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Genetic and epigenetic influences on expression of spermine synthase and spermine oxidase in suicide completers

Laura M. Fiori , Gustavo Turecki
DOI: http://dx.doi.org/10.1017/S1461145709991167 725-736 First published online: 1 July 2010

Abstract

Alterations in the levels of spermine synthase (SMS) and spermine oxidase (SMOX), two enzymes involved in polyamine metabolism, have previously been observed in brains of suicide completers. To characterize the roles played by genetic and epigenetic factors in determining expression levels of these genes, as well as to identify potential mechanisms by which to explain our findings in suicide completers, we (1) assessed the role of promoter polymorphisms in determining expression in the brain and in vitro, and (2) examined CpG methylation and levels of methylated histone H3 lysine-27 in the promoter regions of these genes in the prefrontal cortex of suicide completers and healthy controls. We identified several promoter haplotypes in SMS and SMOX, but found no consistent effects of haplotype on expression levels in either the brain or in reporter gene assays performed in three different cell lines. We also found no overall effects of epigenetic factors in determining expression, with the exception of a relationship between CpG methylation at one site in the promoter of SMOX and its expression in Brodmann area 8/9. In conclusion, the genetic and epigenetic factors examined in this study show little influence on the expression levels of SMS and SMOX, and do not appear to be responsible for the dysregulated expression of these genes in suicide completers.

Key words
  • Polyamines
  • promoter variations
  • spermine oxidase
  • spermine synthase
  • suicide

Introduction

The polyamine system has been identified in all organisms and is essential for life (Minguet et al. 2008). The polyamines comprise agmatine, putrescine, spermidine, and spermine, and are involved in a myriad of essential cellular functions, including growth, division, and signalling cascades (Gilad & Gilad, 2003; Seiler & Raul, 2005; Tabor & Tabor, 1984). The levels of the polyamines are highly regulated and are controlled through the activities of enzymes in both the biosynthetic and interconversion pathways, which are depicted in Fig. 1. Putrescine can be derived from either l-ornithine by the enzyme ornithine decarboxylase (Tabor & Tabor, 1984) or from agmatine by agmatinase (Hillary & Pegg, 2003). Spermidine and spermine are synthesized by spermidine synthase and spermine synthase (SMS), respectively, which transfer aminopropyl groups produced from S-adenosylmethionine (SAM) through SAM decarboxylase (AMD1) (Tabor & Tabor, 1984). The interconversion pathway is responsible for back-conversion of spermine and spermidine to putrescine. Two mechanisms can be used to convert spermine to spermidine of which the better characterized pathway involves acetylation of spermine by spermidine/spermine N1-acetyltransferase (SAT1), followed by oxidative deamination by polyamine oxidase (PAO) to produce spermidine (Bolkenius & Seiler, 1981). In addition to the SAT1/PAO pathway, spermine can also be converted to spermidine through spermine oxidase (SMOX). This enzyme is specific for unacetylated spermine, and does not work upon spermidine or acetylated spermidine (Vujcic et al. 2002). As such, only the first mechanism is used for conversion of spermidine to putrescine.

Fig. 1

Polyamine synthesis and interconversion pathways. Ac, Acetyl; AGMAT, agmatinase; AMD1, S-adenosylmethionine (SAM) decarboxylase; dcSAM, decarboxylated SAM; MTA, 5′ methylthioadenosine; ODC, ornithine decarboxylase; PAO, polyamine oxidase; SAT1, spermidine/spermine N1-acetyltransferase; SMOX, spermine oxidase; SMS, spermine synthase; SRM, spermidine synthase.

Alterations of the polyamine system have been observed in several pathological conditions including cancer (Tabor & Tabor, 1984), ischaemia (Gilad & Gilad, 1991), Alzheimer's disease (Morrison et al. 1993), and mental disorders (Fiori & Turecki, 2008). The first evidence for the involvement of the polyamine system in suicide was the observation of down-regulation of SAT1 expression in brain Brodmann areas (BA) 4, 8/9, and 11, in suicide completers (Sequeira et al. 2006). Following this finding, down-regulation of SAT1 in suicide completers was observed in several additional brain regions by our group and others (Guipponi et al. in press; Klempan et al. 2009a, b; Sequeira et al. 2007), and we identified numerous other polyamine-related genes which were differentially expressed in suicide completers, including SMS, SMOX, ornithine aminotransferase-like 1, AMD1, and ornithine decarboxylase antizymes 1 and 2 (Fiori et al. 2009; Klempan et al. 2009a, b; Sequeira et al. 2007). In addition, we recently characterized several polymorphisms in the promoter region of SAT1, and found three to be associated with expression levels in both the brain and in vitro (Fiori et al. 2009).

SMS is located on Xp22.1, and encodes 11 exons to produce a 366 amino-acid protein. Disruption of the function of this gene can have deleterious effects, and mutations in SMS are responsible for Snyder–Robinson syndrome, a form of X-linked mental retardation which manifests with both intellectual and physical symptoms, including alterations in brain morphology (Becerra-Solano et al. 2009; Cason et al. 2003; de Alencastro et al. 2008; Kesler et al. in press). In addition, mouse Gy mutants, which possess a deletion of a section of the X chromosome containing multiple exons of SMS, display neurological abnormalities and reduced lifespans, which has been attributed to the loss of SMS function (Meyer et al. 1998). Elevated levels of SMS appear to produce far fewer effects, as transgenic mice display only slight increases in spermine and decreases in spermidine levels (Ikeguchi et al. 2004). Under normal conditions, the expression of SMS appears to be regulated primarily by substrate availability although there is evidence for induction under certain conditions (Ikeguchi et al. 2006).

SMOX was isolated much more recently than other enzymes involved in polyamine metabolism (Vujcic et al. 2002). This gene is found at 20p13 and has at least nine known exons, with the translation start site being found in the second exon. Numerous splice forms have been identified although their transcription does not appear to be differentially regulated (Cervelli et al. 2004; Murray-Stewart et al. 2002; Wang et al. 2005). Expression of this gene has been shown to be up-regulated following exposure to polyamines and their analogues (Wang et al. 2005). The increased protein levels appear to be due primarily to increased transcription and stabilization of SMOX mRNA, and the area spanning −55 to −1117 bp from the transcription start site is implicated in this response (Wang et al. 2005).

Based on the strong evidence for a global dysregulation of polyamine metabolism in suicide completers, we chose to further investigate genetic and epigenetic factors which may be involved in the altered expression of SMS and SMOX, two important polyaminergic genes that have been shown to be differentially expressed in suicides compared to controls in our previous studies (Klempan et al. 2009b; Sequeira et al. 2007). To do so, we examined the influence of promoter polymorphisms on brain gene expression as well as in reporter gene assays. Epigenetic differences are another important source of non-coding functional variations, and alterations in the patterns of both CpG methylation and histone modifications in gene promoter regions have been associated with a number of psychiatric disorders, including suicide (Akbarian et al. 2005; de Luca et al. 2009; Ernst et al. 2009b; Huang & Akbarian, 2007; McGowan et al. 2008; Poulter et al. 2008). To determine if methylation is involved in determining the levels of expression of SMS and SMOX, we characterized promoter CpG methylation patterns and levels of trimethylated histone H3 lysine-27 (H3K27me3) in the prefrontal cortex from a sample of suicide completers and healthy controls.

Materials and methods

Genotyping

Subjects

Our sample consisted of 96 male French Canadians. This sample included brain tissues obtained from 40 subjects, comprised of 14 healthy controls, 10 non-depressed, and 16 depressed suicide completers. These samples were obtained from the Quebec Suicide Brain Bank, and the cause of death was assessed by the Quebec Coroner's office. The remaining 56 subjects were obtained from a cohort of individuals representative of the Quebec general population (described in Brezo et al. 2007).

Psychiatric diagnoses of brain tissue donors were obtained using the psychological autopsy method with the Structured Clinical Interview for DSM-IV Axis I (SCID-I; First et al. 2001), as described elsewhere (Dumais et al. 2005). Individuals from the cohort analysed in this study were free of psychopathology and history of suicidal behaviour as assessed by a large number of instruments and measures (Brezo et al. 2007). Written informed consent was obtained for each subject or next of kin, as applicable. This study was approved by our local institutional review board.

Analysis of polymorphisms

We defined the promoter regions of SMS (NM_004595) and SMOX (NM_175842) as comprising the sequence spanning 5000 bp upstream from the transcription start site to 500 bp into the first intron. Single nucleotide polymorphisms (SNPs) from these regions were selected from public databases including the National Centre for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov), Ensembl (www.ensembl.org), and HapMap (www.hapmap.org).

Genomic DNA was extracted from blood, saliva, or frozen brain tissue using standard procedures (Sambrook et al. 1989), and SNP genotyping was performed using the SNaPShot method with an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, USA). Genotypes were determined using GeneMapper version 3.7 (Applied Biosystems). Linkage disequilibrium (LD) and Hardy–Weinberg equilibrium were assessed using Haploview version 4.1 (Barrett et al. 2005).

Expression

Correlation of expression with genotype

Microarray analyses were performed on the 40 brain tissue donors described above in brain regions BA 8/9, BA 47, and the hippocampus. We focused on these regions as previous work indicates polyaminergic dysregulation in prefrontal tissues and the hippocampus in suicide (Klempan et al. 2009b; Sequeira et al. 2007). RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). RNA samples used for analysis possessed A260/A280>1.9 and 28S/18S rRNA peak height ratios >1.6. Samples were analysed using the HG-U133 A/B set, and GeneChip analysis was performed with Microarray Analysis Suite version 5.0 (MAS 5), Data Mining Tool 2.0. All genes were globally normalized and scaled to a signal intensity of 100. RNA quality indicators used to filter samples prior to analysis included β-actin 5′/3′ ratio, glyceraldehyde-3-phosphate dehydrogenase 5′/3′ ratio, RawQ (noise), scale factor and percent of ‘present’ calls per array. Outlier subjects were excluded in regions where they did not pass quality standards.

In order to determine if promoter haplotypes were related to brain expression, ANOVAs were performed with log2-transformed expression values from probe sets 202043_s_at (SMS) and 210357_s_at (SMOX) (www.Affymetrix.com) and haplotypes determined through genotyping, p values were corrected for three tests, with corrected p⩽0.05 considered significant.

Cell culture

Dual-reporter gene assays were performed in three different cell lines: COS7 (green monkey kidney), HTB15 (human glio/astrocytoma), and CRL2137 (human neuroblastoma) (American Type Culture Collection, USA). All cultures were maintained at 37°C with 5% CO2, and grown in Dulbecco's Modified Eagle Medium (Gibco, Canada) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco).

Cloning

A 2.1-kb region of SMS and a 1.4-kb region of SMOX were cloned into the pGL3 Basic Luciferase Reporter Vector (Promega, USA), upstream of the firefly luciferase gene. These regions are depicted in Figs 2 b and 3 b, and primer sequences are given in Supplementary Table S1 (available online). All inserts were sequenced.

Fig. 2

Regions and polymorphisms of the SMS promoter analysed in this study. (a) Location and sequences at polymorphic sites. Exon 1 is shaded in black, and the translation start site is indicated. (b) Region analysed for reporter gene assays. SNPs with asterisks (*) were identified during sequencing of this region. (c) Region analysed for CpG methylation. (d) Haplotypes and frequencies of genotyped polymorphisms. (e) Linkage disequilibrium between polymorphisms plotted as r2.

Fig. 3

Regions and polymorphisms of the SMOX promoter analysed in this study. (a) Location and sequences at polymorphic sites. Exon 1 is shaded in black. (b) Region analysed for reporter gene assays. (c) Region analysed for CpG methylation. (d) Haplotypes and frequencies of genotyped polymorphisms. (e) Linkage disequilibrium between polymorphisms plotted as r2.

Transfection and dual-reporter gene assays

Cells were seeded into 24-well plates and grown at 37°C for 24 h to reach 90–95% confluency. Transfections were then performed using either Lipofectamine 2000 (Invitrogen, Canada) or GeneJuice (Novagen, Canada) systems, as appropriate for each cell line. Each well was treated with equivalent amounts of DNA for each of the vectors of interest, as well as with an internal control vector coding for renilla luciferase, pRL (Promega). Cells were grown for an additional 24 h at 37°C prior to assaying luciferase activity. Reporter gene expression was measured using the Dual-Luciferase Assay system (Promega) using an Orion II Microluminometer (Berthold, Canada). Each vector was analysed in triplicate within individual experiments, and three independent experiments were performed for each cell line. Firefly luciferase expression values were normalized to those of the internal control. ANOVAs and unpaired Student's t tests between haplotypes were performed, with p⩽0.05 considered significant.

Methylation in BA 8/9

Subjects

A subset of 20 subjects taken from our 40 brain samples was examined, and comprised 10 control subjects and 10 suicide completers. Subjects were matched for age, post-mortem interval (PMI), and pH.

CpG methylation mapping

Genomic DNA from BA 8/9 was extracted as above, then treated and purified with the Qiagen EpiTect Bisulfite kit (Qiagen, USA). Bisulfite-treated DNA was amplified by polymerase chain reaction (PCR) using primers shown in Supplementary Table 1. PCR products were purified with the GenElute PCR Clean-up kit (Sigma-Aldrich, USA), ligated into the pDRIVE vector and transformed into EZ Competent cells (PCR Cloning Plus kit, Qiagen). Plasmids were extracted and sequenced using primers SMS-ep-R2 or SMOX-ep-R3. Using percentages of CpG methylation, unpaired Student's t tests and Pearson correlations were used to analyse differences between groups as well as determine the relationship between methylation and expression in BA 8/9, and p values were corrected for the number of methylated CpG sites in each gene.

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Chromatin immunoprecipitation of H3K27me3

Immunoprecipitated DNA was prepared as described by Matevossian & Akbarian (2008). Briefly, tissue from BA 8/9 was treated with micrococcal nuclease (Sigma-Aldrich), which cleaves DNA between adjacent nucleosomes. Intact nucleosomes were extracted, and a portion of sample was treated with anti-H3K27me3 antibody (Upstate Biotechnology, USA), while the remainder was used as an input control. The antibody-treated sample was purified and both input and bound fractions were digested with protease before DNA was purified. Quantitative real-time PCR (RT-PCR) using SYBR Green (Applied Biosystems) was used to measure DNA in each sample, in quadruplicate. Relative quantitation was performed with β-actin as an endogenous control, and ratios of bound:input were calculated for each subject. Student's t tests and Pearson correlations were used to analyse differences between groups and the relationship between methylation and expression in BA 8/9.

Results

We previously observed altered expression of SMS and SMOX in several brain regions of suicide completers (Klempan et al. 2009b; Sequeira et al. 2007). In order to identify molecular mechanisms that may be involved in determining the expression levels of these transcripts, we examined genetic polymorphisms in the promoter regions of these two genes and their relationship to expression in the brain and in vitro, as well as assessing methylation at both the level of DNA and histone.

Genotyping

Our first objective was to characterize the haplotype structures of SMS and SMOX in order to identify haplotypes which may be involved in determining expression in the brain. To this end, we genotyped six and 14 SNPs in the promoter regions of SMS and SMOX, respectively.

The results for SMS are shown in Fig. 2. SMS is located on the X chromosome, and as an all-male sample was used in this study, only one allele was observed in each subject. All of the SNPs except rs7066893 were genotyped as part of the HapMap project (International HapMap Consortium, 2003). Two SNPs, rs7066303 and rs7062138 showed no variability in this sample. The other four SNPs produced three haplotypes, as shown in Fig. 2 d. The LD structure is shown in Fig. 2 e, and reveals that both pairs of non-adjacent SNPs are in LD with each other.

The results for SMOX are depicted in Fig. 3. Eight of these SNPs (rs1741289, rs6052399, rs6139327, rs1741291, rs2422915, rs7265946, rs945772, rs945771) were genotyped in HapMap populations (International HapMap Consortium, 2003). No variability was observed at rs7265946, and only one subject possessed the alternate allele at rs6139327. Four haplotypes were identified with a frequency of at least 5% in our population, and represented ∼96% of all alleles. The LD structure is shown in Fig. 2 e. Two major haplotype blocks were observed, comprising rs1741289 to rs1764998, and rs6052399 to rs945771. Interestingly, rs1631654 is included as part of the second block. Most of the variability at this locus is found in the first LD block, with only two major haplotypes being found in the second.

Brain expression

Our next step was to determine if promoter haplotypes were related to expression levels of SMS and SMOX in the brain. We examined brain regions BA 8/9, BA 47, and the hippocampus as these regions are implicated to be involved in psychiatric disorders (Hercher et al. 2009). In order to determine the relationship between promoter haplotypes and expression, ANOVAs were performed between expression levels and haplotypes of SMS (Table 1) and SMOX (not shown). We found no association between haplotype and expression for either gene in any of the regions, which suggests that promoter haplotypes may not be responsible for the differential expression of SMS or SMOX in suicide completers.

Dual reporter gene assays

To further investigate the functional impact of promoter variability on gene expression, we performed dual reporter gene assays to examine the relative promoter activities of the major haplotypes from both genes.

We selected a 2.1-kb region of SMS, as shown in Fig. 2 b. The region contains 158 bp of exon 1, including the translation start site, as well as all four polymorphic SNPs. Two individuals for each haplotype were sequenced in order to identify additional SNPs in LD with the genotyped SNPs. This revealed two additional SNPs, of which one, rs34507903, has been previously identified. Based upon these results, the three haplotypes examined were TCACGA (haplotype 1), TTACAG (haplotype 2), and CTCAAG (haplotype 3), of which underlined SNPs represent those genotyped in our sample. One vector from each haplotype, as well as the empty pGL3 vector, were used to transfect COS7, HTB15, and CRL2137 cells. As shown in Fig. 4 a, all constructs had promoter activity compared to the empty pGL3 vector, with average fold changes (FC) of 4.1, 1.8, and 11.6 for COS7, HTB15, and CRL2137, respectively. There were significant differences between haplotypes in COS7 cells (ANOVA p=0.04). In this case, haplotype 2 was expressed significantly higher than haplotype 1 (FC=1.15, p=0.007) and haplotype 3 (FC=1.18, p=0.04). This partially agrees with our findings in the brain, where haplotype 1 demonstrated the lowest expression levels. There were no significant differences in HTB15 or CRL2137 cell lines (p=0.69 and 0.39, respectively), which suggests that the haplotype effects may be cell-type specific. Alternatively, as the FCs were relatively small, differences in the other cell lines may have been masked by the increased variability between replicates in these assays.

Fig. 4

Results from reporter gene studies in COS7, HTB15 and CRL2137 cell lines. Normalized relative light units per second (RLU/s) and standard errors were obtained from triplicate replications of each assay. (a) SMS haplotype 1 (■), haplotype 2 (□), haplotype 3 (Embedded Image), and empty pGL3 (Embedded Image), and (b) SMOX haplotype 1 (■), haplotype 4 (□), and empty pGL3 (Embedded Image).

We analysed a 1.4-kb region of SMOX, which contains all of the upstream sequence studied by Wang and colleagues (2005). This region is shown in Fig. 3 b, and contains three of the variable SNPs from the second LD block, giving two haplotypes: CGC (haplotype 1) and GTG (haplotype 4). Sequencing of this region did not reveal any additional SNPs. The results for the two haplotypes are shown in Fig. 4 b. Both constructs demonstrated promoter activity, with FC of 88.5, 4.0, and 36.7 in COS7, HTB15, and CRL2137 cells, respectively. However, there were no significant differences between the two haplotypes in any of the cell lines examined (p=0.70, 0.61, and 0.84, respectively).

CpG methylation

Another possible mechanism for regulation of gene expression is by epigenetic control, which is mediated through different mechanisms, an important one being promoter methylation at CG dinucleotides (Chomet, 1991). In order to determine if CpG methylation was involved in the differential expression of SMS and SMOX in suicide, we analysed CpG methylation in ten suicide completers and ten healthy controls. This analysis was performed in BA 8/9, as previous studies have identified the prefrontal cortex as being an important source of differential patterns of CpG methylation in suicide (de Luca et al. 2009; Ernst et al. 2009b; Poulter et al. 2008).

We examined the 741-bp region of SMS shown in Fig. 2 c. This region has a 62% GC content, and contains all of exon 1 as well as 567 bp of upstream sequence. The results for methylation at individual CpG sites are shown in Fig. 5 a. Overall, there was a low level of methylation, with an average of 2.4% at all CpG sites. Five sites were completely unmethylated, and the majority of other sites were methylated in <5% of all clones. The main exception was the site at −471, which was methylated in 36% of clones. There was no difference in overall methylation between suicide completers and controls (p=0.57), nor was methylation at any individual sites significant after correcting for multiple testing. We also assessed the relationship between methylation and expression of SMS in BA 8/9. These results are shown in Fig. 5 b. We found no relationship between expression and methylation overall (p=0.57), or at specific sites.

Fig. 5

Methylation of CpG sites in the promoter region of SMS. Sites are shown as position relative to the transcription start site. (a) Percentage methylation+s.e.m. for suicide completers (■) and controls (Embedded Image). (b) Pearson correlation (Embedded Image) and –log10p values (–––) for correlation of percentage methylation with SMS expression. The dotted line indicates corrected p=0.05.

The 566-bp region of SMOX is shown in Fig. 3 c, and has a 72% GC content. The results for each site are shown in Fig. 6 a. As with SMS, this region was only sparsely methylated, with an average of 3.1% methylation. We did not identify any relationship between methylation and promoter haplotypes. Similarly to SMS, there were no differences in either overall (p=0.92), or site-specific methylation between suicide completers and control subjects, nor was there a significant correlation between overall methylation and expression of SMOX in BA 8/9 (p=0.19). As shown in Fig. 6 b, we found a significant correlation between methylation and expression at only one site (+73 bp, corrected p=0.035, Pearson=−0.7).

Fig. 6

Methylation of CpG sites in the promoter region of SMOX. Sites are shown as position relative to the transcription start site. (a) Percentage methylation+s.e.m. for suicide completers (■) and controls (Embedded Image). (b) Pearson correlation (Embedded Image) and –log10p values (–––) for correlation of percentage methylation with SMOX expression. The dotted line indicates corrected p=0.05.

H3K27 methylation

Histone modifications are another important form of epigenetic regulation. We chose to focus on H3K27me3, a modification which is associated with gene repression when found in promoter regions (Barski et al. 2007), and which we recently found to display increased levels in the promoter region of the TrkB receptor in the prefrontal cortex of suicide completers (Ernst et al. 2009a). We examined levels of H3K27me3 in the promoter regions of SMS and SMOX in BA 8/9 from the same 20 subjects used for the CpG methylation study. The results of these experiments are depicted in Fig. 7. We found no significant differences between levels of H3K27 methylation at the promoter region of SMS (p=0.80) or SMOX (p=0.89) in suicide completers compared to controls. In addition, there was no relationship between H3K27 methylation and promoter haplotypes, gene expression, or levels of CpG methylation for either gene (not shown).

Fig. 7

Methylation of H3K27 in the promoter regions of SMS and SMOX. Methylation+s.e.m. for controls (Embedded Image) and suicides (■) are expressed as the ratio of immunoprecipitated (IP) DNA to input DNA.

Discussion

In this study, we followed up on our previous findings of differential expression of SMS and SMOX in the brains of suicide completers by examining the role of both genetic and epigenetic factors in determining the expression of these two genes.

We identified three major promoter haplotypes in SMS; however, we found no relationship between haplotypes and expression in the brain, and differential expression of the haplotypes was only observed in one cell line. We also saw no relationship between brain expression and either of the epigenetic mechanisms we examined. Promoter CpG methylation in SMS has previously been examined in bipolar disorder, although findings were inconclusive, and, in agreement with our results, methylation was not found to be significantly correlated with SMS expression (Kuratomi et al. 2008).

The overall percentage CpG methylation and levels of H3K27me3 in the promoter region of SMS did not differ between suicide completers and controls, although this is not overly surprising as we saw no differences in expression between these two groups in BA 8/9. We also found no indication that promoter haplotypes were involved in the increased expression of SMS in the hippocampus of depressed suicide completers.

The haplotype structure of SMOX was more complex than that of SMS. We were unable to identify any relationship between various haplotypes and the expression of SMOX in prefrontal brain regions. However, the complexity at this locus and the fact that unlike SMS, individuals possessed two alleles at each SNP, may have prevented us from identifying any small magnitude effects. Nonetheless, it does appear unlikely that the second LD block plays a role in determining expression under normal conditions, as there were no significant differences between the two haplotypes in this block in reporter gene assays. However, this region has previously been shown to be responsive to polyamine analogues (Wang et al. 2005), and it remains possible that the two haplotypes may differ in their ability to induce SMOX expression in the presence of polyamines.

We also examined CpG methylation of the promoter region of SMOX and found that methylation at one CpG site in exon 1 was significantly negatively correlated with expression, which could indicate that this site plays a regulatory role in determining transcription levels. As with SMS, we did not identify any significant differences in CpG or H3K27 methylation in the promoter region of SMOX between suicides and controls. Interestingly, SMOX has been observed to display monoallelic expression in a human cell line (Gimelbrant et al. 2007), which could have obscured the effects of genetic and epigenetic factors involved in determining expression.

Overall, we failed to identify molecular mechanisms with significant effects in determining the levels of SMS and SMOX transcripts, which could explain the differential expression of these genes in suicide completers. Although we found little evidence for a role for genetic polymorphisms in determining transcript levels, this study focused only on SNPs within the upstream promoter regions of these genes. Introns also have important functions in regulating levels of gene transcripts by providing additional sites for transcriptional regulatory elements, influencing alternative splicing and non-sense mediated mRNA decay, and allowing export of mRNA to the nucleus (Fedorova & Fedorov, 2003); and each of these mechanisms may be influenced by polymorphisms in these genes. In addition to their regulation at the level of gene transcription, sequences in the processed transcript can affect RNA stability and rates of degradation. Indeed, SMOX mRNA has been found to be stabilized by polyamine analogues (Wang et al. 2005), indicating the importance of post-transcriptional mechanisms in regulating levels of SMOX transcripts.

Although we found no evidence for a role of H3K27 methylation in determining the expression levels of SMS or SMOX, numerous other histone modifications are known to play a role in regulating various aspects of transcription (Munshi et al. 2009). Indeed, the expression of two polyamine metabolic genes, ornithine aminotransferase and µ-crystallin, has previously been shown to be influenced by elevated methylation at H3-arginine-17 in prefrontal cortices of schizophrenia patients (Akbarian et al. 2005). We thus cannot rule out the possibility that modifications at the chromatin level are involved in regulating the expression of SMS or SMOX.

There are a number of possible limitations of our study that should be considered. Our study was conducted in a sample composed exclusively of French Canadians, a population with a well-documented founder effect (Simard et al. 1994). It is therefore possible that the frequency and properties of the haplotypes investigated in this study are different in other populations. Similarly, as this study was performed in only male subjects, the applicability of these results to females is unclear, particularly as SMS is found on the X chromosome. Although the subjects used for the epigenetic studies were matched for age, pH and PMI, it is possible that additional factors may have confounded our results. It is equally possible that the sample investigated in this study lacked power to detect methylation effects of discrete magnitude.

In summary, we found little evidence to support roles for either genetic or epigenetic factors in regulating the expression of SMS and SMOX. It is clear that additional mechanisms are involved in determining the levels of SMS and SMOX transcripts in the brain, and further work will be required to identify factors responsible for the dysregulated expression of these genes in suicide completers.

Acknowledgements

This work was supported by the Canadian Institute of Health Research (CIHR) MOP 79253. G.T. is a Fonds de la recherche en santé du Québec (FRSQ) Research Fellow. L.M.F. received a scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC).

Note

Supplementary material accompanies this paper on the Journal's website.

Statement of Interest

None.

References

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