Chemical Genetics of Transcriptional Regulation by CDKs in Human Cells

项目摘要 RNA聚合酶II(POL II)的转录周期依赖于 不同的细胞周期蛋白依赖性激酶(CDK)，但对这些功能进行排序的机制 仍在不断涌现。我们采取了化学遗传的方法-取代野生的- 带有模拟敏感型(AS)突变的CDK--同时显示“早”和“晚”功能 转录中的CDK网络。首先，通过选择性地抑制CDK7-a成分 在人类细胞中，我们发现了两个意想不到的， 和看似对立的功能。需要CDK7活性才能招募以下因素 通过POL II建立启动子-近端停顿，并激活CDK9催化亚基 正转录延伸因子b(P-TEFb)，释放暂停。因此 CDK网络似乎依赖于非相干前馈来引发瞬变 Pol II伸长的动力学障碍，从而创建一个时间窗口来招募mRNA- 加工和染色质修饰机械。与CDK的要求一致- 定向暂停以确保忠实的RNA处理，抑制CDK7或CDK9导致 Pol II转录本的终止和3‘端成熟缺陷。第二，在化学物质中- 对于CDK9底物的遗传筛选，我们鉴定了RNA5‘-参与的多种蛋白质 末端截断和转录终止的“鱼雷”途径，它具有 最近被认为影响停顿和发散的反义转录。我们 将解剖POL II的起始-延伸转变，并阐明其调节 转录终止和极性，以揭示基因调控的新模式 CDK。具体目标是： 1.确定CDKs在起始-伸长转换中的功能和靶点 2.研究P-2对转录终止途径的可能调节作用 在CDK9靶标的化学基因筛查中发现的TEFb 3.从化学遗传学角度剖析CDK7和CDK9在人类细胞中的功能 我们的研究揭示了CDK级联在POL II转录周期的核心； 完成我们的目标将阐明CDK7和CDK9如何合作以确保 该周期各阶段之间的单向转换。通过将CDK转化为化学物质 可以用定制的小分子操纵的开关，我们会区分 它们的特定作用和底物，并揭示新的抗癌或抗病毒药物靶点。

Project Summary The transcription cycle of RNA polymerase II (Pol II) depends on sequential functions of distinct cyclin-dependent kinases (CDKs), but the mechanisms that order those functions are still emerging. We have taken a chemical-genetic approach-replacement of wild- type with analog-sensitive (AS) mutant CDKs-to reveal both "early" and "late" functions of the CDK network in transcription. First, by selective inhibition of Cdk7-a component of transcription initiation factor TFIIH-in human cells, we uncovered two unexpected, and seemingly antagonistic functions. Cdk7 activity is required to recruit factors that establish a promoter-proximal pause by Pol II, and to activate Cdk9, catalytic subunit of positive transcription elongation factor b (P-TEFb), which releases the pause. Therefore the CDK network appears to depend on incoherent feedforward to raise a transient kinetic barrier to Pol II elongation, and thus create a temporal window to recruit mRNA- processing and chromatin-modifying machinery. Consistent with a requirement for CDK- directed pausing to ensure faithful RNA processing, inhibition of Cdk7 or Cdk9 leads to defects in termination and 3'-end maturation of Pol II transcripts. Second, in a chemical- genetic screen for Cdk9 substrates, we identified multiple proteins involved in RNA 5'- end decapping and the "torpedo" pathway of transcription termination, which has recently been suggested to influence pausing and divergent antisense transcription. We will dissect the initiation-elongation transition of Pol II, and elucidate regulation of transcription termination and polarity, to uncover novel modes of gene regulation by CDKs. The specific aims are: 1. To identify functions and targets of CDKs at the initiation-elongation transition 2. To investigate possible regulation of the transcription termination pathway by P- TEFb, uncovered in a chemical genetic screen for Cdk9 targets 3. To dissect functions of Cdk7 and Cdk9 by chemical genetics in human cells Our studies reveal a CDK cascade at the core of the Pol II transcription cycle; completion of our aims will illuminate how Cdk7 and Cdk9 collaborate to ensure unidirectional transitions between phases of that cycle. By turning CDKs into chemical switches that can be manipulated with customized small molecules, we will distinguish their specific roles and substrates, and reveal new anti-cancer or anti-viral drug targets.