title: 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.
authors.affiliation: University of California Los Angeles, David Geffen School of Medicine, Department of Biological Chemistry, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, Bioinformatics Interdepartmental Degree Program, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Los Angeles, California 90095, USA.
authors.forename: Rupa
authors.initials: R
authors.lastname: Sridharan
authors.forename: Michelle
authors.initials: M
authors.lastname: Gonzales-Cope
authors.forename: Constantinos
authors.initials: C
authors.lastname: Chronis
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Bonora
authors.forename: Robin
authors.initials: R
authors.lastname: McKee
authors.forename: Chengyang
authors.initials: C
authors.lastname: Huang
authors.forename: Sanjeet
authors.initials: S
authors.lastname: Patel
authors.forename: David
authors.initials: D
authors.lastname: Lopez
authors.forename: Nilamadhab
authors.initials: N
authors.lastname: Mishra
authors.forename: Matteo
authors.initials: M
authors.lastname: Pellegrini
authors.forename: Michael
authors.initials: M
authors.lastname: Carey
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Garcia
authors.forename: Kathrin
authors.initials: K
authors.lastname: Plath
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics*; D002843:Chromatin / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D002868:Chromosomal Proteins, Non-Histone / Q000037:antagonists & inhibitors / Q000235:genetics*; D019175:DNA Methylation; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D044127:Epigenesis, Genetic; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D023281:Genomics; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics*; D006657:Histones / Q000378:metabolism; D018398:Homeodomain Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051379:Mice; D000071317:Nanog Homeobox Protein; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D024363:Transcription Initiation Site
chemical_list: C453799:Cbx3 protein, mouse; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D018398:Homeodomain Proteins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D011495:Histone-Lysine N-Methyltransferase