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Feedback repression is required for mammalian circadian clock function


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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Figure 1: Mutations in CLOCK and BMAL1 confer insensitivity to CRY-mediated transcriptional repression without affecting CLOCK/BMAL1 transcriptional activity.
Figure 2: Coexpression of CLOCK and BMAL1 desensitized mutants confers synergistic insensitivity to CRY1 in HEK293T cells.
Figure 3: Mutations in the CLOCK PAS domain and BMAL1 C terminus abrogate interactions between the CLOCK/BMAL1 complex and transcriptional repressors CRY1 and PER2.
Figure 4: Coexpression of CLOCK/BMAL1 mutant heterodimers that are insensitive to CRY repression ablates circadian E-box and RORE activities in NIH3T3 cells.
Figure 5: Coexpression of CLOCK/BMAL1 mutant heterodimers impairs circadian rhythmicity in individual cells.

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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.

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Correspondence to Hiroki R Ueda or John B Hogenesch.

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Supplementary information

Supplementary Fig. 1

Single mutations in CLOCK and BMAL1 desensitize CLOCK/BMAL1 heterodimers to CRY1. (PDF 779 kb)

Supplementary Fig. 2

Double mutant circadian heterodimers are synergistically insensitive to CRY-mediated repression. (PDF 425 kb)

Supplementary Fig. 3

Desensitization to CRY is not due to enhanced stability or expression of mutant CLOCK or BMAL1. (PDF 196 kb)

Supplementary Fig. 4

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)

Supplementary Fig. 5

Double CLOCK/BMAL1 mutant heterodimers are insensitive to CRY-mediated activation of BMAL1 expression. (PDF 167 kb)

Supplementary Fig. 6

Expression of wild-type CLOCK/BMAL1 does not alter circadian PER2 expression in individual NIH3T3 fibroblasts. (PDF 326 kb)

Supplementary Fig. 7

Expression of CLOCK1/BMAL1 double mutants causes arrhythmic PER2 expression in individual NIH3T3 fibroblasts. (PDF 281 kb)

Supplementary Fig. 8

Coexpression of Clock-3/Bmal1–4 mutant heterodimers impairs circadian rhythmicity in individual cells. (PDF 1775 kb)

Supplementary Methods (PDF 28 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).

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