41. Materials and methods are available as supplementary materials on Science Online. 42. One of the features of our approach is its ability to distinguish between antibodies targeting overlapping epitopes in a substantially different ways: There is a significant difference in the correlation coefficients for antibodies targeting a similar epitope versus the correlation coefficients for antibodies targeting different epitopes on the same site of vulnerability. Similarly, there is a significant difference for similar epitopes versus different sites of vulnerability; however, there is no significant difference for different epitopes on the same site of vulnerability versus different sites of vulnerability (fig. S1). 43. R. M. Cardoso et al., Immunity 22, 163 (2005). 44. Residue numbering throughout the paper is relative to strain HXB2, unless stated otherwise.

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Rationale Direct reprogramming of cardiac fibroblasts to cardiomyocytes has recently emerged as a novel and promising approach to regenerate the injured myocardium. We have previously demonstrated the feasibility of this approach in vitro and in vivo using a combination of four microRNAs (miR-1, miR-133, miR-208 and miR-499) that we named miR combo. However, the mechanism of miR combo mediated direct cardiac reprogramming is currently unknown. Objective Here we investigated the possibility that miR combo initiated direct cardiac reprogramming through an epigenetic mechanism. Methods and Results Using a qPCR array, we found that histone methyltransferases and demethylases that regulate the tri-methylation of H3K27 (H3K27me3), an epigenetic modification that marks transcriptional repression, were changed in miR combo treated fibroblasts. Accordingly, global H3K27me3 levels were downregulated by miR combo treatment. In particular, the promoter region of cardiac transcription factors showed decreased H3K27me3 as revealed by ChIP-qPCR. Inhibition of H3K27 methyltransferases or of the Polycomb Repressive Complex 2 (PRC2) by pharmaceutical inhibition or siRNA reduced the levels of H3K27me3 and induced cardiogenic markers at the RNA and protein level, similarly to miR combo treatment. In contrast, knockdown of the H3K27 demethylases Kdm6A and Kdm6B restored the levels of H3K27me3 and blocked the induction of cardiac gene expression in miR combo treated fibroblasts. Conclusions In summary, we demonstrated that removal of the repressive mark H3K27me3 is essential for the induction of cardiac reprogramming by miR combo. Our data not only highlight the importance of regulating the epigenetic landscape during cell fate conversion but also provide a framework to improve this technique.