doi: 10.1073/pnas.080469697.
Collaboration of signal transducer and activator of transcription 1 (STAT1) and BRCA1 in differential regulation of IFN-gamma target genes
Affiliations
- PMID: 10792030
- PMCID: PMC25807
- DOI: 10.1073/pnas.080469697
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Collaboration of signal transducer and activator of transcription 1 (STAT1) and BRCA1 in differential regulation of IFN-gamma target genes
Proc Natl Acad Sci U S A.
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Abstract
Most of the activities of IFN-gamma are the result of STAT1-mediated transcriptional responses. In this study, we show that the BRCA1 tumor suppressor acts in concert with STAT1 to differentially activate transcription of a subset of IFN-gamma target genes and mediates growth inhibition by this cytokine. After IFN-gamma treatment, induction of the cyclin-dependent kinase inhibitor, p21WAF1, was synergistically activated by BRCA1, whereas the IRF-1 gene was unaffected. Importantly, the differential induction of p21WAF1 was impaired in breast cancer cells homozygous for the mutant BRCA1 5382C allele. Biochemical analysis illustrated that the mechanism of this transcriptional synergy involves interaction between BRCA1 aa 502-802 and the C-terminal transcriptional activation domain of STAT1 including Ser-727 whose phosphorylation is crucial for transcriptional activation. Significantly, STAT1 proteins mutated at Ser-727 bind poorly to BRCA1, reinforcing the importance of Ser-727 in the recruitment of transcriptional coactivators by STAT proteins. These findings reveal a novel mechanism for BRCA1 function in the IFN-gamma-dependent tumor surveillance system.
Figures
BRCA1 expression enhances IFN-γ-dependent transcription. 2fTGH, U3A, G8, and 293T cells were transfected with a STAT reporter gene (3x Ly6e-LUC) plus BRCA1 expression constructs or empty vectors for 40 h and followed by a 6-h IFN-γ treatment (10 ng/ml) as indicated. Extracts were prepared, and the luciferase assay was performed. The combination of transfected plasmids and IFN-γ treatment is shown at the bottom. Luciferase light units are plotted on the y axis as fold activation in response to IFN-γ. Relative luciferase activity was normalized to a cotransfected β-galactosidase internal control.
IFN-γ responsive genes are differentially regulated by BRCA1. (A) Quantitative RT-PCR analysis of p21WAF1 and IRF-1. 293T cells were transiently transfected with vector (lanes 1 and 2) or BRCA1 expression plasmid for 48 h (lanes 3 and 4). Before total RNA extraction, 293T cells and HCC1937 breast cancer cells were treated with IFN-γ (10 ng/ml, 4 h) (lanes 2, 4, and 6). Fragments of p21WAF1, IRF-1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were PCR amplified with [α-32P]dCTP by appropriate primers (see Materials and Methods). After 5% acrylamide gel electrophoresis, gels were dried and autoradiography performed. No amplification was observed without reverse transcription of the RNA (not shown). Relative induction from phosphorimaging of genes is indicated under the panels. (B) Expression level of p21WAF1 protein in G8 cells treated with recombinant adenovirus plus IFN-γ. Cells were infected with Ad-LacZ or Ad-BRCA1 virus for 2 days. Twelve hours before assay, cells were treated with IFN-γ, and immunoblot analysis was done to study the p21WAF1 protein level.
STAT1α and BRCA1 form a complex both in vitro and in vivo. (A and B) Coimmunoprecipitation of endogenous BRCA1 and STAT1α from IFN-γ-treated 293T cells. BRCA1 was immunoprecipitated from 1.2 mg of IFN-γ-treated (10 ng/ml, 12 h) (lane 3) or untreated (lane 4) cells. Total cell lysates (20 μg) were also analyzed for the positive control of protein expression (lanes 1 and 2). Samples were separated by 6% SDS/PAGE, and immunoblotted with (A) anti-STAT1 antibody or (B) anti-BRCA1 antibody . (C) Copurification of STAT1 with BRCA1. GST-BRCA1 and FLAG-tagged STAT1α or STAT1αSer-727A were cotransfected into 293T cells. For controls, separate plates were transfected with GST vector alone or GST-BRCA1 expression vector with or without FLAG-tagged STAT1α expression vector in the combinations shown at the bottom of the panel. Cells were treated with IFN-γ as indicated (10 ng/ml, 12 h) (lanes 3–6), and GST or GST-BRCA1 was purified from extracts with glutathione beads. Samples were separated by 6% SDS/PAGE, and immunoblotted with anti-FLAG antibody. (D, E, and F) Detection of exogenously expressed proteins. Total cell lysates (20 μg) were separated by 6% (D and E) or 10% (F) SDS/PAGE, and the expression level of FLAG-tagged STAT1α (D), GST-BRCA1 (E), and GST (F) were confirmed by immunoblot analysis by using anti-FLAG or anti-GST antibody.
Purified BRCA1 binds to STAT1α C terminus 38 aa. (A) Purification of full-length BRCA1 and GST-STAT1α C terminus. Full-length BRCA1 was partially purified through a Ni column from Sf9 cells infected with recombinant baculovirus, BVC8B, expressing His-tagged BRCA1. GST-fused STAT1α C terminus was bacterially expressed, and purified through glutathione beads. Coomassie staining of BRCA1 (1 μg, lane 1), GST (5 μg, lane 2), and GST-STAT1C terminus (5 μg, lane 3) are shown. Arrows indicate the purified proteins. (B) Detection of binding of purified BRCA1 to STAT1α C terminus. Purified BRCA1 (500 ng) was incubated with 1 μg of GST (lane 2) or GST-STAT1α C terminus (lane 3). After precipitation with glutathione beads, samples were subjected to immunoblot analysis by using anti-BRCA1 antibody, Ab-1. Purified BRCA1 (200 ng) was loaded as a control (lane 1).
Identification of the STAT1-binding segment of BRCA1. Six GST-BRCA1 fusion proteins (aa 1–324; 260–553; 502–802; 758–1,064; 1,005–1,313; and 1,314–1,863) were generated in E. coli, and used for an in vitro binding assay (from lanes 4 to 9, respectively). GST protein alone was used for lane 3. Approximately equal amounts of each GST-fusion protein, bound to glutathione-Sepharose beads, were incubated with an extract of IFN-γ-treated (10 ng/ml, 15 min) 293T cells. Bound proteins were recovered and separated electrophoretically. Total lysate (20 μg) was loaded as a control (lane 1). Lane 2 contains no protein. The separated proteins were immunoblotted for STAT1. This STAT1 antiserum recognizes a 91-kDa (STAT1α), not 84-kDa (STAT1β), protein bound to GST-BRCA1 (aa 502–802) in lane 6, showing that STAT1α specifically binds to BRCA1.
Mammalian two-hybrid analysis demonstrates functional interaction between BRCA1 and STAT1. Both BRCA1 (502–802) segment fused with GAL4AD (AD-BRCA1) and full-length BRCA1 (BRCA1) can hyperactivate a STAT1α C-terminal transcriptional activation domain fused with GAL4 DBD (DBD-STAT). The reporter plasmids contain four copies of GAL4-binding sites upstream of the luciferase gene. Each construct shown at the bottom of the panel was expressed in 293T cells transiently with (+) or without (−) IFN-γ treatment (10 ng/ml, 6 h). Relative activity was normalized with a β-galactosidase control. In all cases, luciferase activity was determined at 48 h posttransfection.
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