Distinct Cdk9-phosphatase switches act on elongation factor Spt5 at the beginning and end of the RNA polymerase II transcription cycle. Distinct Cdk9-phosphatase switches act on elongation factor Spt5 at the beginning and end of the RNA polymerase II transcription cycle

The Pol II transcription cycle is ordered by CDKs and phosphatases. In fission yeast, Cdk9 phosphorylates carboxy-terminal repeats (CTRs) of Spt5 while inhibiting PP1 during elongation. Transcription past the cleavage and polyadenylation signal (CPS) coincides with PP1-dependent Spt5 dephosphorylation and leads to Pol II pausing with phosphorylated CTD-Ser2 (pSer2). Here we show this switch is conserved in humans: Cdk9 inhibition decreases phosphorylation of both PP1g and Spt5-Thr806 (pThr806), and induces pSer2 upstream of the CPS, whereas PP1 depletion increases pThr806. Moreover, in unperturbed cells, pThr806 is diminished in 3’-paused complexes where pSer2 is maximal. Cdk9 also phosphorylates Spt5 on Ser666, a PP1-refractory site between conserved KOW4 and KOW5 motifs; pSer666 increases upon promoter-proximal pause release and, in contrast to pThr806, persists beyond the CPS. We identify PP4—another target of inhibitory phosphorylation by Cdk9—as a pSer666 phosphatase. PP4 and PP1g are enriched at 5’ and 3’ ends of genes, respectively, consistent with pSer666 and pThr806 distributions. Therefore, distinct Cdk9-phosphatase switches operate on Spt5 at different steps in transcription. Overall design: ChIP-seq: Data represent nineteen different ChIP-seq experiments in human colon carcinoma (HCT116) cells, performed in biological replicates. There are raw and processed files (bigWig) for all biological replicates.

RNA聚合酶II（Pol II）的转录循环进程由细胞周期蛋白依赖性激酶（CDKs）与磷酸酶协同调控。在裂殖酵母中，Cdk9可磷酸化Spt5的羧基末端重复序列（carboxy-terminal repeats, CTRs），并在转录延伸过程中抑制蛋白磷酸酶1（PP1）。当转录穿过切割与多聚腺苷酸化信号（CPS）时，会伴随PP1依赖的Spt5去磷酸化，并使Pol II停留在带有磷酸化CTD丝氨酸2位点（pSer2）的状态。本研究证实该调控开关在人类中保守存在：抑制Cdk9可同时降低PP1γ及Spt5苏氨酸806位点磷酸化形式（pThr806）的水平，并诱导CPS上游区域出现pSer2信号；而敲低PP1则会提升pThr806的水平。此外，在未受干扰的细胞中，3’端暂停复合物内的pThr806水平会降低，而该区域恰是pSer2水平最高的位点。Cdk9还可磷酸化Spt5的丝氨酸666位点（Ser666），该位点位于保守的KOW4与KOW5基序之间，且为PP1不敏感位点；pSer666水平会在启动子近端暂停释放后升高，且与pThr806不同，其信号会持续至CPS下游区域。本研究鉴定出PP4作为pSer666的磷酸酶，而PP4亦是Cdk9抑制性磷酸化的另一作用靶标。PP4与PP1γ分别富集于基因的5’端与3’端，这与pSer666及pThr806的分布特征相符。因此，在转录的不同阶段，Cdk9与不同磷酸酶构成的调控开关可分别作用于Spt5。实验整体设计：染色质免疫共沉淀测序（ChIP-seq）：本数据集包含19组针对人类结肠癌细胞（HCT116）的ChIP-seq实验数据，均设置生物学重复；所有生物学重复样本均提供原始测序文件与处理后的bigWig格式文件。