title: 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.
authors: Biological Engineering and Bindley Bioscience Center, Purdue University; West Lafayette, IN USA.
authors: Yi
authors: Y
authors: Cui
authors: Biological Engineering and Bindley Bioscience Center, Purdue University; West Lafayette, IN USA.
authors: Il-Hoon
authors: IH
authors: Cho
authors: Biological Engineering and Bindley Bioscience Center, Purdue University; West Lafayette, IN USA.
authors: Basudev
authors: B
authors: Chowdhury
authors: Biological Engineering and Bindley Bioscience Center, Purdue University; West Lafayette, IN USA.
authors: Joseph
authors: J
authors: Irudayaraj
mesh_terms: D044503:5-Methylcytosine / Q000378:metabolism / Q000031:analogs & derivatives; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D015687:Cell Hypoxia; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D015254:DNA Modification Methylases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000077209:Decitabine; D006367:HeLa Cells; D006801:Humans; D059010:Single-Cell Analysis; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D004268:DNA-Binding Proteins; C115369:MBD3 protein, human; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D000077209:Decitabine; D003596:Cytosine; D004247:DNA; D015254:DNA Modification Methylases; D001374:Azacitidine
keywords: DNA demethylation; biophysics; dynamics; fluorescence correlation spectroscopy (FCS); methyl-CpG-binding domain protein 3 (MBD3); nuclear protein; ten-eleven-translocation proteins (TETs)
references: Cell Biochem Biophys. 2001;34(3):383-408
references: 11898862
references: Biophys J. 2005 Nov;89(5):2960-71
references: 16113107
references: Cell. 2012 Sep 14;150(6):1135-46
references: 22980977
references: Nat Rev Genet. 2013 Mar;14(3):204-20
references: 23400093
references: Genes Dev. 2011 Dec 1;25(23):2436-52
references: 22156206
references: Cancer Metastasis Rev. 2007 Jun;26(2):225-39
references: 17440684
references: ACS Nano. 2009 Dec 22;3(12):4071-9
references: 19891423
references: Cell. 2007 May 4;129(3):465-72
references: 17482542
references: Nat Rev Genet. 2011 Nov 15;13(1):7-13
references: 22083101
references: Science. 2009 May 15;324(5929):929-30
references: 19372393
references: PLoS Genet. 2012;8(5):e1002692
references: 22615578
references: Nature. 2011 May 19;473(7347):394-7
references: 21552279
references: Oncogene. 2002 Aug 12;21(35):5400-13
references: 12154403
references: J Cell Mol Med. 2010 Nov;14(11):2646-54
references: 19508390
references: PLoS Genet. 2011 Jun;7(6):e1002154
references: 21731508
references: J Nucleic Acids. 2011;2011:870726
references: 21772996
references: Science. 2011 Sep 2;333(6047):1303-7
references: 21817016
references: Biophys J. 2003 Mar;84(3):1977-84
references: 12609900
references: Cell Stem Cell. 2009 Sep 4;5(3):237-41
references: 19716359
references: Brain Res. 2005 Jan 7;1031(1):56-66
references: 15621012
references: Oncogene. 2013 Jan 31;32(5):663-9
references: 22391558
references: Nutr Rev. 2005 Jun;63(6 Pt 1):183-95
references: 16028562
references: PLoS One. 2007 May 02;2(5):e399
references: 17476321
references: Nat Rev Mol Cell Biol. 2003 Sep;Suppl:SS1-5
references: 14587519
references: Ann N Y Acad Sci. 2003 Mar;983:197-207
references: 12724224
references: N Engl J Med. 2008 Mar 13;358(11):1148-59
references: 18337604
references: Cancer. 2006 Apr 15;106(8):1794-803
references: 16532500
references: Nature. 2010 Aug 26;466(7310):1129-33
references: 20639862
references: Nucleic Acids Res. 2005 Dec 02;33(21):6823-36
references: 16326863
references: Cell Oncol (Dordr). 2012 Aug;35(4):231-41
references: 22836879
references: Science. 2013 Jan 25;339(6118):448-52
references: 23223451
references: Cell. 2013 Feb 28;152(5):1146-59
references: 23434322
references: J Biomed Opt. 2009 Mar-Apr;14(2):024013
references: 19405743
references: J Cell Sci. 2012 Jun 15;125(Pt 12):2954-64
references: 22393239
references: Epigenomics. 2009 Dec;1(2):239-59
references: 20495664
references: Nature. 2012 Sep 6;489(7414):57-74
references: 22955616
references: Cell. 2007 Feb 23;128(4):635-8
references: 17320500
references: Science. 2009 May 15;324(5929):930-5
references: 19372391
references: Mol Cell Biol. 1998 Nov;18(11):6538-47
references: 9774669
references: Genes Dev. 2001 Mar 15;15(6):710-23
references: 11274056
references: Epigenetics. 2007 Apr-Jun;2(2):119-25
references: 17965619
references: Oncotarget. 2011 Aug;2(8):627-37
references: 21896958
references: Cancer Res. 2012 Nov 1;72(21):5600-12
references: 22926560
references: Nat Rev Cancer. 2004 Jun;4(6):437-47
references: 15170446
references: Cancer. 2003 Aug 1;98(3):522-8
references: 12879469
references: Science. 2003 Apr 18;300(5618):455
references: 12702868
references: Nat Biotechnol. 2011 Jan;29(1):68-72
references: 21151123
references: PLoS Genet. 2007 Aug;3(8):e137
references: 17708683
references: Cancer Res. 2006 Sep 1;66(17):8469-76
references: 16951158
references: Annu Rev Biochem. 2012;81:97-117
references: 22404632
references: J Cell Sci. 1989 Jun;93 ( Pt 2):221-6
references: 2693468
references: Nucleic Acids Res. 2012 Jun;40(11):4841-9
references: 22362737
references: Genes Dev. 1999 Aug 1;13(15):1924-35
references: 10444591
references: Anal Chem. 2010 Aug 1;82(15):6415-21
references: 20586411
references: J Fluoresc. 2008 Nov;18(6):1093-101
references: 18431548
references: Nat Biotechnol. 2012 Nov;30(11):1107-16
references: 23138310
references: Cell. 2011 Dec 23;147(7):1498-510
references: 22196727
references: J Biomed Opt. 2013 Feb;18(2):25002
references: 23392382
references: Nucleic Acids Res. 2011 Jul;39(12):5015-24
references: 21378125
references: J Neurophysiol. 2002 Aug;88(2):639-49
references: 12163517
references: Annu Rev Biochem. 1998;67:509-44
references: 9759496
references: Biochim Biophys Acta. 2007 Jan;1775(1):138-62
references: 17045745
references: Adv Exp Med Biol. 2013;754:31-56
references: 22956495
references: J Neurosci. 2010 Dec 8;30(49):16409-16
references: 21147979
references: Mol Cancer Ther. 2011 Jun;10(6):1113-23
references: 21460102
references: Nat Rev Cancer. 2011 Jun;11(6):393-410
references: 21606941
references: Nat Methods. 2007 Nov;4(11):963-73
references: 17971781
references: Angew Chem Int Ed Engl. 2011 Jul 11;50(29):6460-8
references: 21688365
references: Int J Mol Sci. 2012 Oct 09;13(10):12890-910
references: 23202928
references: Nat Genet. 2001 Jan;27(1):31-9
references: 11137995
references: Epigenetics. 2010 May 16;5(4):293-6
references: 20418669
references: J Phys Chem B. 2012 Oct 11;116(40):12125-32
references: 22950363
references: J Neurosci. 2001 Feb 1;21(3):788-97
references: 11157065
references: Nat Rev Genet. 2012 May 29;13(7):484-92
references: 22641018
references: Methods Mol Biol. 2009;464:321-41
references: 18951193
references: Nucleic Acids Res. 2013 Apr;41(7):3973-85
references: 23408854
references: Cancer Res. 2006 Sep 1;66(17):8462-9468
references: 16951157
references: Biophys J. 2004 Nov;87(5):3518-24
references: 15339818
references: Prog Mol Biol Transl Sci. 2012;108:129-58
references: 22656376
references: Biochemistry. 1978 Aug 22;17(17):3448-53
references: 28749
references: J Biol Chem. 2011 Dec 30;286(52):44788-98
references: 22084246
references: J Phys Chem A. 2008 Feb 21;112(7):1511-8
references: 18229902