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Embryonic stem cells are characterized by unique epigenetic features including decondensed chromatin and hyperdynamic association of chromatin proteins with chromatin. Here we investigate the potential mechanisms that regulate chromatin plasticity in embryonic stem cells. Using epigenetic drugs and mutant embryonic stem cells lacking various chromatin proteins, we find that histone acetylation, G9a-mediated histone H3 lysine 9 (H3K9) methylation and lamin A expression, all affect chromatin protein dynamics. Histone acetylation controls, almost exclusively, euchromatin protein dynamics; lamin A expression regulates heterochromatin protein dynamics, and G9a regulates both euchromatin and heterochromatin protein dynamics. In contrast, we find that DNA methylation and nucleosome repeat length have little or no effect on chromatin-binding protein dynamics in embryonic stem cells. Altered chromatin dynamics associates with perturbed embryonic stem cell differentiation. Together, these data provide mechanistic insights into the epigenetic pathways that are responsible for chromatin plasticity in embryonic stem cells, and indicate that the genome's epigenetic state modulates chromatin plasticity and differentiation potential of embryonic stem cells.

Embryonic stem cell (ESC) chromatin is characterized by a unique set of histone modifications, including enrichment for H3 lysine 9 acetylation (H3K9ac). Recent studies suggest that histone deacetylase (HDAC) inhibitors promote pluripotency. Here, using H3K9ac ChIP followed by high throughput sequencing analyses and gene expression in E14 mouse ESCs before and after treatment with a low level of the HDAC inhibitor valproic acid, we show that H3K9ac is enriched at gene promoters and is highly correlated with gene expression and with various genomic features, including different active histone marks and pluripotency-related transcription factors. Curiously, it predicts the cellular location of gene products. Treatment of ESCs with valproic acid leads to a pervasive genome-wide and time-dependent increase in H3K9ac, but this increase is selectively suppressed after 4 h in H3K4me3/H3K27me3 bivalent genes. H3K9ac increase is dependent on the promoter's chromatin state and is affected by the binding of P300, various transcription factors, and active histone marks. This study provides insights into the genomic response of ESCs to a low level of HDAC inhibitor, which leads to increased pluripotency. The results suggest that a mild (averaging less than 40%) but global change in the chromatin state is involved in increased pluripotency and that specific mechanisms operate selectively in bivalent genes to maintain constant H3K9ac levels. Our data support the notion that H3K9ac has an important role in ESC biology.

The pluripotent genome is characterized by unique epigenetic features and a decondensed chromatin conformation. However, the relationship between epigenetic regulation and pluripotency is not altogether clear. Here, using an enhanced MEF/ESC fusion protocol, we compared the reprogramming potency and histone modifications of different embryonic stem cell (ESC) lines (R1, J1, E14, C57BL/6) and found that E14 ESCs are significantly less potent, with significantly reduced H3K9ac levels. Treatment of E14 ESCs with histone deacetylase (HDAC) inhibitors (HDACi) increased H3K9ac levels and restored their reprogramming capacity. Microarray and H3K9ac ChIP-seq analyses, suggested increased extracellular matrix (ECM) activity following HDACi treatment in E14 ESCs. These data suggest that H3K9ac may predict pluripotency and that increasing pluripotency by HDAC inhibition acts through H3K9ac to enhance the activity of target genes involved in ECM production to support pluripotency.