Examining the Causes and Consequences of Context-Specific Differential DNA Methylation in Maize

Abstract

DNA methylation is a stable modification of chromatin that can contribute to epigenetic variation through the regulation of genes or transposons. Profiling of DNA methylation in five maize (Zea mays) inbred lines found that while DNA methylation levels for more than 99% of the analyzed genomic regions are similar, there are still 5,000 to 20,000 context-specific differentially methylated regions (DMRs) between any two genotypes. The analysis of identical-by-state genomic regions that have limited genetic variation provided evidence that DMRs can occur without local sequence variation, but they are less common than in regions with genetic variation. Characterization of the sequence specificity of DMRs, location of DMRs relative to genes and transposons, and patterns of DNA methylation in regions flanking DMRs reveals a distinct subset of DMRs. Transcriptome profiling of the same tissue revealed that only approximately 20% of genes with qualitative (on-off) differences in gene expression are associated with DMRs, and there is little evidence for association of DMRs with genes that show quantitative differences in gene expression. We also identify a set of genes that may represent cryptic information that is silenced by DNA methylation in the reference B73 genome. Many of these genes exhibit natural variation in other genotypes, suggesting the potential for selection to act upon existing epigenetic natural variation. This study provides insights into the origin and influences of DMRs in a crop species with a complex genome organization.

Figure 1.

Identification and validation of context-specific DMRs among four maize genotype contrasts. A, The genomewide distribution of DMRs, genes, TEs, and coverage is shown using Integrative Genomics Viewer (IGV; Robinson et al., 2011). Each track displays the number of features per 1-Mbp window of the maize genome. Only data from chromosome 1 (Chr 1) is shown. B, DMR validation and support rates for each genotype contrast (BvC, B73 versus CML322; BvM, B73 versus Mo17; BvO, B73 versus Oh43; BvT, B73 versus Tx303) using an independent sequence capture bisulfite sequencing data set. The subset of the DMR tiles (numbers indicated at the base of each bar) that also have coverage in a sequence capture bisulfite sequencing (SeqCap Epi) data set are analyzed. Each DMR is classified as validated (green), supported (yellow), or not supported (gray). Validated DMRs represent DMRs that show greater than 60% (for CG and CHG) or greater than 20% (for CHH) difference in the same direction in the SeqCap Epi assay. Supported DMRs are the DMRs that show 40% to 60% (for CG and CHG) or 10% to 20% (for CHH) difference in the same direction in the SeqCap Epi assay.

Figure 2.

Context-specific DMRs and their proximity to genomic features. A, Context-specific methylation levels (0%–100%) are plotted for several B73-Mo17 DMRs using IGV (Robinson et al., 2011). The colored region in the center shows context-specific methylation within the DMR, while the black shading for the edges shows methylation levels outside of the DMR. B, Pie charts are used to show the proportion of DMRs that are located near (less than 500 bp) or in genes (red), TEs (blue), both genes and TEs (yellow), or DMRs located more than 500 bp from either a gene or a TE (black). Separate pie charts are shown for CG/CHG DMRs (greater than 60% difference at both CG and CHG contexts), CG-only DMRs (greater than 60% CG difference and less than 20% CHG difference), and CHG-only DMRs (greater than 60% CHG difference and less than 20% CG difference). The pie chart labeled random shows the proportions of each location type for 20,000 randomly selected 100-bp tiles that had coverage in our data set. The size of the pie chart is scaled to the number of DMRs in the specific category, except the random set. C, For the DMRs that are located within or near a TE, we classified the type of TE as terminal inverted repeat (TIR; blue), LTR spreading (red), LTR nonspreading (yellow), or other (black). The spreading/nonspreading classifications are based on Eichten et al. (2012). B and C show the average proportions for the four genotype contrasts; the values for each contrast are available in Supplemental Figure S5. In B and C, an asterisk is used to indicate significant enrichment of the feature relative to 100 random sets at P < 0.01, and a downward arrow indicates significant underrepresentation.

Figure 3.

Characterization of methylation at DMR flanking regions. A to C, Three representative types of DNA methylation at DMR flanking regions are shown for CG methylation in B73-Mo17 using IGV (Robinson et al., 2011). In A, the flanking regions both show low methylation (LL), while in B, the flanking regions both show high DNA methylation (HH) and, in C, high methylation in one flanking region and low methylation for the other flanking region (HL). D and E, Hierarchical clustering (Ward’s method) of methylation levels at the left and right flanking regions of CG (D) or CHG (E) DMRs. The B73 versus Mo17 contrast was shown as an example, and clustering for other genotypes provides similar patterns. F, The flanking pattern characterization of B73-Mo17 DMRs was compared with context specificity of DNA methylation changes and genomic location for B73-Mo17 DMRs (similar plots for the other contrasts are available in Supplemental Fig. S6). The DMR sequence contexts and flanking region types are cross tabulated to give a total of nine different DMR categories. The size of each pie chart is scaled to the number of DMRs in each category. The number above each pie chart shows the DMR number in that specific category. Gene (red) indicates that a DMR is within 500 bp of a filtered gene set gene, TE (blue) indicates that a DMR is within 500 bp of a TE, both (yellow) indicates that a DMR is within 500 bp of both a gene and a TE, and neither (black) indicates that a DMR is more than 500 bp from both annotated genes and TEs.

Figure 4.

Association of DNA methylation variation with differential expression. A to C, The difference in DNA methylation levels were calculated for differentially expressed genes (methylation at high-expressed gene [H)] minus methylation at lower expressed gene [L]) for CG (A), CHG (B), and CHH (C) contexts. Differentially expressed genes were divided into six categories (color code indicated within the plot) based on the fold change between the highest (H) and the lowest (L) genotypes. Methylation levels were calculated for each category as the average of all the genes in that category. The two vertical lines represent the –200 bp upstream of a gene and 600 bp into a gene from 5′ end. The arrow indicates the direction of transcription. D to F, The actual methylation levels for the on-off category at CG (D), CHG (E), and CHH (F) in the genotypes with the highest or the lowest gene expression level. G and H, Percentage of genes in each expression category with CG (G) and CHG (H) DMRs. I, Distribution of CG/CHG difference between off and on genotypes at –200- to 600-bp regions. nDEG, Not differentially expressed genes; DEG, differentially expressed genes.

Figure 5.

Characterizing the potential for cryptic information within the B73 genome. A, The genes with methylcytosine (mC) coverage at the –200 to +600 region (relative to the TSS) were assessed to identify genes that are expressed or have low CG/CHG methylation (group 1), genes that are expressed in other tissues of the B73 expression atlas (group 2), genes that are expressed in seedling tissue of other maize genotypes (group 3), or genes with no detectable expression (group 4). B and C, The distribution of lengths for mRNA transcripts (B) and introns (C) for genes from groups 1 to 4 is shown using a boxplot. D, The proportion of genes within each group located within subgenome 1 (Sub1) or subgenome 2 (Sub2) is shown. E, The proportion of genes in each group that are syntenic with rice is shown. F, The closest TEs for the genes in each group were classified. G, The proportion of genes with Gene Ontology (GO) annotation is shown for each group of genes. H, The proportion of genes located within the low-recombining central portion of each chromosome is assessed for each group of genes. TIR, Terminal inverted repeat.