RORγ directly regulates the circadian expression of clock genes and downstream targets in vivo

Abstract

In this study, we demonstrate that the lack of retinoic acid-related orphan receptor (ROR) γ or α expression in mice significantly reduced the peak expression level of Cry1, Bmal1, E4bp4, Rev-Erbα and Per2 in an ROR isotype- and tissue-selective manner without affecting the phase of their rhythmic expression. Analysis of RORγ/RORα double knockout mice indicated that in certain tissues RORγ and RORα exhibited a certain degree of redundancy in regulating clock gene expression. Reporter gene analysis showed that RORγ was able to induce reporter gene activity through the RORE-containing regulatory regions of Cry1, Bmal1, Rev-Erbα and E4bp4. Co-expression of Rev-Erbα or addition of a novel ROR antagonist repressed this activation. ChIP-Seq and ChIP–Quantitative real-time polymerase chain reaction (QPCR) analysis demonstrated that in vivo RORγ regulate these genes directly and in a Zeitgeber time (ZT)-dependent manner through these ROREs. This transcriptional activation by RORs was associated with changes in histone acetylation and chromatin accessibility. The rhythmic expression of RORγ1 by clock proteins may lead to the rhythmic expression of RORγ1 target genes. The presence of RORγ binding sites and its down-regulation in RORγ−/− liver suggest that the rhythmic expression of Avpr1a depends on RORγ consistent with the concept that RORγ1 provides a link between the clock machinery and its regulation of metabolic genes.

Protein sequestration versus Hill‐type repression in circadian clock models

“…3a and b). On the other hand, in the additional PFL, the activator promotes the transcription of Rors (mammals) [109][110][111][112] or Pdp1 (Drosophila) [107], which upregulates the transcription of the activator (Figs. 3a and b).…”

Dissecting the Rev-erbα Cistrome and the Mechanisms Controlling Circadian Transcription in Liver

“…Although RORs and Rev-erbs recognize the same motifs, they have opposing transcriptional activities and in vitro studies have suggested that the expression of a common target is determined by the outcome of the competition between the two factors (Liu et al 2008;Takeda et al 2012). Comparing gene expression changes in livers of RORa/g double knockout mice at ZT22 (when ROR expression is normally high) with gene expression in Reverba knockout mice at ZT10 (when Rev-erba expression is highest), we found several examples of common targets of Rev-erba and RORs (Zhang et al 2015).…”

“…RAR-related orphan receptors (RORs) are transcriptional activators and recognize the same DNA targets as of Rev-erba (Forman et al 1994;Giguère et al 1994). In addition to active recruitment of NCoR/HDAC3, in vitro studies have suggested that Reverba also represses gene expression by competing with RORs for binding to shared elements (Takeda et al 2012).…”

Gene Model 129 (Gm129) Encodes a Novel Transcriptional Repressor That Modulates Circadian Gene Expression

“…Interestingly, among the target genes with the highest BMAL1 chromatin binding strength, most are well studied circadian genes. The top 10 CLOCK⅐BMAL1 target genes include three core clock gene Per1, Per2, and Cry2 (8,9); three nuclear receptors Rorc, Nr1d1, and Nr1d2 involved in the core clock regulation (10,11), and three PAR domain bZIP transcription factors Dbp, Hlf, and Tef, which function as key genes in the output pathways (12). Only one gene remains uncharacterized, gene model 129 (Gm129), which encodes a novel protein with no predicted functional domains.…”