FACT orchestrates the interplay between chromatin structure and transcription [mNET-Seq]

Transcription by RNA polymerase II (RNA Pol II) depends on transcription factors and chromatin factors. Here we use rapid factor depletion and multiomics analysis to investigate how a histone chaperone, FAcilitates Chromatin Transcription (FACT), influence nascent transcription by RNA PolII in human cells. Depletion of a FACT subunit, SSRP1, led to rapid changes in chromatin structure and concomitantly strongly compromised RNA synthesis. FACT depletion led to a multilayered transcriptional defect, including loss of promoter proximal pausing, deregulated release into elongation and drop-off of RNA Pol II in promoter-distant gene regions. We combined these analyses with biochemical dissection of transcription of a chromatinized template to show that FACT supports both elongation and pausing of RNA Pol II. Our study also provides new evidence how the position of promoter proximal pausing is defined by the +1 nucleosome in human cells. Overall design: Samples for each condition were collected in biological duplicates. Cells were treated with dTAG7 for 1 or 4 hours, the corresponding control cells were treated for the same duration with vehicle only (DMSO) at the same vol/vol dilution.

RNA聚合酶II（RNA polymerase II, RNA Pol II）介导的转录依赖转录因子与染色质调控因子。本研究借助快速因子敲降与多组学分析手段，探究组蛋白伴侣FACT（Facilitates Chromatin Transcription, FACT）对人类细胞中RNA Pol II介导的新生转录的调控作用。敲降FACT的亚基SSRP1后，会快速引发染色质结构改变，并同时显著削弱RNA合成过程。FACT敲降会诱发多层级转录缺陷，具体包括启动子近端暂停（promoter proximal pausing）的丧失、向延伸阶段的释放失调，以及启动子远端基因区域内RNA Pol II的脱落。研究人员将上述多组学分析与染色质化模板转录的生化解析相结合，证实FACT可同时维持RNA Pol II的延伸与暂停过程。本研究还为人类细胞中启动子近端暂停的位置由+1核小体（+1 nucleosome）所决定这一机制提供了全新实验证据。实验设计概述：各实验组样本均设置生物学重复两份。细胞经dTAG7处理1小时或4小时，对应对照组细胞仅以等体积稀释的溶剂二甲基亚砜（DMSO）处理相同时长。