Journal
EPIGENETICS
Volume 8, Issue 10, Pages 1089-1100Publisher
TAYLOR & FRANCIS INC
DOI: 10.4161/epi.25958
Keywords
DNA demethylation; methyl-CpG-binding domain protein 3 (MBD3); ten-eleven-translocation proteins (TETs); dynamics; nuclear protein; fluorescence correlation spectroscopy (FCS); biophysics
Funding
- Purdue Center for Cancer Research
- National Institutes of Health, National Cancer Institute [R25CA128770]
- Indiana Clinical and Translational Sciences Institute
- National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award [TR000006]
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With unprecedented development in technology, epigenetics is recognized as a substantial and flexible regulatory pathway for phenotyping. Cytosine methylation and its subsequent oxidization have attracted significant attention due to their direct impact on gene regulation, in association with methyl-CpG-binding domain proteins (MBDs) and transcription related factors. In this study we record the dynamics of DNA demethylation using the recombinant MBD3-GFP protein in living cells under hypoxia and Decitabine treatment using Fluorescence Correlation Spectroscopy (FCS) by monitoring the diffusion dynamics of MBD3. Our study shows a DNA-replication-independent decrease of 5-methylcytosine (5mC)/5-hydroxymethylcytosine (5hmC) under hypoxia vs. a dependent decrease under Decitabine treatment. Further, we define a significantly faster diffusion of MBD3 in the nucleus as a precursory event for active demethylation rather than the Decitabine induced passive demethylation. By monitoring the diffusion of bound and unbound MBD3 in the nucleus we were able to identify and characterize hypoxia-sensitive cells from insensitive/tolerant cells, as well as the respective contribution to active demethylation in a time-dependent manner. Last, we quantitatively describe the concurrent decreasing trend in all of the three oxidized products of 5mC, which points to the potential involvement of ten-eleven-translocation proteins (TETs) in hypoxia induced active demethylation. Overall, for the first time we correlate the dynamic process of DNA demethylation with the biophysical properties of the corresponding DNA binding proteins in live single cells by single molecule spectroscopy.
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