Enzymatic analysis of Tet proteins: key enzymes in the metabolism of DNA methylation.
One of the most exciting recent advances in the epigenetic field is the discovery that 5-methylcytosine (5mC) in DNA can be iteratively oxidized by a family of proteins known as Tet proteins to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). These 5mC derivatives can be further processed by thymine-DNA glycosylase (TDG) followed by base excision repair or by replication-dependent dilution leading to DNA demethylation. Given the similarity between 5mC and its oxidation derivatives, many of the conventional techniques used for 5mC analysis cannot distinguish between 5mC and 5hmC/5fC/5caC. Here, we describe 2D-TLC and mass spectrometry methods that we have successfully used in differentiating 5mC from its oxidative derivatives as well as in characterizing the enzymatic activity of Tet proteins both in vitro and in vivo.
Howard Hughes Medical Institute; Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Li
L
Shen
Yi
Y
Zhang
D044503:5-Methylcytosine / Q000737:chemistry* / Q000302:isolation & purification; D000818:Animals; D001483:Base Sequence; D002855:Chromatography, Thin Layer; D004247:DNA / Q000737:chemistry; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D049308:Dioxygenases; D057075:Enzyme Assays; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D010084:Oxidation-Reduction; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; C540839:TET1 protein, mouse; D044503:5-Methylcytosine; D004247:DNA; D049308:Dioxygenases; C551914:Tet2 protein, mouse; C551915:Tet3 protein, mouse