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. 2016 Oct;172(2):1131-1141.
doi: 10.1104/pp.16.01238. Epub 2016 Aug 17.

Cooperation Between the H3K27me3 Chromatin Mark and Non-CG Methylation in Epigenetic Regulation

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Free PMC article

Cooperation Between the H3K27me3 Chromatin Mark and Non-CG Methylation in Epigenetic Regulation

Shaoli Zhou et al. Plant Physiol. .
Free PMC article

Abstract

H3K27me3 is a repressive chromatin mark of genes and is catalyzed by homologs of Enhancer of zeste [E(z)], a component of Polycomb-repressive complex 2 (PRC2), while DNA methylation that occurs in CG and non-CG (CHG and CHH, where H is A, C, or T) contexts is a hallmark of transposon silencing in plants. However, the relationship between H3K27me3 and DNA methylation in gene repression remains unclear. In addition, the mechanism of PRC2 recruitment to specific genes is not known in plants. Here, we show that SDG711, a rice (Oryza sativa) E(z) homolog, is required to maintain H3K27me3 of many developmental genes after shoot meristem to leaf transition and that many H3K27me3-marked developmental genes are also methylated at non-CG sites in the body regions. SDG711-binding and SDG711-mediated ectopic H3K27me3 also target genes methylated at non-CG sites. Conversely, mutation of OsDRM2, a major rice CHH methyltransferase, resulted in loss of SDG711-binding and H3K27me3 from many genes and their de-repression. Furthermore, we show that SDG711 physically interacts with OsDRM2 and a putative CHG methylation-binding protein. These results together suggest that the repression of many developmental genes may involve both DRM2-mediated non-CG methylation and PRC2-mediated H3K27me3 and that the two marks are not generally mutually exclusive but may cooperate in repression of developmentally regulated genes in rice.

Figures

Figure 1.
Figure 1.
Function of SDG711 in H3K27me3 reprogramming during SAM-leaf transition. A, Numbers of genes differentially marked by H3K27me3 in wild type and 711OX seedling leaves and in SAMs. B, Enrichment of hormone and transcription factors genes for differential H3K27me3 between SAMs and leaves (upper part) and between wild-type and 711OX leaves (lower part). Fold changes are differences relative to the observed frequencies of specific subsets within the genome. C, Validation of transcript levels (top) and ChIP-seq data (bottom) of 16 randomly selected genes (14 with and 2 without changes of H3K27me3 and expression in leaf compared to SAM). Significance of differences between the samples detected by PCR (from 3 repeats) is indicated by **, P value < 0.01 and *, P value < 0.05. Bars are means ± sd from three biological repeats. D, Validation of transcript levels (top) and ChIP-seq data (bottom) of 10 randomly selected genes (8 with and 2 without changes of H3K27me3 and expression in 711OX compared to wild type). Significance of differences between the samples detected by PCR (from 3 repeats) is indicated by **, P value < 0.01 and *, P value < 0.05. Bars are means ± sd from three biological repeats.
Figure 2.
Figure 2.
Many genes are comarked by H3K27e3 and non-CG methylation. A, Left, numbers of protein-coding genes methylated in the body region at CG, CHG, and CHH sites and their transcript levels in seedlings. Right, expression levels of genes marked by either H3K27me3 or non-CG methylation, or by both marks. B, Expression rates of the genes marked by either H3K27me3 or non-CG methylation or both in different rice organs. C, Overlapping of genes marked by H3K27me3 with those methylated at CG and non-CG sites in the body regions. D, Enrichment of hormone-related and transcription factor genes comarked by H3K27me3 and non-CG methylation. Fold changes are differences relative to the observed frequencies of specific subsets within the genome. Asterisks indicate significant enrichments (P value < 0.05, Fisher’s tests).
Figure 3.
Figure 3.
SDG711-mediated H3K27me3 targets genes methylated at non-CG sites in the body regions. A, Venn map between genes with ectopic H3K27me3 in 711OX and non-CG methylation in wild type plant. B, Genome browser screen shots of BS-seq and anti-H3K27me3 ChIP-seq of genes with (upper four genes) or without (lower two genes) ectopic H3K27me3. C, Wild-type levels and distribution of CG, CHG, and CHH methylation in the 5′ end (2 kb), gene body (thick line), and 3′ end (2 kb) of the genes that showed ectopic H3K27me3 in 711OX compared with genome-wide level. D, Box plots of CG, CHG, and CHH methylation levels in the gene bodies with ectopic H3K27me3 in 711OX. Significances of differences (Student’s t tests) are indicated (**, P value < 0.01).
Figure 4.
Figure 4.
SDG711 protein preferentially targets gene bodies with high non-CG methylation. A, SDG711 binding is enriched in gene body regions. Top, Comparison of anti-SDG711 and anti-H3K27me3 ChIP-Seq reads in gene bodies (thick line) and 5′ and 3′ ends (2 kb); Bottom, comparison of H3K27me3 levels of SDG711-binding genes with the genome-wide averages in genic regions. B, Contour plots of anti-SDG711 and anti-H3K27me3 ChIP-Seq reads. C, Overlap of SDG711-binding genes with those methylated at non-CG sites. D, Consensus sequences of SDG711-binding motifs. E, Genome browser screen shots of BS-seq and anti-SDG711 ChIP-seq of genes with (upper four genes) or without (lower two genes) SDG711-binding.
Figure 5.
Figure 5.
osdrm2 mutation results in a decrease of genome-wide H3K27me3. A, Left, Venn diagram of H3K27me3-marked genes of wild type and osdrm2. Right, Pairwise scatter plots of H3K27me3 levels between wild type and osdrm2. B, Venn diagram of genes that lost H3K27me3 and non-CG methylation. C, McrBC-digestion and PCR tests of methylation of 8 genes that lost methylation in osdrm2. Three replicates are shown. D, Validations of H3K27me3 modification and SDG711-binding and relative transcript levels of the eight genes by quantitative ChIP-PCR and RT-PCR. Significances of differences are indicated by **, P value < 0.01.
Figure 6.
Figure 6.
SDG711 physically interacts with OsDRM2 and SDG703. A, Yeast two-hybrid assays of SDG711 and OsDRM2 or SDG703. B, Pull-down assay of interaction of SDG711 (C5 domain) and the OsDRM2 protein (left) and the SDG703 protein (right). C, Co-IP assay of interaction of SDG711 and OsDRM2 in vivo. Rice nuclear protein extracts were immunoprecipitated by anti-OsDRM2 or IgG and analyzed by western blot using anti-SDG711. D, Co-IP assay of interaction of SDG711 and SDG703 in vivo. Protein extracts of tobacco protoplasts transfected with SDG703-HA alone, SDG703-HA together with GFP or SDG711-GFP, and SDG711-GFP alone were checked first by western blots (WB) with anti-GFP, then immunoprecipitated with anti-HA, followed by western blotting with anti-GFP and anti-HA.

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