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Histone monoubiquitination by Clock–Bmal1 complex marks Per1 and Per2 genes for circadian feedback

Nature Structural & Molecular Biology volume 22, pages 759–766 (2015)Cite this article

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Abstract

Circadian rhythms in mammals are driven by a feedback loop in which the transcription factor Clock–Bmal1 activates expression of Per and Cry proteins, which together form a large nuclear complex (Per complex) that represses Clock–Bmal1 activity. We found that mouse Clock–Bmal1 recruits the Ddb1–Cullin-4 ubiquitin ligase to Per (Per1 and Per2), Cry (Cry1 and Cry2) and other circadian target genes. Histone H2B monoubiquitination at Per genes was rhythmic and depended on Bmal1, Ddb1 and Cullin-4a. Depletion of Ddb1–Cullin-4a or an independent decrease in H2B monoubiquitination caused defective circadian feedback and decreased the association of the Per complex with DNA-bound Clock–Bmal1. Clock–Bmal1 thus covalently marks Per genes for subsequent recruitment of the Per complex. Our results reveal a chromatin-mediated signal from the positive to the negative limb of the clock that provides a licensing mechanism for circadian feedback.

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Figure 1: Identification of Wdr76 and Ddb1 in a Clock–Bmal1 complex.
Figure 2: Clock–Bmal1 recruits the E3 ubiquitin ligase Ddb1–Cul4 to circadian E-box sites.
Figure 3: Clock–Bmal1 recruits Ddb1–Cul4 to many circadian target genes.
Figure 4: Ddb1 and Cul4a have roles in circadian-clock feedback repression.
Figure 5: Ddb1 and Cul4a promote H2B monoubiquitination at circadian E-box sites.
Figure 6: Effects of independent decrease of H2B–Ub mimic circadian phenotypes of Ddb1 and Cul4a.
Figure 7: H2B–Ub decreases Clock–Bmal1 and stabilizes the Per complex at Per E-box sites.
Figure 8: Model showing how Clock–Bmal1 licenses target genes for circadian feedback repression by the Per complex.

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Acknowledgements

We thank M. Liu for mouse colony management and genotyping. This work was supported by an award from the G. Harold & Leila Y. Mathers Charitable Foundation (C.J.W.), a grant from the US National Institute of General Medical Sciences (R01GM095945 to C.J.W.), a US National Institutes of Health Training Grant in Fundamental Neurobiology (T32NS007484 to A.G.T. and H.A.D.), a Dean's Postdoctoral Fellowship at Harvard Medical School (A.G.T.) and a European Community's Seventh Framework Program Marie Curie Fellowship (M.S.R.).

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Authors and Affiliations

  1. Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA

    Alfred G Tamayo, Hao A Duong & Charles J Weitz

  2. Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Martinsried, Germany

    Maria S Robles & Matthias Mann

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  1. Alfred G Tamayo
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  2. Hao A Duong
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  3. Maria S Robles
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Contributions

A.G.T., M.S.R. and H.A.D. performed experiments; A.G.T., M.S.R., H.A.D., M.M. and C.J.W. designed experiments and analyzed data; and C.J.W. oversaw the project.

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Correspondence to Charles J Weitz.

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Integrated supplementary information

Supplementary Figure 1 Scheme for design of E-box oligonucleotide affinity purification of Clock–Bmal1 complexes for mass spectrometric analysis.

The experiment had three conditions: Mouse liver nuclear extracts (CT6) from wildtype mice were incubated with (1) beads linked to a small oligonucleotide containing three copies of the Clock–Bmal1 E-box binding site (CACGTG) or (2) to otherwise identical beads containing mutated E-box sites; (3) liver nuclear extracts from Bmal1−/− mice were incubated with the oligonucleotide containing the E-boxes. Five biological replicates of each condition were analyzed by LC-MS/MS. Statistical comparison of the data for (1) and (2) can identify proteins that are components of specific E-box binding complexes. Comparison of (1) and (3) can identify proteins that are components of DNA-binding complexes that require Bmal1. The overlap of the two significant sets should be highly enriched for components of Clock–Bmal1 complexes. Blue, Clock–Bmal1-associated proteins; light gray, other E-box-binding protein complexes; dark gray, protein complexes that bind to DNA outside the E-box.

Supplementary Figure 2 Ddb1 is a constituent of native Clock–Bmal1 complexes in vivo: additional data.

(a) Immunoblots of liver nuclear extract (CT6) from wildtype mice (Input) and immunoprecipitates (IP) from the extract (antibodies at top) probed with the antibodies indicated at right. Rnf20, a global H2B ubiquitin ligase that is unrelated to Ddb143,44, served as a negative control. IgG light chain (IgG–LC) served as positive control for immunoprecipitation. (b) Immunoblot showing immunoprecipitates (IP) from mouse lung nuclear extract (CT6; antibodies at top) probed with the antibodies indicated at right. IgG heavy chain (IgG–HC) served as positive control.

Supplementary Figure 3 Wdr76 is important for Clock protein stability or expression.

(a) Immunoblots showing the effect of control siRNA or Wdr76 siRNA on the steady-state abundance of the endogenous proteins indicated at right in circadian reporter fibroblasts. β-Actin, loading control. (b) Circadian oscillations of bioluminescence in synchronized reporter fibroblasts after delivery of control siRNA (black) or Wdr76 siRNA (red). Traces from three independent cultures are shown for each. The long period phenotype is the expected from a reduction in stability or expression of the Clock protein.

Supplementary Figure 4 Additional ChIP data.

(a) Circadian cycle of Histone 2B mono-ubiquitination at an additional circadian E-box site. ChIP assays from mouse liver nuclear extracts showing H2B-Ub (normalized to total H2B) across the circadian cycle at a Dbp gene E-box site. (b) No evidence for specific recruitment of Rnf20, a global Histone 2B ubiquitin ligase, to Per2 E-box site. ChIP assays showing (left) Clock and IgG control or (right) Rnf20 and IgG control from liver nuclear extracts (CT6) at Per2 gene E-box site. A similar result was obtained at a Per1 gene E-box.

Supplementary Figure 5 H2B-Ub reduces Clock–Bmal1 and stabilizes the Per complex at Per E-box sites: Per2 E-box site.

(a) ChIP assays from mouse fibroblasts showing Bmal1 and IgG control (left) or Clock and IgG control (right) at Per2 E-box site after introduction of control siRNA (black) or Ddb1 siRNA (gray). (b) ChIP assays as in (a) after introduction of control siRNA (black) or Rnf20 siRNA (white). (c) ChIP assays from mouse fibroblasts showing Per complex proteins Per2 (left), Cry1 (middle), and Hdac1 (right) at Per2 E-box site after introduction of control siRNA (black) or Ddb1 siRNA (gray). (d) ChIP assays as in (c) after introduction of control siRNA (black) or Rnf20 siRNA (white). Shown in (a-d) are mean +/- SD of triplicate experiment; each representative of 3 experiments.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1–3 and Supplementary Note (PDF 719 kb)

Supplementary Data Set 1

Scans of immunoblots (PDF 891 kb)

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Tamayo, A., Duong, H., Robles, M. et al. Histone monoubiquitination by Clock–Bmal1 complex marks Per1 and Per2 genes for circadian feedback. Nat Struct Mol Biol 22, 759–766 (2015). https://doi.org/10.1038/nsmb.3076

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