7SK-BAF axis controls pervasive transcription at enhancers [ChIP-Seq]. Mus musculus

Eukaryotic genomes are extensively transcribed, but unfettered transcription alters gene expression and leads to genome damage by several means. Divergent transcription occurs at active enhancers and promoters, distinct classes of cis-regulatory elements critical for precise control of gene expression. A key step in RNA Polymerase II (Pol II) transcription is promoter-proximal pausing, which occurs bidirectionally ~25-60 nucleotides downstream of transcription start sites (TSS). Promoter-proximal pause release is gated by the positive transcription elongation factor b (P-TEFb)-7SK snRNA pathway; release from 7SK allows P-TEFb phosphorylation of Pol II and subsequent elongation. The 7SK small nuclear ribonucleoprotein (snRNP) is thought to reside in the nucleoplasm, but it has been suggested that 7SK could operate physically on chromatin. Notably, while enhancer transcription is one of the earliest steps of gene activation12 and some enhancer RNAs (eRNAs) participate in gene regulation, far less is known about the control of eRNA transcription. Here we show that 7SK inhibits enhancer transcription by modulating nucleosome position. 7SK occupies enhancers and super enhancers genome-wide, and 7SK is required to limit eRNA initiation and synthesis in a manner distinct from promoter pausing. Clustered elements at super enhancers uniquely require 7SK to prevent convergent transcription of colliding polymerases. 7SK inhibits enhancer transcription by modulating chromatin structure, physically interacts with the BAF chromatin remodeling complex, and is required to recruit BAF to enhancers. Thus, 7SK employs distinct mechanisms to counteract diverse consequences of pervasive transcription that distinguish super enhancers, enhancers, and promoters Overall design: Each experiment was performed in at least biological duplicate with input or other controls when appropriate.

真核基因组会发生广泛转录，但不受控的转录会改变基因表达，并通过多种途径造成基因组损伤。双向转录发生于活性增强子与启动子——这两类是精准调控基因表达的关键顺式调控元件（cis-regulatory elements）。RNA聚合酶II（RNA Polymerase II, Pol II）转录的关键步骤之一是启动子近端暂停（promoter-proximal pausing），该事件在转录起始位点（transcription start sites, TSS）下游约25~60核苷酸处双向发生。启动子近端暂停的释放受正向转录延伸因子b（positive transcription elongation factor b, P-TEFb）-7SK小核RNA（7SK snRNA）通路调控；从7SK中释放的P-TEFb可磷酸化Pol II并启动后续的转录延伸。人们此前认为7SK小核核糖核蛋白（7SK small nuclear ribonucleoprotein, snRNP）定位于核质中，但已有研究提示7SK可直接在染色质上发挥功能。值得注意的是，尽管增强子转录是基因激活的最早步骤之一[12]，且部分增强子RNA（enhancer RNAs, eRNAs）参与基因调控，但目前对eRNA转录调控机制的认知仍十分有限。本研究证实，7SK可通过调控核小体定位抑制增强子转录。7SK在全基因组范围的增强子与超级增强子（super enhancers）上均有富集，且7SK可通过不同于启动子暂停的方式限制eRNA的起始与合成。超级增强子区域的成簇调控元件仅需7SK即可避免相向转录引发的聚合酶碰撞。7SK可通过调控染色质结构抑制增强子转录，可与BAF染色质重塑复合物（BAF chromatin remodeling complex）发生物理互作，并负责将BAF招募至增强子区域。综上，7SK可通过不同机制抵消广泛转录（pervasive transcription）带来的多种不良影响，且该调控方式在超级增强子、增强子与启动子之间存在差异。实验设计：所有实验均至少完成2次生物学重复，必要时设置input对照或其他对照。