Promoter-Wide Hypermethylation of the Ribosomal RNA Gene Promoter in the Suicide Brain Patrick O. McGowan1,2,3, Aya Sasaki1,2,3, Tony C. T. Huang3,4, Alexander Unterberger3,4, Matthew Suderman3,5, Carl Ernst1,6, Michael J. Meaney1,2,3, Gustavo Turecki1,6*, Moshe Szyf3,4* 1 Department of Psychiatry, Douglas Mental Health University Institute, Montreal, Quebec, Canada, 2 Department of Neurology and Neurosurgery, McGill Program for the Study of Behaviour, Genes and Environment, McGill University, Montreal, Quebec, Canada, 3 Sackler Program for Epigenetics & Psychobiology, McGill University, Montreal, Quebec, Canada, 4 Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada, 5 McGill Centre for Bioinformatics, McGill University, Montreal, Quebec, Canada, 6 McGill Group for Suicide Studies, Douglas Mental Health University Institute, Montreal, Quebec, Canada Abstract Background: Alterations in gene expression in the suicide brain have been reported and for several genes DNA methylation as an epigenetic regulator is thought to play a role. rRNA genes, that encode ribosomal RNA, are the backbone of the protein synthesis machinery and levels of rRNA gene promoter methylation determine rRNA transcription. Methodology/Principal Findings: We test here by sodium bisulfite mapping of the rRNA promoter and quantitative real- time PCR of rRNA expression the hypothesis that epigenetic differences in critical loci in the brain are involved in the pathophysiology of suicide. Suicide subjects in this study were selected for a history of early childhood neglect/abuse, which is associated with decreased hippocampal volume and cognitive impairments. rRNA was significantly hypermethylated throughout the promoter and 59 regulatory region in the brain of suicide subjects, consistent with reduced rRNA expression in the hippocampus. This difference in rRNA methylation was not evident in the cerebellum and occurred in the absence of genome-wide changes in methylation, as assessed by nearest neighbor. Conclusions/Significance: This is the first study to show aberrant regulation of the protein synthesis machinery in the suicide brain. The data implicate the epigenetic modulation of rRNA in the pathophysiology of suicide. Citation: McGowan PO, Sasaki A, Huang TCT, Unterberger A, Suderman M, et al. (2008) Promoter-Wide Hypermethylation of the Ribosomal RNA Gene Promoter in the Suicide Brain. PLoS ONE 3(5): e2085. doi:10.1371/journal.pone.0002085 Editor: Jo¨ rg Hoheisel, Deutsches Krebsforschungszentrum, Germany Received October 28, 2007; Accepted March 20, 2008; Published May 7, 2008 Copyright: 2008 McGowan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by a grant from the National Cancer Institute to MS, NICHD to MJM and MS, HSFP to MJM and MS, the Maternal Adversity, Vulnerability and Neurodevelopment Project grant from the Canadian Institutes for Health Research (CIHR) to MJM and MS, as well as grants from CIHR to GT. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gustvao.turecki@mcgill.ca (GT); moshe.szyf@mcgill.ca (MS) Introduction Suicide is a leading cause of death, particularly in males [1,2]. Although many suicide subjects have a diagnosable psychiatric illness, most persons with a psychiatric disorder never attempt suicide [2]. Suicidal behavior aggregates in families [1], and studies of twins show that monozygotic individuals have a greater concordance for suicide completion and suicide attempts com- pared to dizygotic individuals [2–4]. Non-genetic familial factors, including a history of abuse or neglect during childhood, are also risk factors for suicidal behavior [5,6]. Similarly, childhood abuse is associated with an increased risk for psychopathology [7,8] and altered neural development [9]. Several lines of evidence suggest that changes in gene expression in the brain occur in the context of psychiatric disorders and suicide [10–15]. Alterations in gene regulation can be caused by epigenetic programming of gene expression in response to environmental exposure, including social and physical adversity [16]. The genome is epigenetically programmed by changes in the chromatin state and by a pattern of modification of the DNA molecule itself through methylation [17]. DNA methylation is a stable epigenetic mark associated with long-lasting silencing of gene expression [18]. In rodents, genes responsive to differences in the quality of maternal care early in life are altered through epigenetic mechanisms [19,20]. In the human brain, aberrant DNA methylation of specific genes also occurs in the context of psychiatric disorders [21–26]. Decreased expression of ribosomal RNA (rRNA), a bottleneck gene for protein production in the cell, occurs in patients with mild cognitive impairment and early Alzheimer’s disease [27,28]. DNA methylation can regulate gene expression in two ways. One is site-specific methylation, involving direct interference with the binding of transcription factors [29]. The second is site- independent promoter-wide methylation, attracting methylated DNA binding proteins and leading to an inactive chromatin structure. In the latter case, the density of methylated CpGs determines the extent of gene silencing [30]. Both mechanisms can regulate rRNA expression. Previous work in cultured mouse cells indicated that rRNA is regulated by methylation of a single CpG dinucleotide at position 2133 residing at the upstream control element (UCE) [31]. In human cell culture, the transcriptionally active fraction of rRNA promoters associated with RNA polymerase I (pol I) is completely unmethylated whereas the fraction not associated with pol I is almost completely methylated [32], thus determining transcription by defining the fraction of unmethylated rRNA. Mouse and human rRNA promoters show PLoS ONE | www.plosone.org 1 May 2008 | Volume 3 | Issue 5 | e2085