WO1999057137A1 - Compositions et procedes impliquant une regulation des rythmes circadiens de mammiferes - Google Patents

Compositions et procedes impliquant une regulation des rythmes circadiens de mammiferes Download PDF

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WO1999057137A1
WO1999057137A1 PCT/US1999/010072 US9910072W WO9957137A1 WO 1999057137 A1 WO1999057137 A1 WO 1999057137A1 US 9910072 W US9910072 W US 9910072W WO 9957137 A1 WO9957137 A1 WO 9957137A1
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reporter gene
protein
clock
gene expression
cell
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PCT/US1999/010072
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English (en)
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WO1999057137A9 (fr
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Charles J. Weitz
Nicholas Gekakis
David Staknis
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The President & Fellows Of Harvard College
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Priority to AU39760/99A priority Critical patent/AU3976099A/en
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Publication of WO1999057137A9 publication Critical patent/WO1999057137A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • the invention relates in general to the screening of candidate pharmacological compositions and to disease diagnosis and treatment.
  • Circadian clocks are endogenous oscillators that control daily rhythms in physiology and behavior (Pittendrigh, 1993, Ann. Rev. Physiol., 55: 17). Such clocks are phylogenetically widespread (Edmunds, 1988, Cellular and Molecular Rases of Biological Clocks, Springer- Verlag, Berlin) and likely reflect evolutionarily ancient, fundamental mechanisms of timekeeping important for anticipation of daily variations in environmental conditions (Pittendrigh and Daan, 1976, J. Comp. Physiol .. 106: 291). In mammals, the circadian clock driving metabolic and behavioral rhythms is located in the suprachiasmatic nucleus (SCN) of the hypothalamus (D. C.
  • SCN suprachiasmatic nucleus
  • a mouse mutant, Clock has a phenotype affecting both the periodicity and persistence of circadian rhythms (Vitaterna et al., 1994, Science, 264: 719).
  • Positional cloning of the Clock gene revealed that its predicted protein product, CLOCK, is a member of the bHLH-PAS family, some members of which are known to function as transcription factors.
  • the mutant Clock allele acts genetically in a dominant-negative fashion (King et al., 1997, supra; King et al., 1997, Genetics, 146: 1049) and encodes a protein with a 51 -amino acid internal deletion in its putative transcriptional regulatory domain (CLOCK- ⁇ 19). How CLOCK may control the periodicity and persistence of circadian rhythms was heretofore unknown.
  • mperl Small et al., 1997, Cell, 90: 1003; Tei et al., 1997, Nature, 389: 512
  • mper2 Shearman et al., 1997, Neuron, 19: 1261; Albrecht et al., 1997, Cell, 91: 1055
  • mper3 Zylka et al., Neuron 1998), respectively. All three are expressed in the SCN and retina, and, like Drosophila per, their transcripts exhibit a circadian oscillation in abundance.
  • the clock mechanism is constituted in part by a negative feedback loop in which the PER protein, by unknown means, represses transcription of its own gene (Hardin et al., 1990, Nature, 343: 536; Zheng et al., 1994, EMBClX, 13: 3590).
  • Constitutive per mRNA expression has been observed in mutants lacking functional PER protein (Zheng et al., 1994, EMBOJ , 13: 3590; Brandes et al., 1996, Neuron, 16: 687; Stanewsky et al., 1997, FMBO J.. 16: 5006), indicating that there is PER-independent positive regulation oiper transcription.
  • a 69-base pair (bp) "clock control region" located upstream of the Drosophila per gene has been shown to confer tissue-specific, circadian cycling on reporter genes that is dependent on a functional PER protein (Hao et al., 1997, Mol. Cell Biol., 17: 3687).
  • the 69-bp clock control region thus includes sequences sufficient for both negative feedback and positive, PER- independent transcriptional regulation.
  • an E-box element (cacgtg) is required for the positive component of the transcriptional regulation (Hao et al., 1997, Mol. Cell BioJL, 17: 3687).
  • E-box elements are recognition sites for bHLH DNA-binding domains (Murre et al., 1989, Cell, 56: 777; Burbach et al., 1992, Proc. Natl. Acad. Sci. U.S.A., 89: 8185; Zelaer et al., 1997, Genes_Dev., 11: 2079; Sonnenfeld et al, 1997, Development, 124: 4571), and bHLH-PAS proteins are known to heterodimerize in order to bind target sites on DNA molecules.
  • the invention provides a composition comprising isolated BMAJ.1 protein and isolated CLOCK protein.
  • the invention provides another composition comprising isolated BMALl protein, isolated CLOCK protein, and isolated PER protein.
  • the invention provides yet another composition comprising isolated BMALl protein, isolated CLOCK protein, isolated PER protein, and isolated TIM protein.
  • compositions comprising a nucleic acid (DNA or RNA) molecule encoding BMALl protein and a nucleic acid molecule encoding CLOCK protein.
  • the invention further provides a composition comprising a nucleic acid (DNA or RNA) molecule encoding BMALl protein, a nucleic acid molecule encoding CLOCK protein, and a nucleic acid molecule encoding PER protein.
  • the invention also provides a composition comprising a nucleic acid (DNA or RNA) molecule encoding BMALl protein, a nucleic acid molecule encoding CLOCK protein, a nucleic acid molecule encoding PER protein, and a nucleic acid molecule encoding TIM protein.
  • BMALl protein refers to a transcription factor that, when combined with CLOCK protein, activates transcription of the mperl gene.
  • the DNA sequence of human bmallb is provided in Fig. 1, with the predicted amino acid sequence of the human BMALl protein provided in Fig. 2.
  • DNA sequences of alternative splicing variants of the bmall gene are provided in Figs. 3 and 4.
  • the other alternative splicing variant DNA sequences, of bmallc, bmalld, bmalle and bmallf, are provided in Figs. 5, 6, 7 and 8, respectively.
  • the DNA and amino acid sequences for human BMALl proteins known as JAP3 or MOP3 are shown in Figs. 9 and 10, and 11 and 12, respectively.
  • the DNA and amino acid sequences for Drosophila protein dBMALl are shown in Figs. 13 and 14.
  • CLOCK protein refers to a protein having a biological activity which activates trancription of the mammalian er gene (Sun et al., 1997, Cell, 90: 1003; Tei et al., 1997, Nature, 389: 512) when the CLOCK protein is present in combination with BMALl protein.
  • the mouse clock gene sequence and the encoded CLOCK amino acid sequence are presented in Figs. 15 and 16.
  • the human clock DNA sequence and the encoded CLOCK amino acid sequence are presented in Figs. 25 and 26.
  • a CLOCK protein useful in the invention will have a sequence that is substantially similar or identical to either of the amino acid sequences presented in Fig. 16 or Fig.
  • PER protein refers to a protein which forms a heteromeric complex together with TIM protein and has a biological activity which inhibits trancription of the mammalian peri gene when the CLOCK protein is present in combination with BMALl protein.
  • the human er DNA sequence and the encoded PER amino acid sequence are presented in Figs. 27 and 28.
  • the Drosophila per DNA sequence and three encoded PER amino acid sequences (PER A, PER B, and PER C) are provided in Figs. 29-32.
  • a PER protein useful in the invention will have a sequence that is substantially similar or identical to any of the amino acid sequences presented in Figs.
  • TIM protein refers to a protein which forms a heteromeric complex together with PER protein and has a biological activity which inhibits trancription of the mammalian peri gene when the CLOCK protein is present in combination with BMALl protein.
  • the human tim DNA sequence and the encoded TIM amino acid sequence are presented in Figs. 33 and 34.
  • the Drosophila tim DNA sequence and the encoded TIM amino acid sequence are provided in Figs. 35 and 36.
  • a TIM protein useful in the invention will have a sequence that is substantially similar or identical to any of the amino acid sequences presented in Figs. 34, and 36, and will encompass Drosophila TIM as well as human TIM.
  • the invention also encompasses an isolated proteimprotein heterodimer comprising BMALl and CLOCK proteins, in a physiologically compatible solution, e.g., a neutral buffer.
  • a physiologically compatible solution e.g., a neutral buffer.
  • the invention further encompasses a protein complex comprising BMALl, CLOCK, and PER proteins, and another protein complex comprising BMALl, CLOCK, PER, and TIM proteins
  • heterodimer refers to a protei protein complex, wherein the complex comprises two protein which differ in amino acid sequence in at least one position.
  • Each such protein also referred to as a "subunit” or “binding partner”
  • binding partner when present in the heterodimer, may recognize a half-site of a recognition sequence for a protein, which sequence may be found on a DNA molecule.
  • the two protein binding partners of the heterodimeric complex may be bound to each other either reversibly (non-covalently, e.g., by hydrogen bonding) or irreversibly (covalently).
  • the ratio of BMALl protein or DNA encoding BMALl to CLOCK protein or DNA encoding CLOCK is on the order of 1 : 1 , 10:1, 100:1, 1,000:1 or even 10,000:1, 1:10,000, 1:1,000, 1:100, 1:10, or 1:1.
  • the ratio of any protein in the complex or DNA encoding any protein in the complex to any other protein in the complex or any other DNA encoding any other protein in the complex is on the order of 1 : 1 , 10:1, 100:1, 1,000:1 or even 10,000:1, 1:10,000, 1:1,000, 1:100, 1:10, or 1:1.
  • half-site refers to a nucleic acid sequence which is recognized and bound by a targeting amino acid sequence present on one protein subunit of a dimeric protein complex, whether heterodimeric or homodimeric (a homodimer is a protein:protein dimer in which both binding partners are identical in amino acid sequence at all positions). In most cases, neither subunit of the dimeric protein complex will bind its cognate half-site alone (i.e., unless dimerized to the other); therefore, usually either both half-sites are occupied by protein, or neither is.
  • Both half sites of a recognition site for a protein may be identical, whether arranged head-to-tail or as a palindrome (head-to-head or tail-to-tail); if in the latter configuration, the sequence of a recognition site of a protein is said to have "dyad symmetry".
  • a recognition site for a protein bound by a protein homodimer comprises two identical half-sites.
  • the two half-sites comprised by a recognition site for a protein may be unlike in sequence; it is usually true that dissimilar half-sites are bound by different targeting amino acid sequences, as would be found on the two subunits of a protein heterodimer.
  • recognition sites for a protein comprising non-identical, but similar, half-sites may also be said to have dyad symmetry.
  • the invention additionally encompasses an assay for identifying a modulator of period gene expression, comprising providing a gene expression system comprising BMALl protein, CLOCK and a regulatory target DNA comprising an E-box and operatively linked to a reporter gene, wherein the reporter gene is expressed at a basal level in the system; contacting the system with a candidate modulator of the target sequence for a time sufficient to permit reporter gene expression and modulation thereof; and detecting expression of the reporter gene, wherein detection of an increase in reporter gene expression above the basal expression level is indicative of stimulation of reporter gene expression by the candidate modulator and a decrease in reporter gene expression below the basal level is indicative of inhibition of reporter gene expression by the candidate modulator, and wherein stimulation or inhibition of reporter gene expression by a candidate modulator is indicative of the ability of that candidate modulator to modulate period gene expression.
  • regulatory target DNA is a DNA containing an E-box and susceptible to regulation by the BMALl/CLOCK combination of proteins when the regulatory target DNA is operatively linked to a gene (whether that gene is a reporter gene or the natural gene linked to the regulatory DNA, i.e., period gene).
  • operatively linked to usually refers to 5' to 3' covalent linkage of the 3' end of the target DNA to the 5' end of the gene to be regulated; that is, the target DNA is upstream of the gene that it is linked to.
  • E-box and E-box DNA element refer to a DNA hexanucleotide of having the sequence 5'-CACGTG-3'.
  • E-box sequences which are operatively linked to genes whose products regulate the circadian clock of an organism.
  • An E-box DNA element may be present in a single copy and thus constitute a target regulatory sequence which may be modulated according to the invention; alternatively, a plurality of E-box DNA elements, wherein a plurality comprises 2 or more such elements (e.g., 3, 4, 5, 10, 15, 20 or even 5, 75, 100 or more E-box DNA elements).
  • the E-boxes may be spaced apart from each other, for example, by 6-10 nucleotides of random sequence spacer DNA, or they may be present in a contiguous format.
  • the regulatory target DNA will usually be joined to the regulated gene such that the 3'-most nucleotide of the E-box hexanucleotide sequence is within 500 nucleotides, 100 nucleotides, 50 nucleotides, 20 nucleotides 10 nucleotides, or 5 nucleotides of the +1 transcription start site of the gene, or is joined directly to the +1 start site of the gene.
  • the term “modulate” refers to the effect of an agent (such as a drug or other pharmacological composition) or condition (such as an environmental change or a genetic mutation) either to stimulate, enhance or otherwise increase- or to inhibit, repress, depress or otherwise decrease expression of that gene (whether expression is detected via the RNA, protein, or gene product activity) by at least 10% relative to its basal level of expression.
  • the percent change in gene expression relative to the basal level may be greater than 10%, such as 20-50%, or 75-100%.
  • an agent or condition which modulates gene expression may do so such that the resulting expression level is greater than 100%, for example 2- to 10-fold, 20- to 100-fold, 1000-fold, or even 10,000- to-100,000-fold above or below the basal level of expression.
  • basal level of expression or “basal expression level” refers to the level of transcription of that gene in an organism, cell lysate or other gene expression system or relative to its level of expression in a control organism, cell, cell lysate or other gene expression system which has not been treated with- or exposed or otherwise subjected to the candidate modulating agent or condition, or to its level of expression in the subject organism prior to such treatment or exposure.
  • reporter gene expression may be monitored by detecting reporter gene RNA, the encoded protein, or the activity of the RNA or encoded protein. Such detection may be qualitative (i.e., the presence or absence of the transcript, protein product, or activity is detected) and/or quantitative (i.e., the amount of the transcript, protein or activity is measured).
  • gene expression system refers to an expression system which includes, at a minimum, a nucleic acid construct containing a regulatory target sequence comprising an E-box element operatively linked to a reporter gene, wherein the regulatory target sequence is responsive to BMALl and CLOCK protein combination.
  • the invention also provides an assay for identifying a modulator of period gene expression, comprising providing a gene expression system comprising BMALl protein, CLOCK protein and a cell lysate containing a regulatory target sequence comprising an E-box operatively linked to a reporter gene, wherein the reporter gene is expressed at a basal level in the system; contacting the system with a candidate modulator of the target sequence for a time sufficient to permit reporter gene expression and modulation thereof; and detecting expression of the reporter gene, wherein detection of an increase in reporter gene expression above the basal expression level is indicative of stimulation of reporter gene expression by the candidate modulator and a decrease in reporter gene expression below the basal level is indicative of inhibition of reporter gene expression by the candidate modulator, and wherein stimulation or inhibition of reporter gene expression by a candidate modulator is indicative of the ability of that candidate modulator to modulate period gene expression.
  • the above assay for identifying a modulator of period gene expression may alternatively include, in step a), providing a gene expression system comprising a nucleic acid sequence encoding BMALl, a nucleic acid sequence encoding CLOCK and a cell lysate containing a regulatory target sequence comprising an E-box operatively linked to a reporter gene, wherein the reporter gene is expressed at a basal level in the system.
  • the cell lysate is obtained from a cell selected from the group that includes a bacterial cell, a yeast cell, an insect cell and a mammalian cell.
  • the insect cell or mammalian cell is a cell of the central nervous system.
  • the invention additionally encompasses an assay for identifying a modulator of period gene expression, comprising providing a gene expression system comprising BMALl protein, CLOCK protein and a cell comprising a regulatory target sequence comprising an E-box operatively linked to a reporter gene; contacting the system with a candidate modulator of the target sequence for a time sufficient to permit reporter gene expression and modulation thereof; and detecting expression of the reporter gene, wherein detection of an increase in reporter gene expression above the basal expression level is indicative of stimulation of reporter gene expression by the candidate modulator and a decrease in reporter gene expression below the basal level is indicative of inhibition of reporter gene expression by the candidate modulator, and wherein stimulation or inhibition of reporter gene expression by a candidate modulator is indicative of the ability of that candidate modulator to modulate period gene expression.
  • the cell is selected from the group that includes a bacterial cell, a yeast cell, an insect cell and a mammalian cell.
  • the insect cell or mammalian cell is a cell of the central nervous system.
  • the cell is assayed in vitro.
  • the term "in vitro" refers to an isolated cell, or a colony or other population of cells of a unicellular organism which are either in culture or are suspended in an isotonic medium (e.g., an isotonic salt buffer), such that they remain alive under assay conditions; the term “in vitro” additionally refers to a cell or group of cells, such as in a sample of a biological fluid (for example, vertebrate blood or insect hemo lymph), or in an explant, biopsy, swab or scraping of an organ or tissue of a multicellular organism, which cell or group of cells is either maintained in culture or is suspended or otherwise bathed in an isotonic medium (e.g., a physiological salt buffer) such that they remain alive under assay conditions.
  • an isotonic medium e.g., a physiological salt buffer
  • the cell is assayed in vivo.
  • the term "in vivo" refers to a cell which is assayed in its native context, in the body of a whole, multicellular organism. It is contemplated according to the invention that such an organism may be a transgenic animal comprising endogenous BMALl and CLOCK proteins, as defined herein, and bearing a transgene comprising a regulatory target sequence containing an E-box DNA element operatively linked to a reporter gene; also of use in the invention are transgenic mice.
  • a modulator so identified according to the invention in this assay or the assays described herein below may be of use in the diagnosis and treatment of an alteration in- or disruption of the circadian clock in a human or a disease or disorder in a human which involves such an alteration or disruption.
  • Such conditions include, but are not limited to, jetlag, sleep disorders, depression (in .particular, seasonal-affective disorder, or SAD) and infertility.
  • the term "circadian clock” refers to the timing mechanism of an organism which regulates functions which fluctuate or otherwise vary cyclically in a given time period. Such functions which are so regulated include, but are not limited to, the sleep/wake cycle and the timing of production/secretion of hormones and/or other bioactive substances by a cell or tissue of an organism and the resulting actions thereof.
  • the length of the sleep/wake cycle in humans and other mammals is about 24 hours. Diagnostic uses encompassing detection of aberrant BMALl sequences, RNA or protein expression levels, heterodimerization with CLOCK or abnormal binding and/or transcriptional activity with regard to the period gene are contemplated.
  • Another aspect of the present invention is a method of diagnosing a disorder which is linked to an abnormality of the circadian clock comprising assaying performing a step to detect the ability of a biological sample from an organism to regulate the level of period gene expression, comprising a) providing a gene expression system comprising a regulatory target sequence comprising an E-box operatively linked to a reporter gene; b) contacting the system with the biological sample for a time sufficient to permit reporter gene expression and modulation thereof; c) detecting the level of expression of the reporter gene; and d) comparing the level of expression so detected with the expression level of the reporter gene observed when the system is contacted with a biological sample from a control organism, wherein a difference in the levels of expression of the reporter gene is indicative of the disorder.
  • biological sample refers to a whole organism or a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
  • body fluids including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen.
  • Biological sample further refers to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof.
  • biological sample refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or nucleic acid molecules.
  • the term “organism” refers to a unicellular or multicellular life form.
  • the term “organism” refers to a multicellular organism, preferably a mammal, and most preferably a human.
  • the term “organism” refers to a human.
  • the reporter gene expression system further comprises CLOCK protein or nucleic acid encoding this protein.
  • the reporter gene expression system further comprises BMALl protein or a nucleic acid encoding this protein.
  • the detecting step may be performed via detection of reporter gene RNA, encoded protein, or activity of the RNA or protein, such as enzymatic activity.
  • Figure 1 presents the cDNA sequence encoding human BMALlb.
  • Figure 2 presents the amino acid sequence of human BMALlb.
  • Figure 3 presents the cDNA sequence encoding human BMALl a.
  • Figure 4 presents the amino acid sequence of human BMALl a.
  • Figure 5 presents the cDNA sequence encoding human BMALl c.
  • Figure 6 presents the cDNA sequence encoding human BMALld.
  • Figure 7 presents the cDNA sequence encoding human BMALl e.
  • Figure 8 presents the cDNA sequence encoding human BMALl f.
  • Figure 9 presents the cDNA sequence encoding human JAP3.
  • Figure 10 presents the amino acid sequence of human JAP3.
  • Figure 11 presents the cDNA sequence encoding human MOP3.
  • Figure 12 presents the amino acid sequence of human MOP3.
  • Figure 13 presents the cDNA sequence encoding Drosophila BMALl (dBMALl).
  • Figure 14 presents the amino acid sequence of Drosophila BMALl (dBMALl).
  • Figure 15 presents the DNA sequence of the mouse Clock gene.
  • Figure 16 presents the amino acid sequence of the mouse CLOCK protein.
  • Figure 17 presents a yeast two-hybrid screen for CLOCK-interacting proteins.
  • Figure 18 presents localization by in situ hybridization o ⁇ bmall, Clock and mperl transcripts.
  • Figure 19 presents yeast one-hybrid DNA-binding assays showing binding of CLOCK- BMAL1 heterodimer to the Drosophila per E-box.
  • Figure 20(a) presents the 5'-flanking region of the mperl gene, showing location of E-box sites.
  • Figure 20(b) presents the mouse perl 2.0kb 5' untranslated region sequence, the sequences of the 54mer (3 E-boxes), and the sequences of three l ⁇ mer's, the distal, middle and proximal 18mer sequences containing a single E-box.
  • Figure 21 presents transactivation from E-box sites by CLOCK-BMAL1 heterodimer.
  • Figure 22 presents diagramatic schemes showing the role of CLOCK-BMALl heterodimers in the circadian feedback loop.
  • Figure 23 presents the properties of CLOCK- ⁇ 19.
  • Figure 24 presents protein-protein interaction of various combinations of PER, TIM, dCLOCK, and dBMALl in yeast two-hybrid assays. Shown are triplicate yeast patches expressing the LEXA-PER bait in combination with the indicated full-length native proteins and/or the indicated full-length VP16 fusion protein. Blue precipitate indicates cumulative ⁇ - galactosidase activity resulting from activation of the LacZ reporter gene by protein-protein interaction. Each triplicate represents three independent transformants.
  • Figure 25 presents the cDNA sequence encoding human CLOCK protein.
  • Figure 26 presents the amino acid sequence of human CLOCK protein.
  • Figure 27 presents the cDNA sequence encoding human PER protein.
  • Figure 28 presents the amino acid sequence of human PER protein.
  • Figure 29 presents the cDNA sequence encoding Drosophila melanogaster PER A, PER B, and PER C proteins.
  • Figure 30 presents the amino acid sequence o ⁇ Drosophila melanogaster PER A protein.
  • Figure 31 presents the amino acid sequence o ⁇ Drosophila melanogaster PER B protein.
  • Figure 32 presents the amino acid sequence o ⁇ Drosophila melanogaster PER C protein.
  • Figure 33 presents the cDNA sequence encoding human TIM protein.
  • Figure 34 presents the amino acid sequence of human TIM protein.
  • Figure 35 presents the cDNA sequence encoding Drosophila melanogaster TIM protein.
  • Figure 36 presents the amino acid sequence o ⁇ Drosophila melanogaster TIM protein.
  • the invention is based upon the discovery that the protein BMALl is a binding partner with CLOCK protein for activation of expression of the period gene. It is believed that the two proteins heterodimerize and bind a small DNA sequence motif, an E-box, and thereby regulate period gene expression. Regulation o ⁇ period gene expression is believed to be critical in setting the circadian clock of an organism.
  • the relationship between the Drosophila per gene and the circadian clock is well established, a transactivator positively regulating per was heretofore unknown in any organism, including Drosophila and mammals.
  • the present invention represents the first discovery of a pairing partner (BMALl) for CLOCK protein and the first demonstration of interaction of the resulting heterodimer to its cognate recognition/binding sequence in a gene regulatory element.
  • the invention also represents the first discovery o ⁇ a Drosophila BMALl protein, termedBMALl .
  • the invention therefore provides a powerful means by which to perform the screening of compounds which may modulate the circadian rhythms of a patient via the claimed methods, using the combination of BMALl protein and CLOCK protein.
  • BMALl BMAL1 is defined herein above with regard to BMAL/CLOCK-dependent transcription activation of period gene, and also with respect to sequence shown in Figs. 1-14.
  • the present invention is the first discovery that BMALl protein interacts with CLOCK protein to regulate transcription of an E-box linked gene.
  • mice protein which interacts with the mouse CLOCK protein in an interaction-trap screen to regulate transcription of a marker gene in yeast cells. This protein is demonstrated to correspond to the sequence of that encoded by the "b" splicing variant of the human bmall cDNA transcript (Ikeda and Nomura, 1 97, Biochem. Biophys. Res. Cornrrmn., 233: 258-264).
  • CLOCK protein refers to a protein which pairs with BMALl to activate transcription of the period gene, and has an amino acid sequence identical or substantially similar to (i.e., at least 85% identity with) the sequence shown in Fig. 16.
  • the invention encompasses BMALl/CLOCK pairs in which the CLOCK protein or its corresponding DNA is a mouse homolog, or another mammalian homolog, e.g., a human CLOCK protein or corresponding gene, a Drosophila homolog.
  • a regulatory target DNA according to the invention is a DNA that is susceptible to transcriptional activation when linked to a gene.
  • This DNA minimally includes an E-box recognition sequence (cacgtg). Therefore, according to the invention, the regulatory target DNA may include a single E-box or multiple E-boxes, as described above.
  • the nucleotides between multiple E-boxes or between a regulatory DNA and the gene to which it is operatively linked may be of any random or non-random sequence, provided that the susceptibility to
  • BMALl/CLOCK transcriptional activation of the regulatory target DNA with respect to the linked gene is retained. How one determines that activity is described herein in detail.
  • E-box elements present in the 20-kb sequence 5' of the mouse mperl gene, as described below. Also described below is an 18-mer containing a single E-box, a 54-mer containing two E-boxes, and an approximate 2kb sequence containing three E-boxes, all of which are demonstrated to be susceptible to BMALl/CLOCK regulation.
  • Representative regulatory target sequences according to the invention are shown in Fig. 20(a) and 20(b).
  • the invention provides methods for assaying agents which are potentially useful as modulators of BMALl/CLOCK regulation of E-box linked reportere gene expression.
  • BMALl and CLOCK proteins minimally comprise BMALl and CLOCK proteins and a regulatory nucleic acid sequence comprising an E-box DNA element operatively linked to a reporter gene, as described above.
  • Assays according to the invention may be performed in vitro in a cell free gene expression system, in a gene expression system including a cell extract, or in a whole cell expression system, or in vivo.
  • an in vitro assay performed according to the invention will contain BMALl, CLOCK, and a DNA construct containing an E-box linked to a reporter gene.
  • the assay is peformed in a standard in vitro transcription translation system under conditions which permit expression of the reporter gene.
  • the TNT® T7 Quick Coupled Transcription Translation System (Cat. # LI 170; Promega) contains all reagents necessary for in vitro transcription/translation except the DNA of interest and the detection label.
  • TNT® Coupled Reticulocyte Lysate Systems include: TNT® T3 Coupled Reticulocyte Lysate System (Cat. # L4950; Promega); TNT® T7 Coupled
  • An assay involving a cell lysate or a whole cell may be performed in a cell lysate or whole cell preferably eukaryotic in nature (e.g., yeast, fungi, insect (e.g., Drosophila), mouse, or human).
  • a cell lysate or whole cell preferably eukaryotic in nature (e.g., yeast, fungi, insect (e.g., Drosophila), mouse, or human).
  • the reporter gene expression system may operate in vivo, i.e., in an intact, living multicellular organism, such as an insect or a mammal.
  • mice which are transgenic for the regulatory target sequence/reporter gene construct of the invention and that naturally contain bmall, clock and per genes. Methods of generating transgenic mice are well known in the art.
  • a candidate modulator of E-box-regulated gene expression is administered (e.g. by feeding or injection) to the test fly or mouse and expression of the reporter gene is then assayed.
  • detection of reporter gene expression may be performed by a method from the list that includes, but is not limited to, the molecular, biochemical (including enzymatic assay) and histological (including immunohistochemical and enzymatic staining, e.g. using a chromogenic, fluorescent, luminescent or radioactive substrate of the enzyme encoded by the reporter gene) methods described below.
  • the reporter gene is selected such that its expression may be monitored, in terms either of mRNA or protein production, and wherein protein production may be assessed either by the direct measurement of the amount of protein present or by measurement of protein activity (e.g., enzymatic activity).
  • Reporter genes of use in the invention include, but are not limited to, the bacterial genes LacZ, which encodes the enzyme ⁇ -galactosidase, and Cat, which encodes the enzyme chloramphenicol acetyltransferase (CAT), Luc, which encodes luciferase (luc), gfp, which encodes green fluorescent protein (gfp, which fluoresces in vivo when exposed to ultraviolet light), hrp, which encodes horseradish peroxidase (hrp).
  • LacZ which encodes the enzyme ⁇ -galactosidase
  • Cat which encodes the enzyme chloramphenicol acetyltransferase (CAT)
  • Luc which encodes luciferase (luc)
  • gfp which encodes green fluorescent protein (gfp, which fluoresces in vivo when exposed to ultraviolet light)
  • hrp which encodes horseradish peroxidase (hrp).
  • herpesvirus tk gene which encodes the enzyme thymidine kinase (tk), and the Drosophila genes Adh, which encodes alcohol dehydrogenase (Adh) and Rosy, which encodes xanthine dehydrogenase (Xdh).
  • a protein may be detected indirectly, through monitoring its activity, such the activity of an enzyme in the presence of its substrate and, if necessary, an indicator compound which generates a signal upon conversion of the substrate by the enzyme.
  • an indicator may be a chromogenic or fluorescent indicator which is released or otherwise activated in as a result of the catalytic activity of the reporter gene product; the indicator may be either complexed to or separate from the substrate molecule.
  • Biochemical assays for the activity of the enzymes listed above are well known in the art.
  • Detection of an mRNA transcript may be performed by molecular techniques such as are known in the art; these techniques include, but are not limited to, nucleic acid hybridization (such as Northern analysis), affinity .binding to an immobilized nucleic acid molecule having a complementary sequence (i.e., a sequence which will hybridize to the transcript through Watson- Crick base pairing under stringent hybridization conditions), reverse transcription using complementary oligonucleotide primers, and reverse-transcription polymerase chain reaction (RT-PCR).
  • nucleic acid hybridization such as Northern analysis
  • affinity .binding to an immobilized nucleic acid molecule having a complementary sequence i.e., a sequence which will hybridize to the transcript through Watson- Crick base pairing under stringent hybridization conditions
  • reverse transcription using complementary oligonucleotide primers
  • RT-PCR reverse-transcription polymerase chain reaction
  • RT-PCR reverse transcription/polymerase chain reaction
  • the RNA is converted to first strand cDNA, which is relatively stable and is a suitable template for a PCR reaction.
  • the cDNA template of interest is amplified using PCR. This is accomplished by repeated rounds of annealing sequence-specific primers to either strand of the template and synthesizing new strands of complementary DNA from them using a thermostable DNA polymerase.
  • RNA and 75 pmol random hexamer primer e.g., Pd(n)6, supplied by Pharmacia; Piscataway, NJ
  • Pd(n)6 supplied by Pharmacia; Piscataway, NJ
  • This mixture is incubated at 70 °C for 10 minutes and placed on ice for two minutes.
  • MMLV-RT Superscript® reverse transcriptase, BRL, Life Technologies, Gaithersburg, MD
  • 4 ⁇ l 5x reaction buffer BRL, Life Technologies, Gaithersburg, MD
  • 2 ⁇ l 0.1M DTT 1 ⁇ l 10 mM dNTP
  • 1 ⁇ l human placental RNase inhibitor 10 to 50 units per ⁇ l; Boehringer Mannheim, Indianapolis, IN.
  • MMLV-RT omitted (RT negative control).
  • the 19 ⁇ l reaction is incubated for 50 minutes at 42 °C in a programmable thermal cycler (such as is manufactured by MJ Research; Watertown. MA) and inactivated by heating to 90 °C for 5 minutes. After cooling to 37 °C, 1 ⁇ l RNase H (3 units per ⁇ l;BRL, Life Technologies, Gaithersburg, MD) is added, the reaction is incubated at 37°C for 20 minutes, then cooled to 4°C.
  • a programmable thermal cycler such as is manufactured by MJ Research; Watertown. MA
  • RNase H 3 units per ⁇ l;BRL, Life Technologies, Gaithersburg, MD
  • RNA integrity is confirmed by amplification of a transcript of a constitutively-expressed gene (e.g., actin, interleukin-2 or G os ); therefore, it is ensured that a negative result subsequently observed on a test sample can be ascribed to a lack of that specific mRNA and not to degradation of the pool of mRNA or failure of the reverse transcription reaction.
  • a constitutively-expressed gene e.g., actin, interleukin-2 or G os
  • PCR polymerase chain reaction
  • Oligonucleotide primers useful according to the invention are single-stranded DNA or RNA molecules that are hybridizable to a nucleic acid template to prime enzymatic synthesis of a second nucleic acid strand.
  • the primer is complementary to a portion of a target molecule present in a pool of nucleic acid molecules used in the preparation of sets of arrays of the invention. It is contemplated that such a molecule is prepared by synthetic methods, either chemical or enzymatic. Alternatively, such a molecule or a fragment thereof is naturally- occurring, and is isolated from its natural source or purchased from a commercial supplier.
  • Oligonucleotide primers are 15 to 100 nucleotides in length, ideally from 20 to 40 nucleotides, although oligonucleotides of different length are of use.
  • selective hybridization occurs when two nucleic acid sequences are substantially complementary (at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90% complementary). See Kanehisa, M., 1984, Nucleic Acids Res. 12: 203, incorporated herein by reference. Under stringent annealing conditions, longer hybridization probes (of use, for example, in Northern analysis) or synthesis primers hybridize more efficiently than do shorter ones, which are sufficient under more permissive conditions. Stringent hybridization conditions typically include salt concentrations of less than about IM, more usually less than about 500 mM and preferably less than about 200 mM.
  • Hybridization temperatures range from as low as 0°C to greater than 22°C, greater than about 30°C, and (most often) in excess of about 37°C. Longer fragments may require higher hybridization temperatures for specific hybridization. As several factors affect the stringency of hybridization, the combination of parameters is more important than the absolute measure of a single factor.
  • Detection of reporter gene transcripts may advantageously be performed in a single tube reaction for reverse transcription of RNA and specific amplification of transcripts of interest.
  • Commercial kits such as the AccessTM RT-PCR system (Promega; Madison, WI) conveniently assemble all materials (except primers) necessary to carry out the method in this way.
  • the single-tube RT-PCR assay according to this technique may be used to assay serum- or other samples.
  • in situ detection of mRNA transcripts may be performed using either
  • RNA molecules of the sample are reverse-transcribed in situ. Reverse transcription is carried out using reverse transcriptase, (e.g.
  • avian myoblastosis virus reverse transcriptase AMV-RT; Life Technologies/Gibco-BRL or Moloney Murine Leukemia Virus reverse transcriptase, M-MLV-RT, New England Biolabs, Beverly, MA) under the manufacturer's recommended reaction conditions.
  • reagents are pipetted out of the containment ring structure, which is rinsed thoroughly with TE buffer in preparation for amplification of the resulting cDNA molecules.
  • the amplification reaction is then performed, and the amplification product detected.
  • Other measures of restored function include testing of cells for normal mitotic activity, cell viability, cell growth, restored differentiation and normal cell cycle progression, insofar as these functions may exhibit circadian rhythmicity, which is typically assessed according to the invention by measuring the level of per gene expression relative to cells of an untreated control individual. Detection of an alteration of at least 10% in this indicator in the direction of a normal expression level (e.g., that found in an individual not suffering from a circadian clock disorder) is indicative of efficacious treatment according to the invention. Protein Detection
  • the invention also contemplates screening assays in which a reporter gene protein is detected.
  • Detection of a protein may be performed either directly, such as through purification (for example, affinity purification of the protein using a receptor or ligand which will bind the protein, a dimeric pairing partner of the protein, or an antibody directed against the protein), immunological detection (e.g., on a Western blot or immunohistochemically, by in situ binding of an antibody to proteins of a fixed or frozen cell or tissue preparation) or by measurement of energy absorption (for example, spectrophotometrically or fluorimetrically) of the reporter gene expression system before and after sufficient time for protein production to have occurred.
  • purification for example, affinity purification of the protein using a receptor or ligand which will bind the protein, a dimeric pairing partner of the protein, or an antibody directed against the protein
  • immunological detection e.g., on a Western blot or immunohistochemically, by in situ binding of an antibody to proteins of a fixed or frozen
  • Reporter protein detection may be accomplished, e.g., using an antibody specific for the reporter gene product (i.e., antigen).
  • Antibodies are prepared according to conventional methods. i. Preparation of Antibodies
  • Either recombinant proteins or those derived from natural sources can be used to generate antibodies using standard techniques, well known to those in the field.
  • the proteins are administered to challenge a mammal such as a monkey, goat, rabbit or mouse.
  • the resulting antibodies can be collected as polyclonal sera, or antibody-producing cells from the challenged animal can be immortalized (e.g. by fusion with an immortalizing fusion partner) to produce monoclonal antibodies.
  • Monoclonal antibodies e.g. by fusion with an immortalizing fusion partner
  • the antigen protein may be conjugated to a conventional carrier in order to increases its immunogenicity, and an antiserum to the peptide-carrier conjugate is raised. Coupling of a peptide to a carrier protein and immunizations may be performed as described (Dymecki et al, 1992, J. Biol. Chem., 267: 4815-4823).
  • the serum is titered against protein antigen by ELISA (below) or alternatively by dot or spot blotting (Boersma and Van Leeuwen, 1994, J. Neurosci. Methods, 51: 317).
  • the antiserum may be used in tissue sections prepared as described below. The serum is shown to react strongly with the appropriate peptides by ELISA, for example, following the procedures of Green et al., 1982, Cell, 28: 477-487.
  • Monoclonal antibodies Techniques for preparing monoclonal antibodies are well known, and monoclonal antibodies may be prepared using a candidate antigen whose level is to be measured or which is to be either inactivated or affinity-purified, preferably bound to a carrier, as described by Arnheiter et al., Nature, 294, 278-280 (1981).
  • Monoclonal antibodies are typically obtained from hybridoma tissue cultures or from ascites fluid obtained from animals into which the hybridoma tissue is introduced. Nevertheless, monoclonal antibodies may be described as being “raised to” or “induced by” a protein.
  • Monoclonal antibody-producing hybridomas can be screened for antibody binding to the target protein.
  • antibody we include constructions using the binding (variable) region of such an antibody, and other antibody modifications.
  • an antibody useful in the invention may comprise a whole antibody, an antibody fragment, a polyfunctional antibody aggregate, or in general a substance comprising one or more specific binding sites from an antibody.
  • the antibody fragment may be a fragment such as an Fv, Fab or F(ab'), fragment or a derivative thereof, such as a single chain Fv fragment.
  • the antibody or antibody fragment may be non-recombinant, recombinant or humanized.
  • the antibody may be of an immunoglobulin isotype, e.g., IgG, IgM, and so forth.
  • an aggregate, polymer, derivative and conjugate of an immunoglobulin or a fragment thereof can be used where appropriate.
  • immunological tests rely on the use of either monoclonal or polyclonal antibodies and include enzyme-linked immunoassays (ELISA), immunoblotting and immunoprecipitation (see Voller, 1978, Diagnostic Horizons, 2: 1-7, Microbiological Associates Quarterly Publication, Walkersville, MD; Voller et al, 1978, J. Clin. Pathol., 31 : 507-520; U.S. Reissue Pat. No. 31,006; UK Patent 2,019,408; Butler, 1981, Methods Enzymnl , 73: 482-523; Maggio, E.
  • ELISA enzyme-linked immunoassays
  • chromatographic methods such as SDS PAGE, isoelectric focusing, Western blotting, HPLC and capillary electrophoresis.
  • Tissue samples intended for use in in situ detection of either RNA or protein are fixed using conventional reagents; such samples may comprise whole or squashed cells, or may instead comprise sectioned tissue.
  • Fixatives adequate for such procedures include, but are not limited to, formalin, 4% paraformaldehyde in an isotonic buffer, formaldehyde (each of which confers a measure of RNAase resistance to the nucleic acid molecules of the sample) or a multi- component fixative, such as FAAG (85 % ethanol, 4% formaldehyde, 5% acetic acid, 1% EM grade glutaraldehyde).
  • RNAase-free i.e. treated with 0.1% diethylprocarbonate (DEPC) at room temperature overnight and subsequently autoclaved for 1.5 to 2 hours.
  • Tissue is fixed at 4°C, either on a sample roller or a rocking platform, for 12 to 48 hours in order to allow fixative to reach the center of the sample.
  • DEPC diethylprocarbonate
  • samples Prior to embedding, samples are purged of fixative and dehydrated; this is accomplished through a series of two- to ten-minute washes in increasingly high concentrations of ethanol, beginning at 60%- and ending with two washes in 95%- and another two in 100% ethanol, followed two ten-minute washes in xylene.
  • Samples are embedded in one of a variety of sectioning supports, e.g. paraffin, plastic polymers or a mixed paraffin polymer medium (e.g. Paraplast®Plus Tissue Embedding Medium, supplied by Oxford Lab ware).
  • tissue is transferred from the second xylene wash to paraffin or a paraffin/polymer resin in the liquid-phase at about 58 °C, then replace three to six times over a period of approximately three hours to dilute out residual xylene, followed by overnight incubation at 58 °C under a vacuum, in order to optimize infiltration of the embedding medium in to the tissue.
  • the tissue sample is positioned in a sectioning mold, the mold is surrounded by ice water and the medium is allowed to harden.
  • Sections of 6 ⁇ m thickness are taken and affixed to 'subbed' slides, which are those coated with a proteinaceous substrate material, usually bovine serum albumin (BSA), to promote adhesion.
  • BSA bovine serum albumin
  • Other methods of fixation and embedding are also applicable for use according to the methods of the invention; examples of these are found in Humason, G.L., 1979, Animal Tissue Techniques, 4th ed. (W.H. Freeman & Co., San Fransisco), as is frozen sectioning (Serrano et al., 1989, supra).
  • a “candidate modulator” as used herein, is any compound with a potential to modulate the expression of a gene which is either positively regulated (i.e., stimulated) or negatively regulated (i.e., inhibited) via regulatory target sequence containing an E-box DNA element according to the invention.
  • a candidate inhibitor is tested in a concentration range that depends upon the molecular weight of the molecule and the type of assay.
  • small molecules (as defined below) may be tested in a concentration range of lpg - 100 ⁇ g/ml, preferably at about 100 pg - 10 ng/ml; large molecules, e.g., peptides, may be tested in the range of 10 ng - 100 ⁇ g/ml, preferably 100 ng - 10 ⁇ g/ml. .
  • Inhibitors of gene expression may target the novel protein heterodimer described herein, BMALl/CLOCK, or it may target a protein or nucleic acid, such as the E-box DNA element, that interacts with the novel heterodimer so as to prevent or enhance the natural biological interaction that occurs in vivo and leads to transcription.
  • a modulator identified as described herein will possess two properties: 1) at some concentration it will modulate transcription of a gene which is operatively linked to an E-box DNA element; and 2) at the same concentration, it will not signficantly affect the function of a gene regulatory sequence which does not comprise an E- box DNA element.
  • Candidate modulators will include peptide and polypeptide inhibitors having an amino acid sequence based upon the components of the novel heterodimer described herein.
  • a mutant BMALl or CLOCK protein, or a fragment of a mutant or wild-type BMALl or CLOCK protein may act as a competitive inhibitor with respect to heterodimerization or binding of the completed heterodimer to other proteins involved in transcription, e.g., RNA polymerase II, TFIIB, or may inhibit binding of the transcription complex to the DNA template.
  • inhibitor protein CLOCK- ⁇ 19 is determined to be competent for pairing with BMALl and binding to the regulatory target DNA comprising an E-box, but to inhibit transcription of the gene that is operatively linked to the regulatory target DNA comprising an E-box.
  • a nucleic acid molecule such as an oligonucleotide, comprising an E-box element would, when administered in molar excess of the endogenous per-linked E-box DNA element, titrate the BMALl/CLOCK heterodimer away from its natural target, such that the complex would be sequestered in an unproductive association (i.e., one which does not result in the transcription of per).
  • a mutant or fragment may be engineered such that it instead enhances the activity of the heterodimer, e.g., through enhanced binding affinity either to its pairing partner or to the E-box sequence or through constitutive, rather than regulated, activity.
  • Candidate modulator compounds from large libraries of synthetic or natural compounds can be screened.
  • Synthetic compound libraries are commercially available from a number of companies including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT).
  • a rare chemical library is available from Aldrich (Milwaukee, WI).
  • Combinatorial libraries are available and can be prepared.
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g., Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily produceable by methods well known in the art.
  • natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.
  • Useful compounds may be found within numerous chemical classes, though typically they are organic compounds, and preferably small organic compounds. Small organic compounds have a molecular weight of more than 50 yet less than about 2,500 daltons, preferably less than about 750, more preferably less than about 350 daltons. Exemplary classes include heterocycles, peptides, saccharides, steroids, and the like. The compounds may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents.
  • peptide agents may be modified in a variety of ways to enhance their stability, such as using an unnatural amino acid, such as a D-amino acid, particularly D-alanine, by functionalizing the amino or carboxylic terminus, e.g. for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidification, or the like.
  • an unnatural amino acid such as a D-amino acid, particularly D-alanine
  • a candidate modulator of E-box regulated gene function is determined to be effective if its use results in a change of about 10%) or greater of E-box-linked reporter gene expression in the presence of BMALl and CLOCK.
  • the level of modulation by a candidate modulator may be quantified using any acceptable limits, for example, via the following formula, which describes detections performed with a radioactively labeled probe (e.g., a radiolabeled nucleic acid probe in a Northern hybridization, or a radiolabeled antibody in an immunobinding experiment).
  • a radioactively labeled probe e.g., a radiolabeled nucleic acid probe in a Northern hybridization, or a radiolabeled antibody in an immunobinding experiment.
  • CPM Control is the average of the cpm in antibody/ligand complexes or on Northern blots resulting from assays that lack the candidate modulator (in other words, untreated controls)
  • CPM Sample is the cpm in antibody/ligand complexes or on Northern blots resulting from assays containing the candidate modulator.
  • the assay comprises use of a labeling system or system of measuring enzymatic activity in which there is a linear relationship between the amount of label detected and the amount of protein or nucleic acid being represented per unit of label or the amount of protein or nucleic acid represented by a unit of enzymatic activity.
  • a protein or other bioactive agent may be identified according to the invention which modulates per gene expression resulting from the transcriptional regulatory activity of the BMALl/CLOCK heterodimer in a recipient mammal.
  • the amount of a such a protein or other therapeutic agent to be administered to a patient having or suspected of having a circadian disorder is considered, the lowest dose that provides the desired degree of modulation of per gene expression in the target cells should be used; lower doses may be advantageous in order to minimize the likelihood of possible adverse effects.
  • per gene expression includes not only the presence of functional PER, but may also include the presence of the products of genes regulated by PER, regardless of the means by which they have arisen in the cell, as well as normal circadian cycling of cellular functions (e.g., hormone production). It will be apparent to those of skill in the art that the therapeutically-effective amount of a composition administered in the invention will depend, inter alia, upon the efficiency of cellular uptake of a composition, the administration schedule, the unit dose administered, whether the compositions are administered in combination with other therapeutic agents, the health of the recipient, and the therapeutic activity of the particular protein or other pharmaceutical substance.
  • the precise amount of a protein or other pharmaceutical agent identified according to the invention and required to be administered depends on the judgment of the practitioner and may be peculiar to each subject, within a limited range of values.
  • the amount of modulator administered will be determined according to the degree of severity of the circadian clock pathology (e.g., disruption or alteration) and condition of the patient, and will typically be in the range of about lug - 100 mg/kg body weight. Where the modulator is a peptide or polypeptide, it is typically administered in the range of about 100 - 500 ug/ml per dose.
  • a single dose of per modulator identified according to the invention, or multiple doses of such a modulator, daily, weekly, or intermittently, is contemplated according to the invention.
  • Therapeutic agents which are identified according to the invention and are to be administered to a patient also may be formulated in a physiologically acceptable diluent such as water, phosphate buffered saline, or saline, and further may include an adjuvant.
  • Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are materials well known in the art.
  • Administration of a therapeutic agent as described herein may be either localized or systemic.
  • changes in the circadian clock of a patient treated according to the invention may be assessed either by direct clinical observation or by the measurement of a molecular marker (e.g., per or other gene expression) or biochemical markers (e.g., PER protein or a hormone or other substance produced in a human on a 24-hour cycle).
  • a molecular marker e.g., per or other gene expression
  • biochemical markers e.g., PER protein or a hormone or other substance produced in a human on a 24-hour cycle.
  • the methods described below may, additionally, be used to detect reporter gene function in an assay or diagnostic method according to the invention.
  • Disruptions or abnormalities in the circadian clock often require no formal clinical diagnosis; for example, a traveller who has passed rapidly through multiple time-zones is well aware that he or she suffers from jet-lag, as evidenced by the desire to sleep or remain awake at times of the day or night which are inappropriate for those activities.
  • the diagnosis of more complex disorders which may be associated, either as cause or effect, with changes in the circadian cycle of the patient require evaluation by a physician. Such evaluation may take the form of patient interview, or of biochemical- , behavioral- or other testing of the patient.
  • Biochemical testing may comprise the detection/measurement of indicators having levels which are predicted to fluctuate regularly on a circadian (e.g., approximately 24-hour) cycle, such as various hormones whose levels in the bloodstream or other biological fluids peak at specific times of the day. Such testing may be particularly useful in the diagnosis of infertile patients in order to determine whether a link exists between their compromised fertility and, perhaps, asynchronous production of hormones or other molecules necessary to enable conception or the carrying of a pregnancy to term due to abnormalities in the circadian clock.
  • a circadian e.g., approximately 24-hour
  • the invention provides an assay technique by which the ability of a biological sample from a patient to effect transcription of an E-box-linked gene, which transcription occurs in a normal level in a patient with a functional BMALl protein in combination with its human binding partner, is assayed; the claimed diagnostic method requires no prior knowledge of a protein downstream of (i.e., regulated by) the circadian clock mechanism, as it tests the integrity of the mechanism itself. Where a circadian clock abnormality is observed in combination with infertility, therapeutic agents identified according to the claimed methods, which agents will modulate E-box-linked transcription, may be administered in order to restore the proper temporal regulation of these biomolecules.
  • Behavioral testing may involve observation of the patient's sleep/wake cycle or changes in mood in response to changes in duration of light/dark periods. For example, an individual having a sleep disorder may experience unusually long or short waking periods, thereby extending their effective "day” to a period significantly longer than 24 hours or abbreviating it such that they experience more apparent "days” per week than does a normal individual.
  • An individual suffering from Seasonal Affective Disorder (SAD) becomes depressed at high latitutes or during the winter months, where and when the daylight hours are short; observation of changes in the mood of a depressed individual under different periods of daylight (whether real or artificial, the latter as produced by a sun lamp) may be diagnostic of a link between a disruption in the circadian clock and the observed depression.
  • SAD Seasonal Affective Disorder
  • therapeutic agents uncovered by the claimed assay methods may be of use in the treatment of sleep disorders or depression.
  • testing of a patient for a circadian clock disorder may be as simple as taking their temperature throughout the day. Most individuals experience a temperature "low” approximately one hour before waking. In cases of circadian clock disorders, this temperature change may become disengaged from the sleep/wake cycle and occur randomly. Direct correlation of temperature and sleeping or waking may be performed in a controlled environment, such as a hospital or sleep laboratory.
  • CIPs CLOCK-interacting proteins
  • LEXA-CLOCK hybrid as bait, essentially as previously described (Gekakis et al, 1995, Science, 270: 811), except that LEXA-CLOCK yeast transformed with the cDNA library were plated onto THULL/X-gal plates (Gekakis et al., 1995, Science, 270: 811) supplemented with 30mM or 50mM 3-amino triazole, conditions detemined to be optimal for detection of HIS prototrophy resulting from the interaction of LEXA-CLOCK with VP 16-ARNT.
  • LEXA-CLOCK(1-580) which is missing most of the C-terminal glutamine-rich region predicted to function as a transcriptional activation domain (King et al.,.1997, Cell 89: 641) but includes the region deleted from the CLOCK- ⁇ 19 mutant protein, was used.
  • the cDNA library was generated as follows: 32 male Syrian hamsters (Charles River Laboratories) were maintained on a 14: 10 light:dark cycle for __3 weeks, transferred to constant dim light ( ⁇ 1 Lux) at the time of lights off, and killed by decapitation 24-42 hours later, 8 at each of four circadian times (CT 1, 7, 13, 19).
  • control two-hybrid assays were carried out in order to assess the activity of the CLOCK bait and to determine whether CLOCK could act as its own homodimeric partner.
  • control two-hybrid assays show interaction of LEXA-CLOCK with VP16- ARNT but not with VP 16-CLOCK. Background is defined by the signal from LEXA-CLOCK with VP16.
  • Yeast expressing the LEXA-CLOCK(1-580) bait were transformed with VP16 (negative control), VP16-CLOCK(l-389), or VP16-ARNT(74-474) expression plasmids.
  • VP16-CLOCK(l-389) shows a robust interaction with LEXA-ARNT(70-474) (not shown).
  • CLOCK showed clear evidence of interaction with ARNT, a bHLH-PAS protein known to heterodimerize widely (Hoganesch et al., 1997, J. Biol. Chem., 272: 8581), no evidence of CLOCK homodimerization was detected, implying that CLOCK acts in vivo with a heterodimeric partner.
  • Yeast expressing the LEXA-CLOCK( 1-580) bait or the LEXA-p65 (synaptotagmin) control bait were transformed with the indicated VP16 expression plasmids.
  • VP16 and VP16- ARNT plasmids are the negative and positive controls, respectively, for LEXA-CLOCK, as in Fig. 17A.
  • 154 formed colonies that were positive for both HIS3 and LacZ reporter genes.
  • Transcripts encoded by arnt, arnt2, and bmall were examined for co-expression with Clock and mperl transcripts in the mouse SCN by in situ hybridization.
  • coronal brain sections were prepared from mice kept in constant dim light ( ⁇ 1 Lux; killed at CT 7 or CT 19) and in situ hybridizations were performed as described (Morris et al., 1998, Science, 279: 1544). Eyes were removed from perfused mice, the anterior segments were removed by dissection and discarded, and the resulting eye cups were post- fixed and stored as described for brains. Parasaggital sections (12- ⁇ m) were cut on a cryostat, and in situ hybridizations were performed as for brain sections.
  • Riboprobes were synthesized from PCR products that incorporated T3 (for sense strand) and T7 (for antisense strand) polymerase binding sites.
  • the hamster bmall riboprobe cooesponded to full-length BMALlb; the mouse perl riboprobe corresponded to codons 738 to 835; the mouse Clock riboprobe corresponded to codons 1 to 389.
  • Fig. 18 A sets of three neighboring coronal brain sections from a single mouse, each set taken from a different rostrocaudal level, are presented. Within each set, the three sections were hybridized, respectively, to mperl, Clock, or bmall antisense riboprobes, as indicated. Rostrocaudal level of each set within the SCN is indicated at left.
  • EXAMPLE 2 Assay in cells of BMALl /CLOCK-directed regulatory target sequence/reporter gene expression a.
  • the RMAL1 /CLOCK heterodimer hinds an E-hnx DNA element
  • CLOCK-ARNT, CLOCK-ARNT2, and BMAL1/CLOCK1 heterodimers were tested for binding to the E-box element within the Drosophila per clock control region, known to be important for regulation of per gene expression.
  • Yeast one-hybrid assays were performed; by this technique, the binding of test proteins to a given DNA fragment is signalled by activation of the LacZ gene, resulting in an increase in B- galactosidase activity in the yeast.
  • the indicated DNA fragments were ligated into the pBgl-lacZ reporter plasmid (Li and Herskowitz, 1993, Science, 262: 1870), and one-hybrid yeast reporter strains were constructed by targeted integration of recombinant pBgl- lacZ reporter plasmids into the ura3 locus of YPH 499 (Sikorsky and Hieter, 1989, Genetics, 122: 19).
  • Negative control strains were constructed identically with non-recombinant pBgl- lacZ.
  • One-hybrid reporter strains were transformed with the indicated p424-Met25 (Mumberg et al., 1994, Nuc. Acids Res., 22: 5767) and pVP16 expression plasmids, and transformants were patched onto X-gal plates for detection of ⁇ -galactosidase activity.
  • E-box mutant 1 5'-attcgc-3'
  • E-box mutant 2 5'-gtaact-3'
  • yeast one-hybrid DNA-binding assays showing binding of BMALl/CLOCK heterodimer to the E-box site from the Drosophila per gene clock control region are shown in Fig. 19A and Fig. 19B, in which triplicate yeast patches expressing the indicated proteins are presented.
  • DNA-binding is indicated by ⁇ -galactosidase activity, resulting from activation of the LacZ reporter gene. Blue precipitate indicates cumulative ⁇ -galactosidase activity, and each triplicate represents three independent transformants.
  • the 69-bp clock control region was used in the experiment presented in Fig. 19 A.
  • Reporter yeast were transformed with a full-length Clock cDNA expression plasmid (+CLOCK) or with a non-recombinant expression plasmid (- CLOCK) in combination with the indicated VP 16 plasmid.
  • Full-length CLOCK and VP 16- BMAL1 were expressed in four reporter strains, as shown in Fig. 19B (+, reporter gene includes 21-bp sequence.
  • - reporter gene lacking 21-bp sequence; wt, 21-bp sequence, including E-box, from clock control region; mutl and mut2, same 21-bp sequence, but with E-box sequence scrambled two different ways).
  • BMALl and CLOCK together exhibited robust DNA binding to fragments containing the
  • the clone included 3.2 kb of 5'-flanking sequence, from which complete double-stranded sequence was obtained.
  • This transcription start site has not been confirmed experimentally, so it should be regarded as provisional.
  • Complete double- stranded sequence of the 3.2-kb predicted 5' flanking sequence revealed three E-box consensus sites, all identical to that from the Drosophila per clock control region. All three were located within 1.2 kb of the putative transcription start site. These sites are shown in Figure 20(a), in which the numbered axis represents distance in bp from the putative transcription start site, marked as +1.
  • Filled boxes represent the locations of the three E-boxes; the sequence of each E- box with 6 bp of flanking sequence on each side is shown at the top. The sequences of the 54mer is shown in Fig. 20(b), as is the sequence of each 18mer. c.
  • the BMAL1 /CLOCK heterodimer activates transcription from mperl E-box DNA elements Regulation of per gene expression by BMALl/CLOCK heterodimers requires that the heterodimers function to activate rather than suppress transcription, suppressor activity having been documented in the bHLH-PAS family (Moffett et al., 1997, Mol. Cell Biol., 17: 4933).
  • a 2.0 kb fragment of the mperl 5'-flanking region was cloned that includes all three E-boxes as follows:
  • the 2.0-kb mperl upstream fragment (-2122 bp to -129 bp with respect to the putative transcription start site) was generated by PCR amplification using pfu and taq polymerases together on a cloned lambda phage template (primers: 5'-catccgctcgagctctttggtacctggccagcaacc-3' ; 5'-catccgctcgagactgaggtcagggctgtgtcacac-3'; both primers include added Xhol sites at their 5' ends).
  • the 54-mer derived from the mouse perl gene 5'-flanking region consisted of the three 18-bp sequences shown in Fig. 20; they were linked together in the order shown from 5' to 3'.
  • the E-box mutant 54-mer was the same except that it had each E-box site independently scrambled by means of a random number table.
  • luciferase reporter gene assays were used in mammalian cells. In brief, these assays were performed as follows: Mouse NIH-3T3 cells were transfected using Lipofectamine-Plus (GibcoBRL; Gaithersburg, MD).
  • Cells were grown in DMEM with 10% fetal bovine serum (GibcoBRL) in 6-well plates, and cells in each well were transfected with 1 ⁇ g (total) of the pcDNA3 expression plasmid(s) (Invitrogen) with the indicated inserts, 10 ng of the pGL3 firefly luciferase reporter plasmid containing the minimal SV40 promoter (Promega) and the indicated inserts, and 0.5 ng of pRL-CMV ⁇ Renilla) luciferase internal control plasmid (Promega).
  • luciferase activity was corrected for transfection efficiency by dividing the measured firefly luciferase activity (from the reporter construct) by the measured Renilla luciferase activity (from the constitutively-driven construct).
  • a full-length Clock cDNA was constructed by fusion at a Pstl site of clones YZ50 and Dl (7).
  • Clock-A19 cDNA was generated by subcloning of full-length Clock cDNA into pGEX-4-T3 (Pharmacia Biotech) for oligonucleotide-directed deletion of exon 19 with the U.S.E. Mutagenesis kit (Pharmacia Biotech).
  • Figure 21 presents the results of these experiments, in which transcriptional activation in mammalian cells of the luciferase reporter gene from sequences derived from the 5'flanking region of the mperl gene (A-C) or from the Drosophila per gene (D) is assayed.
  • FIG. 21 A 2.0- kb fragment including all three E-boxes.
  • Fig. 2 IB "54-bp", a fragment consisting of the three E-boxes and their immediate flanking sequences linked together (see Fig. 20); "54-bp mut", a 54-bp fragment in which all three E-boxes were scrambled, as described above.
  • Fig. 21 C Each individual E-box with immediate flanking sequences.
  • Fig. 21D The 69-bp Drosophila per clock control region.
  • Figures 21 A-21C + or - denote the presence or absence, respectively, of indicated luciferase reporter plasmid in cell transfection.
  • Fig. 21 A-21C + or - denote the presence or absence, respectively, of indicated luciferase reporter plasmid in cell transfection.
  • Fig. 21 C The 69-bp Drosophila per clock control region.
  • BMALl and CLOCK together produced a substantial increase in transcriptional activity compared to the control (8.7-fold; P ⁇ 0.005) (Fig. 21A), indicating that recognition sites for the BMALl/CLOCK heterodimer are contained within the mperl 5'-flanking region. This result demonstrates that CLOCK acts as a transcriptional activator.
  • BMALl and CLOCK together produced a small increase in transcriptional activity from the distal E-box (2.1 -fold), a moderate increase from the middle E-box (3.6-fold), and a substantial increase from the proximal E-box (8.5-fold) (Fig. 21C).
  • all three E-boxes are potential sites of regulation o ⁇ mperl by BMALl/CLOCK heterodimers. Because the differences in transcriptional activity among them is significant, it appears that the sequences flanking each E-box influence the binding affinity or efficacy of transactivation, or both, of BMALl/CLOCK heterodimers. Together with the co- expression o ⁇ Clock, bmall, and mperl transcripts in the SCN and retina, these results strongly suggest that BMALl/CLOCK heterodimers drive mperl gene expression.
  • BMALl/CLOCK heterodimers directly activate transcription o ⁇ mperl, providing the positive drive into the per transcriptional feedback loop. It is upon this conclusion that the invention is based: Namely, the recognition of the BMALl/CLOCK heterodimer in the direct, positive control of E-box linked genes, the products of which genes regulate the circadian clock of an organism, and the utilization of this knowledge to assay agents which may modulate the expression of circadian clock regulators at the level of BMALl/CLOCK transactivation.
  • Figure 22 offers a schematic presentation of the role of BMALl/CLOCK heterodimers in the circadian clock feedback loop, showing alternating regimes of activation and suppression of per gene transcription.
  • Activation BMALl/CLOCK heterodimer driving per transcription from E-box near transcriptional start site (Fig. 22A).
  • Suppression PER protein, by unknown mechanism, blocking or overriding action of BMALl/CLOCK heterodimer to suppress per transcription (Fig. 22B).
  • Figure 22A and 22B "+" denotes transcriptional activation; "-" denotes transcriptional suppression.
  • a question mark indicates uncertainty regarding the direct or indirect nature of PER action, and arrows from PER denote potential pathways having unknown numbers of steps.
  • a moderate decrease in positive drive would be predicted to slow the accumulation of per transcripts each cycle, delaying negative feedback by PER protein and leading to long-period oscillations, as seen in the stable long-period behavioral rhythms of mice heterozygous for the Clock mutation (Vitaterna et al., 1994, Science, 264: 719).
  • an increase in positive drive would be predicted to quicken the accumulation of per transcripts each cycle, resulting in earlier negative feedback by PER protein and leading to short- period oscillations, as seen in the stable short-period behavioral rhythms of mice overexpressing wildtype Clock (Antoch et al., 1997, Cell, 89: 655).
  • Further strong support comes from the observation that in homozygous Clock mutant mice the peak levels o ⁇ mperl transcript in the SCN are significantly reduced compared to those of wildtype mice (not shown).
  • BMALl as a probable component of the circadian clock mechanism, defined a biochemical function for CLOCK, and implicated a mammalian per homolog as a likely target gene regulated by BMALl/CLOCK heterodimers.
  • This constellation of findings places CLOCK in a specific role within the circadian oscillator, and it mechanistically ties together CLOCK, a genetically, demonstrated component of the circadian clock, and a mammalian per homolog, not yet so demonstrated but likely to be a component.
  • New insights into how the circadian transcriptional feedback loop is generated and regulated will provide the basis for understanding how biological timekeeping evolved, how it operates, and how behavioral and physiological programs are controlled.
  • EX AMPLE 3 Identification of an inhibitor of BMALl/CLOCK transcriptional activation from E-hox DNA elements
  • CLOCK- ⁇ 19 product of this allele is a dominant-negative regulator of transcription acting on mperl E-box sites.
  • CLOCK- ⁇ 19 was further characterized with regard to heterodimerization and DNA binding. In two-hybrid assays, CLOCK- ⁇ 19 showed interactions with ARNT, ARNT2 and BMAL 1 that were similar in signal strength and identical in rank order to those shown by wildtype CLOCK, although the background with the CLOCK- ⁇ 19 bait was somewhat higher (Fig. 23 A; compare with Fig. 17B).
  • CLOCK- ⁇ 19 heterodimerizes, apparently normally, with BMALl and other bHLH-PAS proteins.
  • CLOCK- ⁇ 19 is not, however, equivalent to wildtype in regard to all protein-protein interactions, since several CIPs we isolated in the two-hybrid screen apparently interact only with wildtype CLOCK (for example, CIP8 in Fig. 23 A; compare with Fig. 17B).
  • Fig. 23 A Shown in Fig. 23 A are triplicate yeast patches expressing the LEXA-CLOCK- ⁇ 19(1-580) bait and the indicated VP16 protein. Blue precipitate indicates cumulative ⁇ -galactosidase activity. Each triplicate represents three independent transformants.
  • Fig. 23B a one-hybrid assay showing binding of CLOCK- ⁇ 19-BMAL1 heterodimer to E-box site is presented. Shown are triplicate yeast patches expressing the indicated pairs of proteins. DNA-binding is indicated by ⁇ -galactosidase activity.
  • the reporter gene in the reporter strain was linked to the 69-bp clock control region.
  • the three proteins indicated at left of the figure were LEXA hybrids used above in the one-hybrid system experiments, but the LEXA domain is not relevant to the results in the one-hybrid reporter strain, and it does not effect the function of CLOCK in this assay.
  • CLOCK or CLOCK- ⁇ 19 was paired with BMALl, robust DNA-binding activity was detected, but not when either was paired with negative controls (Fig. 23B).
  • a candidate modulator period gene expression is assayed in a gene expression system comprising BMALl, CLOCK and an E-box DNA element which is operatively linked to a reporter gene.
  • the level of expression of the reporter gene in the presence of the candidate modulator is compared to that observed when the modulator is absent.
  • assay of the candidate modulator is performed in a cell-free reaction (e.g., in a cell lysate) or in cells or even a whole organism.
  • the candidate modulator or a nucleic acid encoding a candidate modulator, is added to a cell extract, administered to cells by inclusion in growth medium or by transfection by methods known in the art, or administered to a whole organism by feeding or by a drug administration method (e.g., injection, inhalation, topical application) known in the art.
  • Transfection-based delivery of the reporter gene construct and/or the candidate modulator to yeast and mammalian cells is performed as described in Example 2, above.
  • Detection of reporter gene function in cells is performed as described in Examples 2 and 3, above, in which the expression of luciferase is detected in mammalian cells and that of ⁇ -galactosidase is detected in yeast cells.
  • reporter gene construct Methods of delivering the reporter gene construct to a whole organism, e.g., by the creation of a transgenic organism (e.g., a transgenic mouse), are well known in the art. Detection of reporter gene function in a multicellular organism is performed either by biochemical methods described above (see “Detection methods") or in Example 1, in which in situ hybridization of a labeled .oligonucleotide probe to mouse brain tissue is performed.
  • Assay of a candidate modulator in a cell-free system is performed as follows: BMAL 1 and CLOCK (or a BMAL 1 -CLOCK heterodimer) are added to a rabbit reticulocyte lysate or to a rabbit reticulocyte lysate-based coupled transcription/translation system (e.g., TNT® from Promega; see above) to which the candidate modulator has been added along with a reporter gene expression construct comprising a regulatory target sequence operatively linked to LacZ, and incubated under conditions and for a time sufficient to permit expression o ⁇ LacZ.
  • a reporter gene expression construct comprising a regulatory target sequence operatively linked to LacZ
  • ly sates are clarified by centrifugation and buffered as previously described; chlorophenol-red- ⁇ -D-galactopyranoside (CPRG; Boehringer Mannheim Biochemicals) is added to the lysates and spectrophotometric readings are taken at intervals of from 2 to 15 minutes for a period of an hour. A curve of absorption over time is plotted, and the rate of change ( ⁇ Abs./hour) is calculated and converted to units of ⁇ -galactosidase activity.
  • CPRG chlorophenol-red- ⁇ -D-galactopyranoside
  • a difference in the number of units of ⁇ -galactosidase activity in the test (+ modulator) reaction of at least about 10% above or below that observed in the control reaction is indicative that the candidate modulator is a modulator o ⁇ period gene expression.
  • yeast two-hybrid assays under conditions that would allow us to select independently for four different expression plasmids (maintained by LEU2, TRPl, ADE2, and HIS3 markers, respectively) in addition to the LacZ reporter gene (maintained by URA3 marker).
  • all four types of plasmids were present in the yeast; for clarity's sake, non-recombinant plasmids that were included when less than four exogenous proteins were expressed are not marked in the figure.
  • the Drosophila proteins were full-length native proteins with N-terminal LEXA- or VP16- fusion domains, as indicated. No interaction signal was detectable in yeast expressing LEXA-PER alone or LEXA-
  • PER in combination with VP16-dBMALl, native dCLOCK, or VP16-dCLOCK by themselves (Fig. 24, top row of panels). This result indicates that the PER monomer binds weakly or not at all to dBMALl or dCLOCK monomers.
  • a yeast two hybrid system similar to the one described in Example 5 was used to examine protein interactions between the dCLOCK-dBMAL heterodimer and PER.
  • a reproducible interaction signal was detected in yeast expressing LexA-PER, dCLOCK, and VP16-dBMAL together (Fig. 24, bottom row, third from left). This result indicates that PER can bind to the dCLOCK-dBMAL heterodimer, apparently much better than it can bind to either monomer alone (compare with top row, second, third, and fourth from left).
  • a yeast two hybrid system similar to the one described in Example 5 was used to examine protein interactions between the PER-TIM heterodimer and dCLOCK.
  • a robust interaction signal was detected in yeast expressing LEXA-PER, TIM, and dCLOCK (Fig. 24, bottom row, fourth from left). This result indicates that the PER-TIM heterodimer binds to the dCLOCK monomer, apparently better than PER can alone (compare with top row, third from left).
  • the configuration of this experiment indicates that dCLOCK bound to TIM while TIM was bound to PER; exclusive binding of dCLOCK to the free TIM monomer would not give a signal here.
  • No VP16 fusion protein was present in this case; dCLOCK is capable of carrying out transactivation activity in yeast.
  • the third panel from left, top row, serves as the control).
  • EXAMPLE S Evidence for the assembly of PER, TIM, CLOCK, and BMAL into a multiprotein complex A yeast two hybrid system similar to the one described in Example 5 was used to examine protein interactions between the dCLOCK-dBMAL heterodimer and the PER-TIM heterodimer. A very strong interaction signal, by far the strongest seen in this series of experiments, was reproducibly observed in yeast expressing LEXA-PER, TIM, dCLOCK, and VP16-BMAL together (Fig. 24, bottom row, rightmost panel). Together with the other results, the configuration of this experiment indicates that all four proteins assembled into a multiprotein complex.
  • EXAMPLE 9 Given the remarkable conservation of mechanism of Drosophila and mammalian circadian clocks (Darlington, eLaL; Gekakis et al., (1998) Science 280: 1564), the results obtained in the experiments of Examples 5-8 strongly suggested that direct contact between the PER-TIM heterodimer and the CLOCK-BMAL1 heterodimer in mammals is the mechanism of Per gene feedback in mammals, including humans.
  • Yeast two-hybrid assays are carried out under conditions that allow us to select independently for four different expression plasmids (maintained by LEU2, TRPl, ADE2, and H7S5 markers, respectively) in addition to the LacZ reporter gene (maintained by URA3 marker).
  • LEU2, TRPl, ADE2, and H7S5 markers, respectively in addition to the LacZ reporter gene (maintained by URA3 marker).
  • all four types of plasmids are present in the yeast.
  • Human proteins are full- length native proteins with N-terminal LEXA- or VP16- fusion domains.
  • a yeast two hybrid system similar to the one described in Example 9 is used to examine protein interactions between the hCLOCK-hBMAL heterodimer and hPER. It is anticipated that a reproducible interaction signal will be detected in yeast expressing LexA-hPER, hCLOCK, and VP16-hBMAL together, indicating that hPER may bind to the hCLOCK-hBMAL heterodimer better than it can bind to either monomer alone.
  • EXAMPLE 11 A yeast two hybrid system similar to the one described in Example 9 is used to examine protein interactions between the hPER-hTIM heterodimer and hCLOCK. A robust interaction signal is expected to be detectable in yeast expressing LEXA-hPER, hTIM, and hCLOCK, indicating that the hPER-hTIM heterodimer may bind to the hCLOCK monomer better than hPER can alone. Even if the binding site for the hCLOCK monomer is largely on hTIM, this experiment would indicate that hCLOCK will bind to hTIM while hTIM is bound to hPER.
  • EXAMPLE 12 A yeast two hybrid system similar to the one described in Example 9 is used to examine protein interactions between the hCLOCK-hBMAL heterodimer and the hPER-hTIM heterodimer. A very strong interaction signal is expected to be observed in yeast expressing LEXA-hPER, hTIM, hCLOCK, and VP 16-hBM AL together. Together with the other results, this experiment would indicate that all four proteins may assemble into a multiprotein complex. Furthermore, this experiment would indicate that the hPER-hTDVI heterodimer directly interacts with the hCLOCK-hBMALl heterodimer. It is very likely that hCLOCK-dependentper gene transcription by hPER and hTIM is causally related to this interaction.
  • the invention based upon the discovery of the transcriptional mechanism regulating genes responsible for the establishment and/or maintenance of the circadian clock of an organism (including a mammal), is useful for the assay of novel drugs aimed at the restoration of a normal circadian cycle in a human or other mammal in whom a disruption or other abnormality of the circadian clock exists and at the cure of a disease which causes- or is caused by such a disruption or abnormality, which drugs are modulators of BMALl -CLOCK-mediated transcription of E- box-linked genes.
  • the invention is further useful in that the therapeutic agents so assayed and identified according to the invention enable treatment of a disease which causes- or is caused by such a disruption or abnormality, which diseases include, but are not limited to, jet-lag, sleep disorders, depression and infertility.
  • the invention is useful, as it provides a compound comprising BMALl and CLOCK with which to stimulate the transcription of an E-box-linked gene which regulates the circadian clock.
  • the invention is useful, as it provides an inhibitor (CLOCK- ⁇ 19) of BMALl -CLOCK-mediated transcription of an E-box-linked gene which regulates the circadian clock.
  • the invention is further useful in that it provides a means of diagnosing a disease which causes- or is caused by such a disruption or abnormality, which diseases include, but are not limited to, jet-lag, sleep disorders, depression and infertility.

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Abstract

L'invention se rapporte à des compositions comportant une protéine BMAL1 isolée et une protéine CLOCK isolée ; à des compositions comportant la protéine BMAL1 isolée, la protéine CLOCK isolée et comportant également la protéine PER isolée et (éventuellement la protéine TIM isolée). L'invention se rapporte aussi à des analyses dépendant de BMAL1/CLOCK en vue de l'identification de modulateurs de l'expression du gène period. Elle se rapporte en outre à des méthodes permettant de diagnostiquer des troubles liés à une anomalie du rythme circadien.
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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0890578A2 (fr) * 1997-07-08 1999-01-13 Smithkline Beecham Plc Polynucleotides codant le géne d'horloge humaine et leur utilisation
WO2000075669A1 (fr) * 1999-06-08 2000-12-14 Aventis Pharmaceuticals Inc. Methodes de criblage visant la modification des proteines du rythme circadien
WO2001036618A2 (fr) * 1999-11-18 2001-05-25 Mitsubishi Pharma Corporation Promoteur period 1 introduit dans des mammiferes transgeniques leur conferant une expression rythmique
WO2001038581A2 (fr) * 1999-11-24 2001-05-31 Neurosciences Research Foundation, Inc. Acides nucleiques en rapport avec la vigilance et methodes connexes diagnostiques, therapeutiques et de criblage
EP1248107A2 (fr) * 2001-03-22 2002-10-09 IPF Pharmaceuticals GmbH Procédé et appareil de criblage à résolution temporelle
WO2003086462A1 (fr) * 2002-03-29 2003-10-23 Celestar Lexico-Sciences,Inc. Inhibiteurs de phosphorylation c-jun
US6730287B1 (en) 2000-04-03 2004-05-04 Neurosciences Research Foundation Inc. Methods for identifying compounds that modulate vigilance states
US7468178B2 (en) 1999-11-24 2008-12-23 Neurosciences Research Foundation, Inc. Methods for identifying compounds that modulate vigilance states
WO2010079284A1 (fr) * 2009-01-09 2010-07-15 Isp Investments Inc. Nouveaux peptides anti-age et composition cosmetique et/ou pharmaceutique les contenant
WO2010079285A1 (fr) * 2009-01-09 2010-07-15 Isp Investments Inc. Nouveaux peptides anti-age et composition cosmetique et/ou pharmaceutique les contenant
WO2010079286A1 (fr) * 2009-01-09 2010-07-15 Isp Investments Inc. Nouveaux peptides anti-age et composition cosmétique et/ou pharmaceutique les contenant
FR2940971A1 (fr) * 2009-01-09 2010-07-16 Isp Investments Inc Nouveaux peptides anti-age et composition cosmetique et/ou pharmaceutique les contenant
WO2010091327A3 (fr) * 2009-02-09 2011-02-03 Elc Management Llc Procédé et compositions de traitement de la peau
US8703161B2 (en) 2007-08-13 2014-04-22 Elc Management, Llc Skin repair compositions comprising circadian gene activators and a synergistic combination of Sirt1 gene activators
US10383815B2 (en) 2012-09-14 2019-08-20 Elc Management Llc Method and compositions for improving selective catabolysis in cells of keratin surfaces

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
IKEDA M, NOMURA M: "CDNA CLONING AND TISSUE-SPECIFIC EXPRESSION OF A NOVEL BASIC HELIX-LOOP-HELIX/PAS PROTEIN (BMAL1) AND IDENTIFICATION OF ALTERNATIVELY SPLICED VARIANTS WITH ALTERNATIVE TRANSLATION INITIATION SITE USAGE", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 233, no. 01, 1 January 1997 (1997-01-01), US, pages 258 - 264, XP002919679, ISSN: 0006-291X, DOI: 10.1006/bbrc.1997.6371 *
REDDY P., ET AL.: "the period clock locus of D.melanogaster codes for a proteoglycan", CELL, CELL PRESS, US, vol. 46., 1 January 1986 (1986-01-01), US, pages 53 - 61., XP002919682, ISSN: 0092-8674, DOI: 10.1016/0092-8674(86)90859-7 *
SANGORAM A.M., ET AL.: "MAMMALIAN CIRCADIAN AUTOREGULATORY LOOP: A TIMELESS ORTHOLOG AND MPER1 INTERACT AND NEGATIVELY REGULATE CLOCK-BMAL1-INDUCED TRANSCRIPTION.", NEURON, CAMBRIDGE, MA, US, vol. 21., 1 January 1998 (1998-01-01), US, pages 1101 - 1113., XP002919683, DOI: 10.1016/S0896-6273(00)80627-3 *
TEL H., ET AL.: "CIRCADIAN OSCILLATION OF A MAMMALIAN HOMOLOGUE OF THE DROSOPHILA PERIOD GENE.", NATURE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 389., 1 January 1997 (1997-01-01), United Kingdom, pages 512 - 516., XP002919681, ISSN: 0028-0836, DOI: 10.1038/39086 *
ZHONG SHENG SUN, ET AL.: "RIGUI, A PUTATIVE MAMMALIAN ORTHOLOG OF THE DROSOPHILA PERIOD GENE", CELL, CELL PRESS, US, vol. 90, 19 September 1997 (1997-09-19), US, pages 1003 - 1011, XP002919680, ISSN: 0092-8674, DOI: 10.1016/S0092-8674(00)80366-9 *

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US7659387B1 (en) 1999-11-18 2010-02-09 University Of Virginia Transgenic mammals introduced a Period 1 promoter that confers rhythmical expression
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CN102272149B (zh) * 2009-01-09 2014-12-17 Isp投资公司 新的抗衰老肽及含有所述肽的化妆品和/或药物组合物
US8193155B2 (en) 2009-02-09 2012-06-05 Elc Management, Llc Method and compositions for treating skin
KR101372130B1 (ko) * 2009-02-09 2014-03-14 이엘씨 매니지먼트 엘엘씨 피부 치료 방법 및 조성물
WO2010091327A3 (fr) * 2009-02-09 2011-02-03 Elc Management Llc Procédé et compositions de traitement de la peau
US8962571B2 (en) 2009-02-09 2015-02-24 Elc Management Method for repairing DNA damage in keratinocytes
US10383815B2 (en) 2012-09-14 2019-08-20 Elc Management Llc Method and compositions for improving selective catabolysis in cells of keratin surfaces

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