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. 2007 Jan;27(1):384-94.
doi: 10.1128/MCB.01528-06. Epub 2006 Oct 16.

The protein arginine methyltransferase Prmt5 is required for myogenesis because it facilitates ATP-dependent chromatin remodeling

Caroline S Dacwag  1 , Yasuyuki Ohkawa, Sharmistha Pal, Saïd Sif, Anthony N Imbalzano
Affiliations

The protein arginine methyltransferase Prmt5 is required for myogenesis because it facilitates ATP-dependent chromatin remodeling

Caroline S Dacwag et al. Mol Cell Biol. 2007 Jan.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Prmt5 is required for skeletal muscle differentiation. NIH 3T3 cells and two independently derived NIH 3T3 lines expressing an antisense vector against Prmt5 (c15 and c12 cells) were mock-differentiated or differentiated by ectopic expression of MyoD. (A) Western blots show that protein levels of Prmt5 were significantly reduced in differentiated and mock-differentiated antisense lines compared to levels in the parental NIH 3T3 cells. Cells were differentiated as described in Materials and Methods for 36 h prior to sample collection. (B) Expression of myogenic target genes myogenin, desmin, and skeletal alpha-actin (Sk. α-actin) and ectopic expression of MyoD were monitored by RT-PCR in mock- and MyoD-differentiated cells collected 36 h postdifferentiation. (C) Quantification of muscle-specific gene expression by quantitative real-time PCR confirms that early and late differentiation markers are significantly decreased in cells containing reduced levels of Prmt5. PI3K, phosphatidylinositol 3-kinase.
FIG. 2.
FIG. 2.
Prmt5 coimmunoprecipitates with Brg1 and MyoD. Mock-differentiated cells, nondifferentiated (time zero [T0]) MyoD-infected cells, or MyoD-infected cells that were exposed to differentiation conditions for 36 h were harvested and used to perform coimmunoprecipitation experiments with the indicated antibodies. (A) Indicated proteins were detected by Western blotting using 10% of the input sample. (B) Whole-cell extracts from mock and differentiated samples were coimmunoprecipitated with Prmt5 antibody (catalog no. 611538; BD Biosciences) or purified immunoglobulin G (IgG). Immunoprecipitated material was run on SDS-PAGE gels, transferred to a membrane, and probed for the presence of Brg1, MyoD, and Prmt5. (C) As a control, the experiment in panel B was repeated using the c15 Prmt5 antisense line mock differentiated or MyoD differentiated for 36 h. Each Western blot panel contains data from the same blot; intervening samples were removed for clarity. (D) Coimmunoprecipitations were performed using Brg1 antisera and probed for Prmt5 and Brg1. (E) Coimmunoprecipitations were performed using MyoD antisera and probed for the presence of Prmt5 and MyoD. WB, Western blot; PI3K, phosphatidylinositol 3-kinase.
FIG. 3.
FIG. 3.
Prmt5 binds to the myogenin promoter and dimethylates H3R8. ChIPs were performed using antibodies against Prmt5 and dimethylated (diMe) H3R8 in mock- and MyoD-differentiated NIH 3T3 and Prmt5 antisense cell lines. (A) ChIPs demonstrate that binding of Prmt5 and dimethylated H3R8 at the myogenin promoter in differentiated cells is significantly reduced in the antisense lines. Amplification of the coding region of elongation factor EF1-alpha was performed as a negative control. (B) Quantification of Prmt5 binding and H3R8 dimethylation at the myogenin promoter was performed by QPCR. (C) Time course of Prmt5 and dimethylated H3R8 association with the myogenin promoter. QPCR analysis of binding in differentiated cells at the indicated times is shown. Values are expressed relative to the values obtained at time zero, which was set at 1.
FIG. 4.
FIG. 4.
Brg1 and MyoD binding at the myogenin promoter require Prmt5. (A) ChIPs were performed using antibodies against Brg1 and MyoD in mock- and MyoD-differentiated cell lines harvested 36 h postdifferentiation, and the myogenin promoter and elongation factor EF1-alpha coding region (input control) were PCR amplified. Data in panel A and Fig. 3A were from the same experiment; thus, the input control bands are the same for both panels. (B) Brg1 and MyoD binding were quantified by QPCR. (C) Western blot showing that Brg1 and MyoD protein levels were unaffected by the reduction in Prmt5 protein levels. (D) An REAA was performed to evaluate the accessibility of a Pvu II site at −320 relative to the myogenin mRNA start site. Chromatin accessibility was dependent upon Prmt5 expression.
FIG. 5.
FIG. 5.
Prmt5 is able to bind to and dimethylate H3R8 at the myogenin promoter in the absence of functional Brg1. ChIPs were performed 36 h postdifferentiation in a cell line containing a Tet-suppressible vector expressing a Flag-tagged, ATPase-deficient, dominant negative Brg1. (A) QPCR shows binding of Prmt5 and dimethylated H3R8 at the myogenin promoter in the presence (+ Tet) and absence (−Tet) of functional Brg1. (B) Protein levels of Brg1, Prmt5, and the Flag-tagged dominant negative mutant Brg1 were evaluated by Western blotting. Expression of Flag in samples without Tet indicates expression of dominant negative Brg1. (C) QPCR evaluation of MyoD mRNA, to demonstrate that differentiated cells expressed MyoD, and myogenin, to demonstrate that MyoD-dependent gene expression was inhibited by dominant negative Brg1. PI3K, phosphatidylinositol 3-kinase.
FIG. 6.
FIG. 6.
Prmt5 binds to and dimethylates H3R8 at the myogenin promoter in muscle satellite cells. (A) The separation and purification of satellite cells and myofibers were monitored by QPCR of marker gene mRNAs. (B) ChIP analysis using nuclei obtained from muscle satellite cells and myofibers indicates that Prmt5 binding and dimethylated H3R8 were present at the myogenin promoter in satellite cells and at reduced levels in myofibers. (C) Transcript levels of Prmt5 in both cell types were quantified by QPCR. (D) Re-ChIP analysis quantified by QPCR. Materials immunoprecipitated with Prmt5 antibodies were subsequently immunoprecipitated with MyoD antibodies. QPCR data are the average ± standard deviation of three independent experiments.
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