BRCA1 targets p53-responsive elements. (A) Basic structure of the minimal reporter plasmid. Two (PGluc, SRFluc, NFκBluc, and E2Fluc) or four (Mycluc and GAL4luc) copies of DNA binding sequences were cloned upstream of c-fos promoter TATA sequence and luciferase genes. (B) BRCA1 activates p53-responsive elements. 293T cells were transfected with empty vector or BRCA1 expression plasmids together with several reporter plasmids indicated, and luciferase activity was measured. Each of the experiments was done in duplicate or triplicate, and normalized by β-galactosidase activity. Lanes: 1, 3, 5, 7, and 9, pcDNA3 vector; 2, 4, 6, 8, and 10, BRCA1/PCDNA3. (C) BRCA1 is not an general coactivator. 293T cells were cotransfected with p53, E2F1, and DP1 expression plasmids and/or BRCA1 to detect activation of PGluc or E2Fluc. Lanes: 1 and 5, pcDNA3 vector; 2, p53; 3 and 7, BRCA1; 4, p53 plus BRCA1; 6, E2F1 plus DP1; 8, E2F1, DP1 plus BRCA1.

BRCA1 requires wild-type p53 to activate p53-responsive elements. (A) 10.1Val5 mouse fibroblasts expressing temperature sensitive forms of murine p53 (wild type at 32°C and mutant at 39°C) were transfected with vector alone or BRCA1/pcDNA3 together with PGluc. Cells were transfected with the indicated DNA for 3 hr at 39°C, and were maintained at 32°C (lane 1 and 2) or 39°C (lane 3 and 4) for an additional 2 days before luciferase assay. Lanes: 1 and 3, pcDNA3 vector; 2 and 4, BRCA1/pcDNA3. (B) Primary fibroblasts prepared from p53 knockout mice were transfected and used for luciferase assays of PGluc. Approximately 2 × 10⁶ p53 (+/+) cells (lane 1 and 2) or p53 (−/−) cells (lanes 3 to 6) were used for each of the assays. Lanes: 1 and 3, pcDNA3 vector; 2 and 5, BRCA1; 4, p53; 6, BRCA1 plus p53. Values shown here are the average of three or four independent experiments (A and B).

Coimmunoprecipitation of BRCA1 and p53. (A) Endogenous BRCA1 protein was immunoprecipitated by anti-BRCA1 polyclonal antibody (C-20) from 300 μg of nuclear extract of human breast epithelial cell line HBL100. Samples were separated by SDS/10% polyacrylamide gel, and immunoblotted by anti-p53 mAb (DO-1). Lane 1, 10 μg of nuclear extract; lane 2, immunoprecipitation by C-20; lane 3, immunopreciptation by normal rabbit IgG. (B) BRCA1 binds to the C-terminal region of p53, in vitro. Lysates prepared from baculovirus-infected Sf9 cells expressing human BRCA1 were incubated with GST (lane 2), GST-p53(1–110) (lane 3), GST-p53(111–270) (lane 4), GST-p53(270–393) (lane 5) and GST-p53(1–393) (lane 6). Ten microliters of Sf9 lysates of baculoviral BRCA1 was loaded as a control of immunoblot (lane 1). Samples were separated by SDS/6% polyacrylamide gel, and blotted with C-20.

BRCA1 stimulates genomic promoters containing p53-responsive elements. (A) Genomic organization of the murine mdm2 promoter. The first intron of the 1.0-kb murine mdm2 and adenovirus major late TATA box/TdT initiation signal were cloned into pGL2 vector to generate mdm2luc. Exons 1–3 (EX1–EX3), and p53-responsive elements (PG) are indicated. (B and D) The procedure for luciferase analysis of mdm2luc (B) or p21luc (D) was basically the same with that of Fig. 1. Lanes: 1, pcDNA3 vector; 2, p53; 3, BRCA1; 4, p53 plus BRCA1. (C) Genomic organization of the human p21^Waf1 promoter. Human p21^Waf1 promoter (2.4 kb) was cloned upstream of luciferase gene of pGL2 vector to generate p21luc. p53-responsive elements (PG1 and 2), interferon-regulatory elements (IRE), MyoD-responsive elements (MyoD), and signal transducers and activators of transcription (STAT) serum-inducible elements (SIE) are indicated. (E) Structure of deletion mutants of mdm2luc. mdm2luc was digested with SmaI or PvuII to generate mdm2smluc or mdm2pvluv, respectively. (F) Activation of mutant mdm2 promoters by BRCA1. Reporter plasmids of mdm2luc (lane 1 and 2), mdm2smluc (lane 3 and 4) and mdm2pvluc (lane 5 and 6) were cotransfected with vector (lane 1, 3, and 5) or BRCA1 (lane 2, 4, and 6). (G) Structure of deletion mutant forms of p21^Waf1 promoter. p21luc was digested with SacI to remove the first p53-responsive element (p21(−)luc). (H) Activation of p21luc or p21(−)luc by BRCA1. Lanes: 1 and 3, vector; 2 and 4, BRCA1.

The second BRCT domain is required for the transactivation of PGluc reporter plasmid. (A) Molecular feature of the mutant form of BRCA1 lacking the second BRCT domain. BRCA1/pcDNA3 was digested by ApaI to generate BRCA1–1769 construct. (B) Relative luciferase activity of PGluc regulated by vector (lane 1), the wild-type (lane 2) or mutant BRCA1 (lane 3) was measured by transfection of 293T cells.