Reprogramming of somatic cells into iPSCs involves a dramatic reorganization of chromatin. To identify posttranslational histone modifications that change in global abundance during this process, we have applied a quantitative mass-spectrometry-based approach. We found that iPSCs, compared to both the starting fibroblasts and a late reprogramming intermediate (pre-iPSCs), are enriched for histone modifications associated with active chromatin, and depleted for marks of transcriptional elongation and a subset of repressive modifications including H3K9me2/me3. Dissecting the contribution of H3K9methylation to reprogramming, we show that the H3K9methyltransferases Ehmt1, Ehmt2, and Setdb1 regulate global H3K9me2/me3 levels and that their depletion increases iPSC formation from both fibroblasts and pre-iPSCs. Similarly, inhibition of heterochromatin-protein-1γ (Cbx3), a protein known to recognize H3K9methylation, enhances reprogramming. Genome-wide location analysis revealed that Cbx3 predominantly binds active genes in both pre-iPSCs and pluripotent cells but with a strikingly different distribution: in pre-iPSCs, but not in ESCs, Cbx3 associates with active transcriptional start sites, suggesting a developmentally-regulated role for Cbx3 in transcriptional activation. Despite largely non-overlapping functions and the association of Cbx3 with active transcription, the H3K9methyltransferases and Cbx3 both inhibit reprogramming by repressing the pluripotency factor Nanog. Together, our findings demonstrate that Cbx3 and H3K9methylation restrict late reprogramming events, and suggest that a dramatic change in global chromatin character is an epigenetic roadblock for reprogramming.

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SUMMARYBone morphogenetic protein (BMP) signaling plays a crucial role in maintaining the pluripotency of mouse embryonic stem cells (ESCs) and has negative effects on ESC neural differentiation. However, it remains unclear when and how BMP signaling executes those different functions during neural commitment. Here, we show that a BMP4-sensitive window exists during ESC neural differentiation. Cells at this specific period correspond to the egg cylinder stage epiblast and can be maintained as ESC-derived epiblast stem cells (ESD-EpiSCs), which have the same characteristics as EpiSCs derived from mouse embryos. We propose that ESC neural differentiation occurs in two stages: first from ESCs to ESD-EpiSCs and then from ESD-EpiSCs to neural precursor cells (NPCs). We further show that BMP4 inhibits the conversion of ESCs into ESD-EpiSCs during the first stage, and suppresses ESDEpiSC neural commitment and promotes non-neural lineage differentiation during the second stage. Mechanistic studies show that BMP4 inhibits FGF/ERK activity at the first stage but not at the second stage; and IDs, as important downstream genes of BMP signaling, partially substitute for BMP4 functions at both stages. We conclude that BMP signaling has distinct functions during different stages of ESC neural commitment.