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: 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
• 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

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.

项目摘要 RNA聚合酶II（Pol II）的转录周期取决于RNA聚合酶的序列功能。 不同的细胞周期蛋白依赖性激酶（CDKs），但这些功能的机制， 仍在浮现我们采取了化学遗传学的方法-取代野生的- 用类似物敏感（AS）突变型CDK-揭示“早期”和“晚期”功能 CDK网络在转录中的作用。首先，通过选择性抑制Cdk 7-a组分 转录起始因子TFIIH-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cdk 7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Cdk 9， 正转录延伸因子B（P-TEF B），其释放暂停。因此 CDK网络似乎依赖于非相干前馈来产生瞬态 Pol II延长的动力学屏障，并因此产生了募集mRNA的时间窗口- 加工和染色质改性机械。符合CDK的要求- 定向暂停以确保忠实的RNA加工，Cdk 7或Cdk 9的抑制导致 Pol II转录物的终止和3 '端成熟缺陷。第二，在一种化学物质中- 通过对Cdk 9底物的遗传筛选，我们鉴定了参与RNA 5 '- 末端去帽和转录终止的“鱼雷”途径， 最近被认为影响暂停和分歧反义转录。我们 将剖析Pol II的起始-延伸转变，并阐明 转录终止和极性，以揭示新的基因调控模式， CDK。具体目标是： 1.确定CDK在起始-延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控作用。 TEFb，在Cdk 9靶点的化学遗传筛选中发现 3.用化学遗传学方法剖析Cdk 7和Cdk 9在人类细胞中的功能 我们的研究揭示了在Pol II转录周期的核心处的CDK级联; 我们的目标的完成将阐明Cdk 7和Cdk 9如何合作，以确保 在该循环的阶段之间的单向转换。通过将CDK转化为化学物质 开关，可以操纵定制的小分子，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。