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Differential Glucocorticoid Receptor Exon 1B, 1C, and 1H Expression and Methylation in Suicide Completers with a History of Childhood Abuse

  • Benoit Labonte
    Affiliations
    McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, Quebec, Canada

    Department of Psychiatry, McGill University, Montreal, Quebec, Canada
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  • Volodymyr Yerko
    Affiliations
    McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, Quebec, Canada
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  • Jeffrey Gross
    Affiliations
    McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, Quebec, Canada

    Department of Psychiatry, McGill University, Montreal, Quebec, Canada
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  • Naguib Mechawar
    Affiliations
    McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, Quebec, Canada

    Department of Psychiatry, McGill University, Montreal, Quebec, Canada
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  • Michael J. Meaney
    Affiliations
    Department of Psychiatry, McGill University, Montreal, Quebec, Canada
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  • Moshe Szyf
    Affiliations
    Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada
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  • Gustavo Turecki
    Correspondence
    Address correspondence to: Gustavo Turecki, M.D., Ph.D., Douglas Mental Health University Institute, 6875 LaSalle Boulevard, Verdun, H4H 1R3, Quebec, Canada
    Affiliations
    McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, Quebec, Canada

    Department of Psychiatry, McGill University, Montreal, Quebec, Canada
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      Background

      Childhood abuse alters hypothalamic-pituitary-adrenal (HPA) function and increases the risk of suicide. Hippocampal glucocorticoid receptor (GR) activation regulates HPA activity, and human GR expression (hGR) is reduced in the hippocampus of suicide completers with a history of childhood abuse compared with controls. The abuse-related decrease in hGR expression associates with increased DNA methylation of the promoter of the hGR1F variant in the hippocampus.

      Methods

      In this study, we investigated the expression and methylation levels of other hGR splice variants in the hippocampus and anterior cingulate gyrus in suicide completers with and without a history of childhood abuse and in controls. Expression levels were quantified using quantitative reverse-transcriptase polymerase chain reaction and promoter methylation was assessed by pyrosequencing.

      Results

      In the hippocampus, the expression of total hGR and variants 1B, 1C, and 1H was decreased in suicide completers with histories of abuse compared with suicides with no histories of abuse and with control subjects. In the anterior cingulate gyrus, however, no group differences in hGR total or variant expression were found. Site-specific methylation in hGR1B and 1C promoter sequences were negatively correlated with total hGR messenger RNA, as well as with hGR1B and 1C expression. Luciferase assay showed that methylation in hGR promoter decreases transcriptional activity. In contrast, total and site-specific methylation in the hGR1H promoter was positively correlated with total hGR messenger RNA and hGR1H expression.

      Conclusion

      These findings suggest that early-life events alter the expression of several hGR variants in the hippocampus of suicide completers through effects on promoter DNA methylation.

      Key Words

      Suicide can occur as a result of the interaction of multiple influences (
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      ), including childhood sexual and physical abuse (
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      ). There is substantial evidence for a link between suicide and hypothalamic-pituitary-adrenal axis (HPA) hyperactivity (
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      ), which in turn, associates with early-life adversity (ELA) (
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      The link between childhood trauma and depression: Insights from HPA axis studies in humans.
      ). Compared to nonabused control subjects, abuse victims with major depressive disorder exhibit higher plasma levels of corticotropin (ACTH) and cortisol following dexamethasone challenge and Trier social stress test (
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      ). In addition, results from animal studies suggest that ELA modifies the functioning of the HPA axis and induces behavioral alterations during adulthood (
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      ). Taken together, these findings suggest that alterations in HPA activity induced by ELA may influence the risk for suicide.
      The glucocorticoid receptor (GR) regulates HPA axis and stress reactivity (
      • Heim C.
      • Newport D.J.
      • Mletzko T.
      • Miller A.H.
      • Nemeroff C.B.
      The link between childhood trauma and depression: Insights from HPA axis studies in humans.
      ) and GR expression in brain is stably influenced by early experience (
      • Meaney M.J.
      • Szyf M.
      Maternal care as a model for experience-dependent chromatin plasticity?.
      ). Variations in quality of the postnatal environment can influence DNA methylation level of specific gene promoters, including that for the GR (
      • Murgatroyd C.
      • Patchev A.V.
      • Wu Y.
      • Micale V.
      • Bockmuhl Y.
      • Fischer D.
      • et al.
      Dynamic DNA methylation programs persistent adverse effects of early-life stress.
      ,
      • Weaver I.C.
      • Cervoni N.
      • Champagne F.A.
      • D'Alessio A.C.
      • Sharma S.
      • Seckl J.R.
      • et al.
      Epigenetic programming by maternal behavior.
      ,
      • Roth T.L.
      • Lubin F.D.
      • Funk A.J.
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      Lasting epigenetic influence of early-life adversity on the BDNF gene.
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      • Oberlander T.F.
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      • Misri S.
      • Devlin A.M.
      Prenatal exposure to maternal depression, neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responses.
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      • Dymov S.
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      • Szyf M.
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      Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse.
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      • Huang T.C.
      • Unterberger A.
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      • Ernst C.
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      Promoter-wide hypermethylation of the ribosomal RNA gene promoter in the suicide brain.
      ). DNA methylation is a covalent modification of cytosines in DNA that regulates genome function. DNA methylation in promoters and gene regulatory regions associates with silencing of gene expression (
      • Klose R.J.
      • Bird A.P.
      Genomic DNA methylation: The mark and its mediators.
      ). For example, in rats, adult offspring of low licking and grooming mothers show decreased hippocampal GR17 expression associated with overall promoter hypermethylation that constrains the binding of the transcription factor nerve growth factor-induced protein A (NGFI-A) (
      • Weaver I.C.
      • Cervoni N.
      • Champagne F.A.
      • D'Alessio A.C.
      • Sharma S.
      • Seckl J.R.
      • et al.
      Epigenetic programming by maternal behavior.
      ). Translational studies in humans (
      • McGowan P.O.
      • Sasaki A.
      • D'Alessio A.C.
      • Dymov S.
      • Labonte B.
      • Szyf M.
      • et al.
      Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse.
      ) show decreased hippocampal hGR1F (hGR) expression, the human GR17 homologue in rats, in abused suicide completers compared with nonabused suicide and control subjects. The decrease in hGR1F expression associates with reduced total hGR messenger RNA and with site-specific promoter hypermethylation in a region that contains an NGFI-A binding site (
      • McGowan P.O.
      • Sasaki A.
      • D'Alessio A.C.
      • Dymov S.
      • Labonte B.
      • Szyf M.
      • et al.
      Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse.
      ). Such patterns of methylation repress NGFI-A binding to hGR1F promoter and decrease transcriptional activity in reporter assays. These results suggest that site-specific hypermethylation in the hGR1F promoter associates with reduced GR expression in the brain of abused, suicide completers (
      • McGowan P.O.
      • Sasaki A.
      • D'Alessio A.C.
      • Dymov S.
      • Labonte B.
      • Szyf M.
      • et al.
      Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse.
      ).
      The human hGR gene is located on chromosome 5 at locus q31–q32 and is composed of eight coding exons preceded by a number of noncoding first exons (
      • Presul E.
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      • Helmberg A.
      Identification, tissue expression, and glucocorticoid responsiveness of alternative first exons of the human glucocorticoid receptor.
      ,
      • Turner J.D.
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      Structure of the glucocorticoid receptor (NR3C1) gene 5' untranslated region: identification, and tissue distribution of multiple new human exon 1.
      ). Each of these first exons contains a transcription start site (TSS) preceded by a promoter (
      • Encio I.J.
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      The genomic structure of the human glucocorticoid receptor.
      ,
      • Breslin M.B.
      • Geng C.D.
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      Multiple promoters exist in the human GR gene, one of which is activated by glucocorticoids.
      ). Because the ATG start codon is located only at the beginning of exon 2, first exons remain untranslated, although it is suggested that they may be involved in the determination of hGR expression in various tissues (
      • Turner J.D.
      • Muller C.P.
      Structure of the glucocorticoid receptor (NR3C1) gene 5' untranslated region: identification, and tissue distribution of multiple new human exon 1.
      ,
      • Pickering B.M.
      • Willis A.E.
      The implications of structured 5' untranslated regions on translation and disease.
      ). Seven (D, J, E, B, F, C, and H) of these first exons are found within a CpG island in a region encompassing 4.5 kb upstream of exon 2. First exon variant expression differs across tissues (
      • Turner J.D.
      • Muller C.P.
      Structure of the glucocorticoid receptor (NR3C1) gene 5' untranslated region: identification, and tissue distribution of multiple new human exon 1.
      ). The 1C and 1B variants are the most expressed first exons in the hippocampus followed by 1F and 1H and finally by 1D, 1J and 1E (
      • Alt S.R.
      • Turner J.D.
      • Klok M.D.
      • Meijer O.C.
      • Lakke E.A.
      • Derijk R.H.
      • et al.
      Differential expression of glucocorticoid receptor transcripts in major depressive disorder is not epigenetically programmed.
      ). The genomic location of these exons within a CpG island renders them susceptible to epigenetic regulation. For instance, numerous transcription factor binding sites lie within the noncoding exon 1 promoters (
      • McGowan P.O.
      • Sasaki A.
      • D'Alessio A.C.
      • Dymov S.
      • Labonte B.
      • Szyf M.
      • et al.
      Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse.
      ,
      • Nunez B.S.
      • Vedeckis W.V.
      Characterization of promoter 1B in the human glucocorticoid receptor gene.
      ,
      • Geng C.D.
      • Vedeckis W.V.
      c-Myb and members of the c-Ets family of transcription factors act as molecular switches to mediate opposite steroid regulation of the human glucocorticoid receptor 1A promoter.
      ,
      • Geng C.D.
      • Schwartz J.R.
      • Vedeckis W.V.
      A conserved molecular mechanism is responsible for the auto-up-regulation of glucocorticoid receptor gene promoters.
      ). Moreover, methylation in the promoter of these exons may regulate differential hGR transcript expression, and there is considerable interindividual heterogeneity in methylation levels of hGR promoters (
      • Turner J.D.
      • Pelascini L.P.
      • Macedo J.A.
      • Muller C.P.
      Highly individual methylation patterns of alternative glucocorticoid receptor promoters suggest individualized epigenetic regulatory mechanisms.
      ).
      Although there is increased methylation of the hGR1F promoter in the hippocampus of abused suicide completers, it remains unclear whether methylation of the promoters of the other noncoding exon 1 variants is also altered by ELA. In this study, we investigated the association of ELA with methylation in the promoters of hGR's noncoding exon 1 variants and the relationship with expression of these transcripts. This study focused on three noncoding exons with the highest expression in the hippocampus besides 1F namely, hGR1B, 1C, and 1H. hGR1F was not included in this study because it was already assessed in a previous study by our group (
      • McGowan P.O.
      • Sasaki A.
      • D'Alessio A.C.
      • Dymov S.
      • Labonte B.
      • Szyf M.
      • et al.
      Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse.
      ). In addition to the hippocampus, we investigated the anterior cingulate cortex (ACC), a brain region implicated in mood regulation (
      • Diorio D.
      • Viau V.
      • Meaney M.J.
      The role of the medial prefrontal cortex (cingulate gyrus) in the regulation of hypothalamic-pituitary-adrenal responses to stress.
      ). Promoter DNA methylation was assessed in regions predicted to be enriched in transcription factor binding sites. Our results suggest that hGR1B, 1C, and 1H expression in the hippocampus, but not in ACC, of abused suicide completers is decreased. This effect was associated with site-specific DNA methylation alterations, and luciferase assay showed that methylation in hGR promoter decreases transcriptional activity. These data support the hypothesis that child abuse is associated with a coordinated DNA methylation response in multiple promoters of the hGR gene contributing to decreased hGR expression and HPA dysregulation.

      Methods and Materials

      Complete methods are described in Supplement 1.

      Sample Selection

      Brain tissue was obtained from the Quebec Suicide Brain Bank (Douglas Mental Health University Institute, Verdun, Quebec). All subjects were male Caucasians of French-Canadian descent, and groups (abused suicides [SA], nonabused suicides [SNA], controls [CTRL]) were matched for age, pH, and postmortem intervals (PMI; Table S1 in Supplement 1). The control group was composed of psychiatrically healthy individuals who died by accidental causes with no history of child abuse. Presence of severe ELA was assessed based on adapted Childhood Experience of Care and Abuse interviews assessing various dimensions of childhood experience including experiences of abuse (
      • Bifulco A.
      • Brown G.W.
      • Harris T.O.
      Childhood Experience of Care and Abuse (CECA): A retrospective interview measure.
      ). Hippocampus (n = 56; 21 SA, 21 SNA, and 14 CTRL) and Brodmann area 24 (n = 50; 22 SA, 14 SNA, and 14 CTRL) were carefully dissected by experienced histopathologists using reference neuroanatomical maps (
      • Nolte J.
      The Human Brain: An Introduction to Its Functional Neuroanatomy.
      ,
      • Haines D.
      Neuroanatomy: An Atlas of Structures, Sections, and Systems.
      ).

      Quantification of Gene Expression Using Reverse-Transcriptase Polymerase Chain Reaction

      Three reverse transcription reactions were performed independently using primers targeting specifically NR3C1 (glucocorticoid receptor, hGR), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and β-Actin. The quantity of complementary DNA was extrapolated from a standard curve, composed of a mix of complementary DNA from all subjects and including six dots, each of which was four times diluted (4:1). Mean quantities from all sample replicates were normalized to the reference gene GAPDH and β-Actin, averaged together and analyzed by mean of one-way analysis of variance followed by Fisher's least significant difference post hoc test.

      Methylation Analysis

      DNA was extracted and sodium bisulfite converted according to the manufacturer's instructions (Qiagen, Germantown, Maryland). Primers and sensitivity assays for pyrosequencing targeting three regions of the hGR variant promoters were designed and optimized by EpigenDx (Worcester, Massachusetts; Tables S3 and S4 in Supplement 1). Pyrosequencing runs were performed by EpigenDx. Mean percentage of methylation at each CpG site, and at all sites (global methylation; Figure 1) was compared across groups and analyzed by two way-mixed model analysis of variance with groups as a fixed factor and CpGs as a repeated measure followed by least significant difference post hoc tests.
      Figure thumbnail gr1
      Figure 1Schematic representation of human glucocorticoid receptor (hGR) noncoding exons organization. Panels A, B, and C represent the promoter regions assessed by pyrosequencing. (A) Sequence of the hGR1B promoter assessed comprising 12 CpGs. Underlined sequences represent predicted Sp1/Sp3 transcription factor binding sites, dotted underlined sequence represent predicted NF-1 transcription factor binding site and dashed underlined sequences represent predicted YY1 transcription factor binding site. (B) Sequence of the hGR1C promoter assessed comprising 18 CpGs. Underlined sequences represent predicted Sp1/Sp3 transcription factor binding sites, and dotted underlined sequences represent predicted AP-1 transcription factor binding sites. (C) Sequence of the hGR1H promoter assessed comprising 13 CpGs. Underlined sequences represent predicted Sp1/Sp3 transcription factor binding sites. Gray and black boxes represent sites of significant hypermethylation and hypomethylation, respectively, in abused and/or nonabused suicide completers compared with controls.

      Luciferase Assays

      hGR1C full length promoter was amplified by polymerase chain reaction from human genomic DNA. hGR1C promoter inserted into pGL3 plasmid was methylated by patch methylation before transfection into Be(
      • Brezo J.
      • Klempan T.
      • Turecki G.
      The genetics of suicide: A critical review of molecular studies.
      )c cells. Firefly renilla plasmid was used as a control for transfection efficiency and to normalize luciferase activity. All experiments were performed in seven replicates. Results were analyzed by t test.

      Results

      hGR Expression in the Hippocampus (HPC) and ACC in Abused Suicide Completers

      The expression of hGR total and the first noncoding exons 1B, 1C, and 1H was compared in the hippocampus and ACC of SA, SNA, and normal control subjects. There was no significant difference across groups for age, pH, PMI, RNA integrity values, and psychiatric medication prescription status (Table S1 in Supplement 1), and analyses of covariance revealed no significant effect of these covariables on gene expression results. The results reveal that relative expression levels of hGR total and first noncoding exons were decreased in SA compared with SNA and CTRL independently of the effect of covariables (Figure 2). Indeed, we found a significant group effect for the mean expression levels of hGR total [F(2,47) = 10.49, p < .001; Figure 2A]. Post hoc analyses revealed significantly decreased hGR total expression in SA compared with CTRL (p < .005) and SNA (p < .001), thus replicating our earlier finding (
      • McGowan P.O.
      • Sasaki A.
      • D'Alessio A.C.
      • Dymov S.
      • Labonte B.
      • Szyf M.
      • et al.
      Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse.
      ). A significant group effect was found for the mean expression levels of hGR1B [F(2,48) = 5.23, p < .01; Figure 2B). Post hoc tests revealed a significant decreased expression in samples from SA compared with those from SNA (p < .05) and CTRL (p < .005), with no difference between SNA and CTRL values. A significant group effect was also found for the relative expression of hGR1C [F(2,46) = 5.82, p < .01; Figure 2C]. Post hoc tests showed that hGR1C relative expression is significantly decreased in SA compared with SNA (p < .005) and CTRLs (p < .05) with no difference between SNA and CTRL. Finally, we found a significant group effect for hGR1H relative expression [F(2,45) = 4.29, p > .05; Figure 2D] with post hoc test revealing significantly decreased relative expression in SA compared to SNA (p < .01) and CTRL (p < .05). No significant differences in expression were observed between SNA and CTRL.
      Figure thumbnail gr2
      Figure 2Relative expression of human glucocorticoid receptor (hGR) first exons in the hippocampus and ACC of abused suicide completers (SA; black), nonabused suicides (SNA; gray), and control subjects (CTRL; white). Ratios of relative expression of hippocampal GR total (second exon) (A), 1B (B), 1C (C), and 1H (D), and anterior cingulate cortex GR total (second exon) (E), 1B (F), 1C (G), and 1H (H). Values are given as hGR mean quantities normalized by glyceraldehyde-3-phosphate dehydrogenase, and β-Actin ± SEM. * p < .05. Hippocampus: hGR total (SA: 19, SNA: 19, and CTRL: 11), hGR1B (SA: 19, SNA: 19 and CTRL: 11), hGR1C (SA: 18, SNA: 19 and CTRL: 10), hGR1H (SA: 18, SNA: 18 and CTRL: 10); Brodmann's area 24: hGR total (SA: 20, SNA: 12, CTRL: 13), hGR1B (SA: 21, SNA: 11 and CTRL: 13), hGR1C (SA: 21, SNA: 14 and CTRL: 12), and hGR1H (SA: 19, SNA: 12 and CTRL: 13).
      In contrast to the hippocampus, analysis of samples from the ACC showed no difference in hGR total relative messenger RNA expression across SA, SNA, and CTRL [hGR total: F(2,44) = .49, p > .1; Figure 2E]. In addition, no significant difference was found across groups in the ACC relative expression of any of the first noncoding exons [hGR1B F(2,44) = .06, p > .1], 1C [F(2,46) = .47, p < .1] and 1H [F(2,43) = .66, p > .1; Figure 2F–2H].

      Differential DNA Methylation in Abused Suicide Completers

      DNA methylation regulates hippocampal hGR expression, and increased methylation associates with a downregulation of hGR1F expression in abused suicide completers (
      • McGowan P.O.
      • Sasaki A.
      • D'Alessio A.C.
      • Dymov S.
      • Labonte B.
      • Szyf M.
      • et al.
      Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse.
      ,
      • Klose R.J.
      • Bird A.P.
      Genomic DNA methylation: The mark and its mediators.
      ). We therefore measured DNA methylation levels across the promoter sequences of differentially expressed nontranslated exon 1 variants. We focused on the promoter sequences enriched with transcription factor binding sites (Figure 1). Because we did not observe any group difference in the hGR or exon 1 variant expression in the ACC, the DNA methylation analysis focused on the hippocampus. We examined methylation levels at 43 individual CpG sites of which 12 were in the hGR1B promoter, 18 in the hGR1C promoter, and 13 in the hGR1H promoter (Figure 1A–1C). Overall methylation levels were generally low, as expected for functional promoter sequences, with mean values ranging from 3.4% to 14.3%. The results did not show differences in methylation levels across groups when total CpG sites were considered (data not shown). However, differential methylation patterns were observed at specific CpG sites as indicated.

      hGR1B

      Methylation levels were analyzed across a 186 bp region composed of 12 CG dinucleotides located 233 bp upstream of hGR1B, including exon 1J (Figure 1A). Methylation levels in this region varied between 1.1% and 18.3% (mean methylation levels: SA = 9.7%, SNA = 9.0%, and CTRL = 10.1%). Two-way analysis of variance performed on percentage of methylation did not reveal a significant main effect of group (Figure 3D), but showed a significant main effect of CpG site [F(11,70) = 79.7; p < .0001] as well as a significant group by CpG site interaction [F(22,70) = 1.9; p < .05; Figure 3A]. Post hoc tests revealed significant hypermethylation in SA and SNA compared with CTRL for CpG6 (SA: p < .05; SNA: p < .005) and CpG8 (SA: p < .05, SNA: p < .05). We also found a significant hypomethylation in SA and SNA compared with CTRL at CpG11 (SA: p < .005; SNA: p < .005; Figure 3A). To assess potential functional role of the differentially methylated sites, we examined the correlation between the level of methylation and the expression of hGR1B and hGR total. Interestingly, methylation levels at CpG8 (hGR1B: r = −.51, p < .001; hGR total: r = −.30, p < .05) were negatively correlated with the expression of both hGR1B and total (Figure 4A and 4B). Analyses of covariance revealed that age, pH, PMI, and psychiatric medication prescription status had no effect on the group differences observed in hGR1B promoter.
      Figure thumbnail gr3
      Figure 3Percentage of methylation of human glucocorticoid receptor (hGR) first noncoding exon promoters in the hippocampus of abused suicide completers (SA; black), nonabused suicides (SNA; gray), and control subjects (CTRL; white). Percentage of methylation at individual CpG dinucleotides in hGR first exon promoter (A) 1B, (B) 1C, and (C) 1H. Values are given as mean percent of methylation for each individual CpG dinucleotides ± SEM. Total percentage of methylation for all CpG dinucleotides in hGR first exon promoter (D) 1B, (E), 1C, and (F) 1H. Values are given as mean total percent of methylation for all CpG dinucleotides found in regions that undergone pyrosequencing ± SEM. Values are given as mean percent of methylation ± SEM. *p < .05 in SA compared with CTRL and p < .05 in SNA compared with CTRL; #p < .05 in SA compared with SNA. hGR1B and 1C: SA: 22, SNA: 21, CTRL: 13; hGR1H: 15 SA, 13 SNA, and 7 CTRL.
      Figure thumbnail gr4
      Figure 4Correlations between hippocampal promoter methylation and expression levels. Methylation in human glucocorticoid receptor (hGR)1B promoter (CpG site 8) is negatively correlated with (A) hGR total expression levels and (B) hGR1B. Methylation in hGR1C promoter is negatively correlated with (C) hGR total (CpG site 8) and (D) hGR1C (CpG site 9) expression levels. Methylation in hGR1H promoter is positively correlated with (E) hGR total (mean) and (F) hGR1H (CpG site 1 and 12) expression levels. Values are given as smoothed correlation lines with spearman coefficient of correlation. p < .05.

      hGR1C

      Methylation levels of the hGR1C promoter were assessed in a region of 186 bp composed of 18 CpGs covering part of 1C exon (Figure 1B). Methylation levels of the hGR1C promoter were generally low, ranging from .6% to 5.5% (SA = 4.2%, SNA = 3.0%, CTRL = 3.3%). There was no main effect of group on percentage of methylation (Figure 3E), although there was a significant main effect of CpG site [F(17,85) = 10.0; p < .0001] and a trend toward a significant group by CpG site interaction effect [F(34,91) = 1.5; p < .1; Figure 3B]. Post hoc tests revealed significant differences in methylation between groups at CpG8, CpG9, CpG12, and CpG13 (Figure 3B), with significant hypermethylation in samples from SA compared with SNA (p < .01) and CTRL (p < .05) for CpG8 and significant hypermethylation in SA and SNA samples compared with CTRL for CpG9 (SA: p < .05; SNA: p < .005) and CpG12 (SA: p < .05; SNA: p < .005) (Figure 3B). In contrast, CpG13 was significantly hypomethylated in samples from SA compared with SNA (p < .05) and CTRL (p < .005; Figure 3B). Methylation at CpG9 was negatively correlated with hGR1C expression levels (r = −.33; p < .05), whereas methylation at CpG8 was negatively correlated with hGR total relative expression (r = −.34, p < .05; Figure 4C and 4D). Analyses of covariance showed that age, pH, PMI, and psychiatric medication prescription status had no effect on the group differences observed in hGR1C promoter.

      hGR1H

      hGR1H promoter methylation was assessed in a region of 77 bp, located 388 bp upstream of the 1H exon and covering 13 CpGs (Figure 1C). Methylation levels in the 1H promoter ranged from 1.8% to 7.1% (SA = 3.6%, SNA = 4.5%, CTRL = 4.5%). A significant main effect of group was found for the mean level of methylation [F(2,94) = 12.45, p < .005; Figure 3F]. Post hoc tests revealed a significant hypomethylation in SA samples compared with those from SNA (p < .01) and CTRL (p < .05). We also found a significant main effect of CpG site [F(12,94) = 37.27, p < .0001] and a significant group by CpG site interaction effect [F(24,94) = 1.86, p < .05; Figure 3C]. Post hoc tests revealed a significant hypomethylation in SA samples compared with those from SNA and CTRL for CpG2 (SNA: p < .005, CTRL: p < .005), CpG5 (SNA: p < .01, CTRLs: p < .05), and CpG10 (SNA: p < .01, CTRL: p < .05; Figure 3C). Methylation in SA was also significantly lower at CpG1 (p < .05) compared with CTRL without any significant difference between SA and SNA. Finally, methylation at CpG3 (p < .005), CpG7 (p < .05), CpG8 (p < .05), and CpG12 (p < .01) was lower in SA compared with SNA, whereas no significant difference was found between SA and CTRL (Figure 3C). In addition, significant correlations between methylation and 1H expression levels were found at CpG1 (r = .44, p < .05) and CpG12 (r = .42, p < .05; Figure 4F). Methylation levels at CpG1 (r = .66, p < .001), CpG2 (r = .65, p < .001), CpG3 (r = .39, p < .05), CpG5 (r = .39, p < .05), CpG8 (r = .44, p < .05), CpG12 (r = .47, p < .05), as well as the total methylation values (r = .54, p < .005) were also positively correlated with hGR total expression (Figure 4F; Figure S3A–3F in Supplement 1). Thus, in contrast with the hGR1B and hGR1C promoters, hGR1H promoter CpG methylation was positively associated with hGR expression. Analyses of covariance revealed that age, pH, PMI, and psychiatric medication prescription status had no effect on the group differences observed in hGR1H promoter.

      Luciferase Assay

      DNA methylation has been previously shown to decrease the expression of hGR1F by interfering with the binding of NGFI-A in the promoter (
      • McGowan P.O.
      • Sasaki A.
      • D'Alessio A.C.
      • Dymov S.
      • Labonte B.
      • Szyf M.
      • et al.
      Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse.
      ), and recent evidence suggest that DNA promoter methylation may regulate the transcriptional activity of various noncoding exon promoters (
      • Cao-Lei L.
      • Leija S.C.
      • Kumsta R.
      • Wust S.
      • Meyer J.
      • Turner J.D.
      • et al.
      Transcriptional control of the human glucocorticoid receptor: Identification and analysis of alternative promoter regions.
      ). To investigate the functional consequences of our findings in hGR promoter and to link our methylation and expression findings, a luciferase assay was performed. We focused on hGR1C promoter (see Results in Supplement 1 for more detailed information). Our data show that the promoter of hGR1C has high intrinsic transcriptional activity compared with empty vector (t = 29.3, p = 3.0E-13; Figure S2 in Supplement 1). Interestingly, promoter activity is completely abolished by methylation (t = 30.3; p = 1.9E-13; Figure S2 in Supplement 1). This suggests that the methylation differences observed in the promoter of hGR1C may be involved in the mechanisms leading to the decreased expression of hGR1C in the HPC of abused suicide completers.

      Discussion

      We investigated expression levels and CpG methylation state of the hGR noncoding exon 1B, 1C, and 1H variants in hippocampal and ACC samples from control subjects or individuals who committed suicide with or without ELA. Our focus was the hGR1B, 1C, and 1H variants that are highly expressed in the hippocampus (
      • Alt S.R.
      • Turner J.D.
      • Klok M.D.
      • Meijer O.C.
      • Lakke E.A.
      • Derijk R.H.
      • et al.
      Differential expression of glucocorticoid receptor transcripts in major depressive disorder is not epigenetically programmed.
      ). Moreover, because hGR1C and 1B are associated with the expression of specific GR protein isoforms (GRα and P, respectively) (
      • Russcher H.
      • Dalm V.A.
      • de Jong F.H.
      • Brinkmann A.O.
      • Hofland L.J.
      • Lamberts S.W.
      • et al.
      Associations between promoter usage and alternative splicing of the glucocorticoid receptor gene.
      ), we sought a comprehensive view of the effects of ELA on the noncoding exons that potentially influence GR expression and function and thus regulation of the HPA axis.
      The results show that the relative expression of total hGR gene and the noncoding exons 1B, 1C, and 1H is decreased in the hippocampus of abused suicide completers compared with both nonabused suicide and control subjects independently of age, brain pH, postmortem interval, RNA integrity values, and psychiatric medication prescription status. These findings suggest an effect of ELA on the expression of these fragments. In contrast to our findings with hippocampal samples, there were no significant differences in the ACC, suggesting that alterations in hGR expression are region-specific.
      GR is involved in the negative feedback regulation of HPA activity in response to elevated glucocorticoid levels. This inhibitory feedback is triggered, in part, by the binding of glucocorticoids to their receptors in the hippocampus and appears to be affected by environmental conditions. For instance, rats raised by low licking and grooming mothers show increased behavioral and hormonal responses to stress (
      • Francis D.
      • Diorio J.
      • Liu D.
      • Meaney M.J.
      Nongenomic transmission across generations of maternal behavior and stress responses in the rat.
      ) that associate with reduced hippocampal GR expression and decreased HPA axis feedback efficacy (
      • Liu D.
      • Diorio J.
      • Tannenbaum B.
      • Caldji C.
      • Francis D.
      • Freedman A.
      • et al.
      Maternal care, hippocampal glucocorticoid receptors, and hypothalamic-pituitary-adrenal responses to stress.
      ). Moreover, conditional GR knockout mice show increased despair-like behavior and anhedonia (
      • Boyle M.P.
      • Brewer J.A.
      • Funatsu M.
      • Wozniak D.F.
      • Tsien J.Z.
      • Izumi Y.
      • et al.
      Acquired deficit of forebrain glucocorticoid receptor produces depression-like changes in adrenal axis regulation and behavior.
      ), as well as an exaggerated anxiety-related hyperlocomotion in response to stressful conditions (
      • Boyle M.P.
      • Kolber B.J.
      • Vogt S.K.
      • Wozniak D.F.
      • Muglia L.J.
      Forebrain glucocorticoid receptors modulate anxiety-associated locomotor activation and adrenal responsiveness.
      ). In humans, depressed subjects with a history of childhood abuse exhibit higher ACTH and cortisol levels following the Trier Social Stress Test and dexamethasone suppression test (
      • Heim C.
      • Mletzko T.
      • Purselle D.
      • Musselman D.L.
      • Nemeroff C.B.
      The dexamethasone/corticotropin-releasing factor test in men with major depression: Role of childhood trauma.
      ,
      • Heim C.
      • Newport D.J.
      • Heit S.
      • Graham Y.P.
      • Wilcox M.
      • Bonsall R.
      • et al.
      Pituitary-adrenal and autonomic responses to stress in women after sexual and physical abuse in childhood.
      ). In addition, hGR1F expression is decreased in the hippocampus of suicide completers with a history of childhood abuse (
      • McGowan P.O.
      • Sasaki A.
      • D'Alessio A.C.
      • Dymov S.
      • Labonte B.
      • Szyf M.
      • et al.
      Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse.
      ). These findings, together with the data presented here, suggest that difference in hGR expression among abused suicide victims can be attributed to the cumulative decrease of expression of noncoding first exons including hGR1B, 1C, and 1H.
      Modifications of GR gene transcription associate closely with variation in GR protein levels. Three major GR isoforms have been characterized. GRα is the functionally active isoform, and GRα appears to function as a dominant-negative inhibitor of GRα action (
      • Hollenberg S.M.
      • Weinberger C.
      • Ong E.S.
      • Cerelli G.
      • Oro A.
      • Lebo R.
      • et al.
      Primary structure and expression of a functional human glucocorticoid receptor cDNA.
      ,
      • Bamberger C.M.
      • Bamberger A.M.
      • de Castro M.
      • Chrousos G.P.
      Glucocorticoid receptor beta, a potential endogenous inhibitor of glucocorticoid action in humans.
      ,
      • Oakley R.H.
      • Sar M.
      • Cidlowski J.A.
      The human glucocorticoid receptor beta isoform Expression, biochemical properties, and putative function.
      ). The third isoform, GR-P, appears to enhance GRα-mediated gene transcription (
      • Krett N.L.
      • Pillay S.
      • Moalli P.A.
      • Greipp P.R.
      • Rosen S.T.
      A variant glucocorticoid receptor messenger RNA is expressed in multiple myeloma patients.
      ,
      • de Lange P.
      • Segeren C.M.
      • Koper J.W.
      • Wiemer E.
      • Sonneveld P.
      • Brinkmann A.O.
      • et al.
      Expression in hematological malignancies of a glucocorticoid receptor splice variant that augments glucocorticoid receptor-mediated effects in transfected cells.
      ). The expression of these isoforms may be linked to the use of particular noncoding exons as GRα and P expression are positively correlated with hGR1C and hGR1B expression, respectively (
      • Russcher H.
      • Dalm V.A.
      • de Jong F.H.
      • Brinkmann A.O.
      • Hofland L.J.
      • Lamberts S.W.
      • et al.
      Associations between promoter usage and alternative splicing of the glucocorticoid receptor gene.
      ). These findings suggest that lower hGR1C and 1B levels, both observed in hippocampal samples from ELA victims, would result in decreased activity of the GRα isoform by decreasing both its expression and potentiation.
      Decreased expression levels of hGR exon 1B, 1C, and 1H transcripts were associated with alterations in site-specific methylation of the respective exon 1 promoters and, in the case of 1H, also with overall promoter methylation. Most differentially methylated sites lie within putative transcription factors binding sites, suggesting that ELA associates with alterations in methylation patterns in particular hGR noncoding exon promoters, with subsequent effects on hippocampal GR expression.
      DNA methylation is a binary signal; an allele can be either methylated or unmethylated at a specific site in a given cell. The percentage of methylation measured in DNA methylation studies represents the fraction of cells in the hippocampus in which the allele is methylated. An increase in methylation levels indicates an increase in number of cells that bear a methylated allele. As expected for functional promoters, methylation levels reported here are low, suggesting that the hGR promoters are hypomethylated in the majority of neurons in the hippocampus. This is in accordance with previous quantifications of methylation levels in promoters of GR noncoding exons (
      • Oberlander T.F.
      • Weinberg J.
      • Papsdorf M.
      • Grunau R.
      • Misri S.
      • Devlin A.M.
      Prenatal exposure to maternal depression, neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responses.
      ,
      • McGowan P.O.
      • Sasaki A.
      • D'Alessio A.C.
      • Dymov S.
      • Labonte B.
      • Szyf M.
      • et al.
      Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse.
      ,
      • Alt S.R.
      • Turner J.D.
      • Klok M.D.
      • Meijer O.C.
      • Lakke E.A.
      • Derijk R.H.
      • et al.
      Differential expression of glucocorticoid receptor transcripts in major depressive disorder is not epigenetically programmed.
      ,
      • Turner J.D.
      • Pelascini L.P.
      • Macedo J.A.
      • Muller C.P.
      Highly individual methylation patterns of alternative glucocorticoid receptor promoters suggest individualized epigenetic regulatory mechanisms.
      ). In contrast, the results suggest the existence of site-specific methylation alterations in hGR1B and 1C promoters in a fraction of cells in the hippocampus and that the fraction of cells that bear methylated alleles is increased in victims of child abuse and/or who died by suicide. We suggest that this will result in inhibited expression of GR in the same cells reducing the overall output of GR expression in the hippocampus as demonstrated by the inverse correlation between percentages of methylation and expression levels of hGR1B and 1C presented here. This is supported by our results from luciferase assay showing complete abolition of transcriptional activity when hGR1C promoter is methylated. Similar findings have recently been reported in the promoter of hGR1F (
      • McGowan P.O.
      • Sasaki A.
      • D'Alessio A.C.
      • Dymov S.
      • Labonte B.
      • Szyf M.
      • et al.
      Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse.
      ) and in various promoters of hGR noncoding exons (
      • Cao-Lei L.
      • Leija S.C.
      • Kumsta R.
      • Wust S.
      • Meyer J.
      • Turner J.D.
      • et al.
      Transcriptional control of the human glucocorticoid receptor: Identification and analysis of alternative promoter regions.
      ). Altogether, this suggests that methylation alterations found in the promoter of hGR noncoding exons, and especially in hGR1C, may be involved in the mechanisms regulating hGR noncoding exon expression.
      In contrast, our results show that hGR1H promoter is significantly hypomethylated in the HPC of abused suicide completers. Excess hypomethylation was observed among abused suicide completers in overall hGR1H promoter sequence, as well as at specific sites. These findings correlated positively with expression of both hGR1H and hGR total expression and are consistent with recent reports of decreased CpG methylation within specific gene regulatory regions associated with environmental adversity. Chronic social stress in rodents decreases methylation at a specific site within CRF promoter (
      • Elliott E.
      • Ezra-Nevo G.
      • Regev L.
      • Neufeld-Cohen A.
      • Chen A.
      Resilience to social stress coincides with functional DNA methylation of the Crf gene in adult mice.
      ). ELA also reduces methylation levels in the intergenic region between AVP and OXT in mice (
      • Murgatroyd C.
      • Patchev A.V.
      • Wu Y.
      • Micale V.
      • Bockmuhl Y.
      • Fischer D.
      • et al.
      Dynamic DNA methylation programs persistent adverse effects of early-life stress.
      ), which associates with increased hypothalamic AVP expression and increased HPA activity. Taken together with our results showing hypomethylation in hGR1H promoter, these findings suggest that an active mechanism triggering hypomethylation may be significantly involved in mediating the effects of environment adversity across multiple sites. Collectively, these effects increase the expression of ACTH secretagogues, CRF an AVP, and decrease hippocampal GR expression, all of which would contribute to increased HPA activity.
      Group differences in methylation found in our study were not associated with covariables such as age, pH, PMI, RNA integrity values, and psychiatric medication prescription. However, medication has already been shown to alter DNA methylation levels in the brain (
      • Elliott E.
      • Ezra-Nevo G.
      • Regev L.
      • Neufeld-Cohen A.
      • Chen A.
      Resilience to social stress coincides with functional DNA methylation of the Crf gene in adult mice.
      ,
      • Uchida S.
      • Hara K.
      • Kobayashi A.
      • Otsuki K.
      • Yamagata H.
      • Hobara T.
      • et al.
      Epigenetic status of Gdnf in the ventral striatum determines susceptibility and adaptation to daily stressful events.
      ). More studies will be required to investigate the role of medication on DNA methylation in human brain.
      As expected, not all sites differentially methylated correlate with expression. Epigenetic regulation represents one mechanism among others acting in the regulation of GR expression. For instance, microRNAs, which have been predicted to bind in the 3′ UTR of GR (
      • Turner J.D.
      • Alt S.R.
      • Cao L.
      • Vernocchi S.
      • Trifonova S.
      • Battello N.
      • et al.
      Transcriptional control of the glucocorticoid receptor: CpG islands, epigenetics and more.
      ), may have a potential role in decreased expression found in this study. Histone modifications represent another form of epigenetic regulation of gene expression. For instance, low H3K9 acetylation levels have been reported in GR17 promoter in rats raised by low licking and grooming mother (
      • Weaver I.C.
      • Cervoni N.
      • Champagne F.A.
      • D'Alessio A.C.
      • Sharma S.
      • Seckl J.R.
      • et al.
      Epigenetic programming by maternal behavior.
      ). Moreover, evidence suggests that DNA methyl binding proteins recruit and/or carry histone modifying enzymes such as histone deacetylases and histone methyltransferases, inducing changes in chromatin conformation (
      • Deplus R.
      • Brenner C.
      • Burgers W.A.
      • Putmans P.
      • Kouzarides T.
      • de Launoit Y.
      • et al.
      Dnmt3L is a transcriptional repressor that recruits histone deacetylase.
      ,
      • Fuks F.
      • Burgers W.A.
      • Brehm A.
      • Hughes-Davies L.
      • Kouzarides T.
      DNA methyltransferase Dnmt1 associates with histone deacetylase activity.
      ,
      • Fuks F.
      • Burgers W.A.
      • Godin N.
      • Kasai M.
      • Kouzarides T.
      Dnmt3a binds deacetylases and is recruited by a sequence-specific repressor to silence transcription.
      ,
      • Fuks F.
      • Hurd P.J.
      • Wolf D.
      • Nan X.
      • Bird A.P.
      • Kouzarides T.
      The methyl-CpG-binding protein MeCP2 links DNA methylation to histone methylation.
      ,
      • Nan X.
      • Campoy F.J.
      • Bird A.
      MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin.
      ,
      • Nan X.
      • Ng H.H.
      • Johnson C.A.
      • Laherty C.D.
      • Turner B.M.
      • Eisenman R.N.
      • et al.
      Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex.
      ). Importantly, DNA methylation patterns are likely to be cell-type-specific. Recent studies suggest that neurons exhibit different DNA methylation patterns and higher variation than nonneuronal cell populations (
      • Iwamoto K.
      • Bundo M.
      • Ueda J.
      • Oldham M.C.
      • Ukai W.
      • Hashimoto E.
      • et al.
      Neurons show distinctive DNA methylation profile and higher interindividual variations compared with non-neurons.
      ). The data presented in this study are representative of the whole cellular population; therefore, future studies should assess GR promoter methylation patterns in specific HPC cell type fractions. Finally, the method used in this study to assess methylation allowed us to quantify low methylation levels. This was possible because of the quality and sensitivity of the assays developed. However, sensitivity of pyrosequencing is sequence-dependent, and one should keep this in mind in the design of future studies as similar levels of sensitivity may not always be reached.
      As expected for promoter regions, methylation levels reported in this study are low, suggesting that these regions are poised for transcriptional regulation. Group differences reported are also low, which may reflect the cellular and functional heterogeneity of the tissue investigated. Indeed, DNA methylation is tissue- and cell-type-dependent (
      • Iwamoto K.
      • Bundo M.
      • Ueda J.
      • Oldham M.C.
      • Ukai W.
      • Hashimoto E.
      • et al.
      Neurons show distinctive DNA methylation profile and higher interindividual variations compared with non-neurons.
      ). The HPC also receives various inputs from different brain regions, which may lead to the establishment of particular neuronal networks involved more specifically in different functions. Overall, it is most likely that these various cellular populations may have distinct epigenetic profiles including their own DNA methylation signature. Although distinction between cell type (neuron [glutamatergic, GABAergic], astrocytes, microglia, oligodendrocytes) was not possible in this study, one may expect that the small differences in methylation levels observed between groups may be indicative of this heterogeneity. Thus, specific cellular subpopulations may be more importantly affected than others, but when taken in the context of a heterogeneous tissue containing millions of cells, these differences may be diluted by the amount of different cells found in the HPC.
      The absence of a group of nonsuicide subjects with a history of childhood abuse presents a limitation in this study. Such a limitation is a consequence of the low incidence of childhood abuse in healthy nonpsychiatric populations and the paucity of such samples. Nevertheless, despite this limitation in our study design, our results are consistent with the hypothesis that ELA alters promoter methylation and expression of hGR variants in the context of suicide. Another potential limitation in this study is the possibility of false-positive results arising from multiple testing. Future studies and independent replications are needed to provide validity to our findings.
      In summary, our results suggest that hGR1B, hGR1C, and hGR1H expression may be significantly decreased in the hippocampus of abused suicide completers. Our data also suggest that alterations in site-specific methylation may be induced by ELA in hGR1C and 1H promoter and may be associated with differences in expression found in the hippocampus of abused suicides. Taken together, our results support the hypothesis that childhood abuse induces long-lasting effects on the HPA axis in suicide completers by affecting the hippocampal expression of hGR variants via modifications in promoter DNA methylation patterns.
      This study was supported by the Canadian Institutes of Health Research (CIHR) (Grant No. MOP84291 ), a National Alliance for Research on Schizophrenia and Depression Independent Investigator Award to GT, and support to the Brain Bank from the Réseau Québécois de Recherche sur le Suicide (RQRS). GT is a chercheur boursier from the FRQS (Fonds de Recherche du Québec - Santé). BL is supported by a CIHR Frederick Banting and Charles Best doctoral fellowship.
      The authors report no biomedical financial interests or potential conflicts of interest.

      Supplementary data

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