Inhibitors of suppressive histone modification promote direct reprogramming of fibroblasts to cardiomyocyte-like cells

Abstract

We recently reported that the combination of Gata4, Hand2, Tbx5, and the fusion gene MM3 between Mef2c and the transactivation domain of MyoD (MM3-GHT) produces 18 times as many clusters of beating cells (induced cardiomyocyte-like cells or iCMs) as the wild-type combination (M-GHT).1 In the current study, we added the chemicals GSK126 and UNC0638 to MM3-GHT to examine whether depletion of suppressive histone markers further increases the efficiency of making iCMs. GSK126 inhibits Enhancer of Zeste Homolog 2 (Ezh2), which induces di- and trimethylation of Lys27 on histone H3 (H3K27me2 and H3K27me3, respectively) as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2).2 UNC0638 inhibits two closely related enzymes, G9a and GLP, which form a heterodimer in vivo and mediate mono- and dimethylation of Lys9 on histone H3 (H3K9me and H3K9me2) and methylation of non-histone substrates such as p53.3 H3K27me3 and H3K9me2 are typically associated with suppressed genes although the underlying mechanisms have not been fully characterized. We prepared fibroblasts from the heads of Day 13.5 mouse embryos and incubated them with 1 μM GSK126 for 2 days to verify a decrease of H3K27me3 (see Supplementary material online, Figure S1 A ). To test the effect of GSK126 on reprogramming, fibroblasts were transduced with MM3-GHT on Day −1 and 0, and GSK126 was added to the culture medium at different time points ( Figure 1 A ). The most effective schedule was to add GSK126 from Day 1 to 4, resulting in 2.1 times as many iCM clusters as negative control without GSK126 ( Figure 1A , encircled and Supplementary material online, Figure S1 B ). Ca2+ oscillations in the cells were monitored using a transgene encoding the Ca2+ indicator protein GCaMP5, which emits stronger green fluorescence in the presence of …