Temporal regulation of gene expression is a hallmark of cellular differentiation pathways, yet the mechanisms controlling the timing of expression for different classes of differentiation-specific genes are not well understood. We previously demonstrated that the class II arginine methyltransferase Prmt5 was required for skeletal muscle differentiation at the early stages of myogenesis (C. S. Dacwag, Y. Ohkawa, S. Pal, S. Sif, and A. N. Imbalzano, Mol. Cell. Biol. 27:384-394, 2007). Specifically, when Prmt5 levels were reduced, the ATP-dependent SWI/SNF chromatin-remodeling enzymes could not interact with or remodel the promoter of myogenin, an essential early gene. Here we investigated the requirement for Prmt5 and the class I arginine methyltransferase Carm1/Prmt4 in the temporal control of myogenesis. Both arginine methyltransferases could bind to and modify histones at late-gene regulatory sequences. However, the two enzymes showed sequential requirements for gene expression. Prmt5 was required for early-gene expression but dispensable for late-gene expression. Carm1/Prmt4 was required for late- but not for early-gene expression. The reason for the requirement for Carm1/Prmt4 at late genes was to facilitate SWI/SNF chromatin-remodeling enzyme interaction and remodeling at late-gene loci. Thus, distinct arginine methyltransferases are employed at different times of skeletal muscle differentiation for the purpose of facilitating ATP-dependent chromatin-remodeling enzyme interaction and function at myogenic genes.

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.

BACKGROUND: Myocyte stress 1 (MS1) is a striated muscle actin binding protein required for the muscle specific activity of the evolutionary ancient myocardin related transcription factor (MRTF)/serum response factor (SRF) transcriptional pathway. To date, little is known about the molecular mechanisms that govern skeletal muscle specific expression of MS1. Such mechanisms are likely to play a major role in modulating SRF activity and therefore muscle determination, differentiation and regeneration. In this study we employed a comparative in silico analysis coupled with an experimental promoter characterisation to delineate these mechanisms. RESULTS: Analysis of MS1 expression in differentiating C2C12 muscle cells demonstrated a temporal differentiation dependent up-regulation in ms1 mRNA. An in silico comparative sequence analysis identified two conserved putative myogenic regulatory domains within the proximal 1.5 kbp of 5' upstream sequence. Co-transfecting C2C12 myoblasts with ms1 promoter/luciferase reporters and myogenic regulatory factor (MRF) over-expression plasmids revealed specific sensitivity of the ms1 promoter to MyoD. Subsequent mutagenesis and EMSA analysis demonstrated specific targeting of MyoD at two distinct E-Boxes (E1 and E2) within identified evolutionary conserved regions (ECRs, alpha and beta). Chromatin immunoprecipitation (ChIP) analysis indicates that co-ordinated binding of MyoD at E-Boxes located within ECRs alpha and beta correlates with the temporal induction in ms1 mRNA. CONCLUSION: These findings suggest that the tissue specific and differentiation dependent up-regulation in ms1 mRNA is mediated by temporal binding of MyoD at distinct evolutionary conserved E-Boxes within the ms1 5' upstream sequence. We believe, through its activation of ms1, this is the first study to demonstrate a direct link between MyoD activity and SRF transcriptional signalling, with clear implications for the understanding of muscle determination, differentiation and regeneration.

Ribosomal RNA (rRNA) genes are down-regulated during osteogenesis, myogenesis, and adipogenesis, necessitating a mechanistic understanding of interrelationships between growth control and phenotype commitment. Here, we show that cell fate-determining factors [MyoD, myogenin (Mgn), Runx2, C/EBPbeta] occupy rDNA loci and suppress rRNA expression during lineage progression, concomitant with decreased rRNA expression and reciprocal loss of occupancy by c-Myc, a proliferation-specific activator of rRNA transcription. We find interaction of phenotypic factors with the polymerase I activator upstream binding factor UBF-1 at interphase nucleoli, and this interaction is epigenetically retained on mitotic chromosomes at nucleolar organizing regions. Ectopic expression and RNA interference establish that MyoD, Mgn, Runx2, and C/EBPbeta each functionally suppress rRNA genes and global protein synthesis. We conclude that epigenetic control of ribosomal biogenesis by lineage-specific differentiation factors is a general developmental mechanism for coordinate control of cell growth and phenotype.

Many studies have examined transcriptional regulation during the initiation of skeletal muscle differentiation; however, there is less information regarding transcriptional control during adult myogenesis and during the maintenance of the differentiated state. MyoD and the mammalian SWI/SNF chromatin-remodeling enzymes containing the Brg1 ATPase are necessary to induce myogenesis in cell culture models and in developing embryonic tissue, whereas myogenin and Brg1 are critical for the expression of the late genes that induce terminal muscle differentiation. Here, we demonstrate that myogenin also binds to its own promoter during the late stages of embryonic muscle development. As is the case during embryonic myogenesis, MyoD and Brg1 co-localize to the myogenin promoter in primary adult muscle satellite cells. However, in mature myofibers, myogenin and Brg1 are preferentially co-localized to the myogenin promoter. Thus, the myogenin promoter is occupied by different myogenic factors at different times of myogenesis. The relevance of myogenin in the continued expression from its own promoter is demonstrated in culture, where we show that myogenin, in the absence of MyoD, is capable of maintaining its own expression by recruiting the Brg1 ATPase to modify promoter chromatin structure and facilitate myogenin expression. Finally, we utilized in vivo electroporation to demonstrate that Brg1 is required for the continued production of the myogenin protein in newborn skeletal muscle tissue. These findings strongly suggest that the skeletal muscle phenotype is maintained by myogenin and the continuous activity of Brg1-based SWI/SNF chromatin-remodeling enzymes.

Skeletal muscle differentiation requires the coordinated activity of transcription factors, histone modifying enzymes, and ATP-dependent chromatin remodeling enzymes. The type II protein arginine methyltransferase Prmt5 symmetrically dimethylates histones H3 and H4 and numerous nonchromatin proteins, and prior work has implicated Prmt5 in transcriptional repression. Here we demonstrate that MyoD-induced muscle differentiation requires Prmt5. One of the first genes activated during differentiation encodes the myogenic regulator myogenin. Prmt5 and dimethylated H3R8 (histone 3 arginine 8) are localized at the myogenin promoter in differentiating cells. Modification of H3R8 required Prmt5, and reduction of Prmt5 resulted in the abrogation of promoter binding by the Brg1 ATPase-associated with the SWI/SNF chromatin remodeling enzymes and all subsequent events associated with gene activation, including increases in chromatin accessibility and stable binding by MyoD. Prmt5 and dimethylated H3R8 were also associated with the myogenin promoter in activated satellite cells isolated from muscle tissue, further demonstrating the physiological relevance of these observations. The data indicate that Prmt5 facilitates myogenesis because it is required for Brg1-dependent chromatin remodeling and gene activation at a locus essential for differentiation. We therefore conclude that a histone modifying enzyme is necessary to permit an ATP-dependent chromatin remodeling enzyme to function.

The activation of muscle-specific gene expression requires the coordinated action of muscle regulatory proteins and chromatin-remodeling enzymes. Microarray analysis performed in the presence or absence of a dominant-negative BRG1 ATPase demonstrated that approximately one-third of MyoD-induced genes were highly dependent on SWI/SNF enzymes. To understand the mechanism of activation, we performed chromatin immunoprecipitations analyzing the myogenin promoter. We found that H4 hyperacetylation preceded Brg1 binding in a MyoD-dependent manner but that MyoD binding occurred subsequent to H4 modification and Brg1 interaction. In the absence of functional SWI/SNF enzymes, muscle regulatory proteins did not bind to the myogenin promoter, thereby providing evidence for SWI/SNF-dependent activator binding. We observed that the homeodomain factor Pbx1, which cooperates with MyoD to stimulate myogenin expression, is constitutively bound to the myogenin promoter in a SWI/SNF-independent manner, suggesting a two-step mechanism in which MyoD initially interacts indirectly with the myogenin promoter and attracts chromatin-remodeling enzymes, which then facilitate direct binding by MyoD and other regulatory proteins.