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Cardiac Lineage Protein 1 (Clp-1), An Inhibitor of Positive Transcription Elongation Factor B (P-Tefb), Plays A Regulatory Role In Skeletal Muscle Cell Differentiation
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Siddiqui, M.A.Q.
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Spring 2010
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2010-04-28
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Doctoral Dissertation
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Emerging evidence suggests that one level of transcriptional regulation in eukaryotic cells is at the stage of transcription elongation in part by the Positive-Transcription Elongation Factor b (P-TEFb) complex. The P-TEFb complex consists of cyclin dependent kinase 9 (cdk9) and its cyclin partner, cyclin T and is dynamically partitioned between the active versus inactive states. The kinase activity is inactivated when P-TEFb associates with Cardiac Lineage Protein-1 (CLP-1), the mouse ortholog of human Hexamethylene bis-acetamide inducible protein 1 (HEXIM1). Recent reports implicate a link between P-TEFb and skeletal muscle in which P-TEFb associates with the skeletal muscle specific transcription factor MyoD. In this study, I present evidence that CLP-1 protein is critical for the regulation of skeletal muscle cell differentiation. I examined the expression of CLP-1 in the mouse myoblast cell line, C2C12, and found that there was a marked increase in CLP-1 association to P-TEFb at the onset of differentiation. I also observed that CLP-1 associates with MyoD, as well as with histone-deacetylases (HDACs), and that this association was maximal in the early stage of C2C12 differentiation. To further investigate the role of CLP-1 in skeletal muscle cell differentiation, I generated CLP-1 +/- knock-down C2C12 cells through homologous recombination. The deficiency in CLP-1 resulted in an inability of C2C12 heterozygote cells to transition into myotubes. I next examined the possibility that the inhibitory complex consisting of CLP-1, MyoD and HDACs targets the promoter of proliferative genes, such as cyclin D1. Chromatin immunoprecipitation (ChIP) analysis confirmed CLP-1/MyoD/HDAC binding to the cyclin D1 gene promoter during differentiation. These data suggest that CLP-1 plays a synergistic role in conjunction with MyoD and HDAC to control the genetic program that ensues in the transition of skeletal myoblasts from proliferative to terminally differentiated cells.
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Galatioto, G. (2010) Cardiac Lineage Protein 1 (Clp-1), An Inhibitor of Positive Transcription Elongation Factor B (P-Tefb), Plays A Regulatory Role In Skeletal Muscle Cell Differentiation. [Doctoral dissertation, SUNY Downstate Health Sciences University]. SUNY Open Access Repository. https://soar.suny.edu/handle/20.500.12648/15914
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Doctoral Dissertation