GSBS Dissertations and Theses

Approval Date

7-9-2008

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Cellular and Molecular Biology

Subjects

Protein-Arginine N-Methyltransferase; Myogenic Regulatory Factors; Muscle Development; Muscle, Skeletal; Academic Dissertations; Dissertations, UMMS

Abstract

Skeletal muscle differentiation requires synergy between tissue-specific transcription factors, chromatin remodeling enzymes and the general transcription machinery. Here we demonstrate that two distinct protein arginine methyltransferases are required to complete the differentiation program. Prmt5 is a type II methyltransferase, symmetrically dimethylates histones H3 and H4 and has been shown to play a role in transcriptional repression. An additional member of the Prmt family, Carm1 is a type I methyltransferase, and asymmetrically methylates histone H3 and its substrate proteins. MyoD regulates the activation of the early class of skeletal muscle genes, which includes myogenin. Prmt5 was bound to and dimethylates H3R8 at the myogenin promoter in a differentiation-dependent fashion. When proteins levels of Prmt5 were reduced by antisense, disappearance of H3R8 dimethylation and Prmt5 binding was observed. Furthermore, binding of Brg1 to regulatory sequences of the myogenin promoter was abolished. All subsequent events relying on Brg1 function, such as chromatin remodeling and stable binding by muscle specific transcription factors such as MyoD, were eliminated. Robust association of Prmt5 and dimethylation of H3R8 at myogenin promoter sequences was observed in mouse satellite cells, the precursors of mature myofibers. Prmt5 binding and histone modification were observed to a lesser degree in mature myofibers. Therefore, these results indicate that Prmt5 is required for dimethylating histone at the myogenin locus during skeletal muscle differentiation in order to facilitate the binding of Brg1, the ATPase subunit of the chromatin remodeling complex SWI/SNF.

Further exploration of the role of Prmt5 during the activation of the late class of muscle genes revealed that though Prmt5 is associated with and dimethylates histones at the regulatory elements of late muscle genes in tissue and in culture, it was dispensable for late gene activation. Previous reports had indicated that Carm1 was involved during late gene activation. We observed that Carm1 was bound to and responsible for dimethylating histones at late muscle gene promoters in tissue and in culture. In contrast to Prmt5, a complete knockout of Carm1 resulted in abrogation of late muscle gene activation. Furthermore, loss of Carm1 binding and dimethylated histones resulted in a disappearance of Brg1 binding and chromatin remodeling at late muscle gene loci. Time course chromatin immunoprecipitations revealed that Carm1 binding and histone dimethylation occurred concurrently with the onset of late gene activation. In vitro binding assays revealed that an interaction between Carm1, myogenin and Mef2D exists. These results demonstrate that Carm1 is recruited to the regulatory sequences of late muscle genes via its interaction with either myogenin or Mef2D and is responsible for dimethylates histones in order to facilitate the binding of Brg1. Therefore, these results indicate that during skeletal muscle differentiation, distinct roles exist for these Prmts such that Prmt5 is required for activation of early genes while Carm1 is essential for late gene induction.

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