rRNA Methylation in Suicide Figure 3. Hypermethylation of the rRNA promoter in suicide subjects relative to controls. (A) (above) Vertical lines indicate locations of CpG dinucleotides on the rRNA promoter relative to the transcription start site, indicated with the solid arrow, with primer pairs used for bisulfite mapping marked by dashed arrows. (below) Average percentage of methylation for each CpG site, for suicide subjects (N = 13; black bars) and controls (N = 11; white bars). Data are expressed as mean 6 S.E.M. *, P,0.05; **, P,0.01; ***, P,0.001, measured by ANOVA. (B) Multiple regression analysis of the number of methylated CpGs per clone and the number of clones shows a significant interaction between suicide subjects (20 clones 6 13 subjects, N = 260 total clones; filled circles) and controls (20 clones 611 subjects, N = 220 total clones; open circles). There are 26 circles per group, as clones are grouped according to methylation status. doi:10.1371/journal.pone.0002085.g003 A greater number of sequenced clones were hypermethylated in the suicide subjects, whereas control subjects showed a greater number of hypomethylated clones (Fig. 3B). An analysis of the regression slopes between groups revealed a significant (F(1) = 7.33, P,0.01) interaction between the number of methylated CpG sites per clone for suicide subjects compared to controls. These data indicate a dramatic increase in the ratio of methylated to unmethylated clones among suicide subjects across most CpG sites. To determine whether the state of methylation of specific CpG methylation sites differed across the rRNA promoter between suicide subjects and controls or whether all CpG sites differed similarly between the two groups, the relationship between groups of the average percentage of methylation for each CpG site was investigated. There was a strong linear relationship between the means of the two groups (R = 0.92, P,0.00001; Fig. 4) demon- strating a similar difference in the state of methylation of all CpG sites in between the groups. No single CpG site stood out as being particularly different between the groups suggesting no site selectivity of methylation in the rRNA of suicide subjects. Instead, the overall level of methylation throughout the promoter and regulatory region differed between the groups. These data showing a lack of site- specificity are consistent with our recent analysis of the state of methylation of rRNA genes in cultured cells [32]. Specificity of Hippocampal rRNA methylation by Analysis of Cerebellum rRNA and Genome-Wide Methylation To examine the anatomical specificity of the differences in rRNA methylation between suicides and controls in the hippo- campus, rRNA promoter methylation was examined in the cerebellum, a region not primarily associated with psychopathol- ogy. Individuals from the suicide subjects group who showed large differences in hippocampal rRNA promoter methylation by contrast to those in the control group (t(6) = 4.12, P,0.01; Fig. 5A) were selected to test whether these differences would be conserved in another brain region. In contrast to the hippocampus, there was no significant difference in the percentage of methylated CpG sites between suicide subjects and controls in the cerebellum (t(6) = 0.55, P = 0.6; Fig. 5B). There was no significant correlation between levels of methylation in the hippocampus and those in the cerebellum (R = 0.11, P = 0.78), showing anatomical specificity in the hypermethylation of the rRNA promoter in hippocampus of suicide subjects. The interaction between the regression slopes for the number of methylated CpG sites per clone was not significant (F(1) = 0.26, P = 0.61; Fig. 5C), indicating that there was no difference in the ratio of unmethylated to methylated clones between groups in the cerebellum. PLoS ONE | www.plosone.org 4 May 2008 | Volume 3 | Issue 5 | e2085