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Effects of Tet-mediated oxidation products of 5-methylcytosine on DNA transcription in vitro and in mammalian cells.

You C, Ji D, Dai X, Wang Y - Sci Rep (2014)

Bottom Line: Recent studies showed that Ten-eleven translocation (Tet) proteins can catalyze the stepwise oxidation of 5-mC to produce 5-hydroxymethylcytosine (5-HmC), 5-formylcytosine (5-FoC) and 5-carboxylcytosine (5-CaC).The exciting discovery of these novel cytosine modifications has stimulated substantial research interests about their roles in epigenetic regulation.These findings provided new evidence for the potential functional interplay between cytosine methylation status and transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California, Riverside, California 92521-0403.

ABSTRACT
5-methylcytosine (5-mC) is a well-characterized epigenetic regulator in mammals. Recent studies showed that Ten-eleven translocation (Tet) proteins can catalyze the stepwise oxidation of 5-mC to produce 5-hydroxymethylcytosine (5-HmC), 5-formylcytosine (5-FoC) and 5-carboxylcytosine (5-CaC). The exciting discovery of these novel cytosine modifications has stimulated substantial research interests about their roles in epigenetic regulation. Here we systematically examined the effects of the oxidized 5-mC derivatives on the efficiency and fidelity of DNA transcription using a recently developed competitive transcription and adduct bypass assay. Our results showed that, when located on the transcribed strand, 5-FoC and 5-CaC exhibited marginal mutagenic and modest inhibitory effects on DNA transcription mediated by single-subunit T7 RNA polymerase or multi-subunit human RNA polymerase II in vitro and in human cells. 5-HmC displayed relatively milder blocking effects on transcription, and no mutant transcript could be detectable for 5-HmC in vitro or in cells. The lack of considerable mutagenic effects of the oxidized 5-mC derivatives on transcription was in agreement with their functions in epigenetic regulation. The modest blocking effects on transcription suggested that 5-FoC and 5-CaC may function in transcriptional regulation. These findings provided new evidence for the potential functional interplay between cytosine methylation status and transcription.

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The effects of 5-FoC and 5-CaC on transcriptional fidelity.(a) The sequences of wild-type and mutant (G→A) transcripts are indicated above the double-stranded DNA construct. The underlined base (i.e., A) indicates an adenosine misincorporation opposite a 5-FoC or 5-CaC. (b). The mutation (G→A) frequencies of 5-FoC and 5-CaC in in vitro transcription systems using T7 RNAP and hRNAPII. (c) The mutation (G→A) frequencies of 5-FoC and 5-CaC in 293T cells treated with siRNA targeting the TDG gene (siTDG).
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f4: The effects of 5-FoC and 5-CaC on transcriptional fidelity.(a) The sequences of wild-type and mutant (G→A) transcripts are indicated above the double-stranded DNA construct. The underlined base (i.e., A) indicates an adenosine misincorporation opposite a 5-FoC or 5-CaC. (b). The mutation (G→A) frequencies of 5-FoC and 5-CaC in in vitro transcription systems using T7 RNAP and hRNAPII. (c) The mutation (G→A) frequencies of 5-FoC and 5-CaC in 293T cells treated with siRNA targeting the TDG gene (siTDG).

Mentions: PAGE analysis showed that, when located on the template DNA strand, a single 5-HmC did not substantially compromise the efficiency of transcription by T7 RNAP, whereas 5-CaC and 5-FoC impeded modestly the T7 RNAP-mediated transcription elongation, with relative bypass efficiencies (RBE) being ~50% and 66%, respectively (Figure 3a–c). Similarly, 5-FoC and 5-CaC, but not 5-HmC, inhibited considerably the transcription by hRNAPII (Figure 3a–c). We also employed LC-MS/MS analysis to detect the potential mutant products induced by these cytosine derivatives during transcription in vitro. We found that transcriptional bypass of 5-HmC with T7 RNAP or hRNAPII did not generate any detectable mutant transcripts; however, 5-FoC and 5-CaC were able to induce at least one type of mutant transcript (G→A), which contains an adenosine misincorporation opposite the 5-FoC and 5-CaC and occurs at frequencies of ~1–1.7% (Figure 4a,b, and Supplementary Figure S1–4).


Effects of Tet-mediated oxidation products of 5-methylcytosine on DNA transcription in vitro and in mammalian cells.

You C, Ji D, Dai X, Wang Y - Sci Rep (2014)

The effects of 5-FoC and 5-CaC on transcriptional fidelity.(a) The sequences of wild-type and mutant (G→A) transcripts are indicated above the double-stranded DNA construct. The underlined base (i.e., A) indicates an adenosine misincorporation opposite a 5-FoC or 5-CaC. (b). The mutation (G→A) frequencies of 5-FoC and 5-CaC in in vitro transcription systems using T7 RNAP and hRNAPII. (c) The mutation (G→A) frequencies of 5-FoC and 5-CaC in 293T cells treated with siRNA targeting the TDG gene (siTDG).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231326&req=5

f4: The effects of 5-FoC and 5-CaC on transcriptional fidelity.(a) The sequences of wild-type and mutant (G→A) transcripts are indicated above the double-stranded DNA construct. The underlined base (i.e., A) indicates an adenosine misincorporation opposite a 5-FoC or 5-CaC. (b). The mutation (G→A) frequencies of 5-FoC and 5-CaC in in vitro transcription systems using T7 RNAP and hRNAPII. (c) The mutation (G→A) frequencies of 5-FoC and 5-CaC in 293T cells treated with siRNA targeting the TDG gene (siTDG).
Mentions: PAGE analysis showed that, when located on the template DNA strand, a single 5-HmC did not substantially compromise the efficiency of transcription by T7 RNAP, whereas 5-CaC and 5-FoC impeded modestly the T7 RNAP-mediated transcription elongation, with relative bypass efficiencies (RBE) being ~50% and 66%, respectively (Figure 3a–c). Similarly, 5-FoC and 5-CaC, but not 5-HmC, inhibited considerably the transcription by hRNAPII (Figure 3a–c). We also employed LC-MS/MS analysis to detect the potential mutant products induced by these cytosine derivatives during transcription in vitro. We found that transcriptional bypass of 5-HmC with T7 RNAP or hRNAPII did not generate any detectable mutant transcripts; however, 5-FoC and 5-CaC were able to induce at least one type of mutant transcript (G→A), which contains an adenosine misincorporation opposite the 5-FoC and 5-CaC and occurs at frequencies of ~1–1.7% (Figure 4a,b, and Supplementary Figure S1–4).

Bottom Line: Recent studies showed that Ten-eleven translocation (Tet) proteins can catalyze the stepwise oxidation of 5-mC to produce 5-hydroxymethylcytosine (5-HmC), 5-formylcytosine (5-FoC) and 5-carboxylcytosine (5-CaC).The exciting discovery of these novel cytosine modifications has stimulated substantial research interests about their roles in epigenetic regulation.These findings provided new evidence for the potential functional interplay between cytosine methylation status and transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California, Riverside, California 92521-0403.

ABSTRACT
5-methylcytosine (5-mC) is a well-characterized epigenetic regulator in mammals. Recent studies showed that Ten-eleven translocation (Tet) proteins can catalyze the stepwise oxidation of 5-mC to produce 5-hydroxymethylcytosine (5-HmC), 5-formylcytosine (5-FoC) and 5-carboxylcytosine (5-CaC). The exciting discovery of these novel cytosine modifications has stimulated substantial research interests about their roles in epigenetic regulation. Here we systematically examined the effects of the oxidized 5-mC derivatives on the efficiency and fidelity of DNA transcription using a recently developed competitive transcription and adduct bypass assay. Our results showed that, when located on the transcribed strand, 5-FoC and 5-CaC exhibited marginal mutagenic and modest inhibitory effects on DNA transcription mediated by single-subunit T7 RNA polymerase or multi-subunit human RNA polymerase II in vitro and in human cells. 5-HmC displayed relatively milder blocking effects on transcription, and no mutant transcript could be detectable for 5-HmC in vitro or in cells. The lack of considerable mutagenic effects of the oxidized 5-mC derivatives on transcription was in agreement with their functions in epigenetic regulation. The modest blocking effects on transcription suggested that 5-FoC and 5-CaC may function in transcriptional regulation. These findings provided new evidence for the potential functional interplay between cytosine methylation status and transcription.

Show MeSH
Related in: MedlinePlus