Peripheral SLC6A4 DNA Methylation Is Associated with In Vivo Measures of Human Brain Serotonin Synthesis and Childhood Physical Aggression

The main challenge in addressing the role of DNA methylation in human behaviour is the fact that the brain is inaccessible to epigenetic analysis in living humans. Using positron emission tomography (PET) measures of brain serotonin (5-HT) synthesis, we found in a longitudinal sample that adult males with high childhood-limited aggression (C-LHPA) had lower in vivo 5-HT synthesis in the orbitofrontal cortex (OBFC). Here we hypothesized that 5-HT alterations associated with childhood aggression were linked to differential DNA methylation of critical genes in the 5-HT pathway and these changes were also detectable in peripheral white blood cells. Using pyrosequencing, we determined the state of DNA methylation of SLC6A4 promoter in T cells and monocytes isolated from blood of cohort members (N = 25) who underwent a PET scan, and we examined whether methylation status in the blood is associated with in vivo brain 5-HT synthesis. Higher levels of methylation were observed in both T cells and monocytes at specific CpG sites in the C-LHPA group. DNA methylation of SLC6A4 in monocytes appears to be associated more reliably with group membership than T cells. In both cell types the methylation state of these CpGs was associated with lower in vivo measures of brain 5-HT synthesis in the left and right lateral OBFC (N = 20) where lower 5-HT synthesis in C-LHPA group was observed. Furthermore, in vitro methylation of the SLC6A4 promoter in a luciferase reporter construct suppresses its transcriptional activity supporting a functional role of DNA methylation in SLC6A4 promoter regulation. These findings indicate that state of SLC6A4 promoter methylation is altered in peripheral white blood cells of individuals with physical aggression during childhood. This supports the relevance of peripheral DNA methylation for brain function and suggests that peripheral SLC6A4 DNA methylation could be a marker of central 5-HT function

The state of DNA methylation of serotonin transporter (SLC6A4) in peripheral T cells and monocytes is associated with aggression and central 5-HT function; DNA methylation as biomarkers of brain function

Aggressive behaviour is a complex phenomenon that often arises in early childhood and typically decreases with age. Studies have shown that adults with severe aggression often have lower serotonin (5-HT) neurotransmission. The hypothesis of this thesis is that 5-HT alterations associated with childhood aggression are also defined by epigenetic mechanisms through differential methylation of critical genes in the 5-HT pathway that can be detected in peripheral white blood cells. Serotonin transporter (SLC6A4) was chosen in this study based on its importance in 5-HT function and preliminary genomic DNA microarray data. We first determined the state of DNA methylation of SLC6A4 promoter using pyrosequencing in T cells and monocytes isolated from blood of adult males with low or high childhood-limited aggression (N=25) and who have been followed since childhood. We then examined whether the state of DNA methylation of SLC6A4 promoter in the blood is associated with in vivo measures of brain 5-HT synthesis by integrating previously obtained Positron emission tomography (PET) data from these participants. Lastly, a luciferase reporter construct was generated to test whether SLC6A4 promoter methylation plays a functional role in its transcriptional activity. Significantly higher levels of methylation in high childhood-limited physical aggression (C-LHPA) group were observed in both T cells and monocytes at specific CpG sites. In addition, greater mean methylation of significantly altered CpGs was associated with lower 5-HT synthesis in the left and right lateral orbitofrontal cortex (OBFC) in both cells (N = 20). Moreover, in vitro methylation of the SLC6A4 promoter dramatically suppressed gene expression suggesting that methylation plays a functional role in gene regulation. Taken all together, these novel findings imply that SLC6A4 is epigenetically modulated by DNA methylation and that DNA methylation is associated with in aggression level observed during childhood. The association between higher DNA methylation and lower brain 5-HT synthesis supports the relevance of using DNA methylation in peripheral white blood cells as a marker for human brain 5-HT function. If these results can be confirmed in a larger sample, the identification of such 5-HT biomarkers may be beneficial for the prediction, prevention and evaluation of treatment of psychiatric disorders with 5-HT involvement.Le comportement agressif est un phénomène complexe qui survient souvent lors de la petite enfance et diminue typiquement avec l'âge. Des études ont montré qu'une agressivité sévère chez l'adulte est associée à une plus faible neurotransmission de sérotonine (5-HT). L'hypothèse de cette thèse est que les modifications de neurotransmission de 5-HT associées à l'agressivité infantile sont également dépendantes de mécanismes epigénétiques, notamment du niveau de méthylation des gènes critiques à la régulation de la neurotransmission de la 5-HT qui peut être mesuré dans les leucocytes périphériques. Le transporteur de la sérotonine (SLC6A4) a été choisi dans cette étude dû à son importance dans la régulation du niveau de 5-HT ainsi qu'au vu de données provenant d'analyses préliminaires de micropuces d'ADN génomique. Nous avons premièrement déterminé les niveaux de méthylation d'ADN du promoteur de SLC6A4 en utilisant la méthode de pyroséquençage dans des cellules T et des monocytes isolés à partir de sang prélevé chez des hommes adultes ayant présenté des niveaux d'agressivité faibles ou importants durant l'enfance (N=25). Nous avons ensuite examiné si les niveaux de méthylation d'ADN du promoteur de SLC6A4 mesurés dans les cellules sanguines sont corrélés aux niveaux de synthèse cérébrale de 5-HT mesurés in vivo par tomographie par émission de positrons (TEP) chez les mêmes participants. Enfin, nous avons évalué in vitro grâce à une technique utilisant le gène rapporteur luciférase si le niveau de méthylation du promoteur de SLC6A4 est directement impliqué dans la modulation de son niveau de transcription. Nous avons mesuré des niveaux de méthylation de CpG spécifiques de SLC6A4 significativement plus importants dans les cellules T ainsi que dans les monocytes du groupe d'hommes ayant présentés une forte agressivité durant l'enfance (C-LHPA) par rapport au groupe ayant présenté une faible agressivité durant l'enfance. De plus, nous avons montré que ces niveaux de méthylation importants sont associés à une synthèse réduite de 5-HT au sein des cortex orbitofrontaux latéraux (OBFC) gauche et droit (N = 20). De plus, nous avons mesuré in vitro que la méthylation du promoteur SLC6A4 réprime fortement son expression. Ces données suggèrent que le niveau d'expression de SLC6A4 est modulé épigénétiquement par la méthylation de l'ADN et que les niveaux de méthylation du gène corrèlent avec les niveaux d'agressivité observés durant l'enfance. L'association entre les niveaux de méthylation d'ADN et de synthèse cérébral de 5-HT renforce la pertinence d'utiliser les niveaux de méthylation d'ADN de leucocytes périphériques comme indicateur du niveau de neurotransmission de 5-HT cérébrale. Ces résultats demandent à être confirmés au sein d'une cohorte plus importante. Cependant, l'identification d'un tel marqueur biologique pourrait être utile à la prédiction, la prévention et l'évaluation de traitements de désordres psychiatriques associés à des problèmes de neurotransmission de 5-HT

Family-based analysis of -675 4G/5G polymorphism in the PAI-1 gene of polycystic ovary syndrome in Chinese population

Previous studies have shown that 4G/5G polymorphism in promoter region of plasminogen activator inhibitor-1 (PAI-1) gene can affect insulin sensitivity by elevating the level and activity of plasma PAI-1. In order to elucidate the relationship between the polymorphism of PAI-1 gene and polycystic ovary syndrome (PCOS), we used transmission disequilibrium test (TDT) to study the family of PCOS. Eight hundred and fifty-five participants consisting of 285 trios (mother, father and offspring with PCOS) were recruited at the Center of Reproductive Medicine, Shandong University from July 2007 to August 2014. 4G/5G polymorphism of PAI-1 gene was genotyped using direct sequencing protocol and TDT was used to analyse the association between PAI-1 gene and PCOS. Though the 5G allele in PAI-1 gene was overtransmitted in families, no statistical significance existed and there was no association between PAI-1 gene and PCOS, indicating that PAI-1 gene was unlikely to play a major role in the aetiology of PCOS in Chinese population.Impact Statement What is already known on this subject? Some studies have shown that 4G/5G polymorphism in promoter region of plasminogen activator inhibitor-1 (PAI-1) gene can affect insulin sensitivity by elevating the level and activity of plasma PAI-1, participating in the formation of insulin resistance (IR). What do the results of this study add? Though the 5G allele in PAI-1 gene was overtransmitted in families, no statistical significance existed and there was no association between PAI-1 gene and polycystic ovary syndrome (PCOS). What are the implications of these findings for clinical practice and/or further research? PAI-1 gene was unlikely to play a major role in the aetiology of PCOS

Differential DNA Methylation Regions in Cytokine and Transcription Factor Genomic Loci Associate with Childhood Physical Aggression

<div><p>Background</p><p>Animal and human studies suggest that inflammation is associated with behavioral disorders including aggression. We have recently shown that physical aggression of boys during childhood is strongly associated with reduced plasma levels of cytokines IL-1α, IL-4, IL-6, IL-8 and IL-10, later in early adulthood. This study tests the hypothesis that there is an association between differential DNA methylation regions in cytokine genes in T cells and monocytes DNA in adult subjects and a trajectory of physical aggression from childhood to adolescence.</p><p>Methodology/Principal Findings</p><p>We compared the methylation profiles of the entire genomic loci encompassing the IL-1α, IL-6, IL-4, IL-10 and IL-8 and three of their regulatory transcription factors (TF) NFkB1, NFAT5 and STAT6 genes in adult males on a chronic physical aggression trajectory (CPA) and males with the same background who followed a normal physical aggression trajectory (control group) from childhood to adolescence. We used the method of methylated DNA immunoprecipitation with comprehensive cytokine gene loci and TF loci microarray hybridization, statistical analysis and false discovery rate correction. We found differentially methylated regions to associate with CPA in both the cytokine loci as well as in their transcription factors loci analyzed. Some of these differentially methylated regions were located in known regulatory regions whereas others, to our knowledge, were previously unknown as regulatory areas. However, using the ENCODE database, we were able to identify key regulatory elements in many of these regions that indicate that they might be involved in the regulation of cytokine expression.</p><p>Conclusions</p><p>We provide here the first evidence for an association between differential DNA methylation in cytokines and their regulators in T cells and monocytes and male physical aggression.</p></div

Characteristics of the chronic physical aggression (CPA) group and control group in the original sample (1) and the selected sample (2) for the methylation analysis.

<p>Include mother and father occupational score, familial status (monoparental vs biparental), mother and father age at birth of first child and the years of schooling of the mother and father.</p

DNA methylation differences between CPA (n = 8) and control (n = 12) groups in pro-inflammatory cytokines IL-6 and IL-1α loci in T cells.

<p>Expanded views from the UCSC genome browser of IL-6 (<b>A</b>) and IL-1α (<b>B</b>) loci located on chromosomes 7 and 2 are depicted (top panel). In both panels, the first track shows the average MeDIP probe log2-fold differences (scale top panel: −0.4 to 0.4, botom panel: −1.0 to 1.0) between chronic physical aggressive (CPA) and control groups for T cells. In black are probes that are more methylated and in gray are those that are less methylated in the CPA group. Highlighted in blue are regions significantly differentially methylated between the groups. The second track shows Pearson’s correlation coefficient values (scale top panel: −0.4 to 0.4, botom panel: −0.7 to 0.7) calculated between the MeDIP microarray probe intensities and the plasma IL-6 (<b>A</b>) and IL-1α (<b>B</b>) levels obtained from the same subjects (n = 20). In red are probes whose methylation levels correlate positively with the cytokine level in plasma and in green are those that correlated negatively. In the top panel, the lowest track shows the average methylation level for all the subjects in T cells estimated from the microarray data. The bottom panel zooms on the closest region upstream of the TSS where the CPA group is found significantly less methylated than the control group in the IL-6 (<b>A</b>) and IL-1α (<b>B</b>) loci. In both regions, an overall positive correlation between methylation and cytokine level in plasma is observed but only reached significance after correcting for multiple testing in IL-1α (<b>B</b>) differentially methylated region. The regulatory elements from ENCODE identified in these regions (see methods) are shown in the additional tracks. First, shown with black lines, is the location of individual CpG sites. Second, is the location of DNase hypersensitive clusters where black indicate strong signal and grey a weaker signal from ChIP-seq data in 24 cell lines. Third is the location of transcription factors (TF) identified from ChIP-seq data in 24 cell lines where black indicate a strong and grey weaker signal occupancy. The letter next to the TF boxes identified the cell line where it was found enriched (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071691#pone.0071691.s008" target="_blank">Table S1</a> for the full legend). The last tracks, identified the level of enrichment of three histone marks determined from ChIP-seq assay, histone 3 lysine 4 tri- and mono-methylation as well as histone 3 lysine 27 acetylation in two cell lines, GM12878 (pink) and K562 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071691#pone.0071691-Armario1" target="_blank">[106]</a>.</p

DNA methylation differences between CPA (n = 8) and control (n = 12) groups in cytokine’s transcription factor STAT6 in T cells.

<p>Expanded views from the UCSC genome browser of STAT6 (<b>A</b>) locus located on chromosomes 12 is depicted (top panel). In both panels, the first track shows the average MeDIP probe log2-fold differences (scale top panel: −0.4 to 0.4, botom panel: −1.0 to 1.0) between chronic physical aggressive (CPA) and control groups for T cells. In black are probes that are more methylated and in gray are those that are less methylated in the CPA group. Highlighted in blue are regions significantly differentially methylated between the groups. The second track shows Pearson’s correlation coefficient (scale top panel: −0.4 to 0.4, botom panel: −0.6 to 0.6) calculated between MeDIP microarray probe intensities and the plasma IL-4 (first) and IL-10 (second) levels obtained from the same subject. These correlations did not reach significance after correcting for multiple testing in T cells. In red are probes whose methylation levels correlate positively with the cytokine level in plasma and in green are those that correlated negatively. In the top panel, the last track shows the average methylation level for all the subjects in T cells estimated from the microarray data (n = 20). The bottom panel shows the two regions close to the TSS of each STAT6 isoform where the CPA group is found significantly more methylated than the control group. The regulatory elements from ENCODE identified in these regions (see methods) are shown in the additional tracks. First, shown with black lines, is the location of individual CpG sites. Second, is the location of DNase hypersensitive clusters where black indicate strong signal and grey a weaker signal from ChIP-seq data in 24 cell lines. Third is the location of transcription factors (TF) identified from ChIP-seq data in 24 cell lines where black indicate a strong presence and grey weaker signal occupancy. The letter next to the TF boxes identified the cell line where it was found enriched (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071691#pone.0071691.s008" target="_blank">Table S1</a> for the full legend). The last tracks, identified the level of enrichment of three histone marks determined from ChIP-seq assay, histone 3 lysine 4 tri- and mono-methylation as well as histone 3 lysine 27 acetylation in two cell lines, GM12878 (pink) and K562 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071691#pone.0071691-Armario1" target="_blank">[106]</a>.</p

DNA methylation differences between CPA (n = 8) and control (n = 12) groups in anti-inflammatory cytokines IL-4 and IL-10 loci in T cells.

<p>Expanded views from the UCSC genome browser of IL-4 (<b>A</b>) and IL-10 (<b>B</b>) loci located on chromosomes 5 and 1 are depicted (top panel). In both panels, the first track shows the average MeDIP probe log2-fold differences (scale top panel: −0.4 to 0.4, botom panel: −1.0 to 1.0) between chronic physical aggressive (CPA) and control groups are shown for T cells. In black are probes that are more methylated and in gray are those that are less methylated in the CPA group. Highlighted in blue are regions significantly differentially methylated between the groups in T cells for IL-4 (<b>A</b>) and IL-10 (<b>B</b>). The second track shows Pearson’s correlation coefficient (scale top panel: −0.4 to 0.4, botom panel: −0.7 to 0.7) calculated between the MeDIP microarray probe intensities and the plasma IL-4 (<b>A</b>) and IL-10 (<b>B</b>) levels obtained from the same subject (n = 20). In red are probes whose methylation levels correlate positively with the cytokine level in plasma and in green are those that correlated negatively. In the top panel, the last track shows the average methylation level for all the subjects in T cells for IL-4 (<b>A</b>) and IL-10 (<b>B</b>) estimated from the microarray data. The bottom panel shows differentially methylated regions located within known Th2 LCRs where the CPA group is found significantly less methylated than the control group in the IL-4 locus (<b>A</b>) and significantly more methylated in the IL-10 locus (<b>B</b>). For the first differentially methylated region in IL-4, an overall positive correlation between methylation and IL-4 level in plasma is observed but did not reach significance after correcting for multiple testing (<b>A</b>, bottom panel). In the region more methylated in the CPA group of the IL-10 locus (<b>B</b>, bottom panel), the overall correlation with IL-10 level in plasma is negative although not significant after corrections for multiple testing. The regulatory elements from ENCODE identified in these regions (see methods) are shown in the additional tracks. First, shown with black lines, is the location of individual CpG sites. Second, is the location of DNase hypersensitive clusters where black indicate strong signal and grey a weaker signal from ChIP-seq data in 24 cell lines. Third is the location of transcription factors (TF) identified from ChIP-seq data in 24 cell lines where black indicate a strong and grey weaker signal occupancy. The letter next to the TF boxes identified the cell line where it was found enriched (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071691#pone.0071691.s008" target="_blank">Table S1</a> for the full legend). The last tracks, identified the level of enrichment of three histone marks determined from ChIP-seq assay, histone 3 lysine 4 tri- and mono-methylation as well as histone 3 lysine 27 acetylation in two cell lines, GM12878 (pink) and K562 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071691#pone.0071691-Armario1" target="_blank">[106]</a>.</p