Conserved epigenetic sensitivity to early life experience in the rat and human hippocampus

Abstract

Early life experience is associated with long-term effects on behavior and epigenetic programming of the NR3C1 (GLUCOCORTICOID RECEPTOR) gene in the hippocampus of both rats and humans. However, it is unlikely that such effects completely capture the evolutionarily conserved epigenetic mechanisms of early adaptation to environment. Here we present DNA methylation profiles spanning 6.5 million base pairs centered at the NR3C1 gene in the hippocampus of humans who experienced abuse as children and nonabused controls. We compare these profiles to corresponding DNA methylation profiles in rats that received differential levels of maternal care. The profiles of both species reveal hundreds of DNA methylation differences associated with early life experience distributed across the entire region in nonrandom patterns. For instance, methylation differences tend to cluster by genomic location, forming clusters covering as many as 1 million bases. Even more surprisingly, these differences seem to specifically target regulatory regions such as gene promoters, particularly those of the protocadherin α, β, and γ gene families. Beyond these high-level similarities, more detailed analyses reveal methylation differences likely stemming from the significant biological and environmental differences between species. These results provide support for an analogous cross-species epigenetic regulatory response at the level of the genomic region to early life experience.

Fig. 1.

Associations of human DNA methylation with early life abuse in the 6.5-Mb NR3C1 locus. Track images obtained from the University of California, Santa Cruz genome browser (human genome assembly hg18) show % 5meC: average methylation levels across all samples estimated from microarray probe intensities; Δ 5meC: mean log₂ fold differences between abused and control sample probe intensities, where positive values are shown in black and indicate higher methylation in abused samples, and gray values indicate higher methylation in control samples; cDMR: locations of cDMRs (significantly higher methylation in control samples); and aDMR: locations of aDMRs (significantly higher methylation in abuse samples). The locations of the protocadherin families of genes and NR3C1 are identified by shading.

Fig. 2.

Associations of human and rat DNA methylation with early life abuse in the 6.5-Mb NR3C1 locus. Top, Middle, and Bottom: Each panel shows the 6.5-Mb NR3C1 locus. Top: Human locus. Bottom: Rat locus. Middle: Human–rat “hybrid” panel created by assigning orthologous genes to positions similar to their relative locations in the human and rat genomes. Each panel is divided into five labeled parts: genes: black horizontal arrows denote genes and the direction of mRNA synthesis; variation: graph indicates regions of high and low methylation variation across all human subjects; % homology: graph shows the percentage of bases in the human genome that were mapped by the lastz alignment tool to the rat genome; 5meC diff: graph shows mean log₂ fold differences between sample groups (i.e., between abused and control humans and between high- and low-LG rats); and % 5meC: graph shows methylation levels estimated from microarray probe intensities. Across each panel, gray vertical lines demark transcription start sites. Black lines between panels link the positions of transcription start sites of orthologous genes. The lack of crossings between these lines illustrates conservation of gene architecture around NR3C1 between rats and humans.

Fig. 3.

Validation of microarray calls. Real-time PCR validation of microarray meDIP data is shown. Eleven of the 28 promoters identified as being differentially methylated by microarray (Table S1) were subjected to real-time PCR quantification of enrichment. The y axis represents concentration values generated by methylation-enriched and input DNA. Left: Concentration levels in the abuse group. Right: Concentration levels in the control group. Each real-time PCR was performed in triplicate. Error bars indicate SEM.

Fig. 4.

Correlation of human DNA methylation associations with early life abuse in the 6.5-Mb NR3C1 locus. Pearson correlations of DNA methylation differences between the subject groups at various genomic distances. Error bars show 95% confidence intervals for the correlation values. The gray highlight shows the expected 95% confidence interval if there is no correlation between methylation differences at different genomic sites. This confidence interval does not overlap with the error bars associated with distances less than 1 Mb, suggesting the existence of systematic dependencies between methylation differences at distances up to 1 Mb.