H3K9me2 regulates early transcription factors to promote mesenchymal stem‑cell differentiation into cardiomyocytes

Abstract

Studies have shown that histone H3 at lysine 9 (H3K9me2) is an important epigenetic modifier of embryonic development, cell reprogramming and cell differentiation, but its specific role in cardiomyocyte formation remains to be elucidated. The present study established a model of 5-Azacytidine-induced differentiation of rat bone mesenchymal stem cells (MSCs) into cardiomyocytes and, on this basis, investigated the dimethylation of H3K9me2 and its effect on cardiomyocyte formation by knockdown of H3K9me2 methylase, euchromatic histone-lysine N-methyltransferase 2 (G9a) and H3K9me2 lysine demethylase 3A (KDM3A). The results demonstrated that, in comparison with the normal induction process, the knockdown of G9a could significantly reduce the H3K9me2 level of the MSCs in the induced model. Reverse transcription-quantitative (RT-q) PCR demonstrated that the expression of cardiac troponin T(cTnT) was significantly increased. In addition, flow cytometry demonstrated that the proportion of cTnT-positive cells was significantly increased on day 21. With the knockdown of KDM3A, the opposite occurred. In order to explore the specific way of H3K9me2 regulating cardiomyocyte formation, western blotting and RT-qPCR were used to detect the expression of key transcription factors including GATA binding protein 4 (GATA-4), NK2 Homeobox 5 (Nkx2.5) and myocyte enhancer factor 2c (MEF2c) during cardiomyocyte formation. The decrease of H3K9me2 increased the expression of transcription factors GATA-4, Nkx2.5 and MEF2c in the early stage of myocardial development while the increase of H3K9me2 inhibited the expression of those transcription factors. Accordingly, it was concluded that H3K9me2 is a negative regulator of cardiomyocyte formation and can participate in cardiomyocyte formation by activating or inhibiting key transcription factors of cardiomyocytes, which will lay the foundation for the optimized induction efficiency of cardiomyocytes in in vitro and clinical applications.

G9a inhibition promotes the formation of pacemaker-like cells by reducing the enrichment of H3K9me2 in the HCN4 promoter region

“…It is well known that H3K9 is widely involved in cell proliferation and differentiation. Our previous study reported that knockdown of the histone methyltransferase G9a increased expression of early transcription factors during the differentiation of MSCs into cardiomyocytes (21). BIX01294 was originally reported to be a G9a inhibitor during a chemical library screen of small molecules and has previously been used in the generation of induced pluripotent stem cells.…”

DNA methylation cooperates with H3K9me2 at HCN4 promoter to regulate the differentiation of bone marrow mesenchymal stem cells into pacemaker-like cells

“…We have previously observed the morphology of cells and detected their surface markers, which were consistent with the characteristics of BMSCs. They have been published in relevant articles [ 21 , 22 ]. The BMSCs were then expanded and used for in vitro and in vivo experiments at passages 3–5.…”

“…According to the sequence of the CDS region of the gene provided by NCBI, the CDS of NKx2.5, DNMT1, DNMT3A and DNMT3B were synthesised was synthesized and cloned into pcDNA3.1 vector, G9a CDS sequence was synthesised and cloned into pcDNA3.1-Myc vector. The sh-G9a lentiviral interference vector was obtained from a previous study in our laboratory [ 18 ]. The DNMT1-shRNA Plasmid was obtained from Origene (Locus ID 84350).…”

“…The sh-G9a lentiviral interference vector was obtained from a previous study in our laboratory [18]. The DNMT1-shRNA Plasmid was obtained from Origene (Locus ID 84350).…”

“…Methylation of H3K4, H3K36, and H3k79 is associated with gene activation, whereas H3K9, H3K27, and H4K20 are associated with gene silencing [ 16 ]. Genetic knockout or BIX01294 treatment to inhibit the histone methyltransferase G9a can induce the expression of cardiac embryonic lineage cell-related genes such as Brachyury and Mesp1in MSCs [ 17 ], and our previous study also found that disruption of G9a and KDM3A had an opposite effect on the differentiation of MSCs into cardiomyocytes [ 18 ]. The above results indicate a potential role of epigenetic modifications in the differentiation of rat MSCs into cardiac pacemaker-like cells.…”