A molecular mechanism for circadian clock negative feedback

Abstract

Circadian rhythms in mammals are generated by a feedback loop in which the three PERIOD (PER) proteins, acting in a large complex, inhibit the transcriptional activity of the CLOCK-BMAL1 dimer, which represses their own expression. Although fundamental, the mechanism of negative feedback in the mammalian clock, or any eukaryotic clock, is unknown. We analyzed protein constituents of PER complexes purified from mouse tissues and identified PSF (polypyrimidine tract-binding protein-associated splicing factor). Our analysis indicates that PSF within the PER complex recruits SIN3A, a scaffold for assembly of transcriptional inhibitory complexes and that the PER complex thereby rhythmically delivers histone deacetylases to the Per1 promoter, which repress Per1 transcription. These findings provide a function for the PER complex and a molecular mechanism for circadian clock negative feedback.

Fig. 1

PSF is a constituent of endogenous PER complexes and is important for clock function. (A) Co-immunoprecipitation of PSF and PER2. Nuclear extracts (CT18) from liver or lung (input) and immunoprecipitates (IP, antibodies at top) from the extracts were probed with antibodies at right. U2AF⁶⁵ and LaminA/C, negative controls; IgG-LC (light chain), positive control. (B) Depletion of PSF increases Per1 transcription. Quantitative RT-PCR assays showing steady-state abundance of indicated pre-mRNAs (normalized to Gapdh mRNA) in mouse fibroblasts after introduction of point-mutant control PSF shRNA (white) or after depletion of PSF by PSF shRNA (black). Shown are mean +/− SEM of triplicate experiments; representative of 3 experiments. (C-E) Short circadian period length caused by depletion of endogenous PSF from fibroblasts. (C) Western blot showing the effect of point-mutant control (Mut) shRNA or PSF shRNA on steady-state level of endogenous PSF. ACTIN, loading control. (D) Circadian oscillations of bioluminescence in synchronized reporter fibroblasts after delivery of control Mut PSF shRNA (yellow) or PSF shRNA (blue). Traces from three independent cultures are shown for each. (E) Circadian periods of fibroblasts in (D) (mean +/− SEM; N = 3 for each condition; t-test, two-tailed).

Fig. 2

PERs, PSF, and the SIN3-HDAC complex. (A) Co-immunoprecipitation of endogenous PER2 and PSF with SIN3A. Nuclear extracts (CT18) from liver or lung (input) and immunoprecipitates (IP, antibodies at top) from the extracts were probed with antibodies at right. U2AF⁶⁵ and LaminA/C, negative controls; IgG-LC (light chain), positive control. (B) Synchronous circadian cycle of PER2, CRY2, PSF, SIN3A, and HDAC1 at Per1 proximal E-box site. ChIP assays from lungs sampled across a circadian cycle (bottom) performed with antibodies at the top left of each panel. ChIP values are relative to the signal at an arbitrary internal control genomic region (SOM methods). (C) Top, diagram of mouse Per1 gene showing positions of proximal E-box and control sites C1, C2, and C3. Bottom, ChIP assays from lungs harvested at CT10-14, performed with antibodies against the indicated proteins. ChIP data show mean +/− SEM of triplicate experiments; representative of 2-4 experiments.

Fig. 3

Presence of SIN3-HDAC1 at Per1 promoter depends on PER and PSF. (A) ChIP assays from lung (CT 10) comparing PSF, SIN3A, and HDAC1 at the Per1 proximal E-box or control promoter in wild-type littermates (white) and Per1^−/−; Per2^−/− mice (black). (B) ChIP assays, as in (A), from mouse fibroblasts after introduction of point-mutant control PSF shRNA (white) or after depletion of PSF by effective PSF shRNA (black). (C) ChIP assays, as in (A), from lung (CT14) comparing acetylation of HDAC1 targets (at bottom) at the Per1 E-box site or control promoter in wild-type littermates (white) and Per1^−/−; Per2^−/− mice (black). (D) ChIP assays, as in (C), from fibroblasts after introduction of point-mutant control PSF shRNA (white) or after depletion of PSF by effective PSF shRNA (black). ChIP values are plotted relative to the signal at an arbitrary internal control genomic region (SOM methods). All show mean +/− SEM of triplicate experiments and are representative of 2-4 experiments.

Fig. 4

Depletion of SIN3A increases Per1 transcription and shortens circadian period length. (A) Quantitative RT-PCR assays, as in Fig. 1B, showing steady-state abundance of indicated pre-mRNAs in fibroblasts after introduction of point-mutant control SIN3A shRNA (white) or after depletion of SIN3A by an effective SIN3A shRNA (black). Shown are means +/− of triplicate experiment; representative of 3 experiments. (B-D) Short circadian period length caused by depletion of endogenous SIN3A from fibroblasts. (B) Western blot showing the effect of mutated control (Mut) siRNA or SIN3A siRNA on steady-state level of endogenous SIN3A. ACTIN, loading control. (C) Circadian oscillations of bioluminescence in synchronized circadian reporter fibroblasts after delivery of control Mut SIN3A siRNA (yellow) or SIN3A siRNA (blue). Traces from three independent cultures are shown for each condition. (D) Circadian period of fibroblasts with control Mut SIN3A siRNA or SIN3A siRNA (mean +/− SEM; N = 3; t-test, two-tailed).