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. 2013 Jul 9;8(7):e68075.
doi: 10.1371/journal.pone.0068075. Print 2013.

DNA Homologous Recombination Factor SFR1 Physically and Functionally Interacts With Estrogen Receptor Alpha

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Free PMC article

DNA Homologous Recombination Factor SFR1 Physically and Functionally Interacts With Estrogen Receptor Alpha

Yuxin Feng et al. PLoS One. .
Free PMC article

Abstract

Estrogen receptor alpha (ERα), a ligand-dependent transcription factor, mediates the expression of its target genes by interacting with corepressors and coactivators. Since the first cloning of SRC1, more than 280 nuclear receptor cofactors have been identified, which orchestrate target gene transcription. Aberrant activity of ER or its accessory proteins results in a number of diseases including breast cancer. Here we identified SFR1, a protein involved in DNA homologous recombination, as a novel binding partner of ERα. Initially isolated in a yeast two-hybrid screen, the interaction of SFR1 and ERα was confirmed in vivo by immunoprecipitation and mammalian one-hybrid assays. SFR1 co-localized with ERα in the nucleus, potentiated ER's ligand-dependent and ligand-independent transcriptional activity, and occupied the ER binding sites of its target gene promoters. Knockdown of SFR1 diminished ER's transcriptional activity. Manipulating SFR1 expression by knockdown and overexpression revealed a role for SFR1 in ER-dependent and -independent cancer cell proliferation. SFR1 differs from SRC1 by the lack of an intrinsic activation function. Taken together, we propose that SFR1 is a novel transcriptional modulator for ERα and a potential target in breast cancer therapy.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. SFR1-A interacts with ERα in mammalian cells and colocalized with ERα in cell nucleus.
(A) FLAG-SFR1-A and ER were co-transfected into Ishikawa cells. Co-IP experiments were performed with anti-ERα and anti-FLAG antibodies in Ishikawa cells in the presence and absence of E2. (B) The interaction of ERα and SFR1-A was further analyzed with mammalian one-hybrid experiments in Ishikawa cells. pM vector, pM-SFR1-A (SFR1-A) and pM-SRC1 were used as baits. Cells co-transfected with pM-SRC1 NR-box (SRC1) and pCMV5-ERα were used as positive control. (n = 4, p<0.005) (C) The localization of SFR1A and ERα was evaluated with immunefluorescent staining. HeLa cells were co-transfected with FLAG-SFR1-A and GFP-ERα. 24 hours after the transfection, cells were treated with E2 or vehicle overnight. The cells were fixed and stained with M2 anti-FLAG antibody and Alexa Fluor 568 anti-mouse secondary antibody. Immunostained cells were photographed using a Zeiss LSM510 confocal microscope with a 63X Zeiss objective. Scale bar = 20 µm. The error bars indicate standard deviations. * = P<0.05; ** = P<0.01; *** = P<0.005.
Figure 2
Figure 2. SFR1-A enhances ERα and AR transcriptional activity.
The effects of SFR1 overexpression on ERα (A and B), ERβ (D) and AR (C) transcriptional activity were assessed by luciferase reporter assays. Ishikawa cells were co-transfected with nuclear receptors (ERα, ERβ or AR), their respective reporter genes (3xERE-luc and ARE-luc), and indicated amount of p3xFLAG-SFR1-A, p3xFLAG-SFR1-B (20 ng) or p3xFLAG-SFR1-C (20 ng) (D). The cells were treated with vehicle or 1 nM E2 or DHT. Transcriptional activity was measured via luciferase assays at 12 hours and 24 hours after ligand treatments. All luciferase reporter assays were conducted at 12 h after the ligand treatment unless it is indicated in the experiment. 20 ng of SFR1A, SFR1B and SFR1C was transfected unless the amount of DNA was indicated in the figures. All experiments were performed multiple times with triplicate samples. The error bars indicate standard deviations. * = P<0.05; ** = P<0.01; *** = P<0.005.
Figure 3
Figure 3. SFR1-A modulates ER transcription activity.
(A) Western Blot was used to determine the effect of siRNA of SFR1 and control siRNA on FLAG-SFR1 expression. MCF7 cells were co-transfected with FLAG-SFR1-A along with indicated control or SFR1 specific siRNA (30 nM). Twenty-four hours after the transfection, cells were exposed to E2 (1 nM) or vehicle overnight. Lysates were harvested 48 hrs post-transfection and subjected to Western Blot analysis with the indicated antibodies. (B) Luciferase reporter assay was performed in MCF7 cells to determine the impact of SFR1 knockdown on ER transcriptional activity. MCF7 cells were co-transfected with the indicated amount of SFR1 siRNA and 3xERE-luc reporter or 40 nM scrambled siRNA and 3xERE-luc reporter. 24 hrs after the transfection, cells were treated with 1 nM E2 or vehicle for 24 h. Transcriptional activity was measured via luciferase assay (n = 4). (C) Luciferase reporter assay was performed in LNCaP cells to determine the impact of SFR1 knockdown on AR transcriptional activity. AR-positive LNCaP cells were co-transfected with the indicated amount of SFR1 siRNA and 3xARE-luc reporter or 40 nM scrambled siRNA and 3xARE-luc reporter. 24 hrs after the transfection, cells were treated with 1 nM DHT or vehicle for 24 h. Transcriptional activity was measured via luciferase assay. (D) Mammalian one-hybrid experiment was performed in Ishikawa cells to determine the intrinsic transcriptional activity of SFR1. GAL4DBD (Vector), GAL4DBD-SFR1-A (SFR1-A), and GAL4DBD-SRC1NR-box plus ER (SRC1+ER) were co-transfected with Gal4-luc reporter. The SRC1+ER one-hybrid was used as positive control. The level of transactivation was represented by luciferase activity (n = 4). (E) SFR1-A was recruited to the promoter of endogenous ER target gene promoters. The in vivo binding of SFR1-A to pS2 and PR promoters was examined by ChIP assay. FLAG-SFR1-A was transiently transfected into MCF7 cells. Soluble chromatin was prepared from the cells treated with 1 nM E2 (+) for 1 h or vehicle (−) and immunoprecipitated with M2 antibody. Co-precipitated DNA was amplified using primers that flank the ERE in the pS2 promoter region or half ERE and Sp1 site in the PR promoter. The presence of total pS2 and PR promoter DNA in the soluble chromatin prior to immunoprecipitation was included as input. The error bars indicate standard deviations. * = P<0.05; ** = P<0.01; *** = P<0.005.
Figure 4
Figure 4. SFR1-A promotes cancer cell proliferation.
Thymidine incorporation (A, B and C) and BrdU incorporation assays (D and E) were used to examine the effect of SFR1 on cell proliferation. In thymidine incorporation assays, SFR1-A cDNA was transfected into HeLa cells (A) to determine the impact of SFR1-A on hormone-independent cell proliferation. SFR1 siRNA (40 nM) or scrambled control, were transfected into LNCaP cells (B) and MCF7 cells (C) to determine the effect of endogenous SFR1 protein on ER-dependent cell proliferation. To determine the long-term effect of loss of SFR1 on cancer cell survival and proliferation, SFR1 knockdown MCF7 cell line was established by transducing MCF7 cells with lentivirus expressing sh-SFR1. MCF7 cells expressing sh-SRC1 were used as positive control and MCF7 cells expressing sh-Luciferase (sh-Luc) were established as the negative control. Sh-Luc, sh-SFR1 and sh-SRC1 cells were treated with BrdU for one hour at 37°C and harvested for antibody staining and FACS analysis. FITC-anti-BrdU and 7AAD were used to distinguish cells in G0/G1 (P5), S (P4), G2/M (P6) and apoptosis phase (P7) in FACS analysis. The results from the FACS analysis were analyzed and plotted with GraphPad Prism5 (E). N ≥3 in all experiments. * = P<0.05; ** = P<0.01; *** = P<0.005.

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