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Plant mobile domain protein–DNA motif modules counteract Polycomb silencing to stabilize gene expression

Nature Plants volume 11, pages 2286–2299 (2025)Cite this article

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Abstract

In plants and animals, Polycomb group proteins are crucial for development, regulating gene expression through the trimethylation of lysine 27 on histone H3 and subsequent gene silencing. While the specification of Polycomb silencing targets is increasingly understood, it remains unclear how certain genes with apparent silencing-attracting features escape this process. Here we show that the plant-mobile-domain-C-containing proteins MAINTENANCE OF MERISTEMS (MAIN), MAIN-LIKE 1 (MAIL1) and MAIL2 oppose Polycomb silencing at numerous actively transcribed genes in Arabidopsis. Mutations in MAIN, MAIL1 or MAIL2 result in Polycomb-group-dependent ectopic H3 K27 trimethylation, often associated with transcriptional repression. We show that MAIL1 (which functions in concert with MAIN) and MAIL2 target distinct gene sets and associate with chromatin at specific DNA sequence motifs. We demonstrate that the integrity of these motif sequences is essential for promoting expression and antagonizing H3 K27 trimethylation. Our results unveil a system opposing Polycomb silencing that involves plant mobile domain C protein–DNA motif modules, expanding our understanding of eukaryotic gene regulation mechanisms.

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Fig. 1: Mutations of NDX or PRC1 RING1A/1B components suppress mail1-induced defects.
Fig. 2: mail1-associated TE and gene misregulation is dependent on NDX or RING1A/1B function.
Fig. 3: MAIL1 and MAIN are mainly co-targeted to M1M at gene proximal promoters.
Fig. 4: NDX and RING1A are enriched at MAIL1/MAIN and MAIL2 binding sites.
Fig. 5: MAIL2 mainly associates with gene promoters and protects numerous actively transcribed genes from H3K27me3-mediated PcG silencing.
Fig. 6: Promoter-associated M1M and M2M are functionally relevant to safeguard genes from PcG-mediated silencing.

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Data availability

The data supporting the findings of this study are available within the article and its Supplementary Information. High-throughput sequencing data have been deposited in the Gene Expression Omnibus database and can be accessed with the accession number GSE278560.

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Acknowledgements

We thank F. Turck (Max Planck Institute for Plant Breeding Research) for providing the pSEP3::GUS construct. We also thank S. Marquardt (Department of Plant and Environmental Sciences, University of Copenhagen) for his help in setting up the CUT&Tag assay. Work in the Mathieu laboratory was supported by Centre National de la Recherche Scientifique (CNRS), Inserm, Université Clermont Auvergne core funding, a grant from the iSITE CAP2025 (to M.O.) and grants from the Agence Nationale de la Recherche (ANR-20-CE12-0009 and ANR-23-CE20-0012 to O.M.). Work in the Moissiard laboratory was supported by CNRS and University of Perpignan Via Domitia core funding, and by a grant from the Agence Nationale de la Recherche (ANR-23-CE20-0012 to G.M.). This study was also supported by ‘Laboratoires d’Excellence’ AGRO 2011-LABX-002 (under the I-Site Muse framework) coordinated by the Agropolis Foundation (ID 2101-009 to G.M.), region Occitanie PhD grant to L.J., and by the ‘Laboratoires d’Excellence (LabEx)’ TULIP (ANR-10-LABX-0041) and ‘École Universitaire de Recherche (EUR)’ TULIP-GS (ANR-18-EURE-0019). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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  1. These authors contributed equally: Thierry Pélissier, Lucas Jarry.

Authors and Affiliations

  1. Institute of Genetics, Reproduction and Development, Université Clermont Auvergne, CNRS-UMR6293, Inserm-U1103, Clermont-Ferrand, France

    Thierry Pélissier, Margaux Olivier, Gabin Dajoux, Marie-Noëlle Pouch-Pélissier, Charles Courtois & Olivier Mathieu

  2. LGDP-UMR5096, CNRS, Université de Perpignan Via Domitia, Perpignan, France

    Lucas Jarry, Julie Descombin, Nathalie Picault & Guillaume Moissiard

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Contributions

T.P., L.J., G.M. and O.M. conceived the study. T.P., L.J., M.O., G.D., M.-N.P.-P., C.C., J.D., N.P., G.M. and O.M. conducted the laboratory experiments. T.P., L.J., G.M. and O.M. interpreted the data. T.P. and O.M. drafted the manuscript. T.P., L.J., G.M. and O.M. edited the manuscript. G.M. and O.M. coordinated the research. The authors read and approved the final manuscript.

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Correspondence to Guillaume Moissiard or Olivier Mathieu.

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Extended data

Extended Data Fig. 1 NDX depletion does not impact overall H3K27me3 distribution.

Metaplots of H3K27me3 accumulation (in RPGC) at protein-coding genes (left) and TEs located in chromosomal arms (middle) or pericentromeric regions (right) in WT and ndx-7 mutant backgrounds.

Extended Data Fig. 2 MAIL1 complexes prevent H3K27me3 incorporation at different loci.

(a) Violin plot inlaid with boxplot showing H3K27me3 levels (in RPKM) at mail1-upregulated (up.) (n = 273) or mail1-downregulated (down.) (n = 95) genes across the indicated genotype. For boxplots, the cross and the center line indicate the mean and median, respectively; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Comparisons (WT vs. ndx, WT vs. mail1, mail1 vs. mail1 ndx and mail1 vs. mail1 ring1a/1b) were performed using Wilcoxon rank sum tests with continuity correction; two sided. Exact P values are shown. (b) Scatter plot showing negative correlation between gain of H3K27me3 and gene expression in mail1. Log2 fold changes were computed for genes with increased H3K27me3 level in mail1 vs WT (DESeq2, P-value < 0.1) and accumulating RNA in at least one of the genotype at this developmental stage. R: Spearman rank correlation coefficient, p: significance level. (c) Genome browser views of RNA-seq (CPM) and H3K27me3 ChIP-seq (RPGC) profiles at mail1 H3K27me3-enriched genes in the indicated genotypes. MAIL1-FLAG and MAIN-FLAG associated patterns are shown together with TRB1-FLAG data (GEO-GSM6895939 retrieved from30). Predicted MAIL1 (M1M) and telobox motifs are indicated. (d) Genome browser view of H3K27me3 levels at SPO11-1 gene in WT and mail1 immature flowers (top). Transcript levels measured by RT-qPCR (bottom) are normalized to ACT2 and further normalized to WT; values represent means from four biological replicates ± s.e.m and statistically significant difference is indicated (unpaired, two-sided Student’s t-test).

Extended Data Fig. 3 Ectopic H3K27me3 deposition is detected at TRBs- enriched genes and correlates with increased levels of H2AUb incorporation.

(a) Metagene plots of H3K27me3 and H2Aub accumulation (in RPGC) in the indicated genotypes at mail1 H3K27me3-enriched genes compared to an equivalent random set of genes or to all Arabidopsis genes. (b) Box plots illustrating H3K27me3 and H2Aub accumulation (in RPGC) at mail1 H3K27me3-enriched genes (upper panel; n = 49) compared to a random set of genes (bottom panel; n = 49) in the indicated genotypes. Cross and center lines indicate the means and medians, respectively; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Statistical significances of mail1 values compared to WT are indicated (Wilcoxon rank sum exact test; two-sided). (c) Sequence logos of most conserved DNA motifs detected by STREME analysis within the 1 kb region surrounding TSS of H3K27me3-enriched genes in mail1. Percentage of regions exhibiting each motif is indicated. (d) Metagene plots showing TRB1/2/3 levels (data from30) at mail1 H3K27me3-enriched genes or at an equivalent random set of genes. (e) Plot heatmaps illustrating TRB1/2/3 enrichment (data from30) at TSS of mail1 H3K27me3-enriched genes.

Extended Data Fig. 4 MAIL1 and MAIN mainly co-localize at M1M-enriched gene TSS.

(a) MAIL1 and MAIN signals (LOG2 FC vs input) at mail1 H3K27me3- enriched or depleted genes. (b) Sequence logo of a highly enriched MEME- predicted motif among the 626 MAIL1/MAIN peaks.

Extended Data Fig. 5 mail2-1 phenotypic defects are partially suppressed by ring1a/1b mutations.

(a) Developmental phenotypes associated to two- week-old seedlings of indicated genotype. (b) Schematic representation of MAIL2 gene showing t-DNA mail2-1 localization and amiR-MAIL2 targeted sequence. Synonymous substitutions creating amiR-MAIL2 insensitive gMAIL2-9xMYC transgene are indicated in red and corresponding amino acids are shown. Positions are given relative to the transcription start site (+1). (c) Two-week-old phenotype of WT, mail2-1 and gMAIL2-1-MYC complemented mail2-1 seedlings. Scale bar is 1 cm.

Extended Data Fig. 6 MAIL1, MAIN and MAIL2 PMD-C domains displays highly similar 3D structures.

Structure predictions of MAIL1, MAIN and MAIL2 were recovered from the Alphafold Protein Structure Database and, individual and merged representation of PMD-specific parts are shown. UniProt IDs and position of PMD-C domains within the proteins are indicated below the structures.

Extended Data Fig. 7 MAIL2 targets a distinct set of genes through recognition of a specific DNA sequence motif.

(a) Overlap between MAIL1/ MAIN and MAIL2-associated peaks. (b) Metagene plot showing mean MAIL2- MYC vs input signal at Arabidopsis gene and TE loci. (c,d) STREME prediction of conserved DNA motifs at MAIL2-associated gene promoters (n = 1059) (c) or at MAIL2 peaks (n = 1775) (d). Relative representation of each motif is reported. (e) Centrimo plot showing strong enrichment of M2M over MAIL2 peaks, within a 45 bp central region delimited by the dashed lines (E-value = 8.7e-143). The 200 bp region surrounding the peak summits is shown. (f) M1M homology to half of the palindromic M2M consensus sequence is highlighted by the dashed box.

Extended Data Fig. 8 Phenotype associated to amiR-mail2 lines.

(a) Examples of developmental phenotype recovered from 10-day-old in vitro growing plants. (b) MAIL2 transcript accumulation in WT and amiR-mail2_4 and _5 lines, detected by RT-qPCR using primers framing the amiRNA-targeted cutting site. Data are normalized to ACT2 and further normalized to WT; values represent means from three biological replicates ± s.e.m and statistical significance for differences to the WT are indicated (unpaired, two-sided Student’s t-test). (c) Photos of five-week-old WT plants compared to amiR-mail2_4 mutant plants containing a single transgene locus at heterozygous (he) or homozygous (ho) state. (d) Two-week-old phenotypes of WT, amiR-mail2 and gMAIL2-1-9xMYC-complemented amiR-mail2 seedlings. Scale bar is 1 cm.

Extended Data Fig. 9 MAIL1/MAIN and MAIL2 pathways target distinct sets of genes.

(a) Venn diagrams showing no overlap between mail1- and amiR-mail2- downregulated genes. (b) Developmental phenotypes observed upon MAIL2 depletion in mail1 mutant background. Scheme of the cross is illustrated with photos of two-week-old plants. Scale bar is 1 cm. Zoomed-in pictures of mail1 F2 progeny expressing or not the amiR-MAIL2 transgene are shown. (c) Metagene plots (top) and heatmaps (bottom) illustrating MAIL2 enrichment at PCGs that display enhanced H3K27me3 levels in amiR-mail2 mutants (d) Metagene plots (top) and heatmaps (bottom) illustrating TRBs (data from30) and MAIL2 enrichment at MAIL2-associated promoter genes downregulated in amiRNA-mail2 lines. For TRBs, only TRB1 data were represented on the heatmap. (e)Venn Diagram showing overlap between MAIL2 and TRB1 peaks at proximal promoter region of amiRNA-mail2 downregulated genes. (f) Venn diagrams showing overlap of M1M and M2M loci with MAIL1 and MAIL2 peaks respectively.

Extended Data Fig. 10 Predicted DNA-binding potential of the PMD-C domains of MAIN, MAIL1 and MAIL2.

(a) Predicted structures of MAIN, MAIL1 and MAIL2 PMD-C domains, with DNA-binding residues mapped onto each structure and colored according to their prediction score. Full length proteins are shown above. (b) Close-up views of the indicated PMD-C regions. DNA-binding residues with a prediction confidence score ≥ 0.8 are colored as in (a). Positively charged residues with a confidence score ≥ 0.8 are highlighted in green. A complete list of DNA-binding prediction scores for amino acid residues of MAIN, MAIL1 and MAIL2 is reported in Supplementary Table 5.

Supplementary information

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Supplementary Figs. 1–6, Tables 1–5 and Doc. 1.

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Pélissier, T., Jarry, L., Olivier, M. et al. Plant mobile domain protein–DNA motif modules counteract Polycomb silencing to stabilize gene expression. Nat. Plants 11, 2286–2299 (2025). https://doi.org/10.1038/s41477-025-02127-1

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