BMAL1 Shuttling Controls Transactivation and Degradation of the CLOCK/BMAL1 Heterodimer

Abstract

CLOCK and BMAL1 are bHLH-PAS-containing transcription factors that bind to E-box elements and are indispensable for expression of core circadian clock components such as the Per and Cry genes. A key step in expression is the heterodimerization of CLOCK and BMAL1 and their accumulation in the nucleus with an approximately 24-h periodicity. We show here that nucleocytoplasmic shuttling of BMAL1 is essential for transactivation and for degradation of the CLOCK/BMAL1 heterodimer. Using serial deletions and point mutants, we identified a functional nuclear localization signal and Crm1-dependent nuclear export signals in BMAL1. Transient-transfection experiments revealed that heterodimerization of CLOCK and BMAL1 accelerates their turnover, as well as E-box-dependent clock gene transcription. Moreover, in embryonic mouse fibroblasts, robust transcription of Per2 is tightly associated with massive degradation of the CLOCK/BMAL1 heterodimer. CRY proteins suppressed this process during the transcription-negative phase and led to nuclear accumulation of the CLOCK/BMAL1 heterodimer. Thus, these findings suggest that the decrease of BMAL1 abundance during the circadian cycle reflects robust transcriptional activation of clock genes rather than inhibition of BMAL1 synthesis.Almost all organisms from bacteria to mammals have developed circadian physiology and behavior to adapt to the environmental changes created by the rotation of our planet. Such daily rhythms are controlled by genetically determined and self-sustaining circadian clocks that are composed of networks of transcription-translation feedback loops involving sets of clock genes (15,30,37). In mammals, a network of feedback loops functions robustly not only in the master circadian pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus (26), but also in most tissues (including liver, heart, lung, and muscle) and even in immortalized cell lines (1, 50, 51). These widely dispersed circadian systems are primarily synchronized by the SCN to coordinate circadian timing in vivo (29,36).One of the main questions in clock biology is how the timekeeping system is controlled at the molecular level. Intensive studies in the mouse using genetic and molecular approaches have largely clarified the structure of the central feedback loop (3,30). Two bHLH-PAS-containing transcription factors, CLOCK and BMAL1, form heterodimers that bind to E-box enhancer elements in the promoters of target genes driving the transcription of three period genes (Per1, Per2, and Per3) and two cryptochrome genes (Cry1 and Cry2) (11,14,17,18). After the PER and CRY proteins have been translated in the cytoplasm, they form heterocomplexes that translocate into the nucleus and inhibit their own transcription. CRY plays a crucial role in this negative feedback process by interacting directly with the CLOCK/BMAL1 heterodimers (12,22).Analysis of Bmal1 defective mice has revealed the indispensable role of BMAL1 as the mainspring of the molecular clockwork; thus, targeted disruption of Bmal1 results...

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