Biochemical fractionation reveals association of DNA methyltransferase (Dnmt) 3b with Dnmt1 and that of Dnmt 3a with a histone H3 methyltransferase and Hdac1.
De novo DNA methyltransferases, Dnmt3a and 3b, were purified by fractionation of S-100 extract from mouse lymphosarcoma cells through several chromatographic matrices followed by glycerol density gradient centrifugation. Dnmt3a was separated from Dnmt3b and Dnmt1 in the first column, Q-Sepharose whereas Dnmt3b co-purified with Dnmt1 after further fractionation through Mono-S and Mono-Q columns and glycerol density gradient centrifugation. Following purification, the majority of de novo DNA methyltransfearse activity was associated with Dnmt3b/Dnmt1 fractions. By contrast, the fractions containing Dnmt3a alone exhibited markedly reduced activity, which correlated with diminished expression of this isoform in these cells. Histone deacetylase 1(Hdac1) cofractionated with Dnmt3a throughout purification whereas Hdac1 was separated from Dnmt3b/Dnmt1 following chromatography on Mono-Q column. Dnmt3a purified through glycerol gradient centrifugation was also associated with a histone H3 methyltransferase (HMTase) activity whereas purified Dnmt3b/Dnmt1 was devoid of any HMTase activity. The activity of this HMTase was abolished when lysine 9 of N-terminal histone H3 peptide was replaced by leucine whereas mutation of lysine 4 to leucine inhibited this activity only partially. This is the first report on the identification of a few key co-repressors associated with endogenous Dnmt3a and of a complex containing Dnmt3b and a minor form of Dnmt1 following extensive biochemical fractionation.
Datta
Jharna
J
Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA.
Ghoshal
Kalpana
K
Sharma
Sudarshana M
SM
Tajima
Shoji
S
Jacob
Samson T
ST
D000818:Animals; D002499:Centrifugation, Density Gradient; D005591:Chemical Fractionation; D002847:Chromatography, Agarose; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry* / Q000302:isolation & purification; D000090205:DNA Methyltransferase 3A; D056284:Histone Deacetylase 1; D006655:Histone Deacetylases / Q000737:chemistry* / Q000302:isolation & purification; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; D008228:Lymphoma, Non-Hodgkin; D008780:Methyltransferases / Q000032:analysis / Q000737:chemistry*; D051379:Mice; D011496:Protein Methyltransferases; D014407:Tumor Cells, Cultured / Q000737:chemistry; D000097584:DNA Methyltransferase 3B
D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D056284:Histone Deacetylase 1; D006655:Histone Deacetylases; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; D008780:Methyltransferases; D011496:Protein Methyltransferases; D000097584:DNA Methyltransferase 3B; C000717967:DNMT3A protein, human; C000715535:Dnmt3a protein, mouse; C000619380:DNMT1 protein, human; C000619381:Dnmt1 protein, mouse; C110550:HDAC1 protein, human