title: Quantitative assessment of Tet-induced oxidation products of 5-methylcytosine in cellular and tissue DNA.
abstract: Recent studies showed that Ten-eleven translocation (Tet) family dioxygenases can oxidize 5-methyl-2'-deoxycytidine (5-mdC) in DNA to yield the 5-hydroxymethyl, 5-formyl and 5-carboxyl derivatives of 2'-deoxycytidine (5-HmdC, 5-FodC and 5-CadC). 5-HmdC in DNA may be enzymatically deaminated to yield 5-hydroxymethyl-2'-deoxyuridine (5-HmdU). After their formation at CpG dinucleotide sites, these oxidized pyrimidine nucleosides, particularly 5-FodC, 5-CadC, and 5-HmdU, may be cleaved from DNA by thymine DNA glycosylase, and subsequent action of base-excision repair machinery restores unmethylated cytosine. These processes are proposed to be important in active DNA cytosine demethylation in mammals. Here we used a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS/MS) method, along with the use of stable isotope-labeled standards, for accurate measurements of 5-HmdC, 5-FodC, 5-CadC and 5-HmdU in genomic DNA of cultured human cells and multiple mammalian tissues. We found that overexpression of the catalytic domain of human Tet1 led to marked increases in the levels of 5-HmdC, 5-FodC and 5-CadC, but only a modest increase in 5-HmdU, in genomic DNA of HEK293T cells. Moreover, 5-HmdC is present at a level that is approximately 2-3 and 3-4 orders of magnitude greater than 5-FodC and 5-CadC, respectively, and 35-400 times greater than 5-HmdU in the mouse brain and skin, and human brain. The robust analytical method built a solid foundation for dissecting the molecular mechanisms of active cytosine demethylation, for measuring these 5-mdC derivatives and assessing their involvement in epigenetic regulation in other organisms and for examining whether these 5-mdC derivatives can be used as biomarkers for human diseases.
authors: Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521, USA.
authors: Shuo
authors: S
authors: Liu
authors: Jin
authors: J
authors: Wang
authors: Yijing
authors: Y
authors: Su
authors: Candace
authors: C
authors: Guerrero
authors: Yaxue
authors: Y
authors: Zeng
authors: Devarati
authors: D
authors: Mitra
authors: Philip J
authors: PJ
authors: Brooks
authors: David E
authors: DE
authors: Fisher
authors: Hongjun
authors: H
authors: Song
authors: Yinsheng
authors: Y
authors: Wang
mesh_terms: D044503:5-Methylcytosine / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001923:Brain Chemistry; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000032:analysis; D049308:Dioxygenases / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D006899:Mixed Function Oxygenases; D010084:Oxidation-Reduction; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D012867:Skin / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D013936:Thymidine / Q000031:analogs & derivatives / Q000032:analysis
chemical_list: C402104:5-formyl-2'-deoxycytidine; C540838:5-hydroxymethyl-2'-deoxycytidine; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D003841:Deoxycytidine; C018080:5-hydroxymethyl-2'-deoxyuridine; D044503:5-Methylcytosine; D004247:DNA; D006899:Mixed Function Oxygenases; C462539:TET1 protein, human; D049308:Dioxygenases; D013936:Thymidine
references: Science. 2009 May 15;324(5929):929-30
references: 19372393
references: Science. 2011 Sep 2;333(6047):1303-7
references: 21817016
references: Cell. 2011 Apr 29;145(3):423-34
references: 21496894
references: Nucleic Acids Res. 2007;35(14):4833-44
references: 17626047
references: Nature. 2010 Aug 26;466(7310):1129-33
references: 20639862
references: Nucleic Acids Res. 2012 Aug;40(15):7368-74
references: 22581771
references: Nature. 2012 Nov 15;491(7424):449-53
references: 23123854
references: Nat Chem Biol. 2012 Sep;8(9):751-8
references: 22772155
references: Nat Chem Biol. 2012 Feb 12;8(4):328-30
references: 22327402
references: Chem Res Toxicol. 2006 May;19(5):614-21
references: 16696563
references: Nat Rev Mol Cell Biol. 2010 Sep;11(9):607-20
references: 20683471
references: Chem Res Toxicol. 2011 Nov 21;24(11):2040-3
references: 22007908
references: Cancer Res. 2011 Mar 1;71(5):1904-11
references: 21239472
references: J Am Chem Soc. 2006 Sep 27;128(38):12510-9
references: 16984202
references: Cancer Res. 2002 Jul 15;62(14):4075-80
references: 12124344
references: J Biol Chem. 2011 Oct 14;286(41):35334-35338
references: 21862836
references: Aging Cell. 2012 Aug;11(4):714-6
references: 22530741
references: Science. 2001 Aug 10;293(5532):1074-80
references: 11498575
references: Cell. 1992 Jun 12;69(6):915-26
references: 1606615
references: Oncogene. 2013 Jan 31;32(5):663-9
references: 22391558
references: J Am Soc Mass Spectrom. 2006 Oct;17(10):1335-1341
references: 16872831
references: Nature. 2010 Dec 9;468(7325):839-43
references: 21057493
references: Cell. 1999 Oct 29;99(3):247-57
references: 10555141
references: Leukemia. 2003 Mar;17(3):637-41
references: 12646957
references: Cancer Res. 2011 Dec 15;71(24):7360-5
references: 22052461
references: Science. 2009 May 15;324(5929):930-5
references: 19372391
references: Cell. 2011 Jul 8;146(1):67-79
references: 21722948
references: Anal Chem. 2011 Mar 15;83(6):2201-9
references: 21323344
references: Nat Genet. 2003 Mar;33 Suppl:245-54
references: 12610534
references: Cell. 2011 Dec 23;147(7):1498-510
references: 22196727
references: Org Lett. 2011 Jul 1;13(13):3446-9
references: 21648398
references: Genes Dev. 2004 Oct 1;18(19):2315-35
references: 15466484
references: Cell. 2008 Jun 27;133(7):1145-8
references: 18585349
references: Science. 2011 Sep 2;333(6047):1300-3
references: 21778364