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

TFIIH 相关激酶 CDK7 在 p53 依赖性转录调控中的机制

• 批准号: 10116163
• 负责人: Jenna Kathleen Rimel
• 金额: $ 3.44万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2022-01-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10116163
• 关键词: Address; Affect; Attention; Binding; Biological Assay; C-terminal; Cancer Biology; Cancerous; Cells; ChIP-seq; Complex; Cyclin-Dependent Kinases; DNA Polymerase II; DNA-Directed RNA Polymerase; Data; Defect; Dependence; Disease; Disseminated Malignant Neoplasm; Exhibits; Gene Expression; Genes; Genetic Transcription; Glioblastoma; HCT116 Cells; Human; In Vitro; Lead; Link; Malignant Neoplasms; Modeling; Pathogenesis; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Positive Transcriptional Elongation Factor B; Process; RNA Processing; RNA Splicing; Reagent; Regulation; Role; Running; Specificity; TP53 gene; Techniques; Testing; Therapeutic; Transcription Elongation; Transcription Initiation; Transcription Process; Transcriptional Regulation; U2 Small Nuclear Ribonucleoprotein; Work; addiction; anti-cancer therapeutic; base; cancer type; experimental study; genome-wide; inhibitor/antagonist; insight; negative elongation factor; novel; promoter; reconstitution; recruit; response; targeted treatment; therapeutic target; transcription factor; transcription factor TFIIH; transcriptome sequencing; transcriptomics; triple-negative invasive breast carcinoma; unpublished works

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 Unpublished work in our lab indicates that CDK7 may also control Pol II promoter-proximal pausing and splicing.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.

项目摘要 RNA聚合酶II（Pol II）转录通过多种因子的协同作用来调节，包括 10-亚基复合转录因子IIH（TFIIH）。TFIIH含有一个激酶亚基CDK 7， 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 阴性乳腺癌。CDK 7表达在这些癌症中上调，并且它们对CDK 7易感。 抑制作用需要彻底了解CDK 7的功能，以进一步了解其在癌症中的作用 及其作为抗癌治疗靶点的前景。CDK 7磷酸化的C-末端结构域（CTD） Pol II，促进从转录起始到延伸的过渡，其方式仍然知之甚少。 Pol II CTD磷酸化对于RNA加工因子的适当募集也至关重要。未发表的作品 我们的实验室表明，CDK 7也可以控制Pol II启动子近端的暂停和剪接。启动子近端 Pol II暂停是涉及暂停因子NELF、DSIF和P-TEFb的主要调节检查点。RNA 剪接是一个高度调节的过程，需要SF 3B 1，U2 snRNP复合物的组分，和 相关系数U2 AF 2，用于适当的分支点选择。这两个转录过程的调控 (Pol II暂停和剪接）通常在癌症中被破坏。在这里，我建议获得机械的洞察力， 1.）的人。CDK 7如何调节Pol II启动子近端暂停和2.）剪接是如何被CDK 7调控的， 结合体外和细胞技术。我的第一个目标是研究CDK 7磷酸化 通过暂停因子NELF、DSIF和P-1的磷酸化调节Pol II启动子近端暂停。 TEFb。我将测试一个由初步数据支持的模型，其中CDK 7 i）通过直接 ii）活化P-TEFb以磷酸化NELF和DSIF。我会利用 重建的体外转录测定（使用纯化的人因子，无提取物）来探测CDK 7激酶如何 活性调节Pol II启动子近端暂停和/或通过暂停的磷酸化的启动子逃逸 Pol II CTD。这些体外发现将通过PRO-Seq实验进一步探讨 p53在人体细胞中的反应。我的第二个目标是研究CDK 7如何通过调节RNA剪接， U2 snRNP剪接因子SF 3B 1及其相关因子U2 AF 2的磷酸化。SILAC-MS实验 （未发表）将SF 3B 1和U2 AF 2确定为高置信度的CDK 7靶点， 体外激酶测定。在p53诱导后使用RNA-Seq和ChIP-Seq，我将测试一个模型，其中CDK 7 可能在活跃剪接位点富集，并可能通过SF 3 B1和U2 AF 2的磷酸化调节剪接。 此外，体外微量热泳（MST）结合测定将提供对细胞内蛋白质结合的机制性洞察。 CDK 7磷酸化对SF 3B 1：U2 AF 2相互作用的潜在作用。从这些中获得的机械论见解 实验将促进对CDK 7调节作用的理解，并可能导致更有针对性的治疗。 治疗表现出CDK 7依赖性的癌症的方法。