Abstract
BackgroundEuchromatic histone-lysine N-methyltransferase 2 (G9a/Ehmt2) is the main enzyme responsible for the apposition of H3K9 di-methylation on histones. Due to its dual role as an epigenetic regulator and in the regulation of non-histone proteins through direct methylation, G9a has been implicated in a number of biological processes relevant to cell fate control. Recent reports employing in vitro cell lines indicate that Ehmt2 methylates MyoD to repress its transcriptional activity and therefore its ability to induce differentiation of activated myogenic cells.MethodsTo further investigate the importance of G9a in modulating myogenic regeneration in vivo, we crossed Ehmt2floxed mice to animals expressing Cre recombinase from the Myod locus, resulting in efficient knockout in the entire skeletal muscle lineage (Ehmt2ΔmyoD).ResultsSurprisingly, despite a dramatic drop in the global levels of H3K9me2, knockout animals did not show any developmental phenotype in muscle size and appearance. Consistent with this finding, purified Ehmt2ΔmyoD satellite cells had rates of activation and proliferation similar to wild-type controls. When induced to differentiate in vitro, Ehmt2 knockout cells differentiated with kinetics similar to those of control cells and demonstrated normal capacity to form myotubes. After acute muscle injury, knockout mice regenerated as efficiently as wildtype. To exclude possible compensatory mechanisms elicited by the loss of G9a during development, we restricted the knockout within adult satellite cells by crossing Ehmt2floxed mice to Pax7CreERT2 and also found normal muscle regeneration capacity.ConclusionsThus, Ehmt2 and H3K9me2 do not play significant roles in skeletal muscle development and regeneration in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-016-0093-7) contains supplementary material, which is available to authorized users.
Highlights
Euchromatic histone-lysine N-methyltransferase 2 (G9a/euchromatic histone-lysine Nmethyltransferase 2 (Ehmt2)) is the main enzyme responsible for the apposition of H3K9 di-methylation on histones
In Fluorescence-activated cell sorting (FACS)-purified satellite cells from control mice (Myodwt/wt Ehmt2floxed/floxed), the Ehmt2 functional allele frequency was 100 %; whereas in the knockout mice (Myodwt/Causes Recombination (Cre) Ehmt2floxed/floxed), the functional allele frequency was reduced to 2.9–7.9 % (Fig. 1a)
Western blot analysis of whole skeletal muscle lysates from the conditional knockout mice showed reduction of H3K9me2 levels compared to those of the wildtype (Fig. 1b), congruent with previous reports that H3K9me2 is diminished in Ehmt2null/null models [14]
Summary
Euchromatic histone-lysine N-methyltransferase 2 (G9a/Ehmt2) is the main enzyme responsible for the apposition of H3K9 di-methylation on histones. A proportion of fetal myoblasts become localized underneath the basal lamina of newly formed myofibers These cells become specified as satellite cells—the quiescent, tissue resident stem cell of the skeletal muscle, identifiable by the expression of paired box transcription factor, paired box 7 (Pax). Despite much knowledge about the key transcription factors regulating myogenesis, the epigenetic landscapes required for the control of gene expression in skeletal muscle differentiation remain less well understood. Chromatin immunoprecipitation (ChIP)-seq analyses have revealed the importance of bivalent domains containing both H3K4me and H3K27ac in regulating muscle enhancers during myogenesis [9] These data point to Myod as playing a key role in the recruitment of chromatin-modifying enzymes and transcription factors to activate such enhancers [11, 12]
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