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Distinct H3K9me3 heterochromatin maintenance dynamics govern different gene programmes and repeats in pluripotent cells

Abstract

H3K9me3 heterochromatin, established by lysine methyltransferases (KMTs) and compacted by heterochromatin protein 1 (HP1) isoforms, represses alternative lineage genes and DNA repeats. Our understanding of H3K9me3 heterochromatin stability is presently limited to individual domains and DNA repeats. Here we engineered Suv39h2-knockout mouse embryonic stem cells to degrade remaining two H3K9me3 KMTs within 1 hour and found that both passive dilution and active removal contribute to H3K9me3 decay within 12–24 hours. We discovered four different H3K9me3 decay rates across the genome and chromatin features and transcription factor binding patterns that predict the stability classes. A ‘binary switch’ governs heterochromatin compaction, with HP1 rapidly dissociating from heterochromatin upon KMT depletion and a particular threshold level of HP1 limiting pioneer factor binding, chromatin opening and exit from pluripotency within 12 h. Unexpectedly, receding H3K9me3 domains unearth residual HP1β peaks enriched with heterochromatin-inducing proteins. Our findings reveal distinct H3K9me3 heterochromatin maintenance dynamics governing gene networks and repeats that together safeguard pluripotency.

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Fig. 1: Acute depletion of H3K9me3 KMTs causes rapid decay of H3K9me3 marks in mES cells.
Fig. 2: H3K9me3 and HP1β decay rates differ genome-wide.
Fig. 3: Heterochromatin states influence kinetics of H3K9me3 decay.
Fig. 4: Maintaining heterochromatin integrity involves a threshold level of H3K9me3 and HP1.
Fig. 5: The critical loss of heterochromatin integrity within 12 h leads to the activation of various lineage genes and DNA repeats.
Fig. 6: Pioneer factors eliciting further heterochromatin loss after KMT decay.
Fig. 7: NFYA acts as a pioneer factor to uniquely target MMERVK10C repeat family.
Fig. 8: H3K9me3 heterochromatin constrains transcription factor Dppa2 to bivalent genes.

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Data availability

All the raw sequencing and processed data are available at the Gene Expression Omnibus under accession number GSE233041. The mouse genome (mm10 assembly), Drosophila genome (dm6 assembly) and yeast (S. cerevisiae) genome (sacCer3 assembly) are downloaded from UCSC genome browser https://hgdownload.soe.ucsc.edu/downloads.html. The raw data supporting the findings of this study are included in this Article and its Supplementary Information. A list of publicly available datasets used in the study can be found in the Article. Source data are provided with this paper.

Code availability

The parameters for next-generation sequencing data analysis are reported in Methods and included in the Gene Expression Omnibus (GEO) upload. All the original code for mathematical modelling of H3K9me3 decay has been deposited to Github at https://github.com/Tomer-Lapidot/H3K9me3_Methylation_Stochastic_Model.

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Acknowledgements

We thank A. Barral and A. Katznelson for comments on the manuscript, A. Hsieh, J. Lerner, R. McCarthy and K. Kaeding for the valuable feedback during the project and the K. Helin lab for the KDM4C antibody. J.Z. was supported by a Human Frontiers Science Program fellowship (LT000761/2019-L) and the research grant by NIH R01GM36477 and R35GM153180 to K.Z.

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Authors

Contributions

J.Z. and K.Z. conceived and designed the experiments and wrote the manuscript. J.Z. carried out the experiments and collected and analysed the data. J.Z. and D.N. established the cTKO mES cell line, M.B.G. carried out MS sample preparations and data analysis. G.D. provided bioinformatic analysis, and T.L. and G.D. performed the mathematical modelling.

Corresponding author

Correspondence to Kenneth S. Zaret.

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The authors declare no competing interests.

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Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Characterization of the dTKO mouse ES cell line.

a, Immunostaining of pluripotent markers, Sox2 and Esrrb in dTKO ES cells under self-renewal conditions. Scale bar, 200 μm. b, Morphologies of the dTKO mouse ES cell and aggregates during the embryonic body differentiation. Scale bar, 200 μm. c, Quantifications of the expression of early lineage markers in dTKO cells over the embryonic body differentiation by RT-qPCR. The expression levels are normalized to the house-keeping gene TBP, values are means ± SEM; n = 3 biologically independent replicates. Two-sided Student’s t-tests were used for D0 vs. each time point. P values at D2, D4 and D6 for each gene are: FGF (0.044, 0.185, 0.002); FoxA2 (0.003, 0.287, 0.053); T (0.055, 0.272, 0.014); Eomes (0.018, 0.297, 0.002); Sox1 (0.025, 0.007, 0.022); Gata6 (0.706, 0.336, 0.050). * P < 0.05. d,e, Scatter plots of H3K9me3 signals (d) and nascent RNAs from TT-seq (e) between dTKO before degradation and parental cTKO mouse ESC line at different families of DNA repeats. f, Western blot with antibodies for HA, V5, Suv39h1, and the loading control, αTubulin in the whole protein extract, over auxin and dTAG13 degrons treatments. Experiments were repeated three times independently with similar results. The molecular weight in kilodaltons were indicated on the left. g, Immunostaining of the V5 (Suv39h1) and HA (Setdb1) in dTKO ESCs at 0 hr and 3 hr of auxin and dTAG degrons treatment. Scale bar, 50 μm. Source numerical data and unprocessed blots are available in Source data.

Extended Data Fig. 2 Acute KMTs’ degradation leads to early depletion of H3K9me3 and later loss of H4K20me3 in dTKO mouse ESCs.

a, Quantifications of H3K9me3 (normalized to total H3), following the auxin and dTAG treatment. H3K9me3 levels are normalized to the 0 hr sample; n = 3 biologically independent replicates. P Values at 1, 3, 6, 12, 24 and 48 h are 0.048, 0.234, 0.082, 0.044, 0.0014, 0.0016. b, Mass spectrometry quantification of histone marks over the degradation time course using acid-extracted histones. c,d, Western blot (c) and quantification (d) of H3K9me2, H4K20me3, H3K27me3, and the loading control, histone H3, over the time (hours) of auxin and dTAG13 degrons addition. The molecular weight in kilodaltons (KD) were indicated on the left. The histone marks’ levels are normalized to the 0 hr sample; n = 3 biologically independent replicates for H4K20me3 and H3K27me3; for H3K9me2, n = 2 biological replicates. P Values at 1, 6, 12, 24 and 48 hr are H3K27me3 (0.12, 0.56, 0.38, 0.42, 0.52); H4K20me3 (0.64, 0.49, 0.67, 0.026, 0.0023). e. Cell growth curve of the dTKO mouse ESC line with or without auxin and dTAG dual degrons treatment for 4 days. The values are log transformed to better visualize the data. Exponential equation fitting the -A-dTAG growth curve is shown above. n = 6 biologically independent replicates for -auxin -dTAG condition; n = 4 biologically independent replicates for +auxin +dTAG condition. P Values at 24, 48, 72 and 96 hr are 0.72, 0.054, 0.079, 0.042. Values in (a,d,e) are means ± SEM, two-sided Student’s t-tests were used for 0 hr vs. other time points. Source numerical data and unprocessed blots are available in Source data.

Extended Data Fig. 3 Kinetics of H3K9me3 decay and recovery.

a, Flow cytometry showing histogram of EdU staining with or without 6 hr of thymidine treatment. b. Line plots showing the exponential decay curve of H3K9me3 measured in (Fig. 1f, h) after fitting the western blot data with the exponential decay model. c, Western blot of KDM4A, KDM4B, and KDM4C and loading control, αTubulin, of dTKOs over the degradation time course with non-targeting (Ctrl) siRNA or KDM4A-C siRNAs. Experiments were repeated three times independently with similar results. d, Western blot of HA (HA-dTAG-Setdb1 fusion protein), V5 (Suv39h1-AID-eGFP-3V5 fusion protein), H3K9me3 and histone H3 of dTKOs over the degradation time course with DMSO or pan-KDM4 inhibitor QC6352 treatment. Experiments were repeated twice independently with similar results. The time (hr) of auxin and dTAG addition are indicated above. The molecular weight in kilodalton (KD) is indicated on the left (c,d). e, Quantifications of H3K9me3 signals over the degradation time course with DMSO or pan-KDM4 inhibitor QC6352 treatment. H3K9me3 signals were normalized to H3 before normalizing to 0 hr sample. Values are means of two biologically independent replicates; KDM4i, pan-KDM4 inhibitor QC6352. f,g, Western blot (f) and quantification (g) of HA (Setdb1) and V5 (Suv39h1), H3K9me3, normalized to the load control H3 or αTubulin over degradation and degrons washout time course. Values are means of two biological replicates. h, Alkaline phosphatase staining of parental untagged cTKO mouse ESCs plated at clonal density after 7 days of treatment with DMSO v.s. +auxin +dTAG, respectively. Source numerical data and unprocessed blots are available in Source data.

Extended Data Fig. 4 Prolonged treatment of dTKO for 2–7 days leads to mESC differentiation and genome instability.

a, RT-qPCR quantifications of the expression of DNA repeats, pluripotent and early differentiation markers in dTKO cells over 7 days of dual degrons treatment in LIF/FCS culture condition. Values are means ± SEM; n = 3 biologically independent replicates. Two-sided Student’s t-tests were used for D0 vs. each time point. P values at D1-D7 for each gene/DNA repeat are: IAP (0.003, 0.035, 0.017, 0.022, 0.013, 0.021, 0.029); Major Satellite (0.012, 0.074, 0.039, 0.014, 0.184, 0.013, 0.058); Mervl (0.009, 0.041, 0.0005, 0.010, 0.008, 0.008, 0.079); LINE1 (0.001, 0.087, 0.04, 0.008, 0.024, 0.002, 0.017); Zscan4 (0.001, 0.012, 0.009, 0.03, 0.022, 0.0002, 0.001); Oct4 (0.052, 0.132, 0.031, 0.009, 0.01, 0.01, 0.01); Nanog (0.001, 0.018, 0.001, 6.8e-06, 0.0003, 0.0003, 3.3e-05); FoxA2 (0.233, 0.305, 0.027, 0.01, 0.011, 0.003, 0.051); T (0.004, 0.016, 0.021, 0.033, 0.008, 0.001, 0.062); Sox1 (0.022, 0.233, 0.984, 0.587, 0.014, 0.012, 0.01); Cdx2 (0.053, 0.801, 0.012, 0.005, 0.014, 0.004, 0.901); Gata6 (0.538, 0.345, 0.007, 0.005, 8.1e-05, 0.008, 0.004).* P < 0.05, b, Cellular morphology changes over the 7 days of dual degrons treatment of dTKO mouse ES cells in LIF/FCS condition. Scale bar, 100 μm. c, DAPI staining showing the nuclear morphology changes over 7 days of dual degrons treatment of dTKO mouse ES cells in LIF/FCS condition. Scale bar, 50 μm d, Flow cytometry showing histograms of PI staining of DNA content over 7 days of dual degrons treatment of dTKO mouse ES cells in LIF/FCS condition. Source numerical data are available in Source data.

Extended Data Fig. 5 The receding of H3K9me3 reveals residual HP1β peaks.

a, Western blot showing nuclear proteins at their expected nuclear fractions, including heterochromatin proteins. The nuclear fractions are indicated above. b,c, Western blot of HP1α/β/γ and loading control, H3, in chromatin fractions (b) or in the total cell lysates (c) of dTKO over the degradation time course. The time (hr) of auxin and dTAG treatments are indicated above. d, Venn diagram showing the number of HP1β residual peaks after 48 hr degradation that falls within H3K9me3 domains before degradation. e, Bar graph showing the number of HP1β residual peaks after 24 hr and 48 hr degradation overlapping with different repeat families. f, Heatmaps of H3K9me3-nucleation proteins, and H3K9me2 within 1 kb window flanking the center of the 48 hr residual HP1β peaks. g, Western blot of TRIM28, Mpp8, Mettl3, YTHDC1, and loading control, H3, in the whole cell extracts of dTKO over the degradation time course. The molecular weight in kilodalton (KD) is indicated on the left. Experiments were repeated three times independently with similar results (a,b,c,g). h–j, ChIP-qPCR of Mettl3 (h), YTHDC1 (i), and TRIM28 (j) at six HP1 residual peaks, and the GAPDH promoter (negative control) over the degrons treatment. Values are means ± SEM; n = 3 biologically independent replicates. Two-sided Student’s t-tests were used for 0 hr vs. each timepoint. P values at GAPDH and residual HP1 peak 1-6 are: Mettl3, 12 hr (0.592, 0.073, 0.156, 0.02, 0.132, 0.081, 0.098), 24 hr (0.479, 0.078, 0.54, 0.018, 0.391, 0.1, 0.101), 48 hr (0.347, 0.047, 0.091, 0.016, 0.101, 0.06, 0.062); YTHDC1, 12 hr (0.493, 0.006, 0.033, 0.01, 0.04, 0.001, 0.017), 24 hr (0.667, 0.004, 0.04, 0.009, 0.043, 0.002, 0.021), 48 hr (0.076, 0.005, 0.003, 0.009, 0.020, 0.002, 0.018); TRIM28, 12 hr (0.523, 0.104, 0.241, 0.037, 0.366, 0.028), 24 hr (0.476, 0.859, 0.129, 0.771, 0.028, 0.520, 0.691, 0.698), 48 hr (0.082, 0.04, 0.103, 0.025, 0.321, 0.008, 0.028). Source numerical data and unprocessed blots are available in Source data.

Extended Data Fig. 6 Impact of H3K9me3 KMTs’ depletion on other heterochromatin marks genome-wide.

a, Venn diagram showing the overlaps between the H3K9me3 domains at 0 hr and all the H3K27me3 domains before and after 48 hr of dual degrons treatment. b, Heatmaps of H3K27me3 signals over 10 kb flanking windows from the center of H3K27me3 domains that do not overlap (top) or overlap (bottom) with H3K9me3 domains at 0 hr and 48 hr of dual degrons treatment, c. Venn diagram showing the overlap between the H3K9me3 domains at 0 hr and all the H3K9me2 domains before and after 48 hr of dual degrons treatment. d, Heatmaps of H3K9me2 signals over 10 kb flanking windows from the center of H3K9me2 domains that do not overlap (top) or overlap (bottom) with H3K9me3 domains at 0 hr and 48 hr of dual degrons treatment. e, Scatter plot of H3K9me3 and H4K20me3 ChIP-seq signals (spike-in normalized ChIP/Input) at 42,890 H3K9me3 domains in dTKO cells before degradation. f, Heatmaps of H4K20me3 ChIP-seq signals over 10 kb flanking windows from the center of 42,890 H3K9me3 domains over the degradation time course. Domains were ranked in descending order of their size. Source numerical data are available in Source data.

Extended Data Fig. 7 Different H3K9me3 stability clusters mark distinct genes and repeat families.

a, b, Heatmaps of H3K9me3 (a) and HP1β (b) ChIP-seq signals over the degradation time course at different H3K9me3 alluvial clusters. The H3K9me3 domains were scaled to 4 kb, with 1 kb extension on each side. c, Heatmap showing differential enrichments of transcription factor motifs at different H3K9me3 alluvial clusters. d, Pathway analysis of genes associated with different H3K9me3 domains clusters. e, Bar graph showing the counts of repeat families that overlap with different H3K9me3 domains clusters. Source numerical data are available in Source data.

Extended Data Fig. 8 Chromatin and genetic features at different heterochromatin states.

a, Heatmap showing ChIP-seq signals of different chromatin features at heterochromatin states 1-4. All chromatin state coordinates are scaled to 3 kb, with 1 kb flanking windows. b, Heatmap showing H3K9me3 levels at heterochromatin states 1-3 from zygote to E6.5 epiblast (GSE97778). c, Neighborhood enrichment of HP1 residual peaks at chromHMM states. d, Relative distances of state 2 and state 3 in relation to state 1. The genomic coordinates of state 1 are positioned at the center, and the presence of state 2 and state 3 within 5 kb windows flanking state 1 were shown in the heatmap. e, Graphic summary of the functions and heterochromatin proteins associated with chromHMM state 1-3. f, Heatmap showing the changes of different heterochromatin associated proteins in chromatin fraction over the degradation time course measured by mass spectrometry. Values are log2 fold changes of mean values of three biological replicates at each time point over the 0 hr. g, Bar graph showing the counts of top 10 repeat families that overlap with heterochromatin state 1 and 4. Source numerical data are available in Source data.

Extended Data Fig. 9 Transcription factor binding at the H3K9me3 domains promote chromatin opening and H3K9me3 decay.

a, Ribbon plots showing the changes of the H3K9me3, HP1β and ATAC signals within 24 hr of degradations. The lines are mean of log2 fold changes normalized to 0 hr (H3K9me3 and HP1β) or 48 hr (ATAC signals), respectively, and ribbons are one standard deviation from the mean. b, Violin plots showing the ATAC peaks signals within different clusters of H3K9me3 domains over the degradation time course. The dots within the violin graph indicate the mean values. c, Heatmap showing footprint score changes of top transcription factors with biggest increase in H3K9me3 domains. The values are log2 fold changes of averaged footprint scores normalized to 0 hr. The total number of motifs are annotated on the left. d, Scatter, and contour plots of H3K9me3 (left) or HP1β (right) vs. the ATAC signals within the H3K9me3 domains at 3 hr of degradation, with the OCT4 and KLF4 footprint scores super-imposed on the dot plots. e, Bar graph showing ChIP-qPCR quantifications of Oct4 signals at four Oct4 footprinted regions, and at the GAPDH promoter (negative control) after dual degrons treatment. n = 3 biologically independent replicates; values are means ± SEM. Two-sided Student’s t-tests were used for 0 hr and each time point. P values at footprint site 1-4 and GAPDH promoters are: 12 hr (0.044, 0.701, 0.040, 0.057, 0.082); 24 hr (0.147, 0.194, 0.069, 0.176, 0.117); * P < 0.05. f,g, Boxplot comparing the decay rates of the H3K9me3 domains that gained ATAC signals (n = 20108) vs. the ones that did not (n = 22931) (f), and H3K9me3 domains with activated transcription of DNA repeats (n = 517) vs. H3K9me3 domains without transcription (n = 29529) (g). The boxes represent interquartile range (IQR) from 25th to 75th percentile, with the median at the center, and whiskers extending to 1.5 times the IQR from the quartiles. Statistical analysis was performed using Wilcoxon test. P values are 2.22e-16 (f) and 1.4e − 309 (g). h, Heatmap showing enrichments of transcription factors-footprinted ATAC peaks in H3K9me3 domains at late-lost H3K9me3 sub-cluster 1 (low decay constant) and 2 (high decay constant), indicated in (Fig. 3e). Source numerical data are available in Source data.

Extended Data Fig. 10 Transcription factors, such as NFYA and Dppa2 target unique repeat families for activation.

a, Heatmap showing the percentage of each activated retrotransposon subfamily with a given transcription factor footprints at 24 hr of degradation. 1.0 in the color keys means 100%. b, Bar graph showing the number of top three families of DNA repeats overlapping with Dppa2 peaks within the H3K9me3 domains. c, Heatmaps of Dppa2 in mouse ESCs (GSE117173), and ATAC signal at the Dppa2 peaks inside (top) vs. outside (bottom) the H3K9me3 domains over the degradation time course. d, Bar graph showing ChIP-qPCR of Dppa2 signals at three Dppa2 footprinted regions in H3K9me3 domains, and at GAPDH promoter (negative control) at 0, 12, and 24 hr of dual degrons treatment. Values are means ± SEM; n = 3 biologically independent replicates. Two-sided Student’s t-tests were used for 0 hr vs. each time point. P values at Dppa2 footprint site 1-3 and GAPDH promoters are: 12 hr (0.007, 0.084, 0.411, 0.38); 24 hr (0.007, 0.044, 0.049, 0.006). * P < 0.05. Source numerical data are available in Source data. e, Graphic summary showing that H3K9me3 heterochromatin normally constrains transcription factor Dppa2 at bivalent genes in wild type cell. Upon H3K9me3 loss, Dppa2 re-distribute to LINE1 elements in the newly de-compacted H3K9me3 domains, at the expense of bivalent genes.

Supplementary information

Supplementary Information

Supplementary Figs. 1–5 and legends.

Reporting Summary

Supplementary Table 1

Supplementary Table 1.

Source data

Source Data Fig. 1

Unprocessed western blots.

Source Data Fig. 2

Numerical source data for all figures.

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Zhang, J., Donahue, G., Gilbert, M.B. et al. Distinct H3K9me3 heterochromatin maintenance dynamics govern different gene programmes and repeats in pluripotent cells. Nat Cell Biol 26, 2115–2128 (2024). https://doi.org/10.1038/s41556-024-01547-z

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