<i>p57Kip2</i> is a methylation target of Dnmt3a and regulates proliferation of SCs.

<p>(A) A schematic diagram of bisulfite sequencing at the <i>p57Kip2</i> locus of <i>Dnmt3a</i>-KO and WT SCs on day 0 and day 2 after inducing differentiation. White circles and black circles indicate unmethylated and methylated CpG sites, respectively. The transcription start site (TSS) is indicated by the bent arrow. The gray bar represents the sequencing target (+1 corresponds to the TSS). (B) Average methylation frequency of CpG sites at the <i>p57Kip2</i> locus; *p<0.05, Student’s t-test. SEMs between sequences are shown. (C) <i>p57Kip2</i> knockdown by siRNA in <i>Dnmt3a</i>-KO and WT SCs; *p<0.01 Student’s t-test. (D) Cell proliferation assay after <i>p57Kip2</i> knockdown. Numbers of SCs 3 days after siRNA transfection are shown. Reduced cell proliferation in <i>Dnmt3a</i>-KO SCs is partially rescued by <i>p57Kip2</i> knockdown; *p<0.05 Student’s t-test. Data represent mean ± SEM. (E) Quantification of PHH3 positive SCs with p57Kip2 knock-down: *p<0.05 Student’s t-test. Data represent mean ± SEM. The sequences of all primers used for PCR are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006167#pgen.1006167.s012" target="_blank">S1 Table</a>.</p

Loss of <i>Dnmt3a</i> leads to elevated expression of <i>p57Kip2</i>.

<p>(A) RT-qPCR analysis of <i>Dnmt3a</i> in Ax-Cre <i>Dnmt3a</i>-KO and WT SCs before differentiation. (B) A heat map showing the expression levels of cell cycle-regulating genes based on the transcriptome analysis of Ax-Cre <i>Dnmt3a</i>-KO and WT SCs. <i>p57Kip2</i> (<i>Cdkn1c</i>) is highly expressed in <i>Dnmt3a</i>-KO SCs. (C) RT-qPCR analysis of <i>p57Kip2</i> in <i>Pax7-Cre; Dnmt3a</i>-KO and WT SCs. (D) Representative photomicrographs of <i>Pax7-Cre; Dnmt3a</i>-KO and WT SCs stained with p57Kip2 and DAPI. Scale bar—30 μm. (E) Dot plots of mean signal intensities of fluorescence in each SC stained with an anti-p57Kip2 antibody; Gray bars represent mean ± SD, ***p<0.001, Student’s t-test.</p

Loss of <i>Dnmt3a</i> leads to impaired proliferation of muscle satellite cells.

<p>(A) A schematic diagram of conditional <i>Dnmt3a</i>-KO in muscle SCs. Triangles represent loxP sites. (B) Genomic <i>Dnmt3a</i> levels in the SCs; <i>Col2a1</i> promoter served as the control; ***p<0.001, Student’s t-test. (C) <i>Dnmt3a</i> mRNA levels in the SCs; <i>Gapdh</i> served as the control. <i>Dnmt3a</i> expression in cKO SCs is below detectable levels. (D) Representative phase-contrast microscopic images of <i>Dnmt3a</i>-KO and WT SCs. Both KO and WT cells were disseminated at the same cell density on Day 0. Scale bar—200 μm. (E) <i>In vitro</i> cell proliferation assay shows significantly reduced proliferation of <i>Dnmt3a</i>-KO SCs compared to WT SCs; ***p<0.001, two-way repeated measures ANOVA. (F) Representative images of PHH3 immunocytochemistry of <i>Dnmt3a</i>-KO and WT SCs. Arrowheads indicate PHH3⁺ cells. Scale bar—30 μm. (G) Quantification of PHH3⁺ cells in <i>Dnmt3a</i>-KO and WT SCs; ***p<0.001, Student’s t-test. Data represent mean ± SEM.</p

Dnmt3a Regulates Proliferation of Muscle Satellite Cells via p57Kip2

<div><p>Cell differentiation status is defined by the gene expression profile, which is coordinately controlled by epigenetic mechanisms. Cell type-specific DNA methylation patterns are established by chromatin modifiers including <i>de novo</i> DNA methyltransferases, such as <i>Dnmt3a</i> and <i>Dnmt3b</i>. Since the discovery of the myogenic master gene <i>MyoD</i>, myogenic differentiation has been utilized as a model system to study tissue differentiation. Although knowledge about myogenic gene networks is accumulating, there is only a limited understanding of how DNA methylation controls the myogenic gene program. With an aim to elucidate the role of DNA methylation in muscle development and regeneration, we investigate the consequences of mutating <i>Dnmt3a</i> in muscle precursor cells in mice. <i>Pax3</i> promoter-driven <i>Dnmt3a</i>-conditional knockout (cKO) mice exhibit decreased organ mass in the skeletal muscles, and attenuated regeneration after cardiotoxin-induced muscle injury. In addition, <i>Dnmt3a</i>-null satellite cells (SCs) exhibit a striking loss of proliferation in culture. Transcriptome analysis reveals dysregulated expression of <i>p57Kip2</i>, a member of the Cip/Kip family of cyclin-dependent kinase inhibitors (CDKIs), in the <i>Dnmt3a</i>-KO SCs. Moreover, RNAi-mediated depletion of <i>p57Kip2</i> replenishes the proliferation activity of the SCs, thus establishing a role for the <i>Dnmt3a</i>-<i>p57Kip2</i> axis in the regulation of SC proliferation. Consistent with these findings, <i>Dnmt3a</i>-cKO muscles exhibit fewer Pax7⁺ SCs, which show increased expression of p57Kip2 protein. Thus, Dnmt3a is found to maintain muscle homeostasis by epigenetically regulating the proliferation of SCs through <i>p57Kip2</i>.</p></div

A schematic diagram of the <i>Dnmt3a-p57Kip2</i> axis in muscle satellite cells.

<p><i>Dnmt3a</i> regulates proliferation of SCs via DNA methylation of the <i>p57Kip2</i> promoter. Loss of <i>Dnmt3a</i> leads to decreased proliferation of SCs, resulting in reduced muscle mass and impaired muscle regeneration.</p

<i>Dnmt3a</i>-cKO mice show impaired muscle regenerative capacity.

<p>(A) A schematic diagram of the muscle injury and regeneration experiment. (B) HE-stained sections of the tibialis anterior muscle of <i>Dnmt3a</i>-cKO and WT mice, 7 and 14 days after cardiotoxin (CTX) injection. Centrally nucleated regenerative myofibers are scarce and thin in the <i>Dnmt3a</i>-cKO muscle 7 days after injury, compared to WT. (C) Distribution of regenerative myofiber CSAs 7 days after injury. (D) Box plots for regenerative myofiber CSAs 7 days after injury indicate significantly smaller myofiber CSA in <i>Dnmt3a</i>-cKO muscle than WT; ***p<0.001, Mann-Whitney U test. Scale bar, 100 μm.</p

Loss of <i>Dnmt3a</i> causes reduced muscle mass in mice.

<p>(A) A schematic diagram of muscle precursor cell-specific <i>Dnmt3a</i> deletion by Cre-loxP recombination. Triangles represent loxP sites. (B) Genomic DNA levels of <i>Dnmt3a</i> in tibialis anterior muscles; <i>Col2a1</i> promoter served as the control; *p<0.05, Student’s t-test. (C) RT-qPCR analysis for <i>Dnmt3a</i> using muscle tissues; <i>Gapdh</i> served as the control; *p<0.05, Student’s t-test. (D) <i>Dnmt3a</i>-cKO mice have smaller body sizes than WT controls. (E) <i>Dnmt3a</i>-cKO mice weigh less than WT controls; *p<0.05, **p<0.01, ***p<0.001, Student’s t-test. (F) X-ray whole body imaging of <i>Dnmt3a</i>-cKO and WT mice; (G, H) Muscle mass in male (G) and female (H) mice. (Left) CT images of the lower hindlimb of <i>Dnmt3a</i>-cKO and WT mice. CT images of the slices that have the maximum muscle CSA in each limb are shown. (Right) The ratio of maximum CSA of the muscle divided by that of the bone at the same slice level demonstrates disproportionately reduced muscle mass in <i>Dnmt3a</i>-cKO mice; *p<0.05, **p<0.01, Student’s t-test. (I) HE staining of the gastrocnemius muscle cross sections of <i>Dnmt3a</i>-cKO and WT mice. (J) Distribution of myofiber CSAs in <i>Dnmt3a</i>-cKO and WT muscles. (K) Box plots for myofiber CSAs in <i>Dnmt3a</i>-cKO and WT muscles; ***p<0.001, Mann-Whitney U test. Data represent mean ± SEM. Hm—<i>Dnmt3a</i> homozygous knockout; Ht—<i>Dnmt3a</i> heterozygous knockout. Scale bar—200 μm.</p

MAML1 promotes Runx2-mediated osteoblastic differentiation.

<p>A, C3H10T1/2 cells were transiently transfected with a Runx2 and/or MAML1 expression plasmids and cultured for 2 days. B, Total RNA was isolated and reverse-transcribed and TaqMan real-time PCR was performed to investigate the expression level of alkaline phosphatase gene, a marker of osteoblast.</p

Analysis of skeletal defects in MAML1^−/− mice.

<p>A, Whole mounted embryos at E16.5 were stained with Alcian Blue and Alizarin Red. B, The femoral sections at E16.5 were stained with Alcian Blue (upper panel). Arrow bar indicates the area of primary spongiosa. The expression of collagen, type 10 alpha 1, a marker of hypertrophic chondrocyte, was shown by in situ hybridization (lower panel). Green bars, primary spongiosa; red bars, hypertrophic zone; blue bars, proliferating zone. Black bars, 200 µm. C, The primary spongiosa length of MAML1 null (KO) embryos and wild type (WT) littermates. D. Femoral sections at E14.5. The expression of type 10 alpha 1 collagen, type 2 alpha 1 collagen and sox9 was shown by in situ hybridization. Bars, 100 µm.</p

MAML1 enhances Runx2 activity in a Notch-independent manner in vitro.

<p>A, The structure of MAML1 and its truncated forms. B, 293T cells were transiently transfected with a p6OSE2-Luc reporter alone or together with truncated forms of MAML1 Error bars represent the standard deviation of triplicate transfections. C, 293T cells were transiently transfected with a pTP1-Luc reporter and NotchΔE expression plasmid in the presence of γ-secretase inhibitor (DAPT). Error bars represent the standard deviation of triplicate transfections. D, 293T cells were transiently transfected with a p6OSE2-Luc reporter with Runx2, MAML1 and NotchΔE expression plasmid in the presence of γ-secretase inhibitor (DAPT). Error bars represent the standard deviation of triplicate transfections.</p