Myocardial cells in culture provide an attractive system for studies of alteration in chromatin that accompany the events of cellular proliferation and differentiation. The High Mobility Group (HMG) class of the nuclear non-histone proteins is well characterized. The HMGs are ubiquitously distributed, and credible evidence exists for their involvement in induction and maintenance of the developmentally regulated genes in chromatin. Using biochemical, molecular, and immunological approaches we will characterize the HMG proteins in the rat ventricular cardiac muscle cells in culture. The neonatal cardiomyocytes in culture exhibit the developmentally regulated profiles of the myosin isoenzymes in the presence of thyroid hormones. We will examine the in vitro and in vivo development stage-specific distribution of the metabolically modified and the unmodified HMG proteins in thyroxine T3 treated and untreated cardiac muscle cells by the high resolution two-dimensional NEPHGE/SDS polyacrylamide gel electrophoresis. High content of HMG 1 and 2 has been correlated to the cell's replicative activity. We will investigate such as role for them in the adult rat cardiomyocyte cultures which reinitiate DNA synthesis. Little is known about how HMG proteins 14 and 17 interact in chromatin during activation of the transcribed gene sequences. I have suggested (Bhorjee, J. S., 1981, PNAS, 78:6944-48) that HMG protein phosphorylation may be an important mechanism by which these proteins may interact in activation/deactivation of gene sequences for the expression of the developmentally regulated phenotype. To investigate the roles of HMG proteins in gene activation we will examine the in vitro transcription of the cardiac muscle myosin heavy chain (MHC) genes in the HMG depleted and undepleted nuclei by the nuclear run-off assays. To test the hypothesis that phosphorylation of HMG 14 is a possible mode by which it may interact in chromatin while inducing activation of such developmentally regulated genes, we will investigate the preferential nuclease sensitivity, ie., the transcriptionally active state, of the cardiac muscle specific MHC- alpha and beta genes in chromatins reconstituted by the 0.35 M NaC1 extracts with and without the phosphorylated HMG 14, where the HMG 14 phosphopeptides will be removed by immunoprecipitation. For assay of MHC gene transcripts in the nuclear run-off experiments and for the presence of the MHC alpha - and beta-gene sequences in the nuclease sensitivity experiments, we will use the highly specific MHC alpha and beta gene oligonucleotide probes available to use from Dr. William Claycomb.
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| Wedrychowski, A; Bhorjee, J S; Briggs, R C (1989) In vivo crosslinking of nuclear proteins to DNA by cis-diamminedichloroplatinum (II) in differentiating rat myoblasts. Exp Cell Res 183:376-87 |
