Context-specific Functions of CDK8

CDK8 的上下文特定功能

• 批准号: 10334536
• 负责人: Jun-yuan Ji
• 金额: $ 33.9万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10334536
• 关键词: Abbreviations; Animal Model; Binding Proteins; Biochemical; Biochemical Genetics; Biological; Biology; Cardiovascular Diseases; Cell Cycle; Cell Line; Child; Clinical; Colorectal Cancer; Complex; Coupled; Cyclin-Dependent Kinases; DNA Binding; DNA Polymerase II; DNA-Directed RNA Polymerase; Data; Development; Disease; Drosophila genus; Drosophila melanogaster; E2F transcription factors; Epidermal Growth Factor; Epidermal Growth Factor Receptor; Eukaryota; Factor Analysis; Funding; G1 Phase; G1/S Transition; Gene Expression; Genes; Genetic; Genetic Screening; Genetic Transcription; Genome Stability; Genomics; Goals; Homeostasis; Human; Immunoprecipitation; Intake; Length; Link; Malignant Neoplasms; Mammals; Mass Spectrum Analysis; Mediator of activation protein; Modeling; Mutate; Nutrient; Pathologic; Pathway interactions; Phase Transition; Phenocopy; Phenotype; Phosphotransferases; Positioning Attribute; Protein Analysis; Proteins; Proteomics; Pupa; Receptor Signaling; Regulation; Reporting; Research; Response Elements; Retrotransposon; Role; Scallop; Serum Response Factor; Signal Pathway; Sterols; System; Telomere Capping; Testing; Trans-Activators; Transforming Growth Factor beta; Up-Regulation; Wing; cofactor; cyclin C; design; ecdysone receptor; fly; genetic analysis; genomic locus; human disease; in vivo; inhibitor; lipid biosynthesis; lipid metabolism; melanoma; mutant; novel; public health relevance; synergism; telomere; transcription factor; transcriptome sequencing

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.

标题：CDK8的特定于上下文的功能 项目总结： 这个项目的长期目标是阐明CDK8模块的功能和调节，CDK8模块是一个关键 在多功能模式生物果蝇中，转录辅助因子介体复合体的组成部分 黑猩猩。CDK8模块的四个亚基-CDK8、CycC、MED12和MED13-是 在心血管疾病和一些人类癌症中发生突变或放大，如黑色素瘤和 结直肠癌。阐明CDK8模块在不同生物环境中的功能和调节 对于理解CDK8模块错误调节的病理后果至关重要，这一点很重要 用于设计治疗这些疾病的临床策略。本项目上一个资金周期中的研究 已经证明CDK8-CycC是一个将营养摄入与脂肪联系起来的关键调控节点 果蝇发育过程中的代谢和发育时序。这些研究表明，CDK8-CycC 是SREBP(类固醇反应元件结合蛋白)依赖的基因表达的直接抑制物，并且 CDK8-CycC正向调节蜕皮激素受体激活的基因表达。这样做的总体目标是 该项目旨在揭示CDK8在不同发育背景下的功能和调节。在美国的研究 之前的资金周期表明，果蝇是实现这一目标的理想和强大的实验系统 长期目标。在这个筹资周期中提出了两个新的目标。目标1将确定CDK8在 调节果蝇端粒反转录转座子和端粒生物学的表达。我们的RNA序列 分析显示，端粒逆转座子的表达特异性上调，并显著增加了 CDK8和cycC突变体的端粒长度。我们也发现了类似的端粒放松调控。 反转录转座子在Med7和扇形突变体中的表达和端粒长度。转录因子 SD在保守的河马途径下游发挥作用。因此，我们建议研究未探索的 CDK8模块和SD在调节端粒逆转座子和端粒表达中的作用 果蝇的长度。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CDK8的效应器。我们已经建立了新的和健壮的翅膀表型，由特定的 CDK8活性的改变，使我们能够进行显性修饰基因筛选来识别因素 在体内与CDK8相互作用。我们已经确定了26个基因组座位，它们的单倍性不足改变了这些 CDK8特有的表型；进一步的遗传分析使我们确定了CDK8和CDK8之间的遗传交互作用 表皮生长因子受体和转化生长因子受体β信号通路的组成以及 几个特定的基因。同时，我们还进行了免疫沉淀与质谱联用(IP-MS)。 MS)分析以确定可以与CDK8相互作用的蛋白质。这些生化和遗传的结合 Screens使我们处于独特的地位，可以系统地识别和验证上游监管机构和下行监管机构- CDK8在体内的流效应，这将影响我们对CDK8特定上下文功能的理解。