The Machenism Of Interaction Between Acetylation And Methylation During The MSCs Differentiation Into Cardiomyocytes Inducing By Islet-1

Objective To confirm that, during MSCs differentiation into cardiomyocyte induced by islet-1, the role of histone methylation/acetylation and DNA methylation in regulating GATA4 and Nkx2.5, to identify the key enzyme of these modification involved in regulating GATA4 and Nkx2.5, and to preliminary verify the mechanism of interactions between these key enzyme in regulating GATA4 and Nkx2.5. This study will lay an important experimental basis for improving the efficiency of MSCs differentiation into cardiomyocyte, which could improve the clinical application of MSCs.Methods 1. Chromatin immunoprecipitation q PCR(Ch IP-q PCR): detect histone acetylation/methylation level in GATA4 and Nkx2.5 promoter, and the key enzyme binding level in these site. 2. Methylation-Specific PCR(MSP): preliminary detect DNA methylation level in GATA4 and Nkx2.5 promoter3. Bisulfite sequencing PCR(BSP): verify the DNA methylation level of the sample which DNA methylation level significant changed in MSP 4. Fluorescence quantitative PCR(FQ-PCR): detect the relative m RNA expression of GATA4 and Nkx2.5 5. Western blot detect the expression of the key enzyme of histone methylation/acetylation and DNA methylation 6. Immunofluorescence detect the expression of cardiac-specific protein c Tn T and Cx43 to confirm the MSCs have differentiated into cardiomyocyte-like cellResults 1. In the process of MSCs differentiation into cardiomyocyte inducing by islet-1, the histone acetylation level of GATA4 promoter site 2 gradually increased, but the histone methylation and DNA methylation level were decreased; the histone methylation level and histone acetylation level of Nkx2.5 promoter as same as GATA4, but there was no change in DNA methylation level throughout the process 2. Use 5-aza to change DNA methylation level after transfection islet-1, the histone acetylation level of GATA4 promoter site2 increased and histone methylation level decreased, but 5-aza could not change the histone methylation/acetylation level both. 3. Use TSA to change histone acetylation level after transfection islet-1, the histone methylation level and DNA methylation level of GATA4 promoter site2 decreased significantly, and only histone methylation level of Nkx2.5 promoter dcreased 4. Western blot detect the key enzyme of the three modifications, and in the the process of MSCs differentiation into cardiomyocyte inducing by islet-1, the significant changed key enzyme were Gcn5, P300, HDAC1, G9 A, DNMT-1 and DNMT-3a 5. Ch IP-q PCR showed Gcn5, HDAC1, G9 A, DNMT-1, HP1-α and HP1-β are bind to promoter of GATA4 and Nkx2.5, but the binding level of DNMT-1, HP1-α and HP1-β were not changed in Nkx2.5 promoter 6. The expression of GATA4 and Nkx2.5 were significantly decreased after Gcn5 inhibition compared with LV-islet-1 group, and there was no expression of c Tn T and Cx43, these results indicated inhibit Gcn5 could block MSCs differentiation into cardiomyocyte-like cell. 7. After inhibition Gcn5, the binding level of HDAC1, G9 A, HP1-α, HP1-β and DNMT-1 to GATA4 promoter significant increased compared with LV-islet-1 group, and the histone acetylation level of this site significant decreased, histone methylation level and DNA methylation level increased significantly; additionally, because the binding level of HP1-α, HP1-β and DNMT-1 to Nkx2.5 promoter were not changed, the DNA methylation level was not changed compared with LV-islet-1Conclusion In the process of MSCs differentiation induced by Islet-1, Islet-1 could guide Histone acetyltransferase Gcn5 binding to cardiac-specific transcription factor, and associate with histone methylation and DNA methylation to form “cycle” regulation in these transcription factor promoter to co-regulate the expression of transcription factor, and induce MSCs differentiation into cardiomyocyte.