A PP2A-Integrator complex fine-tunes transcription by opposing CDK9 [3UTR-seq]

Gene expression by RNA Polymerase II (RNAPII) is tightly controlled by Cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The RNAPII pausing checkpoint, engaged after transcription initiation, is controlled by CDK9 to regulate transcription in metazoans. We discovered that CDK9-mediated RNAPII pause-release is functionally opposed by a protein phosphatase 2A (PP2A) complex. PP2A dynamically competes for key CDK9 substrates, DSIF and RNAPII-CTD, and is recruited to transcription pausing sites by the Integrator complex subunit INTS6. INTS6 depletion confers resistance to CDK9 inhibition in a variety of normal and tumor cell lines. Loss of INTS6 abolishes the Integrator-PP2A association leading to unrestrained CDK9 activity, which amplifies transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill MLL-rearranged leukemias and solid tumors and provide therapeutic benefit in vivo. These data demonstrate that f inely-tuned gene expression relies on the balance of kinase and phosphatase activity at the pausing checkpoint. Overall design: We performed 3'RNA-seq on RNA isolated from THP-1 cells expressing Cas9 and non-targeting or INTS6 targeting sgRNAs which had been treated with LPS to perform differential gene expression analysis. We also performed 3'RNA-seq on RNA isolated from HT-29 cells treated with a CDK9 inhibitor (AZ5576) or a PP2A agonist (DBK-1154) to perform differential gene expression analysis.

RNA聚合酶II（RNAPII）介导的基因表达，在转录周期的多个离散检查点处受细胞周期蛋白依赖性激酶（CDKs）的严格调控。转录起始后激活的RNA聚合酶II暂停检查点，在后生动物中由CDK9调控以控制转录过程。本研究发现，CDK9介导的RNAPII暂停释放，可被一种蛋白磷酸酶2A（PP2A）复合物在功能上拮抗。PP2A通过竞争性结合关键的CDK9底物DSIF与RNAPII-CTD，并由整合子复合物亚基INTS6招募至转录暂停位点。在多种正常及肿瘤细胞系中，INTS6缺失会使细胞对CDK9抑制产生抗性。INTS6的丢失会破坏整合子-PP2A复合物的结合，导致CDK9活性不受调控，进而放大转录应答过程。药物性激活PP2A可与CDK9抑制剂协同作用，杀伤MLL重排白血病与实体瘤，并在体内发挥治疗益处。上述数据表明，精细调控的基因表达依赖于暂停检查点处激酶与磷酸酶活性的动态平衡。

实验设计概述：本研究对携带Cas9、转染非靶向或INTS6靶向sgRNA的THP-1细胞提取RNA，进行3'端RNA测序（3'RNA-seq），以开展差异基因表达分析，其中细胞均经脂多糖（LPS）处理。此外，本研究还对经CDK9抑制剂（AZ5576）或PP2A激动剂（DBK-1154）处理的HT-29细胞提取RNA，进行3'端RNA测序以完成差异基因表达分析。