PMID- 25753672 OWN - NLM STAT- MEDLINE DCOM- 20150626 LR - 20181113 IS - 1362-4962 (Electronic) IS - 0305-1048 (Print) IS - 0305-1048 (Linking) VI - 43 IP - 6 DP - 2015 Mar 31 TI - Dissecting the behavior and function of MBD3 in DNA methylation homeostasis by single-molecule spectroscopy and microscopy. PG - 3046-55 LID - 10.1093/nar/gkv098 [doi] AB - 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 Forster 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. CI - (c) The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. FAU - Cui, Yi AU - Cui Y AD - 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. FAU - Irudayaraj, Joseph AU - Irudayaraj J AD - 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. LA - eng GR - P30CA023168/CA/NCI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, Non-P.H.S. DEP - 20150309 PL - England TA - Nucleic Acids Res JT - Nucleic acids research JID - 0411011 RN - 0 (DNA-Binding Proteins) RN - 0 (MBD2 protein, human) RN - 0 (MBD3 protein, human) RN - 0 (RNA, Small Interfering) RN - 0 (Recombinant Fusion Proteins) RN - EC 2.1.1.- (DNA Modification Methylases) RN - EC 2.1.1.37 (DNA (Cytosine-5-)-Methyltransferase 1) RN - EC 2.1.1.37 (DNA (Cytosine-5-)-Methyltransferases) RN - EC 2.1.1.37 (DNMT1 protein, human) RN - EC 3.5.1.98 (Mi-2 Nucleosome Remodeling and Deacetylase Complex) SB - IM MH - Binding Sites MH - Cell Cycle MH - CpG Islands MH - DNA (Cytosine-5-)-Methyltransferase 1 MH - DNA (Cytosine-5-)-Methyltransferases/metabolism MH - *DNA Methylation MH - DNA Modification Methylases/metabolism MH - DNA-Binding Proteins/antagonists & inhibitors/genetics/*metabolism MH - Fluorescence Resonance Energy Transfer MH - HeLa Cells MH - Humans MH - Kinetics MH - Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism MH - Microscopy, Fluorescence MH - Models, Molecular MH - Promoter Regions, Genetic MH - RNA, Small Interfering/genetics MH - Recombinant Fusion Proteins/metabolism PMC - PMC4381056 EDAT- 2015/03/11 06:00 MHDA- 2015/06/27 06:00 PMCR- 2015/03/09 CRDT- 2015/03/11 06:00 PHST- 2015/01/29 00:00 [accepted] PHST- 2014/04/24 00:00 [received] PHST- 2015/03/11 06:00 [entrez] PHST- 2015/03/11 06:00 [pubmed] PHST- 2015/06/27 06:00 [medline] PHST- 2015/03/09 00:00 [pmc-release] AID - gkv098 [pii] AID - 10.1093/nar/gkv098 [doi] PST - ppublish SO - Nucleic Acids Res. 2015 Mar 31;43(6):3046-55. doi: 10.1093/nar/gkv098. Epub 2015 Mar 9.