Islet-1 induces the differentiation of mesenchymal stem cells into cardiomyocyte-like cells through the regulation of Gcn5 and DNMT-1

Abstract

Previous studies from this group demonstrated that insulin gene enhancer binding protein ISL-1 (Islet-1) specifically induces the differentiation of mesenchymal stem cells (MSCs) into cardiomyocyte‑like cells through histone acetylation. However, the underlying mechanisms remain unclear. In the present study, the role of the histone acetylation and DNA methylation on the regulatory mechanism of the Islet‑1 was further investigated by methylation‑specific polymerase chain reaction (PCR), chromatin immunoprecipitation quantitative PCR and western blot analysis. The results demonstrated that Islet‑1 upregulated expression of general control of amino acid biosynthesis protein 5 (Gcn5) and enhanced the binding of Gcn5 to the promoters of GATA binding protein 4 (GATA4) and NK2 homeobox 5 (Nkx2.5). In addition, Islet-1 downregulated DNA methyltransferase (DNMT)‑1 expression and reduced its binding to the GATA4 promoter. In contrast, the amount of DNMT-1 binding on Nkx2.5 did not match the expression trend. Therefore, it was concluded that Islet‑1 may influence the histone acetylation and DNA methylation of GATA4 promoter region via Gcn5 and DNMT‑1 during the MSC differentiation into cardiomyocyte-like cells, thus prompting the expression of GATA4. The Nkx2.5 was likely only affected by histone acetylation instead of DNA methylation. The present study demonstrated that Islet‑1 induces the differentiation of mesenchymal stem cells into cardiomyocyte‑like cells through a specific interaction between histone acetylation and DNA methylation on regulating GATA4.

Figure 1.

Successful establishment of Islet-1 overexpression model in C3H10T1/2 cells. (A) Fluorescence microscopy. Scale bar=100 µm. (B) Infection efficiency, as GFP detected by flow cytometry, was 91.7%. (C) Islet-1 protein expression detected by western blotting, with β-actin as a loading control. Islet-1, insulin gene enhancer binding protein ISL-1; GFP, green fluorescent protein.

Figure 2.

Islet-1 induces the differentiation of C3H10T1/2 cells into cardiomyocytes. (A) The morphological alterations in C3H10T1/2 cells transfected with Lv-GFP or Lv-islet-1 were observed under a microscope. Scale bar=100 µm. (B) Expression of cTnT detected by immunofluorescence microscopy. Scale bar=100 µm. (C) Reverse transcription-quantitative polymerase chain reaction detected variations in mRNA expression levels of cardiac-specific transcription factors in C3H10T1/2 cells infected with lentiviral vectors containing Islet-1. *P<0.05 vs. blank group. Lv-GFP, lentiviral vector containing green fluorescent protein; Lv-islet-1, lentiviral vector containing Islet-1; cTnT, troponin T2 cardiac type; Nkx2.5, NK2 homeobox 5; GATA4, GATA binding protein 4; Mef2c, myocyte enhancer factor 2C; 1 W, 1 week; 2 W, 2 weeks; 3 W, 3 weeks; 4 W, 4 weeks.

Figure 3.

DNA methylation levels and acetylation levels of the histone H3K9 site in the GATA4 and Nkx2.5 promoter regions during the differentiation process promoted by Islet-1. (A) The detection of methylation levels on the GATA4 promoter (1329–1489 bp) by MSP assay. (B) The detection of the methylation levels at the Nkx2.5 promoter (51–219 bp) by MSP assay. (C) ChIP results demonstrated the levels of histone acetylation on the promoter regions of GATA4 and Nkx2.5. *P<0.05 vs. blank group. GATA4, GATA binding protein 4; Nkx2.5, NK2 homeobox 5; MSP, methylation-specific polymerase chain reaction; Lv-GFP, lentiviral vector containing green fluorescent protein; Lv-islet-1, lentiviral vector containing Islet-1; M, methylated; U, unmethylated; 1 W, 1 week; 2 W, 2 weeks; 3 W, 3 weeks; 4 W, 4 weeks.

Figure 4.

Detection of HATs on the histone H3K9 site that regulate the promoter regions of GATA4 and Nkx2.5. (A) Western blot analysis of Gcn5 and P300 HATs, with quantification relative to β-actin. (B) ChIP analysis of Gcn5 bound to the GATA4 and Nkx2.5 promoter regions. (C) ChIP analysis of P300 bound to the GATA4 and Nkx2.5 promoter regions. *P<0.05 vs. blank control. HATS, histone acetyltransferases; GATA4, GATA binding protein 4; Nkx2.5, NK2 homeobox 5; Lv-GFP, lentiviral vector containing green fluorescent protein; Lv-islet-1, lentiviral vector containing Islet-1; 1 W, 1 week; 2 W, 2 weeks; 3 W, 3 weeks; 4 W, 4 weeks; Gcn5, general control of amino acid biosynthesis protein 5; ChIP, chromatin immunoprecipitation.

Figure 5.

Detection of DNMTs that regulate the GATA4 promoter region. (A) Western blot analysis of DNMT-1 and DNMT-3a expression, with quantification relative to β-actin. (B) ChIP analysis of DNMT-1 bound to the GATA4 and Nkx2.5 promoter regions. (C) ChIP analysis of DNMT-3a bound to the GATA4 and Nkx2.5 promoter regions. (D) ChIP analysis of DNMT-3b bound to the GATA4 and Nkx2.5 promoter regions. *P<0.05 vs. blank control. DNMT, DNA methyltransferase; GATA4, GATA binding protein 4; Nkx2.5, NK2 homeobox 5; Lv-GFP, lentiviral vector containing green fluorescent protein; Lv-islet-1, lentiviral vector containing Islet-1; 1 W, 1 week; 2 W, 2 weeks; 3 W, 3 weeks; 4 W, 4 weeks; ChIP, chromatin immunoprecipitation.