RNA-directed DNA methylation enforces boundaries between heterochromatin and euchromatin in the maize genome

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.T he 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 RNAdirected 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...

Genome-Wide Characterization of Maize Small RNA Loci and Their Regulation in the required to maintain repression6-1 (rmr6-1) Mutant and Long-Term Abiotic Stresses

“…These observations are consistent with previous studies that demonstrated that maize TEs located near genes are targeted by 24-nt siRNA-dependent RdDM, resulting in local deposition of DNA methylation in the asymmetric CHH context in so-called CHH islands (Gent et al, 2013). Maize mutants that disrupt 24-nt siRNA accumulation show loss of CHH islands as well as instances where the flanking regions also lose CG/CHG methylation (Li et al, 2015). Our RNA-seq data show that that major effect on gene expression in the rmr6-1 mutant is a de-repression of genes that are normally lowly-or non-expressed.…”

“…In addition, we found that the smaller numbers of genes that are down-regulated in the rmr6-1 background also show the same loss of gene-proximal siRNAs. Together, these data support the current model by which the geneflanking 24-nt siRNAs in maize primarily function to reinforce transcriptional silencing of TEs located near active genes, avoiding the spreading of euchromatin into the close TEs (Gent et al, 2013(Gent et al, , 2014Erhard et al, 2015;Li et al, 2015). The similar occupancy of RMR6-dependent, gene-flanking, 24-nt siRNA loci found for protein-coding genes and lncRNAs suggests that the CHH island system of RdDM is engaged near active genes independently of their coding or noncoding nature.…”

“…At the genome-wide level, the expression of maize genes positively correlates with the accumulation levels of upstream 24-nt siRNAs (Gent et al, 2013). In the absence of Pol IV-dependent siRNAs, Pol II transcription globally decreases around the transcription start site and increases at 39 end of genes (Erhard et al, 2015), and gene-proximal TEs lose DNA methylation (Li et al, 2015). Thus, the available data suggest that Pol IV-dependent 24-nt siRNAs in maize primarily serve to mark and enforce boundaries between areas of transcriptionally active euchromatin and transcriptionally repressed heterochromatin.…”

The Aegilops tauschii genome reveals multiple impacts of transposons

“…mCHH islands were first identified in maize, and have been proposed to function as 'insulators' that separate the silencing of TEs from that of nearby genes. However, there is as yet no clear conclusion about the relationship between mCHH islands and gene expression [37][38][39] . We found that mCHH islands are obvious in Ae.…”

Creating Order from Chaos: Epigenome Dynamics in Plants with Complex Genomes

“…The CHH methylation region is more often found at the borders between heterochromatin and euchromatin that occur near genes or conserved noncoding sequences (Gent et al, 2014;Li et al, 2015c). The analyses of mutants that affect CHH methylation suggest that the function of these CHH islands is to protect/ensure the silencing of TEs from the open chromatin of nearby genes.…”