Abstract

Epigenetics plays a critical role in SMCs differentiation where histone proteins associated with the myosin heavy chain 11 (Myh11) promoter are post-translationally modified while promoter sequences are de-methylated by the DNA-modifying enzyme ten-eleven translocation-2 (TET2). Di-methylation of lysine 4 on histone 3 (H3K4me2) is generally associated with euchromatin and active gene expression whereas, tri-methylation of lysine 27 on histone 3 (H3K27me3) is generally associated with formation of heterochromatin and gene suppression. Our aim was to determine the level of H3K4me2 and H3K27me3 enrichment on Myh11 promoter of differentiated SMCs and compare that signature to undifferentiated resident vascular stem cells and their stem cell-derived myogenic progeny. Murine Sca1⁺ stem cells (APCs), rat medial Sox10⁺multipotent vascular stem cells (MVSCs) and CD44⁺ bone-marrow derived mesenchymal stem cells (MSCs) were all examined for H3K4me2 and H3K27me3 enrichment on Myh11 promoter, before and after myogenic differentiation and compared to freshly isolated aortic differentiated SMCs and sub-cultured ‘de-differentiated SMCs’ in vitro by Chromatin Immunoprecipitation (ChIP) assay. Differentiated rat and murine SMCs were highly enriched for H3K4me2 mark at the Myh11 promoter compared to the H3K27me3 mark, concomitant with enhanced TET-2 binding. Phenotypical ‘de-differentiated’ sub-cultured rat and murine SMC were also enriched for H3K4Me2 mark when compared to H3K27me3, but to a much lesser extent. In contrast, all three stem cell populations were highly enriched for H3K27me3. The levels of enrichment of H3K27me3 significantly reduced following myogenic differentiation with TGF-β1 concomitant with a significant enrichment of H3K4me2 mark and TET-2 binding to levels that mimicked the level of enrichment in de-differentiated SMCs at the same locus. We conclude that stem-cell derived myogenic progeny enrich for the H3K4me2 mark at the Myh11 promoter and thus may be responsible for the presence of this mark within arteriosclerotic cells following injury.