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

Abstract

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.

The p53-induced lincRNA-p21 derails somatic cell reprogramming by sustaining H3K9me3 and CpG methylation at pluripotency gene promoters

“…Importantly, preiPSCs are stable and can be readily converted to fully reprogrammed iPSCs through a variety of approaches [10,21]. Because reprogramming cells consist of heterogeneous populations progressing at different speeds [3], preiPSCs provide a relatively well-defined model for dissecting the chain of events eventually leading to the full acquisition of pluripotency [10,11]. Using preiPSCs, we thus sought to identify lncRNAs that control the late stage of mouse somatic cell reprogramming.…”

“…LincRNA-p21 was predominantly nuclear ( Figure 3A), consistent with a potential role in epigenetic regulation. Then, we employed RNA immunoprecipitation (RIP) with antibodies against a panel of chromatin modifiers detrimental for reprogramming [9][10][11][12]23] using lysates from MEFs reprogrammed with OSKM on day 12. Notably, there was a substantial enrichment for lincRNA-p21 in DNMT1 RIP and a less substantial enrichment in SETDB1 RIP, but no enrichment in DN-MT3A/B and SUV39H1 RIPs, relative to IgG control ( Figure 3B).…”

“…Two fundamental reprogramming roadblocks are the activation of a p53-mediated response that promotes apoptosis and cell senescence, and the inefficient removal of preexisting somatic-like epigenetic marks. Accordingly, depletion of p53 [8] or the H3K9 methyltransferases Setdb1, Suv39h1 and Suv39h2 [9][10][11], and inhibition of the maintenance DNA methyltransferase DNMT1 [12] improve reprogramming efficiency.…”

Functional Crosstalk Between Lysine Methyltransferases on Histone Substrates: The Case of G9A/GLP and Polycomb Repressive Complex 2

“…Indeed, while PRC2 has been shown to facilitate the acquisition of induced pluripotency in different somatic cells (44,101,114,176), G9a/GLP and H3K9 methylation have been instead suggested as a barrier of cellular reprogramming (25,142). Probably, the requirement of PRC2 for an efficient generation of iPSCs relies on its capacity to maintain the silencing of somatic cells transcriptional programs while being involved in repression of pluripotency genes; H3K9 methylation needs instead to be overcome for the reacquisition of pluripotency and an efficient cellular reprogramming (25).…”

Chromatin module inference on cellular trajectories identifies key transition points and poised epigenetic states in diverse developmental processes

“…Transcription factors alone are sufficient to convert differentiated somatic cells to induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka 2006) albeit at low efficiency. Chemical or genetic modifiers that reduce repressive chromatin levels enhance reprogramming efficiency implicating epigenetic contribution (Onder et al 2012;Apostolou and Hochedlinger 2013;Papp and Plath 2013;Sridharan et al 2013). Reciprocally, during development, the chromatin state at specific loci has to become permissive concomitant with appropriate transcription factor levels for cell-type-specific expression to commence.…”