Redundant Function of REV-ERBα and β and Non-essential Role for BMAL1 Cycling in Transcriptional Regulation of Intracellular Circadian Rhythms

Abstract

The mammalian circadian clockwork is composed of a core PER/CRY feedback loop and additional interlocking loops. In particular, the ROR/REV/Bmal1 loop, consisting of ROR activators and REV-ERB repressors that regulate Bmal1 expression, is thought to ''stabilize'' core clock function. However, due to functional redundancy and pleiotropic effects of gene deletions, the role of the ROR/REV/Bmal1 loop has not been accurately defined. In this study, we examined cell-autonomous circadian oscillations using combined gene knockout and RNA interference and demonstrated that REV-ERBa and b are functionally redundant and are required for rhythmic Bmal1 expression. In contrast, the RORs contribute to Bmal1 amplitude but are dispensable for Bmal1 rhythm. We provide direct in vivo genetic evidence that the REV-ERBs also participate in combinatorial regulation of Cry1 and Rorc expression, leading to their phase-delay relative to Rev-erba. Thus, the REV-ERBs play a more prominent role than the RORs in the basic clock mechanism. The cellular genetic approach permitted testing of the robustness of the intracellular core clock function. We showed that cells deficient in both REV-ERBa and b function, or those expressing constitutive BMAL1, were still able to generate and maintain normal Per2 rhythmicity. Our findings thus underscore the resilience of the intracellular clock mechanism and provide important insights into the transcriptional topologies underlying the circadian clock. Since REV-ERB function and Bmal1 mRNA/protein cycling are not necessary for basic clock function, we propose that the major role of the ROR/REV/Bmal1 loop and its constituents is to control rhythmic transcription of clock output genes.

A Genome-wide RNAi Screen for Modifiers of the Circadian Clock in Human Cells

“…Knockdown of CRY1 , CRY2 and BMAL1 in both lines resulted in phenotypes consistent with previous knockout mouse and cellular knockdown studies (Liu et al, 2007b; Liu et al, 2008; Maier et al, 2009; Baggs et al, 2009) (Figure S1). For instance, CRY1 knockdown shortens period length and compromises rhythm persistence, CRY2 knockdown lengthens period length, and knockdown of both results in arrhythmicity.…”

“…Gratifyingly, many known clock components had phenotypes in this screen consistent with those seen in knockout animals (Liu et al, 2007b; Liu et al, 2008) or dose-dependent siRNA knockdown (Baggs et al, 2009) (Figure 1C). For example, CRY2 knockdown lengthened period, CRY1 knockdown led to rapid loss of rhythmicity.…”

“…We established cell lines harboring a rapidly-degradable form of luciferase, dLuc , driven by the mouse Bmal1 or Per2 gene promoter (Liu et al, 2008) (Figure S1). Knockdown of CRY1 , CRY2 and BMAL1 in both lines resulted in phenotypes consistent with previous knockout mouse and cellular knockdown studies (Liu et al, 2007b; Liu et al, 2008; Maier et al, 2009; Baggs et al, 2009) (Figure S1).…”

Molecular architecture of the mammalian circadian clock

“…TTFL drives rhythmic changes in Bmal1 transcription and introduces a delay in Cry1 mRNA expression that offsets it from genes regulated strictly by CLOCK:BMAL1 [55]. While rhythmic changes in BMAL1 abundance are not required to drive the core TTFL loop [17], the ROR/REV TTFL-induced delay in Cry1 expression is critical for proper circadian timing [55]. The presence of cooperative, interlocking feedback loops provides robustness against noise and environmental perturbations to help keep accurate circadian timing, and also helps to generate phase delays in circadian transcriptional output that optimally time gene expression for local physiology [44].…”

“…Single knockouts of most integral clock genes (Box 1) do not completely disrupt behavioral rhythms due to apparent functional redundancy of paralogs or compensation by the coupled SCN network [9, 15–17], both of which provide resiliency to maintain clock function. For example, both Period genes ( Per 1 and Per2 ) are required for cycling [18], perhaps due to their low abundance as the stoichiometrically limiting factor in forming key clock protein complexes [19].…”