Mediator Kinases and Transcription Regulation

介导激酶和转录调控

• 批准号: 9113194
• 负责人: Dylan J Taatjes
• 金额: $ 38.27万
• 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: 2016-06-28 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9113194
• 关键词: Accounting; Address; Affect; Affinity; Architecture; Behavior; Biological; Biological Assay; Biological Process; Cancer Biology; Cell physiology; Cells; Code; Collaborations; Complement; Complex; DNA Binding; DNA Polymerase I; DNA Polymerase II; DNA-Directed RNA Polymerase; Data; Development; Disease; Enhancers; Enzymes; Foundations; Genes; Genetic Transcription; Goals; Grant; HCT116 Cells; Human; Human Development; In Vitro; Lead; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediator of activation protein; Molecular; Mutagenesis; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Positioning Attribute; Proteins; Publishing; Reagent; Regulation; Research Personnel; Role; Serum; Signal Pathway; Signal Transduction; Solid; Structural Models; Structure; Techniques; Technology; Testing; Therapeutic; Time; Transcript; Transcriptional Regulation; Transfer RNA; Universities; Untranslated RNA; Validation; Work; X-Ray Crystallography; base; cell growth; cortistatin; crosslink; follow-up; genome wide association study; genome-wide; global run on sequencing; human disease; in vitro testing; in vivo; inhibitor/antagonist; innovation; insight; kinase inhibitor; knock-down; mouse model; neuron development; novel therapeutics; paralogous gene; phosphoproteomics; public health relevance; research study; response; small molecule; technology validation; three-dimensional modeling; transcription factor; tumorigenesis

 DESCRIPTION (provided by applicant): This collaborative project seeks to gain a deep mechanistic understanding of the Mediator kinases (CDK8 and CDK19) and the CDK8 module. CDK8 and its paralog, CDK19, are considered "Mediator kinases" because of their stable, but reversible, association with the 26-subunit Mediator complex. Mediator is a global regulator of pol II transcription and appears to be required for expression of perhaps all pol II transcripts, which include all protein-coding and most non-coding RNA genes. CDK8 and CDK19 are linked to a growing number of cancers and developmental diseases, indicative of their essential-yet poorly understood-biological roles. Here, we propose to study CDK8 and CDK19 function in the context of serum response signaling, which is fundamentally important for cell physiology, development, and cancer biology. CDK8 in particular (CDK19 is less studied) has been shown to regulate transcription of serum response genes; however, the mechanisms remain unclear due to inherent limitations with cellular knockdown approaches. Working with our collaborator Dr. Matt Shair (Harvard), we have characterized a small molecule that is an extremely potent and selective Mediator kinase (CDK8 and CDK19) inhibitor. Using this reagent, we can for the first time reliably assess the mechanistic roles of Mediator kinases in human cells. In the context of serum response, we will identify the kinase substrates for CDK8 and CDK19 by using SILAC phosphoproteomics (with/without the Mediator kinase inhibitor). Also during serum response, we will use GRO-Seq to determine how Mediator kinases affect transcription genome-wide. GRO-Seq is uniquely suited to address these questions because, among other reasons, it provides a direct and immediate picture of active transcription (including all classes of transcripts-antisense, divergent, eRNA, lncRNA, tRNA, and so on), which is essential for understanding the rapid (within minutes) cellular response to serum stimulation. CDK8 functions in the context of a four-subunit, 600 kDa "CDK8 module" that also contains CCNC, MED12, and MED13. Because of its genome-wide association with Mediator, the CDK8 module seems poised to broadly regulate pol II transcription. The structure of the CDK8 module is poorly understood, which limits our understanding of the molecular mechanisms that regulate its essential biological functions. In Aim 2, we seek to define the 3D architecture and identify functionally relevant interfaces within the CDK8 module by applying well-tested crosslinking-mass spectrometry (CXMS) approaches and innovative 3D modeling techniques. These data will be supported by detailed in vitro mechanistic studies and cell-based functional validation assays, with a goal of defining the molecular mechanisms by which the CDK8 module regulates pol II transcription and how its kinase activity is regulated. Collectively, the diverse and complementary set of experiments proposed should yield fundamental insights regarding CDK8 module and Mediator kinase function that should drive the field in new directions.

 描述（由适用提供）：该协作项目旨在获得对介体激酶（CDK8和CDK19）和CDK8模块的深刻机械理解。 CDK8及其旁系同源物CDK19被认为是“介体激酶”，因为它们与26个亚基介体综合体具有稳定但可逆的关联。介体是POL II转录的全球调节剂，似乎是表达所有POL II转录物所必需的，其中包括所有蛋白质编码和大多数非编码RNA基因。 CDK8和CDK19与越来越多的癌症和发育疾病有关，这表明其必不可少的生物学作用。在这里，我们建议在血清反应信号传导的背景下研究CDK8和CDK19的功能，这对于细胞生理，发育和癌症生物学至关重要。尤其是CDK8（CDK19较少研究）已显示出调节血清反应基因的转录。然而，由于继承的局限性通过细胞敲低的方法，这些机制仍不清楚。我们与我们的合作者Matt Shair博士（哈佛）合作，我们表征了一个小分子，该分子是极具潜力和选择性的介体激酶（CDK8和CDK19）抑制剂。使用该试剂，我们可以首次可靠地评估介体激酶在人类细胞中的机械作用。在上下文中 在血清反应中，我们将通过使用SILAC磷酸蛋白质组学（使用/没有介体激酶抑制剂）来鉴定CDK8和CDK19的激酶底物。同样在血清反应期间，我们将使用GRO-SEQ来确定介体激酶如何影响整个转录基因组。 GRO-SEQ非常适合解决这些问题，因为除其他原因外，它提供了主动转录的直接和直接图片（包括所有类别的转录本 - 抗抗态，发散，Erna，lncrna，trna等），这对于理解几分钟内的蜂窝响应对血清模拟的快速（内）至关重要。 CDK8在一个四亚基，600 kDa“ CDK8模块”的上下文中发挥作用，其中还包含CCNC，MED12和MED13。由于其全基因组与介体的关联，CDK8模块似乎被中毒以广泛调节Pol II转录。 CDK8模块的结构知之甚少，这限制了我们对调节其基本生物学功能的分子机制的理解。在AIM 2中，我们寻求定义3D体系结构，并通过应用经过经过测试的交联质谱法（CXMS）方法和创新的3D建模技术来识别CDK8模块中与功能相关的接口。这些数据将通过详细的体外机械研究和基于细胞的功能验证测定法支持，以定义CDK8模块调节POL II转录以及其激酶活性如何调节的分子机制。总的来说，提出的潜水员和完整的实验集应产生有关CDK8模块和介体激酶功能的基本见解，该函数应驱动新方向。