Stabilization of Pol II protein, orchestration of transcription cycles, and maintenance of enhancer landscape by general transcription regulator SPT5 [PRO-seq]. Stabilization of Pol II protein, orchestration of transcription cycles, and maintenance of enhancer landscape by general transcription regulator SPT5 [PRO-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（transcription machinery）的进程运行由多任务调控因子所调控，其中包括SPT5——这是一种进化保守的因子，几乎参与从增强子激活到转录终止的全部转录阶段。然而，目前学界对其在人类细胞中的作用机制仍未完全阐明。
本研究借助快速降解系统（rapid degradation system）展开实验，揭示了SPT5在维持细胞内及染色质结合的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，这一表型与负延伸因子（NELF）降解引发的短距离暂停Pol II重分布截然不同。
绝大多数基因呈现表达下调而非上调，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延伸过程性缺陷显著。KOWx-4/5连接域的磷酸化可促进暂停释放，而整合因子-PP2A（Integrator-PP2A，INTAC）通过靶向SPT5与Pol II拮抗该过程。
我们的研究结果表明，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的稳定性、延伸过程性以及转录终止，成为人类细胞全局转录的必需正向调控因子。
总体实验设计：我们开展了精准核运行测序（PRO-seq）、转座酶可及性染色质测序（ATAC-seq）、RNA测序（RNA-seq）、染色质免疫沉淀测序（ChIP-seq）以及时间标记转录测序（TT-seq）实验，以探究SPT5对转录的调控作用。本研究中，各实验组样本均设置生物学重复两次。细胞使用dTAG13分别处理3、12或24小时，对应对照组则以等体积的溶剂对照二甲基亚砜（DMSO）处理相同时长。