Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6875—Nucleoproteins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4703—Regulators; Modulating activity

Definitions

• This invention in general relates to methods for determining the biological effect(s) of a compound. More specifically, this invention discloses methods of examining the effect(s) of compounds by measuring changes in gene expression. Accordingly, the invention can be used to assess compound efficacy, toxicity, mechanism of action, etc. As such, it will have widespread use, for example, in developing novel pharmaceutical compounds as well as in testing effects on gene expression of these and known compounds.
• the regulation of gene expression is critical to the growth, development, proliferation, and maintenance of all living cells and organisms, i most cases, the positive or negative regulation of genes is under the control of signal transduction cascades which transmit information from the cell surface to the nucleus.
• Signal transduction cascades are generally triggered by ligands which may be small molecules, soluble peptides, extracellular matrix, adhesion proteins projected from the exterior surface of neighboring or migrating cells, and even metabolic intermediates.
• ligands interact with a membrane bound, or sometimes soluble intracellular, receptor, thus triggering a cascade of events that ultimately either stimulates or inhibits the expression of one or more genes.
• Such reprogramming of gene expression leads to a, hopefully appropriate, cellular response to the stimuli.
• PNAs protein nucleic acid
• oligonucleotides that are capable of promoting "triple helix” formation, and a class of sequence-specific molecules known as "polyamides” (see, e.g., Dervan, et al., Curr. Opin. Chem. Biol. (1999), vol. 3: 688-693; Bremer, R.E., Baird, E.E. & Dervan, P.B. (1998) Chem. Biol. 5, 119-133).
• PNAs protein nucleic acid
• a first aspect of the invention concerns methods for determining a biological effect (e.g., efficacy, toxicity, resistance, and mechanism of action) of one or more compounds on such gene expression.
• biological effect is meant the influencing of the metabolism or biochemistry of a cell. With respect to the current invention, such effect preferably is one the influences either directly or indirectly expression mechanisms, pathways, etc. of a cells gene pool.
• efficacy is meant the ability of a compound to induce changes in transcription factor binding activities consistent with efficacy for that particular compound.
• toxicity is meant changes in transcription factor binding activities consistent with toxic events in cells.
• stance is meant the ability of a compound to cause changes in transcription factor binding activities consistent with the cell demonstrating resistance to the particular compound.
• the methods of the invention comprise obtaining a nuclear extract from cells that prior to obtaining the nuclear extract were exposed to a compound of interest, and combining the nuclear extract with a nucleic acid containing a cis- binding site (also sometimes referred to as a regulatory element or cis element) under conditions that allow formation of transcription factor I cis site complexes, such complexes being well understood by those of ordinary skill in the art.
• a nucleic acid containing such cw-binding site is a library or plurality of nucleic acids each comprising one more, and preferably different binding sites.
• the transcription factor/c ⁇ complexes so formed are then compared with the transcription factor/cz-?
• cw-binding any cis element of defined nucleotide sequence that can be identified in a nucleic acid molecule and which associates with an endogenous DNA-binding compound of the transcriptional machinery. Such elements include promoters and enhancers.
• a “promoter” is the minimum sequence necessary to initiate transcription of a target gene by an RNA polymerase, for example, in eukaryotic cells, RNA polymerase I (which transcribes ribosomal RNA (rRNA) in eukaryotic cells), RNA polymerase II (which transcribes messenger RNA (mRNA) in eukaryotic cells), and RNA polymerase III (which transcribes transfer RNA (tRNA) in eukaryotic cells).
• RNA polymerase I which transcribes ribosomal RNA (rRNA) in eukaryotic cells
• RNA polymerase II which transcribes messenger RNA (mRNA) in eukaryotic cells
• RNA polymerase III which transcribes transfer RNA (tRNA) in eukaryotic cells.
• An “enhancer” is a c ⁇ -acting sequence that increases the utilization of a eukaryotic promoter.
• Preferred cis elements that are included in an ohgonucleotide are those that occur endogenously in association with the gene whose transcription is to be regulated. As such, promoters from which transcription can be initiated can be targeted.
• “regulate” or “modulate” refers to an ability to alter the level of expression of a particular gene above (i. e. , up-regulate or activate) or below (i.e., down-regulate or repress) the basal level of expression that would occur in the particular system (for example, an in vitro transcription system or a cell) in the absence of a compound of interest under the same conditions.
• a compound that activates transcription is referred to herein as an "activation moiety” or "activator,” whereas a compound that represses transcription is referred to as a "repressor moiety” or “repressor”.
• nucleic acids that are comprised of two completely or partially complementary oligonucleotides that completely or partially overlap with one another.
• an ohgonucleotide used in the practice of a method according to the invention will contain at least one regulatory element.
• the oligonucleotides comprise a plurality of, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, regulatory elements.
• Such an ohgonucleotide may comprise a defined nucleotide sequence.
• Certain preferred oligonucleotides comprise nucleotide sequences that are representative of a genome. Other preferred oligonucleotides comprise nucleotide sequences actually found in genomic DNA.
• nucleotide sequence may be random.
• a "defined nucleotide sequence” refers to a specific sequence of nucleotides, and is typically represented in the 5' to 3' direction using standard single letter notation. Deoxynucleotides, or nucleotides, are referred to according to standard abbreviations: "A”, deoxyadenylate; “C”, deoxycytidylate; “G”, deoxyguanylate; "T”, deoxythymidylate; "M”, A or C; “R”, A or G; “W”, A or T; “S”, C or G; “Y”, C or T; "K”, G or T; and "N", A, C, T or G.
• T bases in DNA molecules are replaced by uridine ("U" bases) in the corresponding RNA molecules.
• U uridine
• an ohgonucleotide having a defined nucleotide sequence may include a different nucleotide at the same position, i.e., is degenerate at that position, with respect to one or more positions in the particular sequence.
• Degenerate bases may be represented by any suitable nomenclature, for example, that which is described in World Intellectual Property Organization Standard ST.25 (1998), Appendix 2.
• Random oligonucleotides may also comprise nucleotide sequences representative of a genome.
• an ohgonucleotide may comprise the same bias for nucleotide representation as a particular genome.
• Oligonucleotides may also contain modified nucleotides, for example, methylated nucleotides, as well as, or alternatively, nucleotide analogs and derivatives.
• the methods of the invention employ libraries of different complementary ohgonucleotide species.
• the members of the library contain various differing -binding sites.
• the double-stranded ohgonucleotide species present in a library contain more than one cw-binding sites, it is preferred that they be different c ⁇ -binding sites, although the invention does contemplate double-stranded oligonucleotides that contain multimers of the same, or several different c ⁇ -binding sites.
• oligonucleotides that comprise a first amplification primer site upstream of the c ⁇ -binding site(s) and a second amplification primer site downstream of the cts-binding site(s).
• the primer sites can be used to amplify the regions disposed therebetween by a suitable amplification process, for example, PCR, strand displacement amplification, and transcription mediated amplification.
• the nucleic acid molecules are attached to or otherwise localized at a solid support.
• nuclear extracts are used in the methods of the invention and can be obtained from any of a variety of cells.
• a "nuclear extract” refers to a preparation obtained from cell nuclei. Preferably, such preparation contains proteins found in the nucleus that retain their biological activities. Preferably, a nuclear extract will be substantially free from naturally occurring lipid and nucleic acid components. Nuclear extracts may be derived from any prokaryotic or eukaryotic plant or animal cell, including cells grown in vitro
• the cells are vertebrate cells, particularly mammalian cells such as canine, equine, feline, murine, ovine, porcine, and primate cells. Particularly preferred are human cells.
• Other preferred vertebrate cells include avian and fish cells.
• Other preferred cells include pathogen cells, for example, yeast and bacterial cells.
• cells infected by a pathogen for example, viruses or bacteria, can also be used in the practice of the invention.
• Other embodiments of the invention concern diseased cells and normal cells. Representative examples of diseased cells include cancer cells, virally infected cells, abnormal T cells, and abnormal neuronal cells.
• Synthetic compounds may be synthesized by solution or solid phase methods. Two or more moieties may also be synthesized together.
• Compounds useful in the practice of the invention can be in unpurified, substantially purified, and purified forms.
• the compounds can be present with any additional component(s) such as a solvent, reactant, or by-product that is present during compound synthesis or purification, and any additional component(s) that is present during the use or manufacture of a compound or that is added during formulation or compounding of a compound.
• a regulatory compound useful in the practice of the invention is any compound that can positively or negatively effect, by either a direct mechanism (i.e., by direct interaction with one or more components of the transcription complex) or an indirect mechanism (i.e., by (i) direct interaction with a repressor protein or (ii) direct interaction with a protein involved in cliromatin or nucleosome structure), transcription of a target gene.
• a direct mechanism i.e., by direct interaction with one or more components of the transcription complex
• an indirect mechanism i.e., by (i) direct interaction with a repressor protein or (ii) direct interaction with a protein involved in cliromatin or nucleosome structure
• compounds may not have a direct effect on gene regulation, but may directly affect one of the many other processes in a cell. Examples include binding to one or more of the numerous cell components other than those involved in gene transcription such as those affecting negatively or positively processes such as cell metabolism, signal transduction, apoptosis, protein secretion, RNA translation, ion transport, respiration, lysosomal makeup, nuclear trafficking, cell cycling, and the myriad of other processes associated with a normal (or diseased) physiologic state of a cell, hi this aspect, the methods of the invention examine the effect a compound may have on a cell that ultimately affects changes in the expression of certain genes.
• Representative embodiments of compounds include peptides, polypeptides (including naturally occurring or synthetic mutant polypeptides), nucleic acids, lipids, carbohydrates, small organic molecules, and any combination thereof.
• a "peptide” is a polymer (i. e. , a linear chain of two or more identical or non-identical subunits joined by covalent bonds) made up of naturally occurring or synthetic D- or L-, or D- and L-, amino acids joined by peptide bonds.
• peptides contain at least two amino acid residues (i.e., the molecule resulting from the formation of a peptide bond between two amino acids, or between an amino acid residue and another amino acid) but fewer than about 50 amino acid residues.
• a “polypeptide” is also a polymer of amino acid residues linked by peptide bonds, but typically contains at least about 50 amino acid residues.
• peptide is used to refer to a regulatory moiety that is less than about 50 amino acid residues in length, and “polypeptide” refers to larger polymers of amino acid residues linked by peptide bonds.
• nucleic acid is any polymer of nucleotides, be they natural (e.g., A, G, C, or T) or synthetic, and whether linked by phosphodiester or other chemical linkages.
• a “lipid” is a substantially water- insoluble molecule that contains as a major constituent an aliphatic hydrocarbon.
• Lipids include fatty acids, neutral fats, waxes, and steroids.
• the hydrocarbon portions of the molecule may be of any length, may be saturated or unsaturated, and may be straight- or branched-chain.
• Carbohydrate refers to any aldehyde or ketone derivative of apolyhydric alcohol, and includes starches, sugars, celluloses, and gums.
• Particularly preferred regulatory compounds are small organic molecules (i.e., a water soluble organic molecule having a molecule weight of less than about 5,000 daltons, preferably less than about 2,500 daltons, more preferably less than about 1,500 daltons, and most preferably less than about 1,000 daltons).
• the methods of the invention are performed in vitro, preferably in a high throughput format, meaning that more than about 10, preferably, more than about 100, 1,000, or 10,000 compounds are screened at once.
• compounds may also be pooled.
• a variety of parameters may be screened, for example, different compound concentrations, nuclear extracts generated after different times following compound addition, etc.
• the regulatable gene is a marker gene, such as a gene encoding a luciferase or green fluorescent protein.
• an expression profile is performed after it is determined which cw-binding sites were bound or unbound by a protein in response to exposure of a cell population (in vivo or in vitro) to one or more compounds.
• the expression profile is determined by performing RNA profiles of the cells cultured or grown in the presence or absence of the compound.
• An RNA profile may be performed by any suitable method, for example, by nucleic acid hybridization.
• Preferred hybridization techniques include the use of a nucleic acid array that comprises probes, or hybridization tags, for the subset of genes expressed in the cells or, preferably, for the genes known to be functionally associated with the particular cM-binding sites determined to change as a result of compound treatment.
• Alternative expression profile embodiments are based on the detection of proteins expressed in the treated cells from genes known to be functionally associated with a particular cis-bmd g site(s).
• Another aspect of the invention concerns a method for determining a biological effect of a compound of interest whereby a nuclear extract from cells exposed to the compound is prepared and then reacted with a solid support to which is attached a nucleic acid molecule containing a cw-binding site for specific interaction with a protein associated with regulating transcription of one or more genes under conditions that allow formation of a transcription factor/cw-binding site complex.
• the complexes formed as a result of the foregoing reaction are then compared with the complexes that are formed using a control nuclear extract obtained from cells not exposed to the compound.
• Another method for determining a biological effect of a compound of interest involves taking a nuclear extract from cells exposed to the compound and reacting that nuclear extract with a DNA library to form transcription factorlcis site complexes.
• the complexes are then characterized by reacting them with a sohd support to which is attached an antibody specific for a protein associated with said complex.
• the analysis would involve analysis of many of the transcription factors likely to be active in the particular cells used for testing the compound.
• the results obtained as a result of the foregoing reaction are then compared with the results obtained using a control nuclear extract obtained from cells not exposed to the compound.
• Figure 1 is a bar graph showing the effects of three different drug compounds, doxorubicin, taxol, and tamoxifen, on the levels of DNA-binding activities for a limited set of transcription factors (names listed on the X-axis) in MCF7 cells.
• the level of DNA binding activity for the individual transcription factors is depicted as a percentage of the total number of DNA fragments sequenced from the "bound" fraction of the DNA library used in the binding reactions and containing the cognate site for that particular protein (shown as a numerical value on the Y-axis).
• the level of binding activities for all the proteins detected as a result of their cognate binding sites being found in the "bound" fraction of the DNA library constitutes the transcription factor activity profile resulting from the specific drug treatment.
• This profile is analogous to a diagnostic fingerprint indicating the effects of any specific drug compound on the overall activities of all transcription factors in the cells being treated. Differences in individual transcription factor DNA-binding activities can be directly correlated to changes in the expression of genes being regulated, either directly or indirectly, by that protein factor.
• Figure 2 is a bar graph showing the effects of nerve growth factor (NGF) treatment on transcription factor DNA-binding activities in PC 12 cells. The identity of the specific transcription factors whose binding activity is being detected are listed on the X-axis.
• NGF nerve growth factor
• the level of DNA binding activity for each individual transcription factor is depicted as a percentage of the total number of DNA fragments sequenced from the "bound" fraction of the DNA library used in the binding reactions and containing the cognate site for that particular protein (shown as a numerical value on the Y-axis).
• the level of binding activities for all the proteins detected as a result of their cognate binding sites being found in the "bound" fraction of the DNA library constitutes the transcription factor activity profiles for PC12 cells and PC12 cells following NGF treatment. The profiles generated provide a useful indicator of the mechanism of action of the compound being used for the cell treatment.
• the present invention concerns novel, useful, and non-obvious methods that allow a biological effect of a compound (e.g., efficacy, resistance, mechanism of action, and toxicity) to be determined.
• RNA hybrid molecules are also produced during the replication of retroviruses due to the action of reverse transcriptase.
• the nucleus of each cell of a multicellular organism contains a full genome complement.
• the full complement of genes is not expressed in any one cell at any one time.
• This difference in gene expression between cells gives rises to the observed differences in cells (e.g., nerve cells are different from muscle cells, normal cells are different from diseased cells, etc.) due to the expression of different genes.
• it is the coordinated pattern of differential expression of only a subset of genes in the nucleus of a given cell type that distinguishes cells of that type (e.g., nerve, muscle, bone, connective tissue, vascular tissue, skin, etc.) from other types of cells.
• the major players in the regulation of gene expression within the nucleus are: the genes and their regulatory sequences which are complexed with structural proteins (e.g., histones) in chromatin; chromatin remodeling activities which allow access to a gene and its regulatory regions; regulatory proteins which instruct the transcription machinery to express (or, as in the case of repressors, prevent the expression of) the relevant genes; and the RNA-synthesizing machinery which decodes the genes.
• a host of other activities play a role in this process, for instance, those that facilitate elongation of paused transcripts, or those that lead to the processing of nascent transcripts and those that play a role in release of full- length transcripts.
• Other components involved in gene expression such as mRNA elongation, processing, termination, or nuclear export, can also be targeted.
• Activators Positive regulation (stimulation) of gene expression requires factors called transcriptional activators.
• An economical 'recruitment' model posits that activator proteins bind to DNA and recruit the transcriptional machinery to the promoter of the gene, thereby stimulating gene expression.
• Most activators are comprised of three functional modules. Of these, specificity in targeting genes is achieved by the DNA recognition module which binds to cognate DNA sequences near a promoter of a gene and in most cases DNA binding specificity is further enhanced by dimerization.
• a key functional module, the activating region is thought to interact, protein-to-protein, with one or more components of the transcriptional machinery.
• Repressors These proteins appear to function to inhibit gene expression at several levels. Some repressors function in part by blocking the activity of activators directly, for example, by binding to an activation domain on an activating protein in order to prevent its interaction with a component of the transcriptional machinery.
• MDM-2 which not only binds to the activating region of p53, but also indirectly attenuates transcriptional activity by stimulating p53 's degradation via a proteolytic pathway. More recently it has been proposed that repressors are recruited to promoters where they serve to inhibit the ability of transcriptional machinery to utilize the proximal promoter by either directly interacting with the machinery and inactivating it, or indirectly by mediating changes in chromatin structure so as to prevent the components of a transcriptional apparatus from interacting with DNA.
• Transcriptional Machinery The general components of the eukaryotic transcription apparatus have been described [Orphanides, G. et al. (1996) Genes Dev. 10, 2657-2683; Conaway. R.C.
• RNA polymerase II (12 subunits), several general transcription factors (TFn -A, B, D, E, F, H), mediator complex (-20 Srb and Med subunits), elongator complex, co-activator proteins and several additional polypeptides, some of which remain to be defined. Most of the these proteins are conserved through evolution and occur in species from plants to yeast to humans. Many of the components of the transcription machinery exist in large multi- subunit complexes which associate with the RNA polymerase II, and are known as the RNA polymerase II holoenzyme.
• RNA-polymerase II holoenzyme can be broadly thought to consist of two functional parts. One part is the "catalytic core" that is required for synthesizing mRNA while the other is the mediator [Bjorkland, S. & Kim, Y.J. (1996) Trends Biochem. Sci. 21, 335-337], a complex of approximately twenty proteins that is required for the holoenzyme to respond to activators. It is believed that the holoenzyme, along with additional factors that do not associate tightly (such as TBP/TFIDD and a class of proteins known as co- activators [Thompson, CM., et al. (1993) Ce/// 73, 1361-1375; Koleske, A.J.
• TFIID [Burley, S.K. & Roeder, R.G. (1996) Ann. Rev. Biochem 65, 769- 799]
• TFIID an essential component of the transcriptional machinery, is not typically found associated with the holoenzyme, and is a target of activators and some repressors as well. It is a protein complex containing about thirteen components, including TBP[Kim, J.L. et al (1993) Nature 365, 520-527; Kim, Y. et al (1993) Nature 365, 512-520] and TBP-associated factors (TAFs) [Dynlacht, B.D. et al. (1991) Cell 66, 563-576].
• TBP TBP-associated factors
• TBP is a sequence-specific D ⁇ A-binding protein that recognizes and binds via the minor groove to a sequence known as the TATA box (consensus: 5'-TATAAAA-3') that exists in the promoters of many genes [Hoopes, B.C. et al. (1992) J. Biol. Chem. 267, 11539-1154; Coleman, R.A. & Pugh, B.F. (1995) J. Biol. Chem. 270, 13850-13859].
• TFIID associates with TFIIA, which is comprised of three polypeptides.
• TFIIA helps TFIID bind to DNA perhaps by competing with repressors as well as displacing inhibitory domains within TAFs away from TBP [Geiger, J.H. et al. (1996) Science 272, 830-836; Thompson, CM., etal. (1993) Cell 13, 1361-1375].
• TFIDB a holoenzyme component, also interacts with the promoter DNA and binds to TBP [Nikolov, D.B., et al. & Burley, S.K. (1995) Nature 377, 119-128; Burley, S.K. (1996) Nature 381, 112-113] and it is proposed to hold the entire complex together as a single unit.
• Chromatin Remodeling Machinery In order for a gene sequestered in chromatin to become available for transcription, the chromatin structure must be remodeled [Felsenfeld, G. (1992) Nature 355, 219-224; Kingston, R.E. et al. (1996) Genes Dev. 10, 905-92; Kadonaga, J.T. (1998) Cell 92, 307-313]. Chromatin remodeling occurs through activator-mediated recruitment of at least two types of chromatin remodeling complexes. The first comprises the histone acetyl transferases that contain proteins that acetylate certain lysine residues in the amino-terminal tails of histone proteins [Brownell, J.E. & Allis, CD. (1996) Curr. Opin.
• chromatin remodeling complex uses energy derived from ATP hydrolysis to facilitate binding of the transcriptional machinery to a particular promoter [Burns, L.G. & Peterson, C.L. (1997) Mol. Cell. Biol. 17, 4811-4819; Quinn, J., et al. (1996) Nature 319, 844-847; Kwon, J., et al. (1994) Nature 370, 477-481; Cote, et al. (1994) Science 265, 65-68].
• Activators can recruit chromatin remodeling complexes through direct binding.
• the viral activator VP16 has been shown to bind to components of both the multi-protein histone acetyl transferase (HAT) complex [Berger, S.L., et al. & Guarente, L. (1992) Cell 70, 251-265; Candau, R., et al. (1997) EMBOJ. 16, 555-565], as well as the Swi/Snf complex, hi fact, TFIID, another target of VP16, was observed to display a weak HAT activity [Mizzen, C .A., et al., & Allis, CD. (1996) Cell 87, 1261-1270; Wilson, C.J., et al. & Roung, R.A. (1996) Cell 84, 235-244].
• HAT multi-protein histone acetyl transferase
• an activator bound to a promoter or enhancer recruits the chromatin remodeling machinery to the adjacent promoter. It then recruits the transcriptional machinery to form a pre-initiation complex at the promoter. It appears that assembly of a pre-initiation complex may require two synchronized steps: TFIID/TBP -TATA binding in concert with the association of the holoenzyme with the complex at the promoter [Stargell, L.A. & Struhl, K. (1996) Trends Genet. 12, 311-315]. For mRNA synthesis to be initiated at a particular gene, the complex must open (melt) the double helix to expose the template strands.
• RNA initiation occurs and after a certain length of transcript is synthesized, the polymerase must move away from the promoter to continue mRNA synthesis.
• Certain activators such as HSF and Tat function to stimulate this stage of transcription process, possibly by recruiting the pTEFB complex which contains a kinase (Cdk9) capable of phosphorylating the largest of the 12 subunits of the polymerase.
• Cdk9 a kinase
• promoter escape appears to involve hyperphosphorylation of the carboxy-terminal domain of the largest subunit of the RNA polymerase II. This hyperphosphorylation achieves two goals: first, it may provide the signal to detach the mediator complex from the catalytic core; and second, it may permit the association of RNA processing and elongator complexes with the rapidly elongating polymerase.
• next transcription complex can be reassembled rapidly by only recruiting the core fragment of the RNA polymerase II holoenzyme. It is postulated that re-initiation is much more likely than initiation alone to contribute significantly to rapid stimulation of gene expression. Also, activators must clearly play a role in [Ho, S.N. et al. (1996) Nature 382, 822-826] facilitating multiple rounds of transcription re-initiation.
• Repression requires the opposite series of events.
• a repressor may first directly engage an activator and mask its activating surface thereby preventing its interactions with the transcriptional and chromatin remodeling machinery.
• the repressor may also directly interrupt the low-level activator-independent assembly of the transcriptional machinery at the exposed promoter, hi the next set of events, the repressor such as Retinoblastoma gene product (Rb) may directly recruit histone deacetylases, which then strip the acetyl groups off the lysine residues on histone tails.
• Rb Retinoblastoma gene product
• deacetylated histone H3 tails are then methylated by methyl transferases, which are also recruited by repressors.
• the methylated histone tails bind to chromatin compacting proteins such as HP-1.
• HP-1 chromatin compacting proteins
• Testing of compounds according to the invention can be conducted as follows. First, the desired compound(s) to be tested is obtained, for example, by purchase or synthesis, for example, by solid state or solution phase synthesis or recombinant techniques, as the case may be.
• the particular compound is typically tested in an in vitro format. For example, samples at one or more concentrations of one or more compounds (including compounds in mixtures of two or more compounds) are exposed to a cultured cell population. After exposure for a period of time appropriate for such compound to have an effect on a cell, nuclear extracts are prepared from the cells by methods well known to those of skill in the art.
• the nuclear extracts are then combined with a nucleic acid molecule, preferably an ohgonucleotide, even more preferably a library of oligonucleotides, to allow formation of transcription factor/czs site complexes between components of the nuclear extract and c ⁇ -binding sites present in the oligonucleotides.
• This reaction is preferably performed under conditions that favor formation of specific transcription factor/cz-y site complexes to approximate those in the cells from which the extract was obtained.
• the profile of transcription factor binding activities is determined for each cell population, both cells exposed to the compound and cells that were not exposed to the compound.
• the profiles comprise a complete profile (i.e., the pattern of all active binding activities altered by the cell's contact with the test compound).
• such profiles can comprise less than a complete profile of all changes in binding activities existing in a cell where the pattern obtained is sufficient to provide useful information such as for example, regarding the efficacy, resistance, mechanism of action and/or toxicity.
• the profiles obtained following treatment of a cell with a compound is then compared with the profile of an untreated cell to determine those transcription factor binding activities that are different between the treated and untreated cell populations. These differences indicate the biological effect(s) of exposure to the compound.
• Particular transcription factor binding activities can be associated with specific molecular and/or cellular effects such as apoptosis or proliferation, or practically any process that can be followed in the cells. For example, an increase in AP-1 binding activity is associated with cell activation.
• binding activities as well as their relative levels can be informative as to which genes are being expressed in the cell populations involved. This information can be used to assess a variety of effects of compounds on cells, including efficacy, mechanism of action, and toxicity of compounds to which the cells had been exposed.
• the screening assays of the invention are conducted in a high throughput format, meaning that more than about 10, preferably more than about 100, 1,000, or 10,000 compounds are tested at once.
• the format may include an array, where either specific detection molecules or combinations thereof are located in specific locations, such as microtiter plates, slides, gels, columns, microarrays, tubes or chips.
• arrays or other solid supports may contain detection elements for transcription factorlcis site complexes, such as antibodies that bind to proteins associated with transcription or chromatin structures, or nucleic acid molecules that hybridize to cz_?-binding sites.
• such methods are performed where the complexes are formed and/or detected in solution, on solid surfaces, on solid supports, in semi-solid media, in gels, in column matrices, in polymer formulations, in aqueous formulations, in organic solutions, or in nonorganic solutions.
• High throughput formats are also often partially or fully automated.
• Cells that may be used to test compounds include animal cells, plant cells, fungal cells, Archaea cells, and bacterial cells.
• Preferred animal cells include avian, bovine, canine, equine, feline, fish, human, murine, ovine, porcine, and primate cells.
• Such cells may be obtained from in vivo or in vitro (including ex vivo) sources, may be normal, diseased, transformed, infected with a virus, pathogen or other exogenous organism, or represent a particular stage of development.
• Cells may further include fibroblasts, epithelial, hematopoietic, CNS-derived, bone-derived, myocytes, stem cells, basal cells, and the like.
• cells to be tested with a compound may be in any state of metabolism or under any physiologic condition.
• cells may be treated with one or more compounds that affect the cell's metabolism or viability. Such compounds may be administered at one or more concentrations.
• the cells may also be pre-treated with other molecules prior to adding the particular compound of interest. Alternatively, other compounds may be added after the cells are exposed to the compound(s). Following the addition of such compounds, the cells of interest are tested for changes in their transcription factor binding activities.
• the methods of the invention employ assays that use libraries of nucleic acids, e.g., oligonucleotides containing fragments representing genomic DNA, comprising one or more c ⁇ -binding sites.
• cells are treated (in vitro or in vivo) with one or more compounds, at one or more concentrations.
• the cells may also be pre-treated with other molecules prior to adding the particular compound of interest.
• other compounds may be added after the cells are exposed to the compound(s), and/or environmental conditions under which the cells are grown may be changed.
• the cells are grown in the presence of a labeled substrate that can be incorporated into a protein.
• a labeled substrate can be incorporated into a protein.
• a radioactively labeled amino acid can be used.
• the methods of the invention employ libraries of nucleic acid molecules
• the library may comprise a population of nucleic acid molecules containing known binding sites for transcription factors.
• the nucleic acid molecules used in the methods according to the invention will each contain at least one binding site.
• the oligonucleotides comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 binding sites.
• Each nucleic acid molecule may contain a different binding site or some binding sites may be in common among multiple nucleic acid molecules.
• Such nucleic acid molecules may comprise defined nucleic acid sequences.
• Certain preferred nucleic acid molecules comprise nucleic acid sequences that are representative of a genome. Other preferred nucleic acid molecules comprise nucleotide sequences found in genomic DNA.
• the nucleic acid sequence may be random.
• a "defined nucleic acid sequence” refers to a specific sequence of nucleotides, and is typically represented in the 5' to 3' direction using standard single letter notation, where "A” represents adenine, "G” represents guanine, "T” represents thymine, and "C” represents cytosine. It will be appreciated that a nucleic acid molecule having a defined nucleotide sequence may include a different nucleotide at the same position, i.e., is degenerate at that position, with respect to one or more positions in the particular sequence.
• Degenerate bases may be represented by any suitable nomenclature, for example, that which is described in World Intellectual Property Organization Standard ST.25 (1998), Appendix 2.
• Random nucleic acid molecules may also comprise nucleotide sequences representative of a genome.
• a nucleic acid molecule may comprise the same bias for nucleotide representation as a particular genome.
• Nucleic acid molecules may be synthetic or isolated from cells, varying in length from about 4 to about 1000 nucleotides in length, comprise purified DNA, partially-purified DNA or unpurified DNA, and may comprise DNA within chromatin, a chromosome, or chromosome segment. Oligonucleotides may be representative of or a part of a genome comprising human, mammalian, vertebrate, animal, plant, fungi, eukaryotic, prokaryotic or viral genomes. Nucleic acid molecules may contain modified nucleotides, for example, methylated nucleotides, as well as, or alternatively, nucleotide analogs and derivatives.
• Nucleic acid molecules may also comprise a first amplification primer site upstream of the transcription factor binding site and a second amplification primer site downstream of the binding site.
• a random DNA library is generated for use in the binding reactions with nuclear or other proteins. For this, a mixture of oligonucleotides, each with a fully randomized central domain flanked by two fixed but different sequences on either side, is synthesized. The fixed sequences are typically at least 15 nucleotides long to allow for efficient primer annealing, while the randomized sequence is typically at least 10 nucleotides in length.
• the double-stranded random DNA library is generated by at least one and up to five cycles of PCR.
• a genomic DNA library containing oligonucleotides representative of human genomic DNA is used.
• the library can be generated by a method similar to the one described by Singer et al. (1997, Nucleic Acids Res. 25, 781-786).
• a primer consisting of a fixed 5'-region (18-22 bp in length) and a 9-nucleotide randomized extension at its 3 '-end is annealed to denatured genomic DNA and extended with Klenow DNA polymerase. Extension products are isolated and the process is repeated with a second primer having a different fixed region.
• the DNA is purified and further amplified by PCR using primers containing only the fixed sequences.
• the amplified DNA is size- fractionated using polyacrylamide gel electrophoresis and then amplified again with primers A and B and gel-purified again to yield genomic libraries containing inserts of defined size ranges.
• a genomic library prepared in this way consists of double-stranded DNA molecules that each contain a genomic DNA sequence in the center (typically 25-250 bp in length) flanked by two different fixed regions (priming sequences) at either end.
• Preferred genomic libraries contain 35-100 bp of center DNA, and even more preferred are genomic libraries containing 45-50 bp of center DNA.
• Nuclear extracts containing nuclear proteins are obtained from the cells exposed to the compound.
• Nuclear extracts can be prepared by any suitable method, including by hypotonic lysis on ice, pelleting of nuclei and extraction of proteins in high salt buffer, and then dialysis or dilution to 100 mM salt, storage at -80°C
• Nuclear extracts may be obtained at a single time point following exposure to the compound, or at different times. Extracts may also be obtained from cells treated at varying concentrations of a compound or with mixtures of more than one compound. These nuclear extracts will exhibit changes in protein composition and concentration according to the type of compound as well as concentration and duration of treatment.
• the nuclear extracts are combined with the DNA library to generate the binding reaction, which typically contains 5-10 ⁇ g of nuclear extract proteins (or various protein fractions or other protein preparations), 5-50 ng of double-stranded library DNA (see above), and non-specific competitor nucleic acids such as polydLdC, salmon sperm DNA, calf thymus DNA, or E.coli total RNA.
• One strand of the library DNA may be biotinylated at its 5'-end, or otherwise modified such that purification from the binding reactions can be carried out using solid phase chemistries.
• the salt and buffer conditions are typically 1-5 mM MgCl 2 , 50- 100 mM KC1, 20-25 mM HEPES-NaOH or Tris-HCl (pH 7.5-8.0), 10-20% glycerol, 0.1 mM EDTA.
• Incubation temperature and time are typically 4 C or room temperature and between 15 minutes and 3 hours, respectively.
• the bound protein/DNA complexes can be partitioned away from unbound components using properties such as molecular weight, charge, or other physical or chemical properties.
• One preferred embodiment involves using the electrophoretic mobility shift assay (EMSA) which allows separating large numbers of bound complexes from unbound nucleic acids and/or binding factors.
• ESA electrophoretic mobility shift assay
• the recovered complexes can then be isolated and the individual nucleic acid and protein components further purified for direct analysis if desired.
• nucleic acids can be purified from the isolated protein/DNA complexes by one of several methods.
• the sample containing the protein/DNA complexes can be extracted by organic solvents (phenol, chloroform) and the nucleic acids can be precipitated by the addition of 2-3 volumes of ethanol and recovered by centrifugation.
• organic solvents phenol, chloroform
• nucleic acids can be captured with streptavidin-coated agarose beads, also making use of magnetic separation.
• Chemical methods for attaching the detectable label biotin i.e., biotinylating
• biotinylating are known in the art. See, e.g.
• Oligonucleotides and other nucleic acids can also be biotinylated using enzymatic systems such as, e.g., nick translation (E. coli DNA Polymerase I and Dnase I; Boyle, Section V of Chapter 3, in Short Protocols in Molecular Biology, Second Edition, Ausubel, et al.
• Proteins can be purified from the isolated protein DNA complexes by dissociation from the DNA in the presence of an ionic detergent (e.g., sodium dodecyl sulfate), concentrated by filtration, or precipitated by the addition of high concentrations (2- 4 M) of ammonium sulfate.
• an ionic detergent e.g., sodium dodecyl sulfate
• the eluted DNA fragments if captured using streptavidin-coated beads, is then recovered from the beads using standard techniques known to those in the field and appropriate to the type of bead.
• the DNA fraction which represents the "protein bound" fraction of the original library, can be amplified by PCR or another nucleic acid amplification method to a moderate level and then used in a binding reaction identical to the first reaction.
• the binding process can be repeated any number of rounds, depending upon the level of selectivity desired. Typically 2 or 3 rounds are sufficient to achieve a significant selection of transcription factor binding activities and a negligible level of background.
• the DNA fraction can also be analyzed directly without amplification.
• the isolated nucleic acid fragments can be analyzed for the presence of transcription factor binding sites and for the level of transcription factor binding activities using a number of methods, including direct DNA sequencing and hybridization techniques.
• direct sequencing the individual oligonucleotides selected in the binding reactions are sequenced and the transcription factor binding sites are identified and counted on the selected oligonucleotides.
• the isolated fragments could be labeled in a way that would allow detection (e.g., by radioactivity, biotin-avidin, enzymatically) and then hybridized to a membrane or array that contains single-stranded DNA oligonucleotides specific for particular c ⁇ -binding sites.
• the nucleic acid fragments could be hybridized to a nucleic acid array comprising a plurality of binding site-specific oligonucleotides, wherein hybridization could be detected using a variety of methods well known in the art.
• nucleic acids when bound to a solid support, e.g., as a nucleic acid array, labeled proteins that interact therewith can be detected.
• labeled proteins that interact therewith can be detected.
• an unlabeled transcription factor bound to its cognate cis-binding site can also be detected in other ways, for example, using detectable antibodies or other epitope-specific moieties.
• control assays concerns obtaining a nuclear extract from cells that have not been exposed to the compound, or which have been exposed to the compound under different conditions, for example, at different concentrations, for differing periods of time, etc. Differences in results reveal which transcription factor binding activities are affected by the compound, which can be used as an indicator of biological effects for that particular compound. Because many particular transcription factor binding activities are involved, for example, with regulating the expression of some, but not all genes, further studies can be undertaken to investigate the compound-mediated effects on expression of such genes.
• the genes with which it is functionally associated i.e., those genes over which it has some regulatory influence, be it activation, repression, sequestering in chromatin, etc.
• This determination can be made, for example, by searching sequence databases to determine which genes the relevant cis-binding site is proximal to in the genome. If desirable, these results can be confirmed experimentally.
• a database of genes whose expression is at least partially controlled by p articular transcription factors and/or cts-binding sites can b e established. Carried to its conclusion, a database of all regulatory elements and the genes whose expression they control can be developed.
• RNA profiling detects and quantifies the transcription factor proteins, as the proteins encoded by particular genes can also be readily determined and detected.
• proteins can be over-expressed in appropriate expressions systems as are understood by those of skill in the art and, for example, high affinity polyclonal, and preferably monoclonal antibodies, raised against each of them.
• Such antibodies can be arrayed on a solid support in a manner analogous to different nucleic acid hybridization probes.
• Cell extracts from treated and untreated cell populations can be used in binding reactions to form the transcription factorlcis site complexes characteristic of each of those cell populations. To characterize the complexes from each of the cell populations, they can be added to such antibody arrays and the level of each transcription factor determined.
• the results of such binding may be detected by any suitable technique, for example, by using a second, labeled antibody specific for a different epitope on the transcription factor so as to create a probe antibody-protein- detection antibody sandwich. This allows the profiling of which particular transcription factor binding activities are present in cells exposed to the compound compared to cells that were untreated.
• Another exemplary technique that can be used in the practice of the invention involves contacting oligonucleotides in a nucleic acid library with transcription factors obtained from nuclear extracts of cells treated with a compound, allowing the factors to form transcription factor 'cis site complexes with specific oligonucleotides of the library, and separating the complexes from free constituents of the reaction using electrophoretic mobility shift assays (EMS A).
• EMS A electrophoretic mobility shift assays
• Cells can be treated with various compounds developed to exert particular effects on cells, e.g., inhibition of growth, inhibition of particular enzymes or other gene products, and production of particular gene products (among many others).
• the effect of the compound can be determined by studying changes in gene expression. This is accomplished by first determining the transcription factor binding activities for both the treated and untreated cell populations and obtaining a binding activity profile for each population. Secondly, the profiles are compared to each other to determine which binding activities change as a result of the compound treatment. Certain binding activities, as well as the relative levels of these activities, can be informative as to which genes are being expressed in the cell populations involved.
• the assay is implemented in a high throughput, preferably automated, format.
• a nuclear extract from each treated cell population, as well as from untreated cells is prepared.
• the profile of transcription factor binding activities identified as having been affected can be used to assess efficacy of the particular compound. Binding activities that become activated or, alternatively, that are repressed in response to compound treatment provide information as to which genes, or subset of genes, are activated or repressed, as the case may be, in response to exposure to the compound. Additional studies on one or more of these genes can then be carried out.
• a nucleic acid array comprising genes known to be regulated by the particular transcription factor can be used to perform RNA expression profiling to further understand the effect of the compound on particular gene(s).
• the methods of the invention can also be used to assess toxicity or other adverse effects of various compounds on cells.
• a preferred method useful in performing such assays is carried out on cells treated with one or more particular compounds at various concentrations.
• the assay is performed on cells at various time points after exposure to the compound(s) on a prepared nuclear extract from each treated cell population, as well as from untreated cells.
• the effect of the compound is determined by first defining the transcription factor binding activities for both the treated cell population and for the untreated cell population. These profiles of binding activities are comprised of both the types of binding activities present in the cells as well as how active they are relative to each other.
• the profiles are then compared to each other to determine those transcription factor binding activities that are different between the treated and untreated cell populations and thus a result of treatment with the compound. Changes in particular binding activities are indicative of certain molecular and cellular changes in the cells.
• the profiles involving transcription factors and their cognate binding sites that are activated or repressed in the treated versus untreated cells and that correlate with toxicity allow toxicity of the particular compound to be assessed. Additional studies on one or more of these transcription factor binding activities shown to be altered can then be carried out.
• a nucleic acid array comprising genes known to be regulated by the particular transcription factor can be used to perform RNA expression profiling to further understand the mechanism of toxicity of the compound.
• RNA expression profiling- The profile of active or silenced gene regulatory elements in cells treated with a particular compound can also give important information concerning mechanism of action. Changes in activity of particular transcription factor binding activities can denote changes in expression of certain genes, which can also be further studied using additional experimental approaches such as RNA expression profiling-
• assays can be carried out on cells treated with a particular compound at various concentrations and at various time points.
• the starting cells may also be varied, e.g., at various levels of confluency, synchronized with regard to cell growth, or serum-starved, before treatment.
• a nuclear extract from each treated cell population, as well as from untreated cells, is prepared and the profile of transcription factor binding activities that result from exposure to the compound are determined and compared according to the embodiments of the invention in order to determine the effect on transcription factor binding activity. Effects of the compound on the expression of particular genes, particularly those regulated by specific transcription factors, can then be assessed.
• Optimizing Lead Compounds The methods of the invention can also be used to correlate the structure/function relationship of families of compounds or particular moieties with activity of specific regulatory elements. Changes in activity of particular transcription factor binding activities, as well as the genes they regulate, can be used as a measure of potential beneficial activity as well as undesired side effects. Assays are carried out on cells treated with the various families or classes of compounds, preferably at various concentrations and at various time points. The profile of transcription factor binding activities after treatment with each compound is determined and compared in order to help determine the optimal compound(s) for each desired effect. Effects of the various compounds on the expression of particular genes regulated by specific transcription factors of interest can then be assessed, e.g. , by RNA expression profiling. This process can be used in an iterative fashion to obtain a compound, or class of compounds, having the desired activity, but having few if any undesired effects on gene transcription. Such methods allow rapid progress to be made with regard to initial lead compound identification and subsequent lead optimization.
• the methods of invention contemplate detecting changes in transcription factor binding activities reflective of gene expression changes induced directly or indirectly by any compound, including but not limited to: proteins, peptides, nucleic acids, lipids, carbohydrates, organic or inorganic molecules, hormones, small molecules, polymers etc.
• any compound including but not limited to: proteins, peptides, nucleic acids, lipids, carbohydrates, organic or inorganic molecules, hormones, small molecules, polymers etc.
• Such compounds can be naturally occurring macromolecules, or synthetic derivatives, analogs ormhnetics of these macromolecules.
• Such a broad array of compounds when in contact with cells, will affect transcription factor binding activities differently, so that when the profiles between cells treated with the various compounds or under various conditions are compared according to the invention hi order to synthesize a compound for testing in the first instance, any suitable method may be employed.
• Such methods include the synthesis of a single compound by traditional methods, up through a massively parallel combinatorial approach.
• the genomic DNA library was generated by a method similar to the one described by Singer et al. (1997, Nucleic Acids Res. 25, 781-786).
• a primer consisting of a fixed 5 '-region (18-22 bp in length) and a 9-nucleotide randomized extension at its 3 '-end was annealed to denatured genomic DNA and extended with Klenow DNA polymerase. Extension products were isolated and the process was repeated with a second primer having a different fixed region.
• the DNA was purified and further amplified by PCR using primers containing only the fixed sequences.
• the amplified DNA was electrophoresed on a native polyacrylamide gel and various size-ranges of DNA were cut out and eluted from the gel (e.g.
• a genomic library prepared in this way consisted of double-stranded DNA molecules that each contained a genomic DNA sequence in the center (typically 25-250 bp in length) flanked by two different fixed regions (priming sequences) at either end.
• the binding reaction typically contained 5-10 ⁇ g nuclear extract proteins, 5-50 ng double-stranded library DNA (see above), and non-specific competitor nucleic acids such as polydhdC, salmon sperm DNA, calf thymus DNA, or E.coli total RNA.
• One strand of the library DNA was biotinylated at its 5'-end.
• the salt and buffer conditions were typically 1-5 mM MgCl 2 , 50-100 mM KCl, 20-25 mM HEPES-NaOH or Tris-HCl (pH 7.5-8.0), 10-20% glycerol, 0.1 mM EDTA.
• Incubation temperature and time are typically 4 C or room temperature and between 15 minutes and 3 hours, respectively.
• the bound protein/DNA complexes were partitioned away from unbound components by electrophoretic mobility shift assay (EMSA).
• ESA electrophoretic mobility shift assay
• the eluted DNA fragments were captured using streptavidin-coated beads and then recovered from the beads, using methods appropriate to the type of bead.
• the DNA fraction which represents the "protein bound" fraction of the original library, was amplified by PCR to a moderate level and then used in binding reaction identical to the first reaction.
• TPA/ionomycin-activated Jurkat cell nuclear extract were sequenced. The presence of known transcription factor binding sites in these fragments was used to form the activity profile and their activity determined by searching for the corresponding consensus motifs for those factors. A partial list of these cw-binding sites can be found in the first column of the Table. The second and third columns show the numbers of the corresponding binding sites identified by the assay using the resting and activated Jurkat nuclear extracts, respectively (expressed as the percentage of DNA fragments containing the sites). The results show that certain cis sites, e.g. AP-1, are strongly induced in activated Jurkat nuclear extracts, while others, e.g., MycMax or CAAT-box, are unchanged between the two cell populations.
• AP-1 e.g. AP-1
• the method of the invention provides profile data regarding aspects of gene expression and reflecting the effect of a compound on a cell population.
• MCF7 cells were grown in high glucose DMEM containing 10% fetal calf serum, antibiotic/antimyotics, and supplemented with 2mM L-glutamine.
• cells were grown to a density of approximately 1 X 10 6 cells/ml and an additional 10% media volume containing 1.85-18.6 ug ml (in 95% EtoH)Tamoxifen, 0.17-8.54 ug/ml (in 95% EtoH) Taxol, or 0.56-2.9 ug/ml (in water)
• Doxorubicin was added and gently mixed. Cells were harvested after 2-6 hr incubation and nuclear extracts were prepared as described above. Toxicity of the drugs was monitored by treating parallel samples and assaying for cell death by Trypan Blue staining at 24 hrs, 48 hrs, and 72 hrs post treatment.
• Nuclear extracts were each mixed with a library of genomic DNA fragments and fragments forming specific complexes with nuclear proteins were sequenced. For each of the four samples, about 800 fragments were sequenced and searched for the presence of cis-binding sites as described for in Example 1. The data are presented in the bar graph shown in Figure 1. It can be seen that the percentage of nucleic acid fragments containing selected cw-binding sites that were isolated in binding assays with nuclear extract from untreated cells (black bars) varies markedly from cells treated with tamoxifen (white bars), taxol (hatched bars) or doxorubicin (gray bars).
• the method of the invention provides a profile of transcription factor binding activities that are useful in establishing a link between toxic effects of compounds (such as those of the example) and changes in gene expression.
• PC 12 cells were grown in high glucose DMEM containing 10% horse serum and 5% fetal calf serum. For differentiation, cells were transferred to serum-free medium containing N-2 Supplement (Life
• NGF-treatment leads to an increase in binding activity for API, ATF, TCF11 (among others), while for example E2F and RFXl activities are reduced after NGF treatment.
• this profile represents only a partial analysis, it indicates that genes regulated by API, ATF, or TCF11 may be activated upon NGF treatment, while genes regulated by E2F and RFXl are expected to be repressed upon NGF treatment.

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