Enhancer of polycomb1 acts on serum response factor to regulate skeletal muscle differentiation

Abstract

Skeletal muscle differentiation is well regulated by a series of transcription factors. We reported previously that enhancer of polycomb1 (Epc1), a chromatin protein, can modulate skeletal muscle differentiation, although the mechanisms of this action have yet to be defined. Here we report that Epc1 recruits both serum response factor (SRF) and p300 to induce skeletal muscle differentiation. Epc1 interacted physically with SRF. Transfection of Epc1 to myoblast cells potentiated the SRF-induced expression of skeletal muscle-specific genes as well as multinucleation. Proximal CArG box in the skeletal alpha-actin promoter was responsible for the synergistic activation of the promoter-luciferase. Epc1 knockdown caused a decrease in the acetylation of histones associated with serum response element (SRE) of the skeletal alpha-actin promoter. The Epc1.SRF complex bound to the SRE, and the knockdown of Epc1 resulted in a decrease in SRF binding to the skeletal alpha-actin promoter. Epc1 recruited histone acetyltransferase activity, which was potentiated by cotransfection with p300 but abolished by si-p300. Epc1 directly bound to p300 in myoblast cells. Epc1+/- mice showed distortion of skeletal alpha-actin, and the isolated myoblasts from the mice had impaired muscle differentiation. These results suggest that Epc1 is required for skeletal muscle differentiation by recruiting both SRF and p300 to the SRE of muscle-specific gene promoters.

FIGURE 1.

Epc1 interacts with SRF. A, immunoprecipitation (IP) showing the physical interaction of Epc1 and SRF in mammalian cells (third panel). pCMV-myc-Epc1 constructs with either pcDNA3.1 (mock) or pCGN-HA-SRF were transiently transfected into 293T cells; anti-HA antibodies were used for immunoprecipitation, and Epc1 was detected with α-Myc antibody. IB, immunoblot. B, reverse immunoprecipitation in 293T cells. Epc1 was pulled down, and SRF was detected in the immunoprecipitates. C, physical interactions of endogenous proteins in C2C12 cells. SRF pulled down Epc1 (third panel), and Epc1 recruited SRF (fourth panel). D, GST pulldown assay. In vitro translated SRF-HA was applied to the columns with either GST or GST-Epc1 chimeric protein. GST-Epc1 successfully pulled down the SRF-HA (upper panel).

FIGURE 2.

Forced expression of Epc1 enhances SRF-induced skeletal muscle differentiation. A, fluorescent immunocytochemistry for MyoD visualization. After transfection of SRF or Epc1, C2C12 cells were subjected to immunocytochemistry with anti-MyoD antibody. B, fluorescent immunocytochemistry for myogenin expression. C and D, quantification of immunoreactive cells for MyoD (C) and myogenin (D). Epc1 further increased the MyoD and myogenin expression. E, Multinucleated C2C12 cells after serum starvation. Epc1 potentiated SRF-induced multinucleation. *, p < 0.05; **, p < 0.01; @, p < 0.05; NS, not significant. Error bars represent S.E.

FIGURE 3.

Epc1 and SRF synergistically induce muscle-specific genes. A, cotransfection of SRF and Epc1 synergistically induced skeletal α-actin, MyoD, and myogenin proteins in C2C12 cells. B, quantification of protein amounts from three independent immunoblots. C, SRF and Epc1 elevated the transcript levels of skeletal α-actin, muscle creatine kinase (MCK), myogenin, and MyoD, but not myf-6 in 10T1/2 cells. D, real-time PCR analysis for quantification of the transcript levels of skeletal α-actin, MCK, myogenin, and MyoD from three independent sets of experiments. Error bars represent S.E.

FIGURE 4.

Epc1 binds to the SRE in the skeletal α-actin promoter. A, Epc1 potentiated SRF-induced transactivation of the minimal promoter of skeletal α-actin. Values are the -fold increase in luciferase activity relative to activation of the reporter alone. *, p < 0.05; **, p < 0.01 compared with mock-transfected group; @@, p < 0.01 compared with SRF-transfected group. B, diagram showing the proximal region of the skeletal α-actin promoter and promoter mutants. Epc1 potentiates SRF-mediated transactivation when the CArG-near box of the skeletal α-actin promoter is intact. C, schematic diagram showing the promoter of rat skeletal α-actin; the arrows show the PCR primers for the chromatin immunoprecipitation assay. D, chromatin immunoprecipitation (ChIP) assay to show the binding of the Epc1-containing complex to the SRE in the skeletal α-actin promoter in H9c2 cells. Chromatin was prepared from H9c2 cells as described under “Experimental Procedures” and was immunoprecipitated with anti-Epc1 antibody. Note that exon 3 and exon 5 regions were not recruited by Epc1. E, quantification of results from three independent chromatin immunoprecipitation assays. Error bars represent S.E. F, sequential chromatin immunoprecipitation assay to show whether Epc1 and SRF can bind simultaneously to the SRE. The Epc1 precipitates (fourth lane) were subjected to immunoprecipitation with anti-SRF antibody and the SRE regions were amplified by PCR (sixth lane). See “Experimental Procedures” for detailed protocols.

FIGURE 5.

Epc1 is required for binding of SRF to the SRE and for acetylation of the SRE. A, reduction of Epc1 expression in antisense Epc1 H9c2 cell lines. Transcripts and protein levels of Epc1 were examined by RT-PCR reaction and Western blot analysis. The protein level of SRF was not significantly altered in Epc1 knockdown. B and C, representative chromatin immunoprecipitation (B) and quantification from three independent experiments (C) of the chromatin immunoprecipitation assay (ChIP) showing the reduction in Epc1 binding to the SRE in Epc1 antisense cell lines. α-Epc1 antibody was used for the assay. D and E, Epc1 is required for the binding of SRF to the SRE. SRF binding to the SRE was examined in Epc1 knockdown cells by chromatin immunoprecipitation assay. Anti-SRF antibody was used for the immunoprecipitation, and rat skeletal α-actin SRE was amplified. SRF/SRE binding was significantly reduced in the absence of Epc1 (third lane in D). Quantification results from three experiments are shown (E). F–I, reduced acetylation of histones associated with the SRE of the skeletal α-actin promoter in Epc1 knockdown cells. A chromatin immunoprecipitation assay was performed with α-acetyl histone H3 (F and G) and H4 (H and I) antibodies, and the SRE in the skeletal α-actin promoter was amplified. Reduction of Epc1 expression dramatically decreased the acetylation of both histones. Quantification of the acetylation of histones H3 (G) and H4 (I) is shown. Error bars represent S.E.

FIGURE 6.

Epc1 acetylates histones associated with the proximal promoter of skeletal α-actin by recruiting p300. A, immunoprecipitation (IP) assay showing the direct interaction of Epc1 with endogenous p300 (first lane in lower panel) in H9c2 cells. Epc1-p300 interaction was reduced in the Epc1-antisense H9c2 cell line (second lane). IB, immunoblot. B, immunoprecipitation assay showing the direct association between endogenous Epc1 and endogenous p300 in C2C12 cells. C, histone acetyltransferase activity of Epc1-containing complex. A histone acetyltransferase activity assay was performed with the Epc1 immunoprecipitates, and histones were used for the substrate. Upper panel, autoradiograph images showing the acetylation. Epc1 immunoprecipitates successfully acetylated histones (second lane), whereas IgG precipitates failed to do so (fifth lane). Cotransfection of p300 potentiated the acetylation in the Epc1 immunoprecipitates in a dose-dependent fashion (third and fourth lanes). Lower panel, Coomassie Blue staining. D, transfection of si-p300 reduced the protein amount of endogenous p300. E, reduction of p300 results in a decrease in Epc1-mediated acetylation of histones. Acetylation in Epc1 immunoprecipitates (fourth lane) was completely blocked by transfection of si-p300 (fifth lane).

FIGURE 7.

Generation of Epc1 knock-out mice and the muscular phenotypes of heterozygous mice. A, diagram showing the genomic structure of gene-trapped embryonic stem cells of Epc1. The first exon of the Epc1 gene was disrupted by insertion of the gene-trap vector. PCR primers for detection of either wild type mice or gene-trap vector are indicated by thick bars. For the detection of wild type mice, sense primer at the 3′-end of exon 1 and antisense primer at the first intron were used. The presence of the gene-trap vector was evaluated by 411-bp amplimers in the neomycin region. B, genotype outcomes of Epc1 knock-out mice at 10 days after birth. Homozygous mice were not seen. C, immunohistochemistry analysis showing desmin expression in longitudinal sections of mouse hamstring muscles. Desmin expression was significantly reduced in Epc1^+/− mice (magnification ×400). d and E, fluorescent immunohistochemistry showing skeletal α-actin. D, in Epc1^+/− mice, skeletal α-actin was clumped and disorganized (magnification ×400). E, images at the higher magnification (×800) were obtained after staining with anti-skeletal α-actin antibody. In Epc1^+/− mice, skeletal α-actin expression was reduced.

FIGURE 8.

Impaired muscle differentiation in Epc1^+/− mice. A, muscle-specific gene expression in hamstring muscles from three wild type and three Epc1^+/− mice. The expression of MRFs such as myogenin and MyoD, as well as of skeletal muscle-specific genes such as skeletal α-actin and desmin, was significantly reduced in heterozygous mice. B, immunoblots of skeletal α-actin, MyoD, and myogenin from four different mice and their quantification results. *, p < 0.05 compared with wild-type mice. C, gene expression in myoblasts obtained from two wild type and five Epc1^+/− mice. The isolated myoblasts underwent serum deprivation to induce differentiation for 2 days. Skeletal muscle markers failed to be increased by serum deprivation in Epc1^+/− mice. D, serum deprivation-induced multinucleation of myoblasts obtained from wild type and Epc1^+/− mice. The myoblasts were deprived of serum and maintained for 3 days. Multinucleated cells were counted. Five different fields were examined per slide, and the experiments were performed in triplicate from three different mice. **, p < 0.01 compared with wild-type mice. Error bars represent S.E.

FIGURE 9.

Diagram showing the mechanism of Epc1/SRF/p300 interaction in the transcriptional regulation of SRF-dependent muscle-specific genes.