Dissecting the behavior and function of MBD3 in DNA methylation homeostasis by single-molecule spectroscopy and microscopy

Nucleic Acids Research
Yi Cui, Joseph Irudayaraj

Abstract

The detailed mechanism for DNA methylation homeostasis relies on an intricate regulatory network with a possible contribution from methyl-CpG-binding domain protein 3 (MBD3). In this study we examine the single-molecule behavior of MBD3 and its functional implication in balancing the activity of DNA methyltransferases (DNMTs). Besides a localization tendency to DNA demethylating sites, MBD3 experiences a concurrent transcription with DNMTs in cell cycle. Fluorescence lifetime correlation spectroscopy (FLCS) and photon counting histogram (PCH) were applied to characterize the chromatin binding kinetics and stoichiometry of MBD3 in different cell phases. In the G1-phase, MBD3, in the context of the Mi-2/NuRD (nucleosome remodeling deacetylase) complex, could adopt a salt-dependent homodimeric association with its target epigenomic loci. Along with cell cycle progression, utilizing fluorescence lifetime imaging microscopy-based Förster resonance energy transfer (FLIM-FRET) we revealed that a proportion of MBD3 and MBD2 would co-localize with DNMT1 during DNA maintenance methylation, providing a proofreading and protective mechanism against a possible excessive methylation by DNMT1. In accordance with our hypothesis, insufficient M...Continue Reading

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Citations

Jul 29, 2016·Tumour Biology : the Journal of the International Society for Oncodevelopmental Biology and Medicine·Jie CuiLuo Gu
Jul 18, 2019·Genetic Testing and Molecular Biomarkers·Mei-Rong LiangOu-Ping Huang
Aug 9, 2019·Cellular Reprogramming·Xingwang WangZicong Li
Jul 9, 2016·Oncotarget·Samrat Roy ChoudhuryJoseph Irudayaraj
Nov 5, 2016·Wiley Interdisciplinary Reviews. Nanomedicine and Nanobiotechnology·Yi CuiJoseph Irudayaraj
Mar 7, 2017·Journal of Biological Engineering·Basudev ChowdhuryJoseph Irudayaraj
Jan 1, 2016·Toxicology Research·Yi CuiJoseph Irudayaraj
Aug 26, 2017·Scientific Reports·Pushpak N BhandariJoseph Irudayaraj
Apr 11, 2018·Frontiers in Cell and Developmental Biology·Qifan ZhuRamiro Alberio
Feb 1, 2019·Clinical Epigenetics·Wenjie LiuJoseph Irudayaraj
Jan 7, 2021·Current Genetics·Anjali PathaniaItay Onn
Oct 23, 2021·British Journal of Pharmacology·Zunaira ShoaibJoseph M K Irudayaraj

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Methods Mentioned

BETA
ChIP-seq
fluorescence correlation spectroscopy
FCS
fluorescence
PCR
transfection
flow cytometry
FRET
pull-down
two-hybrid

Software Mentioned

Primer
Flowjo
FLCS
BLAST
TimeHarp200
SymPhoTime

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