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LC-MS-MS quantitative analysis reveals the association between FTO and DNA methylation
Yuting Zhu1, Guangyu Zhou1, Xuebin Yu1
1Department of Oncology, Second Affiliated Hospital, Institute of Cancer Stem Cell, DaLian Medical University, Dalian, Liaoning, China.
Plos One
|April 29, 2017
Summary
Fat mass and obesity-associated protein (FTO) does not demethylate 5-methyl-2'-deoxycytidine (5mdC) in DNA. This finding highlights FTO
Area of Science:
- Epigenetics
- Molecular Biology
- Biochemistry
Background:
- Fat mass and obesity-associated protein (FTO) is known to demethylate RNA and single-stranded DNA.
- Mammalian DNA contains 5-methyl-2 extquotesingle-deoxycytidine (5mdC) modifications crucial for energy homeostasis and fertility.
- The potential role of FTO in 5mdC demethylation remained unexplored.
Purpose of the Study:
- To investigate whether FTO protein possesses demethylase activity against 5mdC in DNA.
- To determine the substrate specificity and selectivity of FTO demethylase activity.
Main Methods:
- In vitro and in vivo demethylation assays using liquid chromatography-tandem mass spectrometry (LC-MS-MS).
- Immunofluorescence technique to assess 5-hydroxymethyl-2 extquotesingle-deoxycytidine (5hmdC) generation in Hela cells upon overexpression of TET2 and FTO variants.
Main Results:
- FTO demonstrated no significant demethylation activity against 5mdC in DNA, both in vitro and in vivo.
- LC-MS-MS analysis confirmed FTO's high substrate specificity and selectivity.
- Overexpression of wild-type TET2, but not FTO or mutant TET2, led to increased 5hmdC levels.
Conclusions:
- FTO protein does not exhibit potent 5mdC demethylase activity on DNA.
- These findings underscore the specific enzymatic functions of FTO.
- The study may guide future research into novel epigenetic modification proteins.
