Diametrically opposite methylome-transcriptome relationships in high- and low-CpG promoter genes in postmitotic neural rat tissue

Abstract

DNA methylation can control some CpG-poor genes but unbiased studies have not found a consistent genome-wide association with gene activity outside of CpG islands or shores possibly due to use of cell lines or limited bioinformatics analyses. We performed reduced representation bisulfite sequencing (RRBS) of rat dorsal root ganglia encompassing postmitotic primary sensory neurons (n = 5, r > 0.99; orthogonal validation p < 10(-19)). The rat genome suggested a dichotomy of genes previously reported in other mammals: low CpG content (< 3.2%) promoter (LCP) genes and high CpG content (≥ 3.2%) promoter (HCP) genes. A genome-wide integrated methylome-transcriptome analysis showed that LCP genes were markedly hypermethylated when repressed, and hypomethylated when active with a 40% difference in a broad region at the 5' of the transcription start site (p < 10(-87) for -6000 bp to -2000 bp, p < 10(-73) for -2000 bp to +2000 bp, no difference in gene body p = 0.42). HCP genes had minimal TSS-associated methylation regardless of transcription status, but gene body methylation appeared to be lost in repressed HCP genes. Therefore, diametrically opposite methylome-transcriptome associations characterize LCP and HCP genes in postmitotic neural tissue in vivo.

Figure 1. Promoter CpG density defining the dichotomy of LCP vs. HCP genes in the rat genome. (A) Unselected rat genes are (in aggregate) characterized by a high density (frequency) of the CpG motif around the TSS (0 bp). Shown is the CpG density of 17,602 protein-coding genes included in the main analysis. Bins with a width of 500 nucleotides are shown. CpG density peaked in a narrow region of 1000 to 2000 nucleotides around the TSS. CpG density varied, however, considerably between genes as demonstrated by box plots indicating the 5th, 25th, 50th, 75th, 95th percentile. (B) The promoter CpG content of individual genes was bimodally distributed among the total set of genes indicating two distinct classes of promoters. The CpG content of the core promoter region was determined by choosing a 1000bp interval around the TSS (from -500bp to +500bp) as a proxy. Promoter CpG content varied among individual genes from < 0.5% to > 10%. Depiction of the promoter CpG content as histogram demonstrated two peaks at 1% and 5.5% suggesting a mixed distribution resulting from two distinct underlying populations. The position of the valley suggested a cutoff at 3.2% (vertical red line). The resulting dichotomization of genes provided a classification of “LCP” and “HCP” genes resembling that originally proposed by Saxonov et al., which guided subsequent analyses. (C) CpG density in LCP genes was low not only—as expected—at the TSS but also throughout the remaining gene regions suggesting that there were no unrecognized regions of higher CpG density, CGI, farther away from the TSS. CpG density of HCP genes was high at the TSS reflecting how they were defined.

Figure 2. Diametric methylome-transcriptome relationships in LCP vs. HCP genes. (A) LCP genes (20% trimmed mean): Mean CpG methylation levels are shown for highly expressed (red) and repressed (green) LCP genes (low CpG content promoter genes containing < 3.2% CpG). Shown is the 20% trimmed mean in a 1000bp-wide moving window. CpG sites located at the TSS and within several thousand nucleotides 5′ of the TSS differed markedly between highly expressed and repressed LCP genes. Differences were highly significant in the region -6000bp to -2000bp with p≈3.7x10⁻⁸⁸ and in the region -2000bp to +2000bp with p≈1.0x10⁻⁷⁴. In the region +2000bp to +6000bp there was no significant difference with p = 0.42. (B) LCP genes (80th percentile): The 80th percentile rank of CpG methylation levels supported the same observation demonstrating hypomethylation of highly active genes and hypermethylation of silenced genes 5′ of the TSS. Methylation downstream of the TSS was high in LCP genes regardless of gene activity. (C) Gene body methylation in LCP genes: CpG methylation was similar in the gene bodies of highly expressed and silent LCP genes. Shown are boxplots for exons (E) and introns (I) indicating the 10th, 25th, 50th, 75th, and 90th percentile rank of methylation levels for each gene group. (D) HCP genes (20% trimmed mean): Mean CpG methylation levels of highly expressed and repressed HCP genes showed the characteristic deep valley of hypomethylation around the TSS, which is the region of high CpG motif density defining the HCP gene group. Methylation of silenced genes appeared to be only minimally higher at the TSS. (E) HCP genes (80th percentile): The 80th percentile rank of CpG methylation levels further supported the observation that the TSS of HCP genes remained poorly methylated regardless of the level of gene activity. Highly expressed HCP genes were marked by methylation outside of the TSS while the silenced HCP genes appeared to be relatively hypomethylated throughout the whole gene. (F) Gene body methylation in HCP genes: CpG methylation differed between the gene bodies of highly expressed and silent HCP genes. Differences were highly statistically significant with p≈2.6x10⁻¹⁵⁶ for exons and p≈1.2x10⁻⁷⁵ for introns.