Mediator Kinases and Transcription Regulation

 描述（由申请人提供）：该合作项目旨在深入了解介体激酶（CDK 8和CDK 19）和CDK 8模块的机制。CDK 8及其副产物CDK 19被认为是“介体激酶”，因为它们与26亚基介体复合物稳定但可逆地结合。介体是pol II转录的全局调节因子，并且似乎是可能所有pol II转录物（包括所有蛋白质编码和大多数非编码RNA基因）表达所需的。CDK 8和CDK 19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的生物学作用，但人们对其了解甚少。 在这里，我们建议研究CDK 8和CDK 19在血清反应信号传导的背景下的功能，这对细胞生理学，发育和癌症生物学至关重要。特别是CDK 8（CDK 19研究较少）已显示可调节血清应答基因的转录;然而，由于细胞敲低方法的固有限制，其机制仍不清楚。与我们的合作者Matt Shair博士（哈佛）合作，我们已经表征了一种小分子，它是一种非常有效和选择性的介体激酶（CDK 8和CDK 19）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。背景下 我们将通过使用SILAC磷酸化蛋白质组学（有/无介体激酶抑制剂）鉴定CDK 8和CDK 19的激酶底物。此外，在血清应答期间，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是唯一适合解决这些问题，因为，除其他原因外，它提供了一个直接和即时的图片活性转录（包括所有类别的转录物-反义，分歧，eRNA，lncRNA，tRNA等），这是必不可少的理解快速（在几分钟内）细胞对血清刺激的反应。 CDK 8在一个四亚基、600 kDa的“CDK 8模块”中发挥作用，该模块还包含CCNC、MED 12和MED 13。由于其与Mediator的全基因组关联，CDK 8模块似乎准备广泛调节pol II转录。CDK 8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，我们试图定义的3D架构，并确定功能相关的接口内的CDK 8模块，通过应用久经考验的交联质谱（CXMS）的方法和创新的3D建模技术。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试验的支持，目的是定义CDK 8模块调节pol II转录的分子机制及其激酶活性如何调节。总的来说，所提出的多样化和互补的实验集应该产生关于CDK 8模块和介体激酶功能的基本见解，这些见解应该推动该领域朝着新的方向发展。

 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.