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Epigenetic priming by Dppa2 and 4 in pluripotency facilitates multi-lineage commitment

Nature Structural & Molecular Biology volume 27, pages 696–705 (2020)Cite this article

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Abstract

How the epigenetic landscape is established in development is still being elucidated. Here, we uncover developmental pluripotency associated 2 and 4 (DPPA2/4) as epigenetic priming factors that establish a permissive epigenetic landscape at a subset of developmentally important bivalent promoters characterized by low expression and poised RNA-polymerase. Differentiation assays reveal that Dppa2/4 double knockout mouse embryonic stem cells fail to exit pluripotency and differentiate efficiently. DPPA2/4 bind both H3K4me3-marked and bivalent gene promoters and associate with COMPASS- and Polycomb-bound chromatin. Comparing knockout and inducible knockdown systems, we find that acute depletion of DPPA2/4 results in rapid loss of H3K4me3 from key bivalent genes, while H3K27me3 is initially more stable but lost following extended culture. Consequently, upon DPPA2/4 depletion, these promoters gain DNA methylation and are unable to be activated upon differentiation. Our findings uncover a novel epigenetic priming mechanism at developmental promoters, poising them for future lineage-specific activation.

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Fig. 1: DPPA2/4 are required for differentiation and bind developmental bivalent promoters.
Fig. 2: DPPA2/4 are required to maintain bivalent chromatin at a subset of developmental genes.
Fig. 3: DPPA2/4-dependent bivalent genes characterized by low H3K4me3, low expression and initiating but not elongating RNA polymerase II.
Fig. 4: DPPA2/4-dependent promoters gain DNA methylation and fail to be upregulated during differentiation.
Fig. 5: DPPA2/4 are required to target H3K4me3 and prevent DNA methylation at a subset of bivalent genes.
Fig. 6: DPPA2/4 prime the epigenetic landscape at bivalent genes, facilitating cell differentiation.

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Data availability

All sequencing data generated in this study have been submitted to the Gene Expression Omnibus under accession number GSE135841. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE38 partner repository with the data set identifier PXD014981. DPPA2 and DPPA4 ChIP data were reanalyzed from ref. 9 (GSE117173), RNA polymerase II ChIP was reanalyzed from ref. 20 (GSE34520), EZH2 and SUZ12 ChIP data in Fig. 2 were reanalyzed from ref. 39 (GSE49435), ASH2L-GFP ChIP data in Fig. 2 reanalyzed from ref. 1 (GSE52071), MLL2 ChIP data in Fig. 2 reanalyzed from ref. 2 (GSE48172), TIP60/KAT5 ChIP data reanalyzed from ref. 40 (GSE69671), high-confidence bivalent gene list from ref. 14, DNMT TKO ESC RNA-seq data from ref. 21 (GSE67867) and 2C-like ZGA gene list from ref. 41. Uncropped images of immunoblots presented in Fig. 5b are provided as source data.

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Acknowledgements

We thank all members of the Reik laboratory for helpful discussions. We also thank F. di Tullio for help generating overexpression cell lines, F. Krueger for processing sequencing data and for general bioformatics support, S. Wingett for bioinformatic assistance and S. Andrews for bioinformatic advice. We thank B. Hussey and E. Easthope at Sanger Institute and K. Tabbada at Babraham Institute for assistance with high-throughput sequencing, R. Walker for assistance with flow cytometry, and J. Webster and D. Oxley for mass spectrometry. Dnmt TKO cells were a kind gift from D. Schübeler (FMI). M.A.E.-M. is supported by a BBSRC Discovery Fellowship (BB/T009713/1) and was supported by an EMBO Fellowship (ALTF938–2014) and a Marie Sklodowska-Curie Individual Fellowship. A.P. is supported by a Sir Henry Wellcome Fellowship (215912/Z/19/Z). M.B. was supported by an Erasmus Grant. M.N. and Y.I. were supported by a Cancer Research UK Cambridge Institute Core grant (no. C9545/A29580). Research in the Reik laboratory is supported by the Biotechnology and Biological Sciences Research Council (BB/K010867/1) and the Wellcome Trust (095645/Z/11/Z).

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Author notes

  1. Marloes Blotenburg

    Present address: Oncode Institute, Hubrecht Institute–KNAW, University Medical Center Utrecht, Utrecht, the Netherlands

Authors and Affiliations

  1. Epigenetics Programme, Babraham Institute, Cambridge, UK

    Mélanie A. Eckersley-Maslin, Aled Parry, Marloes Blotenburg, Christel Krueger & Wolf Reik

  2. Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK

    Yoko Ito, Valar Nila Roamio Franklin, Masashi Narita & Clive S. D’Santos

  3. Wellcome Trust Sanger Institute, Hinxton, UK

    Wolf Reik

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Contributions

M.A.E.-M. and W.R. conceived, designed and supervised the study. M.A.E.-M. performed experiments, analyzed data and wrote the paper. A.P. helped perform ChIP–qPCR, ChIP–seq, CoIP, inducible knockdown and western blotting experiments, prepared cells for proteomics and analyzed proteomic and ChIP–seq data. M.B. performed embryoid body differentiation assays and qPCRs. C.K. analyzed chomatin state association of DPPA2/4 and generated the random forest model for DPPA2/4 dependency based on promoter features. V.N.R.F. and C.S.D’S. performed qPLEX-RIME experiments. Y.I. and M.N. designed inducible shRNA vectors.

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Correspondence to Mélanie A. Eckersley-Maslin or Wolf Reik.

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W.R. is a consultant and shareholder of Cambridge Epigenetix. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 DPPA2/4 are required for differentiation related to Fig. 1.

a, Western Blotting showing knockout of DPPA2 (top) and DPPA4 (middle) protein in WT (left 3 columns) and Dppa2/4 double knockout (DKO, right 3 columns) cells. β-ACTIN is shown as loading control b, Expression (Log-2 reads per million) of core (orange), naïve (blue), formative (purple) and primed (aqua) pluripotency markers in WT (black) and Dppa2/4 DKO (blue) ESCs as determined by RNA-sequencing. Plotted is the mean + /- standard deviation for 3 individual WT or DKO clones, each prepared in technical triplicate except for DKO clone 43 which was prepared in duplicate (n = 9 WT and 8 DKO total). An individual unpaired two-tailed t-test was performed for each gene to give the indicated p-values (above each pair of bars) where significant (p < 0.05) values are bold. c, Representative immunofluorescence staining of wild type (WT, 3 individual clones) and Dppa2/4 double knockout (DKO, three individual clones) cells showing OCT4 (top), NANOG (middle, red) and SOX2 (bottom, cyan) pluripotency markers. DAPI is included as a DNA counterstain. Scale bar represents 50μm. d–g, Quantitative RT-PCR of pluripotency d, ectoderm e, endoderm f, and mesoderm g, markers during 9-day embryoid body differentiation between wild type (WT, gray, clone 58) and Dppa2/4 DKO (DKO, colored, clone 43) cells. Error bars represent mean plus standard deviation of three independent differentiation experiments. RNA samples are the same that were used for RNA sequencing in Fig. 1a, b.

Extended Data Fig. 2 DPPA2/4 bind developmental bivalent promoters, related to Fig. 1.

a, Genome browser screenshot showing DPPA2 (blue), DPPA4 (purple) binding and H3K4me3 (green) and H3K27me3 (red) histone modifications across a 394 kb region of chromosome 1. Input, DPPA2 and DPPA4 data reanalyzed from9 [GSE117173], H3K4me3 and H3K27me3 data from this study. H3K4me3 promoters highlighted in green, bivalent promoters highlighted in yellow, inactive promoters highlighted in red. b, Distribution of high confidence DPPA2 and 4 peaks. Data reanalyzed from9 [GSE117173]. c, Log2 enrichment of DPPA2 (blue) and DPPA4 (purple) binding at gene promoters for different expression bins. H3K4me3 enrichment (green) is shown for comparison. d, Western blot showing levels of overexpression of DPPA2-GFP (top) and DPPA4-GFP (middle) in different stable clonal cell lines. β-ACTIN (bottom) is shown as loading control. Open arrows denote endogenous protein, filled arrows overexpressed GFP-tagged protein. e,f, Scatter plots showing gene expression between ESCs stably overexpressing GFP and DPPA2-GFP e, or DPPA4-GFP f, (two independent cellular clones each harvested in triplicate, separated by at least one passage). Differentially expressed genes (calculated using DESeq p-value <0.05 after Benjamimi and Hochberg correction) are shown in blue, differentially expressed 2C-like ZGA genes are shown in red. Dppa2 and Dppa4 are shown in green. g, Expression of core, naïve, formative and primed pluripotency markers in E14 + GFP (black, grey), E14 + DPPA2-GFP (blue) and E14-DPPA4-GFP (purple) overexpression clones as determined by RNA-sequencing. Two independent cellular clones per overexpression or control line were harvested in triplicate, separated by at least one passage. h, Overlap in differentially expressed genes in DPPA2-GFP (blue) and DPPA4-GFP (purple) stable overexpression clones with 2C-like (left, orange) and bivalent (right, green) genes. i, Expression of 2C-like genes, bivalent genes and proteomics hits in GFP (grey), DPPA2-GFP (blue), DPPA4-GFP (purple) stable overexpressing cells. 2C-like genes from 48, bivalent genes from13. Two independently targeted clones (containing the average of n = 3 replicates harvested independently) are shown per condition in all subpanels. The central line denotes the median, the yellow box the 25th and 75th percentile of the data and the black whiskers the median + /- the interquartile range (25–75%) multiplied by 2. Circles represent single promoters that fall outside this range. j, qPLEX-RIME results showing log2fold change between DPPA2-GFP and GFP control versus log10 (adjusted p-value). Dotted line represents cut off of p < 0.05. DPPA2 and DPPA4 are shown in orange. Top 3 interactors are shown in black. Members of Polycomb (red), COMPASS (green) and SRCAP/INO-80 (blue) complexes are highlighted. k, Western Blotting showing levels of H3K4me3 (top) and H3K27me3 (middle) in different stable cell lines. β-ACTIN is shown as a loading control. l, Endogenous DPPA4 immunoprecipitation (IP) followed by Western Blot in WT (first two columns) and Dppa2/4 DKO (last two columns) cells. * denotes IgG band. m, Aligned probe plots of a random selection of 5000 genes showing enrichment of different chromatin proteins ordered by DPPA4 levels. Input, DPPA2 and DPPA4 ChIP data reanalysed from9 [GSE11717], ASH2L-GFP data from 1 [GSE52071], MLL2 data from2 [GSE48172], EZH2 and SUZ12 data from39 [GSE49435] and KAT5/TIP60 data from30 [GSE69671].

Extended Data Fig. 3 DPPA2/4 are required to maintain bivalent chromatin at a subset of developmental genes, related to Fig. 2.

a, Western blot showing protein levels of chromatin factors and histone modifications between three wild type (WT) ESC clones (left) and three Dppa2/4 DKO ESC cellular clones (right). Histone H3 and β-ACTIN are included as loading controls. b, Scatter plots showing enrichment at gene promoters for H3K4me3, H3K27me3, H2A.Z, RING1B, EZH2 and ASH2L between WT (x-axis) and Dppa2/4 DKO (y-axis) ESCs. Protein enrichment at DPPA2/4-dependent (apricot), -sensitive (green) and -dependent (teal) genes are shown. c, Box-whisker plots showing enrichment of different chromatin proteins, histone modifications and chromatin accessibility measured by ATAC-seq at DPPA2/4-dependent (left, apricot), -sensitive (middle, green) and -independent (right, blue) promoters (TSS + /-1kb) comparing WT (light) and Dppa2/4 DKO (dark) ESCs. Input, DPPA2 and DPPA4 ChIP data from8. All other data from this study (n = 3 cellular clones for H3K4me3 and H3K27me3 (pooled data) and 1 cellular clone for other marks). The central line denotes the median, the yellow box the 25th and 75th percentile of the data and the black whiskers the median + /- the interquartile range (25–75%) multiplied by 2. Circles represent single promoters that fall outside this range. d, ChIP-qPCR results at DPPA2/4-dependent genes Twist2 and Fermt1, compared to control Klf4 gene promoters in 2–3 replicates (see data points, replicates harvested on different days) of one cell clone each of WT (grey) and Dppa2, 4 DKO (blue) cells for H3K4me3, H3K27me3, H2A.Z, EZH2, RING1B and IgG control. Error bars represent mean + standard deviation. Individual data points are shown. Unpaired two-sided t-tests were used and p-values are shown. e, f, Genome browser view of a DPPA2/4-dependent gene (Fermt1) e, and a -dependent (Nphs2) and -independent (Tdrd5) gene f, showing enrichment of chromatin modifications, proteins, chromatin accessibility (blue), DNA methylation (green/grey) and transcription (bottom two panels) for wild type (WT) and Dppa2/4 double knockout (DKO) ESCs. g, Accessibility measured by ATAC-seq at gene promoters (TSS + /-1kb) comparing DPPA2/4-dependent (apricot), -sensitive (green) and -independent (teal) promoters between WT (x-axis) and Dppa2/4 DKO (y-axis) cells.

Extended Data Fig. 4 DPPA2/4-dependent bivalent genes characterized by low H3K4me3, low expression and initiating but not elongating RNA polymerase II, related to Fig. 3.

a, Scatterplot comparing H3K4me3 peak width (x-axis) and expression of associated gene (y-axis) for Dppa2/4-dependent (orange), -sensitive (green) and -independent (blue) genes (as defined in Fig. 2c). H3K4me3 data is from 3 independent cellular clones. b, Enrichment for different RNA polymerase II modifications across gene bodies. Data reanalysed from20 [GSE34520]. Line indicates the median. c, Enrichment of different chromatin modifications and chromatin proteins along with chromatin accessibility (ATAC-seq) between Dppa2/4-dependent (orange), -sensitive (green) and -independent (blue) gene promoters (TSS + /-1kb). DPPA2 and 4 ChIP-seq data reanalysed from9 [GSE117173], all other data from this study. Line indicates the median value. d, proportion of gene promoters (start of gene + /- 1 kb) sequence masked by repeats. Line indicates the median value. e, CpG density, GC content and DNA methylation levels at Dppa2/4-dependent (orange), -sensitive (green) and -independent (blue) gene promoters (TSS + /-1kb). Line indicates the median value.

Extended Data Fig. 5 DPPA2/4-dependent promoters gain DNA methylation and fail to be upregulated during differentiation, related to Fig. 4.

a, Box-whisker plots of the lowest 20% of expressed genes in WT ESCs (total number 3460), showing all 3460 genes, excluding DPPA2/4-dependent and -sensitive genes, only DPPA2/4-dependent genes and only DPPA2/4 sensitive genes during the embryoid body time course between WT (grey) and DKO (blue) cells. Day of differentiation is denoted below each pair of bars. The central line denotes the median, the yellow box the 25th and 75th percentile of the data and the black whiskers the median + /- the interquartile range (25–75%) multiplied by 2. Circles represent single promoters that fall outside this range. Each bar shows pooled data from three differentiation timecourses ran on different days using 1 WT and 1 DKO clone. b, Mass spectrometry quantification of 5-methylcytosine (5mC, left) and 5-hydroxymethylcytosine (5hmC, right) between WT (grey) and Dppa2/4 DKO (blue) cells. Bars represent mean + /- standard deviation of three independent cellular clones. c, Amplicon-bisulfite-seq analysis of DPPA2/4-dependent gene promotors and 2 control regions (Klf4 and Sox2) in three wild type (WT, first three columns) and Dppa2/4 DKO (last 3 columns) clones. Each row represents an individual CpG included within the amplicon. White rows represent break between different amplicon regions (between 1 and 3 per gene promoter analysed). A minimum of 100-fold coverage per CpG was required to include the CpG in the analysis. d, Mass spectrometry quantification of 5-methylcytosine (5mC, left) and 5-hydroxymethylcytosine (5hmC, right) between Dnmt WT (left two columns) and Dnmt TKO (right two columns) cells following control (grey) or Dppa2/4 siRNA (red) treatment for 4 days. Bars represent mean + /- standard deviation of two replicate experiments harvested on different days. e, Western blot showing levels of DNA methylation machinery (DNMT1, DNMT3A and DNMT3B), DPPA2 and DPPA4 between Dnmt WT (left two columns) and Dnmt TKO (right two columns) following treatment with control siRNA or both Dppa2 and Dppa4 siRNA for 4 days. HSP90 is shown as loading control. f, Expression levels of DPPA2/4-dependent (apricot), -sensitive (green) and -independent (blue) genes as defined in Fig. 2c between Dnmt WT and Dnmt TKO cells. Data reanalysed from21 [GSE67867]. Box plots shown are as described in A. (G) Expression of Dppa2 and Dppa4 mRNAs following control (grey) or Dppa2/4 (red) siRNA treatment in WT (left, dark) and Dnmt TKO (right, light) cells. Relative expression is normalised to the level of control siRNA (dark bars) for WT and Dnmt TKO cells. Dots represent replicates experiments harvested on different days and bars averages plus standard deviation.

Extended Data Fig. 6 DPPA2/4 are required to target H3K4me3 and prevent DNA methylation at a subset of bivalent genes, related to Fig. 5.

a, Western blot for H3K4me3 (top), H3K27me3 (middle) and β-ACTIN (bottom) loading control in inducible shRNA experiment for untreated (-dox, first and fourth column), dox treated (+dox, second and fifth column) and recovery (rec, third and sixth column) cells containing inducible shRNA against Dppa2 (left three columns) or Dppa4 (right three columns). H3K4me3 and H3K27me3 bands are quantified relative to β-ACTIN (below). b, Scatterplots of H3K4me3 peaks (top row) and H3K27me3 peaks (bottom row) for cells containing shRNA against Dppa2 (left two columns) or Dppa4 (right column) highlighting those peaks that are significantly up (blue) or down (red) regulated in +dox versus -dox conditions for the given hairpin. Data is from three replicate experiments harvested on different days using one bulk cell population per inducible hairpin. c, overlap of downregulated H3K4me3 (left) and H3K27me3 (right) peaks between shRNA for Dppa2 (orange) and Dppa4 (green). d, genomic features associated with H3K4me3 (left) and H3K27me3 (right) peaks significantly downregulated with both Dppa2 and Dppa4 shRNAs. e, Table showing number of differentially enriched promoters (DESeq2) and number up- and down-regulated promoters in inducible shRNA experiments. f, Amplicon-bisulfite-seq analysis of DPPA2/4-dependent gene promotors and 2 control regions (Klf4 and Sox2) in inducible shRNA experiment for untreated (-dox, first four columns), dox treated (+dox, middle four columns) and recovery (rec, last four columns) cells containing inducible shRNA against Dppa2 (2) or Dppa4 (4). Two replicate experiments harvested on different days are included per condition. Each row represents an individual CpG included within the amplicon. Crossed rows represent break between different amplicon regions (between 1 and 3 per gene promoter analysed) and crossed columns when insufficient coverage was obtained to accurately measure DNA methylation levels. A minimum of 100-fold coverage per CpG was required to include the CpG in the analysis.

Supplementary information

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Supplementary Table 1

List of all genes, chromosome coordinates, strand and average log2 RPM for embryoid body (EB) differentiation experiments. Three independent differentiations were performed (EBset1, EBset2, EBset3) for one wildtype (clone 58) and one Dppa2/4 DKO (clone 43) clone, and samples were collected at days 1–4, 7 and 9 of differentiation.

Supplementary Table 2

List of differentially expressed genes (DESeq2 + intensity difference filter) for DPPA2-GFP and DPPA4-GFP overexpression lines compared to GFP controls. Columns contain gene name, chromosome coordinates, strand information and classification as differentially expressed in DPPA2 overexpressed cells (Dppa2 O/E, blue), DPPA4-GFP overexpressed cells (Dppa4 O/E, purple), or both DPPA2 and DPPA4 overexpressed cells (Dppa2 O/E and Dppa4 O/E). log2 RPM are give for three biological replicates (_1, _2, _3) for two biological clones for GFP (E14 + GFP_c1, E14 + GFP_c2), DPPA2-GFP (E14 + Dppa2_c3, E14 + Dppa2_c5) or DPPA4-GFP (E14 + Dppa4_c5, E14 + Dppa4_c6) overexpressing cells.

Supplementary Table 3

qPLEX-RIME results showing accession, gene name, description, gene symbol, number of unique peptides detected, peptide intensity for each sample (two biological clones per condition with 3–4 technical replicates each), log2 fold change, average intensity, t, P value, adjusted P value and B value for DPPA-GFP versus GFP (first tab) versus DPPA-GFP versus GFP (second tab).

Supplementary Table 4

List of DPPA2/4-dependent (first tab), DPPA2/4-sensitive (second tab) and DPPA2/4-independent (third tab) gene promoters (TSS ± 1 kb) showing chromosome coordinates, and log2 RPM enrichment for Input, DPPA2, DPPA4, H3K4me3, H3K27me3, H2A.Z, RING1B, ASH2L, EZH2 and chromosome accessibility for WT and DKO cells. Individual clonal replicates are shown. Input, DPPA2 and DPPA4 data reanalyzed from ref. 9, all other data from this study.

Supplementary Table 5

List of attributes used in machine learning, together with their description and data source (first tab) and values (second tab).

Supplementary Table 6

List of differentially enriched H3K4me3 (first tab) and H3K27me3 (second tab) peaks in inducible shRNA experiments for either Dppa2 or Dppa4, showing chromosome coordinates, closest gene within 2 kb, distance to closest gene and log2 RPM values for H3K27me3 and H3K4me3 for all replicates for all conditions.

Supplementary Table 7

Amplicon-bisulfite-sequencing results listing mm10 genomic coordinates of CpG analyzed (columns A–D), gene promoter targeted (column E) average DNA methylation value for inducible shRNA samples (columns G–R), wild-type ESC clones (columns S–U) and Dppa2/4 double knockout cells (columns V–X), and read count for inducible shRNA samples (columns Z–AK), wild-type ESC clones (columns AL–AN) and Dppa2/4 double knockout cells (columns AO–AQ). Each row represents an individual CpG. In red text are CpGs that are omitted due to poor coverage (<100 reads) or quality as they are at the ends of the amplicons. Amplicons are separated by thick borders.

Source data

Source Data Fig. 5

Unprocessed western blots associated with Fig. 5b.

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Eckersley-Maslin, M.A., Parry, A., Blotenburg, M. et al. Epigenetic priming by Dppa2 and 4 in pluripotency facilitates multi-lineage commitment. Nat Struct Mol Biol 27, 696–705 (2020). https://doi.org/10.1038/s41594-020-0443-3

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