Chemical Genetics of Transcriptional Regulation by CDKs in Human Cells

描述(申请人提供)：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)的顺序功能，但对这些功能进行排序的机制仍在研究中。我们采用了化学遗传学的方法--用类似物敏感(AS)突变的CDK替换野生型--来揭示CDK网络在转录中的“早期”和“晚期”功能。首先，通过选择性地抑制 CDK7-转录起始因子TFIIH的一个成分-在人类细胞中，我们发现了两个意想不到的和似乎是拮抗的功能。CDK7的活性需要招募由POL II建立启动子近端停顿的因子，并激活CDK9，正转录延伸因子b(P-TEFb)的催化亚单位，从而释放停顿。因此，CDK网络似乎依赖于非相干前馈来提高Pol II伸长的瞬时动力学障碍，从而创建一个时间窗口来招募mRNA处理和染色质修改机制。与CDK指导的暂停以确保RNA的忠实加工的要求一致，抑制CDK7或CDK9会导致Pol II转录本的终止和3‘端成熟的缺陷。其次，在对CDK9底物的化学遗传筛选中，我们发现了与RNA5‘端解链和转录终止的“鱼雷”途径有关的多种蛋白质，最近被认为影响暂停和发散反义转录。我们将剖析POL II的起始-延伸转变，阐明转录终止和极性的调节，以揭示CDKs基因调控的新模式。其具体目的是：1.确定CDKs在起始-延伸过程中的功能和靶点；2.研究CDK9靶点的化学遗传学筛选中发现的P-TEFb对转录终止途径的可能调控；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；我们目标的完成将阐明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转化为可以用定制的小分子操纵的化学开关，我们将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。

DESCRIPTION (provided by applicant): 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 feed forward 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 elucidte 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.