Dynamic interplay of histone modifications during early embryonic stem cell differentiation

Embyryonic stem (ES) cells are a powerful tool in the study of development and treatment of disease due to two key characteristics: (1) They are capable of unlimited self renewal, and (2) They are pluripotent cells capable of forming any cell in the body under appropriate conditions. We have studied the role of epigenetic histone modifications in regulating early ES cell differentiation. The possible combinatorial interplay of multiple modifications forms the basis of the histone code hypothesis. In an effort to understand how the "histone code" regulates early differentiation, we have analyzed numerous modifications. We have found that during early multilineage differentiation, there is an increase in the active histone H3 phosphorylation-acetylation (phosphoacetylation) modification. This increase is mediated by upstream cell signaling pathways, with Msk1 providing histone kinase activity. Inhibition of these upstream pathways alters differentiation and provides evidence for the role of both cell signaling pathways and histone modifications during early differentiation. We have also found that levels of the repressive modification histone H3 K27 trimethylation (K27me3) and the K27 methylase Ezh2, which are high in the pluripotent state, are globally lost three days after differentiation with the vitamin A derivative retinoic acid. However, there are local changes in K27me3 that occur at a certain class of early response genes after only a few hours of differentiation. We further find that for certain genes, loss of this repressive modification is not sufficient for transcriptional activation, as was previously hyposthesized. Instead, there is a complex interplay of multiple active and repressive modifications that regulate this process. Furthermore, the pattern of these modifications depends on genomic location with early loss of K27me3 initiated at consensus DNA regulatory elements. Finally, we find that early perturbation of these histone modifications with chemical or genetic tools alters later differentiation. These data provide evidence for a combinatorial interplay of histone modifications during early differentiation and point to epigenetic histone modifications as a key tool in utilizing ES cells for the study of development and treatment of disease.