Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been elusive. Here, we developed a molecular genetic screen in mammalian cells to identify mutants of the circadian transcriptional activators CLOCK and BMAL1, which were uncoupled from CRYPTOCHROME (CRY)-mediated transcriptional repression. Notably, mutations in the PER-ARNT-SIM domain of CLOCK and the C terminus of BMAL1 resulted in synergistic insensitivity through reduced physical interactions with CRY. Coexpression of these mutant proteins in cultured fibroblasts caused arrhythmic phenotypes in population and single-cell assays. These data demonstrate that CRY-mediated repression of the CLOCK/BMAL1 complex activity is required for maintenance of circadian rhythmicity and provide formal proof that transcriptional feedback is required for mammalian clock function.
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Dunlap, J.C. Molecular bases for circadian clocks. Cell 96, 271–290 (1999).
Young, M.W. & Kay, S.A. Time zones: a comparative genetics of circadian clocks. Nat. Rev. Genet. 2, 702–715 (2001).
Reppert, S.M. & Weaver, D.R. Coordination of circadian timing in mammals. Nature 418, 935–941 (2002).
Nakajima, M. et al. Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science 308, 414–415 (2005).
Tomita, J., Nakajima, M., Kondo, T. & Iwasaki, H. No transcription-translation feedback in circadian rhythm of KaiC phosphorylation. Science 307, 251–254 (2005).
Etchegaray, J.P., Lee, C., Wade, P.A. & Reppert, S.M. Rhythmic histone acetylation underlies transcription in the mammalian circadian clock. Nature 421, 177–182 (2003).
Gekakis, N. et al. Role of the CLOCK protein in the mammalian circadian mechanism. Science 280, 1564–1569 (1998).
Yoo, S.H. et al. A noncanonical E-box enhancer drives mouse Period2 circadian oscillations in vivo. Proc. Natl. Acad. Sci. USA 102, 2608–2613 (2005).
Griffin, E.A. Jr., Staknis, D. & Weitz, C.J. Light-independent role of CRY1 and CRY2 in the mammalian circadian clock. Science 286, 768–771 (1999).
Sangoram, A.M. et al. Mammalian circadian autoregulatory loop: a timeless ortholog and mPer1 interact and negatively regulate CLOCK-BMAL1-induced transcription. Neuron 21, 1101–1113 (1998).
Kume, K. et al. mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop. Cell 98, 193–205 (1999).
Shearman, L.P. et al. Interacting molecular loops in the mammalian circadian clock. Science 288, 1013–1019 (2000).
van der Horst, G.T. et al. Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. Nature 398, 627–630 (1999).
Vitaterna, M.H. et al. Differential regulation of mammalian period genes and circadian rhythmicity by cryptochromes 1 and 2. Proc. Natl. Acad. Sci. USA 96, 12114–12119 (1999).
Zheng, B. et al. Nonredundant roles of the mPer1 and mPer2 genes in the mammalian circadian clock. Cell 105, 683–694 (2001).
Gu, Y.Z., Hogenesch, J.B. & Bradfield, C.A. The PAS superfamily: sensors of environmental and developmental signals. Annu. Rev. Pharmacol. Toxicol. 40, 519–561 (2000).
Hogenesch, J.B., Gu, Y.Z., Jain, S. & Bradfield, C.A. The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors. Proc. Natl. Acad. Sci. USA 95, 5474–5479 (1998).
McNamara, P. et al. Regulation of CLOCK and MOP4 by nuclear hormone receptors in the vasculature: a humoral mechanism to reset a peripheral clock. Cell 105, 877–889 (2001).
Reick, M., Garcia, J.A., Dudley, C. & McKnight, S.L. NPAS2: an analog of clock operative in the mammalian forebrain. Science 293, 506–509 (2001).
Yildiz, O. et al. Crystal structure and interactions of the PAS repeat region of the Drosophila clock protein PERIOD. Mol. Cell 17, 69–82 (2005).
Eide, E.J., Vielhaber, E.L., Hinz, W.A. & Virshup, D.M. The circadian regulatory proteins BMAL1 and cryptochromes are substrates of casein kinase Iepsilon. J. Biol. Chem. 277, 17248–17254 (2002).
Lowrey, P.L. et al. Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau. Science 288, 483–491 (2000).
Lee, C., Etchegaray, J., Cagampang, F.R., Loudon, A.S. & Reppert, S.M. Posttranslational mechanisms regulate the mammalian circadian clock. Cell 107, 855–867 (2001).
Okamura, H. et al. Photic induction of mPer1 and mPer2 in cry-deficient mice lacking a biological clock. Science 286, 2531–2534 (1999).
Ueda, H.R. et al. A transcription factor response element for gene expression during circadian night. Nature 418, 534–539 (2002).
Ueda, H.R. et al. System-level identification of transcriptional circuits underlying mammalian circadian clocks. Nat. Genet. 37, 187–192 (2005).
Preitner, N. et al. The orphan nuclear receptor REV-ERBalpha controls circadian transcription within the positive limb of the mammalian circadian oscillator. Cell 110, 251–260 (2002).
Sato, T.K. et al. A functional genomics strategy reveals Rora as a component of the mammalian circadian clock. Neuron 43, 527–537 (2004).
Akashi, M. & Takumi, T. The orphan nuclear receptor RORalpha regulates circadian transcription of the mammalian core-clock Bmal1. Nat. Struct. Mol. Biol. 12, 441–448 (2005).
Welsh, D.K., Yoo, S.-H., Liu, A.C., Takahashi, J.S. & Kay, S.A. Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression. Curr. Biol. 14, 2289–2295 (2004).
Panda, S. et al. Coordinated transcription of key pathways in the mouse by the circadian clock. Cell 109, 307–320 (2002).
Lowrey, P.L. & Takahashi, J.S. Mammalian circadian biology: elucidating genome-wide levels of temporal organization. Annu. Rev. Genomics Hum. Genet. 5, 407–441 (2004).
Cook, E. & Peters, K. The smoothing spline: A new approach to standardizing forest interior tree-ring width series for dendroclimatic studies. Tree-Ring Bull. 41, 45–53 (1981).
This research was supported by the Novartis Research Foundation (L.J.M. and J.B.H.), a Rena and Victor Damone Postdoctoral Fellowship from the American Cancer Society (T.K.S.), US National Institute of Health (NIH) grants (D.K.W. and S.A.K.), Scripps Florida (T.K.S., J.E.B., and J.B.H.), RIKEN Center for Developmental Biology (H.R.U.), NIH/Silvio O. Conti Center for Neuroscience grant P50 MH074924-01 (J.E.B. and J.H.), RIKEN Strategic Programs (H.U. and H.R.U.), New Energy and Industrial Technology Organization (NEDO) Scientific Research grant (H.R.U.) and Scientific Research grant and Genome Network Project grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology (H.R.U.). This is manuscript number 17558-CB of The Scripps Research Institute. We thank N. Gekakis, S. Reppert, C. Joazeiro, A. Curtis and G. FitzGerald for plasmids; S. Panda for anti-mCRY1; J. Zhang and T. Orth for robotics support; T. Kondo for high-throughput monitoring systems; M. Ukai-Tadenuma, J. Cartzendafner and J. Geskes for technical support and S. Panda, R. Van Gelder, T. Reyes, M. Pletcher, K. Hayes, B. Miller, M. Conkright and M. Givens for critical reading of the manuscript.
The authors declare no competing financial interests.
Single mutations in CLOCK and BMAL1 desensitize CLOCK/BMAL1 heterodimers to CRY1. (PDF 779 kb)
Double mutant circadian heterodimers are synergistically insensitive to CRY-mediated repression. (PDF 425 kb)
Desensitization to CRY is not due to enhanced stability or expression of mutant CLOCK or BMAL1. (PDF 196 kb)
Overexpression of wild-type CLOCK and BMAL1 differentially affect amplitude of cycling of the PER2 and BMAL1 reporters in real-time bioluminescence assays. (PDF 682 kb)
Double CLOCK/BMAL1 mutant heterodimers are insensitive to CRY-mediated activation of BMAL1 expression. (PDF 167 kb)
Expression of wild-type CLOCK/BMAL1 does not alter circadian PER2 expression in individual NIH3T3 fibroblasts. (PDF 326 kb)
Expression of CLOCK1/BMAL1 double mutants causes arrhythmic PER2 expression in individual NIH3T3 fibroblasts. (PDF 281 kb)
Coexpression of Clock-3/Bmal1–4 mutant heterodimers impairs circadian rhythmicity in individual cells. (PDF 1775 kb)
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Sato, T., Yamada, R., Ukai, H. et al. Feedback repression is required for mammalian circadian clock function. Nat Genet 38, 312–319 (2006). https://doi.org/10.1038/ng1745
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