High histone acetylation and decreased polycomb repressive complex 2 member levels regulate gene specific transcriptional changes during early embryonic stem cell differentiation induced by retinoic acid

Histone modifications play a crucial role during embryonic stem (ES) cell differentiation. During differentiation, binding of polycomb repressive complex 2 (PRC2), which mediates trimethylation of lysine 27 on histone H3 (K27me3), is lost on developmental genes that are transcriptionally induced. We observed a global decrease in K27me3 in as little as 3 days after differentiation of mouse ES cells induced by retinoic acid (RA) treatment. The global levels of the histone K27 methyltransferase EZH2 also decreased with RA treatment. A loss of EZH2 binding and K27me3 was observed locally on PRC2 target genes induced after 3 days of RA, including Nestin. In contrast, direct RA-responsive genes that are rapidly induced, such as Hoxal, showed a loss of EZH2 binding and K27me3 after only a few hours of RA treatment. Following differentiation induced by leukemia inhibitor factor (LIF) withdrawal without RA, Hoxal was not transcriptionally activated. Small interfering RNA-mediated knockdown of EZH2 resulted in loss of K27me3 during LIF withdrawal, but the Hoxal gene remained transcriptionally silent after loss of this repressive mark. Induction of histone hyperacetylation overrode the repressive K27me3 modification and resulted in Hoxal gene expression. Together, these data show that there are multiple temporal phases of derepression of PRC2 target genes during ES cell differentiation and that other epigenetic marks (specifically, increased acetylation of histones H3 and H4), in addition to derepression, are important for gene-specific transcriptional activation. This report demonstrates the temporal interplay of various epigenetic changes in regulating gene expression during early FS cell differentiation.