Adaptation of the CHARM DNA methylation platform for the rat genome reveals novel brain region-specific differences

Abstract

Comprehensive High-throughput Arrays for Relative Methylation (CHARM) was recently developed as an experimental platform and analytic approach to assess DNA methylation (DNAm) at a genome-wide level. Its initial implementation was for human and mouse. We adapted it for rat and sought to examine DNAm differences across tissues and brain regions in this model organism. We extracted DNA from liver, spleen, and three brain regions: cortex, hippocampus, and hypothalamus from adult Sprague Dawley rats. DNA was digested with McrBC, and the resulting methyl-depleted fraction was hybridized to the rat CHARM array along with a mock-treated fraction. Differentially methylated regions (DMRs) between tissue types were detected using normalized methylation log-ratios. In validating 24 of the most significant DMRs by bisulfite pyrosequencing, we detected large mean differences in DNAm, ranging from 33-59%, among the most significant DMRs in the across-tissue comparisons. The comparable figures for the hippocampus vs. hypothalamus DMRs were 14-40%, for the cortex vs. hippocampus DMRs, 12-29%, and for the cortex vs. hypothalamus DMRs, 5-35%, with a correlation of r² = 0.92 between the methylation differences in 24 DMRs predicted by CHARM and those validated by bisulfite pyrosequencing. Our adaptation of the CHARM array for the rat genome yielded highly robust results that demonstrate the value of this method in detecting substantial DNAm differences between tissues and across different brain regions. This platform should prove valuable in future studies aimed at examining DNAm differences in particular brain regions of rats exposed to environmental stimuli with potential epigenetic consequences.

Acknowledgements

The investigators report no competing interests. This work was supported by grants from the NIDDK to Dr. Moran (R01DK077623), and the NICHD to Dr. Tamashiro (R00HD055030), by T32MH015330 supporting Dr. Lee and by The Margaret Ann Price Investigatorship for Bipolar Research supporting Dr. Potash.

Figures and Tables

Figure 1 Clustering analysis based on 5,000 most variably methylated probes. The brain, liver and spleen are clearly distinguished from each, falling into separate branches. Within the branch for brain, the cortex, hippocampus and hypothalamus all cluster separately.

Figure 2 Methylation plots generated by CHARM for 4 brDMRs: (A) Nr4a2, (B) Ntrk2, (C) Tcf4 and (D) Tcfap2c. Chromosomal locations are shown at the top of each part. The upper portion for each panel displays the approximate methylation percentage for all five tissues, represented by the five plot curves with different colors. Although CHARM generates and displays plot curves for all of the five tissues queried, the DMR is chosen and ranked based on the significant differences in DNAm between two selected tissues, represented by black arrows in each part. Displayed below the plot curves are the CpG density, and the genomic organization of the genes associated with the brDMRs. Black boxes represent exons, whereas white boxes represent intronic regions. For instance, brDMRs for Ntrk2 and Tcf4 fall within an intronic region of both genes.

Figure 3 Boxplot of pyrosequencing data obtained from liver (LIV), spleen (SPL) and cerebral cortex (CTX) tDMRs. We assessed and validated percent DNA methylation of three unique tDMRs from each pair of tissues compared by CHARM. For each tDMR, the number of CpGs assayed varied from 4 to 8 CpGs, thus a small subset of the region assayed by the CHARM array. Average percent methylation was determined from all of the assayed CpGs for each tDMR in each tissue type and displayed as a boxplot. Stippled horizontal lines represent the percent DNAm predicted by CHARM for the particular tissue for the entire tDMR region. Types of tissue being compared and the nearest genes associated with the tDMRs are indicated below the X-axis.

Figure 4 Boxplot of pyrosequencing data obtained from the cerebral cortex (CTX), hippocampus (HIP) and hypothalamus (HYP) brDMRs. We assessed and validated percent DNA methylation of five unique DMRs from each pair of brain tissues compared by CHARM. For each brDMR, the number of CpGs assayed varied from 4 to 16 CpGs, thus a small subset of the region assayed by the CHARM array. Average percent methylation was determined from all of the assayed CpGs for each brDMR in each brain tissue type and displayed as a boxplot. Stippled horizontal lines represent the percent DNAm predicted by CHARM for the particular brain tissue for the entire brDMR region. Types of brain tissues being compared and the nearest genes associated with the brDMRs are indicated below the X-axis.

Figure 5 Histogram of the distance of tDMRs and brDMRs to the nearest CpG island. (A) tDMRs annotated by CHARM in liver, spleen and cortex comparisons were tallied and their distance to the nearest CpG island calculated. Distances of DMRs to CpG islands were separated by different distance categories or bins and graphed as a percentage of the total DMRs analyzed. Red bar represents the percentage of DMRs that fall on a CpG island. Distances greater than 50 bp and less than or equal to 3,000 bp from a CpG island are collectively considered as “CpG shores.” (B) Similar analysis was performed for brDMRs annotated by CHARM in cortex, hippocampus and hypothalamus.

Figure 6 Histogram of genomic locations of tDMRs and brDMRs in relation to associated genes. (A) tDMRs annotated by CHARM in the liver, spleen and cortex comparisons were categorized based on their locations to genes, tallied and graphed as a percentage of the total DMRs analyzed. Promoter and “Close to 3′” categories represent DMRs that fall within 2,500 bp of the first and last exons, respectively. (B) Similar analysis was performed for brDMRs annotated by CHARM in the cortex, hippocampus and the hypothalamus.

Table 1 Differentially methylated regions in the liver vs. spleen comparison

Table 2 Differentially methylated regions in the liver vs. cerebral cortex comparison

Table 3 Differentially methylated regions in the spleen vs. cerebral cortex comparison

Table 4 Differentially methylated regions in the hippocampus vs. hypothalamus comparison

Table 5 Differentially methylated regions in the hippocampus vs. cerebral cortex comparison

Table 6 Differentially methylated regions in the cerebral cortex vs. hypothalamus comparison