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

Abstract

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.