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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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Pervasive maintenance of a methylation-free state at TET2 bound promoters. (a) DNA methylation gain in primary tumours was remarkably scarce at the TET2 bound promoters identified in HCT116 cells (P <0.0001, binomial test). (b) Western blot for TET2 and beta TUBULIN from whole cell extracts of HCT116 cells stably transfected with a non-targeting shRNA control (shCtrl.) or with shRNA to TET2 (TET2C) or to TET2 and TET3 (TET2 + 3). Fold change in the knockdown was calculated relative to the shCtrl. (c) qRT-PCR for TET2 and TET3. (d) Global levels of 5hmC and 5mC by LCMS. (e) DNA methylation changes by Infinium arrays after depletion of TET2.
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Fig5: Pervasive maintenance of a methylation-free state at TET2 bound promoters. (a) DNA methylation gain in primary tumours was remarkably scarce at the TET2 bound promoters identified in HCT116 cells (P <0.0001, binomial test). (b) Western blot for TET2 and beta TUBULIN from whole cell extracts of HCT116 cells stably transfected with a non-targeting shRNA control (shCtrl.) or with shRNA to TET2 (TET2C) or to TET2 and TET3 (TET2 + 3). Fold change in the knockdown was calculated relative to the shCtrl. (c) qRT-PCR for TET2 and TET3. (d) Global levels of 5hmC and 5mC by LCMS. (e) DNA methylation changes by Infinium arrays after depletion of TET2.

Mentions: If the TETs bind to DNA and protect against hypermethylation in tumours, then it would be expected that promoters susceptible to DNA methylation gain in colon tumours would form a distinct group with a minimal overlap with TET target promoters. We therefore examined whether loci that gained DNA methylation in our primary tumours (1,597 probes for 1,077 promoters) were likely TET2 target promoters (4,201 probes for 3,144 promoters). This analysis showed less than 1% overlap between loci that gain DNA methylation in tumours and the TET2 bound promoters (Figure 5a). These results could suggest that TET2 might be part of a mechanism that protects promoters from de novo DNA methylation. To examine this we depleted TET2 in HCT116 cells by stable transfection of shRNAs (Figure 5b and c). In one instance we used shRNA against TET2 alone (TET2C) and in the other shRNA against TET2 and TET3 (TET2 + 3 where TET3 mRNA was not affected and therefore treat this sample as a TET2 only knockdown) (Figure 5c). LCMS after TET2 depletion showed a marked reduction in the global level of 5hmC (Figure 5d), confirming TET2 oxygenase activity in HCT116, without changes in global levels of 5mC (Figure 5d) but this could be due to the small contribution of promoter methylation to the methylome. Infinium arrays identified several loci with changes in DNA methylation (Figure 5e) that were for the most part low in magnitude (median of change was 10.4%; not shown). Similar changes in levels of DNA methylation were recently observed after TET1 depletion in differentiated cells [51]. However in our study, methylation levels at TET2 bound CpG islands were largely unaffected after TET2 depletion (less than 1%, Figure 5e), suggesting that these promoters do not require high levels of TET2 to maintain the methylation free state and are intrinsically resistant to methylation changes.Figure 5


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)

Pervasive maintenance of a methylation-free state at TET2 bound promoters. (a) DNA methylation gain in primary tumours was remarkably scarce at the TET2 bound promoters identified in HCT116 cells (P <0.0001, binomial test). (b) Western blot for TET2 and beta TUBULIN from whole cell extracts of HCT116 cells stably transfected with a non-targeting shRNA control (shCtrl.) or with shRNA to TET2 (TET2C) or to TET2 and TET3 (TET2 + 3). Fold change in the knockdown was calculated relative to the shCtrl. (c) qRT-PCR for TET2 and TET3. (d) Global levels of 5hmC and 5mC by LCMS. (e) DNA methylation changes by Infinium arrays after depletion of TET2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4380107&req=5

Fig5: Pervasive maintenance of a methylation-free state at TET2 bound promoters. (a) DNA methylation gain in primary tumours was remarkably scarce at the TET2 bound promoters identified in HCT116 cells (P <0.0001, binomial test). (b) Western blot for TET2 and beta TUBULIN from whole cell extracts of HCT116 cells stably transfected with a non-targeting shRNA control (shCtrl.) or with shRNA to TET2 (TET2C) or to TET2 and TET3 (TET2 + 3). Fold change in the knockdown was calculated relative to the shCtrl. (c) qRT-PCR for TET2 and TET3. (d) Global levels of 5hmC and 5mC by LCMS. (e) DNA methylation changes by Infinium arrays after depletion of TET2.
Mentions: If the TETs bind to DNA and protect against hypermethylation in tumours, then it would be expected that promoters susceptible to DNA methylation gain in colon tumours would form a distinct group with a minimal overlap with TET target promoters. We therefore examined whether loci that gained DNA methylation in our primary tumours (1,597 probes for 1,077 promoters) were likely TET2 target promoters (4,201 probes for 3,144 promoters). This analysis showed less than 1% overlap between loci that gain DNA methylation in tumours and the TET2 bound promoters (Figure 5a). These results could suggest that TET2 might be part of a mechanism that protects promoters from de novo DNA methylation. To examine this we depleted TET2 in HCT116 cells by stable transfection of shRNAs (Figure 5b and c). In one instance we used shRNA against TET2 alone (TET2C) and in the other shRNA against TET2 and TET3 (TET2 + 3 where TET3 mRNA was not affected and therefore treat this sample as a TET2 only knockdown) (Figure 5c). LCMS after TET2 depletion showed a marked reduction in the global level of 5hmC (Figure 5d), confirming TET2 oxygenase activity in HCT116, without changes in global levels of 5mC (Figure 5d) but this could be due to the small contribution of promoter methylation to the methylome. Infinium arrays identified several loci with changes in DNA methylation (Figure 5e) that were for the most part low in magnitude (median of change was 10.4%; not shown). Similar changes in levels of DNA methylation were recently observed after TET1 depletion in differentiated cells [51]. However in our study, methylation levels at TET2 bound CpG islands were largely unaffected after TET2 depletion (less than 1%, Figure 5e), suggesting that these promoters do not require high levels of TET2 to maintain the methylation free state and are intrinsically resistant to methylation changes.Figure 5

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