encoding two zinc finger domains, were ligated to the pool of nucleic acid segments encoding a single finger (prepared as described above by digestion with Xmal and Xhol). The products of this ligation were transfomied into E. coli to obtain about 2.4 x 10⁵ independent transfomiants. Verification of the insert region confirmed that library members predominantly included sequences encoding three zinc finger domains.

To prepare a four-fingered library, DNA segments encoding two fingers were inserted into plasmids encoding two fingers. The two-fingered library was digested with Xmal and Xhol to obtain nucleic acid segments that encode two zinc finger domains. The two-fingered libraiy was also digested with Agel and Xhol to obtain a pool of digested plasmids. The digested plasmids, which retain nucleic acid sequences encoding two zinc finger domains, were ligated to the nucleic acid segments encoding two zinc finger domains to produce a population of plasmids encoding different combination of four fingered proteins. The products of this ligation were transfomied into E. coli and yielded about 7 x 10 independent transfomiants. (7) Construction of libraries for expression in yeast

The three-fingered (3-F) and four-fingered (4-F) libraries were subcloned into the EcoRI and Notl sites of pYTC-Lib, pYTC-Gal4, and pYTC-Ume6. These subcloning procedures produced six different libraries encoding three and four fingered ZFPs with and without transcriptional regulatory domains. After amplification in E. coli, each library was transfomied into the yeast strain YPH499a using lithium acetate. The transfonnation yielded approximately 1.5 x 10 colonies. The size of the insert region of the library was verified by PCR analysis of 50 colonies. 95% of the library members included the correct insert size. The transfomiants were resuspended in TE buffer and stored in glycerol at -80°C.

EXAMPLE 2: GROWTH-DEFECTIVE TRANSFORMANTS ON GALACTOSE MEDIA

The 3-F libraries were screened to identify chimeric zinc fmger proteins that impair the growth of yeast cells. This screen uses the GAL promoter to conditionally express the chimeric zinc fmger proteins. Previous studies have used the GAL promoter to yeast cDNA and genomic DNA sequences to identify genes whose overexpression is lethal (Liu et al, (1992) Genetics 132:665-673; Ramer et al. (1992) Proc. Natl. Acad. Sci. USA 89:11589-11593; Espinet et al, (1995) Yeast 11:25-32; Akada et al, (1997) Mol Gen. Genet 254:267-274; Stevenson et al, (2001) Proc. Natl. Acad. Sci. USA. 98:3946- 3951).

Yeast strain YPH499a was transformed with nucleic acid from the 3-F libraries. Transfomiants were grown for two days at 30°C on plates containing synthetic minimal medium that lack tiyptophan and that include glucose. Each of these glucose plates was replica-plated onto a galactose plate and a second glucose plate. The replica plates were grown overnight at 30°C. Then, each galactose plate was compared to its coιτesponding glucose replica plate. Colonies were identified that did not grow on galactose, but grew on glucose. These colonies were recovered from the glucose plate and retested by streaking selected colonies on galactose media. Libraiy plasmids were rescued from each colony that retested. The plasmids were retransfomied into YPH499a to confinn that the growth defect is caused by expression of the zinc finger protein. The vector plasmids pYTC and pYTC-Gal4, both of which do not encode any zinc finger domains, were analyzed as controls.

As shown in Table 3, 0.7% to 2.8%) of transfoπnants from the 3-F and 4-F libraries were unable to grow on galactose media. These percentages are significantly greater than the similar values obtained with the pYTC-Lib vector control (0.1%) and pYTC-Lib-Gal4 vector control (0.2%).

Table 3. Ratio of growth-defective transfomiants on galactose media

Libraiy No. of colonies on No. of growth- Ratio of growth- glucose media (A) defective colonies on defective mutants galactose media (B) (B/A x lOO)

3 -finger 5,820 42 0.7

3-finger + Gal4AD 7,428 206 2.8

3-finger + UmeβKD 8,400 78 0.93

PYTC-Lib vector 2,690 3 0.1

PYTC-Lib-Gal4 vector 2,750 6 0.2

Plasmids were recovered from ten colonies that were growth-defective on galactose. The plasmids (LI to L10) were retransfomied into yeast cells. All ten plasmids retested. Cells transfomied with the recovered plasmids were unable to grow on galactose media but were able to grow on glucose media. The zinc finger proteins encoded by these ten plasmids were characterized by DNA sequencing (Table 4). The potential target DNA binding sites for these proteins were infeιτed from infonnation about the binding specificities of the component zinc finger domains.

Table 4: ZFPs encoded in plasmids isolated from growth-defective transfomiants

EXAMPLE 3: ANTI-FUNGAL DRUG RESISTANCE

Ketoconazole is an orally absorbed antimycotic imidazole drag. It can be administered for the treatment of certain mucoses. Ketoconazole blocks the biosynthesis of ergosterol in yeasts and other fungi (Burden et al, (1989) Phytochemistry 28:1791- 1804) and has additional effects on cellular metabolism (Kelly et al, (1992) In Femandes, P.B. (Ed.) New Approaches for Antifungal Drug, Birkhauser, Boston, pp.155-187).

To check the fungistatic response of the tester strain YPH499a to ketoconazole, IO⁷ cells of the YPH499a strain were plated onto synthetic media containing different concentrations of the drug. We found that 35 μM ketoconazole inhibited growth of YPH499a cells and used this concentration to screen for ketoconazole-resistant yeast colonies.

1 x IO⁷ yeast cells containing plasmids from the 3-finger and 4-finger libraries were cultivated in synthetic liquid medium with 2%> galactose for 3 hours at 30°C to induce zinc finger protein expression and then were plated onto synthetic galactose agar plates containing 35 μiM ketoconazole (ICN Biomedicals). After fours days of incubation at 30°C, about 120 clones fomied colonies on galactose media containing 35 μM ketoconazole. These ketoconazole resistant yeast colonies were picked and streaked on fresh synthetic galactose agar plates containing 35 μM ketoconazole. 23 clones were randomly selected from the 120 resistant clones. For each of these 23 clones, the resistant phenotype was verified by plasmid rescue. The plasmids were isolated, transfomied into E. coli for amplification, and retransfomied into yeast strain YPH499a (Ausubel, et al. (Eds) (1995) Current Protocols in Molecular Biology⁾ John Wiley and Sons Ltd, New York). Equal numbers of retransfoπnants were spotted onto synthetic galactose agar plates with or without 35 μM ketoconazole. The retransformants were also spotted onto synthetic glucose agar plates with or without ketoconazole to verify that the drag resistance was induced by galactose-inducible expression of zinc finger protein.

In one experiment, 5xl0⁴ cells from each transformant were serially diluted (10^"1, 10^' , and 10^" fold) and spotted on galactose or glucose media supplemented without or with 35 μM ketoconazole. Growth of the cells was monitored after four days at 30°C and compared to controls on the same plate. Controls included a plasmid encoding a zinc finger protein that does not confer ketoconazole resistance and the pYTC-Lib plasmid without an insert encoding a zinc finger protein. In all 23 cases, recovered plasmids retested. In addition, ketoconazole resistance was observed only when the cells were plated on galactose media, confinning that expression of the zinc finger protein confers ketoconazole resistance.

The plasmids isolated from ketoconazole resistant transfomiants were sequenced and their expected target sequences in yeast genome were predicted (Table 5). Eleven unique clones were identified (Table 5).

Table 5: ZFPs that confer ketoconazole resistance

transfomiants. The amino acid sequences of these proteins are listed below (zinc finger domains are underscored and transcriptional regulatory domains are bolded). Kl :QSHV-QFNR- RSHR-Ume6 includes the following amino acid sequence:

MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKPYECDHCGKSFSQSSHLNVHKRTHTGEKP YKCHQCGKAFIQ8FNLRRHERT ΣGEKΕ>YKCMECGKAFNRRSHLTRHQRIHΑΑ]^κj SΑSSSrκτΛ DDLGTAAAVLS]SMRSSPYRTHDKPISNVNDI>1N^ EQQPIFESNNSTACI ( SEQ ID NO : 221 )

K2: RSNR-RSNR-QSSR1-QSHT-Ume6 includes the following amino acid sequence:

MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKP YICRKCGRGFSRKSNLIRHORTΗTGEKP YICRKCGRGFSRKSN lREQR ETGEKΩYKCPDCGKSFSQSSSLIRHORTRΣGEKΕΥKCEECGKA FRQSSHLTTHKIIHΑΑAMiSASSSTKLDDDLGTAAAVLSNMRSSPYRTHDKPISNVNDMNNTNAL GVPASRPHSSSFPSKGVLRPILLRIHNSEQQPIFESNNSTACI (SEQ ID NO: 222)

K3: RSNR-RSNR-QGTR-QSHR5-Ume6 includes the following amino acid sequence:

MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKPYJC^XCG^GFS^ g-V I^fJQ^TJπ'GEKP YICRKCGRGFSRKSNLIRHORTHTGEKPFQCRICMRNFSQRGTLTRHIRTHIGEKPYVCRECGRG FRQHSHLVRHKRTHΆΆΆΆNSAS S STKLDDDLGTAAAVLSNMRS SP YRTHDKP I SNVNDMNNTNAL GVPASRPHSSSFPSKGVLRPILLRIHNSEQQPIFESNNSTACI (SEQ ID NO : 223 )

K4: RSNR-RSNR-QGTR-QTHQ-Ume6 includes the following amino acid sequence: MGKPIPNPLLGLNSTQA GAPPKKKRKVGIRIPGEKP YJCJEKCGRGF.S S -JJRIFQ^RIΠ'GEKP YICRKCGRGFSRKSNLIRHQRTHTGEKPFQCRICMRNFSQRGTLTRHIRTH GEKPYECHDCGKS

FJ?QSTHLTQffi-;j;jHAAAANSASSSTKLDDDLGTAAAVLSNMRSSPYRTHDKPISNVNDi NTNAL GVPASRPHSSSFPSKGVLRPILLRIHNSEQQPIFESNNSTACI (SEQ ID NO: 224)

K5: VSSR-DGNV-VSSR-VDYK-Gal4 includes the following amino acid sequence:

MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKPYRCKQCGI^FGYSGG ^RHETT.Ω'GEKP FQCRICMRNFSDSGNLRVHIRTHTGEKPYTCKQCGKAFSVSSSLRRHETTH GEKPFHCGYCEKS

ID NO : 225 )

K6: MHHE-QSNR1-VSSR-QGDR-Gal4 includes the following amino acid sequence: MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKPYACPVESCDRJΪF.S SHFFLJCEHIRRJΠ'GE KPFECKDCGKAFIQKSNLIRHQRT ΣGEKP YTCKQCGKΆFSVSSSLRRHETTHTGEKPFQCRICM

ITASKIDDGNNSKPLSPGWTDQTAYNAra^^

( SEQ ID NO : 226 )

K7: DGNV-QSHT-QSSR1-DGHR-Gal4 includes the following amino acid sequence:

MGKPIPNPLLGLNSTQA GAPPKKKRKVGIRIPGEKPFQC^JCMR FGDSG T RWFJJΪTHTGEKP YKCEECGKAFRQSSHLTTHKIIHIGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPFQCRICMRN

FgϋP H TOHIRTi-g-AAAKIFt^

SKIDr SΦKKPLSPGE-raD r YNAEGIT CMF^^ (SEQ ID NO -. 227 )

K8: DGAR-RDTN-QTHQ-RDTN includes the following amino acid sequence:

MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKPFQCRICMRNFSDPGALVRHIRTHTGEKP FQCRICMRNFSRSDTLSNHIRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPFQCRICMRN FSRSDTLSNHIRTHAAAARG HLEGRIM (SEQ ID NO: 228)

K9: RDHT-QTHQ-QSHT-DGNV includes the following amino acid sequence:

MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKP YECHDCGKSFRQSTHLTQHRRIHTGEKPYKCEECGKAFRQSSHLTTHKIIHTGEKPFQCRICMRN FSDSGNLRVHIRTHAAAARGMHLEGRIM (SEQ ID NO: 229)

K10: RDHT-QTHQ-QSHT includes the following amino acid sequence:

MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKPFQCKrCQRKFgRgDlfL^THr^rj-JTGEKP YECHDCGKSFRQSTHLTQHRRIHΣGEKΕ>YKCEECGKAFRQSSHLTTHKIIHΑAAΑRGMEIjEGRτM (SEQ ID NO : 230 )

Kl l: RDHT-QSHV-QSHV includes the following amino acid sequence:

MGKPIPNPLLGLNSTQA GAPPKKKRKVGIRIPGEKPFQCJCrCQ^JCFgRgDHLKTHr^rjJTGEKP YECDHCGKSFSQSSHLlWHKRT GEKΕ'YECDHCGKSFSOSSHLNVHKRTmΛΑΑRGME EGRτM ( SEQ ID NO : 231 )

Other zinc finger proteins that include two, three, or more zinc fmger domains that have the same general motifs (i.e., set of four DNA contacting residues) and that are arranged in the same consecutive order as any of the proteins described above may also confer drug resistance to a fungal cell.

Some of clones had included a similar configuration of zinc finger domains. The first and second fingers of K2, K3, and K4 are identical to one another. In these clones, three out of four fingers are identical to those in coιτesponding positions in the other proteins. It is extremely unlikely that these ZFPs are related only by chance (P < 1.6 x 10^" ⁵). Accordingly, the K2, K3, and K4 ZFPs may bind to the same target site in vivo and regulate the same target genes.

In addition, the zinc fmger protein encoded by the K10 clone is closely related to those encoded in the K9 and Kl 1 clones. The QTHQ, QSHT, and QSHV fingers found in K9, K10, and Kl 1 can recognize the same 3-bp DNA site: 5'-HGA-3'. Given their structural similarity, the K9, K10, and Kl 1 ZFPs may bind to the same target site in vivo and regulate the same target genes. Within each of the two groups of related clones (i.e., K2, K3, and K4; and K9, K10, and Kl 1), all the ZFPs include the same type of regulatoiy features. The K2, K3, and K4 clones each include the Ume6 repression domain. The K9, K10, and Kl 1 clones each function without a dedicated transcriptional regulatory domain. Synergistic or additive effects may result when two or more ZFPs are cotransfom ed into cells. For example, when the K4 and K5 ZFPs were co-expressed, yeast cells became completely resistant to ketoconazole. The combination produced an approximately 1, 000-fold enhancement of the phenotype. ZFP mutants were constructed that alter a DNA contacting residue or that remove or replace a regulatoiy domain. In one mutant (VSSR-DGAV-VSSR-VDYK-GAL4AD), the asparagine DNA contacting residue of the second zinc finger of K5 was mutated to alanine. Gel shift assays demonstrated that this mutated ZFP had at least 10-fold decrease in DNA-binding affinity for its expected DNA site. This mutant K5 protein does not confer drag resistance to yeast cells. In another mutant, the Gal4 activation domain of the K5 zinc finger protein was deleted by inserting a stop codon in front of the DNA sequence encoding the activation domain. This protein also does not confer resistance to ketoconazole. Similar results were obtained for the other ketoconazole resistance ZFPs. When the activation domain fused to the K5 ZFP was replaced with the Ume6 repression domain, expression of the Ume6-foπn of the protein reversed the ketoconazole resistance phenotype. The cells that express the Ume6-fomι were more sensitive to ketoconazole relative to control cells. This result indicates that transcription factors can be selected by selecting for transcription factors that exacerbate a phenotype and then altering the attached regulatoiy domain (e.g., by switching the directionality of its function) to produce a transcription factor that has the desired phenotypic effect. Screening for opposite phenotype opposite to the desired phenotype can be more amenable than screening for the desired phenotype itself. Examples of replacements that switch functional directionality include replacing one type of regulatoiy domain with another type or regulatory domain and removing a regulatory domain. In the case of the K5 ZFP, a protein that increases drag sensitivity was obtained by screening for a protein that increased drag resistance and replacing its transcriptional activation domain with a transcriptional repression domain.

Table 6:

DNA microaiTay analysis was used to identify genes associated with the drag resistance phenotype. We reasoned that different ZFPs conferring the identical phenotype may regulate identical gene sets whose differential expression is directly or indirectly associated with the phenotype. Three ZFPs ~ K5, K6, and K7 — were chosen for expression profiling analyses. All three transcription factors contain the Gal4 activation domain. Out of 6,400 yeast open reading frames, ten were activated over 2 fold by at least two different ZFP transcription factors, and four open reading frames were activated by all three tested ZFP transcription factors. The four activated open reading frames are: YLL053C, YJR147W, YLL052C, and YPL091W.

Table 7 lists the number of genes that are regulated by more than one chimeric ZFP. Table 7: Coordinately Regulated Genes

K5 K6 K7 Number of Commonly

Regulated Genes

+ - - 39

+ + - 1

+ - + 2

+ + + 4

- + - 95

- + + 3

- - + 126

PDR5, a gene known to pump out ketoconazole, was activated by two ZFPs, K6 and K7, but not by K5. This result suggests that K5 confers ketoconazole resistance by a PDR5 -independent mechanism and that at least two different pathways can confer ketoconazole resistance in yeast. One pathway depends on activation of PDR, and the other is independent of PDR5.

To identify new genes associated with the drag resistant phenotype, we evaluated the drag resistance phenotype of cells overexpressing one of the four genes activated by all three tested ZFP transcription factors. We found that one of the genes ~ YLL053C ~ induced ketoconazole resistance when overexpressed on its own. See Table 6, above. YLL053C is homologous to plasma membrane and water channel proteins from Candida albicans. The amino acid sequence of YLL053C is as follows:

MWFPQIIAGMAAGGAASAMTPGKVLFTNALGLGCSRSRGLFLEMFGTAVLCLTVL TAVE KRETNFMAALPIGISLFMAHMALTGYTGTGVNPARSLGAAVAARYFPHYH IYWISPLLG AFLAWSV QLLQILDYTTYVNAEKAAGQKKED (SEQ ID NO: 232)

The amino acid sequence of an exemplary homologous channel protein (AQY1) from Candida albicans is as follows: MVAESSSIDNTPNDVEAQRPVYEPKYDDSVNVSPLKNH IAFLGEFFGTFIFLWVAFVIA QIANQDPTIPDKGSDP QLI ISFGFGFGVMMGVF FFRVSGGNLNPAVTLTLVLAQAVP PIRGLFMMVAQMI GMAAAGAASAMTPGPIAFTNGLGGGASKARGVFLEAFGTCILCLTV LM AVEKSRATFMAPFVIGISLFLGHLICVYYTGAGLNPARSFGPCVAARSFPVYHWIY VGPILGSVIAFAI KIFKILKYETCNPGQDSDA (SEQ ID NO: 233)

The YLL053C gene product may confer resistance by pumping out ketoconazole as does the PDR5 gene product. These data demonstrate that genes associated with phenotypic changes can be identified by the analysis of gene expression profiles of cells. Gene identification is further assisted by the use of different chimeric ZFPs that cause the same phenotype (in this example, ketoconazole resistance). EXAMPLE 4: THERMOTOLERANT TRANSFORMANTS SCREENING

We screened libraries encoding chimeric zinc fingers for nucleic acids that encode proteins that confer them otolerance to yeast cells.

1 x 10 yeast cells containing nucleic acids from the 3 -fingered libraries were cultivated on SD synthetic liquid medium with 2% galactose for 3 hrs at 30°C and then incubated at 52°C (air temperature) with slow gyration for 2 hrs. After heat treatment, the culture were plated on galactose media and incubated for 5 days at 30°C. Growing yeast colonies were suspended in galactose liquid media and incubated at 52°C for 2 hrs to confirm the thermotolerant phenotype. Plasmids were isolated from these cells and retransformed into yeast as describe above. The retransfom ants were cultivated in galactose liquid media for 3 hrs at 30°C and incubated at 52°C for 2hrs. The incubated retransfoπnants were spotted in four dilution onto SD galactose agar plates. To verify that the themiotolerance is induced by the expression of zinc fmger protein in the mutants, retransfomiants were cultivated in SD glucose liquid media with the same condition described above and then spotted onto SD glucose agar plates. The transfomiants with pYTC vector and plasmid encoding randomly-picked 3-fingered proteins were used as controls for the procedure.

Thermotolerant yeast cells were identified from the cells transfomied with zinc finger protein expression plasmids. Wild-type cells carrying the pYTC-Lib plasmid or plasmid encoding a randomly-selected zinc fmger protein were used as negative controls. A total of IO⁷ cells were grown in galactose liquid media. Galactose induces zinc finger protein expression. Cells were heat treated at 52°C for 2hrs and then plated on galactose minimal agar plates. After five days of incubation at 30°C, 26 colonies grew on the plates. We rescued plasmids from these colonies and retransformed them into YPH499a. We isolated nine clones that induced varying degrees of themiotolerance. Typically, expression of the zinc finger proteins from these clones caused up to 10% of the cells to survive heat treatment, when cultured in galactose media but not in glucose media. Only about 0.3% of cells transformed with control plasmid survived under the same conditions. Freshly-growing retransfomied cells survive better under the experimental conditions than frozen cell stocks. These results demonstrate that the expression of particular zinc finger proteins can induce themiotolerance in yeast cells. ZFPs were characterized by DNA sequencing of the recovered libraiy plasmids (Table 8). Table 8: ZFPs encoded in plasmids isolated from thermotolerant transfomiants

EXAMPLE 5: NEURITE FORMATION

We screened our nucleic acid libraries for nucleic acids encoding zinc finger proteins that induce neuritogenesis in the mouse neuroblastoma cell line Neuro2A, a cell line capable of differentiating into a neuronal cell type. We identified a novel chimeric ZFP that induces neuronal differentiation, as evidenced by alterations in cell morphology and the expression of neuronal marker genes.

Materials and Methods Libraiy Construction

See Example 1, above. As described, a three- fmger and four-finger ZFP-Tf libraries were constructed using 40 and 25 zinc finger domains, respectively. The three- finger and four-finger libraries recognize approximately 9- and 12-bp DNA binding sites, respectively. ZFPs were expressed as fusion proteins to the p65 transcriptional activation domain and to the KRAB repression domain. Cell culture and neuronal differentiation ofNeuro2A cells

Mouse neuroblastoma Neuro2a cells were maintained in MEM-a media with 10% FBS and antibiotics at 37°C, in a humidified atmosphere containing 95% air and 5%₀ CO . Cells were seeded at 8.0 x IO³ cells per 96-well culture plate and then were transfected using LIPOFECTAMINE PLUS™ reagent (Invitrogen, CA) according to the manufacturer's protocol, with 50 ng of ZFP, together with 20 ng of LacZ reporter plasmid. In vitro differentiation was canied out with or without retinoic acid (RA) (10 μM) respectively, and G418 (1 mg/ml) was treated 24 hours after transfection to reduce the number of untransfected cells. Cells were then cultured for 96 hours and then fixed, stained for β-galactosidase activity, and photographed. Among β-galactosidase-positive cells, cells were regarded as differentiated if the length of neurite extension was at least two times the diameter of the cell body.

Result and Discussion

Screening ZFP-TFs inducing neuritogenesis

To screen for ZFP-TFs that can induce neuronal differentiation, Neuro2A cells were transiently transfected with library plasmids and a reporter plasmid that includes the LacZ gene. LacZ expression was used to visualize the morphology of transfected cells. Because the differentiating cells grow slower than non-differentiating cells and the latter will dominate the cell population, we treated the cell culture with G418 24hr after transfection to reduce the number of untransfected cells. After five days, cells were fixed and LacZ-stained. We then characterized the cells by their morphological characteristics. In particular, we identified cells with increased neurite length and thickness.

We identified several ZFP-TFs that alter neuritogenesis. These ZFP-TFs affect neuritogenesis to varying degrees. The ZFP-TF, named Neurol -p65, had the most prominent effect on differentiation, as measured by the length and thickness of neurites that it produced. A marked acceleration of neuritogenesis in the Neurol -p65 -transfected Neuro2A cell was also observed when cells were treated with 10 μM RA (retinoic acid). The nucleic acid sequence encoding Neurol -p65 and its amino acid sequence is shown in FIG. 14. The Neurol -p65 nucleic acid encodes a chimeric ZFP that includes the zinc fmger domains QSNRl-QSNK-CSNRl. Other ZFPs with the same motifs and/or ZFPs that binding to a site that at least partially overlaps the Neurol -p65 binding site are also expected to modulate differentiation. Based on the zinc finger-DNA binding site directory (see Table 6), the predicted binding site for Neurol is 5'-GACGAAGAA-3'. Neurol -p65 requires the p65 activation domain to induce neurite formation. The same zinc fmger domains fused to the KRAB transcriptional repression domain or without any effector domain do not drive neuritogenesis. See FIG. 8 and Table 9. Also the DNA binding ability of Neurol -p65 is critical, since mutations that are predicted to disable its DNA binding ability of the zinc finger domains of Neurol -p65 (Neurol - p65mut) abolished its ability to support neuritogenesis.

Real-time PCR is used to characterize the expression level of neuronal marker genes during the course of Neurol -p65 expression in cells. Similarly, nucleic acid microarrays are used to compare the pattem of gene expression between RA-treated cells and ZFP-TF treated cells at various time points during differentiation. Neurol -p65 may activate a pathway that is required for neuroblastoma cell differentiation at least in vitro.

Table 9: Percentage of neurite bearing cells

Construct No RA RA lOuM pCDNA3 (vector) 5.77 ±1.99 14.93 ± 7.69

08_Dl-p65 42.39 ± 5.56 41.59 ± 6.46

08_Dlmut-p65 5.25 ± 0.71 NA

08JD1 alone 5.85 ± 4.44 NA

08_D1-KRAB 4.46 ± 0.57 NA

EXAMPLE 6: OSTEOGENESIS

The C2C12 cell line is derived from the myoblast lineage, but can differentiate into osteoblasts upon the addition of bone morphogenic protein-2 (BMP -2) (Katagiri, T. et al. , (1994) J. Cell. Biol. 127, 1755). Several naturally-occunϊng transcription factors have been identified as candidates for controlling this process (Lee, K.-S. et al, (2000) Mol Cell. Biol 20:8783; Nakashima, K. et al, (2002) Cell 108: 17).

We screened for chimeric zinc fmger proteins that induce transdifferentiation of C2C12 myoblasts to osteoblasts in the absence of BMP-2. We transiently transfected a nucleic acid library encoding chimeric zinc finger proteins into C2C12 cells and screened for cells that undergo transdifferentiation in the absence of BMP-2. Seven days after transfection, the transfomied cells were stained for alkaline phosphatase (ALP), a marker for osteoblasts (Katagiri, T. et al, (1994) J. Cell Biol 127:1755). From a screen of about 2,000 ZFP-TFs, we identified one activator, Osteol-p65, that induced strong ALP staining in about 30 % of cells. The percentage of cells stained with ALP was similar to the percentage of transfected cells detected by LacZ staining. From these results, we conclude that the Osteol-p65 transcription factor can trigger the transdifferentiation of C2C12 myoblasts to osteoblasts in the absence of BMP-2.

As a positive control, cells were treated with 1 μg/ml of BMP-2. Nearly 100%_> of the positive control cells strongly stained for ALP upon BMP-2 treatment. Without BMP-2 treatment, negative control cells that were transfected with a control vector had only background staining. Osteol-p65 is a four-finger protein composed of the RDKR- QTHRl-VSTR-RDKR zinc fmger domains (from N- to C-terminus). See FIG. 15. This protein is predicted to recognize the DNA element 5'-GGGGCWRGAGGG-3' (SEQ ID NO:234).

For these experiments, the mouse myoblast cell line, C2C12, was maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 4.5g/L glucose, 10% FBS, and antibiotics, at 37°C, in a humidified atmosphere containing 95%> air and 5% CO₂. Cells were seeded at 1.0 x IO⁴ cells per 96-well culture plates. The cells were transfected with 50 ng of libraiy nucleic acid using LIPOFECTAMINE PLUS™ (Invitrogen) according to the manufacturer's protocol. Twenty-four hours after transfection, the growth medium was replaced with DMEM containing 2% FBS, and the cells were cultured for an additional six days.

To examine the differentiation of C2C12 cells to osteoblasts, ALP staining was perfomied as described in Katagiri, T. et al, (1994) J. Cell. Biol. 127:1755.

In summary (refening to Examples 5 and 6), we have screened artificial transcription factor libraries for proteins that can induce two different cell differentiation processes, neurogenesis and osteogenesis. Again, the method does not require prior knowledge of the biology of particular stem cells.

EXAMPLE 7: INSULIN REGULATION

We stably transfected nucleic acids encoding chimeric zinc fmger proteins in human 293 cell lines, and analyzed transformed cells using DNA microaιτay experiments to characterize genes regulated by each chimeric zinc finger protein (see FIGs. 4, 5, and 9). We have identified a chimeric zinc finger protein (08_D04-p65) that increases the expression of human insulin gene more than 60 fold.

To test whether 08_D04-p65 can induce insulin gene expression in different human cell lines, we transiently transfected a nucleic acid encoding 08_D04-p65 into HeLa cells. Expression of the nucleic acid in HeLa cells caused a 80 fold increase in expression of the insulin gene.

Accordingly, 08_D04-p65, its derivatives, and similarly functional zinc finger proteins can be used as therapeutics for diabetes. DNA encoding 08_D04-p65 or a similarly functional zinc fmger protein can be delivered into diabetic patients by viral delivery or in encapsulated fonn (e.g., a liposome). Once DNA is delivered into cells, the zinc finger protein can be expressed to induce the production of insulin. In some implementations, the nucleic acid encoding the zinc fmger protein can be operably linlced to an inducible promoter, e.g., a Tet-inducible promoter. The use of doxycycline as an inducer enables the level of insulin production to be regulated by a small chemical. Because insulin-inducing zinc finger proteins, such as 08_D04-p65, can function in different human cell lines, it may work in both pancreatic cells (e.g., beta cells and non- beta cells) and non-pancreatic cells.

It is also possible to identify an artificial transcription factor that induces expression of insulin processing enzymes. Cells can be engineered to express such a transcription factor and a transcription factor that induces the insulin gene, e.g., 08 D04- p65. In addition, insulin- inducing zinc finger proteins can be also used for ex vivo cell therapy. In one example, cells from a patient are modified in vitro by the introduction of a nucleic acid encoding an insulin-inducing zinc fmger protein. The modified cells are then transplanted back into patients or into another subject.

Materials and Methods.

DNA microarray analysis of cell lines expressing ZFP-TFs Nucleic acids encoding zinc finger proteins were stably introduced into FlpTRex- 293 cell lines (Invitrogen) essentially as described in the manufacturer's protocols. Briefly, the Hindlll-Xhol fragment from the pLFD-p65 or pLFD-Kid vectors was subcloned into pCDNA5/FRT/TO (Invitrogen CA). This fragment includes a sequence encoding the zinc fmger proteins. Resulting plasmids were cotransfected into FlpTRex- 293 cells with pOG44 (Invitrogen). Stable integrants that express ZFP-TFs upon doxycycline induction were obtained.

DNA microaπays containing 7458 human EST clones were obtained from Genomictree (Korea). FlpTRex-293 cells stably expressing ZFP-TFs were grown with (+Dox) or without (-Dox) 1 μg/ml Doxycycline for 48 hours. Total RNA was prepared from each sample. RNA from the -Dox sample was used as a reference (Cy3), and +Dox as the experimental sample (Cy5). Microarray analysis was perfonned according to the manufacturer's protocols. Spots with sum of median less than 500 were colored red and not analyzed. Nucleic acids encoding certain zinc finger proteins were also expressed in HeLa cells. pLFD-p65 containing 08_D04 ZFP was transiently transfected into HeLa cells using LIPOFECTAMINE™ 2000 (Invitrogen). The pLFD-p65 vector, which does not encode a zinc finger protein, was also transfected into cells in parallel as a control.

EXAMPLE 8

We screened libraries encoding chimeric zinc fingers to identify nucleic acids that encode proteins that alter reporter gene expression in a mammalian cell.

Construction of libraries Two ZFP libraries were constracted as four fmger proteins and three finger proteins in P3, a modified pcDNA3 vector. Constraction of these libraries is described above.

Transfection of ZFP plasmids Human embiyonic kidney 293 cells were cultured with 100 μl of DMEM with

10 % of FBS in a humidified atmosphere containing 95%> air and 5% CO2 and seeded in wells of a 96 well plates 1 day ahead of transfection. The plasmids were isolated from individual colonies of each library separately and transfected into 293 cells LIPOFECTAMINE PLUS™ (Invitrogen) as recommended by the manufacturer. For MTT assay, 50 ng of plasmids containing zinc fmger protein gene fused to the functional domains were transfected into cells cultured at 5 x 10³ cells/well. For the SEAP assay, 10 ng of pSEAP2 -control (Clontech) plasmid was co-transfected with 50 ng of plasmids containing zinc finger protein gene fused to the functional domains into cells cultured at 1 x IO⁴ cells/well. The transfected cells were incubated at 37°C for three days before assays.

SEAP assay

Three days after transfection, medium was transferred to the new tubes and heated for 30 minutes at 65°C to inactivate the endogenous alkaline phosphatase. 25 μl of medium was mixed with the same volume of 2 X SEAP assay buffer (2 M Diethanolamine (pH 9.8) 10 ml, 1 M MgCl₂ 10 μl, IM homoarginine 200 μl, pNitro phenyl phosphate 44.52 mg). Absorbance at 405 nm was measured after the indicated incubation times at 37°C.

Luciferase assay

For the luciferase assay, 5 ng of both CMV-luciferase and pRL-SV40 plasmid were co-transfected with 50 ng of ZFP coding plasmids to 293 cells seeded in wells of a 96 well plate. After 3 days of incubation, cells were harvested and the luciferase assay was done by a Dual Luciferase reporter assay system (Promega) as recommended by the manufacturer.

MTT assay

MTT assay is used to measure cell growth. Tetrazolium MTT reduced by metabolically active cells changes to a yellow color. Measurement of light absorbance by the yellow color indicates the number of viable cells. MTT assay was done using a MTT assay kit (Trevigen). The MTT assay was done at three days after transfection. Briefly, 10 μl of MTT solution was added directly to the culture medium and incubated for two hours in a CO₂ incubator. Then, 100 μl of detergent solution from the MTT assay kit was added and incubated in the dark for two hours before measurement of absorbance at 570 nm was measured using a Power-Wave 340x (Bio-Tele Instrument).

To count cell numbers, 293 cells were seeded at 1.5 x IO⁴ cells/well in a 24 well plate one day before transfection. Three days after transfection with 200 ng of ZFP- coding nucleic acid or control vector plasmids, cells were washed with PBS once and suspended in 30 ul of TE (10 mM Tris 1 mM EDTA, pH 8.0) buffer and counted by the hematocytometer.

Results

Change of protein productivity

We tested whether library nucleic acids could alter expression of a heterologous nucleic acid. SEAP (secretory alkaline phosphatase) was used as a reporter protein to test whether a ZFP can affect amount of foreign protein produced intracellularly. Table 10 shows representative results obtained from 81 wells, each well containing cells transfected with a nucleic acid encoding a random chimeric ZFP from the library and three wells containing reference cells transfected with a control vector. Several wells exhibited increased absorbance relative to other wells that contain cells transfected with nucleic acid encoding other random ZFPs and relative to the wells that contain cells transfected with a control vector.

The P_B08, a zinc fmger protein fused to the p65 domain showing the highest SEAP activity. This ZFP was retested by transfection with the SEAP plasmid in three separate transfections. Table 11 illustrates one such retesting. The P value for P_B08 in this assay is 0.008. P_B08 exhibited approximately 16-fold increase of SEAP activity compared to that of the parental plasmid.

Table 11: SN40-SEAP reporter in 293 cells.

Expression Plasmid average st. dev. P_B08 16.51 0.54

Nector(HAΝLS) 1 0.17

Since the SEAP assay was used to assay the production of foreign proteins, reasons for the observed increased SEAP activity could be various, for example, the direct or indirect activation of a S V40 promoter used for the SEAP gene expression in pSEAP2- Control plasmid. Other reasons include: increased activity of proteins functioning in secretion pathway or activation of the general machinery for the protein production. The mechanism of enhanced activity of SEAP can be elucidated by detennining the effect of P_B08 on the production of other proteins, e.g., using other reporter proteins (e.g., unsecreted reporters) and reporters linlced to other promoters.

To investigate this point, an extensive screening had been earned out using the SEAP reporter under the control of CMV promoter (CMV-SEAP) in 293F cells. Two ZFPs, K44_l 1JD01 and K44_l 1_G12, that increase activity of the SEAP protein were isolated. These two ZFPs were further tested using two other reporter plasmids, SV40- SEAP and the luciferase reporter under the control of CMV promoter (CMV-Luc). Table 12 lists some of the results. K44_l 1JD01 and K44_l 1_G12 exhibited approximately 3- fold increase of SEAP activity compared to that of the parental plasmid (vector). K44_l 1_D01 showed activation of SV40-SEAP but not the CMV-Luciferase, suggesting that the activation may be related to mechanisms specific to SEAP such as secretion. In contrast, K44_l 1_G12 showed approximately 2-3 fold activation for both CMV- luciferase and SV40-SEAP, suggesting that the mechanism is specific to neither promoter nor reporter. Thus, K44_l 1_G12 may be used increase the production of proteins generally in cells, e.g., eukaiyotic cells.

Table 12: CMV-SEAP, CMV-Luc and SV40-SEAP reporter in 293F cells. average st.dev.

Expression CMV- CMV- SV40- CMV- CMV- SV40- plasmid SEAP Luc SEAP SEAP Luc SEAP

K44_11_D01 3.49 0.87 3.66 0.46 0.26 0.28

K44_11_G12 2.82 2.75 2.14 0.50 0.38 0.15

Vector 1 1 1 0.22 0.19 0.17

(HA-NLS)

Zinc finger proteins K44-16-E12 (FIG. 25) and K12_A11 (FIG. 24) may also alter protein production, e.g., as indicated by CMV-SEAP and SMV-Luc expression.

As an analogous example, the overexpression of cyclin Dl has been reported to cause to increase production of some proteins (US patent No. 6,210,924 BI). A zinc finger protein that can directly or indirectly activate the cyclin Dl expression may up- regulate protein production, although the effects of a zinc fmger protein may be different, far more broad ranging, or more potent. Other zinc finger proteins resulting in a physiological effect similar to cyclin Dl overexpression can be isolated by this assay. Such zinc finger proteins can be used to enhance production of the protein of interest.

Change of cellular growth rate Two ZFPs fused to the kid domain, K_D10 and K_F02, were selected as representatives of the ZFPs regulating cell growth since they exhibited the maximum variations compared to the controls based on the MTT assay results. To confinn the MTT assay, the cell numbers were counted after transfection of K_D10 and KJF02 in 293 cells. The result was compared to the cell number of the well transfected with the parental vector, converted to relative percentages and shown in Table 13. Compared to the parental vector, KJD10 exhibited approximately 3-fold inhibition and K_F02 showed 2.7- fold activation of cell growth compared to the control. The P values for KJD10 and K_F02 are 0.001 and 0.01, respectively. For sequence infonnation, see FIGs. 18 and 19.

Table: 13: MTT assay results for two ZFPs. relative cell number average st. dev.

K F02 279.3 6.9

K D10 27.4 4.3

Vector 99.99 3.4

Change of cellular growth rate

We have identified zinc finger proteins that can alter the rate of cell growth and proliferation. These important phenotypes are linked to diseases such as cancer and viral infection and to developmental processes. The ability to use zinc finger protein to control cell growth and proliferation can include, for example, controlling apoptosis, cell differentiation, host cell defenses (e.g., against viral infection), and p53-mediated signaling

A zinc finger protein that produces one or more of these phenotypes may directly or indirectly regulate a gene that regulates cell growth or signaling. The specific zinc finger proteins describe here produce detectable effects to cellular growth as detected by the MTT assay. For example, two zinc finger proteins showed at least a two-fold difference of cell growth compared to the controls. Considering that this assay was based on observations of transiently transfected cells three days after transfection, the two-fold difference is significant. In addition, the change in the cell number confirms that that the difference detected using the MTT assay is not particular to the assay. Since cell growth is such an important parameter of cell physiology, this experiment can be extended to a large screening to isolate sufficient numbers of zinc finger proteins regulating cellular growth. The cDNA microarrays can be used profile the gene expression patterns of cells that expressing each of these zinc finger proteins. The profiles can identify novel genes and pathways that participate in cell proliferation or apoptosis.

These experiments indicate that the zinc finger proteins are able to induce various phenotypes in mammalian cells, e.g., in this case altering cell proliferation. EXAMPLE 9: SOLVENT TOLERANT BACTERIAL CELLS

We screened for bacterial cells that express artificial chimeric zinc fmger proteins for cells that were resistant to an organic solvent as a result of the artificial chimeric zinc finger protein. Three different zinc finger proteins were identified for their ability to confer hexane tolerance to E. coli cells (Table 13). Hexane tolerance was evaluated by comparing the survival rate of transformants expressing one of the zinc finger proteins — HT-1, HT-2, and HT-3 ~ to the survival rate control cells. The control cells either included an empty vector (CI) or ZFP-1. The ZFP-1 construct encodes a zinc fmger protein that does not confer hexane resistance and that includes the fingers RDER-QSSR- DSKR. Bacterial cells that express hexane resistance-confemng zinc fmger proteins exhibited as much as a 200-fold increase in hexane tolerance.

Table 13: Hexane Resistant Zinc Finger Proteins.

Expression Construct Name Survival Rate (%)

Control CI 0.14

Control ZFP-1 0.05

Hexane resistance ZFP HT-1 21.4

Hexane resistance ZFP HT-2 1.85

Hexane resistance ZFP HT-3 28.6

The expression plasmids for these zinc fmger proteins include an IPTG-inducible promoter. Transformants that express each of the hexane resistance inducing ZFPs were characterized in the presence and absence of IPTG. Transfoπnants expressing HT-2 showed higher hexane tolerance in the presence of IPTG, whereas HT-1 or HT-3 expressing cells were hexane tolerant even in the absence of IPTG.

Table 14: Zinc finger proteins that confer hexane tolerance in E. coli

Name FI F2 F3 F4 putative DNA target No. of occun-ences(##)

HT-1 RSHR HSSR ISNR GAH GTT GGG 5

HT-2 ISNR RDHT QTHR VSTR GCT GRA GG GAH 3 (SEQ ID NO:235)

HT-3 QNTQ CSNR ISNR GAH GAV ATA 1 (##) Occurrence of the ZFP in nine colonies that could grow after third round of hexane tolerant screening Construction of a nucleic acid library that encodes ZFPs for E. coli expression. To express zinc fmger proteins conditionally in E. coli, we subcloned the zinc finger proteins cloned in pYTC-Lib yeast vector into a pZLl. pZLl was constracted by the modification of pBT-LGF2 (Clontech, Palo Alto, CA). Plac-UV5 was amplified from pBT-LGF2 by PCR (forward primer: 5 '-GACA ACC GGT CAT CGA TAA GCT AAT TCT CAC-3' (SΕQ ID NO:236); reverse primer: 5'-TTG TCC ATG GAC GCT GTT TCC TG GTG AAA-3' (SΕQ ID NO:237)). The PCR product was cloned into the pYTC Lib vector between Agel and Ncol site. The vector was named as pYTC-lac. pYTC-lac was digested with Clal and Notl to subclone the DNA fragment containing the following elements: Plac promoter- V5 epitope-MCS. The digested DNA fragment was purified after gel-electrophoresis and subcloned into the Clal and Notl sites of pBT-LGF2. The resulting vector was named as pZLl. The 3F- or 4F- ZFP library constructed in pYTC-Lib was digested with ΕcoRI and Notl. The digested DNA fragments were gel- purified and subcloned into ΕcoRI and Notl site of pZLl, thus providing a library for E. coli expression.

Screening for Solvent Tolerance. The E. coli strain DH5α was transformed with the 3-finger or 4-finger ZFP nucleic acid library foπnatted for prokaiyotic expression. Transformants were cultured overnight in LB with chloramphenicol (34 μg/ml). The overnight-culture was diluted to 1:500 in 1 ml fresh LB media with ImM IPTG and chloramphenicol to induce ZFP expression. After a three hour incubation at 30°C, hexane was added to 1.5%> and rapidly vortexed to make emulsion of hexane and E. coli culture. The mixture was incubated for three hours with shaking (250 rpm) at 37°C and plated on LB plates with chloramphenicol μg/ml(34 μg/ml). Plasmids were purified from the pool of growing colonies and transfomied into DH5α. The transformants were treated with hexane as described above. Selection for hexane tolerance was repeated two additional times. Plasmids were recovered from 20 individual colonies that could grow on LB plates with chloramphenicol (34 μg/ml) after the third round of selection. These plasmids were retransfoπned into DH5α. Each transformant was retested for hexane-tolerance as described above. Plasmids that induce hexane tolerance were sequenced to characterize the encoded zinc finger protein. EXAMPLE 10: THERMO-TOLERANT BACTERIAL CELLS

We screened for zinc fmger proteins that confened heat resistance to cells. The nucleic acid library encoding different zinc fmger proteins was transfomied into E. coli cells. The cells were exposed to heat, and heat-resistant cells were recovered. Plasmids were purified from 23 individual colonies that were selected after the third round of phenotypic screening. Ten different zinc finger proteins were identified (Table 15) and the improvement of thermo-tolerance was analyzed by comparing survival rate of ZFP transfomiants and control cells, CI or ZFP-2 upon heat treatment. CI or ZFP-2 represent the transfomiants of empty vector or iπelevant ZFP (QTHQ-RSHR-QTHR1), respectively. More than 99.99% of wild type cells died upon heat treatment at 50°C for 2 hours. In contrast, about 6% of cells transfomied with certain ZFP-TFs survived under these extreme conditions, a 700 fold increase in the themiotolerance phenotype — that is, the percentage of cells expressing ZFP-TFs that survive under stress conditions (6.3%) divided by the percentage of CI that survived under the same conditions (0.0085%>). Since the expression of ZFP is induced by IPTG, transformants of ZFPs that induce thermo-tolerance were analyzed for their phenotype in the presence or absence of IPTG. Transformants of T-l or T-10 showed higher thermo-tolerance in the presence of IPTG.

Table 15: ZFPs that confer themiotolerance.

Name FI F2 F3 F4 putative DNA target Occurrences

T-l QSHV VSNV QSNK QSNK 5' DAA DAA AAT HGA 3'

(SEQ ID NO:238)

T-2 RDHT QSHV QTHR1 QSSR1 5' GYA GRA HGA NGG K 3'

(SEQ ID NO:239)

T-3 WSNR QSHV VSNV QSHV 5' HGA AAT HGA GGT 3'

(SEQIDNO:240)

T-4 QTHR1 RSHR QTHR1 QTHR1 5' GRA GRA GGG GRA 3'

(SEQIDNO:241)

T-5 DSAR RDHT QSHV QTHR1 5' GRA HGA NGG GTC 3'

(SEQIDNO:242)

T-6 QTHQ RSHR QTHR1 QTHR1 5' GRA GRA GGG HGA 3'

(SEQ ID NO:243)

T-7 QSHV VSNV QSNR1 CSNR1 5' GAV GAA AAT HGA 3'

(SEQIDNO:244)

T-8 VSNV QTHR1 QSSR1 RDHT 5' NGG GYA GRA AAT 3'

(SEQ ID NO:245)

T-9 RDHT QSHV QTHR1 QSNR1 5' GAA GRAHGANGG K3'

(SEQIDNO:246)

T-10 DSAR RDHT QSNK QTHR1 5' GRA DAANGG GTC 3'

(SEQ ID NO:247)

Screening for thermotolerance. Libraries for prokaiyotic expression were prepared as described in Example 9. E. coli strain DH5 was transformed with 3-finger or 4-finger ZFP library and cultured overnight in LB with chloramphenicol (34 μg/ml). The overnight-culture was diluted to 1:500 in 1 ml fresh LB media with 1 μM IPTG and chloramphenicol (34 μg/ml) to induce ZFP expression. After a 3 hour incubation at 30°C, lOOul culture was transferred to micro-centrifuge tube and incubated in water bath at 50°C for 2 hrs. The culture was plated on LB plate with chloramphenicol (34 μg/ml). Plasmids were purified from the pool of growing colonies and transfomied into DH5α. Selection for themiotolerance was repeated with retransfomiants. Plasmid was purified from 30 individual colonies that could grow on LB + chloramphenicol plate (34 μg/ml) after third round of selection and retransformed into DH5α. Each transformant was analyzed for themio-tolerance as described above. Plasmids that could induce themiotolerance were sequenced to identify ZFP.

EXAMPLE 11: GENES REGULATED BY ZFP F121-P65

The zinc finger protein F121_p65 was expressed in human embiyonic kidney 293 cells. Transcripts from the cells were profiled and compared transcripts profiled from a corresponding control cell that does not express F121_p65. Examples of transcripts up- regulated by F121_p65 are listed in Table 16. F121 jp65 increases transcription of insulin-like growth factor 2, among other genes.

Table 16: Genes up-regulated by F121_p65

Name ID Norm ratio of medians(ROM) insulin-like growth factor 2 (somatomedi H59614 17.71 insulin-like growth factor 2 (somatomedi H59614 16.88 protein tyrosine phosphatase, receptor t R45941 16.45 insulin-like growth factor 2 (somatomedi N54596 14.40

Putative gene product H09111 12.35 jun B proto-oncogene N94468 11.61

Cellular retinoic acid-binding protein 2 AA598508 10.30 protein phosphatase 2, regulatory subuni R59165 9.89

Nuclear factor of activated T-cells, cyt AA679278 9.64

FK506-binding protein 8 (38kD) N95418 8.66 protein phosphatase 2, regulatoiy subuni R59165 8.60

Cadherin 13, H-cadherin (heart) R41787 7.34

Cysteine-rich protein 2 AA485427 6.71 zinc fmger protein homologous to Zfp-36 R38383 6.46 tumor necrosis factor (ligand) superfami AI347622 6.15

Cadherin 13, H-cadherin (heart) R41787 5.90 brain-specific protein p25 alpha AA133959 5.71 growth arrest and DNA-damage-inducible, AA404666 5.55 lymphocyte antigen 6 complex, locus H AI929550 5.50 ketohexokinase (fructokinase) T61256 5.28 cyclin Dl (PRAD1: parathyroid adenomatos AA487486 5.27

AA045731 5.27

Fnl4 for type I transmembrane protein AI221536 5.06

Example 12: Change of foreign protein solubility in E. coli

We screened for zinc finger proteins that conferred enhanced solubility of the recombinant protein, Aktl expressed in E coli. The mammalian Aktl gene was cloned in pΕT21b vector and GFP was ligated to the C-temiinal of the Aktl ORF. The nucleic acid library encoding different zinc fmger proteins was transfomied into E. coli cells. The Aktl: GFP construct was introduced into ZFP transfomiants. Increased solubility of Aktl was analyzed by FACS sorting, since soluble Aktl expressing cells produce more fluorescent GFP. After the eighth round of "phenotypic screening, individual colonies were selected and the improvement of solubility was analyzed by comparing the amount of Aktl in soluble fraction between ZFP expressed and CI expressed cell by western blot analysis. The westem blot result was quantified by image analysis software, QUANTITY ONE (Bio-Rad, Hercules, CA). CI represent the transfomiants of empty vector, pZLl. Two different zinc finger proteins were identified that increase the solubility to a recombinant Aktl protein expressed inE. coli.

The ratio of the amount of Aktl protein between soluble and insoluble fraction in CI was 32.5:67.5. In contrast, cells transformed with certain zinc finger proteins showed a 2 fold increase of the ratio of Aktl in soluble fraction — that is, the percentage of the amount of Aktl protein in soluble fraction of cells expressing ZFP-TFs (66.1 %) divided by the amount in CI (32.5%>).

Table 19. ZFPs that improve solubility of Aktl protein in E. coli.

Expression Construct Name Ratio of the amount of Aktl protein (%>)

Soluble fraction Insoluble fraction

Control CI 32.5 67.5

Solubility increasing ZFP S-l 66.1 33.9

Solubility increasing ZFP S-2 45.7 54.3

Table 20 : ZFPs that confer increased solubility to Aktl protein in E. coli. Name FΪ F2 F3 F4 putative DNA target S QSTR DSAR RDHT WSNR GGT NGG GTC GYA (SEQ ID NO:248)

S-2 VSTR DGNV QSNR QSNK DAA GAA AAC GCT (SEQ ID NO :249)

Screening for enhanced solubility of recombinant proteins.

Libraries for prokaryotic expression were prepared as described in Example 9. E. coli strain DH5α was co-transformed with ZFP library and Aktl expression vector and cultured overnight in LB with chloramphenicol (34 μg/ml) and ampicillin (50 μg/ml).

The overnight-culture was diluted to 1:500 in 1 ml fresh LB media with 1 mM IPTG and chloramphenicol (34 μg/ml) and ampicillin (50 μg/ml) to induce ZFP expression. After three hour incubation at 30°C, 10° cells were analyzed with FACS on a FACS Vantage flow cytometer and sorted for 5 to 10 % of cells that expressed higher fluorescence. The GFP expressing or pET21b empty vector transfomied cells, were used as a positive or negative control to set the background fluorescence.

Sorted cell were overnight cultured in LB media with chloramphenicol (34 μg/ml) and ampicillin (50 μg/ml). Plasmids were purified from the culture and transfonned into DH5α. Screening for enhanced solubility of recombinant protein was repeated with retransformants. Plasmid was purified from individual colonies that could grow on LB + chloramphenicol (34 μg/ml) + ampicillin (50 μg/ml) plate after eighth round of selection and retransformed into DH5α. Each transformant was analyzed for enhanced solubility of Aktl with western blot analysis. Plasmids that could increase solubility of Alctl were sequenced to identify ZFP.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A modified cell comprising a heterologous nucleic acid encoding an artificial transcription factor that confers stress resistance to the modified cell relative to a reference cell that is substantially identical to the modified cell and that lacks the heterologous nucleic acid and the artificial transcription factor.

2. The modified cell of claim 1 wherein the artificial transcription factor comprises two zinc finger domains.

3. The modified cell of claim 1 wherein the cell is bacterial cell.

4. The modified cell of claim 1 wherein the cell is a eukaiyotic cell.

5. The modified cell of claim 2 wherein the stress resistance includes one or more the following traits: heat resistance, solvent resistance, heavy metal resistance, osmolarity resistance, resistance to extreme pH, chemical resistance, cold resistance, and resistance to a genotoxic agent, resistance to radioactivity.

6. The modified cell of claim 1 wherein the cell expresses the artificial transcription factor and resists stress to a greater extent than a substantially identical culture cell that lacks the artificial transcription factor

7. A method of producing a cellular product, the method comprising: providing the modified cell of claim 1 ; maintaining the modified cell under conditions in which the artificial transcription factor is produced; and recovering a product produced by the cultured cell, wherein the product is other than the artificial transcription factor.

8. The method of claim 7 wherein the modified cell includes a nucleic acid encoding a heterologous protein, other than the artificial transcription factor, and the product is the heterologous protein.

9. The method of claim 7 wherein the product is a metabolite or an endogenous protein.

10. The method of claim 8 wherein the modified cell further includes a second nucleic acid encoding a heterologous protein, and the heterologous protein participates in production of the metabolite.

11. The method of claim 7 wherein the modified cell is maintained at a temperature between 20°C and 40°C.

12. The method of claim 7 wherein the modified cell is maintained under conditions which would inhibit the growth of a substantially identical cell that lacks the artificial transcription factor.

13. The method of claim 7 wherein the artificial transcription factor comprises a zinc finger domain.

14. The method of claim 13 wherein the zinc fmger domain comprises a set of

DNA contacting residues that corcespond to DNA contacting residues of a zinc finger domain listed in Table 15.

15. The method of claim 14 wherein the artificial transcription factor comprises an array of at least three zinc fmger domains, wherein the DNA contacting residues of each of the zinc fmger domains of the aιτay coiτespond respectively to DNA contacting residues of any three consecutive zinc finger domains listed in a row of Table 15.

16. The method of claim 7 wherein the stress resistance includes one or more the following traits: heat resistance, solvent resistance, heavy metal resistance, osmolarity resistance, resistance to extreme pH, chemical resistance, cold resistance, and resistance to radioactivity.

17. A cell that contains a gene encoding a target protein and a heterologous nucleic acid that comprises a sequence encoding an artificial protein chimera that (1) increases the amount of the protein produced by the cell relative to a cell that does not include the heterologous nucleic acid, and (2) does not bind to a transcriptional regulatory region that directly regulates transcription of the gene encoding the target protein.

18. The cell of claim 17 wherein the artificial protein chimera comprises two zinc finger domains and binds to DNA.

19. The cell of claim 17 wherein the cell is a eukaryotic cell, and the artificial protein chimera competes with PB08, K_F02, or K_D10 for binding to a genomic DNA site.

20. The cell of claim 17 wherein the artificial protein chimera alters the rate of cell cycle progression by the cell.

21. The cell of claim 17 wherein the gene is an endogenous gene.

22. An artificial transcription factor that (1) increases the amount of a target protein produced by a eukaryotic cell relative to a cell that does not include artificial transcription factor, and (2) does not bind to a transcriptional regulatory region that directly regulates transcription of the gene encoding the target protein.

23. An artificial transcription factor comprising a plurality of zinc finger domains, wherein the artificial transcription factor alters the rate of cell cycle progression in a eukaryotic cell.

24. A method of producing a protein, the method comprising: providing the cell of claim 17, and maintaining the cell under conditions in which the artificial protein chimera increases the amount of the target protein produced by the cell relative to a cell that does not include the heterologous nucleic acid.

25. The method of claim 24 wherein the protein is a secreted protein.

26. A cell that contains an endogenous gene encoding a secreted protein and a heterologous nucleic acid that comprises a sequence encoding an artificial transcription factor that increases the amount of the secreted protein produced by the cell relative to a cell that does not include the heterologous nucleic acid.

27. The cell of claim 26 wherein the cell is a eukaiyotic cell, and the secreted protein is insulin.

28. The cell of claim 26 wherein the artificial transcription factor specifically binds to an endogenous DNA site that is also specifically bound by 08_D04_p65.

29. The cell of claim 26 wherein the artificial transcription factor specifically binds to an endogenous DNA site and the artificial transcription factor competes with

08_D04_p65 for binding to the endogenous DNA site.

30. The cell of claim 26 wherein the artificial transcription factor comprises an amino acid sequence as follows: CX₍₂.₅₎CXXXBXRXSHJXRHX(₃.₅)HX₍i.₆₎BXCX₍₂.5)CXXXBXRXDHJXTHX(₃.

₅₎H (SEQ ID NO:45); or

CX₍₂.₅₎CXXXBXRXDHJXTHX(₃.₅)HX(i.₆₎BXCX(₂.₅)CXXXBXVXSSJXRHX(₃. ₅₎H (SEQ ID NO:46) where B is phenylalanine or tyrosine; and J is a hydrophobic amino acid.

31. A method of producing insulin, the method comprising: providing the cell of claim 24, and maintaining the cell under conditions in which the artificial transcription factor increases the amount of insulin produced by the cell relative to a cell that does not include the heterologous nucleic acid.

32. An artificial transcription factor comprising at least two zinc finger domain, wherein the artificial transcription factor induces expression of an endogenous insulin gene in a mammalian cell that does not express an endogenous insulin gene in the absence of the artificial transcription factor.

33. An artificial transcription factor that alters sensitivity of the cell to a toxic agent relative to an identical cell that does not contain the nucleic acid.

34. A cell that contains and expresses a nucleic acid encoding an artificial transcription factor that alters sensitivity of the cell to a toxic agent relative to an identical cell that does not contain the nucleic acid.

35. The cell of claim 34 wherein the toxic agent is a drug.

36. The cell of claim 34 wherein the sensitivity is increased.

37. The cell of claim 34 wherein the sensitivity is decreased.

38. The cell of claim 34 wherein the cell is a fungal cell.

39. The cell of claim 38 wherein the toxic agent is ketoconazole.

40. The cell of claim 34 wherein the artificial transcription factor comprises three zinc finger domains.

41. The cell of claim 39 wherein the artificial transcription factor binds to an endogenous DNA site and the artificial transcription factor competes with a zinc fmger protein listed in Table 5 for binding to the endogenous DNA site.

42. A method of altering the drag resistance of a fungal cell, the method comprising: altering the expression or activity of a protein that is at least 70%_> identical to AQY1, YJR147W, YLL052C, YLL053C, or YPL091W in the cell.

43. A method of identifying an artificial chimeric protein that alters the sensitivity of a cell to a toxic agent, the method comprising: providing a nucleic acid library that comprises a plurality of nucleic acids, each nucleic acid of the plurality encoding a chimeric protein that comprises an array of at least three zinc finger domains wherein at least two adjacent zinc finger domains do not occur adjacent to each other in a naturally occuning protein; introducing members of the library into replicates of a test cell to yield transfonned cells; cultivating the transfomied cells in the presence of a toxic agent; and identifying, from the transfomied cells, a cell whose sensitivity to the toxic agent is altered relative to the test cell.

44. The method of claim 43 wherein the test cell is a fungal cell, and the toxic agent is an anti-fungal agent.

45. The method of claim 43 wherein the test cell is a cancer cell, and the toxic agent is an anti-mitotic agent.

46. The method of claim 43 wherein the chimeric protein encoded by each nucleic acid of the plurality comprises a transcriptional regulatory domain.

47. The method of claim 46 further comprising constructing a nucleic acid that encodes a second chimeric protein that comprises the array of zinc finger domains of the chimeric protein encoded by the libraiy member in the selected cell, but does not include the transcriptional regulatory domain of the chimeric protein encoded by the library member in the identified cell.

48. The method of claim 47 further comprising constracting a nucleic acid that encodes a second chimeric protein that comprises (i) the aιτay of zinc finger domains of the chimeric protein encoded by the library member in the selected cell, and (ii) a transcriptional regulatory domain, other than the transcriptional regulatory domain of the chimeric protein encoded by the library member in the identified cell.

49. A nucleic acid comprising a sequence encoding an artificial transcription factor comprising three zinc fmger domains, wherein expression of the artificial transcription factor induces a neuronal phenotype in at least one vertebrate cell.

50. The nucleic acid of claim 49 wherein at least one of the zinc finger domains has the sequence:

Cys-X₂_₅-Cys-X₃-Xa-X-Gln-X_b-X-Ser-Asn-His-X₃.₅-His (SEQ ID NO:250) Cys-X₂_₅-Cys-X₃-X_a-X-Gln-Xb-X-Ser-Asn-His-X₃.₅-His (SEQ ID NO:251); or Cys-X₂.₅-Cys-X₃-X_a-X-Cys-X_b-X-Ser-Asn-His-X₃.₅-His (SEQ ID NO:252), wherein X_a is phenylalanine or tyrosine; and Xb is a hydrophobic amino acid.

51. The nucleic acid of claim 50 wherein the artificial transcription factor comprises the sequence: Cys-X₂-₅-Cys-X₃-X_a-X-Gln-X_b-X-Ser-Asn-His-X₃.₅-His-Xι.g- Cys-X₂.₅-Cys-X₃-X_a-X-Gln-Xb-X-Ser-Asn-His-X₃.5-His-Xi.₆-Cys-X₂.₅-Cys-X3-Xa-X- Cys-X_b-X-Ser-Asn-His-X₃-₅-His (SEQ ID NO:253), wherein X_a is phenylalanine or tyrosine; and X_b is a hydrophobic amino acid.

52. A method of inducing neurite fonnation in a vertebrate cell, the method comprising: providing a vertebrate cell that contains the nucleic acid of claim 44; and maintaining the vertebrate cell under conditions in which the artificial transcription factor is produced and neurite fonnation is induced.

53. The method of claim 52 wherein the vertebrate cell is a mammalian cell.

54. The method of claim 52 wherein the mammalian cell is a human cell.

55. The method of claim 52 wherein the vertebrate cell is a stem cell prior to production of the artificial transcription factor.

56. A nucleic acid comprising a sequence encoding an artificial transcription 5 factor comprised of three zinc fmger domains, wherein expression of the artificial transcription factor induces osteogenesis in at least one vertebrate cell.

57. The nucleic acid of claim 56 wherein at least one of the zinc finger domains has the sequence: o Cys-X₂.₅-Cys-X₃-Xa-X-Arg-X_b-X-Asp-Lys-His-X₃.₅-His (SEQ ID NO:254);

Cys-X₂.₅-Cys-X₃-Xa-X-Gln-X_b-X-Thr-His-His-X₃.₅-His (SEQ ID NO:255); Cys-X₂_₅-Cys-X₃-Xa-X-Val-Xb-X-Ser-Thr-His-X₃.₅-His (SEQ ID NO:256) or; Cys-X₂.₅-Cys-X₃-Xa-X-Arg-Xb-X-Asp-Lys-His-X₃.5-His (SEQ ID NO:257), wherein X_a is phenylalanine or tyrosine; and X is a hydrophobic amino acid. 5

58. The nucleic acid of claim 57 wherein the artificial transcription factor comprises the amino acid sequence:

Cys-X₂.₅-Cys-X₃-X_a-X-Aι-g-X_b-X-Asp-Lys-His-X₃.₅-His-Xι.₆-Cys-X₂.₅-Cys-X₃- X_a-X-Gln-X_b-X-Thr-His-His-X₃.₅-His-Xι.₆-Cys-X₂.5-Cys-X₃-X_a-X-Val-X_b-X-Ser-Thr- 0 His-Xs-s-His-X_Lθ-Cys-Xz.s-Cys-Xs-X_a-X-Arg-X_b-X-Asp-Lys-His-X-j.s-His (SEQ ID

NO:258), wherein X_a is phenylalanine or tyrosine; and Xb is a hydrophobic amino acid..

59. A method of inducing osteogenesis in a vertebrate cell, the method comprising: 5 providing a vertebrate cell that contains the nucleic acid of claim 44; and maintaining the vertebrate cell under conditions in which the artificial transcription factor is produced and osteogenesis is induced.

60. A method of altering the differentiative capacity of a stem cell, the method 0 comprising: providing a stem cell and a nucleic acid that comprises a sequence encoding an artificial transcription factor comprises of a plurality of zinc fmger domains, wherein the artificial transcription factor alters the differentiative capacity of the stem cell; introducing the nucleic into the stem cell; and maintaining the stem cell under conditions in which the artificial transcription factor is produced thereby altering the differentiative capacity of the stem cell.

61. The method of claim 60 wherein the artificial transcription factor induces differentiation of the stem cell.

62. The method of claim 60 wherein the artificial transcription factor enhances self-renewal potential of the stem cell.

63. The method of claim 60 wherein the stem cell is an embryonic stem cell.

64. The method of claim 60 wherein the stem cell is a vertebrate stem cell or a plant stem cell.

65. The method of claim 62 wherein the stem cell is a hematopoietic stem cell, a neuronal progenitor cell or muscular progenitor cell.

66. A method of identifying an artificial transcription factor, the method comprising: providing a nucleic acid library that comprises a plurality of nucleic acids, each encoding a different artificial transcription factor, each artificial transcription comprising an array of at least two zinc finger domains and a regulatory domain that activates or represses transcription; providing cell that have a given trait; introducing members of the nucleic acid library into the cells; identifying a member of the library that alters the given trait; and preparing a coding nucleic acid that comprises a sequence encoding a DNA binding polypeptide that comprises the aι ay of zinc finger domains from the identified member, but does not include a regulatory domain identical to the regulatory domain of the identified member.

67. The method of claim 66 wherein the DNA binding polypeptide is lacks the regulatoiy domain of the identified member.

68. The method of claim 66 wherein the DNA binding polypeptide comprises a regulatory domain that is mutated relative to the regulatory domain of the identified member.

69. The method of claim 67 wherein the DNA binding polypeptide comprises a regulatory domain of opposite functionality relative to the regulatoiy domain of the identified member.

70. The method of claim 66, further comprising introducing the coding nucleic acid into a cell and assessing the given trait of the cell.

71. The method of claim 66 wherein the step of identifying comprises identifying cells having a property selected from the group consisting of: resistance to a given environmental condition; differentiation; dedifferentiation; proliferation; apoptosis; serum-independence; pathogen resistance; and pathogen sensitivity.

72. A nucleic acid libraiy comprising a first plurality of nucleic acids and a second plurality of nucleic acids, wherein (a) each nucleic acid of the first plurality encodes an artificial protein chimera comprising at least two zinc finger domains and a first functional domain that activates transcription, and

(b) each nucleic acid of the second plurality encodes an artificial protein chimera comprising at least two zinc finger domains, and does not include the first functional domain.

73. The nucleic acid library of claim 72 wherein the artificial protein chimera encoded by each nucleic acid of the second plurality comprises a second functional domain, different from the first functional domain.

74. The nucleic acid library of claim 72 wherein the first functional domain is an activation domain and the second functional domain is a repression domain.

75. The nucleic acid libraiy of claim 73 wherein the first functional domain is an activation domain and the second functional domain is an activation domain, with different efficacy from the first activation domain.

76. The nucleic acid library of claim 72 wherein the first functional domain is a histone deacetylase domain.

77. A method of identifying a protein chimera, the method comprising: providing the nucleic acid library of claim 72; introducing members of the nucleic acid library into cells that have a given trait; and identifying a member of the libraiy which alters the given trait.

78. A method of identifying a plurality of protein chimeras, the method comprising: providing replicate cells of a test cell that comprises a nucleic acid encoding a first artificial protein chimera, wherein expression of the first artificial protein chimera alters a given trait of the test cell; introducing a member of a nucleic acid libraiy into each of the replicate cells, the nucleic acid libraiy comprising a plurality of nucleic acids, each encoding a different artificial protein chimera that comprises two zinc finger domains; and screening the replicate cells to identify a cell in which the given trait is further altered, wherein each replicate cell expresses the protein chimera encoded by the first member of the libraiy and a second protein chimera encoded by the introduced member of the nucleic acid library.

79. A method of preparing a modified cell, the method comprising providing a nucleic acid library that comprises a plurality of nucleic acids, each encoding a different artificial protein chimera, each protein chimera comprising at least two zinc finger domains; identifying a first and a second member of the library which alters a given trait of a cell; and preparing a cell that can express first and second polypeptides, wherein the first and second polypeptides are encoded by the first and second identified library members, respectively.

80. The method of claim 79 wherein the step of identifying comprises introducing members of the nucleic acid library into replicates of the cell to provided transfonned cells, and identifying a first and second cell in which the given trait is altered.

81. The method of claim 79 further comprising evaluating the given trait for the prepared cell.

82. The method of claim 79 wherein the preparing comprises introducing a first gene encoding the first polypeptide and a second gene encoding the second polypeptide into the cell.

83. The method of claim 82 wherein the first and second gene are components of the same nucleic acid.

84. The method of claim 79 wherein the preparing comprises fusing a first cell that comprises a first gene encoding the first polypeptide to a second cell that comprises a second gene encoding the second polypeptide.

85. A method of identifying a protein chimera, the method comprising: providing a nucleic acid libraiy that comprises a plurality of nucleic acids, each encoding a different artificial protein chimera that comprises at least two zinc finger domains; introducing members of the nucleic acid library into cells that have a given trait; identifying a member of the library which alters the given trait; and preparing a second library that comprises a plurality of nucleic acids, each encoding (1) a variant that differs from the artificial protein chimera corresponding to the identified member by between one and six amino acid substitutions, insertions, or deletions,

(2) a protein chimera that comprises the zinc finger domains of the artificial protein chimera coιτesponding to the identified member and an additional zinc fmger domain, wherein the additional zinc finger domain varies among the members of the second libraiy,

(3) a variant of the artificial protein chimera conesponding to the identified member, wherein the variant has a subset of the zinc finger domain positions replaced with other zinc finger domains and at least one invariant zinc finger domain that is identical to the zinc finger domain at a conesponding position in the artificial protein chimera coιτesponding to the identified member, or

(4) a variant of the artificial protein chimera coιτesponding to the identified member, wherein, among the members of the second library, one or more zinc finger domain positions are varied such that the particular domain in the artificial protein chimera corresponding to the identified member at that position occurs at a frequency greater than other zinc finger domains at that position.

86. A method of identifying a target of artificial transcription factors, the method comprising: providing a nucleic acid library that comprises a plurality of nucleic acids, each encoding a different artificial transcription factor, each transcription factor comprising at least two zinc finger domains; introducing members of the library into replicates of a test cell to provide a plurality of transfomied cells; maintaining the transformed cells under conditions in which the artificial transcription factor is expressed; identifying a plurality of phenotypically altered cells from the plurality of transfonned cells, wherein each phenotypically altered cell has an altered phenotype relative to the test cell; profiling transcripts or protein abundance in each phenotypically altered cell of the plurality to provide a profile for each phenotypically altered cell; and comparing the profiles to each other to identify one or more transcripts or proteins whose abundance is similarly altered in at least two phenotypically altered cells of the plurality relative to the test cell.

87. A method of evaluating a cell, the method comprising: providing a modified cell that contains a heterologous nucleic acid comprising a sequence encoding an artificial transcription factor comprising at least two zinc finger domains, wherein expression of the artificial transcription factor alters a phenotypic trait of the cell relative to a reference cell that does not express the artificial transcription factor; identifying a target gene whose expression is altered in modified cell relative to the reference cell; and evaluating the phenotypic trait of a test cell wherein the test cell contains the heterologous nucleic acid and expresses the artificial transcription factor, and the activity of the target gene is altered in the test cell.

88. The method of claim 87 wherein the activity of the target gene is altered by a genetic mutation.

89. The method of claim 87 wherein the activity of the target gene is altered by treatment with a double-stranded RNA or an antisense oligonucleotide or a ribozyme.

90. The method of claim 87 wherein the activity of the target gene is altered by overexpression of the target gene.

91. A method of evaluating an artificial transcription factor, the method comprising: providing a modified cell that contains a heterologous nucleic acid comprising a sequence encoding an artificial transcription factor comprising at least two zinc finger domains, wherein expression of the artificial transcription factor alters a phenotypic trait of the cell relative to a reference cell that does not express the artificial transcription factor; and introducing library nucleic acids into replicates of the modified cell; and identifying a library nucleic acid which attenuates the alteration of the phenotypic trait by the artificial transcription factor.

92. The method of claim 91 wherein the library nucleic acids comprise cDNA library nucleic acids.

93. The method of claim 91 wherein the library nucleic acids comprises nucleic acids encoding different artificial protein chimeras.

94. The method of claim 93 wherein the different artificial protein chimeras include different zinc finger domains.

95. A method of identifying a protein chimera, the method comprising: providing a nucleic acid library that comprises a plurality of nucleic acids, each encoding a different artificial protein chimera that comprises at least two zinc finger domains, wherein at least one zinc fmger domain is from a naturally-occurring protein; introducing members of the nucleic acid library into cells that have a given trait; and identifying a cell in which a member of the library alters the given trait.

96. A method of identifying a protein chimera, the method comprising: providing a nucleic acid library that comprises a plurality of nucleic acids, each encoding a different artificial protein chimera that comprises at least two zinc finger domains, wherein at least one zinc finger domain of at least one member of the plurality comprises an amino acid sequence listed in Table 1; introducing members of the nucleic acid library into cells that have a given trait; and identifying a cell in which a member of the library alters the given trait.

97. A method of identifying a protein chimera, the method comprising: providing a library of cells, the library comprising a plurality of cells, wherein each cell includes a nucleic acid encoding a polypeptide comprising a first and a second zinc finger domain, wherein (1) the first and second zinc finger domains of the polypeptide encoded by each nucleic acid of the plurality are each identical to zinc finger domains from naturally occurring proteins and either (i) do not occur in the same naturally occurring protein or (ii) occur in the same naturally occurring protein in a different configuration than in the polypeptide, (2) the first zinc finger domain varies among nucleic acids of the plurality, and (3) the second zinc finger domain varies among nucleic acids of the plurality; identifying a library cell whose phenotype differs from a reference cell; and recovering the nucleic acid from the identified library cell.

98. An isolated nucleic acid encoding an artificial transcription factor that improves solubility of a heterologous, over-expressed protein in a cell that produces the artificial transcription factor.

99. The nucleic acid of claim 98 wherein the artificial transcription factor comprises a plurality of zinc finger domains.

100. The nucleic acid of claim 98 wherein the cell is bacterial cell.

101. The nucleic acid of claim 98 wherein the protein is a mammalian protein.

102. The nucleic acid of claim 101 wherein the protein comprises the AKT protein.

103. The nucleic acid of claim 99 wherein the plurality of zinc fmger domains comprise domains: QSTR-DSAR-RDHT-WSNR or VSTR-DGNV-QSNR-QSNK.

104. A modified cell comprising the heterologous nucleic acid of any one of claims 98 to 103.

105. A method of producing a heterologous target protein, the method comprising: providing the modified cell of claim 104 wherein the modified cell comprises a second nucleic acid that comprises a sequence encoding a heterologous target protein; and maintaining the modified cell under conditions wherein the artificial transcription factor and the heterologous target protein are produced.

106. The method of claim 105 wherein the modified cell is a culture cell.

107. The method of claim 105 wherein the modified cell is in a subject.

108. A method of producing a cellular product, the method comprising: providing a modified cell of claim 1 ; maintaining the modified cell under conditions in which the artificial transcription factor is produced; and recovering a product produced by the cultured cell, wherein the product is other than the artificial transcription factor.

109. A method of transfe ing an altered trait from a first cell to a second cell, the method comprising: providing a nucleic acid library that comprises a plurality of nucleic acids, each encoding a different artificial protein chimera that comprises at least two zinc fmger domains; introducing members of the nucleic acid library into first cells that have a given trait to provide transformed cells; identifying an altered cell from the transformed cells in which a member of the library alters the given trait; recovering a nucleic acid library member from the identified, altered cell; introducing the nucleic acid library member into second cells, wherein the second cells differ from the first cells by a phenotypic trait other than the given trait; and evaluating a second cell that includes the nucleic acid library member and expresses the artificial protein chimera that the nucleic acid library member encodes.

110. The method of claim 109 wherein the first and second cells are eukaiyotic.

111. The method of claim 110 wherein the first and second cells are yeast cells.

112. The method of claim 110 wherein the first and second cells are mammalian cells.

113. The method of claim 112 wherein the first and second cells differ by the proliferative properties.

114. The method of claim 113 wherein the first cells are cancer cells, and the second cells are non-cancerous.

115. The method of claim 109 wherein the evaluated second cell is evaluated for an alteration to the given trait.

116. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 260, 262, and 264.

117. An isolated nucleic acid comprising a nucleic acid sequence that encodes the polypeptide of claim 116.

118. A nucleic acid comprising a sequence encoding an artificial transcription factor comprised of three zinc finger domains, wherein expression of the artificial transcription factor alters a property of at least one eukaiyotic cell, wherein the property is selected from the group consisting of viral replication, virus production, and viral infectivity.

119. A nucleic acid comprising a sequence encoding an artificial transcription factor comprised of three zinc fmger domains, wherein expression of the artificial transcription factor alters an ability of a eukaryotic cell to regulate a stem cell that is co- cultured with the eukaryotic cell or that is cultured in media conditioned by the eukaryotic cell.

120. A nucleic acid comprising a sequence encoding an artificial transcription factor comprised of three zinc fmger domains, wherein expression of the artificial transcription factor alters an ability of a mammalian culture cell to glycosylate a secreted protein.

121. The nucleic acid of claim 120 wherein the secreted protein is a heterologous protein that comprises at least one immunoglobulin variable domain.

122. The nucleic acid of claim 120 wherein the culture cell is a CHO cell.

123. A nucleic acid comprising a sequence encoding an artificial transcription factor comprised of three zinc finger domains, wherein expression of the artificial transcription factor alters the ability of a cell to take up exogenous nucleic acid.