Physical and functional interaction of DNA methyltransferase 3A with Mbd3 and Brg1 in mouse lymphosarcoma cells.
Dnmt3a and Dnmt3b are de novo DNA methyltransferases that also act as transcriptional repressors independent of methyltransferase activity. To elucidate the underlying mechanism of transcriptional repression, Dnmt3a was purified from mouse lymphosarcoma cells (P1798) by extensive fractionation on five different chromatographic matrices followed by glycerol density gradient centrifugation. Liquid chromatography electrospray tandem mass spectrometry analysis of Dnmt3a-associated polypeptides identified the methyl CpG binding protein Mbd3, histone deacetylase 1(Hdac1), and components of Brg1 complex (Brg1, Baf155, and Baf57) in the purified preparation. Association of Dnmt3a with Mbd3 and Brg1 was confirmed by coimmunoprecipitation and coimmunolocalization studies. Glutathione S-transferase pulldown assay showed that the NH2-terminal ATRX homology domain of Dnmt3a interacts with the methyl CpG binding domain of Mbd3 and with both bromo and ATPase domains of Brg1. Chromatin immunoprecipitation assay revealed that all three proteins are associated with transcriptionally silent methylated metallothionein (MT-I) promoter in the mouse lymphosarcoma cells. To understand the functional significance of their association with the promoter, their role on the MT-I promoter activity was analyzed by transient transfection assay. The results showed that Mbd3 and Dnmt3a specifically inhibited the methylated promoter, and the catalytic activity of Dnmt3a was dispensable for the suppression. In contrast, the wild-type but not the ATPase-inactive mutant of Brg1 suppressed MT-I promoter irrespective of its methylation status, implicating involvement of ATP-dependent chromatin remodeling in the process. Coexpression of two of the three interacting proteins at a time augmented their repressor function. This study shows physical and functional interaction of Dnmt3a with components of nucleosome remodeling machinery.
Datta
Jhrana
J
Department of Molecular and Cellular Biochemistry, Ohio State University, Columbus, Ohio 43210, USA.
Majumder
Sarmila
S
Bai
Shoumei
S
Ghoshal
Kalpana
K
Kutay
Huban
H
Smith
David Spencer
DS
Crabb
John W
JW
Jacob
Samson T
ST
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000235:genetics; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism; D000090205:DNA Methyltransferase 3A; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008113:Liver Neoplasms / Q000201:enzymology / Q000235:genetics; D008228:Lymphoma, Non-Hodgkin / Q000201:enzymology*; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000075924:X-linked Nuclear Protein
D004268:DNA-Binding Proteins; C000717967:DNMT3A protein, human; C000715535:Dnmt3a protein, mouse; C492013:Mbd3 protein, mouse; D009687:Nuclear Proteins; D014157:Transcription Factors; D008668:Metallothionein; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; C084108:SMARCA4 protein, human; C513194:Smarca4 protein, mouse; D004265:DNA Helicases; C490462:Atrx protein, mouse; D000075924:X-linked Nuclear Protein