Demethylation of Non-CpG Sites in DNA Is Initiated by TET2 5-Methylcytosine Dioxygenase
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Purification of TET2 Dioxygenase
2.2.1. Purification of hTET2 Catalytic Domain
2.2.2. Purification of GST-Tagged hTET2 Catalytic Domain
2.2.3. Purification of Full-Length Myc-Tagged hTET2
2.3. 5mC Oxidation by TET2 Dioxygenase
2.4. MS/MS Parameters for Nucleosides
2.5. Liquid Chromatographic Conditions for Nucleosides under Different MS/MS Modes
3. Results and Discussion
3.1. Detection of Cytosine Derivatives Produced by Oxidative Demethylation of 5mCpG and 5mCpA Sites
3.2. Quantification of Cytosine Derivatives Produced by TET2-Mediated Oxidation of 5mCpH Sites; Effect of +1 Base
3.3. TET2 Can Initiate Demethylation of 5mCpH Sites
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
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1A | |
---|---|
DNA Substrates | Sequence |
5mCpG Substrate 1 | Forward Strand 5′-GCGCCGGTCGTA/Me-C/GGCCGCTCCCGC-3′ Reverse Strand 5′-GCGGGAGCGGCCGTACGACCGGCGC-3′ |
5mCpA Substrate 1 | Forward Strand 5′-GCGCCGGTCGTA/Me-C/AGCCGCTCCCGC-3′ Reverse Strand 5′-GCGGGAGCGGCTGTACGACCGGCGC-3′ |
1B | |
DNA Substrates | Sequence |
5mCpG Substrate 2 | Forward Strand 5′-GCGCCGGTCCCG/Me-C/GGGCGCTCCCGC-3′ Reverse Strand 5′-GCGGGAGCGCCCGCGGGACCGGCGC-3′ |
5mCpG Substrate 3 | Forward Strand 5′-GCGCCGGTCGTT/Me-C/GTCCGCTCCCGC-3′ Reverse Strand 5′-GCGGGAGCGGACGAACGACCGGCGC-3 |
5mCpG Substrate 4 | Forward Strand 5′-GCGCCGGTCAAC/Me-C/GACCGCTCCCGC-3′ Reverse Strand 5′-GCGGGAGCGGTCGGTTGACCGGCGC-3′ |
5mCpA Substrate 2 | Forward Strand 5′-GCGCCGGTCTAA/Me-C/AAACGCTCCCGC-3′ Reverse Strand 5′-GCGGGAGCGTTTGTTAGACCGGCGC-3′ |
5mCpA Substrate 3 | Forward Strand 5′-GCGCCGGTCGTT/Me-C/ATCCGCTCCCGC-3′ Reverse Strand 5′-GCGGGAGCGGATGAACGACCGGCGC-3′ |
5mCpA Substrate 4 | Forward Strand 5′-GCGCCGGTCGGA/Me-C/AACCGCTCCCGC-3′ Reverse Strand 5′-GCGGGAGCGGTTGTCCGACCGGCGC-3′ |
1C | |
DNA Substrates | Sequence |
5mCpC Substrate | Forward Strand 5′-GCGCCGGTCCTG/Me-C/CCCCGCTCCCGC-3′ Reverse Strand 5′-GCGGGAGCGGGGGCAGGACCGGCGC-3′ |
5mCpT Substrate | Forward Strand 5′-GCGCCGGTCCTG/Me-C/TCCCGCTCCCGC-3′ Reverse Strand 5′-GCGGGAGCGGGAGCAGGACCGGCGC-3 |
2A | |||
---|---|---|---|
DNA Substrate | DNA Sequence | ||
5mCpG | Forward Strand 5′-GCGCCGGTCCCG/Me-C/GGGCGCTCCCGC-3′ Reverse Strand 5′-GCGGGAGCGTTTGTTAGACCGGCGC-3 | ||
Enzyme | Full Length human TET2 | ||
dC Area (Counts) ± Standard Error | 5hmC Area (Counts) ± Standard Error | 5fC Area (Counts) ± Standard Error | 5caC Area (Counts) ± Standard Error |
(2.00 × 107) ± (6.11 × 105) | (3.11 × 104) ± (2.17 × 103) | (2.25 × 104) ± (1.29 × 103) | (6.12 × 103) ± (3.71 × 102) |
Enzyme | GST Tagged human TET2 | ||
dC Area (Counts) | 5hmC Area (Counts) | 5fC Area (Counts) | 5caC Area (Counts) |
(6.63 × 107) ± (3.33 × 105) | (3.17 × 105) ± (2.11 × 104) | (2.50 × 105) ± (2.67 × 103) | (7.37 × 104) ± (1.89 × 103) |
2B | |||
DNA Substrate | DNA Sequence | ||
5mCpA | Forward Strand 5′-GCGCCGGTCCTG/Me-C/ACCCGCTCCCGC-3′ Reverse Strand 5′-GCGGGAGCGTTTGTTAGACCGGCGC-3 | ||
Enzyme | Full Length human TET2 | ||
dC Area (Counts) ± Standard Error | 5hmC Area (Counts) ± Standard Error | 5fC Area (Counts) ± Standard Error | 5caC Area (Counts) ± Standard Error |
(1.56 × 107) ± (6.43 × 105) | (1.91 × 104) ± (1.17 × 103) | (1.67 × 104) ± (1.00 × 103) | (2.89 × 103) ± (4.88 × 102) |
Enzyme | GST Tagged human TET2 | ||
dC Area (Counts) | 5hmC Area (Counts) | 5fC Area (Counts) | 5caC Area (Counts) |
(7.73 × 107) ± (3.33 × 105) | (2.17 × 105) ± (1.18 × 104) | (1.41 × 105) ± (3.67 × 103) | (2.37 × 104) ± (1.65 × 103) |
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Dey, A.S.; Bhattacharya, C.; Guan, Y.; Jha, B.K.; Mukherji, M. Demethylation of Non-CpG Sites in DNA Is Initiated by TET2 5-Methylcytosine Dioxygenase. DNA 2021, 1, 26-36. https://doi.org/10.3390/dna1010004
Dey AS, Bhattacharya C, Guan Y, Jha BK, Mukherji M. Demethylation of Non-CpG Sites in DNA Is Initiated by TET2 5-Methylcytosine Dioxygenase. DNA. 2021; 1(1):26-36. https://doi.org/10.3390/dna1010004
Chicago/Turabian StyleDey, Aninda Sundar, Chayan Bhattacharya, Yihong Guan, Babal Kant Jha, and Mridul Mukherji. 2021. "Demethylation of Non-CpG Sites in DNA Is Initiated by TET2 5-Methylcytosine Dioxygenase" DNA 1, no. 1: 26-36. https://doi.org/10.3390/dna1010004