ChIRC-seq Reveals Promoter Antisense RNAs and HP1a as Key Regulators of Promoter Pause Release and of Enhancer : Architectural RNA Interactions [PRO-seq]

Demonstration of a massive lncRNA RNA transcriptional program in the mammalian genome has engendered lively discussions about their biological roles, particularly the promoter antisense (PAS) transcripts of coding gene transcription units and abundant species localized to specific subnuclear structures with phase separation properties. Here, we report development of an alternative assay to quantitatively detect RNA distribution in the genome, referred to as ChIRC-seq (Chromatin Isolation by RNA-Cas13a Complex Capture by Cas13a), which has revealed that promoter antisense RNAs (PAS-RNAs) serve as a key gatekeeper of a broad transcriptional pause release program, based on decommissioning the 7SK snRNA-dependent inhibitory P-TEFb complex. Induction of PAS-RNA by liganded estrogen receptor ERa (ESR1) releases the basal recruitment of HP1a and KAP1 on target gene promoters, without the spreading characteristic of extended heterochromatic regions, based on PAS-dependent recruitment of H3K9me3 demethylases. Unexpectedly, ERa-bound MegaTrans enhancer activation with features of phase separation events proved to require recruitment of phosphorylated KAP1 to be activated, with its transfer to the cognate promoters licensing estrogen-induced pause release and target gene activation. The ChIRC13a-seq method has proved effective for identification of the cognate promoter for activated enhancers and has revealed that the lncRNAs NEAT1/MALAT1 in phase-separated subnuclear structures interact with robust estrogen-activated MegaTrans TFF1e1 enhancer and other active enhancers, linking acute activation of a subset of enhancers to interactions with specific phase-separated subnuclear architectural structures. Overall design: The changes in the gene expression profiles were investigated by ChIP-seq, PRO-seq, and ChIRC-seq.

哺乳动物基因组中大规模长链非编码RNA（long non-coding RNA, lncRNA）转录程序的相关研究，引发了学界对其生物学功能的热烈讨论，其中尤以编码基因转录单元的启动子反义转录本（promoter antisense, PAS）以及定位于具有相分离特性的特定亚核结构的丰富转录本种类为焦点。本研究报道了一种可定量检测基因组中RNA分布的新型检测方法——染色质RNA-Cas13a复合物Cas13a捕获测序（Chromatin Isolation by RNA-Cas13a Complex Capture by Cas13a, ChIRC-seq），该方法揭示：启动子反义RNA（promoter antisense RNAs, PAS-RNAs）作为广谱转录暂停释放程序的关键守门人，其功能依赖于灭活依赖于7SK小核RNA（7SK snRNA）的抑制性正转录延伸因子b（P-TEFb）复合物。经配体活化的雌激素受体α（estrogen receptor α, ERα/ESR1）诱导产生的PAS-RNA，可解除靶基因启动子上基础募集的异染色质蛋白1α（heterochromatin protein 1α, HP1α）与KRAB相关蛋白1（KRAB-associated protein 1, KAP1），且不会出现延伸异染色质区域的扩散特征，这一过程依赖于PAS介导的组蛋白H3赖氨酸9三甲基化（histone H3 lysine 9 trimethylation, H3K9me3）去甲基化酶的募集。出乎意料的是，带有相分离特征的ERα结合型巨型转录增强子（MegaTrans enhancer）的激活，需要募集磷酸化KAP1才能完成；磷酸化KAP1转移至同源启动子后，可赋予雌激素诱导的转录暂停释放与靶基因激活的能力。ChIRC13a测序（ChIRC13a-seq）方法已被证实可有效鉴定激活型增强子的同源启动子；研究还发现，定位于相分离亚核结构的lncRNA NEAT1/MALAT1，可与强效雌激素激活的巨型转录TFF1e1增强子（MegaTrans TFF1e1 enhancer）及其他活性增强子相互作用，将部分增强子的快速激活与特定相分离亚核结构的相互作用建立关联。实验设计：本研究采用染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）、精确转录延伸测序（precision run-on sequencing, PRO-seq）与ChIRC-seq三种技术，对基因表达谱的变化进行了探究。