rRNA Methylation in Suicide Figure 5. Anatomical and Genomic specificity of rRNA hypermethylation. Average percentage of rRNA promoter methylation for selected subjects with large methylation differences in the hippocampus (A) and in the cerebellum (B) of suicide subjects (N = 4, black bars) and controls (N = 4, white bars) for the same subjects. Data are expressed as mean 6 S.E.M. **, P,0.01, measured by unpaired t-test. (C) Multiple regression analysis shows a similar negative relationship between the number of methylated CpGs per clone and the number of clones in cerebellum samples from suicide subjects (20 clones 6 4 subjects, N = 80 total clones; filled circles) and controls (20 clones 6 4 subjects, N = 80 total clones; open circles). There are 26 circles per group, as clones are grouped according to methylation status. (D) (above) Representative images of genome-wide methylation in the hippocampus for a suicide subject and a control, showing cytosine (C) and 5-methylcytosine (5 mC) content used for nearest neighbor analysis. (below) Quantification of the percentage of methylcytosine, following the formula: [(5-methylcytosine) 6100]/(5-methylcytosine + cytosine), shows no difference between suicide subjects (N = 13, black bar) and controls (N = 11, white bar) in genome-wide levels of methylation (P.0.05), measured by unpaired t-test. doi:10.1371/journal.pone.0002085.g005 cultured cells pharmacological manipulation of the acetylation status of histone 4 influences rRNA expression [31,39]. In this study, we selected suicide subjects with a history of early childhood neglect/abuse. Childhood abuse in humans is associ- ated with decreased hippocampal volume, as well as with cognitive impairments [9]. This influence of childhood adversity and epigenetic aberrations later in life supports the hypothesis that, similar to what was observed in rodents [19,20,40], early childhood events in humans alter epigenetic markings in the brain. It is tempting to speculate that epigenetic processes mediate effects of social adversity during childhood on the brain that persist into adulthood and are known to enhance suicide risk [41,42]. Epigenetic differences might be driven by genetic differences as well as by other environmental and dietary factors. All suicide subjects in our study displayed the same genomic sequence in the rRNA promoter region examined. Additional genetic polymor- phisms in other regions of the rRNA gene, including among individual rRNA gene clusters, may play a role in rRNA function. The structure and length of rRNA gene clusters varies between individuals [43,44], however, the relationship between rRNA promoter methylation or rRNA expression and this additional level of organizational complexity in rRNA is less clear. Genetic abnormalities in rRNA gene cluster organization are associated with increased rRNA methylation during cellular senescence [45]. However, the lack of difference between groups in the cerebellum argues against such genetic differences among these individuals. Another factor that may influence the methylation status of individuals is medication prescribed in the treatment of psychiatric disorders. The mood stabilizing effect of sodium valproate, a potent histone deacetylase (HDAC) inhibitor known to indirectly influence DNA methylation [46,47] via chromatin modification and used in the treatment of bipolar disorder, and the noted antidepressive effect of S-adenosyl methionine, a methyl donor in the DNA methylation reaction and an inhibitor of active demethylation [48] , suggest a role for DNA methylation in mood regulation [49]. Although none of the subjects in the present study had been treated with these pharmacological agents, the possibility of epigenetic regulation by other pharmacological interventions should not be discounted. Our data do not exclude these alternative hypotheses. PLoS ONE | www.plosone.org 6 May 2008 | Volume 3 | Issue 5 | e2085