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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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Experimental outline.(a) Schematic diagrams showing the procedures for the construction of the plasmids harboring a site-specifically incorporated 5-HmC, 5-FoC, or 5-CaC. (b) CTAB assay for assessing the impact of the oxidized 5-mC derivatives on DNA transcription. “X” indicates 5-HmC, 5-FoC or 5-CaC, which was located on the transcribed strand of TurboGFP gene downstream of the CMV and T7 promoters. The +1 transcription start sites are indicated by arrowheads. Oxidized 5-mC derivative-bearing or unmodified control plasmids were mixed individually with the competitor genome as DNA templates for in vitro or in vivo transcription. Although truncated RNA may be produced when transcription arrests at or near a lesion site, only run-off RNA is shown and used for RT-PCR. Among the RT-PCR products, only the wild-type sequence arising from the cytosine derivative-containing vector is shown. The arrows indicate the cleavage sites of Nt.BstNBI, NcoI and SfaNI. The last two enzymes were used to digest the RT-PCR products for subsequent PAGE and LC-MS/MS analyses.
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f2: Experimental outline.(a) Schematic diagrams showing the procedures for the construction of the plasmids harboring a site-specifically incorporated 5-HmC, 5-FoC, or 5-CaC. (b) CTAB assay for assessing the impact of the oxidized 5-mC derivatives on DNA transcription. “X” indicates 5-HmC, 5-FoC or 5-CaC, which was located on the transcribed strand of TurboGFP gene downstream of the CMV and T7 promoters. The +1 transcription start sites are indicated by arrowheads. Oxidized 5-mC derivative-bearing or unmodified control plasmids were mixed individually with the competitor genome as DNA templates for in vitro or in vivo transcription. Although truncated RNA may be produced when transcription arrests at or near a lesion site, only run-off RNA is shown and used for RT-PCR. Among the RT-PCR products, only the wild-type sequence arising from the cytosine derivative-containing vector is shown. The arrows indicate the cleavage sites of Nt.BstNBI, NcoI and SfaNI. The last two enzymes were used to digest the RT-PCR products for subsequent PAGE and LC-MS/MS analyses.

Mentions: We employed a recently developed competitive transcription and adduct bypass (CTAB) assay30 to investigate how the oxidation products of 5-mC affect the efficiency and fidelity of DNA transcription in vitro and in human cells. To this end, we constructed nonreplicative double-stranded plasmids containing a single site-specifically incorporated 5-HmC, 5-FoC, or 5-CaC (Figure 2a), as well as the corresponding unmodified control and competitor plasmids. These cytosine derivatives were located on the transcribed strand 58 and 39 nucleotides downstream of the cytomegalovirus (CMV) and T7 promoters, respectively (Figure 2b). We premixed the cytosine derivative-bearing or control plasmid with a competitor construct and used them as templates for the transcription assays. The run-off transcripts of interest were subjected to reverse transcription PCR (RT-PCR), followed by polyacrylamide gel electrophoresis (PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the restriction digestion mixture of RT-PCR products (Figure 2b).


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)

Experimental outline.(a) Schematic diagrams showing the procedures for the construction of the plasmids harboring a site-specifically incorporated 5-HmC, 5-FoC, or 5-CaC. (b) CTAB assay for assessing the impact of the oxidized 5-mC derivatives on DNA transcription. “X” indicates 5-HmC, 5-FoC or 5-CaC, which was located on the transcribed strand of TurboGFP gene downstream of the CMV and T7 promoters. The +1 transcription start sites are indicated by arrowheads. Oxidized 5-mC derivative-bearing or unmodified control plasmids were mixed individually with the competitor genome as DNA templates for in vitro or in vivo transcription. Although truncated RNA may be produced when transcription arrests at or near a lesion site, only run-off RNA is shown and used for RT-PCR. Among the RT-PCR products, only the wild-type sequence arising from the cytosine derivative-containing vector is shown. The arrows indicate the cleavage sites of Nt.BstNBI, NcoI and SfaNI. The last two enzymes were used to digest the RT-PCR products for subsequent PAGE and LC-MS/MS analyses.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Experimental outline.(a) Schematic diagrams showing the procedures for the construction of the plasmids harboring a site-specifically incorporated 5-HmC, 5-FoC, or 5-CaC. (b) CTAB assay for assessing the impact of the oxidized 5-mC derivatives on DNA transcription. “X” indicates 5-HmC, 5-FoC or 5-CaC, which was located on the transcribed strand of TurboGFP gene downstream of the CMV and T7 promoters. The +1 transcription start sites are indicated by arrowheads. Oxidized 5-mC derivative-bearing or unmodified control plasmids were mixed individually with the competitor genome as DNA templates for in vitro or in vivo transcription. Although truncated RNA may be produced when transcription arrests at or near a lesion site, only run-off RNA is shown and used for RT-PCR. Among the RT-PCR products, only the wild-type sequence arising from the cytosine derivative-containing vector is shown. The arrows indicate the cleavage sites of Nt.BstNBI, NcoI and SfaNI. The last two enzymes were used to digest the RT-PCR products for subsequent PAGE and LC-MS/MS analyses.
Mentions: We employed a recently developed competitive transcription and adduct bypass (CTAB) assay30 to investigate how the oxidation products of 5-mC affect the efficiency and fidelity of DNA transcription in vitro and in human cells. To this end, we constructed nonreplicative double-stranded plasmids containing a single site-specifically incorporated 5-HmC, 5-FoC, or 5-CaC (Figure 2a), as well as the corresponding unmodified control and competitor plasmids. These cytosine derivatives were located on the transcribed strand 58 and 39 nucleotides downstream of the cytomegalovirus (CMV) and T7 promoters, respectively (Figure 2b). We premixed the cytosine derivative-bearing or control plasmid with a competitor construct and used them as templates for the transcription assays. The run-off transcripts of interest were subjected to reverse transcription PCR (RT-PCR), followed by polyacrylamide gel electrophoresis (PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the restriction digestion mixture of RT-PCR products (Figure 2b).

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