| Abstract: | The maize genome is relatively large (∼2.3 Gb) and has a complex organization of interspersed genes and transposable elements, which necessitates frequent boundaries between different types of chromatin. The examination of maize genes and conserved noncoding sequences revealed that many of these are flanked by regions of elevated asymmetric CHH (where H is A, C, or T) methylation (termed mCHH islands). These mCHH islands are quite short (∼100 bp), are enriched near active genes, and often occur at the edge of the transposon that is located nearest to genes. The analysis of DNA methylation in other sequence contexts and several chromatin modifications revealed that mCHH islands mark the transition from heterochromatin-associated modifications to euchromatin-associated modifications. The presence of an mCHH island is fairly consistent in several distinct tissues that were surveyed but shows some variation among different haplotypes. The presence of insertion/deletions in promoters often influences the presence and position of an mCHH island. The mCHH islands are dependent upon RNA-directed DNA methylation activities and are lost in mop1 and mop3 mutants, but the nearby genes rarely exhibit altered expression levels. Instead, loss of an mCHH island is often accompanied by additional loss of DNA methylation in CG and CHG contexts associated with heterochromatin in nearby transposons. This suggests that mCHH islands and RNA-directed DNA methylation near maize genes may act to preserve the silencing of transposons from activity of nearby genes.The cytosine bases in a genome can be modified to 5-methylcytosine by adding a methyl group at the 5′ position. This process, called DNA methylation, is conserved from algae to animals and plants (1, 2). DNA methylation can be separated into different types based on the local sequence context. In plants DNA methylation is found at the symmetric CG or CHG (where H = A, C, or T) sites or at nonsymmetric CHH sites. CG and CHG methylation are maintained at high fidelity following DNA replication due to activity of maintenance methyltransferases such as MET1 or chromomethylase (CMT) 3 (3, 4), whereas CHH methylation (mCHH) requires targeting by either domains rearranged methylase 2 (DRM2) or CMT2 (3–6). The DRM2 targeting occurs via RNA-directed DNA methylation (RdDM) and requires the activity of polymerase IV (PolIV) and polymerase V (PolV) complexes (3, 4). There is evidence that recruitment of PolIV and PolV may require the presence of dimethylation of lysine 9 of histone H3 (H3K9me2) or DNA methylation at the targeted genomic regions (7, 8). The specific mechanisms that recruit CMT2 are not well characterized but may require specific histone modifications (5, 6).Much of our knowledge of DNA methylation in plants is derived from studies of the model plant Arabidopsis thaliana, which has a relatively small genome and relatively few examples of genes with nearby transposons (36.3%; ref. 9). The maize genome is much more complex, with the majority (85.5%) of genes positioned within 1 kb of transposons. In both species, transposons tend to have quite high levels of CG and CHG methylation whereas genes have much lower levels (10). mCHH is often thought to provide an important component for silencing transposons, yet the maize genome has relatively low levels of mCHH despite the high transposon context (11). This is partially attributed to the lack of a CMT2 ortholog in maize (5), which may explain the reduced levels of mCHH in the middle of larger transposons. Although mCHH is low in maize, there are still genomic regions with elevated mCHH (12). Genomic profiles of mCHH in maize revealed that this modification is often found near genes (termed mCHH islands) and is dependent upon RdDM activity (12–14). This elevation of mCHH in regions surrounding genes is much less prevalent in Arabidopsis (10). A recent study showed that high mCHH can also be induced near genes that are up-regulated in plants subjected to phosphate starvation (15).In this study we further probed the basis and function of these mCHH islands. We found that mCHH islands are short regions of elevated mCHH that flank nearly half of the genes in maize and many conserved noncoding sequences (CNSs). These mCHH islands mark a transition for CG and CHG DNA methylation, several histone modifications, and chromatin accessibility. The mCHH islands are relatively stable across different tissues but show some variation among haplotypes that are often associated with sequence insertions/deletions (InDels). The loss of mCHH islands does not strongly affect gene expression, but instead leads to an additional loss of CG and CHG methylation in some transposons flanking maize genes. |
