How do CDK7 and CDK9 regulate Pol II initiation and pausing?

项目摘要 转录抑制已被确定为许多形式的转录调控中的显著弱点和可利用的靶标。 侵袭性转移癌调节转录起始早期阶段的转录激酶， 在侵袭性癌症如三阴性乳腺癌和多发性骨髓瘤中， 来全面上调转录。转录激酶抑制剂的临床前和I期临床试验 已经显示出治疗癌症的前景。这些激酶，CDK 7和CDK 9，磷酸化RNA聚合酶 II（Pol II）C-末端结构域（CTD）和相关因子调节Pol II起始和启动子近端 暂停（暂停）。暂停是转录的限速步骤，在癌症中必须绕过才能上调 转录，将CDK 7和CDK 9带到癌症治疗的最前沿。然而，进一步的药物开发- 由于这些CDK缺乏明确的机制，这一进展受到阻碍。以前的实验未能 定义由于1）使用粗体外系统（例如核提取物）导致的每种激酶的机制贡献 或2）在基于细胞的测定中使用非特异性、混杂激酶抑制剂。在这里，我建议描述 CDK 7和CDK 9调节启动子近端暂停的机制，使用独特的体外重建 转录测定和最先进的基于细胞的测定与特异性的下一代抑制剂组合。我 该项目将有三个不同的目标，1）解剖CDK 7和CDK 9在暂停中的单独贡献，2） 表征影响暂停的激酶共调节事件，以及3）评估 细胞中CDK 7/CDK 9暂停调节。前两个将利用Taatjes实验室的体外转录系统， 是由纯化的人类因子重组而成该系统超越了传统的体外转录实验 它能够明确地询问个体因素。我将确定CDK 7的独立角色， 在体外转录测定中通过抑制每种激酶暂停调节CDK 9。协同调节和 激酶之间的协同作用将用组合联合收割机特异性抑制剂的类似测定法来评价。最后我 将结合联合收割机精确运行测序（PRO-seq）与下一代CDK 7和CDK 9抑制剂， 这些激酶在细胞中的作用，并验证我的体外工作。从这次实验中获得的见解将 提供开发副作用最小的癌症治疗所需的机制理解。

PROJECT SUMMARY Transcription inhibition has been identified as a significant weakness and exploitable target in many forms of aggressive metastatic cancer. Transcriptional kinases that regulate the early stages of transcription initiation and elongation are overexpressed in aggressive cancers such as triple-negative breast cancer and multiple myeloma to globally upregulate transcription. Preclinical and phase I clinical trials with transcriptional kinase inhibitors have shown promise for treating cancer. These kinases, CDK7 and CDK9, phosphorylate the RNA Polymerase II (Pol II) C-terminal domain (CTD) and associated factors to regulate Pol II initiation and promoter-proximal pausing (pausing). Pausing is the rate-limiting step in transcription and must be bypassed in cancer to upregulate transcription, bringing CDK7 and CDK9 to the forefront of cancer therapeutics. However, further drug develop- ment has been impeded by the lack of a defined mechanism for these CDKs. Previous experimentation failed to define the mechanistic contributions of each kinase due to 1) use of crude in vitro systems (e.g. nuclear extracts) or 2) the use of non-specific, promiscuous kinase inhibitors in cell-based assays. Here, I propose to describe the mechanism of CDK7 and CDK9 regulation of promoter-proximal pausing using a unique reconstituted in vitro transcription assay and state-of-the-art cell-based assays combined with specific, next-generation inhibitors. My project will have three distinct goals, 1) dissecting the individual contributions of CDK7 and CDK9 in pausing, 2) characterizing kinase co-regulatory events that influence pausing, and 3) evaluating the mechanism of CDK7/CDK9 pause regulation in cells. The first two will utilize the Taatjes Lab’s in vitro transcription system that is reconstituted from purified human factors. This system surpasses traditional in vitro transcription experiments in its ability to unequivocally interrogate individual factors. I will determine the independent roles of CDK7 and CDK9 in pause regulation through inhibition of each kinase in in vitro transcription assays. Coregulation and cooperation between the kinases will be evaluated with similar assays that combine specific inhibitors. Finally, I will combine precision run-on sequencing (PRO-seq) with next-generation CDK7 and CDK9 inhibitors to assess the role of these kinases in cells and validate my in vitro work. The insights gained from this experimentation will provide the mechanistic understanding needed to develop cancer therapeutics with minimal side effects.