title: 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.
authors.affiliation: Department of Agricultural and Biological Engineering, 225 S. University Street, Purdue University, West Lafayette, IN 47907, USA Bindley Bioscience Center, 1203 W. State Street, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Cui
authors.affiliation: Department of Agricultural and Biological Engineering, 225 S. University Street, Purdue University, West Lafayette, IN 47907, USA Bindley Bioscience Center, 1203 W. State Street, Purdue University, West Lafayette, IN 47907, USA josephi@purdue.edu.
authors.forename: Joseph
authors.initials: J
authors.lastname: Irudayaraj
mesh_terms: D001665:Binding Sites; D002453:Cell Cycle; D018899:CpG Islands; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D056245:Mi-2 Nucleosome Remodeling and Deacetylase Complex / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; C489325:MBD2 protein, human; C115369:MBD3 protein, human; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D015254:DNA Modification Methylases; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; D056245:Mi-2 Nucleosome Remodeling and Deacetylase Complex