Dual modes of CLOCK:BMAL1 inhibition mediated by Cryptochrome and Period proteins in the mammalian circadian clock

Abstract

The mammalian circadian clock is based on a transcription-translation feedback loop (TTFL) in which CLOCK and BMAL1 proteins act as transcriptional activators of Cryptochrome and Period genes, which encode proteins that repress CLOCK-BMAL1 with a periodicity of ∼ 24 h. In this model, the mechanistic roles of CRY and PER are unclear. Here, we used a controlled targeting system to introduce CRY1 or PER2 into the nuclei of mouse cells with defined circadian genotypes to characterize the functions of CRY and PER. Our data show that CRY is the primary repressor in the TTFL: It binds to CLOCK-BMAL1 at the promoter and inhibits CLOCK-BMAL1-dependent transcription without dissociating the complex ("blocking"-type repression). PER alone has no effect on CLOCK-BMAL1-activated transcription. However, in the presence of CRY, nuclear entry of PER inhibits transcription by displacing CLOCK-BMAL1 from the promoter ("displacement"-type repression). In light of these findings, we propose a new model for the mammalian circadian clock in which the negative arm of the TTFL proceeds by two different mechanisms during the circadian cycle.

Keywords: Cryptochrome; DNA binding; Period; circadian; transcription.

Figure 1.

Experimental systems for analysis of the repressive phase of the mammalian clock TTFL. (A) Canonical model of the mammalian circadian clock. In this highly simplified model, only the core TTFL is shown: The CLOCK–BMAL1 heterodimer binds to the E-boxes in Per and Cry genes and activates their transcription. The CRY and PER proteins dimerize in the cytoplasm and, after a time lag, enter the nucleus, bind to CLOCK–BMAL1, and inhibit their own transcription. The cycle starts over after CRY and PER levels decrease by proteolysis. (B) Targeted nuclear delivery system for analysis of CRY1 and PER2 functions. CRY1–ER* or PER2–ER* are expressed in Cry1/2^−/− or Per1/2^−/− cells, respectively. The fusion proteins are retained in the cytoplasm in complex with heat-shock protein (HSP). Upon addition of 4-hydroxytamoxifen (4-OHT), HSP dissociates, and the fusion proteins enter the nucleus. (C,D) 4-OHT-induced nuclear entry of CRY1–ER* and PER2–ER* analyzed by immunofluorescence microscopy (C) and immunoblotting analysis of nuclear extracts (D). Red arrows point to nuclei. A nonspecific band (N.S.) and tubulin protein are shown for loading controls.

Figure 2.

Effect of CRY1 or PER2 nuclear entry on E-box binding and transcription in Cry1/2^−/− or Per1/2^−/− mutants. Nuclear entry of CRY1–ER* inhibits transcription (A) without disrupting the CLOCK–BMAL1–E-box complex (B) and is accompanied by increased binding of CRY1 to the E-box (C). Nuclear entry of PER2–ER* inhibits transcription (D) by displacing CLOCK–BMAL1 (E) and CRY1 (F) from the E-box.

Figure 3.

Effect of CRY1 or PER2 nuclear entry on E-box binding and transcription in the Cry1/2^−/−; Per1/2^-/ mutant. (A) Repressor protein expression in wild-type and mutant mouse cell lines analyzed by immunoblotting. (B) 4-OHT-induced nuclear entry of CRY1–ER* and PER2–ER* in the quadruple mutant cell line analyzed by immunofluorescence microscopy. (C–F) Effects of CRY1 or PER2 nuclear entry on CLOCK–BMAL1 function: CRY1–ER* nuclear entry inhibits transcription (C) without displacing CLOCK–BMAL1 from the promoter (D). In the absence of CRYs, PER2–ER* nuclear entry does not affect E-box transcription (E) or CLOCK–BMAL1 binding to the E-box (F). (EtOH) Ethanol.

Figure 4.

Mapping of PER2 domains necessary for disrupting the CRY–CLOCK–BMAL1–promoter complex. (A) PER2-ER* constructs used in Per1/2^−/− cells. The numbers associated with each mutant represent the amino acids of full-length PER (1257 amino acids) contained in each mutant protein. An SV40 nuclear localization signal is fused to the PER2(882–1257) to assure its nuclear translocation. (B) Nuclear entry of the fusion proteins following 4 h of treatment with 4-OHT analyzed by immunofluorescence. (C) Analyses of BMAL1, CRY1, and PER2 chromatin binding by ChIP. Full-length PER2 and PER2(596–1257) disrupt CRY1–CLOCK–BMAL1 binding to chromatin without measurable PER2 binding. Neither the N-terminal half [PER2(1–916)] nor the C-terminal half [PER2(882–1257)] of the protein have an effect on CRY1–CLOCK–BMAL1 binding to chromatin, but both do weakly associate with the promoter.

Figure 5.

Model for the primary TTFL of the mammalian clock in the mouse liver. (A) Expression patterns of the four core clock proteins in mouse liver nuclei over a circadian period revealing different peak expression times of CRY1 and PER2. (ZT) Zeitgeber time; (ZT0) light on; (ZT12) light off for mice under a 12-h light:12-h dark cycle. (B) New model for the mammalian circadian clock. The figure shows a semiquantitative heat map representation of CRY1 and PER2 protein expression as well as the ChIP data for CLOCK–BMAL1 and CRY1 over a circadian cycle and its consequences with regard to interactions of core clock proteins with the E-box and the effects of these interactions on transcription of genes (Nr1d1 and Dbp) regulated exclusively by the core TTFL.