Mediator Kinases and Transcription Regulation

介导激酶和转录调控

• 批准号: 9306137
• 负责人: Dylan J Taatjes
• 金额: $ 39.56万
• 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/9306137
• 关键词: Address; Affect; Affinity; Architecture; Behavior; Biological; Biological Assay; Biological Process; Cancer Biology; Cell physiology; Cells; Code; Collaborations; Complement; Complex; Crystallization; DNA Binding; DNA Polymerase I; DNA Polymerase II; 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; RNA Polymerase I; 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; X-Ray Crystallography; base; cell growth; cortistatin; crosslink; experimental study; 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; 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(由申请者提供)：这个合作项目寻求获得对介体蛋白激酶(CDK8和CDK19)和CDK8模块的深入机械性理解。CDK8及其类似物CDK19被认为是“介体蛋白激酶”，因为它们稳定但可逆地与26个亚基的介体复合体结合。介体是PolII转录的全球调节因子，似乎是所有PolII转录本表达所必需的，其中包括所有编码蛋白质和大多数非编码RNA基因。CDK8和CDK19与越来越多的癌症和发育疾病有关，这表明它们具有重要的生物学作用，但人们对此知之甚少。在这里，我们建议在血清反应信号的背景下研究CDK8和CDK19的功能，这对细胞生理学、发育和癌症生物学至关重要。尤其是CDK8(CDK19研究较少)已被证明调节血清反应基因的转录；然而，由于细胞敲除方法的固有局限性，其机制尚不清楚。与我们的合作者Matt Shair博士(哈佛大学)合作，我们已经确定了一种小分子的特性，该小分子是一种极其有效和选择性的介体蛋白激酶(CDK8和CDK19)抑制剂。使用这种试剂，我们可以第一次可靠地评估介体蛋白激酶在人类细胞中的机制作用。在上下文中 对于血清反应，我们将使用SILAC磷酸蛋白质组学(使用/不使用介体激酶抑制剂)来确定CDK8和CDK19的激酶底物。此外，在血清反应期间，我们将使用Gro-Seq来确定介体蛋白激酶如何影响基因组范围内的转录。Gro-Seq特别适合于解决这些问题，因为除了其他原因外，它还提供了活性转录的直接和即时图像(包括所有类别的转录本-反义、分歧、Erna、lncRNA、tRNA等)，这对于理解细胞对血清刺激的快速(几分钟内)反应是必不可少的。CDK8在一个四亚单位、600 kDa的“CDK8模块”中发挥作用，该模块还包括Ccnc、MED12和MED13。由于其与Mediator在全基因组范围内的关联，CDK8模块似乎准备广泛地调控PolII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。在目标2中，我们试图通过应用经过良好测试的交联质谱(CXMS)方法和创新的3D建模技术来定义3D架构并确定CDK8模块中功能相关的接口。这些数据将得到详细的体外机制研究和基于细胞的功能验证分析的支持，目的是定义CDK8模块调控PolII转录的分子机制以及其激酶活性是如何调控的。总而言之，所提出的多样化和互补性的一系列实验应该会产生关于CDK8模块和介体蛋白激酶功能的基本见解，这些应该会推动该领域朝着新的方向发展。