The involvement of MORC6 in DNA methylation at a small subset of RdDM target loci.

<p>(A) Box plots showing DNA methylation levels of <i>suvh2/9</i> hypo-DMRs in the indicated mutants. (B) Venn diagram of overlap between hypo-DMRs in <i>morc6</i> and <i>suvh2/9</i>. (C) Heat maps showing DNA methylation changes within <i>suvh2/9</i> hypo-DMRs in the wild type and the mutants. Different scales were used for CG, CHG, and CHH methylation. In the scales, the DNA methylation levels of the wild type were set as 0 (light yellow lines) and absolute DNA methylation changes were represented by lines whose colors are ranged from light yellow to black. (D) DNA methylation levels of <i>morc6</i>-specific hypo-DMRs, <i>suvh2/9</i>-specific hypo-DMRs, and hypo-DMRs shared by <i>morc6</i> and <i>suvh2/9</i> are indicated at three cytosine contexts in the wild type, <i>morc6</i>, and <i>suvh2/9</i>. (E) Validation of bisulfite sequencing results by PCR-based DNA methylation analysis. Genomic DNA was cleaved by McrBC, a restriction enzyme that specifically recognizes methylated DNA. Class I represents hypo-DMRs shared by <i>morc6</i> and <i>suvh2/9</i>; Class II represents <i>suvh2/9</i>-specific hypo-DMRs. The RdDM pathway contributes to CHH methylation and to a lesser extent to CG and CHG methylation [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006026#pgen.1006026.ref005" target="_blank">5</a>], but it is unknown how MORC6 contributes to DNA methylation at the three cytosine contexts. Our analysis indicated that <i>morc6</i> affects DNA methylation at all the three cytosine contexts (CG, CHG, and CHH) in a small subset of the 6514 <i>suvh2/9</i> hypo-DMRs (Fig 1C). In the 527 hypo-DMRs shared in <i>morc6</i> and <i>suvh2/9</i>, DNA methylation is reduced at all three cytosine contexts in both <i>morc6</i> and <i>suvh2/9</i> (Fig 1D). In the 218 <i>morc6</i> specific hypo-DMRs, DNA methylation is reduced at all three cytosine contexts in the <i>morc6</i> mutant (Fig 1D). Although the 218 <i>morc6</i> specific hypo-DMRs do not overlap with the <i>suvh2/9</i> hypo-DMRs, the DNA methylation level of the 218 hypo-DMRs is reduced at CHG and CHH sites in <i>suvh2/9</i> (Fig 1D). However, CG methylation of the 218 hypo-DMRs is markedly reduced in <i>morc6</i> but not in <i>suvh2/9</i> (Fig 1D). Thus, these 218 hypo-DMRs could be either RdDM-independent loci or RdDM-dependent loci that are not dependent on SUVH2/9.