TFIIH and Transcription Regulation

TFIIH 和转录调控

• 批准号: 9177084
• 负责人: JEFFREY A RANISH
• 金额: $ 55.94万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-22 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9177084
• 关键词: ATP phosphohydrolase; Active Sites; Alternative Splicing; Architecture; Biological Assay; C-terminal; CDK9 Protein Kinase; Cells; ChIP-seq; Chemicals; Complex; Crosslinker; DNA; DNA Polymerase II; Data; Defect; Development; Disease; ERCC3 gene; Enzymes; Evaluation; Event; Genes; Genetic Transcription; Goals; Growth and Development function; Heart Diseases; Human; Human Cell Line; Human Development; In Vitro; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediator of activation protein; Messenger RNA; Methods; Molecular; Pharmaceutical Preparations; Phosphotransferases; Play; Polymerase; Positive Transcriptional Elongation Factor B; Process; Protein Complex Subunit; Proteins; Proteomics; RNA; RNA Polymerase II; RNA Processing; RNA Splicing; Regulation; Research Personnel; Resolution; Role; Series; Single-Stranded DNA; Site; Skin Cancer; Staging; Structure; Syndrome; TP53 gene; Techniques; Testing; Therapeutic; Transcription Factor TFIIA; Transcription Factor TFIIB; Transcription Initiation; Transcription Process; Transcriptional Activation; Transcriptional Regulation; Yeasts; base; cell growth; crosslink; genome editing; genome-wide; global run on sequencing; human disease; in vivo; inhibitor/antagonist; insight; melting; mutant; nervous system disorder; next generation; permanganate; promoter; protein protein interaction; reconstitution; research study; response; screening; skin disorder; therapeutic target; three-dimensional modeling; transcription factor TFIIE; transcription factor TFIIF; transcription factor TFIIH; transcriptome sequencing; translocase; yeast genetics

SUMMARY Regulation of transcription by RNA polymerase II (pol II) is essential to control cell growth, differentiation and development. Among the factors required for genome-wide pol II transcription is the evolutionarily conserved, 10-subunit TFIIH complex, which plays fundamental roles in multiple steps of the transcription process including promoter opening and mRNA processing; however, the molecular mechanisms remain unclear. The long term goal of this project is to elucidate the molecular mechanisms by which TFIIH regulates pol II transcription. The rationale for this project is that understanding these mechanisms will reveal the molecular bases for multiple and diverse developmental and somatic diseases, including neurological disorders, progeroid syndromes, skin diseases and cancer. The objectives of this collaborative project are to establish a structural and mechanistic understanding of how TFIIH controls basic transcriptional events such as promoter opening, pol II pausing, and mRNA processing. We recently completed structural analysis of native TFIIH complexes (both human and yeast) using chemical crosslinking/mass spectrometry (CXMS) and 3D modeling. The results predict two distinct, conserved protein-protein interaction networks involving the essential enzymatic subunits XPB/Ssl2 and CDK7/Kin28. The ATPase/translocase XPB/Ssl2 is required for transcription initiation, as it melts the promoter DNA template to allow single-stranded DNA to engage the pol II active site. The CDK7/Kin28 kinase phosphorylates the C-terminal domain (CTD) of the pol II large subunit as well as other key substrates (e.g. P-TEFb) that collectively enable precise regulation of pol II initiation, pausing, and mRNA processing. In Aim 1 we will investigate how the XPB/Ssl2 ATPase/translocase is regulated by an interaction network involving p52/Tfb2 and p8/Tfb5; in Aim 2 we will investigate how specific residues and domains within CCNH/Ccl1 and MAT1/Tfb3 govern CDK7/Kin28 kinase activity. To accomplish these goals, we will utilize a powerful combination of yeast genetics, site-directed insertion of UV-responsive crosslinkers, and in vivo functional screens to structurally refine and functionally validate protein-protein interfaces within TFIIH. These data will guide a series of in vitro and cell-based assays in human cells that will rigorously test the roles of specific molecular interfaces in the regulation of TFIIH function in transcription, genome-wide. We will employ methods that are well-established by the PIs and our collaborators, including reconstituted in vitro transcription, permanganate footprinting (in vitro and in cells), GRO-Seq, ChIP or ChIP-Seq, and RNA-Seq. If successful, these experiments will allow us to link a given protein-protein interface to regulation of specific transcription-associated events such as pol II pausing, promoter opening, or alternate splicing. Finally, potent and specific chemical inhibitors of XPB and CDK7 will be evaluated to more clearly define their roles in general and gene-specific (p53 response will be studied here) transcription while serving as key positive controls for disruption of XPB or CDK7 activity.

概括 RNA聚合酶II（POL II）对转录的调节对于控制细胞的生长，分化和 发展。全基因组POL II转录所需的因素之一是进化保守的， 10-subunit tfiih综合体，在转录过程的多个步骤中扮演基本角色 包括启动子开放和mRNA处理；但是，分子机制尚不清楚。这 该项目的长期目标是阐明TFIIH调节POL II的分子机制 转录。该项目的理由是了解这些机制将揭示分子 多种和多样性发育和体细胞疾病的基础，包括神经系统疾病， 后代综合征，皮肤病和癌症。该协作项目的目标是建立一个 TFIIH如何控制基本转录事件（例如启动子）的结构和机械理解 开放，pol II暂停和mRNA处理。我们最近完成了天然TFIIH的结构分析 使用化学交联/质谱（CXM）和3D建模的复合物（人和酵母）。 结果预测了涉及必需的两个不同的保守蛋白质 - 蛋白质相互作用网络 酶亚基XPB/SSL2和CDK7/KIN28。转录需要ATPase/Clressocase XPB/SSL2 启动时，它融化了启动子DNA模板以使单链DNA参与POL II活性位点。 CDK7/KIN28激酶磷酸化Pol II大亚基的C末端结构域（CTD）以及 其他关键基板（例如P-TEFB）共同启用了Pol II启动，暂停和 mRNA处理。在AIM 1中，我们将研究XPB/SSL2 ATPase/Cranscase如何受到 涉及p52/tfb2和p8/tfb5的相互作用网络；在AIM 2中，我们将研究特定残留和 CCNH/CCL1和MAT1/TFB3中的域控制CDK7/KIN28激酶活性。为了实现这些目标， 我们将利用酵母遗传学的强大组合，紫外线响应交联的位置插入， 并在体内功能性筛选以结构完善并在功能上验证蛋白质 - 蛋白质界面 tfiih。这些数据将指导人类细胞中的一系列体外和基于细胞的测定，这些测定将严格测试 特定分子界面在TFIIH功能转录中的调节中的作用，全基因组。我们 将采用PIS和我们的合作者建立良好建立的方法，包括在体外重组 转录，锰酸足迹（体外和细胞中），gro-seq，芯片或芯片seq和RNA-seq。如果 成功，这些实验将使我们能够将给定的蛋白质蛋白接口与特定的调节联系起来 转录相关的事件，例如Pol II暂停，启动子打开或替代剪接。最后，有效 将评估XPB和CDK7的特定化学抑制剂，以更清楚地定义其角色一般 和基因特异性（将在此处研究p53响应）转录，同时作为关键的阳性对照的转录 XPB或CDK7活性的破坏。