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

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 MBD3 induced by small interfering RNA (siRNA) was found to result in a global DNA hypermethylation as well as increased methylation in the promoter CpG islands (CGIs) of a number of cell cycle related genes.

Analysis of the concentrations and size distributions of cell-free DNA in schizophrenia using fluorescence correlation spectroscopy

“…However, qPCR is only applicable to preselected loci, and paired-end sequencing is time consuming and costly. Fluorescence correlation spectroscopy (FCS) is a technique for single-molecule detection that analyzes the fluctuations in fluorescence resulting from the Brownian motion of fluorescent molecules in a very small detection volume 19–21 . Previous study using FCS has demonstrated DNA molecules of different sizes released from cells following drug-induced apoptosis 22 .…”

Adult Neural Stem Cell Multipotency and Differentiation Are Directed by the Methyl-CpG-Binding Protein MBD1

“…Lack of MBD1 resulted in impaired hippocampal activity and autistic-like behaviors in mice (Zhao et al, 2003;Allan et al, 2008). Since then, it has become clear that, at least in part, MBD1 binds and stabilizes methylated DNA at the promoters of Fgf2 and miR-184 in cultured NSCs, represses the expression of these factors, and thereby enables neuronal differentiation (X.…”

“…For example, in the hypothalamus, MeCP2 was found to activate the vast majority of genes that it affected. Mechanistically, an interaction between MeCP2 and the transcriptional activator CREB1 was deemed responsible for the promoting expression (Chahrour et al, 2008). More recently, MeCP2 was demonstrated to recruit CREB1 to maintain Foxp3 expression to preserve the suppressive functions of regulatory T cells during inflammation (C. Li et al, 2014).…”

“…Furthermore, MBD2 was shown to promote expression of the glucocorticoid receptor in the hippocampus via interaction with Egr1 (Zif268) (Weaver et al, 2014). Interestingly, MBD2 exerts bidirectional influence on transcription by binding with either HDAC2, as part of a repressive complex, or with TACC3 and the histone acetyltransferase pCAF, as part of a reactivating complex (Angrisano et al, 2006). MBD3 behaves in an analogous manner and can bind both DNA methyltransferases and MBD2 to increase or decrease DNA methylation, respectively (Cui and Irudayaraj, 2015).…”

Single-Cell Quantification of Cytosine Modifications by Hyperspectral Dark-Field Imaging

“…For instance, the landscape of DNA methylation can radically fluctuate even in a single cell cycle. 35-37 To uncover epigenomic heterogeneity, modern optical microscopy provides elegant solutions for inspecting a single cell. 18, 38, 39 …”