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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）的转录周期依赖于不同的周期蛋白依赖性激酶（CDKs）的顺序功能，但这些功能的排序机制仍在不断出现。我们采用了化学遗传学方法——用类似物敏感（AS）突变CDK替代野生型CDK——来揭示CDK网络在转录中的“早期”和“晚期”功能。第一，通过选择性抑制