Abstract

Polycomb repressive complex 2 (PRC2) catalyzes methylation on lysine 27 of histone H3 (H3K27) and is required for maintaining transcriptional patterns and cellular identity, but the specification and maintenance of genomic PRC2 binding and H3K27 methylation patterns remain incompletely understood. Epigenetic mechanisms have been proposed, wherein pre-existing H3K27 methylation directs recruitment and regulates the catalytic activity of PRC2 to support its own maintenance. Here we investigate whether such mechanisms are required for specifying H3K27 methylation patterns in mouse embryonic stem cells (mESCs). Through re-expression of PRC2 subunits in PRC2-knockout cells that have lost all H3K27 methylation, we demonstrate that methylation patterns can be accurately established de novo. We find that regional methylation kinetics correlate with original methylation patterns even in their absence, and specification of the genomic PRC2 binding pattern is retained and specifically dependent on the PRC2 core subunit SUZ12. Thus, the H3K27 methylation patterns in mESCs are not dependent on self-autonomous epigenetic inheritance.

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Acknowledgements

We thank members of the Helin laboratory and A. Groth for advice and discussion, and J. Martin and the Transgenic Core Facility staff for assistance with morula injection experiments. H.D. was supported by a postdoctoral fellowship from the Danish Cancer Society. The work in the Helin laboratory was supported by grants to K.H. from The European Research Council (294666_DNAMET), the Seventh Framework Program of the European Union (4DCellFate), the Danish Cancer Society, the Danish National Research Foundation (DNRF82), the Danish Medical Research Council (DFF- 4183-00237), the Novo Nordisk Foundation (NNF16OC0023234), and The Lundbeck Foundation, and through a center grant from the Novo Nordisk Foundation (NNF17CC0027852)).

Author information

Author notes

    • Andrey Tvardovskiy

    Present address: Institute of Functional Epigenetics, Helmholtz Zentrum München, Neuherberg, Germany

Affiliations

  1. Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    • Jonas W. Højfeldt
    • , Anne Laugesen
    • , Helene Damhofer
    • , Lin Hedehus
    • , Faizaan Mohammad
    •  & Kristian Helin
  2. The Novo Nordisk Foundation Center for Stem Cell Biology (DanStem), Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    • Jonas W. Højfeldt
    • , Anne Laugesen
    • , Helene Damhofer
    • , Lin Hedehus
    • , Faizaan Mohammad
    •  & Kristian Helin
  3. Cell Biology and Physiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark

    • Berthe M. Willumsen
  4. Department of Biochemistry and Molecular Biology, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Odense, Denmark

    • Andrey Tvardovskiy
    •  & Ole N. Jensen

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Contributions

J.W.H. and A.L. designed the study, performed the majority of experiments, performed data analysis and wrote the manuscript. K.H. designed the study, performed data analysis and wrote the manuscript. B.M.W. performed experiments regarding the generation of KO cell lines. H.D. performed experiments regarding the inhibitor study. L.H. and F.M. performed experiments regarding the blastocyst injection experiment. A.T. and O.N.J. performed and analyzed experiments critical for development of the project.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Kristian Helin.

Integrated supplementary information

  1. Supplementary Figure 1 Genetic erasure and restoration of H3K27 methylation patterns.

    a, Western blots of extracts from PRC2-subunit knockout cell lines blotted for PRC2 subunits and H3K27 methylation. Experiment has been repeated at least three times with same result. Uncropped blot/gel images are shown in Supplementary Data Set 1. b, ChIP-seq signals generated with H3K27 methylation specific antibodies. The samples and the shown region is the same as Fig. 2b, but the ChIP-seq signals are here shown without spike-in normalization. Values on vertical axis correspond to reads per million mapped mouse reads. c, Heatmaps of sequence-depth normalized ChIP-seq data of H3K27 methylation in genic regions. Vertical axis contains all RefSeq genes > 250bp and horizontal axis is centered on genes represented by relative gene length. TSS: Transcription start site. Arrow ends at end of genes. d, Scatterplots comparing ChIP-seq signal within 5000 bp genomic regions between parental WT mESCs and Ezh1/Ezh2 dKO or EZH2 rescue cells. The entire mouse genome has been divided into 5000 bp regions and spike-in normalized signal quantified for each region. The quantified values are in units of reads per million Drosophila-mapped reads per 1000 bp.

  2. Supplementary Figure 2 Gene deregulation and reversal in Ezh1/Ezh2-dKO and dKO + EZH2 cells.

    a. MA plots of RNAseq data analyzed with DESeq2 for differential expression in either Ezh1/Ezh2 dKO (left) or dKO + EZH2 (right) relative to WT mESCs. Genes that are significantly upregulated (FDR < 0.05 and rlog2-fold change > 1) in Ezh1/Ezh2 dKO cells are colored red and significantly downregulated genes (FDR < 0.05, rlog2-fold change < -1) are colored blue. b. Plot of all genes that are differentially expressed in Ezh1/Ezh2 dKO cells together with their rlog2 fold expression changes (relative to WT mESCs) in Ezh1/Ezh2 dKO cells (red) and dKO + EZH2 cells (blue). The genes are ranked along horizontal axis according to the extent of deregulation in Ezh1/Ezh2 dKO cells.

  3. Supplementary Figure 3 Effect of inhibitor treatment on mESC properties.

    a. Schematic representation E14 and 7d cells, which have been treated with 10 µM Ezh2 inhibitor (EPZ6438) for 7 days. b. Outline of embroid body (EB) differentiation assay used to assess differentiation capacity. c. Quantification of beating clusters following EB differentiation. E14 or 7d cells were each differentiated in both absence or presence of inhibitor. Fractions in graph show number of beating clusters over total clusters observed. d. Proliferation curve for E14 and 7d cells. 7d cells were grown with continuous presence of inhibitor. At each time point, two individual wells are counted for each cell type. This experiment was repeated as a biological replicate 2 (not shown). e. Proliferation rate (doubling time) calculated from proliferation curve in panel d and from replicate experiment 2. Error bars represent 95% confidence interval based on linear regression of proliferation curves (6 duplicate data points per curve). f. Expression level of pluripotency genes. Circles show mean value of two technical replicates for each of three biological replicates. Wide black line marks represent the mean of the biological replicates, and error bars represent the s.e.m. g. Expression level of Hoxa10, which is upregulated in Ezh1/Ezh2 dKO vs WT mESCs, in E14 and 7d cells.

  4. Supplementary Figure 4 Regional de novo methylation kinetics.

    a. ChIP-seq tracks showing kinetics of re-methylation of all three states of H3K27 methylation as well as Suz12 binding in a representative genomic region following treatment with EZH2 inhibitor. ChIPseq signal is spike-in normalized (reads per million mapped Drosophila reads). b. Heatmaps of spike-in normalized ChIP-seq data of H3K27 methylation within random genomic regions. Vertical axis contains 50,000 random 5000 bp regions clustered (k-means clustering, k = 10) according to methylation state in E14 cells. The clusters are ordered according to highest methylation state in untreated cells. c, Biological replicate of experiment presented in Fig. 3c. ChIP-qPCR data probing kinetics of H3K27me3 (first row), H3K27me2 (second row), H3K27me1 (third row) and Suz12 (fourth row) at seven representative loci labeled (above graph) according to nearest gene and in approximate order of decreasing methylation rates (% of input/hr washout). Data values come from a single biological (n=1) experiment, with error bars depicting s.d. of technical duplicates. Source data

  5. Supplementary Figure 5 Accurate H3K27 methylation and restored pluripotency in SUZ12-rescue cells.

    a, Heatmaps of sequence-depth normalized ChIP-seq data of H3K27 methylation in genic regions. Vertical axis contains all RefSeq genes > 250bp and horizontal axis is centered on genes represented by relative gene length. TSS: Transcription start site. Arrow ends at end of genes. b, Images of embryos derived from a morula injection experiment dissected at E13.5. In addition to embryos shown in Fig. 5c, three additional embryos were obtained from morulas injected with Suz12 KO cells and two additional embryos were obtained from injection of SUZ12-rescued cells (KO + SUZ12). Scale bar: 5 mm. c, Side-by-side comparison of embryos shown in Fig. 5b. Scale bar: 5 mm. d, Mouse embryonic fibroblasts (MEFs) and neural stem cells (NSCs) were derived from embryos shown in Fig. 5b and embryo #2 from Suz12 KO cells and SUZ12-rescued cells. These cells were analyzed by FACS to quantify the contribution of mCherry-positive cells.

  6. Supplementary Figure 6 An N-terminal SUZ12 fragment is essential for CGI binding and correct H3K27 methylation patterns.

    a, Heatmaps of sequence-depth normalized ChIP-seq data of H3K27 methylation in genic regions. Vertical axis contains all RefSeq genes > 250bp and horizontal axis is centered on genes represented by relative gene length. TSS: Transcription start site. Arrow ends at end of genes. b, Heatmaps of sequence-depth normalized ChIP-seq data of H3K27 methylation within random genomic regions. Vertical axis contains 50,000 random 5000 bp regions clustered (k-means clustering, k = 10) according to methylation state in E14 cells. Horizontal axis is 25,000 bp wide centered on regions used for clustering. The clusters are ordered according to highest methylation state in untreated cells. c, Heatmaps of sequence-depth normalized H3K27me1 ChIP-seq data clustered with H3K9me3 and H3K36me3. Regions in H3K27me1 positive clusters (8 and 9, 6670 regions) have been submitted to new clustering with published H3K9me3 and H3K36me3 data55 to yield clusters B1 to B6. Horizontal axis is 100,000 bp wide centered on regions (5000 bp) used for clustering.

Supplementary information

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    Supplementary Figures 1–6 and Supplementary Tables 1–5

  2. Life Sciences Reporting Summary

  3. Supplementary Dataset 1

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https://doi.org/10.1038/s41594-018-0036-6

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