Sequential enrichment at the nuclear periphery of H2A.Zac and H3K9me2 accompanies pluripotency loss in human embryonicstem cells

During the transition from pluripotency to a lineage-committed state, chromatin undergoes large-scale changes in structure to effect the required changes to the transcriptional program. This involves covalent modification of histone tails, replacement of histone variants, and alteration in the subnuclear position of genes, including associations with the nuclear periphery. Here, using high-resolution microscopy and quantitative image analysis, we surveyed a panel of histone variants and covalent modifications for changes in nuclear periphery association during differentiation of human embryonic stem cells to a trophoblast-like lineage. This differentiation process is rapid and homogeneous, facilitating the use of a relatively fine timecourse (12h, 24h, and 48h post-initiation) to enable detection of transient changes. With this scheme, we detected two modifications with significant changes in enrichment at the nuclear periphery: acetylation of histone variant H2A.Z, and dimethylation of histone H3 at lysine 9. We show that these chromatin marks increase specifically at the nuclear periphery in a sequential, complementary manner, with a H2A.Z acetylation preceding H3K9 dimethylation. The increase of H3K9 dimethylation occurred coincidentally with but independently of accumulation of Lamin A, since Lamin A-/- hES cells showed no changes in the localization pattern of H3K9 dimethylation. Inhibition of histone deacetylases led to persistent and increased H2A.Z acetylation at the periphery, and failure to differentiate. Our results show that a concerted dynamic change in the nature of peripheral chromatin is required for differentiation into the trophoblast state.