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H3K36 methylation maintains cell identity by regulating opposing lineage programmes

Nature Cell Biology volume 25, pages 1121–1134 (2023)Cite this article

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Abstract

The epigenetic mechanisms that maintain differentiated cell states remain incompletely understood. Here we employed histone mutants to uncover a crucial role for H3K36 methylation in the maintenance of cell identities across diverse developmental contexts. Focusing on the experimental induction of pluripotency, we show that H3K36M-mediated depletion of H3K36 methylation endows fibroblasts with a plastic state poised to acquire pluripotency in nearly all cells. At a cellular level, H3K36M facilitates epithelial plasticity by rendering fibroblasts insensitive to TGFβ signals. At a molecular level, H3K36M enables the decommissioning of mesenchymal enhancers and the parallel activation of epithelial/stem cell enhancers. This enhancer rewiring is Tet dependent and redirects Sox2 from promiscuous somatic to pluripotency targets. Our findings reveal a previously unappreciated dual role for H3K36 methylation in the maintenance of cell identity by integrating a crucial developmental pathway into sustained expression of cell-type-specific programmes, and by opposing the expression of alternative lineage programmes through enhancer methylation.

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Fig. 1: Histone mutant analysis reveals crucial role of H3K36 methylation in cell identity maintenance.
Fig. 2: K36M endows iPS cell intermediates with transcriptional homogeneity and epithelial plasticity.
Fig. 3: K36M acts downstream of TGFβ and Smad2 but upstream of Zeb1.
Fig. 4: K36M decommissions MEF enhancers and activates ES cell enhancers.
Fig. 5: K36M-dependent chromatin rewiring alters transcription factor binding preferences.
Fig. 6: DNA demethylation is limiting for K36M-dependent enhancer activation and reprogramming.

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Data availability

RNA-seq, ATAC-seq and CUT&Tag data have been deposited in the Gene Expression Omnibus under accession code GSE203492. Single-cell RNA-seq data are deposited under GSE203536. RRBS and WGBS data are available under GSE203606. The publicly available datasets used in this study are GSE90893, GSE111172 and GSE77420. All other data supporting the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

Code availability

The code used to analyse the single-cell RNA-seq data presented here, and to generate the corresponding plots, is uploaded to GitHub https://github.com/Michorlab/H3K36_methylation_scRNAseq. Code for additional analyses available upon request.

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Acknowledgements

We thank members of the Hochedlinger laboratory for their suggestions, and members of the MGH CRM/HSCI Flow Core, the Harvard Genome Modification Facility and the MGH Next Generation Sequencing Core for their support. We also thank A. Collier for suggestions and discussions, and H. Kretzmer for her help with DNA methylation data. Support from the Dana-Farber Cancer Institute’s Center for Cancer Evolution and Physical Sciences-Oncology Center (U54CA193461; to F.M.) is gratefully acknowledged. M.S.H. was supported by the German Cancer Aid (Deutsche Krebshilfe). K.P. is supported by the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, the David Geffen School of Medicine, the National Institutes of Health (GM099134) and a Faculty Scholar grant from the Howard Hughes Medical Institute. K.H. was supported by funds from the MGH, the National Institutes of Health (R01 HD058013 and P01 GM099134), the Milky Way Research Foundation and the Gerald and Darlene Jordan Chair in Regenerative Medicine.

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Authors and Affiliations

  1. Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA

    Michael S. Hoetker, Masaki Yagi, Bruno Di Stefano, Lai Ping Wong, Aaron J. Huebner, Ruslan I. Sadreyev & Konrad Hochedlinger

  2. Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA

    Michael S. Hoetker, Masaki Yagi, Bruno Di Stefano, Aaron J. Huebner & Konrad Hochedlinger

  3. Cancer Center, Massachusetts General Hospital, Boston, MA, USA

    Michael S. Hoetker, Masaki Yagi, Bruno Di Stefano, Aaron J. Huebner & Konrad Hochedlinger

  4. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Michael S. Hoetker, Masaki Yagi, Bruno Di Stefano, Aaron J. Huebner & Konrad Hochedlinger

  5. Harvard Stem Cell Institute, Cambridge, MA, USA

    Michael S. Hoetker, Masaki Yagi, Bruno Di Stefano, Aaron J. Huebner, Alexander Meissner & Konrad Hochedlinger

  6. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Michael S. Hoetker, Masaki Yagi, Bruno Di Stefano, Aaron J. Huebner, Alexander Meissner, Franziska Michor & Konrad Hochedlinger

  7. David Geffen School of Medicine, Department of Biological Chemistry, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, USA

    Justin Langerman, Weixian Deng & Kathrin Plath

  8. Department of Data Science, Dana-Farber Cancer Institute, Boston, MA, USA

    Simona Cristea & Franziska Michor

  9. Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA, USA

    Simona Cristea & Franziska Michor

  10. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA

    Jocelyn Charlton, Alexander Meissner & Franziska Michor

  11. Department of Genome Regulation, Max-Planck Institute for Molecular Genetics, Berlin, Germany

    Jocelyn Charlton, Chuck Haggerty & Alexander Meissner

  12. Department of Pathology, Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA

    Ruslan I. Sadreyev

  13. The Center for Cancer Evolution, Dana-Farber Cancer Institute, Boston, MA, USA

    Franziska Michor

  14. The Ludwig Center at Harvard, Boston, MA, USA

    Franziska Michor

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  1. Michael S. Hoetker
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Contributions

M.S.H. and K.H. conceived the study and wrote the manuscript. M.S.H., M.Y., B.D.S., A.J.H. and W.D. performed experiments and analysed the data. S.C. and F.M. analysed single-cell RNA-seq data. A.M. and J.C. performed RRBS. A.M. and C.H. performed WGBS. A.M., J.C., C.H., M.S.H., J.L. and K.P. performed DNA methylation analysis. R.I.S., L.P.W., J.L., K.P. and M.S.H. performed additional bioinformatics analyses.

Corresponding author

Correspondence to Konrad Hochedlinger.

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Competing interests

F.M. is a co-founder of and has equity in Harbinger Health, has equity in Zephyr AI, and serves as a consultant for Harbinger Health, Zephyr AI and Red Cell Partners. F.M. declares that none of these relationships are directly or indirectly related to the content of this manuscript. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Key role of H3K36-methylation in cell identity maintenance.

(a) Colony counts for AP staining of reprogrammable MEFs transduced as indicated (Fig. 1b,c). K36M wells were confluent and could not be counted. P values were determined by two-sided unpaired Student’s t test, error bars indicate mean ± SD (n = 3 biologically independent experiments). (b) Mice with dox-inducible alleles of WT H3.3 or K36M in the Col1a1 locus were crossed with mice harboring a dox-inducible OKSM cassette in the same locus, and an EGFP reporter in the 3’UTR of Pou5f1. (c) Immunofluorescence of MEFs derived as in a). Scale bar 50 µm. (d) Mass spectrometry of histone modifications in day 4 reprogramming intermediates (n = 2 independent biological experiments). (e) MEFs without endogenous OKSM but with inducible H3.3 WT or K36M were doxycycline-treated for 2 days, then replated and transduced with constitutive OKSM virus. Doxycycline was added to the medium for the indicated intervals, intracellular flow cytometry for Nanog was performed on day 8. (f) Quantification of Nanog positivity by flow cytometry on day 8 in K36M cells treated with doxycycline for the indicated time. Cells were either not pre-treated or pre-treated with doxycycline 2 days prior to initiation of reprogramming (see k). Error bars indicate mean ± SD (n = 3 independent biological experiments). (g) Fraction of Oct4-GFP+ cells during reprogramming in FBS/LIF medium supplemented with ascorbic acid (left) and without supplementation (right), error bars indicate mean ± SD (n = 3 independent biological experiments). (h) Membrane dye dilution assay for reprogramming cultures. (i) Percentage of viable cells as assessed by Annexin V/PI negativity on day 2 and 4 of reprogramming. Error bars indicate mean ± SD (n = 3 independent biological experiments). (j) Fraction of Oct4-GFP+ cells after sorting of positive cells and expansion on gelatin (top), and in picked iPSCs passaged on feeders (bottom). (k) Day 10 K36M reprogramming cultures were sorted by Oct4-GFP reporter positivity. Positive cells were maintained in FBS/LIF, negative cells underwent continued reprogramming in AGi medium. (l) Quantification of Oct4-GFP+ cells by flow cytometry in K36M cells sorted by Oct4-GFP reporter status (see i), error bars indicate mean ± SD (n = 3 independent biological experiments).

Source data

Extended Data Fig. 2 K36M enhances the reprogramming of different cell types and generates iPSCs highly similar to control iPSCs.

(a) Reprogramming of GMPs to iPSCs. Alkaline phosphatase staining of iPSC colonies at the indicated timepoints. Quantification of colony counts. P values were determined by two-sided unpaired Student’s t test, n = 3 biologically independent experiments. (b) Reprogramming of keratinocytes to iPSCs. Alkaline phosphatase staining on day 13 of cells cultured for the indicated timeframes. Area percentage of well that is AP positive. P values were determined by two-sided unpaired Student’s t test, error bars indicate mean ± SD (n = 3 independent biological experiments). (c) Immunofluorescence for Nanog, H3K36me3, H3K36me2, and H3K27me3 of passaged iPSC cultures from WT and K36M backgrounds cultured without doxycycline on irradiated feeders. (d) Relative expression (RNA-seq) of key pluripotency genes in passaged iPSCs of both backgrounds, n = 2 biologically independent experiments. (e) DNA methylation at MEF (n = 63,696) and ESC enhancers (n = 72,638) for MEFs and passaged iPSCs of both backgrounds (RRBS, n = 2 biologically independent uninduced MEF replicates were integrated, one replicate each for uninduced iPSCs of each genotype). (f) Representative gene tracks showing RNA-seq, ATAC-seq, and RRBS data for Cdh1 and Pou5f1 in passaged iPSCs of both backgrounds. (g) Correlation matrices for key histone modifications and chromatin accessibility in passaged WT and K36M iPSCs (CUT&Tag and ATAC-seq, n = 2 biologically independent replicates were integrated for this analysis). (h) Correlation of K36M vs. WT derived iPSCs for H3K36me3 over gene bodies (CUT&Tag, n = 2 biologically independent replicates were integrated for this analysis). (i) Correlation of K36M vs. WT derived iPSCs for H3K36me2 in genome-wide 50 kb bins (CUT&Tag, n = 2 biologically independent replicates were integrated for this analysis). (j) Representative gene tracks showing H3K36me3, H3K36me2, H3K27me3, and H3K4me3 at pluripotency gene Nanog (n = 2 biologically independent replicates). (k) Quantification of the diameter of embryoid bodies from passaged iPSCs of both backgrounds (without doxycycline). P value determined by two-sided unpaired Student’s t test, n = 49 for WT, n = 44 for K36M. (l) qPCR for Nanog, Nestin, Sox7, and Gata6 in embryoid bodies derived from passaged iPSCs of both backgrounds (without doxycycline), error bars indicate mean ± SD (n = 3 independent biological experiments). (m) H&E sections of teratomas generated with iPSCs of both backgrounds (without doxycycline). 4/4 WT and 6/6 K36M iPSC lines produced well-differentiated teratomas. Images depict tissue-like structures of all germ layers. (n) Coat-color chimeras generated by blastocyst injection of K36M iPSCs.

Source data

Extended Data Fig. 3 Single-cell RNA-seq reveals main trajectories of WT and K36M reprogramming intermediates.

(a) UMAP embedding of scRNA-seq data (Seurat framework) using MEFs, reprogramming intermediates on days 2, 4, 6, 8 for WT and K36M, as well as passaged iPSCs. For each indicated group, one sample was encapsulated leading to n = 38,743 cells total. (b) Expression of pluripotency gene Nanog projected on the same UMAP embedding as in (a). (c) Expression of mesenchymal gene Prrx1 projected on the same UMAP embedding as in (a). (d,e) Diffusion pseudotime mapping of day 2 to day 8 intermediates undergoing reprogramming. WT cells are colored in blue, K36M cells in red. (f) Expression of pluripotency gene Nanog projected on the same pseudotime embedding as in (d). (g) Expression of epithelial gene Cdh1 projected on the same pseudotime embedding as in (d). (h) Expression of mesenchymal gene Zeb1 projected on the same pseudotime embedding as in (d). (i) Relative expression (RNA-seq) of mesenchymal and epithelial genes in MEFs expressing H3.3 WT or K36M, but not OKSM, n = 2 biologically independent experiments. (j) Gene ontology terms of genes downregulated in K36M MEFs without OKSM. Analysis and p values from geneontology.org. (k) Gene ontology terms of genes upregulated in K36M MEFs without OKSM. Analysis and p values from geneontology.org.

Source data

Extended Data Fig. 4 K36M confers epithelial plasticity on cells undergoing reprogramming.

(a) Gene expression of Twist1 and Cdh1 on the same UMAP embedding as used in Fig. 2d. Dashed circles encompass day-2 and day-4 samples for WT (blue circle) and K36M (red circle). Solid arrow indicates switch-like MET in K36M samples, dashed arrow indicates heterogeneous maintenance/activation of mesenchymal/epithelial programs in WT cells. For each group, one sample was encapsulated leading to n = 38,743 cells total. (b) Immunofluorescence for Vimentin and Epcam in WT and K36M cells on day 4 of reprogramming. Scale bar = 25 µm. Three independent biological experiments with similar results. (c) Correlation plots of single-cell RNA-seq data comparing transcriptional programs within each cell to MEFs (y-axis) and ESCs (x-axis)107. For each sample, the corresponding cells are colored according to their Epcam expression levels, whereas other cells are greyed out. (d) Correlation plots as in (c), expression data of Twist1 is superimposed. (e) Correlation plots as in (c), expression data of Pou5f1 is superimposed.

Extended Data Fig. 5 K36M disrupts TGFβ signaling and modulates epithelial plasticity in diverse contexts.

(a) Flow cytometry histograms displaying Epcam expression in day-4 reprogramming intermediates for WT and K36M samples. Untreated control cells are compared to cells treated with 250 nM Repsox (TGFβi) or 2.5 ng/ml recombinant TGFβ-1 or -2 (rTGFβ-1, rTGFβ-2). (b) Fraction of Oct4-GFP+ cells treated with TGFβi or rTGFβ in day 4 reprogramming intermediates. Error bars indicate mean ± SD (n = 3). (c) Representative tracks for expression of mesenchymal gene Col1a2 on day 8 of reprogramming, WT or K36M cells were treated as indicated. (d) Representative tracks for expression of epithelial gene Cdh1 on day 8 of reprogramming, WT or K36M cells were treated as indicated. (e) Representative tracks for miR-200a and miR-290. (f) Schematic of K36M’s effect on TGFβ signaling and miRNA expression during reprogramming. (g) De-differentiation of MEFs to induced myogenic progenitor cells (iMPCs). qRT-PCR for myotube marker Myh1 and iMPC marker Pax7, P values were determined by two-sided unpaired Student’s t test, error bars indicate mean ± SD (n = 3 biologically independent experiments). Flow cytometry for Pax7-GFP reporter positive cells. (h,i) Immunofluorescence of Nanog and K36M in ES cells of both backgrounds, cultured in S/L (g) or 2iL (h) conditions. Result is representative of three independent biological experiments. (j) Differentiation of ESC aggregates to elongated gastruloids. Representative brightfield images (scale bar = 500 µm) and quantification of long axis diameter (line = mean). P value was determined by two-sided unpaired Student’s t test, n = 19 for WT, n = 20 for K36M. (k) Differentiation of ESCs to pre-somitic mesoderm. Representative immunofluorescence for Cdh2 (scale bar = 50 µm). qRT-PCR for mesodermal transcription factors Tbx6 and Msgn1. P values were determined by two-sided unpaired Student’s t test, error bars indicate mean ± SD (n = 3 independent biological experiments).

Source data

Extended Data Fig. 6 H3K36me2 and H3K36me3 cooperatively safeguard cell identity.

(a) Histone methyltransferases and demethylases implicated in the regulation of H3K36me2 (top) and H3K36me3 (bottom). (b) Fraction of Epcam+ cells on day 4 of reprogramming (top) in WT cells with knockdown of indicated histone methyltransferases. Colony counts after 6 days of doxycycline followed by 4 days of independent growth (bottom), error bars indicate mean ± SD (n = 3 biologically independent experiments). (c,d) Fraction of Epcam+ (c) or Oct4-GFP+ (d) cells on day 8 of reprogramming in WT and K36M cells transduced with either empty vector or dox-inducible Nsd2, error bars indicate mean ± SD (n = 3 biologically independent experiments). (e) Flow cytometry for Epcam on day 8 of reprogramming in WT cells with knockdown of the indicated histone demethylases. (f,g) Fraction of Epcam+ cells on day 4 (f) and day 8 (g) of reprogramming in WT cells with knockdown of the indicated histone demethylases, error bars indicate mean ± SD (n = 3 biologically independent experiments). (h) Fraction of Oct4-GFP+ cells on day 8 of reprogramming in WT cells with knockdown of the indicated histone demethylases. P values were determined by unpaired Student’s t test, error bars indicate mean ± SD (n = 3 biologically independent experiments). (i,j) Flow cytometry for Epcam during reprogramming in WT cells with overexpression of the indicated histone demethylases, error bars indicate mean ± SD (n = 3 biologically independent experiments). (k) Fraction of Oct4-GFP+ cells on day 4 and day 8 of reprogramming in WT cells with overexpression of the indicated histone demethylases, error bars indicate mean ± SD (n = 3 biologically independent experiments). (l) qRT-PCR for mesenchymal genes Vim and Prrx1, epithelial genes Epcam and Cdh1, and pluripotency gene Pou5f1 on day 4 of reprogramming in WT cells overexpressing Kdm2a vs. empty vector control. P values were determined by unpaired Student’s t test, error bars indicate mean ± SD (n = 3 independent biological experiments).

Source data

Extended Data Fig. 7 K36M alters H3K36me2/3 deposition and gene expression.

(a) Median H3K36me3 deposition at expressed genes (RPKM > 0.1, n = 10,251 for WT, n = 10,496 for K36M) of indicated expression quintiles for WT (left) and K36M (right) samples on day 4 of reprogramming (n = 2 biologically independent replicates were integrated for this analysis). (b) Median fold change of H3K36me3 density over gene bodies of differentially expressed genes (upregulated = red, downregulated = blue, n = 1,872) on day 4 (n = 2 biologically independent replicates were integrated for this analysis). (c) Fold change of gene expression (y axis) vs. fold change of H3K36me3 (x axis) between K36M and WT samples on day 4 of reprogramming (n = 2 biologically independent replicates were integrated for this analysis). (d) Representative gene tracks for H3K36me3 and RNA levels at mesenchymal gene Vim, epithelial gene Cdh1, and pluripotency gene Pou5f1 (n = 2 biologically independent replicates). (e) Profile plots of H3K36me2 at promoters, enhancers, and gene bodies (n = 2 biologically independent replicates were integrated for this analysis). (f) Profile plots of H3K36me2 and H3K27ac at H3K36me2 domains containing down- or upregulated enhancers (n = 2 biologically independent replicates were integrated for this analysis). (g) Representative tracks for Prrx1, a mesenchymal gene downregulated in K36M samples on day 4, Krt8, and Pou5f1, epithelial/pluripotency genes upregulated in K36M samples on day 4 (n = 2 biologically independent replicates). Putative regulatory elements highlighted in grey. (h) Gene ontology terms of genes closest to H3K36me2 domain-embedded enhancers that are differentially downregulated in K36M cells. Analysis and p values from geneontology.org. (i) Gene ontology terms of genes closest to H3K36me2 domain-embedded enhancers that are differentially upregulated in K36M cells. Analysis and p values from geneontology.org. (j) Dot plot representing enrichment of ENCODE data for differentially active enhancers within H3K36me2 domains. P values were determined by Fisher’s exact test.

Source data

Extended Data Fig. 8 PRC2 contributes to the K36M-dependent silencing of the somatic program.

(a) H3K27me3 deposition within H3K36me2 domains in K36M vs. WT samples in day-4 reprogramming intermediates. Domains gaining H3K27me3 are colored in red, domains losing H3K27me3 are colored in blue. (b) Ontology terms for genes with promoters overlapping H3K36me2 domains and gaining H3K27me3. Analysis and p values from geneontology.org. (c) Heatmaps for H3K27me3 and H3K4me3 at promoters mesenchymal and epithelial genes in WT and K36M samples. (d,e) Fraction of Epcam+ and Oct4-GFP+ cells in WT (blue) and K36M (red) samples with knockdown of indicated PRC2 components (top). Log2(fold change) of fraction normalized to control siRNA (bottom). Error bars indicate mean ± SD (n = 3 independent biological experiments). (f) Representative histograms of flow cytometry for Epcam in K36M cells with control siRNA and knockdown of Ezh2 or Suz12. (g) qRT-PCR for mesenchymal (Vim, Prrx1, Zeb1), epithelial (Cdh1, Epcam), and pluripotency (Pou5f1) marker genes, error bars indicate mean ± SD (n = 3 biologically independent experiments). (h) Immunofluorescence for H3K27me3 in WT and K36M cells transfected with control siRNA or knockdown of Ezh2 or Suz12. Representative result from three independent biological experiments.

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Extended Data Fig. 9 K36M rewires DNA methylation patterns.

(a) Number of colonies following alkaline phosphatase staining of WT and K36M cell cultures transduced with non-selectable, dox-inducible lentiviruses for the expression of SKM, OSM, or OKM. Cultures were induced for 12 days and stained on day 15, n = 3 independent biological experiments. (b) Correlation plot of log2(fold-change) differences (K36M vs. WT) at Sox2 peaks called in WT and K36M samples. Differences of Sox2 enrichment are correlated with differences in H3K27ac abundance (n = 2 biologically independent replicates were integrated for this analysis). Pearson’s R = 0.56. (c) Profile plots showing H3K36me2 abundance at ectopic and ESC-specific Sox2 sides in WT and K36M cells on day 4 of reprogramming. (d) Sox2 enrichment at Sox2 binding sites as defined in iPSCs, log2(RPKM). (e) Correlation plot of log2(fold-change) differences (K36M vs. WT) at Sox2 peaks called in WT and K36M samples. Differences of Sox2 enrichment are correlated with differences in chromatin accessibility (as measured by ATAC-seq, n = 2 biologically independent replicates were integrated for this analysis). Pearson’s R = 0.68. (f) Median chromatin accessibility (as measured by ATAC-seq, n = 2 biologically independent replicates were integrated for this analysis) at ectopic and ESC-specific Sox2 binding sites in MEFs and passaged iPSCs. (g) Density ridge plots of DNA methylation at differentially methylated regions losing (left, n = 30,294) or gaining (right, n = 28,060) methylation in iPSCs vs. MEFs (n = 2 biologically independent replicates for uninduced MEFs, for d4 and d8 samples were integrated, one sample for each genotype in uninduced iPSCs). (h-k) Representative gene tracks of Cdh1, Krt8, the miR-290 cluster, and Pou5f1 (n = 2 biologically independent replicates). Putative regulatory elements affected by DNA demethylation are highlighted in grey. (l) CpG density at differentially active enhancers in H3K36me2 domains (n = 4,939). P value determined by two-sided Wilcoxon rank sum test. Center line indicates median; lower/upper hinges indicate 25th/75th percentiles; whiskers extend to 1.5x IQR. P value determined by two-sided Wilcoxon rank sum test.

Source data

Extended Data Fig. 10 DNA demethylation is limiting for K36M-dependent enhancer rewiring.

(a) Distribution of DNA methylation (WGBS, n = 2 biologically independent replicates were integrated for this analysis) at differentially active enhancers in H3K36me2 domains (n = 4,939) in day-4 reprogramming intermediates of WT, K36M and K36M + DMOG samples. (b) Distribution of DNA methylation (WGBS, n = 2 biologically independent replicates were integrated for this analysis) at ectopic (n = 45,095) and ESC-exclusive (n = 27,708) Sox2 binding sites in day-4 reprogramming intermediates of WT, K36M and K36M + DMOG samples. (c) Representative histogram plots from flow cytometric analysis for Epcam of K36M cells with knockdown of the indicated Tet demethylases. (d) Fraction of Epcam+ cells in K36M cells with Tet knockdown on day 4 of reprogramming, error bars indicate mean ± SD (n = 3 biologically independent experiments). (e) qPCR of miRNAs miR-200b-3p, miR-205-5p, and miR-290a-5p in untreated (K36M Ctrl) and DMOG-treated K36M cells (K36M DMOG). P values were determined by unpaired Student’s t test, error bars indicate mean ± SD (n = 3 independent biological experiments). (f) Fraction of Epcam+ cells in K36M cultures transduced with either an empty vector or dox-inducible overexpression vectors for Dnmt3a and Dnmt3b, error bars indicate mean ± SD (n = 3 independent biological experiments). (g) Bisulfite-seq of a Cdh1 enhancer in K36M cells transduced with either empty vector (left), or overexpression of Dnmt3a (middle) or Dnmt3b (right). (h) H3K36me2 levels within H3K36me2 domains (n = 7,610) on day 4 of reprogramming in WT, untreated K36M cells (K36M Ctrl), and DMOG-treated K36M cells (K36M DMOG). Crossbars indicate median (n = 2 biologically independent replicates were integrated for this analysis). (i-k) Representative gene tracks of Krt8, Pou5f1, and the miR-290 cluster (n = 2 biologically independent replicates). Putative regulatory elements highlighted in grey.

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Hoetker, M.S., Yagi, M., Di Stefano, B. et al. H3K36 methylation maintains cell identity by regulating opposing lineage programmes. Nat Cell Biol 25, 1121–1134 (2023). https://doi.org/10.1038/s41556-023-01191-z

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