Isolation and initial characterization of the BRCA2 promoter

Abstract

The hereditary breast cancer susceptibility gene, BRCA2, is considered to be a tumor suppressor gene that may be involved in the cellular response to DNA damage. The transcript for this gene is cell cycle regulated with mRNA levels reaching a peak just before the onset of DNA synthesis. In order to de®ne the mechanisms by which BRCA2 is transcriptionally regulated, we have begun to study upstream regulatory sequences. In this report, we de®ne a minimal promoter region that has strong activity in human breast epithelial cells. Deletions of this sequence narrowed the strong basal activity to a region extending from 766 to +129 with respect to the BRCA2 transcriptional start site. This sequence demonstrated cell cycle regulated activity with kinetics similar to the endogenous transcript. Examination of the sequence revealed several consensus binding sites for transcription factors including an E-box, E2F and Ets recognition motifs. Electrohoretic mobility shift assays revealed speci®c protein binding to two sequences upstream of the start site; the palindromic Ebox and an Ets/E2F site. Site-directed mutagenesis of either of these sites reduced both the basal activity in log phase cells and the cell cycle regulated activity of the promoter. Mutational inactivation of both sites within the same construct eectively eliminated promoter activity. Antibodies to candidate transcription factors used in super shift experiments revealed speci®c interactions between the BRCA2 promoter and the basic region/helix ± loop ± helix containing USF-1 and 2 proteins and Elf-1, an Ets domain protein. Binding of these factors depended upon the presence of intact recognition sequences. The USF factors were shown to bind predominantly as a heterodimeric complex of USF-1 and 2 while Elf-1 bound the promoter when it was not occupied by USF. Co-transfection studies with USF proteins and the varicella zoster IE62 protein provide evidence for the involvement of endogenous and exogenous USF in the activation of the BRCA2 promoter. We propose that interactions between USF-1, USF-2 and Elf-1 play an important role in the transcriptional regulation of the BRCA2 gene.

Conformational phenotype of p53 is linked to nuclear translocation

“…However, in most cells expressing wild-type p53, the level of protein is undetectable due to a rapid turnover rate. Therefore, we decided to use a cell line, such as SK-N-SH, a neuroblastoma derived cell line, which expresses wildtype p53 determined by sequencing (Davido et al, 1992) which is cytoplasmically sequestered and more stable (Moll et al, 1996;Zaika et al, 1999). We exposed SK-N-SH cells to 5 Gy IR and observed strong translocation of p53 into the nucleus within 3 h by indirect immuno¯uorescence using PAb 421 ( Figure 4A).…”

Induction of the BRCA2 Promoter by Nuclear Factor-κB

“…USF Regulates BRCA2 Basal Transcription-The Ϫ34 to Ϫ15 region has recently been reported to be responsible for regulation of the basal activity of the BRCA2 promoter (15). The USF binding site was shown to regulate promoter activity in a cell cycle-dependent manner, with binding of USF resulting in 3-fold induction of luciferase activity.…”

“…Low levels of expression are detected in G 0 , G 1 , and M phase (14). Cell cycle-dependent expression has recently been associated with binding of the upstream stimulatory factor (USF) 1 protein and Elf-1 transcription factor to the BRCA2 promoter (15). In addition, BRCA2 expression is elevated indirectly in response to the mitogenic activity of estrogen, which has been associated with progression of the cell cycle (16,17).…”

Pea3 Transcription Factor Cooperates with USF-1 in Regulation of the Murine bax Transcription without Binding to an Ets-binding Site

“…The location of the E box and the EBS at the vicinity of the transcriptional initiation site is documented. A functional E box has been described at position Ϫ7 to Ϫ2 from the transcriptional initiation site of the cathepsin B gene (60), as have an E box and an Ets/E2F site, respectively, at Ϫ18 and Ϫ58 bp on human BRCA2 gene promoter (56). By taking into account the close vicinity of the E boxes to the transcriptional initiation site in the context of the proximal bax promoter and the drastic effect of mutation of these E boxes on the activity of this promoter, we can suggest that the binding of Pea3 with USF-1 to these E boxes is involved in and can facilitate the formation of the transcriptional preinitiation complex.…”

“…Ets-1 and USF-1 are widely known to interact, for example, on the human immunodeficiency virus, type 1, long terminal repeat (55), on the BRCA2 promoter (56) or on the mannose receptor promoter (57). Since Greenall et al (53) have shown by an in vitro glutathione S-transferase pulldown assay, a physical interaction between USF-1 and the ETS domain of Pea3, we then confirmed by co-immunoprecipitation assay in TAC-2.1 cells that these two factors interact (data not shown).…”