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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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Carm1/Prmt4 is required for the induction of myogenic miRNA expression during myogenesis. (A) Immunoblot demonstrating the absence of Carm1/Prmt4 in the knockout (KO) cells. A cross-reacting band (asterisk) demonstrates equal loading between gel lanes. (B–D) Relative expression of MyoD, myogenin and Acta1 mRNAs in the wild-type (WT) and Carm1/Prmt4 KO cells differentiated along the myogenic pathway by expressing MyoD or the empty vector (EV) control. (E–H) Relative expression of primary transcripts of miR-1-1, miR-1-2, miR-133a-1 and miR-133a-2 at various times post-differentiation in the WT and Carm1/Prmt4 knockout (KO) cells. (I and J) Relative expression of Mef2D and SRF mRNAs at various times post-differentiation in the WT and Carm1/Prmt4 knockout (KO) cells. The data represent the average of three independent experiments ± standard deviation. The expression at time 0 in empty vector infected cells was normalized to 1. h, hours.
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Figure 4: Carm1/Prmt4 is required for the induction of myogenic miRNA expression during myogenesis. (A) Immunoblot demonstrating the absence of Carm1/Prmt4 in the knockout (KO) cells. A cross-reacting band (asterisk) demonstrates equal loading between gel lanes. (B–D) Relative expression of MyoD, myogenin and Acta1 mRNAs in the wild-type (WT) and Carm1/Prmt4 KO cells differentiated along the myogenic pathway by expressing MyoD or the empty vector (EV) control. (E–H) Relative expression of primary transcripts of miR-1-1, miR-1-2, miR-133a-1 and miR-133a-2 at various times post-differentiation in the WT and Carm1/Prmt4 knockout (KO) cells. (I and J) Relative expression of Mef2D and SRF mRNAs at various times post-differentiation in the WT and Carm1/Prmt4 knockout (KO) cells. The data represent the average of three independent experiments ± standard deviation. The expression at time 0 in empty vector infected cells was normalized to 1. h, hours.

Mentions: Carm1/Prmt4 is an arginine methyltransferase that methylates arginines 17 and 26 on histone H3 (29,30). Carm1/Prmt4 is required for the expression of late myogenic genes but is dispensable for the expression of myogenin, which is expressed at early times post-differentiation (20). To determine whether Carm1/Prmt4 contributes to myogenic miRNA expression, we utilized cell lines that were derived from wild type and Carm1/Prmt4-deficient mouse embryos via a 3T3 passaging protocol (35). An immunoblot with Carm1/Prmt4 antibody confirmed the absence of Carm1/Prmt4 protein in the knockout (KO) fibroblasts (Figure 4A). We then tested whether Carm1/Prmt4 was required for the proper expression of myogenic miRNA genes. We differentiated wild type and Carm1/Prmt4 KO fibroblasts by expressing MyoD or the empty vector as a control. Cells infected with the MyoD encoding retrovirus expressed similar levels of MyoD (Figure 4B). Differentiated Carm1/Prmt4 KO fibroblasts expressed normal levels of myogenin but failed to express late marker gene Acta1, in agreement with previous results [(20); Figure 4C and D]. When tested for the expression of primary transcripts of miR-1 and miR-133a, no or minimal miRNA induction was observed in Carm1/Prmt4 KO cells at all times post-differentiation, whereas normal induction of miRNA expression was observed in the wild-type fibroblasts upon myogenic differentiation (Figure 4E–H). Analysis of other myogenic regulatory protein such as Mef2D and SRF showed that the absence of Prmt4/Carm1 did not alter the expression of these genes (Figure 4I and J). These results show that Carm1/Prmt4 is required for miR-1 and miR-133a expression during myogenesis.Figure 4.


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)

Carm1/Prmt4 is required for the induction of myogenic miRNA expression during myogenesis. (A) Immunoblot demonstrating the absence of Carm1/Prmt4 in the knockout (KO) cells. A cross-reacting band (asterisk) demonstrates equal loading between gel lanes. (B–D) Relative expression of MyoD, myogenin and Acta1 mRNAs in the wild-type (WT) and Carm1/Prmt4 KO cells differentiated along the myogenic pathway by expressing MyoD or the empty vector (EV) control. (E–H) Relative expression of primary transcripts of miR-1-1, miR-1-2, miR-133a-1 and miR-133a-2 at various times post-differentiation in the WT and Carm1/Prmt4 knockout (KO) cells. (I and J) Relative expression of Mef2D and SRF mRNAs at various times post-differentiation in the WT and Carm1/Prmt4 knockout (KO) cells. The data represent the average of three independent experiments ± standard deviation. The expression at time 0 in empty vector infected cells was normalized to 1. h, hours.
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Figure 4: Carm1/Prmt4 is required for the induction of myogenic miRNA expression during myogenesis. (A) Immunoblot demonstrating the absence of Carm1/Prmt4 in the knockout (KO) cells. A cross-reacting band (asterisk) demonstrates equal loading between gel lanes. (B–D) Relative expression of MyoD, myogenin and Acta1 mRNAs in the wild-type (WT) and Carm1/Prmt4 KO cells differentiated along the myogenic pathway by expressing MyoD or the empty vector (EV) control. (E–H) Relative expression of primary transcripts of miR-1-1, miR-1-2, miR-133a-1 and miR-133a-2 at various times post-differentiation in the WT and Carm1/Prmt4 knockout (KO) cells. (I and J) Relative expression of Mef2D and SRF mRNAs at various times post-differentiation in the WT and Carm1/Prmt4 knockout (KO) cells. The data represent the average of three independent experiments ± standard deviation. The expression at time 0 in empty vector infected cells was normalized to 1. h, hours.
Mentions: Carm1/Prmt4 is an arginine methyltransferase that methylates arginines 17 and 26 on histone H3 (29,30). Carm1/Prmt4 is required for the expression of late myogenic genes but is dispensable for the expression of myogenin, which is expressed at early times post-differentiation (20). To determine whether Carm1/Prmt4 contributes to myogenic miRNA expression, we utilized cell lines that were derived from wild type and Carm1/Prmt4-deficient mouse embryos via a 3T3 passaging protocol (35). An immunoblot with Carm1/Prmt4 antibody confirmed the absence of Carm1/Prmt4 protein in the knockout (KO) fibroblasts (Figure 4A). We then tested whether Carm1/Prmt4 was required for the proper expression of myogenic miRNA genes. We differentiated wild type and Carm1/Prmt4 KO fibroblasts by expressing MyoD or the empty vector as a control. Cells infected with the MyoD encoding retrovirus expressed similar levels of MyoD (Figure 4B). Differentiated Carm1/Prmt4 KO fibroblasts expressed normal levels of myogenin but failed to express late marker gene Acta1, in agreement with previous results [(20); Figure 4C and D]. When tested for the expression of primary transcripts of miR-1 and miR-133a, no or minimal miRNA induction was observed in Carm1/Prmt4 KO cells at all times post-differentiation, whereas normal induction of miRNA expression was observed in the wild-type fibroblasts upon myogenic differentiation (Figure 4E–H). Analysis of other myogenic regulatory protein such as Mef2D and SRF showed that the absence of Prmt4/Carm1 did not alter the expression of these genes (Figure 4I and J). These results show that Carm1/Prmt4 is required for miR-1 and miR-133a expression during myogenesis.Figure 4.

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