Accessible DNA and Relative Depletion of H3K9me2 at Maize Loci Undergoing RNA-Directed DNA Methylation  

Abstract

RNA-directed DNA methylation (RdDM) in plants is a well-characterized example of RNA interference-related transcriptional gene silencing. To determine the relationships between RdDM and heterochromatin in the repeat-rich maize (Zea mays) genome, we performed whole-genome analyses of several heterochromatic features: dimethylation of lysine 9 and lysine 27 (H3K9me2 and H3K27me2), chromatin accessibility, DNA methylation, and small RNAs; we also analyzed two mutants that affect these processes, mediator of paramutation1 and zea methyltransferase2. The data revealed that the majority of the genome exists in a heterochromatic state defined by inaccessible chromatin that is marked by H3K9me2 and H3K27me2 but that lacks RdDM. The minority of the genome marked by RdDM was predominantly near genes, and its overall chromatin structure appeared more similar to euchromatin than to heterochromatin. These and other data indicate that the densely staining chromatin defined as heterochromatin differs fundamentally from RdDM-targeted chromatin. We propose that small interfering RNAs perform a specialized role in repressing transposons in accessible chromatin environments and that the bulk of heterochromatin is incompatible with small RNA production.

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

“…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.…”

“…It was recently demonstrated that at RdDM loci, mop1-1 decreases DNA methylation in all C contexts but does not completely remove CHH methylation (Gent et al, 2014): lower levels of residual CHH methylation might still be sufficient to ensure DNA silencing at most RdDM loci. The down-regulation of the DNA demethylase ROS1/DML1 might also contribute to buffer the effects of siRNA loss (Huettel et al, 2006;Madzima et al, 2014).…”

“…In maize the majority of genes are located within 1 kb of an annotated TE (Baucom et al, 2009;Schnable et al, 2009), and loci undergoing RdDM are primarily located in gene flanking regions (Gent et al, 2013(Gent et al, , 2014. The enrichment of RdDM-associated siRNAs near maize genes (Wang et al, 2009;Gent et al, 2013;Gent et al, 2014;Xin et al, 2014) has been suggested to ensure the continuous silencing of potentially deleterious TEs and TE-like sequences in an active and accessible chromatin environment required for the Pol II transcription of close genes (Gent et al, 2014).…”

“…The enrichment of RdDM-associated siRNAs near maize genes (Wang et al, 2009;Gent et al, 2013;Gent et al, 2014;Xin et al, 2014) has been suggested to ensure the continuous silencing of potentially deleterious TEs and TE-like sequences in an active and accessible chromatin environment required for the Pol II transcription of close genes (Gent et al, 2014). In maize, gene expression can be influenced by gene-proximal TEs and repeats (Erhard et al, 2013), and both genes and TEs can be regulated by the direct competition between Pol IV and Pol II occupancy at their promoters Erhard et al, 2015).…”

Genomic Analysis of the DNA Replication Timing Program during Mitotic S Phase in Maize (Zea mays) Root Tips

“…To investigate the replication times of these biologically important tandem repeat arrays, we used a different approach that took into account all of the Repli-seq sequencing reads, even those that did not uniquely map to the genome assembly. We used consensus sequences for the knob180, TR-1, 5S and 45S rDNA, and CentC tandem repeats (courtesy of J. Gent, K. Dawe, T. Wolfgruber, and G. Presting) to individually query trimmed Repli-seq reads using BLAST software and a nonstringent E value to allow for variants of each repeat (Gent et al, 2014). The resulting read counts for each tandem repeat type in each S phase or the G1 genomic DNA sample were then normalized to the total number of reads in that sample to obtain the percentage of reads that align to each tandem repeat.…”