Hydroxymethylation and DNA methylation profiles in the prefrontal cortex of the non-human primate rhesus macaque and the impact of maternal deprivation on hydroxymethylation

Abstract

5-Hydroxymethylcytosine (5hmC) is abundant in the brain, suggesting an important role in epigenetic control of neuronal functions. In this paper, we show that 5hmC and 5-methylcytosine (5mC) levels are coordinately distributed in gene promoters of the rhesus macaque prefrontal cortex. Although promoter hydroxymethylation and methylation are overall negatively correlated with expression, a subset of highly expressed genes involved in specific cerebral functions is associated with high levels of 5mC and 5hmC. These relationships were also observed in the mouse cortex. Furthermore, we found that early-life maternal deprivation is associated, in the adult monkey cortex, with DNA hydroxymethylation changes of promoters of genes related to neurological functions and psychological disorders. These results reveal that early social adversity triggers variations in brain DNA hydroxymethylation that could be detected in adulthood.

Keywords: brain; early-adversity; hydroxymethylation; maternal deprivation; monkeys; rearing.

Fig. 1.

Correlation between DNA hydroxymethylation, DNA methylation and gene expression in the prefrontal cortex of rhesus macaques. (A) Overall promoter 5hmC (n = 6) and 5mC (n = 8) levels estimated from precipitation microarray data across the entire genome. 5hmC and 5mC are positively correlated. (R = 0.6, p < 2E-16) (Chromosomes 17–10 were not represented in the 5hmC arrays). (B) Correlation between hydroxymethylation and methylation. (C, D) Genomic distribution of 5mC and 5hmC across CpG islands. The levels of both 5mC and 5hmC show an inverse correlation with CpG density. The plots show the interquartile range. Below the box is the first quartile, above the box is the fourth quartiles, inside the box are the second and third quartiles. The whiskers stretch 1.5 times the interquartile range from the box. (E) Repartition of gene expression levels according to CpG density. Gene expression levels are positively correlated with CpG density. (G–J) Inverse correlations between promoter hydroxymethylation (R = −0.09, p < 2E-16) or methylation (R = −0.24; p < 2E-16) and gene expression levels estimated by microarray data. For figures I and J, genes are divided into 20 levels by expression percentiles (0–5, 5–10,…, 95–100). Estimated distributions of promoter hydroxymethylation and methylation levels are shown for each expression percentile. Genes with low or no expression (represented in green) tend to have highly methylated promoters whereas genes with high expression (represented in red) tend to have lower promoter methylation. Gene exceptions (arrow) with high levels of methylation and highly expressed are indicated by an arrow. (LCP, low CpG density, ICG, intermediate CpG density, HCG, high CpG density). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2.

Integration of DNA hydroxymethylation, DNA methylation and gene expression in the prefrontal cortex of rhesus macaques. (A) The 3-D plot shows a general inverse correlation between methylation and expression. However, for highly methylated genes, a positive correlation between hydroxymethylation and expression is observed. (B) The positive correlation between hydroxymethylation and expression increases with methylation level. (C) Highly methylated genes display low and intermediate CpG frequency. Correlation between 5hmC and CpG frequency is mild for highly methylated genes (R = 0.02, p = 0.1). (D) Number of genes according to 5mC and 5hmC levels. An arrow indicates enrichment for a subset of highly hydroxymethylated and methylated genes.

Fig. 3.

DNA hydroxymethylation differences in prefrontal cortex between mother-reared and surrogate peer-reared groups. QPCR analysis of DNA hydroxymethylation differences between rearing groups (SPR, n = 4 – MR, n = 3) in four promoters predicted to be less hydroxymethylated and four predicted to be more hydroxymethylated in the SPR animals by microarray analysis. Relative bound fraction concentrations obtained in triplicate by Q-PCR are shown for the eight genes (see methods). All error bars represent standard error of the mean (SEM). The symbol ‘^#’ denotes a p-value <0.1 and symbols ‘*’ and ‘**’ denote p-values <0.05 and <0.01 respectively from Student’s t test.