Tripartite phosphorylation of SPT5 by CDK9 times pause release and tunes elongation rate of RNA polymerase II (ChIP-Seq)

The RNA polymerase II (RNAPII) transcription cycle is regulated throughout its duration by protein phosphorylation. Previously, two regions phosphorylated by cyclin-dependent kinase 9 (CDK9) in elongation factor SPT5—the linker between Kyrpides-Ouzounis-Woese (KOW) x-4 and 5 domains and carboxy-terminal repeat (CTR) 1—were implicated in promoter-proximal pausing and termination, respectively. Here, we show that phosphorylations in the linker, CTR1, and a third region, CTR2, coordinately control pause release, elongation speed, and termination in HCT116 human colon cancer cells. Pausing was unaffected or increased by mutations preventing CTR1 or CTR2 phosphorylation, respectively, but attenuated when both CTRs were mutated. Whereas loss of CTR1 phosphorylation slowed elongation and repressed nascent transcription, simultaneous CTR2 mutation partially reversed both effects. Nevertheless, mutating both CTRs had additive effects on splicing, termination, steady-state mRNA levels, and cell proliferation. Therefore, tripartite SPT5 phosphorylation times pause release and tunes RNAPII elongation rate to ensure productive transcription and cell viability. Overall design: To ask whether CTR1 and CTR2 mutations had similarly reinforcing effects on chromatin distribution of RNAPII, we performed ChIP-seq analysis in the Flag-SPT5-expressing cells after depletion of endogenous dTAG-SPT5.

RNA聚合酶II（RNAPII）的转录周期全程均受蛋白质磷酸化调控。此前已有研究证实，延伸因子SPT5上被细胞周期蛋白依赖性激酶9（CDK9）磷酸化的两个区域——即Kyrpides-Ouzounis-Woese（KOW）x-4与5结构域之间的连接区段，以及羧基末端重复序列1（CTR1）——分别与启动子近端暂停和转录终止过程存在关联。本研究发现，该连接区、CTR1以及第三个区域CTR2的磷酸化修饰，可在HCT116人结肠癌细胞中协同调控暂停释放、延伸速率与转录终止过程。阻断CTR1磷酸化的突变对启动子近端暂停无显著影响，而阻断CTR2磷酸化的突变则会加剧暂停现象，但同时突变两个CTR区域则会削弱暂停过程。单独丧失CTR1磷酸化会减缓RNAPII延伸进程并抑制新生转录，而同时引入CTR2突变则可部分逆转这两种效应。尽管如此，同时突变两个CTR区域会对剪接、转录终止、稳态mRNA水平以及细胞增殖产生叠加效应。因此，SPT5的三方磷酸化可精准调控暂停释放，并微调RNAPII的延伸速率，以确保高效转录与细胞存活。整体实验设计：为探究CTR1与CTR2突变对RNAPII染色质分布是否具有类似的增强效应，我们在内源性dTAG-SPT5被敲除后，于表达Flag-SPT5的细胞中开展了染色质免疫共沉淀测序（ChIP-seq）分析。