MEF2C/p300-mediated epigenetic remodeling promotes the maturation of induced cardiomyocytes

Figures

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  Figure 1. MEF2C fused with MYOD-TAD promotes cardiac reprogramming (A) Structures of MEF2C isoforms used in this study. (B) Schematic representation of the protocol for cardiac reprogramming. (C, D, K, and L) FACS analyses of cTnT expression after 1 week. Quantitative data are shown in (D and L) (n = 3, independent triplicate experiments). (E, F, M, and N) Immunostaining for αMHC-GFP, cTnT, and DAPI after 4 weeks. High-magnification views in insets show the sarcomeric organization. Quantitative data are shown in (F and N) (n = 3 independent triplicate experiments). (G and Q) Quantification of the number of beating cells after 4 weeks (n = 3 independent triplicate experiments). (H) Schematic representation of the structure of M2TAD. (I) Relative mRNA expression of Mef2c after 1 week (n = 3 independent triplicate experiments). (J) Relative luciferase activity of the Desmin-promoter corresponding with the MEF2C transcriptional activity (n = 3 independent triplicate experiments). (O and P) Spontaneous Ca2+ oscillations in the GT/M2TAD-iCMs after 2 weeks. Maximum and minimum concentrations of Ca2+ signals are shown on the left, and the Rhod-3 intensity trace is shown on the right (O).…

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  Figure 2. GT/M2TAD enhances iCM maturation (A) Hierarchical clustering of microarray data from GT/M1-, M2-, M6-, and M2TAD-induced iCMs after 1 week. Cluster 1 is the upregulated genes in GT/M2TAD-iCMs, whereas clusters 2 and 3 are downregulated genes. (B) GO term analyses of cluster 1, 2, and 3 genes. (C) Relative mRNA expression of cardiac and cell-cycle genes in GT/M2-and M2TAD-transduced MEFs after 1 week (n = 3 independent triplicate experiments). Data were normalized to the values in GT/M2-MEFs. (D) Enrichment plots of developmental stage-specific cardiac genes in GT/M2TAD- and GT/M2-iCMs. (E) PCA analyses for gene expression obtained from RNA-seq data (n = 3 each). (F and G) Immunostaining for α-actinin and DAPI after 4 weeks. The representative sarcomeric structures are enlarged in the right panels (F). Quantitative data of sarcomere length and cell size are shown (G) (n = 3 independent triplicate experiments). (H) Immunostaining for CAV3, cTnI, and DAPI staining after 4 weeks. Quantitative analyses for CAV3+/cTnI+ cells are shown (n = 3 independent triplicate experiments). (I) Electron microscopic images of iCMs. Sarcomere structures and T-tubule formation (arrowhead) were clearly…

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  Figure 3. GT/M2TAD-mediated p300 recruitment promotes chromatin remodeling (A) Schematic representation of the protocol to identify regulators occupying the promoter/enhancer regions in the GT/M2TAD-upregulated genes using ChIP-Atlas Enrichment Analysis. The candidate factors are shown in the right panel. (B) GSEA of MEF2, NKX2-5, and SRF target genes in GT/M2TAD- and GT/M2-iCMs. (C and D) The ChIP-seq signal heatmap for MEF2C, GATA4, TBX5 (in iCMs), SRF, NKX2-5, p300, H3K27ac, and H3K27me3 (in the adult heart) demonstrating co-occupancy of the candidate factors at promoter regions of the GT/M2TAD-upregulated genes (C). ChIP-seq analyses of TF binding, p300, and histone modifications at Nppa and Tmod1 genes (D). (E) ChIP-qPCR analyses of p300, H3K27ac, and H3K27me3 expression at the promoters of Nppa and Tmod1 in control-, GT/M2-, and GT/M2TAD-transduced MEFs after 1 week (n = 3 independent triplicate experiments). qPCR target regions are shown as orange boxes in (D). (F) De novo motif analysis of p300-bound sites using the public p300 ChIP-seq data from the adult heart with HOMER. (G) GO term analyses of the nearest genes to the p300 peaks using the public p300 ChIP-seq data from the…

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  Figure 4. Inhibition of p300 suppresses cardiac reprogramming (A) Schematic representation of the protocol for knockdown experiments using small interfering RNA (siRNA) for p300 (siEp300). (B and C) Immunostaining for p300 and DAPI staining in GT/M2TAD-MEFs transfected with control (Ctrl) or siEp300 after 5 days. Quantitative data are shown in (C) (n = 3 independent triplicate experiments). (D) Relative mRNA expression of Ep300 in GT/M2TAD-MEFs transfected with siEp300 (n = 3 independent triplicate experiments). (E) Heatmap of differentially expressed genes in GT/M2TAD-transduced MEFs induced with p300 knockdown after 1 week. (F) GO term analyses of the upregulated and downregulated genes in GT/M2TAD-MEFs with siEp300. (G) Immunostaining for α-actinin and DAPI staining in GT/M2TAD-iCMs transfected with Ctrl or siEp300 after 4 weeks. The representative sarcomeric structures are enlarged on the right. (H) Quantitative data of sarcomere length and cell size after 4 weeks (n = 3 independent triplicate experiments). (I and J) Quantitative data of the number of spontaneous Ca2+ oscillation+ cells after 2 weeks (I) and beating cells after 4 weeks (J) (n = 3 independent triplicate experiments).…