Differential DNA methylation regions in cytokine and transcription factor genomic loci associate with childhood physical aggression.

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.Expanded views from the UCSC genome browser of IL-6 (A) and IL-1α (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 (A) and IL-1α (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 (A) and IL-1α (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) 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 Table S1 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 [106].

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.Expanded views from the UCSC genome browser of IL-4 (A) and IL-10 (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 (A) and IL-10 (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 (A) and IL-10 (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 (A) and IL-10 (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 (A) and significantly more methylated in the IL-10 locus (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 (A, bottom panel). In the region more methylated in the CPA group of the IL-10 locus (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 Table S1 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 [106].

DNA methylation differences between CPA (n = 8) and control (n = 12) groups in pro-inflammatory chemokine IL-8 locus in monocytes.Expanded views from the UCSC genome browser of IL-8 (A) locus located on chromosomes 4 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 are shown for monocytes. In black are probes that are more methylated and in gray are those that are less methylated in the CPA group. Highlighted in red 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: −0.5 to 0.5) calculated between MeDIP microarray probe intensities (DNA methylation levels) and the plasma IL-8 levels obtained from the same subject (n = 20). In red are probes whose methylation levels correlate positively with the cytokine levels 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 monocytes estimated from the microarray data. The bottom panel shows the closest region upstream of the TSS where the CPA group is found significantly less methylated than the control group in the IL-8 locus. In both regions, an overall negative correlation between methylation and cytokine level in plasma is observed but did not reach significance after correcting 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 Table S1 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 [106].

DNA methylation differences between CPA (n = 8) and control (n = 12) groups in cytokine’s transcription factor STAT6 in T cells.Expanded views from the UCSC genome browser of STAT6 (A) 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 Table S1 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 [106].

Pyrosequencing analyses of differentially methylated sequences in T cells and monocytes between CPA (n = 8) and control (n = 12) groups.DNA methylation differences (%) between the aggressive groups in the IL-6 (A), IL-8 (B) and STAT6 (C) genes as determined by pyrosequencing. CpG sites near the significant probes were analyzed in triplicate. For each gene, the mean methylation per group per CpG with the standard error of the mean (SEM) are shown in the bar graph (top panel). The rightmost bar indicates the mean methylation levels of all CpG sites analyzed in the region. A map of the sites relative to the transcription start site is shown above the bar graph. Each line with a circle represents a CpG site. The location of probes which fold difference is significantly different between the groups is identified by a grey square where dark and light grey indicated more or less methylated in the CPA group respectively. Red arrows delimit the region analyzed by pyrosequencing. Correlations between mean methylation levels obtained by pyrosequencing for each gene and expression levels in plasma obtained by ELISA for each cytokine [32] is shown in the bottom panel. Correlation statistics were performed using Pearson correlation. All error bars represent standard error of the mean (SEM). The symbol ‘#’ denotes a p-value ≤0.1 and the symbol ‘*’ denotes a p-value ≤0.05 as determined by a Student t-test.