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

Abstract

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) involves a marked reorganization of chromatin. To identify post-translational histone modifications that change in global abundance during this process, we have applied a quantitative mass-spectrometry-based approach. We found that iPSCs, compared with 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 H3K9 methylation to reprogramming, we show that the H3K9 methyltransferases Ehmt1, Ehmt2 and Setdb1 regulate global H3K9me2/me3 levels and that their depletion increases iPSC formation from both fibroblasts and pre-iPSCs. Similarly, we find that inhibition of heterochromatin protein-1γ (Cbx3), a protein known to recognize H3K9 methylation, 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 embryonic stem cells, Cbx3 associates with active transcriptional start sites, suggesting a developmentally regulated role for Cbx3 in transcriptional activation. Despite largely non-overlapping functions and the predominant association of Cbx3 with active transcription, the H3K9 methyltransferases and Cbx3 both inhibit reprogramming by repressing the pluripotency factor Nanog. Together, our findings demonstrate that Cbx3 and H3K9 methylation restrict late reprogramming events, and suggest that a marked change in global chromatin character constitutes an epigenetic roadblock for reprogramming.

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Figure 1: Global levels of most histone PTMs differ between MEFs and iPSCs.
Figure 2: Comparison of histone PTM profiles between ESCs, iPSCs, pre-iPSCs and MEFs.
Figure 3: H3K9 methyltransferases, demethylases and methyl binders control the efficiency and kinetics of reprogramming.
Figure 4: Interference with H3K9 methyltransferases or Cbx3 induces reprogramming in pre-iPSCs.
Figure 5: Depletion of the H3K9 HMTases but not of Cbx3 induces a change in PTMs on the histone H3K9/K14 peptide in pre-iPSCs.
Figure 6: Depletion of H3K9 HMTases and Cbx3 promotes reprogramming of pre-iPSCs by inducing Nanog expression.
Figure 7: Cell-type-specific occupancy of Cbx3 at the TSS.
Figure 8: Cbx3 association with mediator components.

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Acknowledgements

We thank V. Pasque for critical reading of the manuscript and M. Grunstein (UCLA) for providing antibodies. K.P. is supported by the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, NIH (DP2OD001686 and P01 GM099134) and CIRM (RN1-00564); R.S. was supported by the Jonsson Comprehensive Cancer Center, C.C. by a Leukaemia and Lymphoma Research Grant (10040), G.B. by the Whitcome Pre-doctoral Training Program, B.A.G. by a National Science Foundation Early Faculty CAREER award, an NIH Innovator award (DP2OD007447) and NIH (P01 GM099134) and M.C. by the NIH (GM074701). R.S., C.C. and S.P. were supported by CIRM training grants.

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R.S., K.P. and B.A.G. planned the project. R.S. and K.P. wrote the manuscript. The following performed experiments, analysed and interpreted data: R.S., C.C., G.B., R.M., S.P. under K.P.’s supervision, M.G-C. under B.A.G.’s supervision, C.H. under M.C.’s supervision, and D.L. under M.P.’s supervision. N.M. generated H3K18me1 antibody.

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Correspondence to Benjamin A. Garcia or Kathrin Plath.

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Sridharan, R., Gonzales-Cope, M., Chronis, C. et al. Proteomic and genomic approaches reveal critical functions of H3K9 methylation and heterochromatin protein-1γ in reprogramming to pluripotency. Nat Cell Biol 15, 872–882 (2013). https://doi.org/10.1038/ncb2768

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