Stabilization of Pol II protein, orchestration of transcription cycles, and maintenance of enhancer landscape by general transcription regulator SPT5 [ChIP-seq]

Transcription machinery progression is governed by multitasking regulators including SPT5, an evolutionarily conserved factor implicated in virtually all transcriptional steps from enhancer activation to termination. Yet its mechanistic understanding in human cells remains incomplete. Here we utilize rapid degradation system and reveal crucial function of SPT5 in maintaining cellular and chromatin Pol II levels. Rapid SPT5 depletion causes a pronounced reduction of paused Pol II at promoters and enhancers, distinct from NELF degradation resulting in short-distance paused Pol II redistribution. Most of genes exhibit down- but not upregulation, accompanied by greatly impaired transcription activation and altered chromatin landscape at enhancers, and severe Pol II processivity defects at gene bodies. Phosphorylation of KOWx-4/5- linker potentiates pause release and is antagonized by Integrator-PP2A (INTAC) targeting SPT5 and Pol II. Our findings position SPT5 as an essential positive regulator of global transcription by controlling cellular Pol II levels, enhancer activation and landscape, paused Pol II stability, elongation processivity and termination in human. Overall design: We performed PRO-seq, ATAC-seq, RNA-seq, ChIP-seq, or TT-seq to investigate the role of SPT5 in regulating transcription. In this study, samples for each condition were collected in biological duplicates. Cells were treated with dTAG13 for 3, 12,or 24 hours, the corresponding control cells were treated for the same duration with vehicle only (DMSO) at the same vol/vol dilution.

转录机器的运转进程由包括SPT5在内的多任务调控因子所调控。SPT5是一类进化保守的蛋白因子，几乎参与从增强子激活到转录终止的全部转录步骤，然而目前学界对人类细胞中其作用机制的认知仍不全面。本研究借助快速降解系统，揭示了SPT5在维持细胞内与染色质结合的RNA聚合酶II（Pol II）水平方面的关键功能。快速降解SPT5会导致启动子与增强子区域的暂停Pol II显著减少，这一表型与NELF降解引发的暂停Pol II短距离重分布截然不同。绝大多数基因呈现表达下调而非上调的趋势，同时伴随转录激活严重受损、增强子区域染色质景观发生改变，以及基因体区域Pol II延伸过程性出现显著缺陷。KOWx-4/5连接区域的磷酸化可促进暂停Pol II的释放，而Integrator-PP2A复合物（INTAC）通过靶向SPT5与Pol II拮抗这一过程。本研究结果表明，SPT5通过调控细胞内Pol II水平、增强子激活与染色质景观、暂停Pol II稳定性、延伸过程性以及转录终止，成为人类细胞中全局转录的关键正向调控因子。总体实验设计：本研究通过PRO-seq、ATAC-seq、RNA-seq、ChIP-seq及TT-seq技术，探究SPT5在转录调控中的作用。本研究中，各实验条件下的样本均设置2次生物学重复。细胞经dTAG13分别处理3、12或24小时，对应的对照组细胞则以相同体积浓度的溶剂（二甲基亚砜，DMSO）处理相同时长。