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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 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.
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Figure 2: 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.

Mentions: In LCP genes, transcriptional activity was linked to the methylation of CpG sites located at the TSS and within up to 8000 nucleotides 5′ of the TSS, a region commonly implicated in gene regulation (Fig. 2A). The most highly expressed genes had a mean level of CpG methylation of 0% in this upstream region, while it was > 40% for silenced genes (Fig. 2A). For this comparison, the mean % of methylation was “trimmed” by removing the highest and lowest 1/5th of values before averaging (“20% trimmed mean”), a common method in descriptive statistics to capture the middle (“central tendency”) of data sets that do not follow a normal distribution. We then performed a separate comparison of the top 1/5th of values by determining the 80th percentile rank of methylation levels. Here the difference in methylation levels between highly expressed and silenced LCP genes became even more evident: for highly expressed genes, the 80th percentile rank of methylation was < 10% in a region from -1000bp to -3000bp from the TSS, while it was > 90% for silenced genes (Fig. 2B). These findings for LCP genes are consistent with the classic model of increased methylation at promoters associated with decreased transcription. However, in the rat DRG this correlation was exclusively found in LCP genes suggesting that the repressive function of DNA methylation was most effective in shutting down genes in regions in which its target motif, the CpG dinucleotide, is sparse.


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 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.
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Figure 2: 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.
Mentions: In LCP genes, transcriptional activity was linked to the methylation of CpG sites located at the TSS and within up to 8000 nucleotides 5′ of the TSS, a region commonly implicated in gene regulation (Fig. 2A). The most highly expressed genes had a mean level of CpG methylation of 0% in this upstream region, while it was > 40% for silenced genes (Fig. 2A). For this comparison, the mean % of methylation was “trimmed” by removing the highest and lowest 1/5th of values before averaging (“20% trimmed mean”), a common method in descriptive statistics to capture the middle (“central tendency”) of data sets that do not follow a normal distribution. We then performed a separate comparison of the top 1/5th of values by determining the 80th percentile rank of methylation levels. Here the difference in methylation levels between highly expressed and silenced LCP genes became even more evident: for highly expressed genes, the 80th percentile rank of methylation was < 10% in a region from -1000bp to -3000bp from the TSS, while it was > 90% for silenced genes (Fig. 2B). These findings for LCP genes are consistent with the classic model of increased methylation at promoters associated with decreased transcription. However, in the rat DRG this correlation was exclusively found in LCP genes suggesting that the repressive function of DNA methylation was most effective in shutting down genes in regions in which its target motif, the CpG dinucleotide, is sparse.

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