Volume 129, Issue 4, Pages 1225-1236 (October 2005) β-Catenin Interacts With the FUS Proto-oncogene Product and Regulates Pre-mRNA Splicing  Satoshi Sato, Masashi Idogawa, Kazufumi Honda, Gen Fujii, Hisashi Kawashima, Kouji Takekuma, Akinori Hoshika, Setsuo Hirohashi, Tesshi Yamada  Gastroenterology  Volume 129, Issue 4, Pages 1225-1236 (October 2005) DOI: 10.1053/j.gastro.2005.07.025 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Assembly of native β-catenin-containing complex in the nucleus of a colorectal cancer cell line. (A) Nuclear extract from DLD-1 cells was immunoprecipitated with anti-β-catenin mouse monoclonal antibody or control mouse IgG and analyzed by SDS-PAGE (7.5%–15%) and silver staining. The arrow (*) indicates β-catenin, and the black bars (1–12) indicate the β-catenin-interacting proteins identified by mass spectrometry: 1, TOP2A (SwissProt accession number P11388); 2, Gemin 5 (Q8TEQ6); 3, DDX9 (Q08211); 4, PARP-1 (P09874); 5, PRPU (O94906); 6, DDX1 (Q92499); 7, hnRNP M (P52272); 8, DDX5 (P17844); 9, FUS (P35637); 10, p54nrb (Q15233); 11, hnRNP G (P38159); and 12, hnRNP A2/B1 (P22626). (B) Nuclear extract from DLD-1 cells was immunoprecipitated with anti-β-catenin mouse monoclonal antibody or control mouse IgG. The immunoprecipitates were blotted with anti-β-catenin, anti-FUS, anti-p54nrb, anti-DDX5, anti-TOP2A, anti-hnRNP A1, anti-hnRNP K, and anti-hnRNP A2/B1 antibodies. Note that FUS appears as double bands. These bands probably reflect posttranslational modification because similar results were obtained by transfecting the cells with an HA-tagged FUS construct (Figure 3G). (C) Nuclear extract from DLD-1 cells was immunoprecipitated with anti-FUS rabbit polyclonal antibody, control rabbit IgG, anti-TCF-4 mouse monoclonal antibody, and control mouse IgG. The immunoprecipitates were blotted with anti-β-catenin, anti-FUS, and anti-TCF-3/4 antibodies. Gastroenterology 2005 129, 1225-1236DOI: (10.1053/j.gastro.2005.07.025) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Two-hybrid analysis of the interaction between FUS and β-catenin proteins. (A) Two-hybrid analysis of the interaction between the full-length FUS protein (amino acids 1–526) and deletion mutants of β-catenin. HEK 293 cells were cotransfected with pACT carrying the entire coding sequence of FUS cDNA (pACT-FUS, black bars) or empty pACT (pACT-control, gray bars) and pBIND carrying a cDNA construct (NM_001904) encoding the indicated amino acid sequence of the β-catenin protein or empty pBIND (Control). The luciferase activity was normalized to that of the control transfectant (pACT-control and pBIND control) and expressed as the fold increase. Bars, SD. (B) Two-hybrid analysis of the interaction between the N-terminal 249 amino acids of β-catenin and deletion mutants of FUS. HEK 293 cells were cotransfected with pBIND carrying a cDNA fragment (NM_004960) encoding 1–249 amino acids of the β-catenin protein (pBIND-β-Cat, black bars) or empty pBIND (pBIND-control, gray bars) and pACT carrying a cDNA fragment encoding the indicated amino acids of the FUS protein or empty pACT (Control). Gastroenterology 2005 129, 1225-1236DOI: (10.1053/j.gastro.2005.07.025) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Colocalization of β-catenin and FUS proteins. (A–C) Immunofluorescence microscopy of DLD-1 cells with anti-β-catenin (A, and green in C) and anti-FUS (B, and red in C) antibodies. (D–F) Immunofluorescence microscopy of HEK293 cells with anti-β-catenin (D, and green in F) and anti-FUS (E, and red in F) antibodies. (G) HEK293 cells cotransfected with pcDNA3.1-HA-FUS and pFlag-β-catenin (left) or pFlag-control (right). Twenty-four hours after transfection, the nuclear extracts were immunoprecipitated with anti-β-catenin antibody or normal mouse IgG and blotted with anti-β-catenin, anti-FUS, or anti-HA antibody. Gastroenterology 2005 129, 1225-1236DOI: (10.1053/j.gastro.2005.07.025) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Immunohistochemistry of normal intestinal and tumor tissues from FAP and sporadic colorectal cancer patients. Expression of the FUS (A–E, G, I, J, and K) and β-catenin (F and H) proteins in the normal large intestine (A–D), adenoma (E–H), and adenocarcinoma (I–K) tissues of FAP (A–I) and sporadic colorectal cancer (J and K) patients. C and D are enlargements of the boxed areas in B. “N” (E, G, I, and J) indicates normal epithelial cells. E and F as well as G and H are from the same blocks of serial sections. Gastroenterology 2005 129, 1225-1236DOI: (10.1053/j.gastro.2005.07.025) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Functional involvement of FUS in the β-catenin and TCF/LEF transcriptional complexes. (A) DLD-1 cells were cotransfected with canonical (TOP, black bars) or mutant (FOP, gray bars) TCF/LEF luciferase reporter and pcDNA3.1-HA-FUS (FUS) or an appropriate mock plasmid (Control) in triplicate. Luciferase activity was measured 24 hours after transfection. Bars, SD. (B) Colony formation of HCT-116 cells transfected with either pcDNA3.1-HA-FUS (FUS) or an appropriate mock plasmid (Control) and selected with G418 for 8 days. (C and D) Phase contrast microscopy of HCT-116 cells that were transfected with either pcDNA3.1-HA-FUS (C) or an appropriate mock plasmid (D) and selected with G418 for 8 days. Gastroenterology 2005 129, 1225-1236DOI: (10.1053/j.gastro.2005.07.025) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Induction of alternative splicing by β-catenin. (A) Schematic representation of possible alternative splice variants (9S-13S and US, unspliced) produced by pCS3-MT-E1A, which carries the adenovirus E1A minigene. The anticipated sizes (bp) of the corresponding RT-PCR products are shown on the right. (B) Cos-7 cells were transiently cotransfected with 0.25 μg pCS3-MT-E1A and 1.0 μg pcDNA3.1-HA-FUS (FUS) or 0.5, 1.0, or 1.5 μg pFLAG-β-catenin as indicated at the bottom. The total amount of DNA used for the transfection was kept constant by adding empty plasmid DNA. Total RNA was extracted 48 hours after transfection and analyzed by RT-PCR. (C) Cos-7 cells were transiently cotransfected with 0.25 μg pCS3-MT-E1A as well as 1.0 μg hnRNP A1, pcDNA3.1-HA-FUS, and/or pFLAG-β-catenin as indicated at the bottom. (D and E) The relative expression levels of 10S and 9S isoforms in Figure 4B (D) and 4C (E) are expressed as the percentage of the sum of all isoforms. Gastroenterology 2005 129, 1225-1236DOI: (10.1053/j.gastro.2005.07.025) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 β-Catenin induces a novel splice variant of ER-β. (A) RT-PCR analysis of ER-β isoforms in HeLa cells transfected with pcDNA3.1-HA-FUS and/or pFlag-β-catenin. (B) cDNA sequence of the ER-β splice variant lacking exons 5 and 6 (Exon Δ5-6). Gastroenterology 2005 129, 1225-1236DOI: (10.1053/j.gastro.2005.07.025) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 Dominant-negative activity of ER-β lacking exons 5 and 6. (A) Transcriptional activity of ER-βΔ5-6. HEK293 cells were cotransfected with pFlag-CMV4 (Control), wild-type (WT) pFlag-ER-β (Exon 1-7), or pFlag-ER-βΔ5-6 (Exon Δ5-6) and pERE-TA-SEAP (ERE-TA, black bars) or pTA-SEAP (Control-TA, gray bars) reporter. The transfected cells were incubated for 48 hours in the absence (−) or presence (+) of 100 nmol/L E2 (17β-estradiol), and the amount of SEAP secreted into the culture media was measured in triplicate. Bar, SD. (B) Dominant-negative activity of ER-βΔ5-6. HEK293 cells were cotransfected with pFlag-CMV4 (−), wild-type pFlag-ER-β (WT), and/or pFlag-ER-βΔ5-6 (Δ5-6) and pERE-TA-SEAP (ERE-TA, black bars) or pTA-SEAP (Control-TA, gray bars) reporter. The transfected cells were incubated for 48 hours in the presence of 100 nmol/L of E2. (C) RT-PCR analysis of ER-α (ESR1), ER-β (ESR2), and GAPD in human colon cancer cell lines RCM-1 (a), SW48 (b), LoVo (c), LS174T (d), COLO320 (e), CCK-81 (f), HT-29 (g), SW1116 (h), HCT-8 (i), SW837 (j), HCT-116 (k), SW403 (l), COLO201 (m), LS513 (n), and SW480 (o) and breast cancer cell lines MCF-7 (p) and MDA-MB-231 (q). The asterisks indicate ER-βΔ5-6. Gastroenterology 2005 129, 1225-1236DOI: (10.1053/j.gastro.2005.07.025) Copyright © 2005 American Gastroenterological Association Terms and Conditions