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    • To the best of our knowledge

    2018-10-20

    To the best of our knowledge, this is the first study to characterize H3K27me3 and H3K4me3 re-patterning accompanying cardiac reprogramming. Recently, it was reported that iPSC reprogramming is characterized by global loss of H3K27me3 in the beginning and later restoration of H3K27me3 at selected sites (Hussein et al., 2014). Here we observed loss of H3K27me3 at cardiac genes early (day 3) in iCM reprogramming (Fig. 3). However, whether the observed removal of H3K27me3 is specific to the cardiac lineage remains to be answered. In iPSC reprogramming, early down-regulation (at day 1–2) of fibroblast markers such as THY1 is one of the hallmarks and prerequisites for reprogramming to proceed (Polo et al., 2012). Here in iCM reprogramming, we showed that H3K27me3 deposition at fibroblast genes was only observed on day 10 and corresponding mRNA expression gradually decreased over time (Fig. 4). More interestingly, Thy1 expression was not completely repressed even at day 10 during iCM reprogramming (Fig. 4A), suggesting that even though both iPSC and iCM reprogramming start from fibroblasts, the routes to pluripotency and to iCM might be different. The difference may reflect differential mechanisms in how the reprogramming factors influence the regulation of histone and DNA modification, or it may suggest that direct cardiac reprogramming allows co-existence of cardiac and certain fibroblast signatures during the early phase of reprogramming. A recent report showed that suppression of pro-fibrotic signaling with small molecule inhibitors led to increased efficiency and accelerated kinetics of iCM reprogramming (Zhao et al., 2015). Yet, future studies of cell reprogramming including iCM reprogramming are required in order to address the necessity and mechanisms of erasure of the fibroblast program, which may provide novel insights for acceleration and improved completeness of the reprogramming process. What\'s more, to completely understand epigenetic landscape dynamics during cardiac reprogramming and how such changes orchestrate the cell fate conversion, genome-wide mapping of a complete set of histone marks is needed in the future. From induced pluripotency to the generation of iCM, iNeuron, iHepatocytes and induced multilineage blood progenitors, fibroblasts have often been chosen as the starting phalloidin for reprogramming (Huang et al., 2011; Ieda et al., 2010; Szabo et al., 2010; Vierbuchen et al., 2010). The rationale behind this choice of starting cell includes easy access to fibroblasts compared with other cell types yet other reasons may apply as well. Studies of ESCs revealed that regulatory genes controlling diverse cell fates were largely marked by both H3K27me3 and H3K4me3 in pluripotent cells, which denotes “poising” of these genes for rapid activation or silencing upon differentiation (Bernstein et al., 2006). Depending on the developmental course, these “bivalent” genes resolve to either only H3K4me3-marked or only H3K27me3-marked in terminally differentiated cells (Mikkelsen et al., 2007). Our data here reveal exclusive marking of cardiac regulatory genes (M, G and T) by H3K4me3 and marking of fibroblast-enriched TFs by both H3K27me3 and H3K4me3 in CMs (Figs. 1, 2). Interestingly, all three types of primary fibroblasts MEF, CF, and TTF were marked by both H3K27me3 and H3K4me3 at cardiac TFs (M, G and T) and at fibroblast-enriched TFs (Figs. 1, 2), similar to findings in ESCs (Wamstad et al., 2012). Our data imply the possibility that fibroblasts are more amenable to being reprogrammed compared to other somatic cell types because of their lower epigenetic barriers and thus higher plasticity. Parallel studies comparing epigenetic re-patterning during cellular reprogramming starting from fibroblasts and other cell types are needed to evaluate this possibility. Even though all fibroblasts are categorized as such, different types of fibroblasts could have distinct origins, functions, gene expression profiles and epigenetic patterns (Sorrell et al., 2004; Baum and Duffy, 2011; Rodemann, 2011). Our ChIP-qPCR results showed that H3K27me3/H3K4me3 states (positive or negative) in the three tested fibroblasts CF, MEF, and TTF were mostly consistent across cardiac and fibroblast loci with a few exceptions, but the levels of methylation were different. At certain cardiac genes, CF has higher H3K4me3 (Mef2c, Gata4) and lower H3K27me3 (Gata4, Tnnt2, Ryr2) compared to MEF and TTF while TTF has higher H3K27me3 and lower H3K4me3 at Tbx5 compared to CF and MEF (Fig. 1A). At fibroblast-enriched TF loci, CF generally shows higher H3K27me3 than MEF and TTF (Fig. 2A). In summary, the H3K27me3/H3K4me3 states in CFs most resemble those found in primary CMs while the H3K27me3/H3K4me3 states in TTFs are least similar to CMs among the three. Interestingly, these findings on epigenetic states of CF, MEF and TTF echo the reprogramming efficiencies from the three fibroblasts. Flow cytometry and immunofluorescence staining demonstrated that CFs led to the highest percentage of αMHC-GFP+ cells among the three fibroblasts while TTFs showed the lowest (Fig. S7). Together, our data suggest that epigenetic states of the starting fibroblast cells determine the permissiveness of cardiac fate acquisition in these cells.