Direct Regulation of CLOCK Expression by REV-ERB

Circadian rhythms play an essential role in coordinating the timing of various physiological processes. In mammals, the master clock for circadian rhythm is maintained in the suprachasmatic nucleus (SCN) in the brain, but many peripheral organs maintain semi-autonomous clocks that can be set using signals from the SCN or other signals, such as nutrient status. The circadian clock is regulated by a transcriptional/translational feedback loop that involves several key proteins including circadian locomotor output kaput (CLOCK). CLOCK was initially discovered in a mutagenesis screen for altered circadian phenotypes in mice [1]. The CLOCK mutant mice had an increased period of circadian activity, as determined by wheel running experiments, and became arrhythmic in constant darkness. The Clock gene was positionally cloned revealing its identity as a member of the basic helix-loop-helix family of transcription factors [2]. Clock is expressed in the SCN of mice as well as in humans, but also displays a wider pattern of expression that includes the liver where it may play a role in regulation of the circadian rhythm in this tissue [3], [4], [5].

CLOCK functions as a heterodimer with another bHLH transcription factor, the Brain and Muscle ARNT (Aryl hydrocarbon receptor nuclear translocator)-like 1 (BMAL1). The CLOCK/BMAL1 heterodimer binds to E-box elements in the promoters of the PERIOD (PER) and CRYTOCHROME (CRY) genes increasing their transcription. The PER and CRY proteins also heterodimerize and inhibit CLOCK/BMAL1 activity leading to reduction in PER and CRY expression, thereby completing the core feedback loop responsible for the circadian rhythm. Two nuclear receptors that are expressed in a circadian pattern modulate the activity of this core loop. The retinoic acid receptor-related orphan receptor α (RORα) and REV-ERBα directly regulate the expression of BMAL1 by binding to a specific ROR/REV-ERB response element in the BMAL1 promoter [6]. RORα stimulates BMAL1 whereas REV-ERBα represses transcription. Studies using genetically modified mice show that RORα and REV-ERBα play a role in maintaining circadian rhythm. The RORα staggerer mutant mice (RORα^sg/sg) and RORα^−/− mice have shortened circadian periods [7]. The REV-ERBα null mice also have a shortened circadian period and greater light-induced phase responsiveness [8]. Given that RORα and REV-ERBα have been shown to regulate BMAL1 expression, we investigated whether another BMAL1 heterodimer partner, NPAS2, was regulated by these 2 nuclear receptors and found that it was a direct target gene [9]. Thus, we next sought to determine if RORα and/or REV-ERBα might regulate the expression of CLOCK.

A ChIP/chip screen was performed to determine regions where REV-ERBα is bound within the genome as previously described [9]. Significant REV-ERBα occupancy was observed with the CLOCK gene, as diagrammed in Figure 1a. Using the Evolutionarily Conserved Region Browser [15], it was determined that a putative RevRE is located within the CLOCK gene that is conserved between mice and humans, as shown in Figure 1b. The same site was detected using MatInspector [16]. An alignment between the CLOCK RevRE site to a characterized RevRE in the BMAL1 promoter is shown in Figure 1c.

We suppressed RORα (including RORα1 and RORα4), RORγ, and REV-ERBα expression in HepG2 cells by transfecting the cells with specific siRNAs. When RORα expression was decreased by ∼78% using siRNA, the expression of CLOCK was unchanged (Figure 2a). A similar lack of effect was observed when we suppressed RORγ expression by ∼50% using siRNA (Figure 2b). However, when REV-ERBα expression was decreased by ∼57% using siRNA, the expression of CLOCK was increased 3.2-fold, as shown in Figure 2c, indicating that endogenous REV-ERBα exerts a repressive effect on CLOCK expression. We confirmed occupancy of REV-ERBα at the site identified by the ChIP-on-chip screen by ChIP and also noted that the corepressor NCoR was also recruited to the site (Figure 2d). When we examined occupancy following knock-down of REV-ERBα expression as indicated above we noticed loss of REV-ERBα occupancy as well as loss of NCoR occupancy (Figure 2d) indicating that NCoR recruitment was mediated by REV-ERBα and consistent with the loss of repressive effects of this receptor and the increase in CLOCK gene expression we noted in Figure 2c. We next assessed the ability of a synthetic REV-ERBα agonist to modulate the expression of CLOCK expression in HepG2 cells. GSK4112 (SR6452) binds directly to the ligand binding domain of REV-ERBα and increases the recruitment of NCoR leading to increased repression of target genes [17], [18], [19], [20]. As shown in Figure 2e, treatment of the HepG2 cells with GSK4112 results in repression (∼55%) of CLOCK gene expression. Interestingly, overexpression of REV-ERBα in HepG2 cells did not result in any change in CLOCK gene expression (data not shown), suggesting that the endogenous REV-ERBα levels may be saturating.

The putative RevRE was examined for REV-ERBα binding using an EMSA. Synthetic oligonucleotides encoding the putative CLOCK RevRE were radiolabeled, and then incubated with REV-ERBα protein produced in vitro. The sequences of the oligonucleotides are shown in Figure 3a. Direct binding of REV-ERBα to the wild-type CLOCK RevRE is shown in Figure 3b. Addition of unlabeled wild-type CLOCK RevRE probe was able to displace the radiolabeled CLOCK RevRE from REV-ERBα, as shown in Figure 3b while an excess of unlabeled mutant CLOCK RevRE was unable to displace the radiolabeled CLOCK RevRE probe from the REV-ERBα protein demonstrating specificity. We also assessed the ability of the CLOCK RevRE probe to compete with the well-characterized BMAL1 RevRE. The BMAL1 RevRE was radiolabeled and then 100-fold molar excess of unlabeled CLOCK RevRE was included as a competitor. As illustrated in Figure 3c, the CLOCK RevRE was able to compete for binding of REV-ERBα to the BMAL1 RevRE.

Due to the location of the putative RevRE within the 1^st intron of the CLOCK gene, we generated a three-times repeat of the RevRE to clone upstream of the firefly luciferase gene (Figure 4a). When the wild-type 3×RevRE reporter construct was transfected into HepG2 cells, the co-transfection of REV-ERBα suppressed luciferase expression relative to reporter alone (Figure 4b). The expression of luciferase from the mutant 3×RevRE reporter construct was unaffected by co-transfection of REV-ERBα when transfected into HepG2 cells. Similar results were observed in HEK293 cells (data not shown).

CLOCK heterodimerizes with BMAL1 and maintains the circadian expression of target genes containing E-boxes in their promoters. BMAL1 is a known direct target gene of RORα and REV-ERBα, whose circadian expression influences the expression of BMAL1. However, little is known about how the dimerization partner of BMAL1, CLOCK, is regulated at the transcriptional level. We recently demonstrated that another BMAL1 heterodimerization partner, NPAS2, is regulated by RORα and REV-ERBα providing for a potential mechanism for coordinated expression of these two bHLH factors that regulate the circadian rhythm [9]. In this study, we found that another important factor that heterodimerizes with BMAL1, CLOCK, is also a target of REV-ERBα. BMAL1 cannot function independently of a dimer partner such as CLOCK or NPAS2, thus it is logical that both components of the heterodimer may share some regulatory components. Coordination of their regulation by simultaneous modulation by REV-ERBα may be one mechanism that this can occur. However, it is intriguing that RORα regulates BMAL1 expression but does not appear to play a role in regulation of CLOCK. We also noted that CLOCK is not responsive to RORγ, indicating that this particular response element in the CLOCK gene is selective for REV-ERBα. Given the similarity between the BMAL1 and CLOCK RevREs this is surprising and it is currently unclear what differences may be driving the selectivity. It is possible that the REV-ERBα selectivity for regulating the CLOCK gene may be a function of the HepG2 cell line used in this study and in other cell types RORα may play a significant role. Alternatively, other BMAL1 partners such as NPAS2 may play a significant role in maintaining the proper ratio of BMAL1 to heterodimerization partner. It is interesting to note that in the HepG2 cells we previously observed circadian oscillations in the expression of BMAL1, RORα and NPAS2 but not REV-ERBα following a serum shock [9]. We also observed that CLOCK does not oscillate in the HepG2 cells (data not shown), which is consistent with the lack of oscillation in REV-ERBα and the lack of responsiveness to the oscillating RORα. Again, this pattern of regulation may be specific for HepG2 cells or in cells or tissues where CLOCK does not exhibit a strong circadian pattern of expression. Clearly, REV-ERBα plays an important role in the circadian pattern of expression of CLOCK in some circumstances since the REV-ERBα null mouse exhibits a loss in the pattern [8]. Our data suggests that at least a component of the effect may be mediated by a direct effect of REV-ERBα on expression of the CLOCK gene. REV-ERBα mediated regulation of CLOCK adds to the complexity of the feedback loop that maintains circadian rhythms. The BMAL1/CLOCK dimer is capable of up-regulating REV-ERBα, which will then repress BMAL1 and CLOCK expression. This coordinate regulation of BMAL1 and CLOCK by REV-ERBα may help maintain the circadian oscillations of various genes, including REV-ERBα itself (Figure 4c).