Mechanisms of the TFIIH-associated kinase CDK7 in p53-dependent transcriptional regulation

项目总结 RNA聚合酶II(Pol II)转录受多种因素的协同作用调节，包括 10亚基复合转录因子IIH(TFIIH)。TFIIH含有一个激酶亚基CDK7，它已经获得了 注意作为侵袭性、转移性癌症的治疗靶点，包括多形性胶质母细胞瘤和三重胶质母细胞瘤 阴性乳腺癌。CDK7在这些癌症中表达上调，它们对CDK7易感 抑制力。为了进一步了解CDK7在癌症中的作用，需要对CDK7的功能进行彻底的了解 以及它作为抗癌治疗靶点的前景。CDK7使C端结构域(CTD)磷酸化 POL II，以仍然知之甚少的方式促进从转录起始到延伸的转变。 POL II CTD的磷酸化对于RNA加工因子的正确招募也是至关重要的。未出版的作品在 我们的实验室表明，CDK7也可能控制Pol II启动子-近端的暂停和剪接。启动子-近端 POL II暂停是一个主要的调节检查点，涉及暂停因素NELF、DSIF和P-TEFb。核糖核酸 剪接是一种高度调控的过程，需要SF3B1，U2 SnRNP复合体的一种成分，以及 关联系数U2AF2，用于正确选择分支点。对这两个转录过程的调控 (Pol II停顿和剪接)在癌症中通常被破坏。在这里，我建议从机械的角度来洞察 1)CDK7如何调控POL II启动子-近端停顿和2。)CDK7如何通过使用 体外技术和基于细胞的技术相结合。我的第一个目标是研究CDK7的磷酸化 通过暂停因子NELF、dSIF和P-2的磷酸化来调节POL II启动子近端的暂停 TEFb.我将测试一个有初步数据支持的模型，在该模型中，CDK7)通过直接释放Pol II暂停 NELF和dSIF的磷酸化以及II)激活P-TEFb使NELF和dSIF磷酸化。我会利用 重组的体外转录分析(含纯化的人因子，不含提取物)以探讨CDK7激酶如何 Pol II启动子--近端停顿和/或启动子逃逸通过停顿的磷酸化调节活性 因素和波尔II的CTD。这些体外实验结果将在下文中通过PRO-SEQ实验进一步探讨 人类细胞中的P53反应。我的第二个目标是研究CDK7如何通过 U2 SnRNP剪接因子SF3B1及其相关因子U2AF2的磷酸化。SILAC-MS实验 (未发表)确定SF3B1和U2AF2为高置信度CDK7目标，这一点得到了In 体外激活酶分析。在P53诱导后，使用RNA-Seq和ChIP-Seq，我将测试CDK7 可能在活跃的剪接位点上富含，并可能通过SF3B1和U2AF2的磷酸化来调节剪接。 此外，体外微量热浸(MST)结合分析将提供对 CDK7磷酸化在SF3B1：U2AF2相互作用中的潜在作用。从这些中获得的机械洞察力 实验将促进对CDK7调节作用的理解，并可能导致更有针对性的治疗 治疗依赖CDK7的癌症的方法。

PROJECT SUMMARY RNA Polymerase II (Pol II) transcription is regulated through the concerted action of a variety of factors including the 10-subunit complex transcription factor IIH (TFIIH). TFIIH contains a kinase subunit, CDK7, which has gained attention as a therapeutic target in aggressive, metastatic cancers, including glioblastoma multiforme and triple negative breast cancer. CDK7 expression is upregulated in these cancers, and they are vulnerable to CDK7 inhibition. A thorough understanding of CDK7 function is needed to gain further insight into its roles in cancer and its prospects as an anti-cancer therapeutic target. CDK7 phosphorylates the C-terminal domain (CTD) of Pol II, facilitating the transition from transcription initiation to elongation in ways that remain poorly understood. Pol II CTD phosphorylation is also vital for proper recruitment of RNA processing factors. Unpublished work in our lab indicates that CDK7 may also control Pol II promoter-proximal pausing and splicing. Promoter-proximal Pol II pausing is a major regulatory checkpoint involving the pausing factors NELF, DSIF, and P-TEFb. RNA splicing is a highly regulated process that requires SF3B1, a component of the U2 snRNP complex, and the associated factor U2AF2 for proper branch point selection. Regulation of these two transcriptional processes (Pol II pausing and splicing) is commonly disrupted in cancer. Here, I propose to gain mechanistic insight into 1.) how CDK7 regulates Pol II promoter-proximal pausing and 2.) how splicing is regulated by CDK7 using a combination of in vitro and cell-based techniques. My first aim will examine how CDK7 phosphorylation regulates Pol II promoter-proximal pausing through phosphorylation of the pausing factors NELF, DSIF, and P- TEFb. I will test a model, supported by preliminary data, in which CDK7 i) releases Pol II pausing through direct phosphorylation of NELF and DSIF and ii) activates P-TEFb to phosphorylate NELF and DSIF. I will utilize reconstituted in vitro transcription assays (with purified human factors, no extracts) to probe how CDK7 kinase activity regulates Pol II promoter-proximal pausing and/or promoter escape through phosphorylation of pausing factors and the Pol II CTD. These in vitro findings will be probed further with PRO-Seq experiments in the context of p53 response in human cells. My second aim will investigate how CDK7 may regulate RNA splicing through phosphorylation of the U2 snRNP splicing factor SF3B1 and its associated factor U2AF2. SILAC-MS experiments (unpublished) identified SF3B1 and U2AF2 as high-confidence CDK7 targets, which was further confirmed by in vitro kinase assays. Using RNA-Seq and ChIP-Seq following p53 induction, I will test a model in which CDK7 may be enriched at actively spliced loci and may regulate splicing through phosphorylation of SF3B1 and U2AF2. Furthermore, in vitro microscale thermophoresis (MST) binding assays will provide mechanistic insight into the potential role of CDK7 phosphorylation on SF3B1:U2AF2 interactions. The mechanistic insight gained from these experiments will advance understanding of CDK7 regulatory roles and may lead to more targeted therapeutic approaches to treat cancers that exhibit a CDK7 dependence.