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：
8630081
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 2017-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8630081
关键词：
Antineoplastic Agents Antiviral Agents Archaeal RNA Binding Catalytic Domain Cell Cycle Progression Cell Proliferation Cell division Cells Chemicals Chromatin Colon Carcinoma 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 Screening Genetic Transcription Genome Human Human Development Human Engineering Human Identifications 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 Polymerase Positive Transcriptional Elongation Factor B Process Property Proteins RNA RNA Polymerase II RNA Processing RNA chemical synthesis Reading Recruitment Activity Regulation Role Signal Transduction Staging Testing Time Torpedo Transcript Transcription Elongation 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 feeding genome-wide human disease in vivo mutant novel novel strategies prevent promoter public health relevance reconstitution research study small molecule transcription factor TFIIE transcription factor TFIIH transcription termination

项目摘要

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.

项目概要 RNA 聚合酶 II (Pol II) 的转录周期取决于以下功能的顺序：不同的细胞周期蛋白依赖性激酶 (CDK)，但控制这些功能的机制仍在不断涌现。我们采取了化学遗传方法——替代野生—— 具有模拟敏感 (AS) 突变体 CDK 的类型 - 揭示“早期”和“晚期”功能 CDK 网络在转录中的作用。一、通过选择性抑制Cdk7-a成分在人类细胞中转录起始因子 TFIIH 的研究中，我们发现了两个意想不到的、和看似对立的功能。 Cdk7 活性是招募因子所必需的通过 Pol II 建立启动子近端暂停，并激活 Cdk9（催化亚基）正转录延伸因子 b (P-TEFb)，可释放暂停。所以 CDK 网络似乎依赖于不相干的前馈来引发瞬态 Pol II 延伸的动力障碍，从而创建一个时间窗口来招募 mRNA- 加工和染色质修饰机械。符合 CDK 的要求- 定向暂停以确保忠实的 RNA 加工，抑制 Cdk7 或 Cdk9 会导致 Pol II 转录本的终止和 3' 端成熟缺陷。其次，在化学品中—— 通过对 Cdk9 底物的遗传筛选，我们鉴定了多种参与 RNA 5'- 的蛋白质末端脱帽和转录终止的“鱼雷”途径，最近被认为影响反义转录的暂停和发散。我们将剖析 Pol II 的起始-延伸转变，并阐明转录终止和极性，通过以下方式揭示基因调控的新模式 CDK。具体目标是： 1. 确定CDK在起始-延伸转变过程中的功能和靶标 2. 研究P-转录终止途径的可能调控 TEFb，在 Cdk9 靶标的化学遗传筛选中发现 3. 通过化学遗传学剖析Cdk7和Cdk9在人体细胞中的功能我们的研究揭示了 Pol II 转录周期核心的 CDK 级联；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 转化为化学物质可以用定制的小分子操纵的开关，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。