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Histone editing elucidates the functional roles of H3K27 methylation and acetylation in mammals

Abstract

Posttranslational modifications of histones (PTMs) are associated with specific chromatin and gene expression states1,2. Although studies in Drosophila melanogaster have revealed phenotypic associations between chromatin-modifying enzymes and their histone substrates, comparable studies in mammalian models do not exist3,4,5. Here, we use CRISPR base editing in mouse embryonic stem cells (mESCs) to address the regulatory role of lysine 27 of histone H3 (H3K27), a substrate for Polycomb repressive complex 2 (PRC2)-mediated methylation and CBP/EP300-mediated acetylation6,7. By generating pan-H3K27R (pK27R) mutant mESCs, where all 28 alleles of H3.1, H3.2 and H3.3 have been mutated, we demonstrate similarity in transcription patterns of genes and differentiation to PRC2-null mutants. Moreover, H3K27 acetylation is not essential for gene derepression linked to loss of H3K27 methylation, or de novo activation of genes during cell-fate transition to epiblast-like cells (EpiLCs). In conclusion, our results show that H3K27 is an essential substrate for PRC2 in mESCs, whereas other PTMs in addition to H3K27 acetylation are likely involved in mediating CBP/EP300 function. Our work demonstrates the feasibility of large-scale multicopy gene editing to interrogate histone PTM function in mammalian cells.

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Fig. 1: Substitution of lysine 27 with arginine in variant and canonical histone H3 phenocopies loss of PRC2 during long-term differentiation.
Fig. 2: H3K27 is dispensable for binding of transcriptional machinery to chromatin in mESCs.
Fig. 3: H3K27 mutant mESCs share transcriptional similarity with SUZ12 KO mESCs.
Fig. 4: EpiLC transcripts are activated in H3K27R mutant ESCs during differentiation.
Fig. 5: H3K27 acetylation is dispensable for transcriptional activation during EpiLC differentiation.

Data availability

All related raw sequencing and related processed data are deposited in and publicly available from the GEO under the accession numbers GSE160974 and GSE160975. These data include processed data such as genome region sets for genes and cell-type-specific enhancers, H3.3 peak sets, ChIP signal and RNA-seq quantification data that serve as source data for Figs. 25 and Extended Data Figs. 2, 3 and 58. Numerical source data not covered in the GEO processed data deposition are provided separately along with source data for immunoblots in Fig. 1 and Extended Data Fig. 2. Source data are provided with this paper.

Code availability

Only publicly available tools were used in data analysis as described wherever relevant in Methods and Reporting Summary.

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Acknowledgements

We thank A. Laugesen and R. L. Armstrong for critical reading of the manuscript and all members of the Helin laboratory for discussions and support. The work in the Helin laboratory was supported by the Neye Foundation (K.H.), the Brain Tumor Charity (GN-00358, K.H.), a center grant from the Novo Nordisk Foundation to the NNF Center for Stem Cell Biology (NNF17CC0027852, K.H.), the European Union’s Horizon research and innovation programme (MSCA-ITN-2018-813327, K.H.) and a Memorial Sloan Kettering Cancer Center Support Grant (National Institutes of Health P30 CA008748, K.H.).

Author information

Authors and Affiliations

Authors

Contributions

F.M. conceived the mutagenesis strategy. A.S., F.M. and A.K.S. designed and performed the experiments. A.K.S. and T.T. imaged EBs. A.S., H.W. and M.L. performed bioinformatic analysis. A.S. and K.H. wrote the manuscript, followed by review and editing by all authors. K.H. supervised the study and acquired funding.

Corresponding author

Correspondence to Kristian Helin.

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Competing interests

K.H. is a co-founder of Dania Therapeutics ApS, a consultant for Inthera Bioscience and a scientific advisor for MetaboMed and Hannibal Innovation. All other authors have no competing interests.

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Nature Genetics thanks Shelley Berger and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Base editing strategy to generate canonical H3K27 mutant mESCs.

(a) Schematic showing step-wise strategy to generate complete canonical H3K27R (cK27R) base edited mESCs from wild-type mESCs. (b) Global and gene-specific quantitative PCR genomic DNA screening strategy to detect cK27R base edited genes targeted in ABE7.10 mESCs. (c) Bar plot showing gene-specific quantitative PCR for canonical histone genes showing difference in Ct values for wild-type and K27R-specific primer sets in ABE7.10 and two independent cK27R clones. Each data point represents two independent biological repeats. (d) Sanger sequencing verification of H3K27R conversion at canonical histone genes in two independent mESC clones.

Source data

Extended Data Fig. 2 Generation and characterization of canonical and pan-H3K27R mutant mESCs.

(a) Immunoblots showing H3S28 phosphorylation in cK27R mESCs at steady state and after Colcemid-induced mitotic arrest. (b) Immunoblots showing SUZ12, H3K27me1, H3K27me2, H3K27me3, H3K27ac and H3S28P in SUZ12 KO mESCs generated in ABE7.10 background. (c) Violin plot of endogenous H3f3a and H3f3b mRNA in indicated mESCs with median and quartiles shown. n represents three independent biological replicates per sample. (d) Immunoblot comparing global levels of histone acetylation marks in indicated mESCs; with known EP300/CBP substrates highlighted in red. Blots (a), (b) and (d) are representative of three independent experiments; Ponceau staining is used as loading control. (e) Average ChIP-Seq signal abundance at all H3.3 peaks (n) found in ABE7.10 relative to H3.3 KO (4-fold increase) for the indicated addback mESCs. Signal is quantified in FPKM and representative ChIP-Seq tracks for H3.3 at Esrrb and Ctcf locus. (f) Alkaline phosphatase staining of the ABE7.10, cK27R, pK27WT and pK27R mESCs. Scale bar represents 100 µm. (g) Cell cycle profiles of ABE7.10, cK27R, pK27WT and pK27R mESCs following EdU incorporation on y-axis and DNA content (DAPI) on x-axis. Data is representative from two independent experiments. (h) Left, representative images from three independent experiments of embryoid bodies (EBs) on day 4 after induction of differentiation in the indicated cell lines. Scale bar represents 0.5 mm. Right, quantification of day 4 EB area. Statistical significance was calculated using an unpaired two-tailed Student’s t-test; n, number of EBs examined; the violin plot shows data distribution with median and quartiles. (i) Heatmap representation of pluripotency and germ layer marker gene expression at 0, 4 and 8 days along EB differentiation. Each square contains data derived from three independent biological replicates. (j) Violin plot showing endogenous H3f3a and H3f3b expression in ABE7.10 mESCs during short-term EpiLC and long-term EB differentiation; n represents three independent biological replicates. Data distribution with median and quartiles are shown.

Source data

Extended Data Fig. 3 Genome-wide H3K27 methylation and acetylation status in H3K27R mutant mESCs.

(a) Heatmaps showing enrichment of H3K27me3 as determined by ChIP-Seq in ABE7.10 and H3K27R mutant mESCs at genes including TSS, gene bodies, TTS and surrounding loci. All non-redundant genes annotated in the UCSC database (n = 24402) were adjusted to fit the same visual space and ordered according to decreasing signal in ABE7.10 sample with signal enrichment shown as spike-in adjusted FPKM. 50% of gene length is shown as offset for TSS and TTS. (b) Heatmaps (left) and line graph (right) showing H3K27me2 ChIP-Seq enrichments in ABE7.10 and H3K27R mutant mESCs at genes visualized as in (a). (c) Representative genome browser track for H3K27me2 in ABE7.10 and H3K27R mutant mESCs. (d) Average ChIP-Seq enrichment profiles and representative genome browser track for H3K27ac using a mouse monoclonal antibody at genes in ABE7.10 and H3K27R mutant mESCs quantified as in (a). (e) Heatmaps showing H3K27ac ChIP-Seq enrichments using two different H3K27ac monoclonal antibodies in ABE7.10 and H3K27R mutant mESCs at genes as in (a). (f) Average ChIP-Seq signal profiles and representative genome browser track for H3K27ac using a mouse monoclonal antibody at mESC enhancers (n = 8394) and surrounding loci in a 20 kilobase window. Enhancers are ordered in heatmap according to decreasing signal in ABE7.10 with signal enrichment shown as spike-in adjusted FPKM. (g) Heatmaps showing ChIP-Seq signal enrichment for H3K27ac using two different H3K27ac monoclonal antibodies in ABE7.10 and H3K27R mutant mESCs at enhancers quantified and represented as in (f).

Extended Data Fig. 4 Specificity testing of H3K27ac monoclonal antibodies.

(a) ELISA test performed for H3K27ac (Rb) against single and double modified histone PTM peptides of histone H3 on an in-house histone PTM peptide array as indicated. On target substrates are highlighted in green and off-target substrates are highlighted in red. (b) ELISA test performed for H3K27ac (Ms) against single and double modified histone PTM peptides of histone H3 on an in-house histone PTM array as indicated. On target substrates are highlighted in blue and off-target substrates are highlighted in red.

Source data

Extended Data Fig. 5 Genome-wide profiling of RPB1 (RNA Pol-II) and MED1 binding in H3K27R mutant mESCs.

(a) Heatmaps showing RPB1(RNA Pol-II) ChIP-Seq enrichments in ABE7.10 and H3K27R mutant mESCs at genes including TSS, gene bodies, TTS and surrounding loci. All non-redundant genes annotated in the UCSC database (n = 24402) were adjusted to fit the same visual space and ordered according to decreasing signal in ABE7.10 sample with signal enrichment shown as spike-in adjusted FPKM. 50% of gene length is shown as offset for TSS and TTS. (b) Heatmaps (left) and average line plot (right) showing RPB1(RNA Pol-II) ChIP-Seq enrichments in ABE7.10 and H3K27R mutant mESCs at mESC enhancers (n = 8394) and surrounding loci in a 20 kilobase window. Enhancers are ordered according to decreasing signal in ABE7.10 with signal enrichment shown as spike-in adjusted FPKM. (c) Representative genome browser track for RPB1(RNA Pol-II) at miR290 super-enhancer locus. (d) Heatmaps showing MED1 ChIP-Seq enrichments in ABE7.10 and H3K27R mutant mESCs at genes visualized as in (a). (e) Heatmaps showing MED1 ChIP-Seq enrichments in ABE7.10 and H3K27R mutant mESCs at mESC enhancers visualized as in (b). (f) 2-D histograms showing read count comparison between individual cK27R and pK27R clones at enhancers and genes for MED1 and RPB1(RNA Pol-II); n represents number of genes or enhancers in comparison. (g) 2-D histograms showing read count comparison between ABE7.10 and individual cK27R or pK27R clones at enhancers for MED1 and RNA Pol-II (RPB1); n represents number of enhancers in comparison. (h) 2-D histograms showing read count comparison between ABE7.10 and individual cK27R or pK27R clones at genes for MED1 and RNA Pol-II (RPB1); n represents number of genes in comparison.

Extended Data Fig. 6 Gene expression profiles of H3K27R mutant mESCs.

(a) Bar plot summarizing number of significantly up- and downregulated genes in SUZ12 KO and H3K27R mutant mESCs versus ABE7.10 mESCs using the data filtering parameters highlighted following DeSeq2 (see Methods for statistical testing). (b) Venn diagrams showing overlap in identity of up- and downregulated genes between SUZ12 KO and cK27R mESC clones using parameters from (a). (c) Venn diagrams showing overlap in identity of up- and downregulated genes between SUZ12 KO and pK27R mESC clones using parameters from (a). (d) Gene Ontology analysis for biological processes in genes commonly upregulated (left) or downregulated (right) between SUZ12 KO and cK27R mESCs from (b). (e) Gene Ontology analysis for biological processes in genes commonly upregulated (left) or downregulated (right) between SUZ12 KO and pK27R mESCs from (c). (f) Heatmap showing promoters of significantly deregulated genes from DeSeq2 in cK27R_2 mESCs (left) and H3K27me3 (center) and H3K27ac (right) ChIP-Seq signals at these genes in ABE7.10, cK27R_1 and SUZ12 KO mESCs, sorted according to H3K27me3 (top) or H3K27ac (bottom) in ABE7.10 mESCs. All genes with adjusted p-value < 0.05 in cK27R_2 mESCs following DeSeq2 were represented in the plot. (g) Heatmap showing promoters of significantly deregulated genes from DeSeq2 in pK27R_2 mESCs (left) and H3K27me3 (center) and H3K27ac (right) ChIP-Seq signals at these genes in ABE7.10, cK27R_1 and SUZ12 KO mESCs, sorted according to H3K27me3 (top) or H3K27ac (bottom) in ABE7.10 mESCs. All genes with adjusted p-value < 0.05 in pK27R_2 mESCs following DeSeq2 were represented in the plot.

Source data

Extended Data Fig. 7 Transcriptional profiling of H3K27R EpiLCs.

(a) Representative images from three independent biological replications of 48-hour EpiLCs generated from ABE7.10, SUZ12 KO and H3K27 mutant mESCs. Scale bar represents 30 µm. (b) Bar plot showing RT-qPCR analysis of naïve, pluripotent and EpiLC marker gene mRNAs in the indicated cell lines. Circles represent data from three independent biological replicates where bar graph shows mean value with s.d. (c) MA plots showing average changes in gene expression from DESeq2 (see Methods for statistical testing parameters) between EpiLCs and their respective mESCs based on three independent biological replicates. DeSeq2 data were filtered for a log 2 fold change in gene expression >=1 and adjusted p-value <0.05. Red and blue dots represent significantly EpiLC and mESC genes, respectively; n represents number of genes. (d) Venn diagrams of filtered data from (c) showing the degree of overlap between cK27R or pK27R EpiLCs with ABE7.10 or SUZ12 KO with number of genes indicated.

Source data

Extended Data Fig. 8 Chromatin profiling of H3K27R EpiLCs.

(a) Heatmaps showing ChIP-Seq enrichments for RPB1(RNA Pol-II) in indicated EpiLCs at all genes including TSS, gene bodies, TTS and surrounding loci. All gene bodies were adjusted to fit the same visual space with 50% of gene body length offset in either side and ordered according to decreasing signal in ABE7.10 with signal enrichment shown in spike-in adjusted FPKM; n represents number of genes. 50% of gene length is shown as offset for TSS and TTS. (b) Heatmaps showing ChIP-Seq enrichments for H3K27ac in indicated EpiLCs at all genes as shown in (a). (c) Heatmaps showing ChIP-Seq enrichments for H3K27ac in indicated samples at EpiLC-specific active enhancers and surrounding loci in a 20 kilobase window. Enhancers are ordered in heatmap according to decreasing signal in ABE7.10 with signal enrichment shown as spike-in adjusted FPKM; n represents number of enhancers. (d) Representative genome browser track of H3K27ac and associated RPB1(RNA Pol-II) in EpiLCs at Myc locus. (e) Representative genome browser track of H3K27ac and associated RPB1(RNA Pol-II) in EpiLCs at Dnmt3b locus.

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Reporting Summary

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Supplementary Table

Table 1. Primers, oligos and sgRNA sequences Table 2. Antibody list

Source data

Source Data Fig. 1

Numerical source data associated with Fig. 1.

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Unprocessed western blots.

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Source Data Extended Data Fig. 2

Unprocessed western blots.

Source Data Extended Data Fig. 4

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Sankar, A., Mohammad, F., Sundaramurthy, A.K. et al. Histone editing elucidates the functional roles of H3K27 methylation and acetylation in mammals. Nat Genet 54, 754–760 (2022). https://doi.org/10.1038/s41588-022-01091-2

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