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5-hydroxymethylcytosine marks promoters in colon that resist DNA hypermethylation in cancer.

Uribe-Lewis S, Stark R, Carroll T, Dunning MJ, Bachman M, Ito Y, Stojic L, Halim S, Vowler SL, Lynch AG, Delatte B, de Bony EJ, Colin L, Defrance M, Krueger F, Silva AL, Ten Hoopen R, Ibrahim AE, Fuks F, Murrell A - Genome Biol. (2015)

Bottom Line: In vitro studies of TET2 in cancer cells confirm that these promoters are resistant to methylation gain independently of sustained TET2 expression.We also find that a considerable number of the methylation gain-resistant promoters marked by 5hmC in normal colon overlap with those that are marked with poised bivalent histone modifications in embryonic stem cells.Our study highlights the potential of cytosine modifications as biomarkers of cancerous cell proliferation.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The discovery of cytosine hydroxymethylation (5hmC) as a mechanism that potentially controls DNA methylation changes typical of neoplasia prompted us to investigate its behaviour in colon cancer. 5hmC is globally reduced in proliferating cells such as colon tumours and the gut crypt progenitors, from which tumours can arise.

Results: Here, we show that colorectal tumours and cancer cells express Ten-Eleven-Translocation (TET) transcripts at levels similar to normal tissues. Genome-wide analyses show that promoters marked by 5hmC in normal tissue, and those identified as TET2 targets in colorectal cancer cells, are resistant to methylation gain in cancer. In vitro studies of TET2 in cancer cells confirm that these promoters are resistant to methylation gain independently of sustained TET2 expression. We also find that a considerable number of the methylation gain-resistant promoters marked by 5hmC in normal colon overlap with those that are marked with poised bivalent histone modifications in embryonic stem cells.

Conclusions: Together our results indicate that promoters that acquire 5hmC upon normal colon differentiation are innately resistant to neoplastic hypermethylation by mechanisms that do not require high levels of 5hmC in tumours. Our study highlights the potential of cytosine modifications as biomarkers of cancerous cell proliferation.

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Promoters marked by 5hmC in normal colon resist DNA methylation gain in tumours. (a) DNA methylation changes in adenocarcinoma (n = 17) relative to matched normal tissues (n = 17) (Infinium arrays). Each dot represents a CpG (grey dots are changes with P <0.01). (b) 5hmC read content measured in windows around the Infinium probes (black bars). CpG island (CpGi) as orange bar. (c) Overlay of 5hmC high or 5hmC low promoters on the methylation states. (d) Left panel: 5hmC content around the Infinium probes of promoters with a significant change in methylation. High 5hmC promoters are prone to loss of DNA methylation in tumours whereas low 5hmC promoters are prone to methylation gain in tumours (limma geneSetTest). Middle panel: 5hmC content in normal and levels of DNA methylation in normal to show that methylation gain or loss occurs across a range of methylation levels in normal (P values from a Wilcox test). Right panel: 5hmC content in normal and expression levels in normal. DNA methylation prone genes (5hmC low) have low expression in the normal tissue (P values from a Wilcox test). (e) Heatmap comparing 5hmC and 5mC levels in normal to the 5mC changes in tumours at selected loci.
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Fig3: Promoters marked by 5hmC in normal colon resist DNA methylation gain in tumours. (a) DNA methylation changes in adenocarcinoma (n = 17) relative to matched normal tissues (n = 17) (Infinium arrays). Each dot represents a CpG (grey dots are changes with P <0.01). (b) 5hmC read content measured in windows around the Infinium probes (black bars). CpG island (CpGi) as orange bar. (c) Overlay of 5hmC high or 5hmC low promoters on the methylation states. (d) Left panel: 5hmC content around the Infinium probes of promoters with a significant change in methylation. High 5hmC promoters are prone to loss of DNA methylation in tumours whereas low 5hmC promoters are prone to methylation gain in tumours (limma geneSetTest). Middle panel: 5hmC content in normal and levels of DNA methylation in normal to show that methylation gain or loss occurs across a range of methylation levels in normal (P values from a Wilcox test). Right panel: 5hmC content in normal and expression levels in normal. DNA methylation prone genes (5hmC low) have low expression in the normal tissue (P values from a Wilcox test). (e) Heatmap comparing 5hmC and 5mC levels in normal to the 5mC changes in tumours at selected loci.

Mentions: Methylation changes in our patient cohort showed both gain and loss of promoter DNA methylation (Figure 3a). To refine our analysis of 5hmC content to changes in DNA methylation at the promoters assessed by the Infinium platform, we counted the hmeDIP-seq reads from normals in 200 bp windows around the Infinium probes (Figure 3b). After ranking by read content we identified the top 3,000 5hmC enriched loci (5hmC-high) as well as 3,000 loci where 5hmC was low or undetected (5hmC-low). Interestingly, by this measurement of read counts around the Infinium probes, we observed that promoters with high 5hmC in normal are either resistant to methylation change or are prone to methylation loss (79% loss vs. 21% gain from 676 probes with significant change out of 3,000) and that 5hmC marked promoters more frequently associate with a range of intermediate levels of methylation in normal (Figure 3c left panel and d). 5hmC low promoters more frequently associated with low levels of methylation in normal and showed an increased propensity to methylation gain, albeit methylation loss was also observed (56% gain vs. 44% loss from 379 probes with significant change out of 3,000) (Figure 3c right panel and d). We also find that the methylation-prone genes that lack 5hmC in normal have a low level of expression in the normal tissue (Figure 3d, right panel), in agreement with a recent report where propensity to methylation gain in tumours is frequent at promoters of genes with low expression in the normal tissue [47].Figure 3


5-hydroxymethylcytosine marks promoters in colon that resist DNA hypermethylation in cancer.

Uribe-Lewis S, Stark R, Carroll T, Dunning MJ, Bachman M, Ito Y, Stojic L, Halim S, Vowler SL, Lynch AG, Delatte B, de Bony EJ, Colin L, Defrance M, Krueger F, Silva AL, Ten Hoopen R, Ibrahim AE, Fuks F, Murrell A - Genome Biol. (2015)

Promoters marked by 5hmC in normal colon resist DNA methylation gain in tumours. (a) DNA methylation changes in adenocarcinoma (n = 17) relative to matched normal tissues (n = 17) (Infinium arrays). Each dot represents a CpG (grey dots are changes with P <0.01). (b) 5hmC read content measured in windows around the Infinium probes (black bars). CpG island (CpGi) as orange bar. (c) Overlay of 5hmC high or 5hmC low promoters on the methylation states. (d) Left panel: 5hmC content around the Infinium probes of promoters with a significant change in methylation. High 5hmC promoters are prone to loss of DNA methylation in tumours whereas low 5hmC promoters are prone to methylation gain in tumours (limma geneSetTest). Middle panel: 5hmC content in normal and levels of DNA methylation in normal to show that methylation gain or loss occurs across a range of methylation levels in normal (P values from a Wilcox test). Right panel: 5hmC content in normal and expression levels in normal. DNA methylation prone genes (5hmC low) have low expression in the normal tissue (P values from a Wilcox test). (e) Heatmap comparing 5hmC and 5mC levels in normal to the 5mC changes in tumours at selected loci.
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Fig3: Promoters marked by 5hmC in normal colon resist DNA methylation gain in tumours. (a) DNA methylation changes in adenocarcinoma (n = 17) relative to matched normal tissues (n = 17) (Infinium arrays). Each dot represents a CpG (grey dots are changes with P <0.01). (b) 5hmC read content measured in windows around the Infinium probes (black bars). CpG island (CpGi) as orange bar. (c) Overlay of 5hmC high or 5hmC low promoters on the methylation states. (d) Left panel: 5hmC content around the Infinium probes of promoters with a significant change in methylation. High 5hmC promoters are prone to loss of DNA methylation in tumours whereas low 5hmC promoters are prone to methylation gain in tumours (limma geneSetTest). Middle panel: 5hmC content in normal and levels of DNA methylation in normal to show that methylation gain or loss occurs across a range of methylation levels in normal (P values from a Wilcox test). Right panel: 5hmC content in normal and expression levels in normal. DNA methylation prone genes (5hmC low) have low expression in the normal tissue (P values from a Wilcox test). (e) Heatmap comparing 5hmC and 5mC levels in normal to the 5mC changes in tumours at selected loci.
Mentions: Methylation changes in our patient cohort showed both gain and loss of promoter DNA methylation (Figure 3a). To refine our analysis of 5hmC content to changes in DNA methylation at the promoters assessed by the Infinium platform, we counted the hmeDIP-seq reads from normals in 200 bp windows around the Infinium probes (Figure 3b). After ranking by read content we identified the top 3,000 5hmC enriched loci (5hmC-high) as well as 3,000 loci where 5hmC was low or undetected (5hmC-low). Interestingly, by this measurement of read counts around the Infinium probes, we observed that promoters with high 5hmC in normal are either resistant to methylation change or are prone to methylation loss (79% loss vs. 21% gain from 676 probes with significant change out of 3,000) and that 5hmC marked promoters more frequently associate with a range of intermediate levels of methylation in normal (Figure 3c left panel and d). 5hmC low promoters more frequently associated with low levels of methylation in normal and showed an increased propensity to methylation gain, albeit methylation loss was also observed (56% gain vs. 44% loss from 379 probes with significant change out of 3,000) (Figure 3c right panel and d). We also find that the methylation-prone genes that lack 5hmC in normal have a low level of expression in the normal tissue (Figure 3d, right panel), in agreement with a recent report where propensity to methylation gain in tumours is frequent at promoters of genes with low expression in the normal tissue [47].Figure 3

Bottom Line: In vitro studies of TET2 in cancer cells confirm that these promoters are resistant to methylation gain independently of sustained TET2 expression.We also find that a considerable number of the methylation gain-resistant promoters marked by 5hmC in normal colon overlap with those that are marked with poised bivalent histone modifications in embryonic stem cells.Our study highlights the potential of cytosine modifications as biomarkers of cancerous cell proliferation.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The discovery of cytosine hydroxymethylation (5hmC) as a mechanism that potentially controls DNA methylation changes typical of neoplasia prompted us to investigate its behaviour in colon cancer. 5hmC is globally reduced in proliferating cells such as colon tumours and the gut crypt progenitors, from which tumours can arise.

Results: Here, we show that colorectal tumours and cancer cells express Ten-Eleven-Translocation (TET) transcripts at levels similar to normal tissues. Genome-wide analyses show that promoters marked by 5hmC in normal tissue, and those identified as TET2 targets in colorectal cancer cells, are resistant to methylation gain in cancer. In vitro studies of TET2 in cancer cells confirm that these promoters are resistant to methylation gain independently of sustained TET2 expression. We also find that a considerable number of the methylation gain-resistant promoters marked by 5hmC in normal colon overlap with those that are marked with poised bivalent histone modifications in embryonic stem cells.

Conclusions: Together our results indicate that promoters that acquire 5hmC upon normal colon differentiation are innately resistant to neoplastic hypermethylation by mechanisms that do not require high levels of 5hmC in tumours. Our study highlights the potential of cytosine modifications as biomarkers of cancerous cell proliferation.

Show MeSH
Related in: MedlinePlus