Cell Biology Section, Systems Biology Group, Biostatistics Branch, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Nucleic Acids Res. 2012 Sep 1;40(17):8519-35. doi: 10.1093/nar/gks630. Epub 2012 Jun 29.
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.
在这项研究中，我们证明了在缺乏视黄酸相关孤儿受体（ROR）γ或α的情况下，小鼠 Cry1、Bmal1、E4bp4、Rev-Erbα 和 Per2 的峰值表达水平显著降低，这种降低方式具有 ROR 同型和组织选择性，而不影响其节律表达的相位。对 RORγ/RORα 双敲除小鼠的分析表明，在某些组织中，RORγ和 RORα在调节时钟基因表达方面具有一定程度的冗余性。报告基因分析表明，RORγ 能够通过 Cry1、Bmal1、Rev-Erbα 和 E4bp4 中含 RORE 的调节区诱导报告基因活性。Rev-Erbα 的共表达或添加新型 ROR 拮抗剂抑制了这种激活。ChIP-Seq 和 ChIP-定量实时聚合酶链反应（QPCR）分析表明，RORγ 可以通过这些 RORE 在体内直接且依赖 Zeitgeber 时间（ZT）调节这些基因。RORs 的这种转录激活与组蛋白乙酰化和染色质可及性的变化有关。时钟蛋白对 RORγ1 的节律表达可能导致 RORγ1 靶基因的节律表达。RORγ-/-肝脏中存在 RORγ 结合位点及其下调表明，Avpr1a 的节律表达依赖于 RORγ，这与 RORγ1 将时钟机制与其对代谢基因的调节联系起来的概念一致。
