Browsing by Author "Ji, Jun-Yuan"

Now showing 1 - 3 of 3

Context-specific Functions of CDK8
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/229; DHHS-NIH-National Institute of General Medical Science
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/TGF? 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.
Context-specific Functions of CDK8
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/229; DHHS-NIH-National Institute of General Medical Science
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.
Role of Alpha-Catenin and Wnt Signaling in Regulating Lipid Homeostasis
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/229; DHHS-NIH-National Institute of General Medical Science
Wnt signaling, normally limited to embryogenesis, stem cell renewal and wound healing, is inappropriately re- employed in a variety of human cancers, such as hepatocellular carcinoma and colorectal cancer, as well as other diseases. Aberrant Wnt signaling and altered lipid metabolism are both signs of oncogenesis, and recent data suggest that Wnt control of adipogenesis and lipid metabolism may occur through separate mechanisms. Currently, the mechanisms remain poorly understood, and so remain outside of our ability to monitor, mitigate, prevent, or correct. It has been impossible to clearly delineate separate functions of Wnt in adipogenesis, lipid anabolism, and lipid catabolism, because these processes are inextricably interconnected in mammals. To circumvent this limitation, we use Drosophila as a primary experimental system, which provides unparalleled sophistication in manipulating Wnt (Wingless in Drosophila) activity in vivo. More importantly, the unique temporal separation of adipogenesis, lipogenesis, lipolysis, and fatty acid ?-oxidation during the Drosophila life cycle allows us to precisely monitor and manipulate these fundamental processes. Our genetic analyses of Axin and Alpha-catenin, two components of the Wnt signaling pathway, have revealed that Wnt signaling regulates lipid homeostasis during the late larval stage, separately from adipogenesis completed during embryogenesis. We have confirmed that the phenotypes of Axin mutants are caused by a gain of the canonical Wnt activity, elevated expression of Beta-catenin target genes, and altered expression of genes encoding enzymes involved in lipid catabolism. By screening a library of diverse FDA-approved drugs, we discovered that both the defective lipid homeostasis and the hyperactive Wnt signaling are potently suppressed by peptide boronic acids, a class of proteasome inhibitors. The suppressive effects of these inhibitors are dependent on Alpha-catenin. Despite the important role of Alpha-catenin in Wnt signaling, the precise mechanisms that normally regulate the stability of Alpha-catenin remain unclear. Thus the objective of this proposal is to determine how Alpha-catenin stability in particular, and Wnt signaling in general, regulates lipid catabolism. We will identify the molecular and cellular mechanisms that control the stability of Alpha-catenin in Drosophila by analyzing fat deposition and lipid accumulation. Our investigations will define the molecular mechanism(s) that control the stability of Alpha-catenin and reveal how Wnt signaling regulates lipid mobilization and lipid catabolism, thereby advancing our understanding of the tumor suppressive effects of Alpha-catenin and how Wnt signaling regulates lipid homeostasis.