The Role of Histone Modifications in Pluripotency

Abstract

Pluripotent cells have the potential to differentiate into different cell types while having the same genetic material. Fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs) by the addition of reprogramming factors that alter the gene expression pattern of the cells, and at the same time, chromatin changes occur. Successful reprogramming occurs at a low efficiency, so in these experiments we sought to determine the differences in histone post-translational modifications (PTMs) between pluripotent cells and non-pluripotent cells. These epigenetic marks can alter gene expression, and histone PTMs can alter chromatin structure. In the first set of experiments, we probed the process of cellular reprogramming by analyzing the histone PTM levels during reprogramming. The results showed differences in PTM levels between MEFs and iPSCs, but also between successful reprogramming (iPSCs) and unsuccessful reprogramming (pre-iPSCs). The most interesting difference was that methylation on histone H3 lysine 9 must decrease for successful reprogramming, implying that H3K9 methylation is an epigenetic barrier to reprogramming. Knockdown of the euchromatic histone methyltransferases (EHMT1, EHMT2, and ESET) improved reprogramming efficiency and facilitated pre-iPSC to iPSC transition. Depletion of heterochromatin protein 1 gamma (Cbx3), which binds H3K9 methylation, recapitulated the effects. Genome-wide mapping showed that Cbx3 represses Nanog expression in pre-iPSCs. In the second set of experiments, we looked at the process of differentiation. The results were consistent with the first set of experiments in that H3K9 methylation increased upon differentiation. In addition, in both sets of experiments, histone H4 acetylation was consistent with pluripotency. Both H4 acetylation and Brd4 decrease upon differentiation. Inhibition of Brd4 by the BET domain inhibitor (+)-JQ1 in mESCs results in differentiation to the endodermal lineage. Consistent with those results, (+)-JQ1 also inhibits cellular reprogramming, which suggests that Brd4 is necessary for pluripotency. Genome-wide mapping showed that Brd4 and H4 acetylation are found upstream of core pluripotency genes.