Proteomic and genomic approaches reveal critical functions of H3K9 methylation and heterochromatin protein-1γ in reprogramming to pluripotency

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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.