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

Hartung T, Zhang L, Kanwar R, Khrebtukova I, Reinhardt M, Wang C, Therneau TM, Banck MS, Schroth GP, Beutler AS - Epigenetics (2012)

Bottom Line: 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)).HCP genes had minimal TSS-associated methylation regardless of transcription status, but gene body methylation appeared to be lost in repressed HCP genes.

View Article: PubMed Central - PubMed

Affiliation: Mayo Clinic, Rochester, MN, USA.

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.

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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.,6 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.
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Figure 1: 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.,6 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.

Mentions: CpG dinucleotides are unevenly scattered across the genome. We found that CpG density in the rat genome peaked in the promoter region around the TSS consistent with findings in other genomes and with the observation that many promoters overlap with CGI (Fig. 1A). Next, we examined how the feature of promoter CpG density varied throughout the genome of the rat. Specifically, we wished to determine whether promoter CpG density was bimodally distributed in the rat like it is in the human genome.6 Taking the region from –500 bp to +500 bp around the TSS as a proxy, a bimodal distribution was noted, indicating two distinct groups (Fig. 1B). Dichotomizing the genes with a cutoff at a CpG density of 3.2% led to the classification of low CpG content (< 3.2%) promoter, “LCP,” genes and high CpG content (≥ 3.2%) promoter, “HCP,” genes (Figs. 1B and C). Of 17,602 protein-coding genes included in this study, 8644 were LCP genes and 8958 were HCPs.


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

Hartung T, Zhang L, Kanwar R, Khrebtukova I, Reinhardt M, Wang C, Therneau TM, Banck MS, Schroth GP, Beutler AS - Epigenetics (2012)

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.,6 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.
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Related In: Results  -  Collection

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Figure 1: 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.,6 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.
Mentions: CpG dinucleotides are unevenly scattered across the genome. We found that CpG density in the rat genome peaked in the promoter region around the TSS consistent with findings in other genomes and with the observation that many promoters overlap with CGI (Fig. 1A). Next, we examined how the feature of promoter CpG density varied throughout the genome of the rat. Specifically, we wished to determine whether promoter CpG density was bimodally distributed in the rat like it is in the human genome.6 Taking the region from –500 bp to +500 bp around the TSS as a proxy, a bimodal distribution was noted, indicating two distinct groups (Fig. 1B). Dichotomizing the genes with a cutoff at a CpG density of 3.2% led to the classification of low CpG content (< 3.2%) promoter, “LCP,” genes and high CpG content (≥ 3.2%) promoter, “HCP,” genes (Figs. 1B and C). Of 17,602 protein-coding genes included in this study, 8644 were LCP genes and 8958 were HCPs.

Bottom Line: 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)).HCP genes had minimal TSS-associated methylation regardless of transcription status, but gene body methylation appeared to be lost in repressed HCP genes.

View Article: PubMed Central - PubMed

Affiliation: Mayo Clinic, Rochester, MN, USA.

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.

Show MeSH