Targeted reprogramming of H3K27me3 resets epigenetic memory in plant paternal chromatin

Abstract

Epigenetic marks are reprogrammed in the gametes to reset genomic potential in the next generation. In mammals, paternal chromatin is extensively reprogrammed through the global erasure of DNA methylation and the exchange of histones with protamines1,2. Precisely how the paternal epigenome is reprogrammed in flowering plants has remained unclear since DNA is not demethylated and histones are retained in sperm3,4. Here, we describe a multi-layered mechanism by which H3K27me3 is globally lost from histone-based sperm chromatin in Arabidopsis. This mechanism involves the silencing of H3K27me3 writers, activity of H3K27me3 erasers and deposition of a sperm-specific histone, H3.10 (ref. 5), which we show is immune to lysine 27 methylation. The loss of H3K27me3 facilitates the transcription of genes essential for spermatogenesis and pre-configures sperm with a chromatin state that forecasts gene expression in the next generation. Thus, plants have evolved a specific mechanism to simultaneously differentiate male gametes and reprogram the paternal epigenome.

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Fig. 1: H3K27me3 marks are globally lost from Arabidopsis sperm chromatin.
Fig. 2: Sperm-specific histone H3.10 is immune to K27 methylation.
Fig. 3: H3.10 deposition in sperm correlates with the loss of H3K27me3.
Fig. 4: Paternal resetting of H3K27me3 facilitates sperm specification.
Fig. 5: Sperm chromatin state forecasts gene expression in the next generation.

Data availability

Deep-sequencing data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession code GSE120669. Egg cell transcriptomic data have been deposited at the DNA Data Bank of Japan (BioProject; PRJDB8211). Previously published RNA-Seq35,74,75,76,77,78,79,80,81 and ChIP-Seq11,89 datasets re-analysed in this study are detailed in Supplementary Table 6. Source data for Figs. 1, 2, 4 and 5 and Extended Data Figs. 2, 4 and 5 are presented with the paper. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

Code availability

Custom code used to process and analyse the genomic data, as detailed in the Methods, are available upon request.

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Acknowledgements

We thank P. Andersen and J. M. Watson for critical reading of the manuscript, Z. Lorkovic and S. Akimcheva for guidance and technical support, T. Suzuki for sequencing the egg cell transcriptome, and Life Science Editors for editing services. We also thank the Vienna BioCenter Core Facilities for Next Generation Sequencing, Plant Science, HistoPathology, the IMP/IMBA BioOptics Facility and the MENDEL High-Performance Computing team. This work was supported through core funding from the Gregor Mendel Institute, and external grants from the FWF (P 26887 and I 4258) and ERA-CAPS (EVO-REPRO I 2163). M.B. was supported through an FWF Lise Meitner fellowship (M 1818). Y.J., C.L. and R.M. were supported by the Howard Hughes Medical Institute and NIH funding (R01 GM067014). D.S. and T.H. were supported by the Japan Society for the Promotion of Science (18J01963 to D.S. and 16H06464, 16H06465 and 16K21727 to T.H.). P.V. was supported by the Wellcome Trust (104175/Z/14/Z; Sir Henry Dale Fellowship), ERC EU Horizon 2020 research and innovation programme (ERC-STG grant agreement 639253) and core funding from the Wellcome Trust (203149).

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Contributions

M.B. and F.B. conceived of the project. M.B. performed the main experimental work. Y.J., C.L. and P.V. performed the histone methyltransferase assays under the supervision of R.M. D.B. helped with the immunostaining. T.K., E.A., L.B. and J.B. generated sperm cell RNA-Seq data. D.S. generated the egg cell transcriptome under the supervision of T.H. M.B. performed the bioinformatics analysis. M.B. and F.B. interpreted the data and wrote the manuscript.

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Correspondence to Frédéric Berger.

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Extended data

Extended Data Fig. 1 Dynamics of histone H3.1 and H3.3 during pollen development.

Expression of H3.1 a–d, and H3.3 e, f, isoforms during pollen development. Histone H3.1 is encoded by five isoforms: HTR1 (a), HTR2 (b), HTR3 (c), HTR9 and HTR13 (d). Histone H3.3 is encoded by three isoforms: HTR4, HTR5 (e) and HTR8 (f). Two pairs of genes (HTR4-HTR5 and HTR9-HTR13) are found in tandem at the same locus so a single reporter for each pair was used to monitor expression. Histone H3.1 (HTR2, HTR3 and HTR13) were detectable in the microspore and sperm precursor but this signal disappeared rapidly before sperm mitosis. No pollen expression was detected for HTR1. Histone H3.3 (HTR5 and HTR8) were detected throughout pollen development but had a much reduced (HTR5) or absent (HTR8) signal in sperm. Arrows indicate expression in the microspore or VN while arrowheads distinguish expression in the sperm lineage. The marker line analysis was repeated twice with independent inflorescences. Scale, 5 µm.

Extended Data Fig. 2 Specificity of anti-H3K27 methylation antibodies used in this study.

a, Peptide sequences of H3.1, H3.3 and H3.10 surrounding K27 used for testing antibody specificity. Different forms with no methylation (me0), mono-methylation (me1), di-methylation (me2), and tri-methylation (me3) at K27 were used in all dot blots. b, c, Dot blots with serial dilutions of the different forms of histone H3 peptides described in a. The resulting membranes were probed with (b) α-H3K27me1 from Millipore #07-448 and (c) α-H3K27me3 from Millipore #07-449. Importantly, both α-H3K27 methylation antibodies cross react with the correct methylated form of H3.10 peptides, confirming that a lack of H3K27me3 detection in sperm chromatin (Fig. 1c,f) or on ectopically expressed H3.10-3xHA (Fig. 2e) is not due to poor antibody affinity. The experiment was repeated twice on two independent blots. e, Representative image of T1 htr4;htr5;htr8 plants expressing either untagged H3.10 under control of an H3.3 promoter (left) or endogenous H3.3 (right). Plants devoid of endogenous H3.3 and expressing only H3.10 and H3.1 (left) were developmentally stunted and completely sterile. This was evident in two independent experiments with individual htr4;htr5;htr8 T1 lines. Raw blots are provided in Source Data Extended Data Fig. 2. Source data

Extended Data Fig. 3 Epigenomic profiling of Arabidopsis sperm chromatin.

a, Pearson correlation matrix of the ChIP-seq datasets generated in this study. Each ChIP-seq replicate is indicated in the matrix, which was performed with three biological replicates; two for H3K27me1 and H3K27me3. b, Distribution of repressive (left panel) and active marks (right panel) over Arabidopsis chromosome one. Plotted is the ChIP-seq log2 enrichment of immunoprecipitated (IP) DNA relative to input calculated in 10kb bins. Pericentromeric heterochromatin is indicated with grey shading. c, Genome browser view of the sperm ChIP-seq datasets. Coverage is represented as the log2 ratio of IP DNA relative to input. Coloured and grey shading indicate an enriched or depleted signal, respectively. Genes (light grey) and transposable elements (dark grey) are shown below. d, Distribution of sperm histone marks over transposable elements. Plotted is the ChIP-seq log2 enrichment relative to input. e, Distribution of sperm histone marks over genes sorted by expression level in sperm. f, Genomic distribution of histone mark peaks in sperm. As expected, H3K27ac and H3K4me3 peaks were mostly enriched over the 5′UTR of genes. H3K27me1 and retained H3K27me3 peaks were mostly enriched over exons, while H3K27me1 peaks were also enriched in intergenic regions. g, Overlap of the retained sperm H3K27me3 peaks with somatic H3K27me3 domains. Statistical analysis is based on a one-sided permutation overlap test (n = 100 permutations) compared with random genomic regions. h, Estimated library complexity curves confirmed a sufficient sequencing depth for two independent biological replicates of sperm H3K27me3. The red curve represents the interpolated and extrapolated increase in complexity (that is distinct reads) with increased sequencing depth. The grey shading represents the upper and lower 95% confidence interval of the extrapolation. The dashed grey line represents the final sequencing depth of each sample. i, Plot of the pairwise correlation between sperm H3K27me3 biological replicates, which showed high reproducibility. Pearson’s correlation coefficient is shown.

Extended Data Fig. 4 Dynamics of the Polycomb machinery during sperm development.

a–f, Expression of MEA-YFP (a), CFP-CLF (b), SWN-GFP (c), LHP1-YFP (d), EMF2-GFP (e) and FIE-VENUS (f) during pollen development. All markers were absent from sperm at mature pollen stage. FIE had an appreciable signal in the sperm precursor but was excluded from the nucleus. Arrows indicate expression in the microspore or VN while arrowheads distinguish expression in the sperm lineage. Marker line analysis was repeated twice with independent inflorescences. Scale, 5 µm. g, Expression of Arabidopsis PRC2 (top panel) and PRC1 (bottom panel) subunits. Expression represents the inverse hyperbolic sine (asinh) transform of the mean RNA-seq TPM values obtained from previously published datasets detailed in Supplementary Table 6. Sperm and eggs were profiled with three and four biological replicates, respectively. h, Ectopic expression of SWN-Clover under control of the sperm lineage-specific DUO1 promoter. Predicted insertions were estimated from T2 segregation of RFP fluorescent seeds arising from the pAlligatorR43 selection marker. Expression of SWN-GFP in T1 lines was barely detectable in pollen and well below that predicted from the T2 segregation data. i, Schematic of the action of JMJ proteins, which can demethylate H3K27 di- and tri-methylation but not mono-methylation. Statistical source data are provided in Source Data Extended Data Fig. 4. Source data

Extended Data Fig. 5 Transcriptional profiling of htr10, elf6;ref6;jmj13 and elf6;ref6;jmj13;htr10 pollen.

a, Principal component analysis illustrating the high reproducibility of replicates and variation among the RNA-seq datasets generated from WT (n = 3 replicates), htr10 (n = 4 replicates), elf6;ref6;jmj13 (n = 3 replicates) and elf6;ref6;jmj13;htr10 (n = 3 replicates) pollen. All the biological replicates indicated (n) were used in the analysis that follows in panels b,c,d of this figure. b,c, Expression of (a) Arabidopsis histone H4 variants and (b) H3K27 demethylases in WT, elf6;ref6;jmj13, htr10 and elf6;ref6;jmj13;htr10 pollen. Expression represents the inverse hyperbolic sine (asinh) transform of the mean RNA-seq TPM values. The mean value of the biological replicates in a is shown, while the asterisks (*) indicate significantly different expression relative to WT pollen (p < 0.001) using DESeq differential expression analysis and Benjamin-Hochberg correction to control for multiple comparisons. See source data for p-values. d, Volcano plots summarising significantly (adjusted p-value < 0.1) up-regulated (log2 FC > 0, red) and down-regulated (log2 FC < 0, blue) genes in htr10, elf6;ref6;jmj13 and elf6;ref6;jmj13;htr10 pollen relative to WT. DESeq analysis was used to determine differentially expressed genes from the biological replicates detailed in a and multiple comparisons controlled for using Benjamin-Hochberg correction. See Supplementary Table 3. e, Differentially-expressed genes (DEGs) in htr10 (n = 73) and elf6;ref6;jmj13 (n = 194) significantly overlap each other. Significance of the enriched overlap (p-value) was determined using a two-sided Fisher’s exact test. f, Clustered heatmap displaying enriched gene ontology (GO) terms associated with the DEGs in htr10 (n = 73), elf6;ref6;jmj13 (n = 194) and elf6;ref6;jmj13;htr10 (n = 468) pollen relative to WT. Significant enrichment was assessed using g:Profiler and controlled for the multiple testing problem using the in-built g:SCS (sets counts and sizes) correction. Source data

Extended Data Fig. 6 Sperm-specific accumulation of H3K4me3 is enriched at somatic H3K27me3 domains.

a, Heatmaps centred on H3K4me3 peaks in sperm and leaf. Regions are split based on peaks being sperm-specific, leaf-specific or common to both sperm and leaf. The number of peaks and relative percentage are indicated in the labels to the left. Plotted is the ChIP-seq log2 ratio relative to input or H3 for sperm and leaf, respectively. ChIP-seq was performed with three biological replicates for sperm; four for leaf. b, Expression of the Arabidopsis SET-domain family of proteins. Expression represents the inverse hyperbolic sine (asinh) transform of the mean RNA-seq TPM values obtained from previously published datasets detailed in Supplementary Table 6. Sperm and egg were profiled with three and four biological replicates, respectively. c, Overlap of somatic H3K27me3 domains with sperm-specific H3K4me3 peaks. Statistical analysis is based on a one-sided permutation overlap test (n = 100 permutations) compared with random genomic regions.

Extended Data Fig. 7 Reprogramming of Polycomb-silenced genes in sperm.

a, Heatmap illustrating the developmentally regulated expression of somatic H3K27me3-marked genes. Expression represents z-score normalised RNA-seq TPM values. b, Heatmap of the expression of the genes marked in Figure 4a. Expression represents the inverse hyperbolic sine (asinh) transform of RNA-seq TPM values. c, Averaged DNA methylation signal over MEGs and PEGs in sperm. Plotted is the proportion of methylated cystosines in all contexts (that is CG, CHG and CHH). d, Averaged H3K4me3 signal over PEGs with detectable expression (TPM>1, black line) or no expression (TPM<1, grey line) in sperm. Plotted is the ChIP-seq log2 enrichment relative to input. PEGs accumulate H3K4me3 regardless of expression in sperm, although the level of H3K4me3 enrichment was expectedly higher at sperm-expressed PEGs.

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1: Somatic H3K27me3-silenced genes reprogrammed in sperm. This table provides all of the genes marked by H3K27me3 in somatic tissues (seedlings and leaf) used in this study. RNA-Seq expression data (TPM values) are provided for multiple tissues, along with the reprogrammed cluster each gene belongs to, as defined in Fig. 4a. The table is ordered by AGI code. Supplementary Table 2: Sperm-enriched genes defined in this study. This table provides all of the genes with sperm-enriched expression used in this study. RNA-Seq expression data (TPM values for sperm, seedlings, roots, leaf and dry seeds) are provided for reference. The table is ordered by highest to lowest expression in sperm. Supplementary Table 3: RNA-seq analysis of pollen compromised of H3K27 demethylase activity and H3.10 deposition. Differential gene expression analysis of htr10 (n = 4 replicates), elf6;ref6;jmj13 (n = 3 replicates) and elf6;ref6;jmj13;htr10 (n = 3 replicates) mutant pollen relative to WT (n = 3 replicates). DESeq analysis was used to determine differentially expressed genes from the biological replicates and multiple comparisons controlled for using Benjamin-Hochberg correction. The table is ordered by AGI code. Supplementary Table 4: PEGs and MEGs used in this study. This table provides a stringent list of PEGs and MEGs used in this study and details whether each gene is marked by H3K27me3 in somatic tissues. The table is ordered by AGI code. Supplementary Table 5. Gene ontology terms enriched in reprogrammed H3K27me3 target gene clusters. Significant enrichment was assessed using g:Profiler and controlled for multiple testing using the in-built g:SCS (sets counts and sizes) correction. The number of genes considered were n = 1,866 (cluster 1), 2,220 (cluster 2) and 3,105 (cluster 3). Supplementary Table 6: Publicly available genomic datasets used in this study. This table details the GEO accession codes for all published ChIP-seq and RNA-seq datasets re-analysed in this study.

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Borg, M., Jacob, Y., Susaki, D. et al. Targeted reprogramming of H3K27me3 resets epigenetic memory in plant paternal chromatin. Nat Cell Biol (2020). https://doi.org/10.1038/s41556-020-0515-y

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