Context-specific Functions of CDK8

标题：CDK 8的上下文特定功能 项目概要： 本项目的长期目标是阐明CDK 8模块的功能和调节，这是一个关键的 多功能模式生物果蝇中转录辅因子介体复合物的组成部分 黑腹菌CDK 8模块的四个亚基-CDK 8，CycC，MED 12和MED 13-是 在心血管疾病和许多人类癌症中突变或扩增，例如黑素瘤和 结肠直肠癌阐明CDK 8模块在不同生物学背景中的功能和调节 对于理解CDK 8模块失调的病理后果至关重要， 设计治疗这些疾病的临床策略。本项目上一个供资周期的研究 已经证明CDK 8-CycC是连接营养摄入和脂肪的关键调节节点， 果蝇发育中的代谢和发育时间。这些研究表明，CDK 8-CycC 是SREBP（固醇反应元件结合蛋白）依赖性基因表达的直接抑制剂， CDK 8-CycC正调控蜕皮激素受体激活基因表达。本报告的总体目标 该项目旨在揭示CDK 8在不同发育背景下的功能和调控。的研究 先前的资助周期表明，果蝇是实现这一目标的理想和强大的实验系统 长期目标。本供资周期提出了两个新目标。目的1将确定CDK 8在以下方面的作用： 调节果蝇端粒反转录转座子的表达和端粒生物学。我们的RNA测序 分析显示端粒逆转录转座子表达的特异性上调和端粒长度的显著增加。 cdk 8和cycC突变体的端粒长度。我们还发现了类似的端粒调控失调， 反转录转座子表达和端粒长度在med 7和扇贝（Sd）突变体。转录因子 Sd在保守的Hippo通路下游发挥作用。因此，我们建议研究未探索的 CDK 8模块和Sd在调节端粒逆转录转座子和端粒表达中的作用 果蝇的长度该提案的目标2将确定和验证上游监管机构和下游监管机构。 果蝇中CDK 8的效应子。我们已经建立了新的和强大的翅膀表型所造成的具体 CDK 8活性的改变，使我们能够进行显性修饰基因筛选，以确定 在体内与CDK 8相互作用。我们已经确定了26个基因组位点，其单倍不足改变了这些基因组位点。 CDK 8特异性表型;进一步的遗传分析使我们确定了CDK 8和 表皮生长因子受体（EGFR）和Dpp/TGFβ信号通路的组分，以及 几个特定的基因。与此同时，我们进行了免疫沉淀结合质谱（IP-100）。 MS）分析以鉴定可与CDK 8相互作用的蛋白质。这些生物化学和遗传学的结合 筛选使我们处于独特的位置，可以系统地识别和验证上游监管机构和下游监管机构， CDK 8在体内的流效应器，这将影响我们对CDK 8的上下文特异性功能的理解。

Title: Context-specific functions of CDK8 Project Summary: The long-term goal of this project is to elucidate the function and regulation of the CDK8 module, a key component of the transcription cofactor Mediator complex, in the versatile model organism Drosophila melanogaster. The four subunits of the CDK8 module – CDK8, CycC, MED12, and MED13 – are either mutated or amplified in cardiovascular diseases and a number of human cancers, such as melanoma and colorectal cancers. Elucidating the function and regulation of the CDK8 module in different biological contexts is essential to understanding the pathological consequences of CDK8 module misregulation, which is important for the design of clinical strategies to treat these diseases. Studies in the previous funding cycle of this project have demonstrated that CDK8-CycC serves as a critical regulatory node linking nutrient intake to fat metabolism and developmental timing in Drosophila development. These studies have shown that CDK8-CycC is a direct inhibitor of SREBP (sterol response element binding protein)-dependent gene expression, and that CDK8-CycC positively regulates ecdysone receptor-activated gene expression. The overall objective of this project is to unravel the function and regulation of CDK8 in different developmental contexts. Studies in the previous funding cycle illustrate that Drosophila is an ideal and powerful experimental system to achieve this long-term goal. Two new aims are proposed in this funding cycle. Aim 1 will determine the role of CDK8 in regulating the expression of telomeric retrotransposons and telomere biology in Drosophila. Our RNA-seq analyses revealed a specific upregulation of telomeric retrotransposon expression and a significant increase in telomere length in cdk8 and cycC mutants. We have also discovered similar deregulation of telomeric retrotransposon expression and telomere length in med7 and Scalloped (Sd) mutants. The transcription factor Sd functions downstream of the conserved Hippo pathway. Thus we propose to examine the unexplored functions of the CDK8 module and Sd in regulating the expression of telomeric retrotransposons and telomere length in Drosophila. Aim 2 of the proposal will identify and validate upstream regulators and downstream effectors of CDK8 in Drosophila. We have established novel and robust wing phenotypes caused by specific alterations of CDK8 activities, allowing us to perform a dominant modifier genetic screen to identify factors interacting with CDK8 in vivo. We have identified 26 genomic loci whose haploinsufficiency modifies these CDK8-specific phenotypes; further genetic analysis led us to identify genetic interactions between CDK8 and the components of the epidermal growth factor receptor (EGFR) and Dpp/TGFβ signaling pathways, as well as several specific genes. In parallel, we have performed immunoprecipitation coupled to mass spectrometry (IP- MS) analyses to identify proteins that can interact with CDK8. Combination of these biochemical and genetic screens puts us in a unique position to systematically identify and validate upstream regulators and down- stream effectors of CDK8 in vivo, which will impact our understanding of the context-specific functions of CDK8.