Giehr, Pascal and Kyriakopoulos, Charalampos and Lepikhov, Konstantin and Wallner, Stefan and Wolf, Verena and Walter, Joern (2018) Two are better than one: HPoxBS - hairpin oxidative bisulfite sequencing. NUCLEIC ACIDS RESEARCH, 46 (15): e88. ISSN 0305-1048, 1362-4962
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The controlled and stepwise oxidation of 5mC to 5hmC, 5fC and 5caC by Tet enzymes is influencing the chemical and biological properties of cytosine. Besides direct effects on gene regulation, oxidised forms influence the dynamics of demethylation and re-methylation processes. So far, no combined methods exist which allow to precisely determine the strand specific localisation of cytosine modifications along with their CpG symmetric distribution. Here we describe a comprehensive protocol combining conventional hairpin bisulfite with oxidative bisulfite sequencing (HPoxBS) to determine the strand specific distribution of 5mC and 5hmC at base resolution. We apply this method to analyse the contribution of local oxidative effects on DNA demethylation in mouse ES cells. Our method includes the HPoxBS workflow and subsequent data analysis using our developed software tools. Besides a precise estimation and display of strand specific 5mC and 5hmC levels at base resolution we apply the data to predict region specific activities of Dnmt and Tet enzymes. Our experimental and computational workflow provides a precise double strand display of 5mC and 5hmC modifications at single base resolution. Based on our data we predict region specific Tet and Dnmt enzyme efficiencies shaping the distinct locus levels and patterns of 5hmC and 5mC.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | SINGLE-BASE RESOLUTION; EMBRYONIC STEM-CELLS; ACUTE MYELOID-LEUKEMIA; NON-CPG METHYLATION; DNA METHYLATION; MAMMALIAN DNA; THYMINE DNA; 5-FORMYLCYTOSINE; 5-METHYLCYTOSINE; 5-HYDROXYMETHYLCYTOSINE; |
| Subjects: | 600 Technology > 610 Medical sciences Medicine |
| Divisions: | Medicine > Lehrstuhl für Klinische Chemie und Laboratoriumsmedizin |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 13 Dec 2019 08:47 |
| Last Modified: | 13 Dec 2019 08:47 |
| URI: | https://pred.uni-regensburg.de/id/eprint/13874 |
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