br Materials and methods br Results br

Materials and methods
Results
Discussion Since the initial report of iCM reprogramming in vitro (Ieda et al., 2010) and in vivo (Inagawa et al., 2012; Qian et al., 2012; Song et al., 2012), there have been considerable efforts to enhance the efficiency of direct cardiac reprogramming. One of the strategies is to optimize reprogramming efficiency through identifying enhancers of iCM generation or new combinations of reprogramming cocktails. Jayawardena et al. reported iCM induction using a microRNA cocktail consisting of miR-1, -133, -208, and -499 (Jayawardena et al., 2012). Others found that different TF/microRNA combinations could efficiently generate iCMs depending on the starting Caspase-3/7 Inhibitor type, the viral-delivery system and the cardiac reporter(s) being used. These various combinations of reprogramming factors include wild-type G, T, Hand2 in combination with Mef2c fused with the transactivation domain of MyoD (Hirai et al., 2013), the combination of M, T, and Myocd (Protze et al., 2012), the combination of G, M, T, Hand2, and Nkx2.5 (Addis et al., 2013), the combination of G, M, T, Myocd, and Srf (Christoforou et al., 2013), and the G, M, T cocktail in combination with miR-1 or miR-133 (Muraoka et al., 2014). Additionally, growth factors and small molecules have been used to enhance the efficiency of iCM reprogramming. Pre-conditioning infarcted hearts with VEGF and inhibition of TGFβ signaling has been shown to improve cardiac reprogramming (Ifkovits et al., 2014; Mathison et al., 2012). Furthermore, we recently demonstrated that stoichiometry of G, M, T could greatly affect iCM reprogramming and a relative high level of M and moderate levels of G and T in a MGT polycistronic construct gave rise to enhanced reprogramming (Wang et al., 2015). During the review process of our manuscript, two studies were published, reporting enhanced reprogramming efficiency based on the G, M, T and Hand2 cocktail by overexpression of the protein kinase Akt1 (Zhou et al., 2015) or by suppression of pro-fibrotic signaling such as TGFβ or ROCK kinase pathways with small molecule inhibitors (Zhao et al., 2015). Nevertheless, how the fibroblast epigenome is reset during direct cardiac reprogramming remains largely unknown. Ieda et al. examined the epigenetic states of three cardiac promoters (Tnnt2, Ryr2 and Actn2) in 4-week FACS-sorted αMHC-GFP+ iCMs. ChIP –qPCR analysis revealed decreased H3K27me3 and increased H3K4me3 at these three promoters in late-stage reprogrammed cells. However, epigenetic re-patterning has been shown to occur rapidly after the overexpression of reprogramming factors, for example, within a few days for iPSCs (Papp and Plath, 2013; Koche et al., 2011). In this study, we intended to characterize early epigenetic changes when the cell fate transition was initiated. Therefore, we took advantage of our puro-selectable polycistronic system to enrich cells that have taken up all reprogramming factors, and determined the epigenetic resetting at two critical time points at early stages of iCM reprogramming (day 3 and day 10). In addition, in order to gain insights into how fibroblast fate is suppressed during iCM reprogramming, we also expanded the testing loci to include both cardiac promoters and fibroblast promoters; particularly some largely unexplored fibroblast regulatory genes in other iCM studies. As critical control experiments, we characterized H3K27me3 and H3K4me3 patterns at cardiac and fibroblast loci in CMs and fibroblasts. Our results revealed that in addition to cardiac regulatory genes, fibroblast regulatory gene promoters were also marked by both H3K27me3 and H3K4me3 in fibroblasts, suggesting the plasticity of fibroblasts. Analysis of H3K27me3 and H3K4me3 dynamics during iCM reprogramming revealed early re-patterning of H3K27me3 and H3K4me3 at cardiac loci and later alterations at fibroblast loci paralleled by a dramatic increase in cardiac markers expression and a gradual decrease in fibroblast markers expression. These results suggest an early activation of the cardiac program and a progressive suppression of fibroblast fate. Additionally, specific CpGs in the promoters of Myh6 and Nppa were found to be possible main contributors to total CpG demethylation during iCM reprogramming and their methylation states were correlated with transcriptional activation of Myh6 and Nppa. The methylation states of these CpGs might have important regulatory roles that contribute to the control of DNA accessibility of these two loci during iCM reprogramming. Our study thus reveals a differential re-patterning of H3K27me3, H3K4me3 at cardiac and fibroblast loci, and establishes the platform and conditions that may be used in future genome-wide studies.