An Intrinsic Disorder Region Controlling Condensation of a Circadian Clock Component and Rhythmic Transcription in the Liver [ChIA-PET]

Circadian gene transcription is fundamental to metabolic physiology. Here we report that the nuclear receptor REV-ERBalpha, a repressive component of the molecular clock, forms circadian condensates in the nuclei of mouse liver. These condensates are dictated by an intrinsically disordered region (IDR) located in the protein's hinge region which specifically concentrates nuclear receptor corepressor 1 (NCOR1) at the genome. IDR deletion diminishes the recruitment of NCOR1 and disrupts rhythmic gene transcription in vivo. REV-ERBalpha condensates are located at high-order transcriptional repressive hubs in the liver genome that are highly correlated with circadian gene repression. Deletion of the IDR disrupts transcriptional repressive hubs and diminishes silencing of target genes by REV-ERBalpha. This work demonstrates physiological circadian protein condensates containing REV-ERBalpha whose IDR is required for hub formation and the control of rhythmic gene expression. Overall design: In-situ Chromatin Interaction Analysis with Paired-End Tag (In-situ ChIA-PET) for REV-ERBalpha at different circadian time points (ZT22/ZT10) or different AAV virus combinational injection (DKO+WT, DKO+?hIDR)

节律基因转录是代谢生理学的核心基础。本研究报道，分子钟的阻遏组分核受体REV-ERBα（nuclear receptor REV-ERBalpha）可在小鼠肝脏细胞核中形成昼夜节律性凝聚体。此类凝聚体的形成依赖于位于该蛋白铰链区的内在无序区域（intrinsically disordered region, IDR），该区域可在基因组层面特异性富集核受体辅阻遏因子1（nuclear receptor corepressor 1, NCOR1）。敲除该IDR会减少NCOR1的招募，并在体内破坏节律性基因转录。REV-ERBα凝聚体定位于肝脏基因组中的高阶转录阻遏枢纽，该枢纽与节律基因的阻遏过程呈现高度相关性。敲除IDR会破坏转录阻遏枢纽，并削弱REV-ERBα对靶基因的沉默效应。本研究证实了包含REV-ERBα的生理性昼夜节律蛋白凝聚体的存在，其IDR是凝聚体枢纽形成与节律性基因表达调控所必需的。

实验整体设计：针对不同昼夜节律时间点（ZT22/ZT10）或不同腺相关病毒（adeno-associated virus, AAV）联合注射组（DKO+WT、DKO+?hIDR）的样本，开展REV-ERBα的原位配对末端标签染色质相互作用分析（In-situ Chromatin Interaction Analysis with Paired-End Tag, In-situ ChIA-PET）