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Nucleic Acids Research 2005 33(13):4243-4254; doi:10.1093/nar/gki729
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Published online 28 July 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Article

Specific binding of the methyl binding domain protein 2 at the BRCA1-NBR2 locus

Emilie Auriol, Lise-Marie Billard, Frédérique Magdinier1 and Robert Dante*

Laboratoire de Génétique et Cancer, FRE2692 CNRS, UCBL1 8 avenue Rockefeller, 69373 Lyon cedex 08, France 1Laboratory of Molecular Embryology, NIH/NICHD Bethesda, MD, USA

*To whom correspondence should be addressed. Tel: +33 4 78 78 59 22; Fax: +33 4 78 78 27 20; Email: dante{at}univ-lyon1.fr

Received April 15, 2005. Revised July 5, 2005. Accepted July 5, 2005.

The methyl-CpG binding domain (MBD) proteins are key molecules in the interpretation of DNA methylation signals leading to gene silencing. We investigated their binding specificity at the constitutively methylated region of a CpG island containing the bidirectional promoter of the Breast cancer predisposition gene 1, BRCA1, and the Near BRCA1 2 (NBR2) gene. In HeLa cells, quantitative chromatin immunoprecipitation assays indicated that MBD2 is associated with the methylated region, while MeCP2 and MBD1 were not detected at this locus. MBD2 depletion (~90%), mediated by a transgene expressing a small interfering RNA (siRNA), did not induce MeCP2 or MBD1 binding at the methylated area. Furthermore, the lack of MBD2 at the BRCA1-NBR2 CpG island is associated with an elevated level of NBR2 transcripts and with a significant reduction of induced-DNA-hypomethylation response. In MBD2 knockdown cells, transient expression of a Mbd2 cDNA, refractory to siRNA-mediated decay, shifted down the NBR2 mRNA level to that observed in unmodified HeLa cells. Variations in MBD2 levels did not affect BRCA1 expression despite its stimulation by DNA hypomethylation. Collectively, our data indicate that MBD2 has specific targets and its presence at these targets is indispensable for gene repression.

Present addresses: Emilie Auriol and Robert Dante, Unité INSERM 590, Laboratoire d'Oncologie Moléculaire, Centre Léon Bérard, 28 rue Laennec, 69373 Lyon Cedex 08, France

Frédérique Magdinier, Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR 5161-ENS, Lyon, France


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