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

• 批准号: 8806563
• 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
• 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/8806563
• 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; Health; 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; genetic approach; genome-wide; human disease; in vivo; mutant; novel; novel strategies; prevent; promoter; reconstitution; research study; small molecule; transcription factor TFIIE; transcription factor TFIIH; transcription termination

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的细胞周期蛋白依赖性激酶 (CDK) 的顺序功能，但排序这些功能的机制仍在出现。我们采用化学遗传学方法——用类似物敏感（AS）突变体CDK替代野生型——以揭示CDK网络在转录中的“早期”和“晚期”功能。首先，通过选择性抑制 Cdk7——转录起始因子TFIIH的一个组成部分——在人类细胞中，我们发现了两个意想不到的、看似对立的功能。 Cdk7 活性需要招募通过 Pol II 建立启动子近端暂停的因子，并激活 Cdk9（正转录延伸因子 b (P-TEFb) 的催化亚基，从而释放暂停）。因此，CDK 网络似乎依赖于不相干的前馈来提高 Pol II 延伸的瞬时动力学障碍，从而创建一个时间窗口来招募 mRNA 加工和染色质修饰机制。与 CDK 指导的暂停以确保忠实的 RNA 加工的要求一致，抑制 Cdk7 或 Cdk9 会导致 Pol II 转录物的终止和 3' 端成熟缺陷。其次，在 Cdk9 底物的化学遗传筛选中，我们鉴定了多种参与 RNA 5' 端脱帽和转录终止“鱼雷”途径的蛋白质，最近有人认为该途径会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的调节，以揭示 CDK 基因调节的新模式。具体目标是： 1. 确定 CDK 在起始-延伸转变时的功能和靶标 2. 研究 Cdk9 靶标化学遗传筛选中发现的 P-TEFb 对转录终止途径的可能调节 3. 通过化学遗传学剖析人类细胞中 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；我们目标的完成将阐明 Cdk7 和 Cdk9 如何协作以确保该周期各阶段之间的单向过渡。通过将 CDK 转变为可以用定制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。