A) Chromosomal distribution of H3K27me3 regions. The H3K27me3 regions per 200 kb and genes per 200 kb (y-axis, right-side scale) and number of transposons (y-axis, left-side scale). Numbers on top indicate chromosome number. B) Average H3K27me3 profiles (red line) over H3K27me3 targets. The black line represents the H3K27me3 profile over genes not marked by H3K27me3. The blue bar represents the annotated gene body from transcription start (left) to transcription end (right). Profiles are shown for 5% length intervals along the gene body and for 100 bp sequence intervals for the 2-kb regions upstream and downstream of each gene. C) Length distribution of H3K27me3 regions. D) Comparison of ChIP-chip results with Arabidopsis genes (red boxes, where arrows indicate direction of transcription) that were previously shown to be H3K27me3 targets [6], [7], [11], [57]. Genes are shown as yellow boxes (exons) and lines (introns), and H3K27me3 is shown as vertical light blue bars [MAT score ranging from −1 (bottom) to 6 (top)].

A) Expression level of shared (green) and endosperm-specific (orange) H3K27me3 target genes compared to all genes (black) in the peripheral endosperm of seeds containing globular stage embryos. B) Box plot of H3K27me3 MAT scores of shared (green) and endosperm-specific (orange) target genes. H3K27me3 MAT scores of endosperm-specific target genes with low expression levels (log₂<4.2) and moderate expression levels in the endosperm (log₂>4.2) are symbolized by striped and dotted fill patterns, respectively. C) Cluster analysis of endosperm-specific H3K27me3 target genes. H3K27me3 target genes are grouped into five mutually exclusive clusters based on their expression patterns. Each row represents a gene, and each column represents a tissue type. Tissue types are: seedlings, leaves, stems, roots, flowers, siliques containing seeds with embryos in the heart or torpedo stage, seeds with embryos in the torpedo, walking stick, curled cotyledon, early green, and green cotyledon stage and endosperm derived from seeds with embryos in the preglobular, globular and heart stage. Red or green indicate tissues in which a particular gene is highly expressed or repressed, respectively.

A) Venn diagram showing overlap of H3K27me3 target genes with genes deregulated in fis2 seeds at 3 DAP and 6 DAP. Only genes present on the ATH1 microarray were included in the analysis. B) Cluster analysis of H3K27me3 target genes that are deregulated in fis2 mutant. H3K27me3 target genes are grouped into two main clusters based on their expression patterns in different domains of the endosperm. Each row represents a gene, and each column represents a tissue type. Tissue types are: seedlings, leaves, stems, roots, flowers, siliques containing seeds with embryos in the heart or torpedo stage, seeds with embryos in the torpedo, walking stick, curled cotyledon, early green, and green cotyledon stage and endosperm derived from seeds with embryos in the preglobular, globular and heart stage. Red or green indicate tissues in which a particular gene is highly expressed or repressed, respectively. Colors at the right side symbolize genes deregulated in fis2 at 3 DAP (orange), 6DAP (yellow), or at both time points (yellow with orange stripes). C) Quantitative RT-PCR analysis of TEGs in wild-type, fis2/FIS2, met1/MET1 and fis2/FIS2;met1/MET1 seeds. Error bars, s.e.m.

A) PHE1::PHE1-EGFP is specifically expressed in endosperm nuclei from 1 DAP to 4 DAP. First five images are fluorescence images overlaid with bright-field images. The chalazal endosperm (CZE) is indicated. Last image of the row shows a confocal image. The position of the embryo (EMB) is indicated by a white line. Scale bars, 50 µM. B) Quantitative RT-PCR expression analysis of seed coat marker genes BANYULS (BAN) and TRANSPARENT TESTA2 (TT2), and embryo marker genes WUSCHEL-RELATED HOMEOBOX (WOX) 2 and WOX9 in sorted endosperm nuclei and total nuclei isolated from PHE1::PHE1-EGFP expressing 1–4 DAP-old seeds. Values are shown relative to PHE1 expression. Error bars, s.e.m. C) Transcript levels of seed coat marker genes BAN and TT2 and embryo marker genes WOX2 and WOX9 in peripheral endosperm and seed coats of seeds from 1 DAP to 3 DAP, corresponding to seeds containing preglobular to globular stage embryos. Values are based on ATH1 microarray signals after RMA normalization. Error bars, s.e.m.

A) Venn diagram showing overlap of H3K27me3 target genes in seedlings [11], [19] and endosperm. B) Distribution of different types of H3K27me3 targets (genes, pseudogenes, transposable elements, transposable element genes) in endosperm and seedling tissues. Upper panel shows distribution in the endosperm (this study) and seedlings [11], [19], lower panel shows genome-wide distribution of genes, pseudogenes, transposable elements and transposable element genes in comparison to the distribution of shared and specific endosperm H3K27me3 targets. Number in parenthesis reflect number of detectable targets.

A) Median DNA methylation levels of TEs in vegetative tissues (green) and endosperm (orange). CG and CHG methylation levels were analyzed for all TEs, TEs marked specifically by H3K27me3 in the endosperm (“Specific”) and TEs marked by H3K27me3 in the endosperm and in seedlings [11], [19] (“Shared”). Median DNA methylation levels of shared TEs are close to zero. Green and orange dotted horizontal lines mark CG methylation levels of specific H3K27me3 TEs in vegetative and endosperm tissues, respectively. B) Median DNA methylation levels of TEGs in vegetative tissues (green) and endosperm (orange). CG and CHG methylation levels were analyzed for all TEGs, TEGs marked specifically by H3K27me3 in the endosperm (“Specific”) and TEGs marked by H3K27me3 in the endosperm and in seedlings [11], [19] (“Shared”). Median DNA methylation levels of shared TEGs are close to zero. Green and orange dotted horizontal lines mark CG methylation levels of specific H3K27me3 TEGs in vegetative and endosperm tissues, respectively. C) Median DNA methylation levels of protein coding genes in vegetative tissues (green) and endosperm (orange). CG and CHG methylation levels were analyzed for all protein coding genes, protein coding genes marked specifically by H3K27me3 in the endosperm (“Specific”) and protein coding genes marked by H3K27me3 in the endosperm and in seedlings [11], [19] (“Shared”). Green and orange dotted horizontal lines mark CG methylation levels of specific H3K27me3 protein coding genes in vegetative and endosperm tissues, respectively. D) Cluster analysis of DNA methylated H3K27me3 target genes and transposons. Genes and transposons are grouped into two mutually exclusive clusters based on their expression patterns in different tissues. Each row represents a gene, and each column represents a tissue type. Tissue types are: seedlings, leaves, stems, roots, flowers, siliques containing seeds with embryos in the heart or torpedo stage, seeds with embryos in the torpedo, walking stick, curled cotyledon, early green, and green cotyledon stage and endosperm derived from seeds with embryos in the preglobular, globular and heart stage. Red or green indicate tissues in which a particular gene is highly expressed or repressed, respectively.