Epigenetic regulation of spermidine/spermine N1-acetyltransferase (SAT1) in Suicide
Introduction
Suicide is one of the leading causes of death in Western countries (Nock et al., 2008), which results from the interaction of social, environmental, and genetic factors. While there is strong evidence for the involvement of the monoaminergic neurotransmitter systems in the neuropathology of suicide, it has become increasingly evident that additional systems are involved in this complex condition. In recent years, evidence has emerged implicating dysregulation of the polyamine system as an important factor in suicide and other psychiatric disorders (Fiori and Turecki, 2008). To date, alterations of spermidine/spermine N1-acetyltransferase (SAT1), the rate-limiting enzyme in polyamine catabolism, have been one of the most robust findings implicating this system in the neurobiology of suicide. Decreased expression of SAT1 has now been observed in a number of brain regions in suicide completers (Guipponi et al., 2009, Sequeira et al., 2007, Sequeira et al., 2006, Klempan et al., 2009a, Klempan et al., 2009b), and two promoter polymorphisms, rs6526342 and rs6151267, have been associated with suicide in French Canadians (Fiori and Turecki, 2010a, Sequeira et al., 2006). Several single nucleotide polymorphisms (SNPs) in the promoter were recently found to be involved in determining expression levels in vitro as well as in the brain, and interestingly two SNPs, rs6526342 and rs928931, produced haplotype-specific effects on expression (Fiori et al., 2009).
In addition to DNA sequence variants, epigenetic regulation is another important determinant of gene expression, and can involve methylation of DNA as well as modification of histones. DNA methylation is a covalent modification at the 5′ position of cytosine, occurring at CG dinucleotides (CpG), and is often associated with gene repression when found in promoter regions (Klose and Bird, 2006). Histones are the core proteins involved in packaging of DNA into nucleosomes, and can be covalently modified at specific residues by acetylation, methylation, phosphorylation, SUMOylation, and ubiquitinylation (Berger, 2007). The effect of these modifications on gene expression depends upon the histone protein (H2A, H2B, H3, H4), specific amino acid residue, the nature of the modification, and its positioning within the gene.
Interestingly, polyamine metabolism has been shown to influence both DNA methylation and histone modifications (for examples, see (Ara et al., 2008, Duranton et al., 1998, Frostesjo et al., 1997, Schipper et al., 2007)) and increased S-adenosylmethionine, a precursor in polyamine biosynthesis, has been implicated in the downregulation of several genes in the prefrontal cortex of psychotic patients through altering levels of promoter methylation (Guidotti et al., 2007). The expression of several polyamine-related genes has also been shown to be influenced by DNA methylation, including polyamine-modulated factor-1 (PMF-1) (Aleman et al., 2008), and methionine adenosyltransferase (MAT1A) (Ikeda et al., 2008), although a recent study performed by our group found no relationship between DNA methylation or histone modifications and expression of spermine synthase or spermine oxidase in the brains of suicide completers (Fiori and Turecki, 2010b). However, the expression of ornithine aminotransferase and μ-crystallin has been shown to be influenced by histone methylation in the prefrontal cortices of schizophrenia patients (Akbarian et al., 2005), suggesting that histone modifications are important modulators for the expression of polyamine genes. Several studies have now identified altered levels of CpG methylation and histone modifications in gene promoter regions in a number of psychiatric disorders, including suicide (Akbarian et al., 2005, De Luca et al., 2009, Ernst et al., 2009a, Huang and Akbarian, 2007, Keller et al., 2010, McGowan et al., 2008, Poulter et al., 2008). Overall, SAT1 appears to be a compelling target for assessing the involvement of epigenetic factors in the regulation of the polyamine system and in conferring risk for suicide.
In this study, we were interested in determining the role of epigenetic modifications in determining the expression of SAT1, and to assess their involvement in down regulating the expression of SAT1 in suicide completers. In addition, we were interested in investigating if epigenetic mechanisms were responsible for the haplotype-specific effects of the functional promoter variants, particularly as rs6526342 and rs928931 create polymorphic CpG sites. Using prefrontal brain tissue from a sample of suicide completers and controls, we focused on promoter CpG methylation and levels of tri-methylated histone-3-lysine 27 (H3K27), epigenetic modifications which are both associated with gene repression (Barski et al., 2007, Klose and Bird, 2006). Both of these modifications have been shown to be relatively unaffected by complications arising in post-mortem tissues such as altered pH and post-mortem interval (Ernst et al., 2008, Huang et al., 2006), and are thus well-suited for this type of analysis. Additionally, tri-methylated H3K27 was recently implicated in the downregulation of the TrkB receptor in the prefrontal cortex of suicide completers (Ernst et al., 2009a).
Section snippets
Subjects
We examined 20 male subjects, described in Table 1, comprised of 10 non-suicide controls and 10 suicide completers. Subjects were matched for age, post-mortem interval (PMI), and brain pH. Cause of death was assessed by the Quebec Coroner’s office. Samples were obtained from the Quebec Suicide Brain Bank, where they were processed and dissected at 4 °C, then snap-frozen in liquid nitrogen before storage at −80 °C, following standard procedures (Bird and Vonsattel, 1993). All subjects died
CpG methylation
In order to determine if CpG methylation is involved in the differential expression of SAT1 in suicide completers and the relationship between promoter haplotype and expression, we analyzed methylation in BA 8/9 of 20 subjects (10 suicide completers and 10 controls). This brain region was selected due to previous findings in suicide completers demonstrating both decreased expression of SAT1 (Sequeira et al., 2006), as well as elevated levels of putrescine and spermidine (Chen et al., 2010), in
Discussion
In this study, we examined the role of two epigenetic modifications, CpG methylation and histone methylation, in determining the expression of SAT1 in the brain, and assessed their involvement in the decreased expression of SAT1 previously observed in the brains of suicide completers (Sequeira et al., 2006, Sequeira et al., 2007, Klempan et al., 2009a, Klempan et al., 2009b).
We found a strong relationship between CpG methylation and SAT1 expression, such that increased overall levels of CpG
Conflict of interest
All authors declare that they have no conflicts of interest.
Funding
This work was supported by the Canadian Institute of Health Research (CIHR) MOP 79253. GT is a Fonds de la recherche en santé du Québec (FRSQ) research fellow. LMF received a scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC). These agencies had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
Contributors
Both authors designed the experiments and wrote the manuscript. All experimental and data analysis steps were performed by LMF.
Acknowledgements
None.
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