Direct Regulation of CLOCK Expression by REV-ERB

Circadian rhythms are regulated at the cellular level by transcriptional feedback loops leading to oscillations in expression of key proteins including CLOCK, BMAL1, PERIOD (PER), and CRYPTOCHROME (CRY). The CLOCK and BMAL1 proteins are members of the bHLH class of transcription factors and form a heterodimer that regulates the expression of the PER and CRY genes. The nuclear receptor REV-ERB alpha plays a key role in regulation of oscillations in BMAL1 expression by directly binding to the BMAL1 promoter and suppressing its expression at certain times of day when REV-ERB alpha expression levels are elevated. We recently demonstrated that REV-ERB alpha also regulates the expression of NPAS2, a heterodimer partner of BMAL1. Here, we show that REV-ERB alpha also regulates the expression another heterodimer partner of BMAL1, CLOCK. We identified a REV-ERB alpha binding site within the 1(st) intron of the CLOCK gene using a chromatin immunoprecipitation microarray screen. Suppression of REV-ERB alpha expression resulted in elevated CLOCK mRNA expression consistent with REV-ERB alpha's role as a transcriptional repressor. A REV-ERB response element (RevRE) was identified within this region of the CLOCK gene and was conserved between humans and mice. Additionally, the CLOCK RevRE conferred REV-ERB responsiveness to a heterologous reporter gene. Our data suggests that REV-ERB alpha plays a dual role in regulation of the activity of the BMAL1/CLOCK heterodimer by regulation of expression of both the BMAL1 and CLOCK genes.