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（由申请人提供）：该合作项目旨在深入了解介体激酶（CDK8 和 CDK19）和 CDK8 模块。 CDK8 及其旁系同源物 CDK19 被认为是“介导激酶”，因为它们与 26 亚基介导复合物具有稳定但可逆的关联。 Mediator 是 pol II 转录的全局调节因子，似乎是所有 pol II 转录本（包括所有蛋白质编码基因和大多数非编码 RNA 基因）表达所必需的。 CDK8 和 CDK19 与越来越多的癌症和发育性疾病有关，这表明它们具有重要但仍知之甚少的生物学作用。 在这里，我们建议在血清反应信号传导背景下研究 CDK8 和 CDK19 的功能，这对于细胞生理学、发育和癌症生物学至关重要。尤其是 CDK8（CDK19 研究较少）已被证明可以调节血清反应基因的转录；然而，由于细胞敲低方法的固有局限性，其机制仍不清楚。我们与我们的合作者 Matt Shair 博士（哈佛大学）合作，鉴定了一种小分子，它是一种极其有效的选择性介导激酶（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激酶在人类细胞中的机制作用。在上下文中 为了观察血清反应，我们将使用 SILAC 磷酸蛋白质组学（有/无介导激酶抑制剂）来鉴定 CDK8 和 CDK19 的激酶底物。此外，在血清反应期间，我们将使用 GRO-Seq 来确定介体激酶如何影响全基因组转录。 GRO-Seq 特别适合解决这些问题，因为除其他原因外，它提供了活性转录的直接、即时图像（包括所有类别的转录物 - 反义、发散、eRNA、lncRNA、tRNA 等），这对于了解细胞对血清刺激的快速（几分钟内）反应至关重要。 CDK8 在四个亚基、600 kDa“CDK8 模块”的背景下发挥作用，该模块还包含 CCNC、MED12 和 MED13。由于其与 Mediator 在全基因组范围内的关联，CDK8 模块似乎准备广泛调节 pol II 转录。人们对 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，我们寻求通过应用经过充分测试的交联质谱 (CXMS) 方法和创新的 3D 建模技术来定义 3D 架构并识别 CDK8 模块内功能相关的接口。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测定的支持，目的是定义 CDK8 模块调节 pol II 转录的分子机制以及如何调节其激酶活性。总的来说，所提出的多样化和互补的实验应该会产生关于 CDK8 模块和介体激酶功能的基本见解，从而推动该领域朝着新的方向发展。