Fkbp52 Regulates N6-methyladenine DNA Modification Under Hypoxia

Mouse FK506 binding protein 52(Fkbp52)is an important molecular chaperone protein in epigenetic modification.It has been reported that Fkbp52 can phosphorylate Dnmtl and increases the methyltransferase activity of Dnmt1.Whether the Fkbp52 has an effect on epigenetic modification under hypoxia condition is unclear.Increasing evidences has shown that DNA methylation plays a significant part in embryonic development,maintaining normal cell growth,tumorigenesis development.While,it's very interesting to see that DNA methylation(5mC)modification of the cell can be reduced by 15%-20%under hypoxic condition.Recent reports have found that 6mA DNA methylation of higher eukaryotic regulate gene expression.However,it has not been reported that the hypoxia stimulation can change the 6mA DNA modification.This project aim to explore the effects of the 6mA DNA modification and the mechanism of action of Fkbp52 under hypoxia.Our results showed that the 6mA modification was significantly decreased under hypoxia treatment in mouse embryonic fibroblasts(MEFs)cells.Meanwhile,Fkbp52 depletion led to significantly increased of genomic DNA 6mA level.Then,we generated a mutant cell of FKBP4 which is the homologous protein of Fkbp52 by CRISPR-cas9 technology in HEK293T cells.As we expected,the mutant cells produced similar results under hypoxia treatment.Recent studies have suggested that Dmad is a N6-methyladenine DNA demethylase in Drosophila.Thus,we speculated that the change of DNA 6mA modification in mice is caused by its homologous protein-Tet.To further detect whether the 6mA changes in DNA were caused by Tetl and Tet2,the expression levels of Tetl and Tet2 were detected after wild-type MEF and Fkbp52 depletion treatment under hypoxia.The results showed that hypoxia-induced increased Tetl and Tet2 mRNA levels in the wild type MEF,while decreased Tetl and Tet2 mRNA levels in Fkbp52 knockout MEFs,and showed the same phenotype in HEK293T cells knocked out by FKBP4.Therefore,we conclude that Tet1 and Tet2 may be 6mA demethylases in mice.In summary,our experiments shown that Tetl and Tet2 have certain effects on the DNA 6mA methylation modification controlled by Fkbp52 under hypoxic conditions.Studies have shown that Fkbp52 positively regulates the transcriptional activities of various steroid hormone receptors,mainly relying on the activity of Fkbp52 peptidyl-proline ci-trans isomerase(PPIase),and this activity is affected by hypoxia.Whether fat differentiation is related to DNA 6mA modification or not,the specific mechanism needs to be further studied.This study aims to supplement the regulatory mechanism of 6mA DNA modification in higher eukaryotes and explore the effect of 6mA DNA modification on fat differentiation.We hope our findings will be helpful to the study of DNA 6mA modification and its effect on fat formation.