Among the more than different histone

Among the more than 100 different histone modifications, histone3–lysine27–trimethylation (H3K27me3) and histone3–lysine4–trimethylation (H3K4me3) are widely used histone marks associated with inactive and active promoters, respectively (Zhou et al., 2011). The H3K27me3 methyltransferase polycomb repressive complex 2 is an important regulator of normal heart development (He et al., 2012a; Delgado-Olguin et al., 2012). Mutations in MLL2, one of the H3K4 methyltransferases, are a major cause of congenital heart defects in Kabuki Syndrome in human (Ng et al., 2010). Recently, two epigenetic studies on embryonic stem cell (ESC) differentiation into CMs revealed distinct temporal patterns of H3K27me3 and H3K4me3 that are coordinated with changes in mRNA expression of functionally related genes (Wamstad et al., 2012; Paige et al., 2012). Another important regulator of order tegaserod state is the methylation status of CpG dinucleotides in DNA, particularly in the promoter regions (Suzuki and Bird, 2008). Global lack of DNA methylation in ESCs precludes differentiation (Jackson et al., 2004) while DNA methylation-silencing of pluripotency-associated promoters allows differentiation to proceed (Feldman et al., 2006). Dynamic DNA methylation is also associated with normal cardiac development, such as myocyte maturation and contractile functioning during postnatal transition of CMs to mature adult CMs (Gilsbach et al., 2014). Together, epigenetic regulation plays an essential role in a variety of important cellular processes like cardiac development, yet its role in direct cardiac reprogramming remains unknown. Recently, we established a selectable polycistronic system for the delivery of iCM reprogramming factors G, M, and T, where the complete set of constructs resulted in a wide range of reprogramming efficiencies, while infection with each construct led to a relatively homogenous cell population (Wang et al., 2015). Since these constructs consistently give rise to differential reprogramming consequences, parallel comparisons among them may reveal important epigenetic changes and identify key regulators and events in iCM reprogramming. In this study, we first characterized H3K27me3 and H3K4me3 patterns in primary neonatal CMs, the HL-1 CM cell line, and three different types of primary fibroblasts to determine their levels at cardiac and fibroblast loci in the starting and target cell types of iCM reprogramming. Interestingly, our data revealed that both H3K27me3 and H3K4me3 marks were present at fibroblast regulatory loci in CMs and at cardiac and fibroblast regulatory loci in fibroblasts. We then harnessed our polycistronic system and utilized chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) to interrogate cardiac and fibroblast promoters for possible H3K27me3 and H3K4me3 re-patterning at two critical time points during iCM reprogramming, and also sought to answer whether and how observed changes in histone marks corresponded to mRNA expression of these genes and reprogramming outcomes. Our data revealed early changes in both histone marks at cardiac promoters and later alterations at fibroblast marker genes. This re-patterning of histone marks coincided with rapid activation of cardiac gene expression and gradual suppression of fibroblast marker genes expression. Next, we generated a list for fibroblast-enriched TFs based on previously published data and examined changes in histone marks at their promoters during iCM reprogramming. We also found a late deposition of H3K27me3 in about one third of the tested fibroblast-enriched TFs, and observed a decrease in mRNA expression in a few of these TFs. Additionally, we determined the DNA methylation states of two select cardiac loci and found that both promoters were demethylated at an early stage of reprogramming. Importantly, we discovered that specific CpGs in these promoters were main contributors to total demethylation and their methylation states closely correlated with transcription activation.