Stabilization of Pol II protein, orchestration of transcription cycles, and maintenance of enhancer landscape by general transcription regulator SPT5 [TT-seq]. Stabilization of Pol II protein, orchestration of transcription cycles, and maintenance of enhancer landscape by general transcription regulator SPT5 [TT-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）的运行进程受多任务调控因子的调控，其中包括SPT5——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所有转录步骤。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。
本研究利用快速降解系统，揭示了SPT5在维持细胞内及染色质结合的RNA聚合酶II（RNA polymerase II, Pol II）水平中的关键功能。快速降解SPT5会导致启动子和增强子区域的暂停Pol II显著减少，这与负延伸因子（Negative Elongation Factor, NELF）降解后引发的短距离暂停Pol II重分布现象截然不同。
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改变，以及基因体区域Pol II延伸过程性缺陷。KOWx-4/5连接域的磷酸化可促进暂停释放，而Integrator-PP2A复合物（INTAC）通过靶向SPT5和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，成为人类细胞全局转录的必需正向调控因子。
整体实验设计：本研究通过PRO-seq（Precision Run-On Sequencing）、ATAC-seq（Assay for Transposase-Accessible Chromatin using sequencing）、RNA-seq（RNA Sequencing）、ChIP-seq（Chromatin Immunoprecipitation sequencing）及TT-seq（Transient Transcriptome Sequencing）技术，探究SPT5在转录调控中的作用。本研究中各实验条件的样本均设置生物学重复两次。细胞使用dTAG13处理3、12或24小时，对应对照组细胞则以相同体积/体积比稀释的溶剂（二甲基亚砜，DMSO）处理相同时长。