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 1(st) 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.

Figure 1. Identification of a REV-ERBα binding site within the CLOCK gene.

(a) Significant REV-ERBα occupancy was observed within the CLOCK gene within the 1st intron. The genomic structure of CLOCK is shown with REV-ERBα occupancy illustrated as the gray line. The arrow indicates the direction of transcription. The raw ChIP/chip data is shown in a window beneath the gene as is a screen shot from the integrative genome browser. (b) The region of REV-ERBα occupancy was scanned for conserved RevRE using the Evolutionarily Conserved Region Browser and MatInspector. A RevRE was found to be conserved between mice and humans, the alignment of the RevRE is shown. (c) Alignment of the CLOCK RevRE to a characterized RevRE in the Bmal1 promoter.

Figure 2. REV-ERBα is required for normal expression of CLOCK.

HepG2 cells were transfected with siRNAs targeting RORα (78% reduction), RORγ (50% reduction) and REV-ERBα (57% reduction) to reduce their expression. CLOCK expression was then examined by RT-PCR. CLOCK expression was elevated when REV-ERBα expression was reduced (c), but unaffected by alteration of RORα (a) expression or RORγ (b). Data shown is mean ± SEM where n = 4. (d) ChIP assay illustrating REV-ERBα and NCoR occupancy of the 1^st intron of the CLOCK gene. (e) The synthetic REV-ERBα agonist GSK4112 suppresses CLOCK gene expression in HepG2 cells. *, p<0.05.

Figure 3. Identification of a functional RevRE within the CLOCK gene by EMSA.

(a) The sequences of the wild-type CLOCK RevRE and mutant CLOCK RevRE are shown. (b) Demonstration of direct binding of REV-ERBα to radiolabeled CLOCK RevRE DNA. Binding of REV-ERBα to labeled DNA decreased with addition of unlabeled (cold) CLOCK RevRE, but not mutant CLOCK RevRE. The arrow indicates increases amounts of cold DNA added (10×, 50×, and 100× molar excess). (c) EMSA illustrating that REV-ERBα binds to radiolabeled BMAL1 RevRE and that 100-fold molar excess of CLOCK RevRE DNA can compete for binding to REV-ERBα.

Figure 4. Identification of a functional RevRE within the CLOCK gene using reporter-luciferase assays.

(a) The reporter constructs contain a three-times repeat of the putative CLOCK RevRE cloned upstream of the firefly luciferase gene. (b) REV-ERBα was co-transfected with a luciferase reporter containing the wild-type 3×RevRE leading to reduced expression of luciferase relative to the reporter alone. The expression of luciferase from the mutant 3×RORE was unaffected by REV-ERBα co-transfection. Data shown is mean ± SEM, n = 8. *, p<0.05. (c) Proposed model for coordinated regulation of BMAL1/CLOCK heterodimers.