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 psychopathology and methylation, these data are consistent with growing evidence suggesting that alterations in cytosine methylation mediate biological processes associated with psychopathology 
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 
, 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 Our data indicate that post mortem pH does not affect DNA methylation (CE POM VD MJM MS and GT, unpublished observations).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 
. 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 
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 influencing 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 cultured cells pharmacological manipulation of the acetylation status of histone 4 influences rRNA expression 
In this study, we selected suicide subjects with a history of early childhood neglect/abuse. Childhood abuse in humans is associated with decreased hippocampal volume, as well as with cognitive impairments 
. This influence of childhood adversity and epigenetic aberrations later in life supports the hypothesis that, similar to what was observed in rodents 
, 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 
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 polymorphisms 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 
, 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 
. 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 
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 
, suggest a role for DNA methylation in mood regulation 
. 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.
In summary, these data reveal increased promoter-wide methylation of the rRNA promoter as well as decreased rRNA gene expression in suicide subjects. The results of the psychological autopsy suggest a developmental origin, however, at time this is speculation based on samples that differ along a wide range of experiential and potentially genetic dimensions. To date, our data are merely consistent with the hypothesis that early life events can alter the epigenetic status of genes that mediate neural functions, and thus contribute to individual differences in the risk for suicide.