Chemical Genetics of Transcriptional Regulation by CDKs in Human Cells

人类细胞中 CDK 转录调控的化学遗传学

基本信息

批准号：
9198169
负责人：
ROBERT P FISHER
金额：
$ 32.21万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-02-15 至 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9198169
关键词：
Antineoplastic Agents Antiviral Agents Archaeal RNA Binding Catalytic Domain Cell Cycle Progression Cell Proliferation Cell division Cells Chemicals Chromatin Colon Carcinoma Custom Cyclin A Cyclin-Dependent Kinases DNA Polymerase II DNA-Directed RNA Polymerase Defect Development Drug Targeting Elongation Factor Ensure Enzymes Eukaryota Evaluation Fission Yeast Gene Expression Gene Expression Regulation Genes Genetic Genetic Screening Genetic Transcription Genome Human Human Development Human Engineering Impairment In Vitro Individual Kinetics Label Malignant Neoplasms Messenger RNA Modeling Normal tissue morphology Organism Orthologous Gene Pathway interactions Pattern Peptide Initiation Factors Pharmaceutical Preparations Phase Phosphorylation Phosphotransferases Play Positive Transcriptional Elongation Factor B Property Proteins RNA RNA Processing RNA chemical synthesis Recruitment Activity Regulation Role Signal Transduction Testing Time Torpedo Transcript Transcription Initiation Transcriptional Regulation Variant Viral Yeasts adenine analog analog base cancer cell chemical genetics chemotherapy chromatin modification chromatin protein crosslink cyclin T1 cyclin-dependent kinase-activating kinase experimental study feeding genetic approach genome-wide human disease in vivo mutant novel novel strategies prevent promoter public health relevance reconstitution small molecule transcription factor TFIIE transcription factor TFIIH transcription termination

项目摘要

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.

描述（由申请人提供）：RNA聚合酶II（POL II）的转录周期取决于不同依赖细胞周期蛋白依赖性激酶（CDKS）的顺序函数，但是订购这些功能的机制仍在出现。我们采用了对野生型的化学遗传方法的替代，具有模拟敏感的（AS）突变cdks，揭示了CDK网络在转录中的“早期”和“晚”功能。首先，通过选择性抑制 CDK7-转录起始因子TFIIH-IN人类细胞的组成部分，我们发现了两个意外且看似拮抗的功能。需要CDK7活性来募集通过POL II建立启动子抗性暂停的因子，并激活CDK9，即阳性转录伸长因子B（P-TEFB）的催化亚基，该因子释放了停顿。因此，CDK网络似乎取决于不连贯的馈电，从而将瞬时动力学屏障引起到Pol II的伸长率，从而创建了一个时间窗口，以募集mRNA-GOCRIDENT和CHROMATIN-MOTIFERIDIDENS机制。一致的与CDK定向暂停的要求，以确保忠实的RNA处理，抑制CDK7或CDK9会导致终止缺陷和POL II转录本的3'-End成熟。其次，在CDK9底物的化学遗传筛选中，我们确定了与RNA 5'-末端脱皮和转录终止的“鱼雷”途径有关的多种蛋白质，最近有人建议它影响暂停和不同的反义转录。我们将剖析POL II的启动 - 延长跃迁，并调节转录终止和极性的Elucidte调节，以发现CDK的新型基因调节模式。具体目的是：1。要在启动 - 延误转换2中识别CDK的功能和目标2。研究可能通过P-TEFB对转录终止途径进行调控，在化学遗传筛选中发现CDK9目标3。以化学细胞中的CDK7和CDK9的功能在人类细胞中剖析CDK7和CDK9的功能。我们的研究揭示了Pol II转录周期核心的CDK级联反应。我们的目标的完成将阐明CDK7和CDK9如何协作以确保该周期阶段之间的单向过渡。通过将CDK变成可以用定制的小分子来操纵的化学开关，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标。