Direct Regulation of CLOCK Expression by REV-ERB

Abstract

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-ERBα 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-ERBα expression levels are elevated. We recently demonstrated that REV-ERBα also regulates the expression of NPAS2, a heterodimer partner of BMAL1. Here, we show that REV-ERBα also regulates the expression another heterodimer partner of BMAL1, CLOCK. We identified a REV-ERBα binding site within the 1st intron of the CLOCK gene using a chromatin immunoprecipitation – microarray screen. Suppression of REV-ERBα expression resulted in elevated CLOCK mRNA expression consistent with REV-ERBα'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-ERBα 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.

Circadian regulation of lipid metabolism

“…Additionally, ROR genes (RORα, RORβ and RORγ) are rhythmically expressed and their protein products activate Bmal1 and Npas2 transcription by binding to ROR response elements, thus competing with and having the opposite effect of REV-ERB proteins (43)(44)(45) . REV-ERBα might also act as a transcriptional repressor for Clock by binding to a REV-ERB response element, although ROR proteins do not appear to regulate Clock gene expression (46) . The aforementioned feedback loops constitute part of the core molecular mechanism for circadian gene expression; however, there are other pathways by which circadian timing can be regulated at the cellular level, as reviewed in detail elsewhere (47,48) .…”

Circadian rhythms in glucose and lipid metabolism in nocturnal and diurnal mammals

“…PERs and CRYs accumulate in the cytoplasm and form complexes which translocate into the nucleus to inhibit their own transcription along with other BMAL1/CLOCKdriven transcription, such as that of the clock-controlled genes, thereby forming a negative loop (Mohawk et al, 2012). An additional negative loop in this molecular network is contributed by the nuclear receptors REB-ERBa and -b. REV-ERBs bind to the ROR response element (RRE) of BMAL1 and CLOCK promoters and repress their transcription, whereas in counterbalance to REV-ERBs inhibition, RORa/b/g also bind on the RRE of BMAL1 and induce its transcription (Crumbley and Burris, 2011;Guillaumond et al, 2005;Preitner et al, 2002). The circadian rhythms generated by this molecular clock machinery get fine-tuned by environmental cues.…”

Heme Sensor Proteins

“…In vertebrates, the expression of several day/night cycle genes, as well as Rev-erb␣, is controlled by binding a heterodimer of Clock (or NPAS2 (neuronal PAS domain protein 2)) and Bmal1 to their 5Ј-UTR, thus switching on transcription. Expression of Bmal1 and NPAS2/Clock is repressed in turn by binding of Rev-erb␣ to a homodimeric partner (5). Rev-erb␣ and homologs (Rev-erb␤ and E75, with the latter involved in insect ecdysone signaling) are heme-regulated NRs (6,7).…”