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The expression of myogenic microRNAs indirectly requires protein arginine methyltransferase (Prmt)5 but directly requires Prmt4.

Mallappa C, Hu YJ, Shamulailatpam P, Tae S, Sif S, Imbalzano AN - Nucleic Acids Res. (2010)

Bottom Line: Both Prmts are required for myogenic microRNA induction during differentiation.By contrast, Prmt4 binds to the upstream regulatory regions of myogenic microRNAs and is required for dimethylation of the Prmt4 substrate, H3R17, at microRNA regulatory sequences.Deletion of Prmt4 does not alter MyoD binding at myogenic microRNA regulatory sequences but prevents the binding of both myogenin and the Brg1 ATPase that catalyzes SWI/SNF-dependent chromatin remodeling, resulting in an inhibition of microRNA expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA.

ABSTRACT
Myogenic microRNAs are important regulators of muscle development and differentiation. To better understand the roles of chromatin-modifying and remodeling enzymes in the activation of myogenic microRNA expression, we have functionally analyzed two different protein arginine methyltransferases, Prmt5 and Prmt4, both of which have previously been implicated in the regulation of myogenic mRNA expression. Both Prmts are required for myogenic microRNA induction during differentiation. Prmt5 is indirectly required due to the necessity of Prmt5 for expression of the transcriptional regulator, myogenin, as ectopic expression of myogenin eliminates Prmt5 dependency. By contrast, Prmt4 binds to the upstream regulatory regions of myogenic microRNAs and is required for dimethylation of the Prmt4 substrate, H3R17, at microRNA regulatory sequences. Deletion of Prmt4 does not alter MyoD binding at myogenic microRNA regulatory sequences but prevents the binding of both myogenin and the Brg1 ATPase that catalyzes SWI/SNF-dependent chromatin remodeling, resulting in an inhibition of microRNA expression.

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The myogenic regulatory factors MyoD and myogenin and the chromatin remodeling enzyme Brg1 interact with the proximal E-boxes containing sequences upstream of miR-1-1. ChIP experiments demonstrated the binding of MyoD, myogenin and Brg1 at regions containing the proximal E-boxes (A–C) but not to the distal E-boxes (D and E) of miR-1-1 at the indicated times in MyoD differentiated WT and Carm1/Prmt4 KO cells. Values at time 0 in the empty vector (EV) control were normalized to 1. The experiment was repeated three times, and the data represent the average of three experiments ± standard deviation. h, hours.
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Figure 8: The myogenic regulatory factors MyoD and myogenin and the chromatin remodeling enzyme Brg1 interact with the proximal E-boxes containing sequences upstream of miR-1-1. ChIP experiments demonstrated the binding of MyoD, myogenin and Brg1 at regions containing the proximal E-boxes (A–C) but not to the distal E-boxes (D and E) of miR-1-1 at the indicated times in MyoD differentiated WT and Carm1/Prmt4 KO cells. Values at time 0 in the empty vector (EV) control were normalized to 1. The experiment was repeated three times, and the data represent the average of three experiments ± standard deviation. h, hours.

Mentions: Brg1-based SWI/SNF chromatin remodeling enzyme function is required for the expression of miR-1 and miR-133a primary transcripts during myogenesis (13). In addition, MyoD has been shown to interact with upstream regulatory regions of miR-1 and miR-133a in differentiated C2C12 myoblasts (14) as well as in MyoD-differentiated fibroblasts and in skeletal muscle tissue (13). The myogenin factor also binds to some of these sequences in differentiated C2C12 myoblasts (14). To address the relationship between Carm1/Prmt4, the dimethylation of H3R17, and the binding of Brg1, MyoD and myogenin, we carried out ChIP assays on wild-type and Carm1/Prmt4 KO cells that were differentiated by MyoD or the empty vector. Consistent with our previous report (13), MyoD was found bound to the proximal but not distal regulatory regions of miR-1 and miR-133a in wild-type cells both at the onset of and during differentiation (Figures 8 and 9). Binding of MyoD at these sites was not affected in the Carm1/Prmt4 KO cells (Figures 8 and 9), indicating that MyoD could access these E boxes in the absence of Prmt4 and in the absence of dimethylated H3R17. By contrast, myogenin binding was not observed at any region upstream of the microRNAs at the onset of differentiation. However, myogenin did bind to the same sequences bound by MyoD, Carm1/Prmt4 and diMeH3R17 at 12 h post-differentiation and beyond in MyoD-differentiated cells (Figures 8 and 9). Intriguingly, myogenin binding was completely inhibited in the absence of Carm1/Prmt4 (Figures 8 and 9). This was not due to a change in myogenin expression in the Carm1/Prmt4-deficient cells (Figure 4C), suggesting instead that the local chromatin environment is insufficient to permit myogenin binding to the miRNA regulatory regions of miR-1 and miR-133a in the absence of Carm1/Prmt4.Figure 8.


The expression of myogenic microRNAs indirectly requires protein arginine methyltransferase (Prmt)5 but directly requires Prmt4.

Mallappa C, Hu YJ, Shamulailatpam P, Tae S, Sif S, Imbalzano AN - Nucleic Acids Res. (2010)

The myogenic regulatory factors MyoD and myogenin and the chromatin remodeling enzyme Brg1 interact with the proximal E-boxes containing sequences upstream of miR-1-1. ChIP experiments demonstrated the binding of MyoD, myogenin and Brg1 at regions containing the proximal E-boxes (A–C) but not to the distal E-boxes (D and E) of miR-1-1 at the indicated times in MyoD differentiated WT and Carm1/Prmt4 KO cells. Values at time 0 in the empty vector (EV) control were normalized to 1. The experiment was repeated three times, and the data represent the average of three experiments ± standard deviation. h, hours.
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Figure 8: The myogenic regulatory factors MyoD and myogenin and the chromatin remodeling enzyme Brg1 interact with the proximal E-boxes containing sequences upstream of miR-1-1. ChIP experiments demonstrated the binding of MyoD, myogenin and Brg1 at regions containing the proximal E-boxes (A–C) but not to the distal E-boxes (D and E) of miR-1-1 at the indicated times in MyoD differentiated WT and Carm1/Prmt4 KO cells. Values at time 0 in the empty vector (EV) control were normalized to 1. The experiment was repeated three times, and the data represent the average of three experiments ± standard deviation. h, hours.
Mentions: Brg1-based SWI/SNF chromatin remodeling enzyme function is required for the expression of miR-1 and miR-133a primary transcripts during myogenesis (13). In addition, MyoD has been shown to interact with upstream regulatory regions of miR-1 and miR-133a in differentiated C2C12 myoblasts (14) as well as in MyoD-differentiated fibroblasts and in skeletal muscle tissue (13). The myogenin factor also binds to some of these sequences in differentiated C2C12 myoblasts (14). To address the relationship between Carm1/Prmt4, the dimethylation of H3R17, and the binding of Brg1, MyoD and myogenin, we carried out ChIP assays on wild-type and Carm1/Prmt4 KO cells that were differentiated by MyoD or the empty vector. Consistent with our previous report (13), MyoD was found bound to the proximal but not distal regulatory regions of miR-1 and miR-133a in wild-type cells both at the onset of and during differentiation (Figures 8 and 9). Binding of MyoD at these sites was not affected in the Carm1/Prmt4 KO cells (Figures 8 and 9), indicating that MyoD could access these E boxes in the absence of Prmt4 and in the absence of dimethylated H3R17. By contrast, myogenin binding was not observed at any region upstream of the microRNAs at the onset of differentiation. However, myogenin did bind to the same sequences bound by MyoD, Carm1/Prmt4 and diMeH3R17 at 12 h post-differentiation and beyond in MyoD-differentiated cells (Figures 8 and 9). Intriguingly, myogenin binding was completely inhibited in the absence of Carm1/Prmt4 (Figures 8 and 9). This was not due to a change in myogenin expression in the Carm1/Prmt4-deficient cells (Figure 4C), suggesting instead that the local chromatin environment is insufficient to permit myogenin binding to the miRNA regulatory regions of miR-1 and miR-133a in the absence of Carm1/Prmt4.Figure 8.

Bottom Line: Both Prmts are required for myogenic microRNA induction during differentiation.By contrast, Prmt4 binds to the upstream regulatory regions of myogenic microRNAs and is required for dimethylation of the Prmt4 substrate, H3R17, at microRNA regulatory sequences.Deletion of Prmt4 does not alter MyoD binding at myogenic microRNA regulatory sequences but prevents the binding of both myogenin and the Brg1 ATPase that catalyzes SWI/SNF-dependent chromatin remodeling, resulting in an inhibition of microRNA expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA.

ABSTRACT
Myogenic microRNAs are important regulators of muscle development and differentiation. To better understand the roles of chromatin-modifying and remodeling enzymes in the activation of myogenic microRNA expression, we have functionally analyzed two different protein arginine methyltransferases, Prmt5 and Prmt4, both of which have previously been implicated in the regulation of myogenic mRNA expression. Both Prmts are required for myogenic microRNA induction during differentiation. Prmt5 is indirectly required due to the necessity of Prmt5 for expression of the transcriptional regulator, myogenin, as ectopic expression of myogenin eliminates Prmt5 dependency. By contrast, Prmt4 binds to the upstream regulatory regions of myogenic microRNAs and is required for dimethylation of the Prmt4 substrate, H3R17, at microRNA regulatory sequences. Deletion of Prmt4 does not alter MyoD binding at myogenic microRNA regulatory sequences but prevents the binding of both myogenin and the Brg1 ATPase that catalyzes SWI/SNF-dependent chromatin remodeling, resulting in an inhibition of microRNA expression.

Show MeSH
Related in: MedlinePlus