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Multilayered epigenetic control of persistent and stage-specific imprinted genes in rice endosperm

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Abstract

In angiosperms, epigenetic profiles for genomic imprinting are established before fertilization. However, the causal relationships between epigenetic modifications and imprinted expression are not fully understood. In this study, we classified ‘persistent’ and ‘stage-specific’ imprinted genes on the basis of time-course transcriptome analysis in rice (Oryza sativa) endosperm and compared them to epigenetic modifications at a single time point. While the levels of epigenetic modifications are relatively low in stage-specific imprinted genes, they are considerably higher in persistent imprinted genes. Overall trends revealed that the maternal alleles of maternally expressed imprinted genes are activated by DNA demethylation, while the maternal alleles of paternally expressed imprinted genes with gene body methylation (gbM) are silenced by DNA demethylation and H3K27me3 deposition, and these regions are associated with an enriched motif related to Tc/Mar-Stowaway. Our findings provide insight into the stability of genomic imprinting and the potential variations associated with endosperm development, different cell types and parental genotypes.

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Fig. 1: Imprinted genes at the developmental stages of F1 endosperm.
Fig. 2: Allelic DNA methylation patterns for persistent and stage-specific imprinted genes in F1 endosperm.
Fig. 3: Types of DNA methylation and control regions related to PEGs.
Fig. 4: Demethylation of gbM and trimethylation at H3K27 in maternally inherited alleles of PEGs.
Fig. 5: Cis-motifs associated with H3K27me3 enrichment on the maternally inherited alleles of PEGs.

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Data availability

All sequencing data generated in this study have been deposited in the DDBJ Sequence Read Archive with accession numbers DRA015294, DRA015356, DRA018338 and DRA018337. The genes can be found in GenBank or Rice Genome Annotation Project database (http://rice.plantbiology.msu.edu/analyses_search_locus.shtml) under the following accession numbers: OsMADS77, LOC_Os09g02780; OsEMF2a, LOC_Os04g08034; Waxy, LOC_Os06g04200; GE1, LOC_Os07g41240; Oryza;CycB1;1, LOC_Os01g59120; OsIAA29, LOC_Os11g11430; OsNF-YB1, LOC_Os02g49410; and OsYUCCA12, LOC_Os02g17230. Source data are provided with this paper.

Code availability

The scripts are available upon reasonable request from the corresponding authors.

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Acknowledgements

This work was partly supported by Grants-in-Aid for Transformative Research Areas (22H05172, 22H05175 and 21H02170 to T. Kinoshita, 23H04756 to T. Kawakatsu) and Grants-in-Aid for Scientific Research (20K15504 to K.T., 22K15145 and 23H04749 to D.S., 22K05575 to A.O., 23K23585 to T. Kawakatsu, 22H02320 to T. Kinoshita) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Computations were partially performed on the NIG supercomputer at ROIS National Institute of Genetics, Japan. We thank K. Yamaguchi for expert technical assistance and T. Okamoto for providing the H2B-GFP reporter line.

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Authors and Affiliations

  1. Kihara Institute for Biological Research, Yokohama City University, Yokohama, Kanagawa, Japan

    Kaoru Tonosaki, Daichi Susaki, Akemi Ono, Hiroki Nagata & Tetsu Kinoshita

  2. Faculty of Agriculture, Iwate University, Morioka, Iwate, Japan

    Kaoru Tonosaki & Katsunori Hatakeyama

  3. RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, Japan

    Hatsune Morinaka & Keiko Sugimoto

  4. Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka, Japan

    Hiroyasu Furuumi & Yutaka Sato

  5. Plant Cytogenetics, National Institute of Genetics, Mishima, Shizuoka, Japan

    Ken-Ichi Nonomura

  6. Department of Plant Biology and Genome Center, University of California, Davis, CA, USA

    Luca Comai

  7. Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan

    Taiji Kawakatsu

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  1. Kaoru Tonosaki
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Contributions

K.T. and T. Kinoshita designed the research; K.T., A.O., K.S., L.C., T. Kawakatsu and T. Kinoshita supervised the experiments; K.T., D.S., H.M. and H.N. performed the experiments; K.T. analysed the data; H.F., K.-I.N., Y.S. and K.H. provided research materials; K.T., L.C., T. Kawakatsu and T. Kinoshita wrote the paper.

Corresponding authors

Correspondence to Kaoru Tonosaki, Taiji Kawakatsu or Tetsu Kinoshita.

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Extended data

Extended Data Fig. 1 Maternal rates of accession-specific imprinted genes in F1 endosperm.

a, b, Box plots showing the average maternal rate of the accession-specific MEGs (a) and PEGs (b) in two cross combinations at 3, 5, and 7 DAP. Boxes surrounded by broken rectangles represent accession-specific imprinted genes in the corresponding cross. These genes showed relatively higher average maternal rates than did those of accession-specific imprinted genes identified from the other cross. Colored boxes indicate maternal rates of accession-specific imprinted genes: purple and blue, NIP-KAS specific; yellow and green, NIP-9311 specific; gray, 200 randomly selected genes (as control) from F1 endosperm at 3, 5, and 7 DAP from NIP-KAS (left panel) and NIP-9311 (right panel) crosses. All data are from eight independent crosses, each consisting of two biological replicates of two combinations of reciprocal crosses between NIP-KAS and NIP-9311. The box plots show the median (line within the box), the lower and upper quartiles (box), margined by the largest and smallest data points that are still within the interval of 1.5 times the interquartile range from the box (whiskers); outliers are not shown. P-values were calculated using Kruskal-Wallis tests followed by a pairwise two-sided Wilcoxon rank-sum test with Bonferroni correction.

Extended Data Fig. 2 Comparisons between persistent and stage-specific imprinted genes.

a, Log2 fold change of maternal ratio (maternal/paternal) of persistent and allele-specific MEGs (left panel) and PEGs (right panel). All data are from eight independent crosses, each consisting of two biological replicates of two combinations of reciprocal crosses between NIP-KAS and NIP-9311. The box plots show the median (line within the box), the lower and upper quartiles (box), margined by the largest and smallest data points that are still within the interval of 1.5 times the interquartile range from the box (whiskers); outliers are not shown. P values were calculated using Kruskal-Wallis tests followed by pairwise two-sided Wilcoxon rank-sum test and adjustment by the Bonferroni methods. b, c, Proportions of different classifications of imprinted genes among stage-specific (b) and persistent imprinted genes (c).

Extended Data Fig. 3 Allelic DNA methylation profiles of imprinted genes.

a–d, Metagene plots showing average weighted allelic DNA methylation levels in CG (a), CHG (b), and CHH (c) contexts in embryos and in the CHH context in endosperm at 5 DAP (d) for MEGs (left panels) and PEGs (right panels). Colored lines represent the different classes of genes: gray, all genes; blue, persistent imprinted genes; green, stage-specific imprinted genes. Solid lines indicate paternal alleles, and broken lines represent maternal alleles.

Extended Data Fig. 4 Distribution of allelic DNA methylation profiles of persistent and stage-specific imprinted genes.

a–d, Heatmap showing allelic weighted DNA methylation of CG (a,b) and CHG (c,d) contexts for persistent and stage-specific MEGs (a,c) and PEGs (b,d) in the endosperm.

Extended Data Fig. 5 Correlations between allelic DNA methylation and imprinted expression.

a–d, Comparison between the difference in allelic weighted CG (a,b) and CHG (c,d) methylation levels (paternal − maternal) and the log2 maternal ratio (maternal/paternal) of the persistent and stage-specific MEGs (a,c) and PEGs (b,d). Differently colored points indicate different classes of imprinted genes. Regression lines for all imprinted genes are indicated. The P values were obtained from two-sided tests for association between paired samples based on Pearson’s correlation coefficient.

Extended Data Fig. 6 Hypo-DMRs associated with maternally inherited alleles of imprinted genes.

a, b, Venn diagrams showing the overlaps of maternal hypo-DMRs in CG and CHG contexts for MEGs (a) and PEGs (b). c, d, UpSet plots representing the combinations of the four genomic regions, promoter, downstream, gene body, and TSS, for maternal CG hypo-DMRs (left) and CG/CHG hypo-DMRs (right) in MEGs (c) and PEGs (d). Significant enrichment for maternal hypo-DMRs in combinations of genomic regions was tested using a hypergeometric test. P-values were adjustment by Bonferroni methods, *P < 1.0E-04, **P < 1.0E-08, ***P < 1.0E-16. e, Heatmap showing distribution of allelic weighted DNA methylation in CG (left) and CHG (right) contexts for maternal CG or CG/CHG hypomethylated imprinted genes. Colors in the left box represent different classes of imprinted genes: blue, persistent; yellow, stage-specific; gray, other genes.

Source data

Extended Data Fig. 7 Allelic H3K27me3 enrichment of MEGs.

a, Metagene plots showing allelic Z-scores for H3K27me3 levels in MEGs. Differently colored lines represent different classes of genes: gray, all genes; blue, persistent imprinted genes; green, stage-specific imprinted genes. Solid lines indicate paternal alleles, and broken lines indicate maternal alleles. b, Differences in average allelic Z-scored H3K27me3 levels between paternal and maternal alleles (maternal – paternal). All data are from eight independent crosses, each consisting of two biological replicates of two combinations of reciprocal crosses between NIP-KAS and NIP-9311. The box plots show the median (line within the box), the lower and upper quartiles (box), margined by the largest and smallest data points that are still within the interval of 1.5 times the interquartile range from the box (whiskers); outliers are not shown. Significant differences were not detected in MEGs. c, Proportions of different epigenetic marks in the gene body region of each class of MEGs. Different colors indicate different combinations of epigenetic marks. Significant enrichment for combinations of epigenetic marks was tested using a hypergeometric test. P values were adjustment by Bonferroni methods, *P < 1.0E-08.

Source data

Extended Data Fig. 8 Imprinting profiles in different cell types of 5-DAP endosperm.

a, Expression and log2 maternal bias (maternal/paternal) of persistent and 5-DAP-specific MEGs (left) and PEGs (right) in pseudo-bulk data sets of UMAP clusters. Left panels show hierarchical clustering of average expression patterns. Expression levels are represented by Z-scored TPM values. Right panels represent log2 maternal bias (maternal/paternal) for each imprinted gene. Gray color represents genes below the threshold of detectable expression. b, Bar plots showing the number of detectable MEGs and PEGs from pseudo-bulk data sets of UMAP clusters. Different colors represent different classes of imprinted genes: blue, persistent; green, 5-DAP-specific.

Extended Data Fig. 9 Epigenetic state of genes overlapping Stowaway motifs.

a–d, Differences in allelic weighted CG methylation (a,c) and Z-scored H3K27me3 levels (b,d) in four regions harbouring Stowaway motifs (a,b) and Stowaway motifs overlapping with hypo-DMRs of maternally inherited regions (c,d) in 5-DAP endosperm. P values were calculated by two-sided Wilcoxon rank-sum test with adjustment by Bonferroni methods. Statistical analyses were performed using 2,000 randomly selected samples from each category. e, Proportion of Stowaway motifs overlapping with hypo-DMRs of maternally inherited alleles in six genomic categories. f, Histograms showing distribution of the Stowaway motif sequence in the three categories of genes shown in the Venn diagram (Fig. 5f). Different colors indicate whether the Stowaway motif overlaps with hypo-DMRs of maternally inherited alleles (red) or not (blue). g, h, Differences in average allelic weighted CG methylation (g) and Z-scored H3K27me3 levels (h) between paternal and maternal alleles in 5-DAP endosperm are indicated according to three categories of genes shown in the Venn diagram (Fig. 5f). Colors of boxes represent different classes of imprinted genes. P values were calculated by two-sided Wilcoxon rank-sum test and adjustment by Bonferroni methods. In a–d, g, h, all data are from eight independent crosses, each consisting of two biological replicates of two combinations of reciprocal crosses between NIP-KAS and NIP-9311. The box plots show the median (line within the box), the lower and upper quartiles (box), margined by the largest and smallest data points that are still within the interval of 1.5 times the interquartile range from the box (whiskers); outliers are not shown.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–8.

Reporting Summary

Supplementary Data 1

Summary of our NGS data sets and public data sets used in this study.

Supplementary Data 2

List of statistical values and maternal rates of imprinted genes in F1 endosperm and embryo.

Supplementary Data 3

List of imprint classes for MEGs and PEGs.

Supplementary Data 4

Comparative list of the reported imprinted genes.

Supplementary Data 5

List of differential methylated regions between parental alleles in F1 endosperm.

Supplementary Data 6

List of the maternal enrichment regions for H3K27me3.

Supplementary Data 7

List of imprinted genes detected from UMAP clusters of snRNA-seq.

Supplementary Data 8

List of clustered imprinted genes and their epigenetic marks and Stowaway motif.

Source data

Source Data Fig. 2

Statistical test results with exact P values.

Source Data Fig. 3

Statistical test results with exact P values.

Source Data Fig. 4

Statistical test results with exact P values.

Source Data Fig. 5

Statistical test results with exact P values.

Source Data Extended Data Fig. 6

Statistical test results with exact P values.

Source Data Extended Data Fig. 7

Statistical test results with exact P values.

Source Data Extended Data Fig. 9

Statistical test results with exact P values.

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Tonosaki, K., Susaki, D., Morinaka, H. et al. Multilayered epigenetic control of persistent and stage-specific imprinted genes in rice endosperm. Nat. Plants 10, 1231–1245 (2024). https://doi.org/10.1038/s41477-024-01754-4

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