Multifunctional transcription factor TFII-I is an activator of BRCA1 function

Abstract

Background: The TFII-I is a multifunctional transcriptional factor known to bind specifically to several DNA sequence elements and to mediate growth factor signalling. A microdeletion at the chromosomal location 7q11.23 encoding TFII-I and the related family of transcription factors may result in the onset of Williams-Beuren syndrome, an autosomal dominant genetic disorder characterised by a unique cognitive profile, diabetes, hypertension, anxiety, and craniofacial defects. Hereditary breast and ovarian cancer susceptibility gene product BRCA1 has been shown to serve as a positive regulator of SIRT1 expression by binding to the promoter region of SIRT1, but cross talk between BRCA1 and TFII-I has not been investigated to date.

Methods: A physical interaction between TFII-I and BRCA1 was explored. To determine pathophysiological function of TFII-I, its role as a transcriptional cofactor for BRCA1 was investigated.

Results: We found a physical interaction between the carboxyl terminus of TFII-I and the carboxyl terminus of BRCA1, also known as the BRCT domain. Endogenous TFII-I and BRCA1 form a complex in nuclei of intact cells and formation of irradiation-induced nuclear foci was observed. We also showed that the expression of TFII-I stimulates the transcriptional activation function of BRCT by a transient expression assay. The expression of TFII-I also enhanced the transcriptional activation of the SIRT1 promoter mediated by full-length BRCA1.

Conclusion: These results revealed the intrinsic mechanism that TFII-I may modulate the cellular functions of BRCA1, and provide important implications to understand the development of breast cancer.

Figure 1

In vivo and in vitro association between TFII-I and BRCA1, and mapping of the BRCT-interacting region of TFII-I. (A) Identification of the interaction between BRCT and TFII-I using GST-BRCT. Bacterially expressed GST fusion proteins immobilised on beads were used in in vitro pull-down assays. Nuclear extracts of HeLa cells were incubated with GST-BRCT. The beads were extensively washed, and followed by immunoblotting (IB) using anti-TFII-I antibodies. (B) The complex formation of TFII-I and BRCA1 in HeLa cells was analysed by co-immunoprecipitation (IP) with the antibodies to BRCA1 (epitope mapping at the carboxyl-terminus of BRCA1). The immunoblotting analysis using anti-TFII-I antibodies revealed the existence of TFII-I in cell lysate immunoprecipitates (Figure 1B, 1), which indicates that TFII-I physically associates with BRCA1 in living cells. Reciprocal immunoprecipitation analysis confirmed the association of TFII-I and BRCA1 (Figure 1B, 3). The whole-cell extracts of HCC1937 cells known to lack last BRCT domain were also immunoprecipitated with anti-BRCA1 antibodies (epitope mapping at the amino-terminus of BRCA1). The immunoblotting analysis revealed the absence of TFII-I in cell lysate immunoprecipitates (Figure 1B, 2), indicating the importance of BRCT as a binding surface of TFII-I. (C) Mapping of the BRCT-interaction region of TFII-I. COS7 cells were transfected with GST-tagged TFII-I (wild type, ΔN90, p70, and p46) and Flag-tagged BRCA1 expression vectors. Nuclear extracts of transfected COS7 cells were prepared and the complex formation of TFII-I and BRCA1 was analysed by IP with the anti-Flag M2 agarose beads, followed by IB using anti-GST antibodies. (D) A schematic diagram of the structure of TFII-I (wild type, ΔN90, p70, p46, and β isoform) is shown.

Figure 2

Colocalisation of BRCA1 and TFII-I in HeLa cells. (A–D) HeLa cells were either treated by 8-Gy of gamma-irradiation or none, fixed, and permeabilised. The cells were incubated with primary antibodies and subsequently with secondary antibodies. The expression of BRCA1 (green) and TFII-I (red) was investigated under the confocal fluorescence microscopy (Carl-Zeiss). Representative immunofluorescence studies are shown (A, control cells; B, irradiated cells; 1, TFII-I; 2, BRCA1; 3, merge; 4, 4′, 6-diamino-2-phenylindole staining). Arrows in B3 indicate a cell showing nuclear foci formation of TFII-I and BRCA1. Bars indicate 10 μm.

Figure 3

TFII-I stimulates transcription of GAL4-BRCT through its carboxyl-terminal domain. Transient transfection assays were performed to examine the cofactor activity of TFII-I in the transactivation function of GAL4-fused BRCT. COS7 cells were transfected with the indicated combinations of mammalian expression plasmids. At 24 h after transfection, the cells were harvested, and transfected whole-cell lysates were assayed for luciferase activity produced from the reporter plasmid (17M8-AdMLP-luc). TFII-I showed a specific stimulation of the transactivation function of BRCT. Carboxyl-terminus of TFII-I was indispensable for this stimulation of BRCT. The phRL Renilla CMV-luc vector was transfected as a control of transfection efficiency. Each experiment was repeated at least three times in triplicate. Error bars represent s.d.

Figure 4

TFII-I stimulates transcription by BRCA1 through its carboxyl-terminal domain. (A) Transient transfection assays were performed to examine the influence of TFII-I using an artificial luciferase reporter constructs. COS7 cells were transfected with the indicated combinations of mammalian expression plasmids. At 24 h after transfection, cells were harvested, and transfected whole-cell lysates were assayed for luciferase activity produced from the reporter plasmids. Full-length TFII-I and TFII-I ΔN90 showed specific upregulation of SIRT1 (1-2852)-luciferase activity mediated by BRCA1, while TFII-I p70 and TFII-I p46, lacking BRCT-interaction region, had no effect on SIRT1-luciferase activity. TFII-I showed no effect on SIRT1 (1-202)-luciferase activity that lack the binding domain of BRCA1. (B) The siRNA-mediated knockdown of BRCA1 decreased the expression of SIRT1. Knockdown of TFII-I also resulted in downregulation of SIRT1. Expression of BRCA1 and p53 was decreased by depletion of TFII-I. HeLa cells were transfected with indicated siRNA. At 48 h after transfection, cells were harvested and analysed by western blotting. (C) Chromatin immunoprecipitation assay was performed to confirm the recruitment of BRCA1 and TFII-I at the SIRT1 gene promoter and the p21 gene promoter. Both promoter regions are known to recruit BRCA1.