Impact of Gata4, Mef2c and Tbx5 Stoichiometry on Epigenetic Status of Direct Cardiac Reprogramming

Reprogramming from fibroblasts into induced cardiomyocytes (iCMs) offers alternative strategies for cardiac disease modeling and cardiac regeneration. Cellular reprogramming is closely associated with global re-patterning of the epigenetic landscape. The reprogramming cells must overcome the epigenetic barriers to acquire the target cell-like epigenetic pattern. We have recently demonstrated that stoichiometry of iCM reprogramming factors Gata4 (G), Mef2c (M) and Tbx5 (T) has profound impact on reprogramming efficiency and quality, yet the underlying mechanism remains unclear. Here, we demonstrate that the optimal stoichiometry combination MGT resulted in a reduced binding of the repressive marker histone 3 lysine 27 trimethlytion (H3K27me3) and an enhanced binding of the active marker histone 3 lysine 4 trimethlytion (H3K4me3) on the cardiac gene loci, compared to the least optimal combination GTM. We found that this epigenetic change occurred as early as day 3 when cardiac marker genes started to be induced in the reprogramming fibroblasts. Additionally, we determined the DNA methylation status in two cardiac gene loci during MGT and GTM induced reprogramming. Subsets of the CpG islands in these promoters were significantly more demethylated in MGT than GMT-transduced cells. We thus propose these CpGs islands as the “regulatory” CpGs critical for iCM reprogramming. In conclusion, we demonstrated that stoichiometry of G, M, T influences the epigenetic status of the iCM cells, suggesting a potential mechanism of how stoichiometry of G,M,T influences reprogramming.