Figure 4. Site-independent hypermethylation of the rRNA promoter in suicide subjects. Positive correlation between suicide and control rRNA promoter methylation percentage across CpG sites (N = 26), showing a conserved hypermethylation in suicide subjects throughout the promoter region. Each data point is labeled with the position of each CpG dinucleotide in the rRNA promoter, relative to the transcription start site. doi:10.1371/journal.pone.0002085.g004 To determine whether the observed differences in methylation of the rRNA promoter in the hippocampus reflect genome-wide differences in methylation between suicide subjects and controls, nearest neighbor analysis of the overall percentage of methylated cytosines was performed for each subject. Nearest neighbor analysis revealed no significant difference between suicide subjects and controls in the overall percentage of methylated cytosines (t(22) = 0.54, P = 0.59), and there was no significant correlation between the percentage of rRNA promoter methylation and genome-wide methylation (R = 0.07, P = 0.78), revealing specific- ity in the regulation of the rRNA promoter by methylation in the suicide brain (Fig. 5D). rRNA Methylation Does Not Vary With Psychiatric Diagnoses Next, the relationship between methylation and psychiatric diagnoses was examined (Table 1). Mood disorders and substance abuse disorders are risk factors for suicide and have also been linked to alterations of DNA methylation in several genes [24,25,36,37]. There were no significant differences within the suicide subjects or the controls when the overall percentages of rRNA methylation of those with mood disorders were compared to those without mood disorders as well as among those with substance abuse disorders compared to those without substance abuse disorders (all P’s.0.05). rRNA Expression is Downregulated in Suicide Because it has been established in cell culture that methylation of the rRNA promoter regulates the transcription of rRNA [31– 34], rRNA expression in suicide subjects and controls was rRNA Methylation in Suicide investigated. rRNA expression was significantly higher in controls than in suicide subjects (t(23) = 2.16, P,0.05; Fig. 6A). Correla- tional analysis revealed a trend for a relationship between the overall percentage of methylation and rRNA expression (R = 20.21, P.0.05; Fig. 6B), indicating that rRNA expression may be regulated by methylation and additional epigenetic factors. There was no relationship between and PMI, brain pH, or age and rRNA expression (P’s.0.05). Discussion The data reveal evidence for DNA hypermethylation of the rRNA promoter region in the hippocampus of suicide subjects with histories of childhood abuse or severe neglect relative to controls (victims of sudden, accidental death with no history of abuse or neglect). Although our findings are largely based on correlational studies indicating an association between psychopa- thology and methylation, these data are consistent with growing evidence suggesting that alterations in cytosine methylation mediate biological processes associated with psychopathology [38]. Since DNA methylation is a highly stable mark, the bond between a methyl group and cytosine ring being one of the most stable chemical bonds [18], the differences in methylation are unlikely to be a consequence of conditions immediately preceding death or during the postmortem interval. No reaction which could spontaneously demethylate 5-methylcytosine in DNA has ever been described. Our data indicate that post mortem pH does not affect DNA methylation (CE POM VD MJM MS and GT, unpublished observations). Indeed, post-mortem interval, brain pH, or age did not differ between suicide subjects and controls. The increase in hippocampal rRNA promoter methylation among suicide subjects appears to occur in the absence of obvious site-specific effects on particular CpG sites. The results are consistent with data in cell culture showing that transcriptionally active rRNA promoters are completely unmethylated while transcriptionally inactive molecules are completely methylated in a promoter-wide manner without any obvious site selectivity [32]. In contrast to the situation in humans reported here and previously reported in human cells in culture, the situation in mouse is different. In the mouse, a site-specific change in methylation is sufficient to mediate silencing of the rRNA promoter [31]. Importantly, the changes in rRNA promoter methylation do not reflect a genome-wide change in methylation, as nearest neighbor analysis revealed no differences in overall levels of methylation in suicide subjects relative to controls. Moreover, this difference in the methylation of the rRNA promoter shows anatomical specificity. When the rRNA methylation status for subjects with large methylation differences in hippocampus was assessed in the cerebellum, suicide subjects and controls showed similar levels of rRNA methylation. In contrast to the hippocampus, the number of methylated CpG sites observed per clone was similar between suicide subjects and controls in the cerebellum. As a part of the brain not primarily associated with neuroplastic changes influenc- ing psychopathology, this result indicates that rRNA methylation differences between the groups are specific to the hippocampus. In addition to the difference in methylation, suicide subjects showed impaired hippocampal rRNA expression compared to controls. The decrease in gene expression was associated with increased methylation of the rRNA promoter sequence, as indicated by a trend for a linear correlation between the overall percentage of methylation and gene expression. Although a trend was evident, the results do not exclude other known epigenetic mechanisms influencing rRNA gene expression. For example, in PLoS ONE | www.plosone.org 5 May 2008 | Volume 3 | Issue 5 | e2085