CTCF blocks anti-sense transcription initiation at divergent gene promoters [ChIP-Seq]

Transcription is typically divergent, initiating at closely spaced oppositely oriented core promoters to produce sense and unstable upstream antisense transcripts (uasTrx). How antisense transcription is regulated and coordinated with sense transcription is largely unknown. Here by combining acute degradation of the multi-functional transcription factor CTCF and nascent transcription measurements, we find that CTCF specifically suppresses antisense but not sense transcription at hundreds of divergent promoters, the great majority of which bear proximal CTCF binding sites. Genome editing, chromatin conformation studies and 5' transcript mapping revealed that CTCF directly suppresses uasTrx initiation in manner independent of its chromatin architectural function. Primary transcript RNA FISH revealed co-bursting of sense and anti-sense transcripts is disfavored, suggesting CTCF-regulated competition for transcription initiation. In sum, CTCF shapes the noncoding transcriptional landscape by suppressing upstream antisense transcription. Overall design: Transient endogenous CTCF/Nipbl depletion coupled with genome wide RNA Pol II binding profiling (ChIP-seq) and nascent transcription quantification (PRO-seq)

转录通常以双向模式发生：在紧密间隔、方向相反的核心启动子处起始，分别生成有义转录本与不稳定的上游反义转录本（upstream antisense transcripts, uasTrx）。目前，反义转录的调控机制及其与有义转录的协同方式仍未完全明确。
本研究结合多功能转录因子CCCTC结合因子（CTCF）的急性降解技术与新生转录定量检测手段，发现CTCF可在数百个双向启动子处特异性抑制反义转录，而非有义转录；其中绝大多数启动子均带有近端CTCF结合位点。
全基因组编辑、染色质构象研究与5'端转录本定位实验表明，CTCF可直接抑制uasTrx的起始过程，且该调控作用不依赖于其染色质架构功能。
初级转录本RNA荧光原位杂交（RNA FISH）实验显示，有义与反义转录本的共爆发式转录并不受青睐，这提示CTCF调控了转录起始过程中的竞争关系。
综上，CTCF通过抑制上游反义转录，塑造了非编码转录组的调控景观。
实验设计概述：通过瞬时内源CTCF/Nipbl降解，结合全基因组RNA聚合酶II结合谱分析（染色质免疫共沉淀测序，ChIP-seq）与新生转录定量分析（PRO-seq）