H3K4me1 directs H3K36me2 and H3K36me3 deposition in land plants

Domain: chemistry
Published: 2026-02-16
Editorial outcome: minor_revision

Abstract
Monomethylation of histone 3 lysine 4 (H3K4me1) marks enhancers in mammals. However, the function of H3K4me1 in plants remains largely unclear. Here, we present the genome-wide distribution of H3K4me1 in diverse species across evolution, revealing a distinctive H3K4me1 distribution pattern in land plants. To explore the function of H3K4me1 in plants, we identified an H3K4me1-specific reader protein, Early heading date 3 (Ehd3), and solved the structure of Ehd3 in complex with the H3K4me1 peptide, revealing a unique binding module differing from the previously reported PHD finger proteins. We further identified an Ehd3-binding protein, SET domain group 724 (SDG724), and the deletion of either Ehd3 or SDG724 caused similar defects in plant phenotype and changes in transcriptome and epigenome profiles. Both Ehd3 and SDG724 are enriched at chromatin regions marked by H3K4me1 but not H3K4me2 or H3K4me3. Ehd3 activates the H3K36 methyltransferase SDG724, and H3K36me2/me3 are colocalized with H3K4me1 in the genomes of the examined land plants. Collectively, our results reveal that H3K4me1 directs the establishment of H3K36me2 and H3K36me3 in land plants.

Introduction
Histone post-translational modifications (PTMs) contribute to transcriptional regulation and play essential roles in diverse biological processes, including development and response to environmental cues1,2. These modifications, including methylation, acetylation, phosphorylation, and ubiquitination, are dynamically established and removed by specific writer and eraser enzymes, respectively, and are recognized by distinct reader proteins3,4. Methylation of histone 3 lysine 4 (H3K4) is among the most extensively studied PTMs that confers active or repressive transcription depending on its status (mono-, di-, or trimethylation)5. H3K4 trimethylation (H3K4me3) is distributed around the transcription start site (TSS) and is associated with gene activation in yeast6, animals7,8, and plants9. H3K4 dimethylation (H3K4me2) is diversely distributed in eukaryotes: it is enriched at the TSS and associated with gene activation in yeast6 and humans7,8, whereas it is located in the gene body region and linked to gene repression in plants9. H3K4 monomethylation (H3K4me1) is enriched over the gene body in yeast6 and plants9, but tends to be concentrated around the TSS in mammals7,8. In addition, H3K4me1 is associated with other PTMs that mark distinct states of enhancers in mammals. Active enhancers are enriched with H3K4me1 and histone 3 lysine 27 acetylation (H3K27ac)10. During the intermediate state, primed enhancers primarily exhibit enrichment of H3K4me111. Poised enhancers in pluripotent cells are marked by H3K4me1 and H3K27me312. However, the deposition of H3K4me1 at enhancers is not conserved in plants13 and the role of H3K4me1 in plants has yet to be elucidated.

Reader proteins possess specific domains that recognize H3K4 methylation, including the plant homeodomain (PHD), double chromodomain, tandem Tudor domain, and zinc finger CW3. To date, a number of structures of PHD finger–H3K4me2/me3 complexes have been determined in animals and plants, whereas the PHD finger in complex with H3K4me1 has not been reported in plants and exclusive readers for H3K4me1 are lacking in animals14. The structures of CW or Tudor domains complexed with H3K4me1 have been reported in plants, including the CW domain of H3K36 methyltransferase SET DOMAIN GROUP 8 (SDG8)15 and the Tudor domain of RNA-directed DNA methylation15 (RDM15)16 in Arabidopsis. Nevertheless, the biological function of H3K4me1 decoding in plants remains largely uncharacterized.

Here, we identified a distinct distribution pattern of H3K4me1 in land plants that differs from that observed in yeast and animals. A newly identified PHD finger domain-containing protein, Early heading date 3 (Ehd3), specifically recognizes H3K4me1 and recruits the H3K36 methyltransferase SDG724 to facilitate H3K36me2/me3 deposition. The colocalization of H3K4me1 and H3K36me2/me3 is conserved in the examined land plants, revealing a specific role of H3K4me1 in directing H3K36me2/me3 deposition in plants.

Results
H3K4me1 exhibits a distinct genome-wide profile in land plants
To determine the function of H3K4me1 in plants, we analyzed the genome-wide distributions of H3K4me1/me2/me3, and the correlation between H3K4 methylations and gene transcription by conducting chromatin immunoprecipitation followed by sequencing (ChIP-seq) and RNA-sequencing (RNA-seq) assays in a variety of plant species, comprising the unicellular aquatic Chlamydomonas reinhardtii, the early land plant Physcomitrium patens, and the flowering plants Arabidopsis thaliana and Oryza sativa. By integrating previously published ChIP-seq and RNA-seq data for the unicellular yeast Saccharomyces cerevisiae, the invertebrate Drosophila melanogaster, and the vertebrates Mus musculus and Homo sapiens17–21, we analyzed the genome-wide H3K4me1/me2/me3 profiles across diverse organisms (Fig. 1 and Supplementary Fig. 1).

We observed that H3K4me1 exhibited particularly distinct characteristics across the examined species. In animals, comprising Drosophila melanogaster, Mus musculus, and Homo sapiens, H3K4me1 was enriched near the TSS and gradually decreased along the gene body. In contrast, H3K4me1 was mainly enriched in gene bodies in Chlamydomonas reinhardtii, Physcomitrium patens, Arabidopsis thaliana, and Oryza sativa as previously reported22 (Fig. 1a, b). In yeast and animals, a positive correlation was observed between H3K4me1 and gene transcription (Fig. 1c). In Chlamydomonas reinhardtii, a weak correlation between H3K4me1 and gene transcription was detected. In contrast, the land plants Physcomitrium patens, Arabidopsis thaliana, and Oryza sativa displayed a distinctive relationship between H3K4me1 and gene transcription: a high percentage of genes displayed a positive correlation between H3K4me1 and gene transcription, but the most highly expressed genes exhibited lower levels of H3K4me1 (Fig. 1c). Thus, land plants displayed a unique genome-wide distribution pattern for H3K4me1, and a distinct correlation between H3K4me1 and gene transcription.

Unlike animals, in which two peaks around the TSS were observed, H3K4me2 was distributed over the entire gene body in land plants (Supplementary Fig. 1a, b). Consistent with a previous study, a positive correlation between H3K4me2 level and gene transcription level was observed in yeast and animals (Supplementary Fig. 1c). However, H3K4me2 was negatively correlated with gene transcription in land plants, consistent with our previous report9 (Supplementary Fig. 1c). The H3K4me3 distributions in all investigated species were similar, peaking close to the TSS (Supplementary Fig. 1d, e). Moreover, H3K4me3 consistently displayed a positive correlation with gene transcription in all species examined, highlighting the conserved association of H3K4me3 with gene activation in eukaryotes (Supplementary Fig. 1f).

Ehd3, a PHD finger domain-containing protein, specifically recognizes H3K4me1
The unique distribution pattern and distinct correlation with gene transcription suggested that H3K4me1 may play a unique role in land plants. Given that the epigenetic information encoded by histone PTMs is interpreted by specialized reader proteins, we thus investigated the function of H3K4me1 in land plants by searching for its specific reader proteins. There are multiple plant homeodomain (PHD) domain-containing proteins in plants and PHD domains are widely reported to recognize H3K4 methylations, thus we analyzed the PHD domain-containing proteins in Chlamydomonas reinhardtii and Physcomitrium patens, aiming to discover the potential H3K4me1 readers conserved in land plants but not in Chlamydomonas reinhardtii. We identified 52 and 107 proteins containing at least one PHD domain in Chlamydomonas reinhardtii and Physcomitrium patens, respectively. Notably, this expansion in Physcomitrium patens was particularly pronounced for proteins containing multiple PHD domains (n≥2) (Supplementary Fig. 2a). Proteins containing triple-tandem PHD domains were presented in both Chlamydomonas reinhardtii and Physcomitrium patens, however, those with double-tandem PHD were unique in Physcomitrium patens (Supplementary Fig. 2b). Therefore, our searching focused on the proteins containing double-tandem PHD domains. Our phylogenetic analysis showed that a protein (A0A2K1L966) evolved a double-tandem PHD domain in Physcomitrium patens, which was absent in Chlamydomonas reinhardtii but conserved in land plants (Fig. 2a). For in-depth functional study, we selected a homolog of A0A2K1L966, Ehd3, which is known to regulate flowering time in rice23 (Fig. 2b). To explore the histone modification decoding mode of Ehd3, we purified the full-length recombinant Ehd3 protein and performed an in vitro binding assay using a histone peptide microarray with diverse histone PTMs. Ehd3 exhibited a strong preference for binding to H3K4me1 over other modifications (Supplementary Fig. 2c). A biotinylated histone peptide pulldown assay similarly showed that Ehd3 interacted exclusively with the H3K4me1 peptide, but not with other tested modifications, including H3K4me0, H3K4me2, and H3K4me3 (Fig. 2c and Supplementary Data 1). Further investigation showed that individual PHD1, PHD2, or PHD3 failed to bind to H3K4me1, whereas the tandem PHD finger domain (PHD2–PHD3) was essential and sufficient for recognition of H3K4me1 (Fig. 2d). Isothermal titration calorimetry (ITC) analysis further confirmed robust interaction between Ehd3 and the H3K4me1 peptide with a dissociation constant (Kd) of 1.7 μM (Fig. 2e and Supplementary Data 1–2). In summary, Ehd3 specifically recognized H3K4me1 through its tandem PHD finger domain.

Structure of Ehd3 tandem PHD finger domain in complex with H3K4me1 peptide
To clarify the molecular mechanism underlying Ehd3 recognition of H3K4me1, we solved the crystal structure of Ehd3 417–547 bound to a H3K4me1 peptide (ART-Kme1-QTARKSTG) at atomic resolution (1.5 Å, Supplementary Data 3). Ehd3 418–540 adopts an L-shaped overall architecture and the peptide binds across an acidic groove formed by the PHD2 and PHD3 domains (Fig. 3a). Both PHD2 and PHD3 of Ehd3 adopt interleaved topologies chelating two Zn2+ ions (Supplementary Fig. 2d, e), while the H3K4me1 peptide adopts an extended conformation (Supplementary Fig. 2f, g). Partial atoms of Gln5, Arg8, and Lys9 are invisible in the map, suggesting flexible disposition of these residues (Supplementary Fig. 2f, g). The side chains of Ala1 and Arg2 of the H3K4me1 peptide reside in two surface channels separated by Asp493 of Ehd3. The monomethylated Lys4 inserts into a deep pocket adjacent to the Arg2-binding channel, forming a ‘Y’-shaped conformation with Ala1 and Arg2 that anchor the binding interface (Fig. 3b). The main-chain amino group of Ala1 forms two stable hydrogen bond (H-bond) interactions with the main-chain carbonyl groups of Pro512 and Gly514, respectively, whereas the methyl group of Ala1 forms a hydrophobic interaction with the aromatic ring of Pro512 (Supplementary Fig. 2h). The main-chain amide group and carbonyl group of Arg2 form two water-mediated H-bonds and one direct H-bond interaction with Met491 and Asp493, respectively. Via the guanidyl group, the side chain of Arg2 forms three stable H-bond interactions with Tyr446, Cys492, and Asp496 (Fig. 3c and Supplementary Fig. 2i, j).

In contrast to trimethylated Lys4-binding pockets present in many PHD proteins, which are composed of several conserved aromatic residues within a single PHD domain (Supplementary Fig. 3), the monomethylated Lys4-binding pocket of Ehd3 is formed by residues from both PHD2 (His441, Tyr471, and Cys475) and PHD3 (Asp483, Asp486, and Ile489) (Fig. 3d and Supplementary Fig. 2j, k). The side-chain primary amino group of monomethylated Lys4 forms H-bond interactions with the main-chain carbonyl group of PHD2 Cys475 and the side-chain carbonyl group of PHD3 Asp486. The side-chain methyl and alkyl groups of Lys4 hydrophobically interact with the side chains of PHD2 Tyr471 and His441, respectively. The main chains of Lys4 and PHD3 Ile489 form a H-bond and the side chain of Ile489 hydrophobically interacts with the side-chain alkyl groups of Lys4. The phenolic hydroxyl group of PHD2 Tyr471 forms a H-bond interaction with the side-chain carbonyl group of PHD3 Asp483 to stabilize the binding pocket (Fig. 3d and Supplementary Fig. 2j). As a result, the side chain of monomethylated Lys4 perpendicularly and compactly inserts into the pocket, which harbors a hydrophobic environment created by PHD2 and a polar environment created by PHD3 (Fig. 3e). Our modeling study suggests that the introduction of a second or third methyl group to Lys4 will lead to a clash with Cys475 and/or Asp486, which will prevent the binding of Ehd3 to H3K4me2 or H3K4me3, implying the H3K4me1 specificity of Ehd3 (Fig. 3f). In addition to the monomethylated Lys4, the side-chain imidazole ring of PHD2 His441 forms one stable H-bond interaction with the main-chain carbonyl group of Gln5 of the peptide. Residues 6–9 of the peptide mainly form direct or water-mediated H-bond interactions with symmetry-related Ehd3 molecules, whereas residues 10–12 of the peptide are completely disordered in the structure (Supplementary Fig. 2j, l, m). Consistent with the structural observation, mutation of H3R2-interacting residues (Tyr446 and Asp496) or H3K4me1 binding-pocket residues (His441, Tyr471, Asp486, and Ile489) either abolished or markedly reduced the binding affinity of the Ehd3 tandem PHD finger to the H3K4me1 peptide (Fig. 3g and Supplementary Data 2), which further confirmed that both H3R2-binding and H3K4me1-binding residues are critical for H3K4me1 recognition by Ehd3. The dissociation constant of the wild-type Ehd3 tandem PHD to H3K4me1 peptide was comparable to that of the full-length Ehd3 (Figs. 2e and 3g), excluding the potential contribution of PHD1 to specific recognition of H3K4me1.

Given that Double PHD Finger 3b (DPF3b) is a well-studied epigenetic factor that recognizes histone modification through a tandem PHD finger domain24, we generated a sequence alignment of the tandem PHD of Ehd3 and DPF3b. Most H3K4me1-binding sites in Ehd3 were not conserved in DPF3b (Supplementary Fig. 3a). The tandem PHD finger domain in Ehd3 forms a narrow and sealed pocket, whereas DPF3b relies on a single PHD finger domain to create an open pocket for H3K14ac binding25 (Fig. 3e and Supplementary Fig. 3c, d). In addition, we compared the reported structures of the H3K4me3-binding PHD finger domains in the Bromodomain PHD finger transcription factor (BPTF)26, PHD finger protein 2 (PHF2)27, YNG128, and Inhibitor of growth protein 2 (ING2)29 with that of Ehd3–H3K4me1. The interaction between the PHD finger domain and H3K4me3 is highly conserved in these proteins and the critical residues are not conserved in the three PHD finger domains of Ehd3 (Supplementary Fig. 3b, e–h). In most cases, a single PHD finger domain is sufficient for recognition of histone modifications, highlighting a novel binding mode of the tandem PHD finger domain within Ehd3 to specifically recognize H3K4me1.

Ehd3–H3K4me1 interaction is required for proper timing of flowering in rice
To further investigate the functional significance of Ehd3–H3K4me1 binding in vivo, we first generated CRISPR/Cas9 mutants targeting Ehd3 in the ‘Nipponbare’ (NiP) background. Two single guide RNAs (sgRNAs) targeting the second exon of Ehd3 were designed. We selected mutants with two independent transgene-free mutations, one with a 1-bp deletion at target site 1 and a 9-bp deletion at target site 2 (ehd3-1), and the other with a 4-bp deletion at target site 1 and a 1-bp insertion at target site 2 (ehd3-4), for further analysis (Supplementary Fig. 4a). Mutations in the ehd3-4 allele resulted in an net 3-bp deletion and amino acid changes within the residues 52–92 of Ehd3. The ehd3-1 and ehd3-4 mutant plants displayed a delayed-flowering phenotype under both natural long-day (LD) and short-day (SD) photoperiods (Supplementary Fig. 4b), consistent with the reported phenotypes of ehd3 mutants in the Oryza sativa subsp. japonica ‘Tohoku IL9’ background23,30. We then generated the plants HA-Ehd3/ehd3-1-1 and HA-Ehd3/ehd3-1-2, expressing Ehd3 fused with the 4×HA tag driven by its native promoter in the ehd3-1 mutant background (Supplementary Fig. 4c). The delayed-flowering phenotype of ehd3-1 was fully rescued upon the expression of HA-Ehd3 (Fig. 3h), indicating that the delayed-flowering phenotype is attributable to mutations in Ehd3.

In addition, we generated transgenic plants expressing a mutated version of Ehd3 by substituting two critical Ehd3–H3K4me1-interacting residues, Tyr471 and Asp496, with Ala under the control of its native promoter in the ehd3-1 mutant background (Supplementary Fig. 4c). The transgenic line (HA-Ehd3mu/ehd3-1-1) carrying these site-specific mutations exhibited a delayed-flowering phenotype similar to that of the ehd3-1 mutant (Fig. 3i), suggesting that the mutation of critical residues responsible for Ehd3–H3K4me1 interaction within Ehd3 failed to rescue the phenotype of the ehd3-1 mutant. All of the aforementioned findings showed that binding to H3K4me1 is critical for the biological function of Ehd3 in rice.

Ehd3 physically interacts with the H3K36 methyltransferase SDG724
To further investigate the role of H3K4me1 through Ehd3 in plants, we identified the co-factors of Ehd3 by immunoprecipitation followed by mass spectrometry (IP-MS). Transgenic plants were generated harboring Enhanced Green Fluorescent Protein (EGFP) fused to Ehd3 driven by the rice Ubiquitin promoter (Supplementary Fig. 4d). Among the candidates for Ehd3-binding proteins identified in the IP-MS experiment, the H3K36-specific methyltransferase SDG724 was identified31 (Fig. 4a and Supplementary Data 4). Therefore, transgenic plants expressing Yellow Fluorescent Protein (YFP) fused to SDG724 driven by the Ubiquitin promoter were generated (Supplementary Fig. 4e). Ehd3 was among the SDG724-binding candidates identified by IP-MS (Fig. 4a and Supplementary Data 5), suggesting the possible interaction between Ehd3 and SDG724. While transgenic plants expressing YFP alone driven by the Ubiquitin promoter were served as the control, neither Ehd3 nor SDG724 was detected in the IP-MS experiment (Supplementary Data 6). Yeast two-hybrid, glutathione-S-transferase (GST) pulldown assays, co-immunoprecipitation, and bimolecular fluorescence complementation (Fig. 4b–e and Supplementary Fig. 4f) assays consistently confirmed the physical interaction between Ehd3 and SDG724 in rice. Furthermore, the Arabidopsis homolog of rice Ehd3, Q1JPM3 (named as AtEHD3), also interacted with the homolog of rice SDG724, SDG7, suggesting a potentially conserved interaction between H3K4me1 readers and H3K36 methyltransferases in plants (Supplementary Fig. 4g, h).

Deletion of Ehd3 or SDG724 leads to similar changes in plant phenotype, transcriptome, and epigenome
Both Ehd3 and SDG724 promote flowering under SD and LD photoperiods31 (Fig. 4f). To investigate the genetic relationship between Ehd3 and SDG724, we crossed the ehd3-1 and sdg724-1 mutants to generate the ehd3 sdg724 double mutant. The ehd3 sdg724 double mutant displayed a similar late-flowering phenotype to that of each single mutant under both SD and LD photoperiods (Fig. 4f), implying that Ehd3 and SDG724 promote flowering through the same genetic pathway.

To evaluate how Ehd3 and SDG724 regulate gene transcription at the genome scale, we conducted RNA-seq analyses of the wild-type NiP and the ehd3-1, sdg724-1, and ehd3 sdg724 mutants. Hundreds of differentially expressed genes (DEGs) were identified in the ehd3-1, sdg724-1, and ehd3 sdg724 mutants compared with NiP (Supplementary Fig. 5a and Supplementary Data 7). Nearly half of the up- and down-regulated genes among the single and double mutants were overlapped (Supplementary Fig. 5b, c). The overlapping DEGs displayed similar transcription changes in the ehd3-1, sdg724-1, and ehd3 sdg724 mutants (Fig. 4g). Furthermore, pairwise scatterplot analyses indicated a strong correlation in the DEGs among the ehd3-1, sdg724-1, and ehd3 sdg724 mutants (Fig. 4h), implying cooperative regulation of gene transcription by Ehd3 and SDG724.

Given that Ehd3 is an H3K4me1 reader and SDG724 is an H3K36 methyltransferase, we investigated the global distributions of H3K4me1/me2/me3, H3K36me1/me2/me3, and H3 control in NiP and the ehd3-1, sdg724-1, and ehd3 sdg724 mutants via ChIP-seq analysis. Gene-by-gene scatterplots and heatmaps revealed no alterations in the distributions of H3K4me2/me3, similarly subtle changes for H3K4me1 and H3K36me1, and similarly pronounced changes in H3K36me2/me3 distributions in the ehd3-1, sdg724-1, and ehd3 sdg724 mutants compared with those of NiP (Fig. 4i and Supplementary Fig. 6a, b). As a control, the H3 distribution in these mutants was comparable to that in NiP (Supplementary Fig. 6c, d). Therefore, Ehd3 cooperates with SDG724 to regulate H3K36me2/me3 distributions, gene transcription, and plant development.

The Ehd3 homologs containing tandem PHD finger domain recognize H3K4me1 in land plants
In mammals, H3K4me1 marks enhancers but fewer H3K4me1 reader proteins have been identified to date14. The present analysis revealed the prevalence of Ehd3 homologs in both plants and animals (Fig. 5a). Distinct domain architecture and protein length were observed among Ehd3 homologs across plant and animal species (Fig. 5a). Sequence alignment of the tandem PHD finger domains within Ehd3 homologs in land plants revealed a remarkable conservation of residues responsible for recognition of H3K4me1, particularly His441 and Tyr471 (Supplementary Fig. 7a). The tandem PHD finger domains within Ehd3 homologs in various species were predicted with AlphaFold (Fig. 5b–e and Supplementary Fig. 7b–g)32; the interleaved topology and residues crucial for H3K4me1 binding were largely identical to those of Ehd3 in land plants, including Zea mays, Triticum aestivum, Arabidopsis thaliana, and Daucus carota (Fig. 5b–e). However, the tandem PHD finger domains in Physcomitrium patens showed only partial similarity, and the overall conformations of those in Chlamydomonas reinhardtii, Penaeus vannamei, Elysia chlorotica, Mus musculus, and Homo sapiens (Supplementary Fig. 7b–g) differed significantly from the tandem PHD finger domain within Ehd3.

The ITC analysis for the tandem PHD finger domains within Ehd3 homologs in Chlamydomonas reinhardtii, Physcomitrium patens, Arabidopsis thaliana, and Mus musculus indicated their diverse binding affinities towards H3K4 methylations. Specifically, the tandem PHD finger domains in Mus musculus showed binding affinity exclusively with the H3K4me0 peptide (PHD23, Kd = 6.5 μM; PHD56, Kd = 13.5 μM) (Fig. 5f). The single PHD finger domain in Chlamydomonas reinhardtii failed to bind to any methylated H3K4. In contrast, the tandem PHD finger domain in Physcomitrium patens exhibited a stronger affinity for H3K4me1, with even greater binding observed in Arabidopsis thaliana, similar to that in Oryza sativa (Fig. 5f). Collectively, the tandem PHD finger domain initiates H3K4me1 recognition in land plants, and with highly exclusive affinity in flowering plants.

H3K4me1 directs H3K36me2 and H3K36me3 deposition in land plants
Genome-wide occupancies of Ehd3 and SDG724 were analyzed by ChIP-seq using the EGFP-Ehd3 and YFP-SDG724 transgenic plants. 15,967 Ehd3-occupied genes and 17,070 SDG724-occupied genes were identified, respectively. Among them, 10,325 genes showed significant co-occupancy by Ehd3, SDG724 and H3K4me1 modification (Fig. 6a and Supplementary Data 8–9). Gene-by-gene analysis confirmed that Ehd3 and SDG724 were preferentially enriched at genes marked with H3K4me1, but not H3K4me2 or H3K4me3, compared with the random control genes, supporting the hypothesis that Ehd3 specifically recognizes H3K4me1 in vivo (Fig. 6b, c, Supplementary Fig. 8a, b, and Supplementary Data 8–9). Importantly, the enrichment of SDG724 at its target genes was dramatically abolished when Ehd3 was deleted, highlighting an essential role of Ehd3 in recruiting SDG724 to H3K4me1-marked genes (Supplementary Fig. 8c–f). Furthermore, gene-by-gene heatmaps revealed that loss of either Ehd3 or SDG724 caused similar changes in H3K36me2/me3 distributions within the genes enriched of H3K4me1 (Fig. 6d). In detail, H3K36me2 occupancy decreased at the 3′ regions, whereas H3K36me3 decreased across the gene body in the ehd3-1, sdg724-1, and ehd3 sdg724 mutants compared with NiP (Fig. 6d). We thus examined whether the introduction of site-mutated Ehd3 can rescue the genome-wide occupancy of H3K36me2/me3 in the HA-Ehd3mu/ehd3-1-1 transgenic plants. Comparative analysis revealed that the site-mutated Ehd3 (Ehd3mu), which lost its binding capacity to H3K4me1, failed to recover the H3K36me2/me3 profiles in the HA-Ehd3mu/ehd3-1-1 plants, while the wild-type Ehd3 did in the HA-Ehd3/ehd3-1-1 plants (Supplementary Fig. 9a). Gene-by-gene analysis further confirmed that the site-mutated Ehd3 failed to rescue the aberrant H3K36me2/me3 distribution patterns in the ehd3-1 mutant background (Supplementary Fig. 9b, c). Together, all these results proved that Ehd3-binding to H3K4me1 is necessary for SDG724 to establish H3K36me2/me3 in planta, supporting that Ehd3 recognizes H3K4me1 and recruits SDG724 to H3K4me1-marked chromatin to promote the deposition of H3K36me2 and H3K36me3 in rice (Fig. 6e and Supplementary Fig. 9d).

Different from human, correlation analyses showed that H3K4me1 was positively correlated with H3K36me2 (R=0.53) and H3K36me3 (R=0.63), but not with other tested histone modifications, implying that H3K4me1 is associated with H3K36me2/me3 in rice33–38 (Supplementary Fig. 10a). SDG724 is required for deposition of H3K36me2/me3 at H3K4me1-enriched genes, supporting the positive correlation between H3K4me1 and H3K36me2/me3 in rice (Fig. 6e and Supplementary Fig. 10a). In addition, gene-by-gene heatmaps showed that genes enriched with H3K4me1 exhibited significant enrichment of H3K36me2/me3 (Fig. 6f and Supplementary Data 8–9), supporting the observation that H3K4me1 specifically colocalizes with H3K36me2/me3 in rice. To investigate if the colocalization of H3K4me1 and H3K36me2/me3 is conserved in plants, we performed ChIP-seq analysis of H3K36me2/me3 in Chlamydomonas reinhardtii and Physcomitrium patens, and integrated published H3K36me2/me3 ChIP-seq data for Saccharomyces cerevisiae, Drosophila melanogaster, Mus musculus, Homo sapiens, and Arabidopsis thaliana21,39–42 (Fig. 6g and Supplementary Fig. 10b, c). In contrast to the diverse patterns of H3K4me1 and H3K36me2/me3 distributions in Chlamydomonas reinhardtii, Drosophila melanogaster, Mus musculus, and Homo sapiens, the distributions of H3K4me1 and H3K36me2/me3 displayed high similarity in Physcomitrium patens, Arabidopsis thaliana, and Oryza sativa (Fig. 6g and Supplementary Fig. 10b, c). Moreover, the published ChIP-seq data22 also presented similar distributions of H3K4me1 and H3K36me3 in other land plants including Eutrema salsugineum, Populus trichocarpa, Phaseolus vulgaris, Asparagus officinalis, Brachypodium distachyon, Setaria viridis, Sorghum bicolor, and Zea mays (Supplementary Fig. 10d). Therefore, a distinct evolutionary trend for colocalization of H3K4me1 and H3K36me2/me3 in land plants was implied.

To investigate the molecular mechanism linking H3K4me1 and H3K36me2/me3, a histone methyltransferase assay was performed to test the activity of SDG724 and SDG725, which are H3K36-specific methyltransferases in rice, by using H3K4me0- or H3K4me1-modified mononucleosomes. The CW domain of Arabidopsis SDG8, a homolog of rice SDG725, is responsible for binding to H3K4me115, and the critical residues within the CW domain of SDG8 involved in H3K4me1 binding are conserved in SDG725 (Supplementary Fig. 10e), supporting the hypothesis that SDG725 is able to recognize H3K4me1 through its CW domain. Thus, the truncated rice H3K36 methyltransferase SDG725 containing the CW and SET domains, and GST were used as positive and negative controls, respectively. When incubated with H3K4me0 nucleosomes, neither SDG724 nor SDG725 showed obvious methyltransferase activity (Fig. 6h and Supplementary Fig. 10f). However, when using H3K4me1-modified nucleosomes as the substrate, the activity of SDG725 greatly increased. Importantly, the SDG724 activity obviously increased in the presence of Ehd3, indicating that Ehd3 is required to stimulate the catalytic activity of SDG724 (Fig. 6h and Supplementary Fig. 10f). Collectively, recognition of H3K4me1-modified nucleosomes by the interacting reader protein (Ehd3) or internal reading domain (the CW domain of SDG725) is essential for the activation of rice H3K36 methyltransferases, leading to H3K36me2/me3 deposition on chromatin. Taken together, all these results revealed that H3K4me1 directs H3K36me2/me3 deposition in land plants.

Methods
Plant material and growth conditions
The background rice material used in this study was Oryza sativa subsp. japonica ‘Nipponbare’ (NiP). Genomic DNA-edited mutants ehd3-1, ehd3-4, and osldl3 were generated using the CRISPR/Cas9 system and obtained from BIORUN (Wuhan, China). The sdg724-1 mutant was described previously31. The ehd3 sdg724 double mutant was produced through a genetic cross between the single mutants ehd3-1 and sdg724-1. The rice seedlings were cultured on Murashige and Skoog (MS) solid medium (comprising 2.2g/L MS medium (M0222; Duchefa, Haarlem, The Netherlands), 3% (w/v) sucrose, and 0.4% (w/v) phytagel, adjusted to pH 5.8) under a 14-h photoperiod (30°C light/28°C dark) in an artificial climate chamber. Rice plants were grown in paddy fields at two distinct locations: Shanghai, under long-day (LD) conditions, and Sanya, under short-day (SD) conditions. For all experiments, 20 plants were used to evaluate the heading date, defined as the date at which the panicle emerged from the flag leaf. Arabidopsis seedlings were grown on MS solid medium (consisting of 4.9 g/L MS medium (M0255, Duchefa), 1% (w/v) sucrose, and 0.5% (w/v) agarose, adjusted to pH 5.7) under a 16-h photoperiod at 22°C in an artificial climate chamber. Chlamydomonas reinhardtii strains were cultured in Tris-acetate-phosphate (TAP) medium70 on a rotary shaker at 25°C in an artificial climate chamber. Physcomitrium patens were propagated on BCD medium71 under a 16-h photoperiod at 25°C in an artificial climate chamber.

Vector construction and transgenic plants
To generate the Ubi::EGFP-Ehd3 construct, the Ehd3 cDNA sequence was amplified by PCR and subsequently cloned into the pRHVnGFP vector72. For the Ubi::YFP-SDG724 construct, the SDG724 cDNA sequence was amplified by PCR and cloned into the pU1301 plant expression vector. For the Ubi::SDG724-4×HA construct, the SDG724 cDNA sequence was amplified by PCR and cloned into the pRHVcHA vector72. For the pEhd3::4×HA-Ehd3 and pEhd3::4×HA-Ehd3mu constructs, the native Ehd3 promoter (2,005-bp upstream of ATG), the coding sequence for 4×HA, and the genomic DNA sequence of either wild-type Ehd3 or the mutated Ehd3 (Y471A and D496A) were amplified by PCR and cloned into the pCAMBIA1300 vector (CAMBIA, https://cambia.org). The primers used in cloning are listed in Supplementary Data 10.

The Ubi::EGFP-Ehd3 and Ubi::YFP-SDG724 constructs were transformed into NiP to generate transgenic overexpression plants, designated as EGFP-Ehd3/NiP and YFP-SDG724/NiP. The Ubi::SDG724-4×HA construct was transformed into the sdg724-1 single mutant to obtain SDG724-HA/sdg724−1-1, then SDG724-HA/ehd3 sdg724−1 was obtained by a genetic cross with the ehd3 sdg724 double mutant. In addition, HA-Ehd3/ehd3-1−1 and HA-Ehd3mu/ehd3-1-1 plants were generated by transforming the pEhd3::4×HA-Ehd3 and pEhd3::4×HA-Ehd3mu constructs into the ehd3-1 mutant. Genetic transformations were conducted by BIORUN and stable transgenic plants were selected using hygromycin B (Roche, Mannheim, Germany). The stable transgenic plants were confirmed by western blot using Rabbit anti-GFP (ab290, Abcam, Cambridge, UK, 1:2000), Rabbit anti-HA (ab9110, Abcam, 1:2000), Rabbit anti-H3 (ab1791, Abcam, 1:2000), or Mouse anti-Actin (M20009, Abmart, 1:2000) antibodies. In total, we obtained eight EGFP-Ehd3/NiP lines, five YFP-SDG724/NiP lines, two HA-Ehd3/ehd3-1 lines, and two HA-Ehd3mu/ehd3-1 lines. EGFP-Ehd3/NiP-1 and YFP-SDG724/NiP-1 were selected for the following experiments. In addition, HA-Ehd3/ehd3-1-1, HA-Ehd3/ehd3-1-2, and HA-Ehd3mu/ehd3-1-1, which demonstrated comparable abundance of HA-Ehd3 and HA-Ehd3mu proteins (Supplementary Fig. 4c), were selected for subsequent analysis.

Phylogenetic analysis
PHD domain encoding genes were identified using Phytozome database (https://phytozome-next.jgi.doe.gov/). For each identified gene, a representative annotated protein sequence was analyzed using the SMART database (http://smart.embl-heidelberg.de/) to characterize domain architectures. Homologs in different species were identified using BLAST tool searches against the UniProtKB database (https://www.uniprot.org/). Multiple sequence alignment and neighbor-joining dendrograms were constructed using MEGA version 10.2.673.

Protein expression and purification
The coding sequences of Ehd3 (1–563 amino acids (aa), 281–563 aa, 281–405 aa, 406–563 aa, 406–473 aa, 473–563 aa, and 417–547 aa), OsLDL3 (1263–1832 aa), SDG724 (1–394 aa), SDG725 (1240–1882 aa), and Arabidopsis Q1JPM3 (named as AtEHD3) were amplified by PCR using primers (Supplementary Data 10) and subsequently cloned into the pGEX-6P-1 vector (GE Healthcare, Milwaukee, WI, USA) to produce glutathione-S-transferase (GST)-fusion proteins. In addition, DNA fragments encoding Ehd3 (1–563 aa and 417–547 aa), the Ehd3 417–547 aa mutants (H441A, Y446A, Y471A, D486A, I489A, and D496A), OsLDL3 (1744–1828 aa), SDG725 (1240–1898 aa), A0A2K3D188 (173–217 aa), A0A2K1L966 (364–473 aa), AtEHD3 (342–474 aa), Q8BRH4 (338–468 aa), and Q8BRH4 (911–1043 aa) were amplified by PCR using primers (Supplementary Data 10) and cloned into the pSUMO vector74. The coding sequence of Arabidopsis SDG7 was amplified by PCR using primers (Supplementary Data 10) and cloned into the pColdTF vector. All recombinant proteins were expressed in Escherichia coli strain Rosetta (DE3) cells and induced with isopropyl-β-D-thiogalactoside. ZnCl2 was added during expression of the Ehd3, SDG724, SDG725, AtEHD3, and SDG7 proteins.

To purify GST-tagged proteins, cells were harvested and suspended in phosphate-buffered saline (1× PBS) containing 10% (v/v) glycerol, 0.1% (v/v) Triton X-100, 2 mM 1,4-dithiothreitol (DTT), and 0.1 mM ZnCl2, pH 7.4. After sonicating and centrifuging the lysed cells, the supernatant was incubated with Glutathione Sepharose 4 Fast Flow (Cytiva, Uppsala, Sweden) at 4°C for 2 h. Subsequently, the supernatants were discarded and the beads were washed three times with 1× PBS containing 10% (v/v) glycerol, 2 mM DTT, and 0.1 mM ZnCl2, pH 7.4. The GST-tagged proteins were eluted with an elution buffer (150 mM NaCl, 20 mM Tris-HCl, and 20 mM reduced glutathione, pH 7.4). The flow-through was concentrated and substituted with the buffer containing 150 mM NaCl, 20 mM Tris-HCl, 2 mM DTT, and 0.1 mM ZnCl2, pH 7.4, using an Amicon 10-KDa cutoff (Millipore, Burlington, MA, USA).

To purify the proteins cloned into the pSUMO vector, cells were harvested and suspended in the binding buffer (500 mM NaCl, 20 mM Tris-HCl, and 25 mM imidazole, pH 8.0). The lysed cells, processed using a high-pressure disruptor, were centrifuged, and the supernatant was loaded onto a Ni-NTA column (GE Healthcare). The proteins were gradually eluted using an elution buffer (500 mM NaCl, 20 mM Tris-HCl, and 500 mM imidazole, pH 8.0). Subsequently, the eluted proteins were incubated with ULP1 protease and then diluted for 3 h in buffer containing 500 mM NaCl and 20 mM Tris-HCl, pH 8.0. The diluted proteins were then loaded onto a Ni-NTA column, and the flow-through was further purified using a Superdex 75 column (GE Healthcare) equilibrated with buffer containing 100 mM NaCl, 20 mM Tris-HCl, and 1 mM Tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP), pH 8.0. Finally, the proteins were concentrated using an Amicon 10-KDa cutoff (Millipore).

To purify the protein expressed in the pColdTF vector, cells were harvested and suspended in the binding buffer (20 mM Tris-HCl, 500 mM NaCl, 25 mM Imidazole, 5% (v/v) Glycerol, pH 8.0). After sonicating and centrifuging the lysed cells, the supernatant was incubated with cOmplete His-Tag Purification Resin (Roche) at 4°C for 2 h. Subsequently, the supernatants were discarded and the beads were washed three times with binding buffer. The His-tagged proteins were eluted with the elution buffer (20 mM Tris-HCl, 500 mM NaCl, 500 mM Imidazole, 5% v/v Glycerol, pH 8.0). The flow-through was concentrated using an Amicon 10-KDa cutoff (Millipore).

Crystallization and structure determination
The purified Ehd3 protein (417–547 aa) was incubated with H3(1–12) K4me1 histone peptide (Supplementary Data 1) at a molar ratio of 1:1.2 at 4°C for 1 h. Crystallization was performed by a Gryphon crystallization robot system with a sitting-drop vapor diffusion method. Specifically, 0.2 μl Ehd3 protein (10 mg/ml) and peptide mixture was combined with an equal volume of a crystallization kit solution (Hampton, Aliso Viejo, CA, USA) and incubated at 18°C. The optimization procedure was performed using the hanging-drop vapor diffusion method at 18°C. X-ray data for the crystals were collected at the B18U1 beamline of the Shanghai Synchrotron Radiation Facility and were processed using HKL300075. The crystal structure was solved by the autosol and refined by the phenix.refine programs from Phenix76. The 2Fo–Fc and Fo–Fc electron density maps and unbiased omission map were regularly calculated and used as guides for the building of the H3K4me1 peptide, Zn2+ ions, and water molecules in COOT77. The data collection and refinement statistics are summarized in Supplementary Data 3. All molecular graphics were generated using PyMOL (https://pymol.org/).

Structure prediction by AlphaFold
AlphaFold version 2.1.032 was used to predict the structures of Ehd3 tandem PHD finger homologs, i.e., A0A2K3D188 (117–217 aa) in Chlamydomonas reinhardtii, A0A2K1L966 (364–464 aa) in Physcomitrium patens, A0A1D6LDK6 (401–498 aa) in Zea mays, A0A3B6RKR3 (432–532 aa) in Triticum aestivum, Q1JPM3 (354–450 aa) in Arabidopsis thaliana, A0A162A0W2 (424–519 aa) in Daucus carota, A0A3R7Q333 (793–889 aa) in Penaeus vannamei, A0A433TT05 (688–784 aa) in Elysia chlorotica, O14686 (1379–1475 aa) in Homo sapiens, and Q8BRH4 (914–1010 aa) in Mus musculus. AlphaFold version 3.055 was used to predict the structures of SRI domain-containing homologs, i.e., A0A2K3DEA3 (1–1870 aa) in Chlamydomonas reinhardtii, A0A2K1LAV4 (1–1709 aa) and A0A2K1L6H4 (1–1814 aa) in Physcomitrium patens, SK05G17270.mRNA1 (1–1295 aa) in Selaginella kraussiana, Ceric.12G094400.1 (1–1600 aa) in Ceratopteris richardii, F4JLS1 (1–1628 aa) in Arabidopsis thaliana, and Q336Y0 (1–1832 aa) in Oryza sativa. The protein codes correspond to the UniProt entries (https://www.uniprot.org), except for SK05G17270.mRNA1 and Ceric.12G094400.1, which were obtained from https://sk.ccgg.fun/blast.

Peptide microarray
The MODified™ Histone Peptide Array (Active Motif, Carlsbad, CA, USA) was blocked with TTBS–milk buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% (v/v) Triton X-100, and 5% (w/v) non-fat dried milk, pH 7.4) at room temperature for 2 h. After washing three times with TTBS buffer (20 mM Tris-HCl, 150 mM NaCl, and 0.1% (v/v) Triton X-100, pH 7.4), the peptide array was incubated overnight at 4°C with 500 nM GST-tagged full-length Ehd3 protein in TTBS–milk buffer. Subsequently, the peptide array was washed three times with TTBS buffer and incubated with the Mouse anti-GST antibody (M20007, Abmart, Shanghai, China, 1:2000) in the TTBS–milk buffer at room temperature for 1 h. After washing three times with TTBS buffer, the peptide array was incubated with the Goat anti-Mouse IgG HRP antibody (M21001, Abmart, 1:5000) in the TTBS–milk buffer at room temperature for 1 h. The peptide array was washed three times with TBS buffer (20 mM Tris-HCl and 150 mM NaCl, pH 7.4) and imaged using a luminescence imaging workstation (Tanon, Shanghai, China). The results were analyzed using Array Analyze software (Active Motif).

Peptide pulldown
Biotinylated histone peptides (2 μg) (H3(1–21), H3(1–21)K4me1, H3(1–21)K4me2, H3(1–21)K4me3, H3(1–21)K9me2, H3(1–21)K9ac, H3(1–21)K14ac, H3(21–41)K27me3, H3(16–34)K27ac, and H3(21–41)K36me3) (Supplementary Data 1) were incubated with 20 μl pre-washed streptavidin magnetic beads (NEB, Ipswich, MA, USA) at 4°C for 2 h in 500 μl binding buffer (150 mM NaCl, 20 mM Tris-HCl, and 0.1% (v/v) Nonidet P-40). After washing three times, the peptide-bound beads were incubated with 2 μg GST-tagged full-length or truncated Ehd3 proteins at 4°C for 2 h in 500 μl binding buffer. The beads were then washed three times with the binding buffer and boiled in 1× SDS loading buffer (50 mM Tris-HCl, 2% (w/v) sodium dodecyl sulfate, 0.01% (w/v) bromophenol blue, 2 mM DTT, and 2 mM β-Mercaptoethanol, pH 6.8). The resulting products were subjected to SDS-PAGE and detected using the Mouse anti-GST antibody (M20007, Abmart, 1:2000).

Semi-in vivo pulldown
Total nuclear proteins were extracted from wild-type NiP using a buffer containing 50 mM Tris-HCl, 5 mM MgCl2, 60 mM NaCl, 60 mM KCl, 2 mM DTT, and proteinase inhibitor cocktail (Roche), pH 7.5. The nuclear protein supernatant was incubated at 4°C for 2 h with beads precoated with GST, GST-OsLDL3 SRI, GST-SDG724, or GST-SDG725 CW-SET proteins. After washing three times, the samples were analyzed by SDS-PAGE and western blot using Rabbit anti-Ser2P RNAPII (ab5095, Abcam, 1:2000) and Rabbit anti-Ser5P RNAPII (ab5131, Abcam, 1:2000) antibodies.

Isothermal titration calorimetry (ITC)
The ITC experiments were conducted using an iTC200 Micro-Calorimeter (Malvern Panalytical, Malvern, UK) at the National Center for Protein Science Shanghai. The interactions involving the full-length Ehd3 (100 μM), wild-type or mutated Ehd3 truncations (417–547 aa, 100 μM), A0A2K3D188 (173–217 aa, 100 μM), A0A2K1L966 (364–473 aa, 100 μM), Q1JPM3 (342–474 aa, 100 μM), Q8BRH4 (338–468 aa, 50 μM), and Q8BRH4 (911–1043 aa, 50 μM) were analyzed in the presence of the peptides H3(1–12)K4me0, H3(1–12)K4me1, H3(1–12)K4me2, or H3(1–12)K4me3 (1 mM), as well as OsLDL3 (1744–1828 aa, 100 μM) with the peptides NP CTD, S2P CTD, or S5P CTD (1 mM) (Supplementary Data 1). All reactions were performed at 25°C in buffer containing 100 mM NaCl, 20 mM Tris-HCl, and 1 mM TCEP, pH 8.0. The titration data were fitted using Origin version 7.0 software (Supplementary Data 2).

Immunoprecipitation followed by mass spectrometry (IP-MS)
Transgenic seedlings (2 g) were ground in liquid nitrogen and homogenized in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 5 mM MgCl2, 10% (v/v) glycerol, 0.1% (v/v) Nonidet P-40, 5 mM DTT, 0.1 mM PMSF, and proteinase inhibitor cocktail (Roche), pH 7.6). After sonication and centrifugation, the supernatant was incubated with the Rabbit anti-GFP antibody (ab290, Abcam, 1:100) at 4°C for 3 h. The mixture was then incubated with protein A magnetic beads (Thermo Fisher Scientific, Waltham, MA, USA) at 4°C for 3 h. Subsequently, the beads were washed with the same buffer and subjected to SDS-PAGE. The protein bands were then excised and subjected to in-gel digestion with trypsin. The resulting peptides were collected and analyzed using a nanoflow EASY-nLC 1200 system (Thermo Fisher Scientific) coupled with an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Raw data analysis was performed using Proteome Discoverer version 2.4 (Thermo Fisher Scientific) and an in-house Mascot server version 2.7 (Matrix Science, London, UK). The rice protein database was obtained from RGAP (http://rice.uga.edu). Data were searched using the following parameters: trypsin/P as the enzyme; allowing up to two missed cleavage sites; 10 ppm mass tolerance for MS and 0.05 Da for MS/MS fragment ions; carbamidomethylation on cysteine as fixed modifications; and protein N-terminal acetylation, oxidation on methionine, and N-terminal pyroQ as variable modifications. The incorporated Percolator in Proteome Discoverer was used for validation, accepting only the hits with false discovery rate ≤ 0.01 for the following analysis.

GST pulldown
The GST, GST-SDG724, or GST-AtEHD3 beads-coated protein was incubated with the full-length Ehd3 or His-SDG7 proteins at 4°C for 2 h in buffer containing 75 mM NaCl, 75 mM KCl, 50 mM HEPES, 5% (v/v) glycerol, 8 mM DTT, and 2 mM MgCl2, pH 8.0. After washing three times, the beads were boiled in 1× SDS loading buffer, subjected to SDS-PAGE, stained with Coomassie Brilliant Blue, and analyzed by western blot using Mouse anti-His antibody (M20001, Abmart, 1:2000).

Yeast two-hybrid
The Ehd3, SDG724, Arabidopsis AtEHD3, or SDG7 cDNA sequence was cloned into the pGADT7 and pGBKT7 vectors (Clontech, Kyoto, Japan), respectively, using specific primers (Supplementary Data 10). The resulting constructs were transformed into the Y2H Gold yeast strain following the manufacturer’s instructions (Weidi Bio, Shanghai, China). The interaction was screened on a synthetic defined medium lacking leucine, tryptophan, and histidine (SD/−L/−W/−H), or on medium additionally lacking adenine (SD/−L/−W/−H/−A).

Co-immunoprecipitation (Co-IP)
Rice protoplast isolation and transformation were performed as described previously78. The Ehd3 and SDG724 cDNA sequences were cloned into the pRTVnMyc and pRTVnHA vectors72, respectively. After transformation of the constructs, the protoplasts were incubated at 28°C for 20 h. The protoplasts were subsequently harvested in buffer containing 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 2 mM DTT, 0.5% (v/v) Triton X-100, and proteinase inhibitor cocktail (Roche), pH 8.0. The harvested protoplasts were sonicated at 4°C. After centrifugation, the supernatant was incubated with the agarose-conjugated Mouse anti-Myc antibody (M20012, Abmart, 1:100) at 4°C for 2 h. The immunoprecipitants were washed three times, subjected to SDS-PAGE, and detected with the Mouse anti-Myc (M20002, Abmart, 1:2000) and Rabbit anti-HA (ab9110, Abcam, 1:2000) antibodies.

Bimolecular fluorescence complementation (BiFC)
The Ehd3 and SDG724 cDNA sequences were cloned into the pRTVnVC and pRTVnVN vectors using the primers listed in Supplementary Data 1072, respectively. After transformation of the constructs, the protoplasts were incubated at 28°C for 20 h. Fluorescence in the protoplasts was observed using an LSM710 confocal laser scanning microscope (Carl Zeiss, Jena, Germany; https://www.zeiss.com).

Histone methyltransferase assay
Either 500 nM recombinant Ehd3, GST-SDG724, GST-SDG724 mixed with Ehd3, or 100 nM SDG725 (CW-SET domain, 1240–1898 aa) were incubated with 1 μM unmodified (31467, Active Motif) or H3K4me1-modified (31585, Active Motif) mononucleosomes in histone methyltransferase buffer (50 mM Tris-HCl, 0.02% (v/v) Triton X-100, 1 mM TCEP, and 160 μM SAM, pH 8.5) at room temperature for 15 h. Reactions were stopped by adding 1× SDS loading buffer, then were subjected to SDS-PAGE and detected by western blot using the Rabbit anti-H3K36me2 (ab9049, Abcam, 1:2000) and Rabbit anti-H3K36me3 (ab9050, Abcam, 1:2000) antibodies.

RNA extraction, library construction and sequencing
Fourteen-day-old Oryza sativa and Arabidopsis thaliana seedlings (100 mg) grown on MS medium, 7-day-old Physcomitrium patens grown on BCD medium, and Chlamydomonas reinhardtii cultured in TAP medium to a density of 4–8×106 cells/ml were used for mRNA extraction. The mRNA was extracted using the RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China). Three independent biological replicates of RNA-seq libraries were prepared using the KAPA Stranded mRNA-seq Kit (Kapa Biosystems, Wilmington, MA, USA) and sequenced on an Illumina NovaSeq 6000 instrument by NEO BIO (Shanghai, China). Raw reads were cleaned with Cutadapt version 3.579 to remove low-quality bases and sequencing adapters. Trimmed reads were aligned to the reference genomes of different species using HISAT2 version 2.1.080. Samtools version 1.1781 was used to remove reads with low mapping quality (MAPQ<20), and then featureCounts version 2.0.182 was used to quantify the number of reads mapped to exons. RPKM (reads per kilobase per million reads) was used to characterize the transcription level of RNA. For rice RNA-seq, the DESeq2 version 1.36.083 package for R (version 4.2.1) was used to screen DEGs (|fold of change|≥1.5 and Benjamini–Hochberg adjusted p-value≤0.05, by two-sided Wald test). Gene ontology (GO) functional enrichment analysis for specific gene sets was conducted through the online platform CARMO84 with a modified one-sided Fisher’s exact test (EASE score)85, which was applied to identify significantly enriched GO terms. The reference genomes used for rice and Arabidopsis were MSU7 (http://rice.uga.edu/) and TAIR10 (https://www.arabidopsis.org/), respectively. The reference genomes for other species were obtained from Ensembl (https://www.ensembl.org/). The genomes correspond to specific species: R64-1-1 for Saccharomyces cerevisiae, BDGP6.32 for Drosophila melanogaster, GRCm39 for Mus musculus, GRCh38.p14 for Homo sapiens, Chlamydomonas_reinhardtii_v5.5 for Chlamydomonas reinhardtii, and Phypa_V3 for Physcomitrium patens.

Chromatin immunoprecipitation (ChIP)
Fourteen-day-old Oryza sativa and Arabidopsis thaliana seedlings (2 g) grown on MS medium, and 7-day-old Physcomitrium patens grown on BCD medium, were fixed in cross-linking buffer (0.4 M sucrose, 10 mM Tris-HCl, 1 mM PMSF, 1 mM EDTA, and 1% (v/v) formaldehyde, pH 8.0). Chlamydomonas reinhardtii grown in 1 L TAP medium to a density of 4–8×106 cells/ml were harvested and fixed in cross-linking buffer (20 mM HEPES-KOH, 80 mM KCl, and 0.4% (v/v) formaldehyde, pH 7.6). The ground seedlings or cells were resuspended in isolation buffer (0.25 M sucrose, 15 mM PIPES, 5 mM MgCl2, 60 mM KCl, 15 mM NaCl, 1 mM CaCl2, 0.9% (v/v) Triton X-100, 1 mM PMSF, and proteinase inhibitor cocktail (Roche), pH 6.8). Chromatin was sonicated in lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1% (w/v) SDS, 0.1% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100, and proteinase inhibitor cocktail (Roche), pH 7.5) using a Bioruptor™ Plus sonication device (Diagenode, Liege, Belgium). After centrifugation, the supernatant was incubated with antibodies including Rabbit anti-H3K4me1 (ab8895, Abcam, 1:200), Rabbit anti-H3K4me2 (07-030, Millipore, 1:200), Rabbit anti-H3K4me3 (07-473, Millipore, 1:200), Rabbit anti-H3K36me1 (ab9048, Abcam, 1:200), Rabbit anti-H3K36me2 (ab9049, Abcam, 1:200), Rabbit anti-H3K36me3 (ab9050, Abcam, 1:200), Rabbit anti-H3 (ab1791, Abcam, 1:200), or Rabbit anti-GFP (ab290, Abcam, 1:200) overnight at 4°C. The prewashed protein A magnetic beads (Thermo Fisher Scientific) were added to the mixture and incubated at 4°C for 2 h. The immunoprecipitates were washed sequentially: once with low-salt buffer (150 mM NaCl, 20 mM Tris-HCl, 0.1% (w/v) SDS, 1% (v/v) Triton X-100, and 2 mM EDTA, pH 8.0), once with high-salt buffer (500 mM NaCl, 20 mM Tris-HCl, 0.1% (w/v) SDS, 1% (v/v) Triton X-100, and 2 mM EDTA, pH 8.0), once with LiCl buffer (10 mM Tris-HCl, 0.25 M LiCl, 1% (w/v) sodium deoxycholate, 1% (v/v) Nonidet P-40, and 1 mM EDTA, pH 8.0), and twice with Tris-EDTA buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0). The immunoprecipitates were eluted with elution buffer (0.5% (w/v) SDS and 0.1 M NaHCO3), followed by reverse crosslinking overnight at 65°C. Subsequently, the samples were treated with RNase A and proteinase K. DNA extraction and ChIP-seq library construction were conducted as described previously86.

ChIP sequencing (ChIP-seq) analysis
DNA fragments for ChIP assays were prepared for sequencing on an Illumina NovaSeq 6000 instrument by NEO BIO (Shanghai, China). Raw reads trimmed by Cutadapt version 3.579 were aligned to genomes using Bowtie2 version 2.4.587. Reads with low-quality mapping (MAPQ<20) and duplicate reads were filtered out using Samtools version 1.1781. Macs2 version 2.2.7.1/Macs3 version 3.0.0b388 (q-value≤0.05) and MAnorm version 1.3.089 (with a Bayesian model for significance testing) were used for peak-calling and differential peaks detection for histone modifications, respectively. SICER version 1.190 was used for peak-calling for Ehd3- and SDG724-enriched peaks (W200-G600-FDR0.05), which were subtracted by the corresponding NiP control. The ChIPpeakAnno version 3.30.191 package for R version 4.2.1 was used to annotate peaks to genes. BEDtools version 2.31.092 was used to perform data format conversion. deepTools version 3.5.393 was used to calculate the normalized signals across the whole genome or on the selected genes. Genomic track files, generated by the bamCoverage tool of deepTools version 3.5.393 (with the parameter ‘--normalizeUsingRPKM’), were visualized in the genome browser IGV version 2.11.194. Metagene plots and heatmaps were plotted using the computeMatrix and plotHeatmap tools in deepTools version 3.5.393. Each ChIP-seq sample included at least two independent biological replicates.

Cleavage under targets and tagmentation (CUT&Tag) and data analysis
Fourteen-day-old Oryza sativa seedlings (2 g) grown on MS medium were ground in liquid nitrogen and resuspended in ChIP isolation buffer containing 0.1% (v/v) formaldehyde. Cross-linking was quenched by adding 2.5 M Glycine. The extracted nuclei were used for CUT&Tag assay with the Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro (Vazyme, Nanjing, China), according to the manufacturer’s protocol. Briefly, nuclei were bound to Concanavalin A (ConA) beads and incubated overnight at 4°C with a Rabbit anti-HA (AT1747, Engibody, Shanghai, China, 1:50) antibody. After washing, samples were incubated with a Goat anti-rabbit IgG H&L (ab6702, Abcam, 1:50) antibody for 1 h at room temperature, followed by incubation with Tn5 transposase for 1 h at room temperature. Target chromatin was fragmented at 37°C for 1 h, and reverse cross-linking was performed at 55°C for 3 h. DNA was purified, and libraries were constructed and amplified. Sequencing was carried out on an Illumina NovaSeq 6000 instrument by NEO BIO (Shanghai, China). The raw reads from CUT&Tag libraries were processed using a bioinformatic pipeline largely consistent with that applied to the ChIP-seq data described above. Due to the inherent affinity of the transposome for exposed DNA95, an additional correction step was implemented: genomic regions corresponding to NiP peaks (identified by SICER version 1.190 with parameters W200-G200-E100) were excluded from the coverage tracks via the --blackListFileName option in bamCoverage (deepTools version 3.5.393). Each CUT&Tag sample included two independent biological replicates.

Statistics and reproducibility
For rice heading date analysis, 20 individual plants grown in Shanghai or Sanya were randomly selected, with no data excluded. Statistical analyses were conducted using GraphPad Prism (version 9.5.0). For multigroup comparisons in rice heading date analysis, one-sided Ordinary one-way ANOVA test followed two-sided Tukey’s Honestly Significant Difference test was applied. An adjusted p-value<0.01 was considered statistically significant. No statistical method was used to predetermine sample size. For RNA-seq, ChIP-seq and CUT&Tag data, the independent biological replicates for each sample were analyzed to be consistent and no data were excluded. But not all the independent biological replicates of ChIP-seq or CUT&Tag data were shown in the figures. Statistical analyses were performed using R (version 4.2.1), including two-sided correlation tests and one-sided Fisher’s exact tests. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "I reviewed this paper in another journal before transferred to Nature Communications. Because the comments I raised before\nhave not been incorporated to the manuscript, I mostly copy the previous comments below.\nThis paper by Wu et al describes a PHD domain protein Ehd3 in rice, which specifically binds to histone H3K4me1. They\nanalyzed structure of Ehd3-H3K4me1 peptide and showed a unique binding mode of Ehd3 to H3K4me1. They further\nshowed that Ehd3 bind to a H3K36 methyltransferase SDG724, and the loss of function of these genes showed similar loss\nof H3K36me3 where H3K4me1 localizes in gene body. Overall this paper is interesting and shows novel mechanism\npossibly connecting H3K4me1 and H3K36me3, co-localizing histone marks in land plants. The data are solid and well\nwriten. However, I have concern about the main conclusion of this paper “H3K4me1 directs H3K36me2/3 in land plants” is\nstill overstatement. Especially the role of Ehd3 and SDG724 in this potential pathway. I summerize my specific comments\nbelow.\n1. In the beginning of the discussion section the authors stated that “In this study, we identified a specific H3K4me1 reader\nprotein, Ehd3, and revealed a crucial role of H3K4me1 in facilitating H3K36me2/me3 deposition in rice.” However, authors\ndid not show direct evidence that H3K4me1 is facilitating H3K36me2/3 in rice. Experimental evidence should be presented\nor tone down the statement. Same statement is in several places such as title and abstract.\n2. In the next sentense “Ehd3 recognizes H3K4me1 through its tandem PHD finger domain and recruits the H3K36\nmethyltransferase SDG724 to H3K4me1-enriched chromatin regions.” To state this, the authors should perform SDG724\nChIP-seq in ehd3 mutant background, and hopefully, in the mutant that lose H3K4me1.\n3. L308-310, “Collectively, these results indicated that Ehd3 and SDG724 together bind to genes enriched with H3K4me1\nand promote deposition of H3K36me2 and H3K36me3 (Fig. 5d and Extended Data Fig. 6e).” To claim that the binding of\nEhd3 to H3K4me1 is important for H3K36me2/3 deposition, the authors should perform H3K36me2/3 ChIP-seq in HA-\nEhd3mu/ehd3-1 plants and compare it with wild type and ehd3-1 plants.\n4. This paper presents a lot of data in the discussion section, such as Extended data 8-10. I suggest to move the description\nof data in the results section.\n5. The patterns of Ehd3 and SDG724 localizations are bit different (Fig. 5a and Extended Data Fig. 6d). Again, the effect of\nehd3 mutation on SDG724 localization may support the authors’ claim.\n6. Similar to point 5, L293 says “SDG724 exhibited a notable co-occupancy with Ehd3 at H3K4me1-enriched genes”, but\nheatmaps are not enough to show co-occupancy. Formal analysis of co-occupancy is needed.\n7. Does IP-MS experiment in Fig. 4a contain negative control such as non-transgenic plants to show that the detected\nproteins are specifically bound to the bait proteins?\n8. How is the experiment in Fig. 5g reproduced? Results of replicate experiments should be presented in Supplementary\ndata.\n9. The author describes the potential role of H3K36me3 in repressing cryptic transcription start. To test this possibility,\nstudying transcription start by such as TSS-seq in sdg724 and ehd3 mutants will be needed.\nOther comments\n1. Around L184-189, a lot of abbreviation such as NZ or OD1 without explanation. Explanation is needed.\n2. Line 201-203, “the Ala substitution for each H3K4me1 binding-pocket residue markedly reduced the affinity of the Ehd3\ntandem PHD finger for the H3K4me1 peptide (Fig. 3g and Supplementary Table 2).” Are D496 and Y446 H3K4me1 binding-\npocket? Looks like they are interacting with H3R2. Explanation for this results will be helpful.\n3. Line 229, “a 4-bp deletion at target site 1 and a 1-bp insertion at target site 2 (ehd3-4)” This will make 3-bp deletion in total\nand frame will be normal. Explanation of why this mutant show loss-of-function phenotype will be helpful.\n4. Line 277, “Significant overlap”. Please indicate statistical significance for overlap if state so.\n5. Line 290-291, “As a control, the H3 distribution in these mutants was comparable to that in NiP (Extended Data Fig. 6c).”\nWhy H3 data is shown only by heatmaps and all gene metaplots, which do not tell if pattern is unaltered or not in mutants?\nShowing scatter plots like Fig. 4i will be preferable.\n6. Extended Data Fig. 7a. Is this based on gene-by-gene? Or based on fixed length bin such as 100bp?", "reviewer_2": "In this manuscript, Wu et al. investigate the connection between H3K4me1 and the deposition of H3K36me2 and\nH3K36me3 in plants. The authors show that rice Ehd3 interacts with H3K4me1 and the H3K36 methyltransferase SDG724,\nand that Ehd3 and SDG724 act in the same pathway and co-localize with H3K4me1. They further demonstrate that the\ninteraction between H3K4me1 and Ehd3 homologs is conserved in land plants. Overall, the results are convincing, and the\nfindings are both interesting and significant. My suggestions for improving the manuscript are as follows:\n1. Results from Extended Data Figures 8 and 9 should be described in the Results section rather than the Discussion.\n2. The authors are suggested to test the interaction between Ehd3 homolog proteins in Arabidopsis and the H3K36\nmethyltransferases (e.g., SDG8) to examine whether the interaction between the H3K4me1 reader and H3K36\nmethyltransferase is conserved in other plant species.\n3. Lines 277–279: Please provide P-values to support the claim of “significant overlap”.\n4. Lines 326–330: In addition to the genome browser view, a correlation analysis should be performed to strengthen the\nconclusion.", "reviewer_3": "Histone modifications play critical roles in eukaryotic gene regulation. Among the multiple common histone modifications,\nH3K4me1 is relatively less studied. It is associated with enhancers in animals but not plants. To investigate the functional\nroles of H3K4me1 in plants, Wu et al. performed comprehensive studies in rice and later extended to land plants in general.\nThe authors first analyzed the H3K4me1 patterns in multiple species and found a unique distribution of H3K4me1 in land\nplants. A PHD finger protein Ehd3 was later identified as the H3K4me1 reader. The structural studies successfully revealed\nthe molecular basis for the specific recognition of H3K4me1 by Ehd3, and the structure-based mutagenesis studies\nconfirmed the H3K4me1 reading is essential for the in vivo function of Ehd3. Further, the H3K36 MTase SDG724 was\nidentified as an Ehd3 binding factor that links H3K4me1 reading to H3K36me deposition. Overall, this study revealed an\nimportant and unique interplay between two histone modifications in land plants. I support the publication of this paper with\nthe following minor concerns.\nL31 and 53, H3 means histone 3. So, please change histone H3 directly to histone 3.\nL171-172, it is not rigorous to say that the hydrogen bond exists between the N and O atoms, as there is no hydrogen. I\nwould like to suggest that the author change the N and O atoms here to the amino and carbonyl groups, respectively. The\nfollowing descriptions should also be revised to prevent using the atom but use the group to describe the hydrogen bonds.\nFig. 4f, it looks like there is no significant difference between II and IV, and III and IV. Please analyze the differences to\nconfirm.\nL284-285, why analyze H3K36me here? Because SDG724 is an H3K36 MTase? Please add the short sentence here to\ndescribe the logic for analyzing H3K36me. Or else, the introduction of H3K36 becomes strange here.\nL334, why SDG725? Please have a very brief introduction for SDG725, especially about the CW domain. A potential way is\nto move the sentence of L341-345, the introduction of the CW domain of SDG8 and SDG725, to L334.\nDoes SDG724 have a CW domain like SDG725? If yes, please give the details. If not, why could the H3K4me1 NCP\nstimulate the activity of SDG724? Please have a short discussion about this issue."}, "author_response": "Response: We thank you for your interest in this work. And we thank you for your\nhelpful comments that have improved our manuscript and led to the inclusion of new\ndata. We have addressed the specific comments as presented below.\n1. In the beginning of the discussion section the authors stated that “In this study, we\nidentified a specific H3K4me1 reader protein, Ehd3, and revealed a crucial role of\nH3K4me1 in facilitating H3K36me2/me3 deposition in rice.” However, authors did not\nshow direct evidence that H3K4me1 is facilitating H3K36me2/3 in rice. Experimental\nevidence should be presented or tone down the statement. Same statement is in several\nplaces such as title and abstract.\nResponse: Following your suggestion, we have toned down our statements in the\nrevised abstract and discussion. In the revised manuscript, we clarified that our findings\nsuggest a crucial role for H3K4me1 in directing H3K36me2/me3 deposition in the\nexamined land plants (Lines 43 and 83).\nAs you suggested, we have provided new experimental evidence in the revised\nmanuscript, and we think our data strongly support the function of H3K4me1 in\ndirecting H3K36me2/me3 establishment in rice. 1) Loss of the H3K4me1 reader Ehd3\nor the H3K36 methyltransferase SDG724 leads to similar changes in plant phenotype,\ntranscriptome, and epigenome, especially genome-wide changes of H3K36me2/me3\ndistributions (Figure 4). 2) As you suggested, we performed CUT&Tag assay to test the\ngenome-wide distribution of SDG724 in the ehd3-1 mutant background and found that\nSDG724 failed to enrich at its target regions due to the absence of Ehd3, indicating that\nEhd3 is required for SDG724 to bind to chromatin (Extended Data Fig. 8 c-f). 3) Also\nfollowing your comments, we performed ChIP-seq of H3K36me2/me3 in HA-\nEhd3mu/ehd3-1-1, HA-Ehd3/ehd3-1-1, and WT plants, showing that the site-mutated\nEhd3 (Ehd3mu), which lost its binding capacity to H3K4me1, failed to recover the\nH3K36me2/me3 profiles in the HA-Ehd3mu/ehd3-1-1 plants, while the wild-type Ehd3\ndid in the HA-Ehd3/ehd3-1-1 plants (Extended Data Fig. 9a). Gene-by-gene analysis\nfurther confirmed that the site-mutated Ehd3 failed to rescue the aberrant\nH3K36me2/me3 distributions in the ehd3-1 mutant background (Extended Data Fig.\n9b, c). 4) We have also examined the published ChIP-seq data of plants and confirmed\nthe colocalization of H3K4me1 and H3K36me3 in other land plants including Eutrema\nsalsugineum, Populus trichocarpa, Phaseolus vulgaris, Asparagus officinalis,\nBrachypodium distachyon, Setaria viridis, Sorghum bicolor, and Zea mays, further\nsupporting our conclusion (Extended Data Fig. 10d).\nTogether, all these results proved that Ehd3-binding to H3K4me1 is necessary for\nSDG724 to establish H3K36me2/me3 in planta, supporting that Ehd3 recognizes\nH3K4me1 and recruits SDG724 to H3K4me1-marked chromatin to promote the\ndeposition of H3K36me2 and H3K36me3 in rice.\n2. In the next sentense “Ehd3 recognizes H3K4me1 through its tandem PHD finger\ndomain and recruits the H3K36 methyltransferase SDG724 to H3K4me1-enriched\nchromatin regions.” To state this, the authors should perform SDG724 ChIP-seq in ehd3\nmutant background, and hopefully, in the mutant that lose H3K4me1.\nResponse: We thank you for your helpful comments. We have generated the SDG724-\nHA/sdg724-1-1 and SDG724-HA/ehd3 sdg724-1 transgenic plants (Extended Data Fig.\n8 c, d) and performed CUT&Tag assay to examine the genome-wide distributions of\nSDG724 in the sdg724-1 and ehd3 sdg724 mutant plants, respectively. In SDG724-\nHA/sdg724-1-1 plants, SDG724 was predominantly enriched at H3K4me1-marked\ngenes, similarly as in the YFP-SDG724/NiP plants. However, in SDG724-HA/ehd3\nsdg724-1 plants, which lack Ehd3, SDG724-binding to chromatin was almost abolished\n(Extended Data Fig. 8 e, f). These results support the conclusion that Ehd3 is required\nfor recruiting SDG724 to H3K4me1-occupied chromatin regions. We have now\nincluded the new data in our revised manuscript (Lines 350–353, Extended Data Fig. 8\nc-f).\n3. L308-310, “Collectively, these results indicated that Ehd3 and SDG724 together bind\nto genes enriched with H3K4me1 and promote deposition of H3K36me2 and\nH3K36me3 (Fig. 5d and Extended Data Fig. 6e).” To claim that the binding of Ehd3 to\nH3K4me1 is important for H3K36me2/3 deposition, the authors should perform\nH3K36me2/3 ChIP-seq in HA-Ehd3mu/ehd3-1 plants and compare it with wild type\nand ehd3-1 plants.\nResponse: We thank you for your helpful comments. Following your suggestion, we\nperformed H3K36me2/me3 ChIP-seq in the HA-Ehd3/ehd3-1-1 and HA-Ehd3mu/ehd3-\n1-1 plants, showing that the introduction of HA-Ehd3mu, encoding site-mutated Ehd3\nprotein lacking the binding capacity to H3K4me1, failed to rescue the H3K36me2/me3\nprofiles in the ehd3-1 background, while HA-Ehd3 did, and HA-Ehd3/ehd3-1-1 plants\ndisplayed similar H3K36me2/me3 profiles as WT. All these results proved that Ehd3-\nbinding to H3K4me1 is necessary for H3K36me2/me3 establishment in planta,\nsupporting that Ehd3 recognizes H3K4me1 and recruits SDG724 to H3K4me1-marked\nchromatin to promote the deposition of H3K36me2 and H3K36me3 in rice. We have\nincluded the new data in our revised manuscript (Lines 357–365, Extended Data Fig.\n9a-c).\n4. This paper presents a lot of data in the discussion section, such as Extended data 8-\n10. I suggest to move the description of data in the results section.\nResponse: Following your suggestion, we have moved the description of Extended data\n8-9 to the Results section, and we prefer to keep the former Extended data 10 in the\ndiscussion section in our revised manuscript (Lines 311–339, Figure 5).\n5. The patterns of Ehd3 and SDG724 localizations are bit different (Fig. 5a and\nExtended Data Fig. 6d). Again, the effect of ehd3 mutation on SDG724 localization\nmay support the authors’ claim.\nResponse: Following your suggestion, we performed CUT&Tag assay in the SDG724-\nHA/sdg724-1-1 and SDG724-HA/ehd3 sdg724-1 transgenic plants. Introduction of\nSDG724-HA rescued the phenotypes of the sdg724-1 single mutant but not of the ehd3\nsdg724 double mutant. CUT&Tag data showed that due to the absence of Ehd3,\nSDG724-binding to chromatin was nearly abolished in the SDG724-HA/ehd3 sdg724-\n1 plants compared with the SDG724-HA/sdg724-1 plants. These results strongly\nsupport the notion that Ehd3 is necessary for recruiting SDG724 to chromatin. We have\nincluded the new data in our revised manuscript (Lines 350–353, Extended Data Fig. 8\nc-f).\n6. Similar to point 5, L293 says “SDG724 exhibited a notable co-occupancy with Ehd3\nat H3K4me1-enriched genes”, but heatmaps are not enough to show co-occupancy.\nFormal analysis of co-occupancy is needed.\nResponse: Following your suggestion, we have now performed a formal analysis of the\nco-occupancy of Ehd3, SDG724, and H3K4me1 modification. We identified 15,967\ngenes occupied by Ehd3 and 17,070 genes occupied by SDG724, of which 10,325 genes\nwere co-occupied by both Ehd3 and SDG724. Notably, these overlapping genes were\nsignificantly enriched of H3K4me1 modification. We have included the analysis in our\nrevised manuscript (Lines 342–346, Fig. 6a).\n7. Does IP-MS experiment in Fig. 4a contain negative control such as non-transgenic\nplants to show that the detected proteins are specifically bound to the bait proteins?\nResponse: Indeed, we have used the transgenic plants expressing YFP alone driven by\nthe Ubiquitin promoter as the negative control in IP-MS experiments. This control\nconfirmed that the detected proteins are specifically bound to the bait proteins. We have\nnow included the negative control information in the revised manuscript (Lines 270–\n272 and Supplementary Table 6).\n8. How is the experiment in Fig. 5g reproduced? Results of replicate experiments should\nbe presented in Supplementary data.\nResponse: Following your suggestion, we have repeated the histone methyltransferase\nassay and included the data in our revised manuscript (Fig. 6h and Extended Data Fig.\n10f). The results confirmed that both Ehd3 and H3K4me1 modification are required to\nstimulate the catalytic activity of SDG724.\n9. The author describes the potential role of H3K36me3 in repressing cryptic\ntranscription start. To test this possibility, studying transcription start by such as TSS-\nseq in sdg724 and ehd3 mutants will be needed.\nResponse: We agree with you that analyzing transcription start sites using approaches\nsuch as TSS-seq in the ehd3-1 and sdg724-1 mutants will be insightful to understand\nthe potential role of H3K36me3 in regulation of cryptic transcription initiation in plants.\nHowever, this is not the main scope of the current study, and we hope to report the\nrelated results in our future work.\nOther comments\n1. Around L184-189, a lot of abbreviation such as NZ or OD1 without explanation.\nExplanation is needed.\nResponse: Following your suggestion, we have revised the relevant descriptions by\nreferring to the functional groups instead of individual atoms in our revised manuscript\n(Lines 175–203), which will be more approachable for all readers. We would like to\nexplain that ‘NZ’ and ‘OD1’ are not context-specific abbreviations, but standard atomic\nnomenclature in structural biology, as defined by IUPAC-IUBMB\n(https://iupac.qmul.ac.uk/AminoAcid/).\n2. Line 201-203, “the Ala substitution for each H3K4me1 binding-pocket residue\nmarkedly reduced the affinity of the Ehd3 tandem PHD finger for the H3K4me1 peptide\n(Fig. 3g and Supplementary Table 2).” Are D496 and Y446 H3K4me1 binding-pocket?\nLooks like they are interacting with H3R2. Explanation for this results will be helpful.\nResponse: We thank you for your helpful comments. Indeed, both Y446 and D496 are\ninteracting with H3R2. To avoid possible confusion, we revised the description as\n“Consistent with the structural observation, mutation of H3R2-interacting residues\n(Tyr446 and Asp496) or H3K4me1 binding-pocket residues (His441, Tyr471, Asp486,\nand Ile489) either abolished or markedly reduced the binding affinity of the Ehd3\ntandem PHD finger to the H3K4me1 peptide (Fig. 3g and Supplementary Table 2),\nwhich further confirmed that both H3R2-binding and H3K4me1-binding residues are\ncritical for H3K4me1 recognition by Ehd3” in our revised manuscript (Lines 206–211).\n3. Line 229, “a 4-bp deletion at target site 1 and a 1-bp insertion at target site 2 (ehd3-\n4)” This will make 3-bp deletion in total and frame will be normal. Explanation of why\nthis mutant show loss-of-function phenotype will be helpful.\nResponse: Following your suggestion, we have added an explanation in our revised\nmanuscript (Lines 238–239). Indeed, in the ehd3-4 allele, a 4-bp deletion combined\nwith a 1-bp insertion resulted in a net 3-bp deletion. However, this frameshift led to\namino acid changes within the Ehd3 protein (residues 52–92; see Fig. R1 below), which\ncould abolish the function of Ehd3.\n4. Line 277, “Significant overlap”. Please indicate statistical significance for overlap if\nstate so.\nResponse: Following your suggestion, we have performed Fisher’s exact test and\nprovided the p-values in the revised figures to support significance for overlap\n(Extended Data Fig. 5b, c).\n5. Line 290-291, “As a control, the H3 distribution in these mutants was comparable to\nthat in NiP (Extended Data Fig. 6c).” Why H3 data is shown only by heatmaps and all\ngene metaplots, which do not tell if pattern is unaltered or not in mutants?\nShowing scatter plots like Fig. 4i will be preferable.\nResponse: Following your suggestion, we have added the scatter plots of H3 data,\nsupporting that H3 distribution in the mutants was comparable to that in NiP (Extended\nData Fig. 6d).\n6. Extended Data Fig. 7a. Is this based on gene-by-gene? Or based on fixed length bin\nsuch as 100bp?\nResponse: Following your suggestion, we have revised the figure legend to provide a\ndetailed description for the analysis (Extended Data Fig. 10a). Specifically, all genes\nwere scaled to fixed length and then processed for histone modification quantification,\nZ-normalized, and Pearson correlation coefficients calculation.\nResponse: We thank you for your helpful comments that have improved our manuscript\nand led to the inclusion of new data. We have addressed the specific comments as\npresented below.\n1. Results from Extended Data Figures 8 and 9 should be described in the Results\nsection rather than the Discussion.\nResponse: Following your suggestion, we have described Extended Data Figures 8 and\n9 in the Results section and have combined part of the data into a main figure (Lines\n311–339, Figure 5).\n2. The authors are suggested to test the interaction between Ehd3 homolog proteins in\nArabidopsis and the H3K36 methyltransferases (e.g., SDG8) to examine whether the\ninteraction between the H3K4me1 reader and H3K36 methyltransferase is conserved\nin other plant species.\nResponse: Following your suggestion, we examined the interaction between AtEHD3\n(the Arabidopsis homolog of rice Ehd3) and SDG7 (the Arabidopsis homolog of rice\nSDG724) using yeast two-hybrid and GST pulldown assays. Our results proved that\nAtEHD3 interacted with SDG7, supporting that the interaction between the H3K4me1\nreader and the H3K36 methyltransferase is conserved in Arabidopsis. We have now\nincluded the data in our revised manuscript (Lines 276–279, Extended Data Fig. 4g, h).\n3. Lines 277–279: Please provide P-values to support the claim of “significant overlap”.\nResponse: We thank you for your comments. We have performed Fisher’s exact test\nand provided the p-values in the revised figures to support our claim of “significant\noverlap” (Extended Data Fig. 5b, c).\n4. Lines 326–330: In addition to the genome browser view, a correlation analysis should\nbe performed to strengthen the conclusion.\nResponse: Following your suggestion, we have performed correlation analyses across\ndifferent species, supporting that land plants exhibit stronger positive correlation\nbetween H3K4me1 and H3K36me2/me3 compared to animals and C. reinhardtii. We\nhave now included the data in our revised manuscript (Extended Data Fig. 10c).\nResponse: We thank you for your helpful comments that have improved our manuscript.\nWe have addressed the specific comments as presented below.\nL31 and 53, H3 means histone 3. So, please change histone H3 directly to histone 3.\nResponse: We have changed it in our revised manuscript (Lines 31, 53, and 64).\nL171-172, it is not rigorous to say that the hydrogen bond exists between the N and O\natoms, as there is no hydrogen. I would like to suggest that the author change the N and\nO atoms here to the amino and carbonyl groups, respectively. The following\ndescriptions should also be revised to prevent using the atom but use the group to\ndescribe the hydrogen bonds.\nResponse: We thank you for your helpful suggestion. We have revised the descriptions\nof individual atoms to functional groups in our revised manuscript (Lines 175–203).\nFig. 4f, it looks like there is no significant difference between II and IV, and III and IV.\nPlease analyze the differences to confirm.\nResponse: Following your suggestion, we have performed the statistical analysis to\ncompare groups II and IV, as well as III and IV. As expected, there is no significant\ndifference between these groups. We have now revised Fig. 4f in our revised manuscript.\nL284-285, why analyze H3K36me here? Because SDG724 is an H3K36 MTase? Please\nadd the short sentence here to describe the logic for analyzing H3K36me. Or else, the\nintroduction of H3K36 becomes strange here.\nResponse: Following your suggestion, we have added a brief introduction to clarify that\nSDG724 is a known H3K36 methyltransferase in our revised manuscript (Line 300).\nL334, why SDG725? Please have a very brief introduction for SDG725, especially\nabout the CW domain. A potential way is to move the sentence of L341-345, the\nintroduction of the CW domain of SDG8 and SDG725, to L334.\nResponse: Thank you for your helpful comments. Now we have added a brief\nintroduction for SDG725 and moved the introduction of the CW domain of SDG8 and\nSDG725 as you suggested in our revised manuscript (Lines 396–402).\nDoes SDG724 have a CW domain like SDG725? If yes, please give the details. If not,\nwhy could the H3K4me1 NCP stimulate the activity of SDG724? Please have a short\ndiscussion about this issue.\nResponse: SDG724 does not contain a CW domain (Extended Data Fig. 11a). We have\nrepeated the histone methyltransferase experiment and proved that H3K4me1-NCP did\nnot stimulate SDG724 activity. We have now included the data in our revised\nmanuscript (Line 406, Fig. 6h)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors provided satisfactory new data addressing my previous comments. The paper is now very much suited for the\npublication in Nature Communications. Congratulations for the great piece of work. I only have a suggestion on the title of\nthe paper. I suggest to make title more informative and accurate; such as \"The H3K4me1 reader protein EHD3 mediates\nH3K36me2 and H3K36me3 by recruiting the H3K36me writer SDG724 in rice\" or \"The H3K4me1 reader protein EHD3\nmediates H3K4me1–H3K36me3 co-occupancy in land plants\" or similar.", "reviewer_2": "The authors have addressed my concerns. I have no further comments.", "reviewer_3": "Most of my concerns have been precisely addressed. A small error is listed below.\nLine 175, it is correct that here is “amino group”. But in Line 178, it should be “amide group” of Arg2, but not “amino group”."}, "author_response": "Response: We sincerely appreciate your positive comments on our revised manuscript.\nAt the same time, we thank you for your thoughtful suggestion regarding the title of the\nmanuscript. After careful consideration, we hope to keep the title “H3K4me1 directs\nH3K36me2 and H3K36me3 deposition in land plants”, as we think it directly and\nclearly conveys the core conclusion of our work: H3K4me1 has a completely different\nfunction in plants compared to animals. Once again, we thank you for your constructive\nsuggestions throughout the review process.\nResponse: We thank you for your constructive comments throughout the review process.\nResponse: We thank you for your constructive comments throughout the review process.\nWe have now corrected the mistake in our revised manuscript (Line 178)."}]

Decision letter