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Molecular and Cellular Biology, March 2003, p. 1656-1665, Vol. 23, No. 5
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.5.1656-1665.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

MBDin, a Novel MBD2-Interacting Protein, Relieves MBD2 Repression Potential and Reactivates Transcription from Methylated Promoters

Francesca Lembo,1,2 Raffaela Pero,1 Tiziana Angrisano,1 Carmen Vitiello,1 Rodolfo Iuliano,3 Carmelo B. Bruni,1* and Lorenzo Chiariotti1,3*

Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano," Istituto di Endocrinologia ed Oncologia Sperimentale del C.N.R.,1 Dipartimento di Chimica Farmaceutica e Tossicologica, Università degli Studi di Napoli "Federico II," 80131 Naples,2 Dipartimento di Medicina Sperimentale e Clinica "G. Salvatore," Università degli Studi "Magna Graecia" di Catanzaro, 88100 Catanzaro, Italy3

Received 26 August 2002/ Returned for modification 26 September 2002/ Accepted 2 December 2002

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.

* Corresponding author. Mailing address: Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano," Università degli Studi di Napoli "Federico II," Via S. Pansini 5, 80131 Naples, Italy. Phone: 081-7462056. Fax: 081-7703285. E-mail for Carmelo B. Bruni: brucar{at}unina.it. E-mail for Lorenzo Chiariotti: chiariot{at}unina.it.

Molecular and Cellular Biology, March 2003, p. 1656-1665, Vol. 23, No. 5
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.5.1656-1665.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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