Specific binding of the methyl binding domain protein 2 at the BRCA1-NBR2 locus.
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 (approximately 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.
Auriol
Emilie
E
Laboratoire de Génétique et Cancer, FRE2692 CNRS, UCBL1, 8 avenue Rockefeller, 69373 Lyon cedex 08, France.
Billard
Lise-Marie
LM
Magdinier
Frédérique
F
Dante
Robert
R
D001665:Binding Sites; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D018899:CpG Islands; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020868:Gene Silencing; D019398:Genes, BRCA1; D006367:HeLa Cells; D006801:Humans; D051783:Methyl-CpG-Binding Protein 2; D009363:Neoplasm Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D062085:RNA, Long Noncoding; D012097:Repressor Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C066141:MBD1 protein, human; C489325:MBD2 protein, human; C497392:MECP2 protein, human; D051783:Methyl-CpG-Binding Protein 2; C107422:NBR2 lncRNA, human; D009363:Neoplasm Proteins; D062085:RNA, Long Noncoding; D012097:Repressor Proteins; D014157:Transcription Factors