rRNA Methylation in Suicide likely that these differences reflect population-specific variants with regards to the reference sequence. For each subject, sequences overlapping the region targeted by primers after bisulfite conversion were identical to the published sequence, except for the presence of a G/T conversion in the forward primer that was present in all subjects, thus eliminating potential primer bias between subjects in sodium bisulfite mapping. The rRNA Promoter is Hypermethylated in the Hippocampus of Suicide Subjects DNA methylation can affect expression via specific methylation of distinct CpG sites [29] and/or via regional site-independent changes in the overall density of methylation [30]. Previous work in cell culture on the state of human rRNA promoter methylation showed regional differences in methylation between active and inactive rRNA promoters [32]. To determine whether the pattern of methylation of single nucleotides and/or the overall methylation of rRNA differed between suicide subjects and controls, the rRNA promoter was examined at single nucleotide resolution by sodium bisulfite mapping (Fig. 2). The rRNA promoter was heavily methylated throughout the promoter and 59 regulatory region in the hippocampus of suicide subjects in comparison with that of controls (F(1) = 191.04, P,0.0001, Fig. 2, 3A). Twenty-one out of 26 CpG sites were significantly more methylated in suicide subjects compared to controls, whereas no CpG was more methylated in controls relative to suicide subjects (F(25) = 11.01, P,0.001; Fig. 3A). An analysis of the effect size for each CpG site revealed that these 21 sites did not differ in the magnitude of the methylation difference between groups at each CpG site (P’s.0.05). Figure 2. Sodium bisulfite mapping of the rRNA promoter in suicide subjects and controls. Twenty clones were sequenced for each suicide subject (left side) and control (right side), from 2 to 3 independent PCR reactions. Each line represents one clone. Circles representing CpG dinucleotides follow the 59 to 39 order of the rRNA promoter sequence for methylated CpG dinucleotides (filled circles), and unmethylated CpG dinucleotides (open circles). doi:10.1371/journal.pone.0002085.g002 PLoS ONE | www.plosone.org 3 May 2008 | Volume 3 | Issue 5 | e2085