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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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Prmt4/Carm1 binds and dimethylates H3R17 at regulatory regions upstream of myogenic miR-1-1 miRNA. (A) A schematic diagram showing the location of consensus E-boxes, the cis elements that mediate the interaction of myogenic regulatory factors, upstream of miR-1 and miR-133a genes. This schematic diagram is a modified version of the diagram published in Figure 6 of ref. (13), that was amended with permission from the American Society for Microbiology. (B–F) ChIP experiments demonstrating the interaction of Carm1/Prmt4 and the presence of dimethylated (diMe) H3R17 at the three proximal E-boxes but not at the two distal E-boxes of miR-1-1 in WT but not in the Carm1/Prmt4 KO cells at various times during myogenic differentiation. No binding of IgG was found at any of the E-boxes at any time point. The binding at time 0 in the empty vector (EV) control in WT and Carm1/Prmt4 KO cells was normalized to 1. Data represent the average of three independent experiments ± standard deviation. h, hours.
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Figure 6: Prmt4/Carm1 binds and dimethylates H3R17 at regulatory regions upstream of myogenic miR-1-1 miRNA. (A) A schematic diagram showing the location of consensus E-boxes, the cis elements that mediate the interaction of myogenic regulatory factors, upstream of miR-1 and miR-133a genes. This schematic diagram is a modified version of the diagram published in Figure 6 of ref. (13), that was amended with permission from the American Society for Microbiology. (B–F) ChIP experiments demonstrating the interaction of Carm1/Prmt4 and the presence of dimethylated (diMe) H3R17 at the three proximal E-boxes but not at the two distal E-boxes of miR-1-1 in WT but not in the Carm1/Prmt4 KO cells at various times during myogenic differentiation. No binding of IgG was found at any of the E-boxes at any time point. The binding at time 0 in the empty vector (EV) control in WT and Carm1/Prmt4 KO cells was normalized to 1. Data represent the average of three independent experiments ± standard deviation. h, hours.

Mentions: We next addressed whether Carm1/Prmt4 binds to the upstream regulatory regions of miRNA genes and whether the Carm1/Prmt4 substrate, dimethylated H3R17, is also present at these sequences. A schematic diagram illustrates the positions of E boxes upstream of the miR-1-1, miR-133a-2, miR-1-2 and miR-133a-1 stem–loop sequences (Figure 6A). Open rectangles indicate E box containing regions that we previously showed were bound by both MyoD and the Brg1 ATPase that is the enzymatic subunit of the SWI/SNF ATP-dependent chromatin remodeling enzyme. Solid rectangles indicate E box containing sequences that we previously showed were not bound by either MyoD or Brg1 [Figure 6A; (13)]. ChIP experiments for Carm1/Prmt4 and diMeH3R17 at each of these putative miRNA regulatory regions in MyoD-differentiated wild-type and Carm1/Prmt4 KO fibroblasts showed that Carm1/Prmt4 binds to the proximal three E box containing regions of the miR-1-1 upstream region but not to the two more distal E-boxes (Figure 6B–F). Carm1/Prmt4 also interacted with the proximal E box containing sequences of the miR-1-2, miR-133a-1 and miR-133a-2 upstream regions. However, no binding was observed at the distal E box containing sequences upstream of miR-133a-2 (Figure 7A–D). Carm1/Prmt4 binding at these regions correlated precisely with the presence of diMeH3R17, and the presence of diMeH3R17 was entirely Prmt4/Carm1 dependent (Figures 6B–F and 7A–D). Binding of both Carm1/Prmt4 and diMeH3R17 was first observed at 12 h post-differentiation, consistent with the onset of miRNA expression (Figure 1) and was maintained through the 48-h post-differentiation time point. These results demonstrate that Carm1/Prmt4 and diMeH3R17 are present in the upstream regulatory regions of myogenic miRNAs, miR-1 and miR-133a, that dimethylation of H3R17 at these sequences required Carm1/Prmt4, and that the onset of Carm1/Prmt4 and diMeH3R17 binding occurred at the time of myogenic miRNA gene induction. In combination with studies from our previous work (13), we have demonstrated that each of the sites bound by Carm1/Prmt4 and diMeH3R17 also are bound by MyoD and by Brg1.Figure 6.


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)

Prmt4/Carm1 binds and dimethylates H3R17 at regulatory regions upstream of myogenic miR-1-1 miRNA. (A) A schematic diagram showing the location of consensus E-boxes, the cis elements that mediate the interaction of myogenic regulatory factors, upstream of miR-1 and miR-133a genes. This schematic diagram is a modified version of the diagram published in Figure 6 of ref. (13), that was amended with permission from the American Society for Microbiology. (B–F) ChIP experiments demonstrating the interaction of Carm1/Prmt4 and the presence of dimethylated (diMe) H3R17 at the three proximal E-boxes but not at the two distal E-boxes of miR-1-1 in WT but not in the Carm1/Prmt4 KO cells at various times during myogenic differentiation. No binding of IgG was found at any of the E-boxes at any time point. The binding at time 0 in the empty vector (EV) control in WT and Carm1/Prmt4 KO cells was normalized to 1. Data represent the average of three independent experiments ± standard deviation. h, hours.
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Figure 6: Prmt4/Carm1 binds and dimethylates H3R17 at regulatory regions upstream of myogenic miR-1-1 miRNA. (A) A schematic diagram showing the location of consensus E-boxes, the cis elements that mediate the interaction of myogenic regulatory factors, upstream of miR-1 and miR-133a genes. This schematic diagram is a modified version of the diagram published in Figure 6 of ref. (13), that was amended with permission from the American Society for Microbiology. (B–F) ChIP experiments demonstrating the interaction of Carm1/Prmt4 and the presence of dimethylated (diMe) H3R17 at the three proximal E-boxes but not at the two distal E-boxes of miR-1-1 in WT but not in the Carm1/Prmt4 KO cells at various times during myogenic differentiation. No binding of IgG was found at any of the E-boxes at any time point. The binding at time 0 in the empty vector (EV) control in WT and Carm1/Prmt4 KO cells was normalized to 1. Data represent the average of three independent experiments ± standard deviation. h, hours.
Mentions: We next addressed whether Carm1/Prmt4 binds to the upstream regulatory regions of miRNA genes and whether the Carm1/Prmt4 substrate, dimethylated H3R17, is also present at these sequences. A schematic diagram illustrates the positions of E boxes upstream of the miR-1-1, miR-133a-2, miR-1-2 and miR-133a-1 stem–loop sequences (Figure 6A). Open rectangles indicate E box containing regions that we previously showed were bound by both MyoD and the Brg1 ATPase that is the enzymatic subunit of the SWI/SNF ATP-dependent chromatin remodeling enzyme. Solid rectangles indicate E box containing sequences that we previously showed were not bound by either MyoD or Brg1 [Figure 6A; (13)]. ChIP experiments for Carm1/Prmt4 and diMeH3R17 at each of these putative miRNA regulatory regions in MyoD-differentiated wild-type and Carm1/Prmt4 KO fibroblasts showed that Carm1/Prmt4 binds to the proximal three E box containing regions of the miR-1-1 upstream region but not to the two more distal E-boxes (Figure 6B–F). Carm1/Prmt4 also interacted with the proximal E box containing sequences of the miR-1-2, miR-133a-1 and miR-133a-2 upstream regions. However, no binding was observed at the distal E box containing sequences upstream of miR-133a-2 (Figure 7A–D). Carm1/Prmt4 binding at these regions correlated precisely with the presence of diMeH3R17, and the presence of diMeH3R17 was entirely Prmt4/Carm1 dependent (Figures 6B–F and 7A–D). Binding of both Carm1/Prmt4 and diMeH3R17 was first observed at 12 h post-differentiation, consistent with the onset of miRNA expression (Figure 1) and was maintained through the 48-h post-differentiation time point. These results demonstrate that Carm1/Prmt4 and diMeH3R17 are present in the upstream regulatory regions of myogenic miRNAs, miR-1 and miR-133a, that dimethylation of H3R17 at these sequences required Carm1/Prmt4, and that the onset of Carm1/Prmt4 and diMeH3R17 binding occurred at the time of myogenic miRNA gene induction. In combination with studies from our previous work (13), we have demonstrated that each of the sites bound by Carm1/Prmt4 and diMeH3R17 also are bound by MyoD and by Brg1.Figure 6.

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