1. Chromosomes and Gene Expression
  2. Plant Biology

A new role for histone demethylases in the maintenance of plant genome integrity

  1. Javier Antunez-Sanchez
  2. Matthew Naish
  3. Juan Sebastian Ramirez-Prado
  4. Sho Ohno
  5. Ying Huang
  6. Alexander Dawson
  7. Korawit Opassathian
  8. Deborah Manza-Mianza
  9. Federico Ariel
  10. Cecile Raynaud
  11. Anjar Wibowo
  12. Josquin Daron
  13. Minako Ueda
  14. David Latrasse
  15. R Keith Slotkin
  16. Detlef Weigel
  17. Moussa Benhamed  Is a corresponding author
  18. Jose Gutierrez-Marcos  Is a corresponding author
  1. School of Life Science, University of Warwick, United Kingdom
  2. Université Paris-Saclay, CNRS, INRAE, Univ Evry, Institute of Plant Sciences Paris-Saclay (IPS2), France
  3. Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Japan
  4. Department of Molecular Biology, Max Planck Institute for Developmental Biology, Germany
  5. Department of Molecular Genetics, The Ohio State University, United States
  6. Institute of Transformative Bio-Molecules, Nagoya University, Japan
  7. Division of Biological Science, Graduate School of Science, Nagoya University, Japan
  8. Donald Danforth Plant Science Center, United States
  9. Division of Biological Sciences, University of Missouri, United States
  10. Université de Paris, Institute of Plant Sciences Paris-Saclay (IPS2), F-75006, France
https://doi.org/10.7554/eLife.58533
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Cite this article
  1. Javier Antunez-Sanchez
  2. Matthew Naish
  3. Juan Sebastian Ramirez-Prado
  4. Sho Ohno
  5. Ying Huang
  6. Alexander Dawson
  7. Korawit Opassathian
  8. Deborah Manza-Mianza
  9. Federico Ariel
  10. Cecile Raynaud
  11. Anjar Wibowo
  12. Josquin Daron
  13. Minako Ueda
  14. David Latrasse
  15. R Keith Slotkin
  16. Detlef Weigel
  17. Moussa Benhamed
  18. Jose Gutierrez-Marcos
(2020)
A new role for histone demethylases in the maintenance of plant genome integrity
eLife 9:e58533.
https://doi.org/10.7554/eLife.58533
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Abstract

Histone modifications deposited by the Polycomb repressive complex 2 (PRC2) play a critical role in the control of growth, development, and adaptation to environmental fluctuations of most multicellular eukaryotes. The catalytic activity of PRC2 is counteracted by Jumonji-type (JMJ) histone demethylases, which shapes the genomic distribution of H3K27me3. Here, we show that two JMJ histone demethylases in Arabidopsis, EARLY FLOWERING 6 (ELF6) and RELATIVE OF EARLY FLOWERING 6 (REF6), play distinct roles in H3K27me3 and H3K27me1 homeostasis. We show that failure to reset these chromatin marks during sexual reproduction results in the transgenerational inheritance of histone marks, which cause a loss of DNA methylation at heterochromatic loci and transposon activation. Thus, Jumonji-type histone demethylases play a dual role in plants by helping to maintain transcriptional states through development and safeguard genome integrity during sexual reproduction.

Introduction

In eukaryotes, chromatin accessibility is modified by DNA methylation, the covalent modification of histone proteins and the deposition of histone variants. These epigenetic modifications allow the establishment of specific transcriptional states in response to environmental or developmental cues. While in most cases environmentally-induced chromatin changes are transient, epigenetic changes induced during development are often stably inherited through mitotic divisions, so that cell identity is maintained and individual cells or tissues do not revert to previous developmental states. A key chromatin modification implicated in these responses is the post-translational modification of histone tails, which are associated with active or inactive transcriptional states. Among these, the methylation of lysine 9 of histone H3 (H3K9me2) and H3K27me1 have been associated with the repression of transposable elements (TEs) in constitutive heterochromatin (Lindroth et al., 2004; Lippman et al., 2004; Mathieu et al., 2005), whereas other types of methylation, including H3K27me3, have been associated with the repression of genes in euchromatic genome regions (Berger, 2007; Pfluger and Wagner, 2007). H3K27me3 methylation is deposited by PRC2 and plays a crucial role in the development of most multicellular eukaryotes (Laugesen et al., 2019). In plants, this modification is found in approximately one quarter of protein-coding genes and is dynamically regulated during growth and development (Lafos et al., 2011; Roudier et al., 2011; Zhang et al., 2007). The activity of PRCs is counterbalanced by JMJ demethylases, which catalyze the specific removal of H3K27me3 (Liu et al., 2010). In Arabidopsis, five histone demethylases [RELATIVE OF EARLY FLOWERING 6 (REF6); EARLY FLOWERING 6 (ELF6); JUMONJI 13 (JMJ13); JUMONJI 30 (JMJ30); and JUMONJI 32 (JMJ32)] have been implicated in the demethylation of H3K27 (Crevillén et al., 2014; Gan et al., 2014; Lu et al., 2011). These proteins are thought to mediate the temporal and spatial de-repression of genes necessary for a wide range of plant processes such as flowering, hormone signaling, and control of the circadian clock. Inactivation of REF6 results in the ectopic accumulation of H3K27me3 at hundreds of loci, many of them involved in developmental patterning and environmental responses (Lu et al., 2011; Yan et al., 2018). It has been proposed that REF6 is recruited to a specific sequence motif through its zinc-finger domain (Cui et al., 2016; Lu et al., 2011); however, others have shown that it is also recruited by specific interactions with transcription factors (Yan et al., 2018). Moreover, it has been shown that the affinity of REF6 to chromatin is hindered by DNA methylation, which could explain why its activity is primarily found at euchromatic loci (Qiu et al., 2019).

Previous studies have suggested that REF6 acts redundantly with ELF6 and JMJ13 to restrict the accumulation of H3K27me3 in gene regulatory regions, thereby unlocking tissue-specific expression (Yan et al., 2018). Importantly, REF6, ELF6, JMJ30 and JMJ32 appear to specifically remove methyl groups from H3K27me3 and H3K27me2 but not from H3K27me1 (Crevillén et al., 2014; Gan et al., 2014; Lu et al., 2011). Previous investigations have shown that H3K27me1 in Arabidopsis is associated with constitutive heterochromatin, where it is deposited by ARABIDOPSIS TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATRX6 (Jacob et al., 2009; Jacob et al., 2010). However, several studies in mammals and plants have shown that H3K27me1 is also found in euchromatin (Fuchs et al., 2008; Jacob et al., 2009; Vakoc et al., 2006). The presence of H3K27me3 in euchromatin is thought to be actively re-set during sexual reproduction – a view supported by studies in Arabidopsis showing that ELF6, REF6 and JMJ13 are necessary to reset and prevent the inheritance of this epigenetic mark to the offspring (Crevillén et al., 2014; Liu et al., 2019; Zheng et al., 2019). However, the extent to which these epigenetic imprints are reset during sexual reproduction remains unknown.

Here, we show that the histone demethylases REF6 and ELF6 play distinct roles in the demethylation of histones in Arabidopsis, and that REF6 plays a major role in H3K27me1 dynamics in active chromatin. We also found that failure to reset H3K27me3 marks during sexual reproduction results in the inheritance of these epigenetic imprints even in the presence of fully functional histone demethylases. The ectopic inheritance of H3K27me3 is associated with the loss of DNA methylation at heterochromatic loci, leading to activation of TEs. Moreover, genetic and epigenetic mutations arising in histone demethylase mutants are stably inherited over multiple generations and result in pleiotropic developmental defects. Collectively, our work has uncovered a hitherto unrecognized role for histone demethylases in maintaining genetic and epigenetic stability of plant genomes.

Results

Arabidopsis REF6 and ELF6 play distinct roles in H3K27me3 homeostasis

The deposition of H3K27me3 by PRCs correlates with transcriptional repression in plants and animals. The dynamic regulation of this epigenetic mark enables the reactivation of genes primarily implicated in developmental programs; thus, any disruption to these regulatory networks results in major developmental aberrations (Kassis et al., 2017; Lewis, 1978; Molitor et al., 2016). The demethylation of H3K27me3 has been linked to the enzymatic activity of five JMJ-type proteins, which act antagonistically to SET-domain histone methyltransferases from the PRC2 complex (Yan et al., 2018). To gain further knowledge about these processes in Arabidopsis, we investigated the function of two sequence-related histone demethylases, ELF6 and REF6. To aid this analysis, we isolated a loss-of-function T-DNA insertion of REF6 (ref6-5) and a targeted CRISPR/Cas9 deletion in the first exon of ELF6 (elf6-C) (See Materials and methods and Figure 1—figure supplement 1). Similar to previous reports, we found that our elf6-C plants displayed an early flowering phenotype characterized by a reduced number of rosette leaves at bolting (Jeong et al., 2009; Noh et al., 2004). Conversely, ref6-5 plants displayed a late flowering phenotype and an increased number of rosette leaves at bolting stage (Figure 1A and Figure 1—figure supplement 2A–B). By manual crossing, we generated elf6-C/ref6-5 double mutant plants, which displayed pleiotropic growth phenotypes, including increased number of petals and pleiotropic defects in leaf morphology, such as serrations and downward curling (Figure 1A and Figure 1—figure supplement 2C–D). These phenotypes were akin to those recently reported for independently generated double mutants of the same histone demethylases (Yan et al., 2018), and their stability was confirmed by generating double mutants using different mutant allele combinations (Figure 1—figure supplement 2E). In all combinations tested, double mutant plants displayed a reduction in silique length (Figure 1B and Figure 1—figure supplement 2E), thus suggesting that these mutations redundantly affect plant fertility. Microscopy analysis of developing seeds revealed that while embryo development in elf6-C was normal, seeds from ref6-5 and elf6-C/ref6-5 contained embryos with patterning defects (Figure 1B). However, these embryonic abnormalities did not affect seed germination rates (Figure 1—figure supplement 2F).

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Arabidopsis Histone demethylases ELF6 and REF6 play distinct roles in development and H3K27me3 homeostasis.

(A) Arabidopsis wild-type (WT) and histone demethylase mutants (elf6-C, ref6-5 and elf6-C/ref6-5). Scale bars, 1 cm. (B) Siliques and embryos from Arabidopsis wild-type (WT) and different mutant alleles of histone demethylase ELF6 and REF6. Numbers show the frequency of the abnormal embryos (n = 250). Scale bars 1 cm and 10 μm, respectively. (C) Venn diagram showing the overlap between genes accumulating H3K27me3 in wild-type (WT) and histone demethylase mutants (elf6-C, ref6-5 and elf6-C/ref6-5). (D) Genome browser views of background subtracted ChIP-seq signals as normalized reads per genomic content (RPGC). Shaded red boxes, genes targeted exclusively by REF6. Shaded grey boxes, genes targeted by REF6 and ELF6. Shaded purple boxes, genes targeted by both REF6 and ELF6, and only hyper-methylated in double mutant elf6-C/ref6-5. (E) Venn diagram showing overlap between differentially expressed genes (DEGs) and H3K27me3 differentially methylated genes in histone demethylase mutants. To the left metaplot for H3K27me3 levels for genes both up-regulated and hypo-methylated and to the right metaplot of H3K27me3 levels in genes both down-regulated and hyper-methylated. Top panel, ref6-5; Bottom panel, elf6-C. p-values for Fisher’s exact test are shown in brackets, N.S. Not Significant. 

REF6 is thought to act as a H3K27me3 demethylase and a positive regulator of gene expression (Hou et al., 2014; Li et al., 2016; Lu et al., 2011; Wang et al., 2019), while the role of ELF6 remains poorly understood. To shed light on the function of these two proteins, we analyzed the distribution of H3K27me3 in elf6-C, ref6-5 and elf6-C/ref6-5 seedlings through a ChIP-seq assays and compared them to that in wild-type plants. Overall, the accumulation of H3K27me3 within genes was more pronounced in ref6-5 than in elf6-C (Figure 1C). Most of the hyper-methylated genes found in elf6 (75%) were hyper-methylated to a greater extent in both ref6-5 and elf6-C/ref6-5, suggesting that these histone demethylases have partially overlapping yet distinct roles in the control of H3K27me3 homeostasis in Arabidopsis (Figure 1D and Figure 1—figure supplements 3–4). In order to further understand the role of ELF6 and REF6 in transcriptional regulation, we performed an RNA-seq analysis. When combining transcriptomic and H3K27me3 ChIP-seq data, we found a strong correlation primarily between genes that were both hyper-methylated at H3K27me3 and down-regulated, thus indicating that this epigenetic mark contributes to their transcriptional repression (Figure 1E and Figure 1—figure supplements 5–6). We also found genes that were hypo-methylated and up-regulated, which could be linked to the global transcriptional deregulation observed in these mutants. Taken together, our data point to the essential, yet distinct, roles of REF6 and ELF6 in H3K27me3 homeostasis at genic regions of the Arabidopsis genome.

REF6 controls H3K27me1 homeostasis in euchromatin

Biochemical analyses have revealed that REF6 can remove both tri- and di-methyl groups but not mono-methyl groups at lysine 27 on histone 3 (Lu et al., 2011). We therefore hypothesized that, in addition to controlling H3K27me3 homeostasis, REF6 and ELF6 may be also implicated in H3K27me1 homeostasis. To test this hypothesis, we determined the distribution of H3K27me1 through ChIP-seq assays and found that most of the genes targeted by REF6 accumulate high levels of H3K27me1 in wild-type (Figure 2A–C and Figure 2—figure supplement 1). Because the deposition of H3K27me1 in Arabidopsis is thought to be mediated by ATXR5 and 6 (Jacob et al., 2009; Jacob et al., 2010), we determined the genomic distribution of H3K27me1 in the hypomorphic atxr5/atxr6 mutant. As previously described, large-scale H3K27me1 accumulation in these mutants was significantly reduced at pericentromeric heterochromatin (Figure 2—figure supplement 2). However, we noticed that in atxr5/atxr6 lines the levels of this histone mark increased in gene-rich regions, pointing to the existence of an alternative pathway for H3K27me1 deposition in euchromatin. These data led us to postulate that the maintenance of H3K27me1 at euchromatin could be mediated by REF6. We therefore investigated the relationship between H3K27me1 and H3K27me3 at genes targeted by REF6. We found that the loss of REF6 activity results in both the accumulation of H3K27me3 and a drastic reduction in H3K27me1 at those loci, while the loss of ELF6 did not have an effect (Figure 2B). We next assessed genomic regions directly targeted by REF6 (Cui et al., 2016; Li et al., 2016) and found that the accumulation of H3K27me3 in ref6-5 was associated with a complete loss of H3K27me1 (Figure 2C). Taken together these data revealed that the maintenance of H3K27me1 in euchromatin is dependent on REF6. To test if the H3K27me1 present in REF6 binding sites is produced by the sequential methylation by PRC2 and partial demethylation by REF6, we performed ChIP-seq analyses for H3K27me1 in the PRC2 methyltransferase double mutant clf/swn. We found a strong reduction of H3K27me1 in these mutants at REF6 binding sites (Figure 2C), thus confirming that H3K27me1 at these euchromatic sites is PRC2-dependant.

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Arabidopsis REF6 plays an essential role in the deposition of H3K27me1 in active chromatin.

(A) Genome browser views of background subtracted ChIP-seq signals for H3K27me3 and H3K27me1 as normalized reads per genomic content (RPGC) in wild-type (WT) and histone demethylase mutants (elf6-C, ref6-5 and elf6-C/ref6-5). Shaded boxes, genes targeted exclusively by REF6. (B) Violin plots showing the distribution of H3K27me3 and H3K27me1 on genes targeted by REF6. Genes were categorised as targeted if a H3K27me3 peak was annotated on them in ref6-5 and in elf6-C/ref6-5 but not in WT. (C) Heatmap showing the distribution of H3K27me3 and H3K27me1 on genomic sequences targeted by REF6 for wild-type (WT), ref6-5, and clf/swn plants. Sample size n = 3385. (D) Bar charts showing the number of genes for different expression quantiles predicted to be targeted by PRC2 and REF6. (E) Heatmap showing the distribution of H3K27me3 and H3K27me1 present on genes corresponding to low-expression (1-5) quantiles. .

Although it is well known that H3K27me1 in Arabidopsis contributes to the repression of heterochromatic TEs, its role in euchromatin remains unknown. To address this gap in our knowledge, we examined the relationship between REF6-dependent H3K27me1 deposition and transcription. To aid this analysis, we divided the transcriptome into 10 equal deciles according to their transcriptional state (Figure 2—figure supplement 3). We found that while H3K27me3 was primarily associated with strongly repressed genes in wild-type (first three quantiles), in ref6-5, the ectopic accumulation of H3K27me3 primarily affected genes that displayed low levels of expression (second to fifth quantiles) (Figure 2D and Figure 2—figure supplement 4). Moreover, we found that the activity of REF6 was required for low-level expression genes (third to fifth quantiles) (Figure 2E and Figure 2—figure supplement 5). Collectively, these data support the view that REF6 contributes to gene activation, by the removal of PRC2-dependent repressive marks, and to the maintenance of low-level basal expression, by maintaining H3K27me1 in transcriptionally active chromatin.

Inheritance of ectopic H3K27me3 imprints alters the epigenome

It has been shown in Arabidopsis that histone demethylases are critical for the resetting of H3K27me3 across generations (Crevillén et al., 2014; Gan et al., 2014; Liu et al., 2019; Zheng et al., 2019). To understand the biological significance of this epigenetic resetting thought to take place during plant sexual reproduction, we generated reciprocal crosses between single and double histone demethylase mutants and wild-type plants. We found that F1 plants from these crosses were indistinguishable from wild-type, however some F2 plants displayed unexpected developmental phenotypes that were not present in either single or double mutants (4.65% paternal and 4.42% maternal transmission, n = 1500 each) (Figure 3A). On the other hand, the frequency of plants displaying developmental abnormalities in the F2, resulting from F1 hybrids between wild-type and single histone demethylase mutants, was markedly lower (0.58% for elf6-C and 0.63% for ref6-5; n > 300 each). Intriguingly, abnormal plants arising from the elf6-C/+;ref6-5/+ hybrids segregated for the different mutant allele combinations (Figure 3A), thus indicating that these novel phenotypes are not genetically linked to either elf6-C or ref6-5 mutations. These plants displayed an array of developmental abnormalities, including abnormal leaf and inflorescence development and a severe reduction in fertility. We therefore reasoned that these defects could be caused by epimutations arising in elf6-C/ref6-5 due to defects in H3K27me3 resetting during sexual reproduction (Figure 3A). In support of this hypothesis, we identified among these F2 progenies one line (A5) that was genetically wild-type for ELF6 and REF6, but had enlarged rosette leaves and partial fertility. From this line, the viable seeds were used to propagate single-seed descent progenies by self-pollinating for over two generations. Plants from F3 progenies displayed a wide spectrum of developmental abnormalities ranging from plants with wild-type characteristics (21.7%, n > 100 each) to partially infertile plants with abnormal rosette leaves (78.3%, n > 100 each). Notably, F4 and F5 progenies continued to segregate the broad range of developmental phenotypes (Figure 3—figure supplement 1). These data indicate that loss of histone demethylase activity causes the accumulation of epimutations. To test this working hypothesis, we grew seedlings from two different F5 progenies (A5.B1 and A5.C6) and performed a ChIP-seq analysis to determine H3K27me3 distribution across the genome. This analysis revealed 544 euchromatic loci with elevated levels of H3K27me3, of which one third were also found to be hyper-methylated in the parental double mutant line used in reciprocal crosses (Figure 3B). These data suggest that some of the H3K27me3 imprints present in epimutants were established in elf6-C/ref6-5 and were then stably transmitted over the five generations, even after the wild-type function of these histone demethylases had been restored (Figure 3C and Figure 3—figure supplement 2). We herein named these lines epiERs (epimutants arising from elf6-C/ref6-5). Notably, for both characterized lines we found that the ectopic accumulation of H3K27me3 was particularly common and strong in constitutive heterochromatin within the pericentromeric regions (Figure 3D). Taken together, our data suggest that ELF6 and REF6 are necessary to limit the transmission of H3K27me3 imprints to offspring, and that failure to do so results in epigenomic and developmental abnormalities.

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Pleiotropic developmental abnormalities associated with the inheritance of ectopic H3K27me3 imprints in Arabidopsis.

(A) The F2 hybrids from reciprocal crosses between wild-type (WT) and elf6-C/ref6-5 display novel abnormal plant growth phenotypes. Frequency of abnormal phenotypes according to parental transmission of mutant alleles is indicated. Pedigree of an epimutant that was genetically wild-type for ELF6 and REF6 and selected for genomic analysis after propagation by selfing. Scale bars, 1 cm. (B) Venn diagram showing the overlap in genes accumulating H3K27me3 in elf6-C/ref6-5 and F5 progenies from A5.B1. p-values for Fisher’s exact test are shown in brackets, N.S. Not Significant. (C) Genome browser views of background subtracted ChIP-seq signals for H3K27me3 as normalized reads per genomic content (RPGC) in wild-type (WT), elf6-C, ref6-5, elf6-C/ref6-5, and F5 progenies from A5.B1 and A5.C6. Shaded boxes, genes showing transgenerational inheritance of H3K27me3. (D) Top panel: Differences in the chromosomal distribution of H3K27me3 as normalized reads per genomic content (RPGC) between F5 progenies from A5.B1 and A5.C6 and wild-type (WT). Grey shaded boxes, pericentromeric regions. Bottom panel: Genome browser view of ChIP-seq signal for H3K27me3 as normalized reads per genomic content (RPGC) in wild-type (WT), and F5 progenies from A5.B1 and A5.C6 in a pericentromeric region. 

Accumulation of ectopic H3K27me3 at centromeric heterochromatin is linked to DNA hypomethylation in epiERs

Loss of DNA methylation has been linked to the abnormal deposition of H3K27me3 in heterochromatin (Batista and Köhler, 2020; Mathieu et al., 2005). However, mutants defective in H3K27me3 deposition do not display altered global DNA methylation levels (Stroud et al., 2014). To test if the ectopic accumulation of H3K27me3 found in epiERs could affect DNA methylation, we performed a BS-seq analysis on the two F5 epimutant progenies used for the ChIP-seq analysis. We found that both epiER lines displayed global reductions in DNA methylation, primarily at pericentromeric regions (Figure 4A). This global reduction in methylation occurred despite there being no ectopic accumulation of H3K27me3 at any genes involved in the DNA methylation pathway, including MET1, DDM1, CMT2 and CMT3, in the parental mutant. In addition, we found that the two analysed epiERs displayed notable differences in DNA methylation levels between lines and among chromosomes (Figure 4A).

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Ectopic accumulation of H3K27me3 is associated with the loss of DNA methylation at pericentromeric heterochromatin and affects chromatin condensation.

(A) Distribution of DNA methylation across chromosomes of wild-type (WT) and progenies from epiERs A5.B1 and A5.C6. Grey shaded boxes, pericentromeric regions. (B) Distribution of DNA methylation across chromosomes of individual plants from wild-type (WT), ddm1, and epiERs A5.B1.3.B1, A5.B5.C5, A5.C5.A6, A5.C5.D6 and A5.C6.C3. Grey shaded boxes, pericentromeric regions. (C) Distribution of DNA methylation across Transposable Elements (TEs) and Transposable Element Genes (TEGs) of individual plants from wild-type (WT) and epiERs A5.B1.3.B1, A5.B5.C5, A5.C5.A6, A5.C5.D6 and A5.C6.C3. Black box, centromeric regions. (D) Correlation between DNA methylation changes and H3K27me3 changes on euchromatic and heterochromatic TEs, in wild-type (WT) and epiER A5.B1. (E) Immunolocalization showing the distribution of H3K27me3 and H3K27me1 in interphase nuclei of wild-type, A5.C5.A2 and A5.C5.B4 plants. Scale bars, 5 μm.

To test whether the reduction in DNA methylation found in epiERs could arise from defects already present in histone demethylation mutants, we analyzed the DNA methylation levels in the genomes of elf6-C, ref6-5 and elf6-C/ref6-5 mutants. Importantly, we did not find any significant changes in DNA methylation in any of these mutants (Figure 4—figure supplement 1A). We therefore investigated variation in DNA methylation between plants within each population by performing a BS-seq analysis on individual F4 epiER plants. This analysis revealed that while some plants were consistently devoid of DNA methylation at pericentromeric regions, similar to the ddm1 mutant, others displayed intermediate states that varied from chromosome to chromosome, thus providing evidence for a partial resetting of DNA methylation (Figure 4B). Notably, the loss of DNA methylation in constitutive heterochromatic regions in epiERs was associated with a decrease in methylation at TEs and genes located therein (Figure 4C and Figure 4—figure supplement 2). Moreover, these defects in heterochromatin methylation were also observed in F1 hybrids derived from reciprocal crosses between wild-type and histone demethylase mutants (Figure 4—figure supplement 1B). Given that these pericentromeric regions in epiERs failed to fully recover DNA methylation to wild-type levels and displayed elevated H3K27me3 levels, we hypothesized that they may be partially protected from the DNA methylation activity commonly used to target transposons and repetitive DNA elements in plants (Matzke and Mosher, 2014). We therefore investigated the relationship between DNA methylation and H3K27me3 on transposons located in euchromatic and constitutive heterochromatic genome regions. We found that in epiERs, heterochromatic TEs that gained H3K27me3 had a proportional loss of DNA methylation whereas euchromatic TEs showed no change in DNA methylation (Figure 4D). These data support the view that a gain in H3K27me3 has a negative effect on the deposition and/or the maintenance of DNA methylation at heterochromatic transposons. To evaluate the extent to which these defects may affect chromatin compaction, we performed immunostaining assays on interphase nuclei using specific antibodies. We found that in epiERs, heterochomatin compaction is strongly affected and manifest as a higher proportion of decondensed nuclei compared to wild type (Figure 4E; Figure 4—figure supplement 3). Collectively, these data suggest that the ectopic accumulation of H3K27me3 in epiERs results in pericentromeric heterochromatin defects.

Epigenomic defects result in transcriptional activation of pericentromeric loci and genome instability

We next investigated whether the abnormal distribution of epigenetic marks in epiERs could be responsible for the developmental abnormalities observed in these plants. To this end, we performed a RNAseq analysis and found that 1240 and 1128 genes were misregulated in epiERs A5.B1 and A5.C6, respectively (Supplementary file 1). A fraction of the upregulated in epiERs (483 and 544) were also upregulated in elf6-C/ref6-5 plants (Figure 5A; Figure 5—figure supplement 1A). Gene ontology analysis revealed that most upregulated genes in the epimutants were involved in biotic stress responses (Figure 5B; Figure 5—figure supplement 1B). When we investigated the chromosomal distribution of these deregulated genes, we found that some were located in constitutive pericentromeric heterochromatin and showed the strongest upregulation effect (Figure 5C). These data suggest that the abnormal distribution of epigenetic marks in epiERs results in transcriptional activation of euchromatic and heterochromatic loci. Given that pericentromeric heterochromatin in plants is rich in TEs and is tightly regulated by DNA methylation and other epigenomic modifications (Dubin et al., 2018), we tested whether the epigenomic perturbations found in epiERs could result in the activation of transposons. We initially used our transcriptome data to determine the transcriptional state of different TEs in the two epiER progenies. We found that both RNA and DNA transposon families were significantly upregulated in epiERs (Figure 6A; Figure 6—figure supplement 1). We next determined their copy number in different epiER lines (see Materials and methods) to assess whether the transcriptional activation of TEs in these epimutants could result in increased mobility. We found that one heterochromatic transposon, CACTA1 (At2TE20205), and one euchromatic retrotransposon, EVADE (EVD) (At5TE20395), showed a significant increase in copy number in both epiERs (Figure 6B). Further analysis revealed that these TEs were depleted in DNA methylation and significantly upregulated (Figure 6C; Figure 6—figure supplement 2). We then determined the precise location of some of the transposons that had newly mobilized in the different epimutants. Most novel insertions accumulated in euchromatin, continued to be active over multiple generations, and sometimes disrupted expression of genes that could be linked to the observed developmental phenotypes (Figure 6D–F and Supplementary file 2). Collectively, our data demonstrate that the developmental abnormalities found in epiER lines arise from heritable epigenetic changes and from genetic mutations caused by TE mobilization and reinsertion into the genome.

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Global upregulation of centromeric gene expression in epiERs.

(A) Heatmap showing scaled expression levels of Differentially Expressed Genes between wild-type and progeny of epiER A5.B1 in wild-type (WT) elf6-C, ref6-5, elf6-C/ref6-5, and progenies of epiERs A5.B1 and A5.C6. (B) Gene Ontology analysis showing the functional categories enriched in genes upregulated in progeny of epiERs A5.B1. (C) Differential gene expression across each Arabidopsis chromosome for genes upregulated and downregulated in progenies of epiERs A5.B1 and A5.C6. Grey shaded boxes, pericentromeric regions.

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Transposon mobilization in epiERs results in heritable genetic lesions.

(A) Differential expression of DNA and RNA transposon families grouped by superfamily in progenies of epiERs A5.B1 and A5.C6. (B) Copy number variation of transposons in progenies of epiER A5.C6. Blue dots, euchromatic TEs; Red dots, heterochromatic TEs. (C) Genome browser views of normalized sequencing coverage (RPGC), DNA methylation frequency (%) and RNAseq coverage (RPGC) in wild-type (WT) and progenies of epiERs A5.B1 and A5.C6. Grey box, AT2TE20205 (CACTA1). (D) Map of transposon insertion in AT3G11330 and its segregation in epiER A5.B1.3 progenies. P1-3, primers used for PCR amplification and sequencing. (E) Map of transposon insertion in AT5G10770 and sequence footprint resulting from re-mobilization in epiER A5.B1.3 progenies. P4-6, primers used for PCR amplification and sequencing. (F) Seed pigmentation defects caused by a sequence insertion in AT5G13930 (TRANSPARENT TESTA4/CHALCONE SYNTHASE) resulting from transposon re-mobilization in in epiER A5.B1.3 progenies. P7-8, primers used for PCR amplification and sequencing.

Discussion

In plants, histone modifications deposited by PRC2 play a critical role in growth and development, and in the adaptation of these processes to environmental fluctuations. Previous studies in Arabidopsis have shown that the activity of a distinct group of JmJ-type demethylases shape the genomic distribution of H3K27me3 (Yan et al., 2018). Three of these proteins – JMJ13, ELF6 and REF6 – have been shown to play important roles in development and in the regulation of environmental perception (Noh et al., 2004; Zheng et al., 2019). Our data show that REF6 and ELF6 regulate the removal of H3K27me3 at different genomic loci; while REF6 has a large repertoire of target genes, ELF6 activity is restricted to a small subset of genes, most of which can also be targeted by REF6. Combined with our genetic analysis, these data collectively suggest that despite the structural similarities between these two proteins, they are able to carry out distinct functions in H3K27me3 homeostasis. Our data also support the view that although REF6 restricts the spreading of H3K27me3 to the genomic regions flanking PRC2 targets (Yan et al., 2018), it also has a hitherto unrecognized function in the regulation of H3K27me1 homeostasis in euchromatin. This view is also supported by the overlap between REF6 genomic targets and H3K27me1 accumulation in euchromatic regions in wild-type plants, as well as by the complete loss of H3K27me1 in PRC2 target loci in plants without REF6 activity, and the partial loss of H3K27me1 in plants without PRC2 activity. Therefore, the accumulation of H3K27me1 in Arabidopsis relies both on the activity of ATXR5 and ATXR6 in heterochromatin (Jacob et al., 2009; Jacob et al., 2010) and on the activity of REF6 in transcriptionally active euchromatin (Figure 7). ATXR5 and 6 have been shown to target only newly synthesized H3.1 histone variants, which are deposited during replication and replaced by H3.3 in euchromatin during later stages of the cell cycle (Jacob et al., 2014). In mammals, the histone demethylases UTX and JMJD3, also known as KDM6A and KDM6B, have been shown to catalyze the conversion of H3K27me3 and H3K27me2 into H3K27me1 (De Santa et al., 2007; Lan et al., 2007; Lee et al., 2007; Swigut and Wysocka, 2007). Moreover, defects in PRC2 methyltransferase activity in mammals completely abolishes the accumulation of H3K27me1 in embryonic stem cells (Ferrari et al., 2014; Montgomery et al., 2005), suggesting a conserved PRC2-mediated mechanism for H3K27me1 homeostasis in euchromatin, in both animals and plants. The precise mechanism responsible for the deposition of this chromatin mark in Arabidopsis is currently unknown, but our data support the view that the deposition of H3K27me1 in euchromatin is dependent on the activity of PRC2 and REF6 (Figure 7). In mammals the presence of H3K27me1 in actively transcribed genomic regions has been associated with the promotion of transcription (Ferrari et al., 2014), a fact that may explain why Arabidopsis genes associated with H3K27me1 display moderate levels of expression, whereas the conversion of this mark into H3K27me3 negatively impacts their transcriptional rates.

Figure 7
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Proposed model for the role of histone demethylases in the accumulation of H3K27me1 and the formation of epimutations arising in ELF6 and REF6 mutants (epiERs).

(Top panel) Model for the mechanisms implicated in the accumulation of H3K27me1. In pericentromeric heterochromatin of somatic cells, ATRX5/6 deposits in a single-step H3K27me1 on histones containing H3K9me2. In euchromatin of somatic cells, the PRC2 complex deposits H3K27me3, which is converted to H3K27me1 by the catalytic activity of REF6. (Bottom panel) Model for the origin of epiERs. In constitutive heterochromatin of wild type somatic cells, DNA methylation obstructs the PRC2 complex from depositing H3K27me3. In gametes of wild-type plants, reprogramming of DNA methylation facilitates the deposition of H3K27me3 by the PRC2 complex, but these imprints are actively removed by histone demethylases, thus permitting DNA methylases to establish normal levels of methylation. In gametes of elf6-C/ref6-5, the H3K27me3 deposited by the PRC2 complex accumulates during the reprogramming of DNA methylation and interfere with the activity of DNA methyltransferases. The ectopic accumulation of H3K27me3 spreads to flanking genomic regions by recruitment of LHP1 and the PRC2 complex. Dark blue circles, methyl groups. C, Cytosines. H3.X, H3 variant that is not H3.1. Coiled lines represent closed and inactive chromatin. Wavy lines represent Polycomb-repressed chromatin. DNA MT: DNA methyltransferases. Faded shapes represent low amount of enzyme.

Plant somatic cells accumulate H3K27me3 primarily at protein-coding genes; however, in reproductive tissues and mutants where DNA methylation is reduced, this mark also accumulates at TEs (Deleris et al., 2012; Weinhofer et al., 2010). Other studies have also reported the accumulation of H3K27me3 at transposon sites in plant species with reduced levels of DNA methylation (Montgomery et al., 2020), as well as in mammal somatic and reproductive tissues which also show reduced levels of DNA methylation (Hanna et al., 2019; Reddington et al., 2014; Saksouk et al., 2014). However, our data do not fully support the idea that the deposition of this chromatin mark acts as a compensatory system to silence hypomethylated TEs (Deleris et al., 2012; Hanna et al., 2019). Instead, our results suggest that the homeostasis and function of H3K27me1 and H3K27me3 in plants is more complex than previously anticipated. The stable inheritance of de novo acquired DNA methylation imprints in plants is well documented. Mutations in the machinery involved in the deposition of DNA methylation, such as the cytosine DNA METHYLTRANSFERASE 1 (MET1) and the chromatin-remodeling DEFICIENT IN DNA METHYLATION 1 (DDM1), frequently induce epimutations caused by DNA hypomethylation (Johannes et al., 2009; Kakutani et al., 1996; Mathieu et al., 2007). These epimutations are maintained during sexual reproduction and remain stable over several generations, even after the function of MET1 or DDM1 is restored. Moreover, natural epimutations created during asexual propagation and associated with DNA hypomethylation involving TEs, can be stable over multiple generations, thus contributing to a variety of novel heritable phenotypes (Ong-Abdullah et al., 2015; Wibowo et al., 2018). Additionally, analysis of the Arabidopsis met1 mutant has shown that a large number of TEs that lose DNA methylation gain H3K27me3 (Deleris et al., 2012). Similarly, TEs in Arabidopsis gain H3K27me3 in response to ddm1-induced loss of DNA methylation (Rougée et al., 2020). Collectively, our data together with these reports suggest that DNA methylation and H3K27me3 act antagonistically to mediate the transcriptional silencing of transposons.

There is accumulating evidence for the active role of histone demethylases in resetting H3K27me3 at specific loci during sexual reproduction (Crevillén et al., 2014; Noh et al., 2004) and for a global depletion of H3K27me3 during spermatogenesis (Borg et al., 2020). While the precise mechanism(s) governing this phenomenon remains poorly understood, our finding that the failure to reset H3K27me3 during sexual reproduction resulted in its trans-generational inheritance in euchromatin, even when functional demethylase activity was restored, might suggest that some of the H3K27me3 imprints that are ectopically deposited in histone demethylase mutants cannot be reset if they are distal to the target sequences recognized by the demethylases. Furthermore, once established, these H3K27me3 imprints could be maintained across generations as epimutations through the recruitment of LHP1-PRC2 complexes (Derkacheva et al., 2013). Our data revealed that the inheritance of H3K27me3 imprints causes defects on the maintenance of DNA methylation at constitutive heterochromatic. However, these defects are not caused by the ectopic accumulation of H3K27me3 at genes implicated in the DNA methylation machinery. The ectopic deposition of H3K27me3 in constitutive heterochromatin may instead be linked to defects in the resetting of DNA methylation thought to take place during gametogenesis (Calarco et al., 2012; Ibarra et al., 2012; Slotkin et al., 2009) and/or during early embryo development (Bouyer et al., 2017), where epigenetic modifications have been shown to be anti-correlative (Mathieu et al., 2005; Roudier et al., 2011). Under this scenario, an active resetting of H3K27me3 in gametes would be critical for the re-establishment of DNA methylation, while the ectopic deposition of H3K27me3 in histone demethylase mutants could antagonize the deposition of DNA methylation at TEs (Figure 7). Mutants defective in histone demethylases could accumulate epigenomic alterations in gametes that could explain the heritable, yet unstable, phenotypes observed in epiERs. Similar epimutations and phenotypic variation have been shown to arise from crosses between wild-type plants and mutants defective in the machinery that maintains DNA methylation (Kakutani et al., 1996; Kato et al., 2004; Marí-Ordóñez et al., 2013; Mirouze et al., 2012). As in these studies, we also found that epiERs have defects in the silencing of some transposons resulting in an increase in genetic lesions associated with their mobilization. Taken together our data reveal novel, critical roles for histone demethylases in maintaining both genome integrity and transcriptional states during plant development.

Materials and methods

Plant material and growth conditions

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All plant lines used in this study were derived from Arabidopsis thaliana, Col-0 accession. The T-DNA insertion lines ref6-1 (SALK_001018), elf6-3 (SALK_074694), atxr5 (SALK_130607) and atxr6 (SAIL_240_H01) have been previously described. The ref6-5 mutant (GABI_705E03) was obtained from the GABI-Kat collection (Kleinboelting et al., 2012) and a genomic deletion line for elf6-C was produced using two sgRNAs (Supplementary file 3) and CRISPR/Cas9 (Durr et al., 2018). New mutant alleles were backcrossed twice to wild-type plants and homozygous plants were identified from F2 progenies by molecular genotyping (Supplementary file 3). Double mutants between different mutant alleles were produced by manual pollination. The plant materials used for crossing and flowering time measurements were grown in chambers under long day conditions (16 hr light, 8 hr dark) with 120 μ mol m−2 s−1 light intensity (22°C daytime, 20°C at night). Plants for the screening were grown in a climate-controlled greenhouse under long day conditions (20°C daytime, 20°C at night,16 h light plus 8 hr dark). The seeds were mixed in 0.1% agarose and underwent 2 d cold treatment at 4°C in the dark. After treatment, seeds were directly sown on soil and transferred to growth a chamber or greenhouse.

Genotyping and phenotyping

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Primary transformants were identified using the seed-specific RFP reporter under a Leica MZ-FL III stereomicroscope (Leica Camera AG). Genotyping of CRISPR/Cas9-based mutations and T-DNA insertions were performed using KAPA-Taq (Sigma-Aldrich) following the manufacturer’s instructions. PCR product size was selected using gel electrophoresis and the introduced genetic lesion was determined by sequencing (Figure 1—figure supplementary file 1). The phenotypes of whole plants, leaf number and rosette size were scored at bolting. Silique length measurement were carried out on the 6th-15th siliques of main stems, when the last flowers of the inflorescence started producing siliques. The mean value of the 10 siliques represented the silique length of a plant. For embryo analysis, ovules from self-pollinated plants were cleared with a chloral hydrate solution, observed with a light microscope (Zeiss AxioImager A2) and photographed with a digital camera (Zeiss AxioCam HRm).

ChIP-seq assay

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ChIP-seq assays were performed on 14 day-old in vitro shoot seedlings using anti-H3K27me3 (Millipore 07–449) or anti- H3K27me1 (Millipore 07–448), following a procedure modified from Gendrel et al., 2005. Five grams of plantlets were cross-linked in 1% (v/v) formaldehyde at room temperature for 15min. Crosslinking was then quenched with 0.125 M glycine for 5 min. The crosslinked plantlets were ground and nuclei were isolated and lysed in Nuclei Lysis Buffer (1% SDS, 50 mM Tris-HCl pH 8, 10 mM EDTA pH 8). Cross-linked chromatin was sonicated using a water bath Bioruptor UCD-200 (Diagenode, Liège, Belgium) (15 s on/15 s off pulses; 15 times). The complexes were immunoprecipitated with antibodies, overnight at 4°C with gentle shaking, and incubated for 1 hr at 4°C with 40 µL of Protein AG UltraLink Resin (Thermo Scientific). The beads were washed 2 × 5 min in ChIP Wash Buffer 1 (0.1% SDS, 1% Triton X-100, 20 mM Tris-HCl pH 8, 2 mM EDTA pH 8, 150 mM NaCl),2 × 5 min in ChIP Wash Buffer 2 (0.1% SDS, 1% Triton X-100, 20 mM Tris-HCl pH 8, 2 mM EDTA pH 8, 500 mM NaCl), 2 × 5 min in ChIP Wash Buffer 3 (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate,10 mM Tris-HCl pH 8, 1 mM EDTA pH 8) and twice in TE (10 mM Tris-HCl pH 8, 1 mM EDTA pH 8).ChIPed DNA was eluted by two 15 min incubations at 65°C with 250 μL Elution Buffer (1% SDS, 0.1 M NaHCO3). Chromatin was reverse-crosslinked by adding 20 μL of NaCl 5M and incubated over-night at 65°C. Reverse-cross-linked DNA was submitted to RNase and proteinase K digestion, and extracted with phenol-chloroform. DNA was ethanol precipitated in the presence of 20 μg of glycogen and resuspended in 50 μL of nuclease-free water (Ambion) in a DNA low-bind tube. 10 ng of IP or input DNA was used for ChIP-Seq library construction using NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer’s recommendations. For all libraries, 12 cycles of PCR were used. The quality of the libraries was assessed with Agilent 2100 Bioanalyzer (Agilent).

Computational analysis of ChIP-seq

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Single-end sequencing of ChIP samples was performed using Illumina NextSeq 500 with a read length of 76 bp. Reads were quality controlled using FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Trimmomatic was used for quality trimming. Parameters for read quality filtering were set as follows: Minimum length of 36 bp; Mean Phred quality score greater than 30; Leading and trailing bases removal with base quality below 5. The reads were mapped onto the TAIR10 assembly using Bowtie (Langmead, 2010) with mismatch permission of 1 bp. To identify significantly enriched regions, we used MACS2 (Zhang et al., 2008). Parameters for peaks detection were set as follows: Number of duplicate reads at a location:1; mfold of 5:50; q-value cutoff:0.05; extsize 200; broad peak. Visualization and analysis of genome-wide enrichment profiles were done with IGB. Peak annotations such as proximity to genes and overlap on genomic features such as transposons and genes were performed using BEDTOOLS INTERSECT. To identify regions that were differentially enriched in the H3K27me3 or H3K27me1 histone modification between WT and mutants, we used DIFFREPS (Shen et al., 2013) with parameters of pvalue 0,05; z-score cutoff 2; windows 1000 (Supplementary file 4).

Expression profiling by RNA-seq

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Leaf samples were collected from five plants at the 4 week growth stage. Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen) according to manufacturer’s instructions and used to produce libraries using TruSeq RNA library Prep Kit v2 (Illumina). Pooled libraries were sequenced in a NextSeq550 sequencing platform (Illumina). Two biological replicates were generated for each genotype, and at least 20 million reads were produced per replicate.

Generation of epimutations using histone demethylase mutants

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Five independent homozygous elf6-C, ref6-5 elf6-C/ref6-5 plants were selected for reciprocal crosses with wild-type plants (Col-0). All the F1 progenies were self-pollinated to generate F2 seeds that were grown in individual pots. The frequency of developmental phenotypes, not observed in the histone demethylase mutants, was scored 8 week old plants and fertility was determined according to the production of viable seeds. Plants that displayed developmental phenotypes not found in elf6-C, ref6-5 or elf6-C/ref6-5 mutants where genotyped by PCR (Supplementary file 3) to determine their zygosity.

Bisulfite sequencing

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Rosette leaves from five 4 week old plants were pooled for each sample. Genomic DNA was extracted with the DNeasy Plant Mini Kit (Qiagen, Germany). DNA libraries were generated using the Illumina TruSeq Nano kit (Illumina, CA, USA). DNA was sheared to 350 bp. The bisulfite treatment step using the Epitect Plus DNA Bisulfite Conversion Kit (Qiagen, Germany) was inserted after the adaptor ligation; incubation in the thermal cycler was repeated once before clean-up. After clean-up of the bisulfite conversion reaction, library enrichment was done using Kapa Hifi Uracil+ DNA polymerase (Kapa Biosystems, USA). Libraries were sequenced with 2 × 150 bp paired-end reads on an HiSeq 4000 (Illumina), with conventional gDNA libraries in control lanes for base calling calibration. Sixteen to 24 libraries with different indexing adapters were pooled in each lane.

Computational analysis of paired end BS-seq

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Paired-end quality was assessed using FASTQC (Andrews et al., 2010). Trimmomatic (Bolger et al., 2014) was used for quality trimming. Parameters for read quality filtering were set as follows: Minimum length of 40 bp; sliding window trimming of 4 bp with required Phred quality score of 20. Trimmed reads were mapped to the Arabidopsis thaliana TAIR10 genome assembly using bwa-meth (Pedersen et al., 2014) with default parameters. Mapped reads were deduplicated using picardtools (Picard toolkit, 2019), and numbers of methylated/unmethylated reads per position were retrieved using MehtylExtract (Oliver et al., 2014).

Pericentromeric heterochromatic regions

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Heterochromatin regions were defined as in Qiu et al., 2019 (Chr1:12,500,000–17,050,000, Chr2:2,300,000–6,300,000, Chr3: 12,800,000–14,800,000, Chr4: 1,620,000–2,280,000; 2,780,000–5,804,000, Chr5: 10,680,000–14,000,000).

Gene expression and ontology analysis

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We used agriGO v2.0 (Tian et al., 2017) to classify significantly enriched Gene Ontology (GO) terms associated with differential expression.

Immunostaining of chromatin

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Leaf protoplasts were isolated from 14 day old seedlings and fixed. After rehydration in PBS, slides were blocked in 2% BSA in PBS (30 min, 37°C) and incubated overnight at 4°C in 1% BSA in PBS containing antibodies (Upstate Biotechnology) specific to lysine-27-monomethylated H3 (1:100 dilution), and lysine-27-trimethylated H3 (1:100 dilution). Detection was carried out with an FITC-coupled antibody to rabbit IgG (Molecular Probes; 1:100 dilution, 37°C, 40 min) in 0.5% BSA in PBS. DNA was counterstained with 4,6 diamidino-2-phenylindole (DAPI) in Vectashield (Vector Laboratories).

Data visualisation

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For visualising BS-seq, RNA-seq and ChIP-seq genomic data we used Integratice Genomic Viewer (IGV) (Thorvaldsdóttir et al., 2013), And R version 3.5.1 (www.r-project.org) with packages ggplot2 (Wickham, 2016), eulerr (Larsson, 2019), pheatmap (Kolde, 2015) and EnrichedHeatmap (Gu et al., 2018).

Prediction of new TE insertion sites and molecular validation

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We analysed Bisulfite-seq data using Bismark (Krueger and Andrews, 2011) using the following parameters:–bowtie2 –ambiguous –unmapped –R 10 –score_min L,0,–0.6 -N 1. Identification of new TE insertion sites was performed using epiTEome (Daron and Slotkin, 2017). For the validation of new transposon insertions, we designed primers outside of predicted TE insertion site and inside the transposon based on physical reads identified by epiTEome. We used KAPA Taq Polymerase and PCR conditions of 95°C for 5 min, followed by 30–35 cycles of 95°C for 30 s, 58°C for 15 s, and 72°C for 2 min. The list of primers employed for this analysis are listed (Supplementary file 3).

Major datasets

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The following dataset was generated: ‘Arabidopsis H3K27 demethylases contribute to genomic integrity’. Dataset URL https://www.ebi.ac.uk/ena/browser/view/PRJEB36508.

The following previously published datasets were used: DDM1 and RdDM are the major regulators of transposon DNA methylation in Arabidopsis’. Dataset URL https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41302.

Data availability

Sequence data (BS-seq, RNA-seq and ChiP-seq) that support the findings of this study have been deposited at the European Nucleotide Archive (ENA) under the accession code PRJEB36508.

The following data sets were generated
    1. Sanchez JA
    (2020) European Nucleotide Archive
    ID PRJEB36508. Arabidopsis H3K27 demethylases contribute to genomic integrity.
    https://www.ebi.ac.uk/ena/browser/view/PRJEB36508
The following previously published data sets were used
    1. Qiu Q
    (2019) NCBI Gene Expression Omnibus
    ID GSE111830. DNA methylation prevents REF6 binding in Arabidopsis.
    https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE111830
    1. Zemach A
    (2013) NCBI Gene Expression Omnibus
    ID GSE41302. DDM1 and RdDM are the major regulators of transposon DNA methylation in Arabidopsis.
    https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41302

References

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    1. Pedersen BS
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    4. Yang IV SDA
    (2014)
    Fast and Accurate Alignment of Long Bisulfite-Seq Reads.
    Fast and Accurate Alignment of Long Bisulfite-Seq Reads..
  76. Book
    1. Wickham H
    (2016)
    ggplot2: Elegant Graphics for Data Analysis
    Springer-Verlag, New York. ISBN 978-3-319-24277-4.

Decision letter

  1. Christian S Hardtke
    Senior Editor; University of Lausanne, Switzerland
  2. Pil Joon Seo
    Reviewing Editor; Seoul National University, Republic of Korea
  3. Yannick Jacob
    Reviewer

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This study illustrates a novel role of H3K27me2/3 demethylases in homeostasis of H3K27me1 level in plants. It is also noteworthy that resetting the chromatin marks is particularly important for maintaining genetic and epigenetic stability in the next generation.

Decision letter after peer review:

Thank you for submitting your article "A new role for histone demethylases in the maintenance of plant genome integrity" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Christian Hardtke as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Yannick Jacob (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.

Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in eLife, either of which would be linked to the original paper.

All reviewers agree that this work looks interesting, but all three reviewers also raised major issues about biological relevance of H3K27me1 in plants and mechanisms behind epimutation and linkages of DNA methylation and H3K27me3. Reviewer 1 and 3 also critisized that paper has two main findings, which are not cohesive. Essential revisions:

1) The most important issue is how ectopic accumulation of H3K27me3 leads to impaired DNA methylation. The mechanistic linkage is poor in the current manuscript, and this should be substantialized in the revision.

2).Please address the biological relevance of H3K27me1, which is not clear. It could be indirectly linked to gene expression control and genome stability.

3) The paper could be separated. The first part covers a new biochemical function of REF6 and ELF6, but the second part mainly deals with epimutation in progenies. It is not clear that changes in H3K27me1/H3K27me3 are relevant for the phenotypic and epigenetic alterations in next generations. The manuscript should be more connected.

However, please thoroughly check the reviewer comments (you find the individual reviews below) and address them to the best of your capacity. All comments are critical for publication in eLife.

Reviewer #1:

The contribution by Antunez-Sanchez et al. demonstrates that histone demethylases REF6 and ELF6 have distinct roles in the histone demethylation in Arabidopsis. REF6 has a broader impact in reducing H3K27me3 and is also a major player in the deposition of H3K27me1 in active euchromatin regions. The maintenance of H3K27me1 in active chromatin tends to allow a low-level basal expression. In addition, this study also showed that the failure to reset H3K27me marks during sexual reproduction results in the inheritance of H3K27me3 imprints, which is associated with the loss of DNA methylation at heterochromatic loci, leading to TE activations. Overall, the manuscript covers new roles of REF6 and ELF6 and biological relevance of H3K27me1/me3 homeostasis in genome integrity and transcription activity. However, several flaws should be addressed to show the importance of this study.

1) What tissues were used for each analysis? Genetic interactions between ELF6 and REF6 vary as shown in phenotypic analysis. Therefore, plant tissues and age should be described in figure legends.

2) They suggested the importance of H3K27me1 homeostasis in gene expression. I am wondering if the H3K27me1 imprints can be transmitted to offspring in wild type. Otherwise, how the histone mark could be reset. Is there another responsible histone demethylase that removes H3K27me1? In addition, the relevance of H3K27me1 in genome stability is limited, and interpretation about epi-mutants relies mainly on ectopic accumulation of H3K27me3. Although they emphasize their finding about REF6-catalyzed H3K27me1 as shown in Figure 7, the analysis of H3K27me1 was not extensive.

3) Do REF6 and ELF6 have critical roles in silencing pericentromoric regions? How about H3K27me3 and H3K27me1 levels in pericentromeric regions in ref6 and elf6 mutants?

4) Ectopic accumulation of H3K27me3 leads to global reductions in DNA methylation at pericentromeric regions. To support the idea that hypomethylation caused by ectopic accumulation of H3K27me3 depends on RdDM pathways, they should use mutants of RdDM pathways. In addition, if REF6 and ELF6 are particularly relevant in histone modification at active chromatin, what mechanisms could be involved in changes in DNA methylation at pericentromeric regions? I think that DNA methylation could be indirect and stochastic results.

5) Reduction of H3K27me1 could be linked to global reductions in DNA methylation? There is no experiment to rule out the possibility.

6) Individual epi-mutants have variable phenotypes, and genetic backgrounds are not likely uniform. I am wondering if GO analysis is meaningful in this situation. It could be very different depending on individuals.

7) Figure 4E: Please quantify the results, rather than showing representative images.

8) Figure 7: The summary figure should show main finding of this paper. I think only the fraction of this study is shown, and this makes reader confusing. The authors should rearrange the manuscript and figures and give a more focused view of the conclusion.

Reviewer #2:

General Assessment

The manuscript by Antunez and colleagues provide important biological insights into the role of DNA demethylase in maintaining genetic and epigenetic stability between different generations. In addition, this study also shed light on the contribution of H3K27me2/3 demethylases toward in vivo levels of H3K27me1 in plants. I think this is important work that should be of general interest to the plant epigenetic community. The manuscript is well-written and the conclusions are substantiated.

Substantive concern:

1) One concern with the manuscript is the mechanism leading to epimutations that is proposed by the authors, which is that ectopic inheritance of H3K27me3 in gametes leads to the different phenotypes observed. The work of Olivier Mathieu and his colleagues in 2005 has shown that loss of DNA methylation in ddm1 mutants leads to ectopic gains of H3K27me3 in heterochromatin. Transcriptional reactivation of transposons and transposition (e.g. Tsukahara et al., Nature 2009) is also observed in ddm1 mutants like in the EpiER lines. Therefore, it would be useful for the authors to investigate if DNA methylation is impaired because of genome-wide gains in H3K27me3 in gametes, or whether one or a few affected loci are responsible for the loss of DNA methylation. The authors try to address this point (e.g. subsection “Accumulation of ectopic H3K27me3 at centromeric heterochromatin is linked to DNA hypomethylation”), however, more information should be provided. They should indicate which genes in the DNA methylation pathways have been assessed for H3K27me3 levels. In addition, the transcriptional status of these genes should be assessed (the data is already available from this study) to determine if loss of DNA methylation is due to down-regulation of DNA methylation genes. Transcriptional silencing of these genes could be achieved indirectly, if no gains in H3K27me3 are observed at the genes.

2) Results: It's not clear to me looking at Figure 2E and Figure 2—figure supplement 5 that REF6 is "…required for low-level expression genes." I think those figures indicate that these genes (3rd and 5th quantiles) gains H3K27me3, but the transcriptional impact of the increase of H3K27me3 at these genes is not provided. Since they have transcriptional data for ref6 mutants, the authors could look if gains in H3K27me3 at these genes decrease their expression.

Reviewer #3:

The paper looks at the effects of the Arabidopsis H3K27me3 histone demethylases (HDM) REF6 and ELF6 on the plant epigenome and on the generation of novel epialleles. There are two fairly distinct parts to the paper. In the first part, they generate elf6 ref6 double mutants using likely null alleles, one generated by genome editing. They use genomic approaches to show that many target genes gain H3K27me3 methylation and have decreased expression in the mutants. This part is consistent with what has been shown by several other studies, notably the Kaufman group ( Nature Plants, 4:681). Of greater novelty, they then show that REF6/ELF6 targets are enriched for H3K27me1 in the wild type background, but lose this mark in ref6 elf6 double mutants, consistent with a role for REF6 and ELF6 in generating this mark by demethylating H3K27me3 to H3K27me1 but being unable to demethylate H3K27me1 substrates. Using ChIP seq they show that ATXR5 and ATXR6 (H3K27me1 monomethylases required for heterochromatic H3K27me1) are not required for the H3K27me1 that they find at euchromatic locations. They correlate H3K27me1 in euchromatin with intermediate level gene expression, although whether this is an indirect effect of a lack of H3K27me3 is hard to disentangle. In the second part they describe the appearance of novel phenotypes at low frequency in F2 progeny of WT X elf6 ref6. They identify REF6+ ELF6+ homozygous progeny in F2 and show that these can transmit novel phenotypes from F2 to F5 generations. Profiling of selected F5 individuals with novel phenotypes reveal various changes, including H3K27me3 hyper and hypomethylation, loss of DNA methylation, and mobilisation of transposable elements (TE). The suggestion is that the novel phenotypes are caused by epialleles, i.e. heritable alterations in histone or DNA methylation, although DNA sequence based changes due to TE insertion or imprecise excision are also possible causes.

Overall I found this a very interesting paper. The role of HDM in euchromatic H3K27me1 is novel and intriguing, perhaps difficult to assess the significance as the mechanism of H3K27me1 action is not known in plants, i.e. readers etc for this mark have not been identified. The observation that ref elf mutants may give rise to epialleles that persist even when REF6 ELF6 activity is restored is of broad interest, and builds on studies such as Crevillen et al. (Nature 515: 587) which showed that elf6 impairment can give rise to heritable epigenetic changes (but in this case only in the elf6 mutant background). On the negative side, the two parts of the paper are not very strongly connected, for example it is not very clear if changes in H3K27me1 described in part one are relevant for the phenotypic and epigenetic alterations described in part two. Also, there is very limited description of the phenotypes found and the causes are not very clear i.e. alterations in which genes are causal and what are the genetic or epigenetic changes involved? It is not very clear why ref6/elf6 depletion gives rise to the effects observed, for example one of the major changes in hyper H3K27me3 methylation and loss of DNA methylation at pericentromeric heterochromatin, yet ref6 elf6 mutants do not seem to show this change. Overall, the observations are interesting, they have done a lot of analysis and it is unreasonable to expect a complete explanation but the overall picture remains confusing. As such I think the paper is borderline for eLife, and would at least require some revision along the lines below.

1) Figure 3A shows plants with aberrant phenotypes and their transmission, however there is no description of the phenotypes, or of their inheritance i.e. what kind of segregation ratios are observed in the different generations. For example, A5 looks to be broad leaved but gives rise to plants with narrow curled leaves in F3 generation, it is not clear if these are transmitted into F4 generation or at what frequency, or if traits are dominant or recessive.

2) It's not very clear which generation is used for the various profiles of A5 progeny, possibly F4 or F5 plants?

3) Much of the data is not accessible or presented. For example, no lists of the peaks found for the different marks profiled are provided, nor are there lists of differential peaks found, or how these related to genes showing altered expression.

4) In Figure 3D the wild type trace is invisible, either omitted or masked by another trace?

5) The recent paper from Berger group on reprogramming in sperm (Borg et al., 2020) could be referenced and discussed. This study would suggest that H3K27me3 hypermethylation should not be transmitted paternally once REF6/ELF6 activity is restored. It would be interesting to know if the phenotypes and or epigenetic changes seen in A5 can be transmitted maternally, paternally or both once RER6/ELF6 activity is restored. Given that some or all of the effects of REF6/ELF6 depletion may be indirect effects of H3K27me3 changes, for example TE mobilisation or DNA hypomethylation, it is possible that the phenotypes would be transmitted but this might give some indication of whether H3K27me3 changes are causal for the phenotypes or not, or indirect. If the phenotypes are recessive, then this would require both paternal and maternal transmission in self progeny.

6) Subsection “REF6 controls H3K27me1 homeostasis in chromatin” states that H3K27me1 is not affected at euchromatic gene in atxr5 atxr6 mutants, however in Figure 2—figure supplement 2 it looks as if levels are actually increased in the mutant. This should be commented on and discussed. It should also be mentioned that the particular atxr5 atxr6 double mutant combination is hypomorphic and not null.

7) Figure 6F shows that line A5.B1.3 which has a seed pigmentation phenotype has a 5 bp insertion in the tt4 gene. It is stated that this is due to transposon remobilisation but it is not clear if this is inferred as a likely imprecise excision, or whether they are able to show that a TE is present in this location in a progenitor plant?

8) Given that a lot of sequence data is available for some of the lines, I wondered if authors should check that there are no mutations present in genes associated with RdRM etc? That is not intended as a slight, but since changes in DNA methylation are observed that don't readily correlate with what is seen in the ref6 elf6 parental lines it could presumably be easily checked and eliminated as a potential cause.

9) The correlation between pericentromeric H3K27me3 increase and DNA methylation decrease is an interesting observation, but they provide no mechanism why this should arise in elf6 ref6 backgrounds, which they show seem normal for pericentromeric DNA methylation and H3K27me3. The Qiu et al. (2019) Nat Comm paper, which the authors here mention in the Introduction, reported that REF6 cannot bind efficiently on methylated DNA, so it is puzzling.

10) Lines were picked based on aberrant phenotypes, propagated, and then analysed and found to contain various epigenetic and genetic changes. It is difficult to know whether the changes cause the phenotypes seen, and if so which particular changes. This made me wonder to what extent ELF6 REF6 F2 progeny with normal phenotypes would also show epigenomic changes, or whether this is specific to the plants with phenotypic abnormalities?

11) The fact that ref6 and elf6 have opposite effects on flowering time is curious. The elf6c mutant is early flowering, consistent with a role for ELF6 in binding the floral repressor FLC and promoting its expression by H3K27me3 demethylation (Mol Plant 11:1135). The ref6 mutant is late flowering, and has been correlated with increased FLC expression (Plant Cell 16: 2601), which seems at odds with the hypermethylation for H3K27me3 and decreased expression more typical of these mutants. This might be commented on, and whether the various profiling experiments offer any explanation.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "A new role for histone demethylases in the maintenance of plant genome integrity" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Christian Hardtke as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Yannick Jacob (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.

Reviewer #3:

The authors have provided some new data, ChIP seq of H3K27me1 in clf swn mutants, which provides some support for their model that euchromatic H3K27me1 derives from demethylation by REF6 of H3K27me3 deposited by PRC2 and is a good addition. Overall I find it an interesting paper of broad general interest but don't feel that the revision greatly addresses the comments from before as to mechanism and significance of H3K27me1 being unclear. That said, there is a lot of data in the paper and it is an interesting story that is perhaps complementary to the Borg et al., 2020 paper.

I have issues with a couple of the points made by authors.

3) Much of the data is not accessible or presented. For example, no lists of the peaks found for the different marks profiled are provided, nor are there lists of differential peaks found, or how these related to genes showing altered expression. All the raw data has been uploaded in a public repository. We don't think it is necessary to upload all of the peak data as supplemental information.

It is a given that the raw data will be provided. Since there is a lot of discussion of the ChIP seq data and selected peaks etc are shown I think it reasonable that the authors provide lists of enriched genes for the various ChIP seq experiments, as is usually provided in other papers.

8) Given that a lot of sequence data is available for some of the lines, I wondered if authors should check that there are no mutations present in genes associated with RdRM etc? That is not intended as a slight, but since changes in DNA methylation are observed that don't readily correlate with what is seen in the ref6 elf6 parental lines it could presumably be easily checked and eliminated as a potential cause.

We have investigated the chromatin (H3K27me3 and H3K27me1) and expression profile of gene implicated in DNA methylation genes, both in our histone demethylase mutants and also in epiERs (see heatmap in response to reviewer 2). We only found one differentially expressed gene (FDM2) that acts redundantly with five other genes.

This does not answer the question. A mutation in one of the RdRM genes will not necessarily alter expression level (nonsense mediated decay could occur, but not necessarily) or chromatin.

https://doi.org/10.7554/eLife.58533.sa1

Author response

Reviewer #1:

The contribution by Antunez-Sanchez et al. demonstrates that histone demethylases REF6 and ELF6 have distinct roles in the histone demethylation in Arabidopsis. REF6 has a broader impact in reducing H3K27me3 and is also a major player in the deposition of H3K27me1 in active euchromatin regions. The maintenance of H3K27me1 in active chromatin tends to allow a low-level basal expression. In addition, this study also showed that the failure to reset H3K27me marks during sexual reproduction results in the inheritance of H3K27me3 imprints, which is associated with the loss of DNA methylation at heterochromatic loci, leading to TE activations. Overall, the manuscript covers new roles of REF6 and ELF6 and biological relevance of H3K27me1/me3 homeostasis in genome integrity and transcription activity. However, several flaws should be addressed to show the importance of this study.

1) What tissues were used for each analysis? Genetic interactions between ELF6 and REF6 vary as shown in phenotypic analysis. Therefore, plant tissues and age should be described in figure legends.

In the revised Materials and methods, we have included information about the age and type of plant tissues used.

2) They suggested the importance of H3K27me1 homeostasis in gene expression. I am wondering if the H3K27me1 imprints can be transmitted to offspring in wild type. Otherwise, how the histone mark could be reset. Is there another responsible histone demethylase that removes H3K27me1? In addition, the relevance of H3K27me1 in genome stability is limited, and interpretation about epi-mutants relies mainly on ectopic accumulation of H3K27me3. Although they emphasize their finding about REF6-catalyzed H3K27me1 as shown in Figure 7, the analysis of H3K27me1 was not extensive.

The reviewer makes an interesting point. Although it is not yet known how removal of H3K27me1 occurs and which proteins are involved in this process, it has been recently proposed that this might occur during replacement by the histone H3.10 variant (Borg et al., 2020).

We have investigated the relationship between H3K27me3, H3K27me1 and DNA methylation in epiER plants and found a negative correlation between H3K27me3 and the other two epigenetic marks (see heatmap analysis in Author response image 1). We do not think these results affect our current hypothesis but are happy to include these data if deemed necessary.

Author response image 1
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3) Do REF6 and ELF6 have critical roles in silencing pericentromoric regions? How about H3K27me3 and H3K27me1 levels in pericentromeric regions in ref6 and elf6 mutants?

Analysis of H3K27me3 and H3K27me1 in ref6-5, elf-C and elf6-C/ref6-5 did not reveal significant changes in pericentromeric regions compared to WT, thus indicating that these histone demethylases do not play a major role in the regulation of chromatin at these genomic regions. Moreover, the binding of these histone demethylases to chromatin is thought to be prevented by DNA methylation (Qui et al., 2019), therefore their role in silencing pericentromeric regions in somatic tissues appears to be limited.

4) Ectopic accumulation of H3K27me3 leads to global reductions in DNA methylation at pericentromeric regions. To support the idea that hypomethylation caused by ectopic accumulation of H3K27me3 depends on RdDM pathways, they should use mutants of RdDM pathways. In addition, if REF6 and ELF6 are particularly relevant in histone modification at active chromatin, what mechanisms could be involved in changes in DNA methylation at pericentromeric regions? I think that DNA methylation could be indirect and stochastic results.

We thank the reviewer for this comment. We clarified these points in the revised Discussion and explained briefly below:

Deleris et al., 2012 have shown that in the Arabidopsis met1 mutant impaired for CG methylation, hundreds of TEs that lost DNA methylation also gained H3K27me3. Recently, Rougée et al. (2020) have also shown that numerous TEs gain H3K27me3 in response to ddm1-induced loss of DNA methylation. Collectively, these studies suggest that DNA methylation can antagonize H3K27me3 deposition at TEs and that transposon sequences can be marked by H3K27me3 through the activity of the PRC2 complex. In addition, our data show that a gain on H3K27me3 can induce a loss of DNA methylation at TEs. Collectively, our data combined with these reports suggest that these two epigenetic pathways act antagonistically at TE sites. The precise mechanism(s) implicated in changes in DNA methylation at pericentromeric regions are not yet fully understood but we hypothesize that these histone demethylases may be required to protect DNA demethylated regions from the activity of the PRC2 complexes during the reprogramming of heterochromatic marks thought to take place during plant gametogenesis.

5) Reduction of H3K27me1 could be linked to global reductions in DNA methylation? There is no experiment to rule out the possibility.

H3K27me1 is linked to DNA methylation primarily in centromeric and pericentromeric transposon-related sequences but not on euchromatin. Our data shows that the accumulation of H3K27me3 and reduction of H3K27me1 in ref6-5 mutant is primarily on genic regions (REF6 targets) usually lacking DNA methylation. For these reasons, we did not consider it necessary to perform the experiment suggested by the reviewer.

6) Individual epi-mutants have variable phenotypes, and genetic backgrounds are not likely uniform. I am wondering if GO analysis is meaningful in this situation. It could be very different depending on individuals.

We believe the GO analysis is informative because if specific gene networks are affected in epimutants, which differ in their epigenetic background, it suggests that the genomic regions affected are non-random.

7) Figure 4E: Please quantify the results, rather than showing representative images.

Quantification of nuclear chromatin condensations was provided in Figure 4—figure supplement 3.

8) Figure 7: The summary figure should show main finding of this paper. I think only the fraction of this study is shown, and this makes reader confusing. The authors should rearrange the manuscript and figures and give a more focused view of the conclusion.

We have revised the Discussion and summary figure (Figure 7) to highlight the two main findings of our study.

Reviewer #2:

General Assessment

The manuscript by Antunez and colleagues provide important biological insights into the role of DNA demethylase in maintaining genetic and epigenetic stability between different generations. In addition, this study also shed light on the contribution of H3K27me2/3 demethylases toward in vivo levels of H3K27me1 in plants. I think this is important work that should be of general interest to the plant epigenetic community. The manuscript is well-written and the conclusions are substantiated.

Substantive concern:

1) One concern with the manuscript is the mechanism leading to epimutations that is proposed by the authors, which is that ectopic inheritance of H3K27me3 in gametes leads to the different phenotypes observed. The work of Olivier Mathieu and his colleagues in 2005 has shown that loss of DNA methylation in ddm1 mutants leads to ectopic gains of H3K27me3 in heterochromatin. Transcriptional reactivation of transposons and transposition (e.g. Tsukahara et al., Nature 2009) is also observed in ddm1 mutants like in the EpiER lines. Therefore, it would be useful for the authors to investigate if DNA methylation is impaired because of genome-wide gains in H3K27me3 in gametes, or whether one or a few affected loci are responsible for the loss of DNA methylation. The authors try to address this point (e.g. subsection “Accumulation of ectopic H3K27me3 at centromeric heterochromatin is linked to DNA hypomethylation”), however, more information should be provided.

Our data support the view that DNA methylation is impaired because gametes gain H3K27me3. This hypothesis is supported by a recent report that has shown that histone demethylase mutants cause a significant increase in H3K27me3 in male gametes (Borg et al., 2020).

They should indicate which genes in the DNA methylation pathways have been assessed for H3K27me3 levels. In addition, the transcriptional status of these genes should be assessed (the data is already available from this study) to determine if loss of DNA methylation is due to down-regulation of DNA methylation genes. Transcriptional silencing of these genes could be achieved indirectly, if no gains in H3K27me3 are observed at the genes.

We have checked the chromatin (H3K27me3 and H3K27me1) and expression profiles of genes implicated in DNA methylation in our histone demethylase mutants and in two epiERs. We only found one common differentially expressed gene (FDM2), which is thought to act redundantly with five other genes (FDM1, FDM3, FDM4 and FDM5) in RNAdirected DNA methylation (Xie et al., 2012). Moreover, although this gene is downregulated in ref6-5 and elf6-C/ref6-5, it does not cause significant changes in DNA methylation.

Author response image 2
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2) Results: It's not clear to me looking at Figure 2E and Figure 2—figure supplement 5 that REF6 is "…required for low-level expression genes." I think those figures indicate that these genes (3rd and 5th quantiles) gains H3K27me3, but the transcriptional impact of the increase of H3K27me3 at these genes is not provided. Since they have transcriptional data for ref6 mutants, the authors could look if gains in H3K27me3 at these genes decrease their expression.

When we select the genes hypermethylated at H3K27me3 in ref6-5 mutants and split them by decile of expression in WT, we observed a significant decrease (Bonferroni adjusted p-value < 0.0001) in the expression between WT plants and ref6-5 mutants for all deciles; deciles 3 to 5 included.

Author response image 3
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Reviewer #3:

[…]

Overall I found this a very interesting paper. The role of HDM in euchromatic H3K27me1 is novel and intriguing, perhaps difficult to assess the significance as the mechanism of H3K27me1 action is not known in plants, i.e. readers etc for this mark have not been identified. The observation that ref elf mutants may give rise to epialleles that persist even when REF6 ELF6 activity is restored is of broad interest, and builds on studies such as Crevillen et al. (Nature 515: 587) which showed that elf6 impairment can give rise to heritable epigenetic changes (but in this case only in the elf6 mutant background). On the negative side, the two parts of the paper are not very strongly connected, for example it is not very clear if changes in H3K27me1 described in part one are relevant for the phenotypic and epigenetic alterations described in part two. Also, there is very limited description of the phenotypes found and the causes are not very clear i.e. alterations in which genes are causal and what are the genetic or epigenetic changes involved? It is not very clear why ref6/elf6 depletion gives rise to the effects observed, for example one of the major changes in hyper H3K27me3 methylation and loss of DNA methylation at pericentromeric heterochromatin, yet ref6 elf6 mutants do not seem to show this change. Overall, the observations are interesting, they have done a lot of analysis and it is unreasonable to expect a complete explanation but the overall picture remains confusing. As such I think the paper is borderline for eLife, and would at least require some revision along the lines below.

1) Figure 3A shows plants with aberrant phenotypes and their transmission, however there is no description of the phenotypes, or of their inheritance i.e. what kind of segregation ratios are observed in the different generations. For example, A5 looks to be broad leaved but gives rise to plants with narrow curled leaves in F3 generation, it is not clear if these are transmitted into F4 generation or at what frequency, or if traits are dominant or recessive.

We have added a description of the phenotypes observed and the ratios at which they appear. We have also added a figure (Figure 3—figure supplement 1 phenotypes of segregating plants).

2) It's not very clear which generation is used for the various profiles of A5 progeny, possibly F4 or F5 plants?

We have clarified in the text that F5 plants were used for the ChIP-seq, RNA-seq and bulk BS-seq, and that F4 plants were used for the BS-seq of individual plants.

3) Much of the data is not accessible or presented. For example, no lists of the peaks found for the different marks profiled are provided, nor are there lists of differential peaks found, or how these related to genes showing altered expression.

All the raw data has been uploaded in a public repository. We don’t think it is necessary to upload all of the peak data as supplemental information.

4) In Figure 3D the wild type trace is invisible, either omitted or masked by another trace?

Figure 3D represents the difference in H3K27me3 levels between the epiER lines and WT plants, 0 in the y axis represents no difference to WT. We have changed the y axis label to clarify this.

5) The recent paper from Berger group on reprogramming in sperm (Borg et al., 2020) could be referenced and discussed. This study would suggest that H3K27me3 hypermethylation should not be transmitted paternally once REF6/ELF6 activity is restored. It would be interesting to know if the phenotypes and or epigenetic changes seen in A5 can be transmitted maternally, paternally or both once RER6/ELF6 activity is restored. Given that some or all of the effects of REF6/ELF6 depletion may be indirect effects of H3K27me3 changes, for example TE mobilisation or DNA hypomethylation, it is possible that the phenotypes would be transmitted but this might give some indication of whether H3K27me3 changes are causal for the phenotypes or not, or indirect. If the phenotypes are recessive, then this would require both paternal and maternal transmission in self progeny.

We have discussed the data that was published by Borg et al., 2020 while our manuscript was under review. Their data support our view that the accumulation of H3K27me3 could have profound consequences in the reprogramming of other epigenetic marks. Our genetic data suggest that histone methylases are required in the male and female germline to prevent the accumulation of epigenetic defects in the offspring.

6) Subsection “REF6 controls H3K27me1 homeostasis in chromatin” states that H3K27me1 is not affected at euchromatic gene in atxr5 atxr6 mutants, however in Figure 2—figure supplement 2 it looks as if levels are actually increased in the mutant. This should be commented on and discussed. It should also be mentioned that the particular atxr5 atxr6 double mutant combination is hypomorphic and not null.

We have clarified these points in the revised manuscript.

7) Figure 6F shows that line A5.B1.3 which has a seed pigmentation phenotype has a 5 bp insertion in the tt4 gene. It is stated that this is due to transposon remobilisation but it is not clear if this is inferred as a likely imprecise excision, or whether they are able to show that a TE is present in this location in a progenitor plant?

The 5 bp insertion in TT4 resembled a footprint caused by CACTA remobilisation. A similar footprint is shown in Figure 6E. In the case of TTG4, we did not find the intact transposon in plants from previous generations.

8) Given that a lot of sequence data is available for some of the lines, I wondered if authors should check that there are no mutations present in genes associated with RdRM etc? That is not intended as a slight, but since changes in DNA methylation are observed that don't readily correlate with what is seen in the ref6 elf6 parental lines it could presumably be easily checked and eliminated as a potential cause.

We have investigated the chromatin (H3K27me3 and H3K27me1) and expression profile of gene implicated in DNA methylation genes, both in our histone demethylase mutants and also in epiERs (see heatmap in response to reviewer 2). We only found one differentially expressed gene (FDM2) that acts redundantly with five other genes (FDM1, FDM3, FDM4 and FDM5) in RNA-directed DNA methylation (Xie et al., 2012).

9) The correlation between pericentromeric H3K27me3 increase and DNA methylation decrease is an interesting observation, but they provide no mechanism why this should arise in elf6 ref6 backgrounds, which they show seem normal for pericentromeric DNA methylation and H3K27me3. The Qiu et al. (2019) Nat Comm paper, which the authors here mention in the Introduction, reported that REF6 cannot bind efficiently on methylated DNA, so it is puzzling.

Our data combined with recent work (Borg et al., 2020) supports the hypothesis that an increase in H3K27me3 levels in histone demethylase mutants causes defects on the epigenomic reprogramming that takes place during gametogenesis, primarily at pericentromeric regions.

10) Lines were picked based on aberrant phenotypes, propagated, and then analysed and found to contain various epigenetic and genetic changes. It is difficult to know whether the changes cause the phenotypes seen, and if so which particular changes. This made me wonder to what extent ELF6 REF6 F2 progeny with normal phenotypes would also show epigenomic changes, or whether this is specific to the plants with phenotypic abnormalities?

For practical reasons we did not perform epigenomic analyses in plants from ELF6 REF6 progenies that were indistinguishable from wild-type. We cannot exclude that “normal-looking” plants may contain a small number of epigenomic modifications, but this does not affect any of our conclusions.

11) The fact that ref6 and elf6 have opposite effects on flowering time is curious. The elf6c mutant is early flowering, consistent with a role for ELF6 in binding the floral repressor FLC and promoting its expression by H3K27me3 demethylation (Mol Plant 11:1135). The ref6 mutant is late flowering, and has been correlated with increased FLC expression (Plant Cell 16: 2601), which seems at odds with the hypermethylation for H3K27me3 and decreased expression more typical of these mutants. This might be commented on, and whether the various profiling experiments offer any explanation.

We agree that it would be interesting to investigate why ref6-5 and elf6-C have opposing effects on flowering but in order to define the precise mechanism(s) it would be desirable to conduct a detailed analysis, which is beyond the scope of the current study.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Reviewer #3:

The authors have provided some new data, ChIP seq of H3K27me1 in clf swn mutants, which provides some support for their model that euchromatic H3K27me1 derives from demethylation by REF6 of H3K27me3 deposited by PRC2 and is a good addition. Overall I find it an interesting paper of broad general interest but don't feel that the revision greatly addresses the comments from before as to mechanism and significance of H3K27me1 being unclear. That said, there is a lot of data in the paper and it is an interesting story that is perhaps complementary to the Borg et al., 2020 paper.

I have issues with a couple of the points made by authors.

3) Much of the data is not accessible or presented. For example, no lists of the peaks found for the different marks profiled are provided, nor are there lists of differential peaks found, or how these related to genes showing altered expression. All the raw data has been uploaded in a public repository. We don't think it is necessary to upload all of the peak data as supplemental information.

It is a given that the raw data will be provided. Since there is a lot of discussion of the ChIP seq data and selected peaks etc are shown I think it reasonable that the authors provide lists of enriched genes for the various ChIP seq experiments, as is usually provided in other papers.

We have included in the revised manuscript a table with a list of genes enriched for the different ChIP-seq experiments.

8) Given that a lot of sequence data is available for some of the lines, I wondered if authors should check that there are no mutations present in genes associated with RdRM etc? That is not intended as a slight, but since changes in DNA methylation are observed that don't readily correlate with what is seen in the ref6 elf6 parental lines it could presumably be easily checked and eliminated as a potential cause.

We have investigated the chromatin (H3K27me3 and H3K27me1) and expression profile of gene implicated in DNA methylation genes, both in our histone demethylase mutants and also in epiERs (see heatmap in response to reviewer 2). We only found one differentially expressed gene (FDM2) that acts redundantly with five other genes (FDM1,

This does not answer the question. A mutation in one of the RdRM genes will not necessarily alter expression level (nonsense mediated decay could occur, but not necessarily) or chromatin.

In our previous response, we provided evidence that the expression of RdDM genes is not affected in our histone demethylase mutants nor in the epimutants. We have also shown in our previous response that RdDM genes do not accumulate chromatin changes (H3K27me1 and H3K27me3) in these plants. We have conducted a thorough mutation analysis in our histone demethylase mutants and epiERs but we have found no evidence of mutations in any of the genes implicated with RdDM or other DNA methylation pathways.

In addition, the fact that some epiER plants display partial restoration of DNA methylation at transposons and pericentromeric chromatin regions (See Figure 3B-C for details) lends further support that the changes in DNA methylation observed in epiERs are not caused by mutations in RdDM genes.

https://doi.org/10.7554/eLife.58533.sa2

Article and author information

Author details

  1. Javier Antunez-Sanchez

    School of Life Science, University of Warwick, Coventry, United Kingdom
    Contribution
    Conceptualization, Investigation, Writing - original draft
    Contributed equally with
    Matthew Naish and Juan Sebastian Ramirez-Prado
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7485-7061

  2. Matthew Naish

    School of Life Science, University of Warwick, Coventry, United Kingdom
    Present address
    Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom
    Contribution
    Conceptualization, Investigation, Writing - original draft
    Contributed equally with
    Javier Antunez-Sanchez and Juan Sebastian Ramirez-Prado
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8977-1295

  3. Juan Sebastian Ramirez-Prado

    Université Paris-Saclay, CNRS, INRAE, Univ Evry, Institute of Plant Sciences Paris-Saclay (IPS2), Orsay, France
    Present address
    Centre of Microbial and Plant Genetics, KU Leuven, PSB-VIB.Center for Plant Systems Biology, Leuven, Belgium
    Contribution
    Investigation, Visualization, Methodology, Writing - original draft
    Contributed equally with
    Javier Antunez-Sanchez and Matthew Naish
    Competing interests
    No competing interests declared

  4. Sho Ohno

    1. School of Life Science, University of Warwick, Coventry, United Kingdom
    2. Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan
    Contribution
    Investigation, Visualization, Writing - original draft
    Competing interests
    No competing interests declared

  5. Ying Huang

    Université Paris-Saclay, CNRS, INRAE, Univ Evry, Institute of Plant Sciences Paris-Saclay (IPS2), Orsay, France
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  6. Alexander Dawson

    School of Life Science, University of Warwick, Coventry, United Kingdom
    Contribution
    Investigation, Visualization
    Competing interests
    No competing interests declared

  7. Korawit Opassathian

    School of Life Science, University of Warwick, Coventry, United Kingdom
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  8. Deborah Manza-Mianza

    Université Paris-Saclay, CNRS, INRAE, Univ Evry, Institute of Plant Sciences Paris-Saclay (IPS2), Orsay, France
    Contribution
    Data curation, Formal analysis
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3374-0200

  9. Federico Ariel

    Université Paris-Saclay, CNRS, INRAE, Univ Evry, Institute of Plant Sciences Paris-Saclay (IPS2), Orsay, France
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  10. Cecile Raynaud

    Université Paris-Saclay, CNRS, INRAE, Univ Evry, Institute of Plant Sciences Paris-Saclay (IPS2), Orsay, France
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  11. Anjar Wibowo

    Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany
    Present address
    Faculty of Science and Technology, Airlangga University, Kampus C, Mulyorejo, Indonesia
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  12. Josquin Daron

    Department of Molecular Genetics, The Ohio State University, Columbus, United States
    Present address
    Laboratoire MIVEGEC (Université de Montpellier-CNRS-IRD), Montpellier, France
    Contribution
    Software, Investigation
    Competing interests
    No competing interests declared

  13. Minako Ueda

    1. Institute of Transformative Bio-Molecules, Nagoya University, Nagoya, Japan
    2. Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan
    Present address
    Tohoku University, Graduate School of Life Sciences, Sendai, Japan
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  14. David Latrasse

    Université Paris-Saclay, CNRS, INRAE, Univ Evry, Institute of Plant Sciences Paris-Saclay (IPS2), Orsay, France
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared

  15. R Keith Slotkin

    1. Donald Danforth Plant Science Center, St. Louis, United States
    2. Division of Biological Sciences, University of Missouri, Columbia, United States
    Contribution
    Resources, Project administration
    Competing interests
    No competing interests declared

  16. Detlef Weigel

    Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany
    Contribution
    Resources, Supervision, Funding acquisition, Project administration, Writing - review and editing
    Competing interests
    Senior editor, eLife
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2114-7963

  17. Moussa Benhamed

    1. Université Paris-Saclay, CNRS, INRAE, Univ Evry, Institute of Plant Sciences Paris-Saclay (IPS2), Orsay, France
    2. Université de Paris, Institute of Plant Sciences Paris-Saclay (IPS2), F-75006, Paris, France
    Contribution
    Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    moussa.benhamed@u-psud.fr
    Competing interests
    No competing interests declared

  18. Jose Gutierrez-Marcos

    School of Life Science, University of Warwick, Coventry, United Kingdom
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    j.f.gutierrez-marcos@warwick.ac.uk
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5441-9080

Funding

European Commission (AUREATE)

  • Jose Gutierrez-Marcos

Biotechnology and Biological Sciences Research Council (BB/L003023/1)

  • Jose Gutierrez-Marcos

Japan Society for the Promotion of Science (JP19H05676)

  • Minako Ueda

Agence Nationale de la Recherche (EpiGen)

  • Moussa Benhamed

Biotechnology and Biological Sciences Research Council (BB/N005279/1)

  • Jose Gutierrez-Marcos

Biotechnology and Biological Sciences Research Council (BB/N00194X/1)

  • Jose Gutierrez-Marcos

Biotechnology and Biological Sciences Research Council (BB/P02601X/1)

  • Jose Gutierrez-Marcos

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Gary Grant for help with plant husbandry; Xiaofeng Cao and Caroline Dean for seed stocks and data. Ranjth Papareddy for the identification of ref6-5 and Liliana M Costa for discussions and comments on the manuscript. Supported by ERA-CAPS Program (Project AUREATE), and the Max Planck Society (DW), ANR/CNRS grant (EpiGEN) to MB, JSPS grant (JP19H05676) to MU and BBSRC grants (BB/L003023/1, BB/N005279/1, BB/N00194X/1 and BB/P02601X/1) to JG-M.

Senior Editor

  1. Christian S Hardtke, University of Lausanne, Switzerland

Reviewing Editor

  1. Pil Joon Seo, Seoul National University, Republic of Korea

Reviewer

  1. Yannick Jacob

Version history

  1. Received: May 4, 2020
  2. Accepted: October 26, 2020
  3. Accepted Manuscript published: October 27, 2020 (version 1)
  4. Version of Record published: November 17, 2020 (version 2)

Copyright

© 2020, Antunez-Sanchez et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Javier Antunez-Sanchez
  2. Matthew Naish
  3. Juan Sebastian Ramirez-Prado
  4. Sho Ohno
  5. Ying Huang
  6. Alexander Dawson
  7. Korawit Opassathian
  8. Deborah Manza-Mianza
  9. Federico Ariel
  10. Cecile Raynaud
  11. Anjar Wibowo
  12. Josquin Daron
  13. Minako Ueda
  14. David Latrasse
  15. R Keith Slotkin
  16. Detlef Weigel
  17. Moussa Benhamed
  18. Jose Gutierrez-Marcos
(2020)
A new role for histone demethylases in the maintenance of plant genome integrity
eLife 9:e58533.
https://doi.org/10.7554/eLife.58533

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