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

The circadian rhythm is the cyclic change in biochemical, physiological, and behavioral functions of organisms with a periodicity of ∼24 h. In mammalian organisms, this cell-autonomous and self-sustained rhythm is generated by a transcription–translation feedback loop (TTFL) (Reppert and Weaver 2002; Hardin and Panda 2013; Partch et al. 2014). According to the commonly accepted model (“canonical model”), the core clock circuitry is composed of four genes/proteins and their paralogs (Clock [Npas2], Bmal1, Cry [Cry1 and Cry2], and Per [Per1 and Per2]), which generate rhythmicity in the following manner: CLOCK and BMAL1 transcriptional activators bind to E-box sequences in the promoters of Cry and Per genes and activate their transcription; CRY and PER proteins then accumulate in the cytoplasm and, after a time delay, enter the nucleus as a heterodimer and inhibit their own transcription (Kume et al. 1999; Vitaterna et al. 1999; Zheng et al. 1999, 2001; Shearman et al. 2000) as well as the transcription of other output genes controlled by CLOCK–BMAL1 (Fig. 1A; Hughes et al. 2009). The core clock circuitry is stabilized by secondary Ror/Rev-Erb (Nr1d1, Nr1d2) loops that are controlled by the core loop and in turn regulate the activation and repression of Bmal1 and Cry1 transcription, respectively (Preitner et al. 2002; Etchegaray et al. 2003; Liu et al. 2008; Ukai-Tadenuma et al. 2011; Bugge et al. 2012; Cho et al. 2012). Further robustness of the rhythm and stability of the clock are ensured by post-translational modifications and proteolysis of the clock proteins (Lee et al. 2001; Masri and Sassone-Corsi 2010; Partch et al. 2014). 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 ... The canonical model is largely based on genetic data with mutant mice, reporter gene assays, and protein–protein interaction analysis (Kume et al. 1999; Vitaterna et al. 1999; Shearman et al. 2000; Bae et al. 2001; Zheng et al. 2001; Ishikawa et al. 2002; Tamai et al. 2007). Although the model has provided a framework for molecular clock research, the mechanism of action of the core clock proteins—in particular the roles of PERs and CRYs, which make up the negative arm of the loop—has remained ill-defined. A recent comprehensive biochemical study with the four core clock proteins and in vivo chromatin immunoprecipitation (ChIP) and transcription analyses revealed an unexpected fact that is inconsistent with the canonical model: CRY binds to the CLOCK–BMAL1–E-box complex in vitro and in vivo independent of PER and inhibits transcription (Ye et al. 2011). A subsequent genome-wide ChIP study (Koike et al. 2012) and a small molecule inhibitor/computational modeling study (St John et al. 2014) supported this finding; namely, that CRY is the dominant repressor in the TTFL. In contrast to these findings that define a role of CRY in the feedback loop, the role of PER in repression has remained unclear. PER heterodimerizes with CRY, protects it from ubiquitylation and proteolysis (Czarna et al. 2013; Hirano et al. 2013; Xing et al. 2013; Yoo et al. 2013), and promotes nuclear entry of CRY, thus contributing to repression (Lee et al. 2001). However, its physical participation in the repressive complex has been controversial (Zheng et al. 1999; Miki et al. 2012). The biochemical study (Ye et al. 2011) revealed that PER (PER1 or PER2) does not bind to the CLOCK–BMAL1–E-box complex and, importantly, provided the first evidence for a ternary CLOCK–BMAL1–CRY complex in vitro and in vivo that is incompatible with PER binding (Partch et al. 2014). Most surprisingly, in vitro, it was found that PER causes the dissociation of CRY from the CLOCK–BMAL1–E-box complex (Ye et al. 2011), suggesting that it may actually interfere with E-box repression by CRY. However, genome-wide ChIP experiments have indicated that PER actively participates in repression by binding to E-box promoters in a multiprotein complex that includes PER and CRY in addition to other transcription factors that interact with PERs (Brown et al. 2005; Duong et al. 2011; Koike et al. 2012; Padmanabhan et al. 2012; Duong and Weitz 2014). Furthermore, it was reported that the associations of CRY and the PER multiprotein complex with E-boxes are temporally separated, and thus it was proposed that each mode of binding confers unique regulatory properties (Koike et al. 2012; Gustafson and Partch 2014; Partch et al. 2014). To reconcile these seemingly conflicting findings and define more precisely the roles of CRYs and PERs in the core clock mechanism, we generated a mouse fibroblast cell line lacking CRYs and PERs and derivative lines that express CRY or PER that can be targeted to the nucleus in a controllable manner. Using this system, we were able to analyze the effects of CRY alone, PER alone, and CRY plus PER on the binding of CLOCK–BMAL1 to chromatin and on transcription of genes exclusively controlled by CLOCK–BMAL1. We found that CRY alone binds to CLOCK–BMAL1 on chromatin and inhibits the transcriptional activation without affecting the binding of CLOCK–BMAL1 to chromatin. In contrast, PER alone had no effect on the binding of CLOCK–BMAL1 to cognate promoters or on CLOCK–BMAL1-activated transcription. Unexpectedly, however, in cells expressing CRY, nuclear entry of PER resulted in removal of CLOCK–BMAL1 from chromatin and inhibition of CLOCK–BMAL1-mediated transcription. We propose a new model for the core mammalian molecular clock that incorporates these new findings and previously described properties of CRY and PER proteins.