Real-time dynamics of methyl-CpG-binding domain protein 3 and its role in DNA demethylation by fluorescence correlation spectroscopy

Abstract

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.

Key words: :

• DNA demethylation
• methyl-CpG-binding domain protein 3 (MBD3)
• ten-eleven-translocation proteins (TETs)
• dynamics
• nuclear protein
• fluorescence correlation spectroscopy (FCS)
• biophysics

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr Adrian Bird for providing the MBD3-GFP plasmid, and Dr Amy C Lossie and Dr Chiao-Ling Lo for plasmid maintenance and technical assistance. Support from the Purdue Center for Cancer Research through the innovative grant and the W.M. Keck Foundation is appreciated. Partial student support for BC by the National Institutes of Health, National Cancer Institute R25CA128770 Cancer Prevention Internship Program administered by the Oncological Sciences Center and the Discovery Learning Research Center at Purdue University is appreciated. This publication was made possible in part, with support from the Indiana Clinical and Translational Sciences Institute funded, in part by Grant Number (TR000006) from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award.

Supplemental Materials

Supplemental materials may be found here: www.landesbioscience.com/journals/epigenetics/article/25958