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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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.
Custom code used to process and analyse the genomic data, as detailed in the Methods, are available upon request.
Braun, R. E. Packaging paternal chromosomes with protamine. Nat. Genet. 28, 10–12 (2001).
Reik, W., Dean, W. & Walter, J. Epigenetic reprogramming in mammalian development. Science 293, 1089–1093 (2001).
Calarco, J. P. et al. Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA. Cell 151, 194–205 (2012).
Borg, M. & Berger, F. Chromatin remodelling during male gametophyte development. Plant J. 83, 177–188 (2015).
Ingouff, M. et al. Zygotic resetting of the HISTONE 3 variant repertoire participates in epigenetic reprogramming in Arabidopsis. Curr. Biol. 20, 2137–2143 (2010).
Reinberg, D. & Vales, L. D. Chromatin domains rich in inheritance. Science 361, 33–34 (2018).
Xu, M. et al. Partitioning of histone H3–H4 tetramers during DNA replication-dependent chromatin assembly. Science 328, 94–98 (2010).
Reverón-Gómez, N. et al. Accurate recycling of parental histones reproduces the histone modification landscape during DNA replication. Mol. Cell 72, 239–249.e5 (2018).
Laprell, F., Finkl, K. & Müller, J. Propagation of Polycomb-repressed chromatin requires sequence-specific recruitment to DNA. Science 356, 85–88 (2017).
Coleman, R. T. & Struhl, G. Causal role for inheritance of H3K27me3 in maintaining the off state of a Drosophila HOX gene. Science 356, eaai8236 (2017).
Jiang, D. & Berger, F. DNA replication-coupled histone modification maintains Polycomb gene silencing in plants. Science 357, 1146–1149 (2017).
Jacob, Y. et al. Selective methylation of histone H3 variant H3.1 regulates heterochromatin replication. Science 343, 1249–1253 (2014).
Grossniklaus, U. & Paro, R. Transcriptional silencing by polycomb-group proteins. Cold Spring Harb. Perspect. Biol. 6, a019331 (2014).
Crevillén, P. et al. Epigenetic reprogramming that prevents transgenerational inheritance of the vernalized state. Nature 515, 587–590 (2014).
Tao, Z. et al. Embryonic epigenetic reprogramming by a pioneer transcription factor in plants. Nature 551, 124–128 (2017).
Sano, Y. & Tanaka, I. Distinct localization of histone H3 methylation in the vegetative nucleus of lily pollen. Cell Biol. Int. 34, 253–259 (2010).
Houben, A., Kumke, K., Nagaki, K. & Hause, G. CENH3 distribution and differential chromatin modifications during pollen development in rye (Secale cereale L.). Chromosome Res. 19, 471–480 (2011).
She, W. & Baroux, C. Chromatin dynamics in pollen mother cells underpin a common scenario at the somatic-to-reproductive fate transition of both the male and female lineages in Arabidopsis. Front. Plant Sci. 6, 294 (2015).
Zhang, K., Sridhar, V. V., Zhu, J., Kapoor, A. & Zhu, J.-K. Distinctive core histone post-translational modification patterns in Arabidopsis thaliana. PLoS ONE 2, e1210 (2007).
Okada, T., Endo, M., Singh, M. B. & Bhalla, P. L. Analysis of the histone H3 gene family in Arabidopsis and identification of the male-gamete-specific variant AtMGH3. Plant J. 44, 557–568 (2005).
Moritz, L. E. & Trievel, R. C. Structure, mechanism, and regulation of Polycomb-repressive complex 2. J. Biol. Chem. 293, 13805–13814 (2018).
Wollmann, H. et al. The histone H3 variant H3.3 regulates gene body DNA methylation in Arabidopsis thaliana. Genome Biol. 18, 94 (2017).
Friedman, W. E. Expression of the cell cycle in sperm of Arabidopsis: implications for understanding patterns of gametogenesis and fertilization in plants and other eukaryotes. Development 126, 1065–1075 (1999).
Lu, F. et al. Comparative analysis of JmjC domain-containing proteins reveals the potential histone demethylases in Arabidopsis and rice. J. Integr. Plant Biol. 50, 886–896 (2008).
Yan, W. et al. Dynamic and spatial restriction of polycomb activity by plant histone demethylases. Nat. Plants 4, 681–689 (2018).
Min, G. L. et al. Demethylation of H3K27 regulates polycomb recruitment and H2A ubiquitination. Science 318, 447–450 (2007).
Lu, F., Cui, X., Zhang, S., Jenuwein, T. & Cao, X. Arabidopsis REF6 is a histone H3 lysine 27 demethylase. Nat. Genet. 43, 715–719 (2011).
Zheng, S. et al. The Arabidopsis H3K27me3 demethylase JUMONJI 13 is a temperature and photoperiod dependent flowering repressor. Nat. Commun. 10, 1303 (2019).
Wu, S.-F., Zhang, H. & Cairns, B. R. Genes for embryo development are packaged in blocks of multivalent chromatin in zebrafish sperm. Genome Res. 21, 578–589 (2011).
Borg, M. et al. The R2R3 MYB transcription factor DUO1 activates a male germline-specific regulon essential for sperm cell differentiation in Arabidopsis. Plant Cell 23, 534–549 (2011).
Gehring, M. & Satyaki, P. R. Endosperm and imprinting, inextricably linked. Plant Physiol. 173, 143–154 (2017).
Moreno-Romero, J., Del Toro-De León, G., Yadav, V. K., Santos-González, J. & Köhler, C. Epigenetic signatures associated with imprinted paternally expressed genes in the Arabidopsis endosperm. Genome Biol. 20, 41 (2019).
Makarevitch, I. et al. Genomic distribution of maize facultative heterochromatin marked by trimethylation of H3K27. Plant Cell 25, 780–793 (2013).
Inoue, A., Jiang, L., Lu, F., Suzuki, T. & Zhang, Y. Maternal H3K27me3 controls DNA methylation-independent imprinting. Nature 547, 419–424 (2017).
Zhao, P. et al. Two-step maternal-to-zygotic transition with two-phase parental genome contributions. Dev. Cell 49, 882–893.e5 (2019).
Bayer, M. et al. Paternal control of embryonic patterning in Arabidopsis thaliana. Science 323, 1485–1488 (2009).
Khanday, I., Skinner, D., Yang, B., Mercier, R. & Sundaresan, V. A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds. Nature 565, 91–95 (2019).
Horstman, A. et al. The BABY BOOM transcription factor activates the LEC1–ABI3–FUS3–LEC2 network to INDUCE somatic embryogenesis. Plant Physiol. 175, 848–857 (2017).
Boscá, S., Knauer, S. & Laux, T. Embryonic development in Arabidopsis thaliana: from the zygote division to the shoot meristem. Front. Plant Sci. 2, 93 (2011).
Vermeulen, M. et al. Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4. Cell 131, 58–69 (2007).
Zhao, P., Begcy, K., Dresselhaus, T. & Sun, M.-X. Does early embryogenesis in eudicots and monocots involve the same mechanism and molecular players? Plant Physiol. 173, 130–142 (2017).
Chen, J. et al. Zygotic genome activation occurs shortly after fertilization in maize. Plant Cell 29, 2106–2125 (2017).
Hammoud, S. S. et al. Distinctive chromatin in human sperm packages genes for embryo development. Nature 460, 473–478 (2009).
Brykczynska, U. et al. Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa. Nat. Struct. Mol. Biol. 17, 679–687 (2010).
Sachs, M. et al. Bivalent chromatin marks developmental regulatory genes in the mouse embryonic germline in vivo. Cell Rep. 3, 1777–1784 (2013).
Zheng, H. et al. Resetting epigenetic memory by reprogramming of histone modifications in mammals. Mol. Cell 63, 1066–1079 (2016).
Murphy, P. J., Wu, S. F., James, C. R., Wike, C. L. & Cairns, B. R. Placeholder nucleosomes underlie germline-to-embryo DNA methylation reprogramming. Cell 172, 993–1006.e13 (2018).
Tabuchi, T. M. et al. Caenorhabditis elegans sperm carry a histone-based epigenetic memory of both spermatogenesis and oogenesis. Nat. Commun. 9, 4310 (2018).
Kaneshiro, K. R., Rechtsteiner, A. & Strome, S. Sperm-inherited H3K27me3 impacts offspring transcription and development in C. elegans. Nat. Commun. 10, 1271 (2019).
Zenk, F. et al. Germ line-inherited H3K27me3 restricts enhancer function during maternal-to-zygotic transition. Science 357, 212–216 (2017).
Maehara, K. et al. Tissue-specific expression of histone H3 variants diversified after species separation. Epigenetics Chromatin 8, 35 (2015).
Wang, D., Tyson, M. D., Jackson, S. S. & Yadegari, R. Partially redundant functions of two SET-domain Polycomb-group proteins in controlling initiation of seed development in Arabidopsis. Proc. Natl. Acad. Sci. USA 103, 13244–13249 (2006).
De Lucas, M. et al. Transcriptional regulation of Arabidopsis Polycomb repressive complex 2 coordinates cell-type proliferation and differentiation. Plant Cell 28, 2616–2631 (2016).
Zhou, Y. et al. Ctf4-related protein recruits LHP1–PRC2 to maintain H3K27me3 levels in dividing cells in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 114, 4833–4838 (2017).
Sun, B. et al. Timing mechanism dependent on cell division is invoked by Polycomb eviction in plant stem cells. Science 343, 1248559 (2014).
Yang, H., Howard, M. & Dean, C. Physical coupling of activation and derepression activities to maintain an active transcriptional state at FLC. Proc. Natl Acad. Sci. USA 113, 9369–9374 (2016).
Kawashima, T. et al. Dynamic F-actin movement is essential for fertilization in Arabidopsis thaliana. eLife 3, e04501 (2014).
Brownfield, L. et al. A plant germline-specific integrator of sperm specification and cell cycle progression. PLoS Genet. 5, e1000430 (2009).
Wang, Z.-P. et al. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 16, 144 (2015).
Twell, D. & Brownfield, L. Analysis of fluorescent reporter activity in the male germline during pollen development by confocal microscopy. in. Methods Mol. Biol. 1669, 67–75 (2017).
Borg, M., Buendía, D. & Berger, F. A simple and robust protocol for immunostaining Arabidopsis pollen nuclei. Plant Reprod. 32, 39–43 (2019).
Galbraith, D. W. et al. Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220, 1049–1051 (1983).
Glöckle, B. et al. Pollen differentiation as well as pollen tube guidance and discharge are independent of the presence of gametes. Development 145, dev152645 (2018).
Jacob, Y. et al. ATXR5 and ATXR6 are H3K27 monomethyltransferases required for chromatin structure and gene silencing. Nat. Struct. Mol. Biol. 16, 763–768 (2009).
Voigt, P. et al. Asymmetrically modified nucleosomes. Cell 151, 181–193 (2012).
Jacob, Y. & Voigt, P. in Plant Chromatin Dynamics. Methods in Molecular Biology Vol 1675 (eds Bemer, M. & Baroux, C.) 345–360 (Humana Press, 2018).
Johnson-Brousseau, S. A. & McCormick, S. A compendium of methods useful for characterizing Arabidopsis pollen mutants and gametophytically-expressed genes. Plant J. 39, 761–775 (2004).
Borges, F. et al. FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei. Plant Methods 8, 44 (2012).
Picelli, S. et al. Full-length RNA-Seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014).
Hamamura, Y. et al. Live-cell imaging reveals the dynamics of two sperm cells during double fertilization in Arabidopsis thaliana. Curr. Biol. 21, 497–502 (2011).
Ikeda, Y. et al. HMG domain containing SSRP1 is required for DNA demethylation and genomic imprinting in Arabidopsis. Dev. Cell 21, 589–596 (2011).
Dobin, A. et al. STAR: ultrafast universal RNA-Seq aligner. Bioinformatics 29, 15–21 (2013).
Bray, N. L., Pimentel, H., Melsted, P. & Pachter, L. Near-optimal probabilistic RNA-Seq quantification. Nat. Biotechnol. 34, 525–527 (2016).
Slotte, T. et al. The Capsella rubella genome and the genomic consequences of rapid mating system evolution. Nat. Genet. 45, 831–835 (2013).
Martínez-Fernández, I. et al. The effect of NGATHA altered activity on auxin signaling pathways within the Arabidopsis gynoecium. Front. Plant Sci. 5, 210 (2014).
Niederhuth, C. E., Patharkar, O. R. & Walker, J. C. Transcriptional profiling of the Arabidopsis abscission mutant hae hsl2 by RNA-Seq. BMC Genomics 14, 37 (2013).
Kang, J. et al. Suppression of photosynthetic gene expression in roots is required for sustained root growth under phosphate deficiency. Plant Physiol. 165, 1156–1170 (2014).
Nozue, K. et al. Shade avoidance components and pathways in adult plants revealed by phenotypic profiling. PLoS Genet. 11, e1004953 (2015).
Dowen, R. H. et al. Widespread dynamic DNA methylation in response to biotic stress. Proc. Natl Acad. Sci. USA 109, E2183–E2191 (2012).
Narsai, R. et al. Extensive transcriptomic and epigenomic remodelling occurs during Arabidopsis thaliana germination. Genome Biol. 18, 172 (2017).
Hofmann, F., Schon, M. A. & Nodine, M. D. The embryonic transcriptome of Arabidopsis thaliana. Plant Reprod. 32, 77–91 (2019).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-Seq data with DESeq2. Genome Biol. 15, 550 (2014).
Brind’Amour, J. et al. An ultra-low-input native ChIP-Seq protocol for genome-wide profiling of rare cell populations. Nat. Commun. 6, 6033 (2015).
Schoft, V. K. et al. SYBR Green-activated sorting of Arabidopsis pollen nuclei based on different DNA/RNA content. Plant Reprod. 28, 61–72 (2015).
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Ramírez, F., Dündar, F., Diehl, S., Grüning, B. A. & Manke, T. deepTools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Res. 42, W187–W191 (2014).
Robinson, J. T. et al. Integrative Genomics Viewer. Nat. Biotechnol. 29, 24–26 (2011).
Baerenfaller, K. et al. Diurnal changes in the histone H3 signature H3K9ac|H3K27ac|H3S28p are associated with diurnal gene expression in Arabidopsis. Plant. Cell Environ. 39, 2557–2569 (2016).
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
Gu, Z., Eils, R., Schlesner, M. & Ishaque, N. EnrichedHeatmap: an R/Bioconductor package for comprehensive visualization of genomic signal associations. BMC Genomics 19, 234 (2018).
Daley, T. & Smith, A. D. Predicting the molecular complexity of sequencing libraries. Nat. Methods 10, 325–327 (2013).
Zhu, L. J. et al. ChIPpeakAnno: a Bioconductor package to annotate ChIP-seq and ChIP-chip data. BMC Bioinformatics 11, 237 (2010).
Reimand, J., Arak, T. & Vilo, J. g:Profiler—a web server for functional interpretation of gene lists (2011 update). Nucleic Acids Res. 39, W307–W315 (2011).
Belmonte, M. F. et al. Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed. Proc. Natl Acad. Sci. USA 110, E435–E444 (2013).
Schon, M. A. & Nodine, M. D. Widespread contamination of Arabidopsis embryo and endosperm transcriptome data sets. Plant Cell 29, 608–617 (2017).
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).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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
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.
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.
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 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