MBDin, a novel MBD2-interacting protein, relieves MBD2 repression potential and reactivates transcription from methylated promoters.
We have identified a human gene encoding a novel MBD2-interacting protein (MBDin) that contains an N-terminal GTP-binding site, a putative nuclear export signal (NES), and a C-terminal acidic region. MBDin cDNA was isolated through a two-hybrid interaction screening using the methyl-CpG-binding protein MBD2 as bait. The presence of the C-terminal 46-amino-acid region of MBD2 and both the presence of the acidic C-terminal 128-amino-acid region and the integrity of the GTP-binding site of MBDin were required for the interaction. Interaction between MBD2 and MBDin in mammalian cells was confirmed by immunoprecipitation experiments. Fluorescence imaging experiments demonstrated that MBDin mainly localizes in the cytoplasm but accumulates in the nucleus upon disruption of the NES or treatment with leptomycin B, an inhibitor of NES-mediated transport. We also found that MBDin partially colocalizes with MBD2 at foci of heavily methylated satellite DNA. An MBD2 deletion mutant lacking the C-terminal region maintained its subnuclear localization but failed to recruit MBDin at hypermethylated foci. Functional analyses demonstrated that MBDin relieves MBD2-mediated transcriptional repression both when Gal4 chimeric constructs and when in vitro-methylated promoter-reporter plasmids were used in transcriptional assays. Southern blotting and bisulfite analysis showed that transcriptional reactivation occurred without changes of the promoter methylation pattern. Our findings suggest the existence of factors that could be targeted on methylated DNA by methyl-CpG-binding proteins reactivating transcription even prior to demethylation.
Lembo
Francesca
F
Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano," Istituto di Endocrinologia ed Oncologia Sperimentale del C.N.R., Università degli Studi di Napoli "Federico II," 80131 Naples, Italy.
Pero
Raffaela
R
Angrisano
Tiziana
T
Vitiello
Carmen
C
Iuliano
Rodolfo
R
Bruni
Carmelo B
CB
Chiariotti
Lorenzo
L
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D001665:Binding Sites; D015152:Blotting, Northern; D015139:Blotting, Southern; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D019175:DNA Methylation; D018076:DNA, Complementary / Q000378:metabolism; D004276:DNA, Satellite / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D017353:Gene Deletion; D015723:Gene Library; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013447:Sulfites / Q000494:pharmacology; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D020798:Two-Hybrid System Techniques
D000935:Antifungal Agents; D018076:DNA, Complementary; D004276:DNA, Satellite; D004268:DNA-Binding Proteins; D005231:Fatty Acids, Unsaturated; C052130:GAL4 protein, S cerevisiae; C115368:MBD2 protein; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013447:Sulfites; D014157:Transcription Factors; D006160:Guanosine Triphosphate; C472669:GPN1 protein, human; D019204:GTP-Binding Proteins; C038753:leptomycin B