The circadian clock component BMAL1 is a critical regulator of p21WAF1/CIP1 expression and hepatocyte proliferation

Most living organisms show circadian (similar to 24 h) rhythms in physiology and behavior. These oscillations are generated by endogenous circadian clocks, present in virtually all cells where they control key biological processes. Although circadian gating of mitosis has been reported for many years in some peripheral tissues, the underlying molecular mechanisms have remained poorly understood. Here we show that the cell cycle inhibitor p21(WAF1/CIP1) is rhythmically expressed in mouse peripheral organs. This rhythmic pattern of mRNA and protein expression was recapitulated in vitro in serum-shocked differentiated skeletal muscle cells. p21(WAF1/CIP1) circadian expression is dramatically increased and no longer rhythmic in clock-deficient Bmal1(-/-) knock-out mice. Biochemical and genetic data show that oscillation of p21(WAF1/CIP1) gene transcription is regulated by the antagonistic activities of the orphan nuclear receptors REV-ERB alpha/beta and ROR alpha 4/gamma, which are core clock regulators. Importantly, p21(WAF1/CIP1) overexpressing Bmal1(-/-) primary hepatocytes exhibit a decreased proliferation rate. This phenotype could be reversed using small interfering RNA-mediated knockdown of p21(WAF1/CIP1). These data establish a novel molecular link between clock and cell cycle genes and suggest that the G(1) progression phase is a target of the circadian clock during liver cell proliferation.