Induction of the BRCA2 promoter by nuclear factor-kappa B.
BRCA2 is a tumor suppressor gene that has been implicated in response to DNA damage, cell cycle control, and transcription. BRCA2 has been found to be overexpressed in many breast tumors, suggesting that altered expression of the BRCA2 gene may contribute to breast tumorigenesis. To determine how BRCA2 is overexpressed in tumors, we investigated the transcriptional regulation of the BRCA2 promoter. Deletion mapping of the BRCA2 promoter identified three regions associated with 3-fold activation or repression and one upstream stimulatory factor binding site associated with 20-fold activation. Gel shift and cotransfection studies verified the role of USF in regulation of BRCA2 transcription. Analysis of the -144 to -59 region associated with 3-fold activation identified a putative NFkappaB binding site. Cotransfection of the p65 and p50 subunits of NFkappaB up-regulated the BRCA2 promoter 16-fold in a luciferase reporter assay, whereas mutations in the binding site ablated the effect. Gel shift and supershift assays with anti-p65 and -p50 antibodies demonstrated that NFkappaB binds specifically to the NFkappaB site. In addition, ectopic expression of NFkappaB resulted in increased levels of endogeneous BRCA2 expression. Thus, NFkappaB and USF regulate BRCA2 expression through the BRCA2 promoter.
Wu
K
K
Departments of Laboratory Medicine and Pathology, and Biochemistry and Molecular Biology and the Endocrinology Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Jiang
S W
SW
Thangaraju
M
M
Wu
G
G
Couch
F J
FJ
D024682:BRCA2 Protein; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D005786:Gene Expression Regulation; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D009363:Neoplasm Proteins / Q000235:genetics*; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D051996:Transcription Factor RelA; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D024682:BRCA2 Protein; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D009363:Neoplasm Proteins; D051996:Transcription Factor RelA; D014157:Transcription Factors; D012313:RNA; D008156:Luciferases