DACH1 Inhibits Transforming Growth Factor-β Signaling through Binding Smad4
2003; Elsevier BV; Volume: 278; Issue: 51 Linguagem: Inglês
10.1074/jbc.m310021200
ISSN1083-351X
AutoresKongming Wu, Ying Yang, Chenguang Wang, Maria Antonietta Davoli, Mark D’Amico, Anping Li, Květa Cveklová, Zbyněk Kozmík, Michael P. Lisanti, Robert G. Russell, Aleš Cvekl, Richard G. Pestell,
Tópico(s)Developmental Biology and Gene Regulation
ResumoThe vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, we hypothesized that DACH1 might play a similar cellular function. Herein, DACH1 was found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-β (TGF-β)-induced apoptosis. DACH1 repressed TGF-β induction of AP-1 and Smad signaling in gene reporter assays and repressed endogenous TGF-β-responsive genes by microarray analyses. DACH1 bound to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localized with NCoR in nuclear dotlike structures. NCoR enhanced DACH1 repression, and the repression of TGF-β-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH was sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibited the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-β signaling by interacting with NCoR and Smad4. The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, we hypothesized that DACH1 might play a similar cellular function. Herein, DACH1 was found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-β (TGF-β)-induced apoptosis. DACH1 repressed TGF-β induction of AP-1 and Smad signaling in gene reporter assays and repressed endogenous TGF-β-responsive genes by microarray analyses. DACH1 bound to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localized with NCoR in nuclear dotlike structures. NCoR enhanced DACH1 repression, and the repression of TGF-β-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH was sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibited the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-β signaling by interacting with NCoR and Smad4. The pleiotropic transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF-βtransforming growth factor-βFACSfluorescence-activated cell sortingTBSTris-buffered salineSBESmad-binding elementCREBcAMP-response element-binding protein.1The abbreviations used are: TGF-βtransforming growth factor-βFACSfluorescence-activated cell sortingTBSTris-buffered salineSBESmad-binding elementCREBcAMP-response element-binding protein. family of cytokines regulate diverse biological functions through transmembrane Ser/Thr kinase receptors. The inhibition of epithelial cell proliferation, production of extracellular matrix components, regulation of differentiation, and apoptosis by TGF-β involve a tightly coordinated signaling pathway (1Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Peptide Growth Factors and Their Receptors. Springer, Berlin1990: 419-472Google Scholar). Numerous components of the TGF-β pathway are tumor suppressors that are functionally mutated in cancer (2Massague J. Blain S.W. Lo R.S. Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2053) Google Scholar), and TGF-β1, the first member of the TGF-β family, plays an important role in cancer, including breast cancer, functioning both as an antiproliferative factor and as a tumor suppressor (3Attissano L. Wrana J l. Science. 2002; 296: 1646-1647Crossref PubMed Scopus (1121) Google Scholar, 4Xie L. Law K.B. Aakre M.E. Edgerton M. Shyr Y. Bhowmick N.A. Moses H.L. Breast Cancer Res. 2003; 5: R187-R198Crossref PubMed Scopus (105) Google Scholar). Upon ligand binding, a heterodimeric complex forms between the type I and type II receptor, with the type II receptor transphosphorylating the type I receptor. The activated type I receptor interacts with an adaptor protein, SARA, which recruits Smad2 and Smad3 to serve as phosphorylation substrates of the type 1 receptor.The Smad family of transcription factors participates in TGF-β signaling at multiple levels (3Attissano L. Wrana J l. Science. 2002; 296: 1646-1647Crossref PubMed Scopus (1121) Google Scholar). Phosphorylated pathway-restricted Smad2 and Smad3 form heterodimers with the common mediator Smad4 in the cytoplasm and translocate into the nucleus, where they bind Smad-binding elements (SBEs) at specific promoters of genes regulating cell growth (5Derynck R. Zhang Y. Feng X.H. Cell. 1998; 95: 737-740Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar, 6Heldin C.-H. Miyazono K. Ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3317) Google Scholar). Alternatively, Smad complexes bind DNA in conjunction with other DNA-binding proteins such as FAST1 and FAST2. DNA-bound Smad complexes regulate transcription, either positively through recruitment of coactivators of the p300/CREB-binding protein class (7Janknecht R. Wells N.J. Hunter T. Genes Dev. 1998; 12: 2114-2119Crossref PubMed Scopus (434) Google Scholar) or negatively by recruiting the Sno/Ski family (8Sun Y. Liu X. Eaton E.N. Lane W.S. Lodish H.F. Weinberg R.A. Mol. Cell. 1999; 4: 499-509Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 9Stroschein S.L. Wang W. Zhou S. Zhou Q. Luo K. Science. 1999; 286: 771-774Crossref PubMed Scopus (436) Google Scholar). Interaction of Smad3 with Ski and Sno allows formation of a DNA-binding complex that represses transcription of TGF-β-responsive genes (9Stroschein S.L. Wang W. Zhou S. Zhou Q. Luo K. Science. 1999; 286: 771-774Crossref PubMed Scopus (436) Google Scholar, 10Xu W. Angelis K. Danielpour D. Haddad M.M. Bischof O. Campisi J. Stavnezer E. Medrano E.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5924-5929Crossref PubMed Scopus (180) Google Scholar). Thus, overexpression of Ski antagonizes the normal response to TGF-β signaling (i.e. inhibition of cell growth) and enables the cells to grow in the presence of TGF-β (11Liu J. Stevens J. Rote C.A. Yost H.J. Hu Y. Neufeld K.L. White T.L. Matsunami N. Mol. Cell. 2001; 7: 927-936Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar).Understanding the specificity of TGF-β signal transduction pathway is critically dependent upon identifying factors in the cellular environment and key cellular components within this signal transduction pathway. To identify and functionally characterize the candidate co-regulators contributing to TGF-β signaling, we hypothesized that proteins structurally related to important components mediating the TGF-β signal transduction pathway are likely to play significant roles. The Ski/Sno proteins share structural homology with the Dach protein (12Kozmik Z. Pfeffer P. Kralova J. Paces J. Paces V. Kalousova A. Cvekl A. Dev. Genes Evol. 1999; 209: 537-545Crossref PubMed Scopus (58) Google Scholar). The founding member of the DACH subfamily of nuclear proteins, Drosophila dachshund (dac), is an essential gene regulating the development of eye and leg (13Mardon G. Solomon N.M. Rubin G.M. Development. 1994; 120: 3473-3486Crossref PubMed Google Scholar). The Dach N-BOX (the dac and ski/sno DS domain) (12Kozmik Z. Pfeffer P. Kralova J. Paces J. Paces V. Kalousova A. Cvekl A. Dev. Genes Evol. 1999; 209: 537-545Crossref PubMed Scopus (58) Google Scholar, 14Hammond K.L. Hanson I.M. Brown A.G. Lettice L.A. Hill R.E. Mech. Dev. 1998; 74: 121-131Crossref PubMed Scopus (120) Google Scholar, 15Caubit X. Thangarajah R. Theil T. Wirth J. Nothwang H.-G. Ruther U. Krauss S. Dev. Dyn. 1999; 214: 66-80Crossref PubMed Scopus (71) Google Scholar) consists of ∼100 amino acids conserved with various Sno/Ski family members, predicted to form a highly organized structure of α-helices and β-strands (12Kozmik Z. Pfeffer P. Kralova J. Paces J. Paces V. Kalousova A. Cvekl A. Dev. Genes Evol. 1999; 209: 537-545Crossref PubMed Scopus (58) Google Scholar). This domain comprises the critical region of Ski responsible for its oncogenic potential. From the crystallographic analysis of the DACH1 N-BOX (DS domain), it has been proposed that DACH1 might have a general and/or specific DNA binding activity (16Kim S.-S. Zhang R.-G. Braustin S.E. Joachimiak A. Cvekl A. Hedge R.S. Structure. 2002; 10: 787-795Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), an idea further supported by studies of DACH1 interaction with chromatin-complexed and naked DNA (17Ikeda K. Watanabe Y. Sno H. Ohto I. Kawakami K. Mol. Cell. Biol. 2002; 22: 6759-6766Crossref PubMed Scopus (119) Google Scholar).Drosophila dac is a component of a genetic network, including eyeless (ey), sine oculis (so), and eyes absent (eya) that regulates proliferation and differentiation of the eye imaginal disk epithelium. Two vertebrate homologues (DACH1 and DACH2 in humans, Dach1 and Dach2 in mouse and chicken) have been cloned, and their expression patterns have been characterized (12Kozmik Z. Pfeffer P. Kralova J. Paces J. Paces V. Kalousova A. Cvekl A. Dev. Genes Evol. 1999; 209: 537-545Crossref PubMed Scopus (58) Google Scholar, 14Hammond K.L. Hanson I.M. Brown A.G. Lettice L.A. Hill R.E. Mech. Dev. 1998; 74: 121-131Crossref PubMed Scopus (120) Google Scholar, 15Caubit X. Thangarajah R. Theil T. Wirth J. Nothwang H.-G. Ruther U. Krauss S. Dev. Dyn. 1999; 214: 66-80Crossref PubMed Scopus (71) Google Scholar, 18Heanue T.A. Reshef R. Davis R.J. Mardon G. Oliver G. Tomarev S. Lassar A.B. Tabin C.J. Gene Dev. 1999; 13: 3231-3243Crossref PubMed Scopus (302) Google Scholar, 19Davis R.J. Shen W. Sandler Y.I. Amoui M. Purcell P. Maas R. Ou C.N. Vogel H. Beaudet A.L. Mardon G. Mol. Cell. Biol. 2001; 21: 1484-1490Crossref PubMed Scopus (73) Google Scholar, 20Ayres J.A. Shum L. Akarsu A.N. Dashner R. Takahashi K. Ikura T. Slavkin H.C. Nuckolls G.H. Genomics. 2001; 77: 18-26Crossref PubMed Scopus (27) Google Scholar, 21Heanue T.A. Davis R.J. Rowitch D.H. Kispert A. McMahon A.P. Mardon G. Tabin C.J. Mech. Dev. 2002; 111: 75-87Crossref PubMed Scopus (50) Google Scholar, 22Szele F.G. Chin H.K. Rowlson M.A. Cepko C.L. Mech. Dev. 2002; 112: 179-182Crossref PubMed Scopus (17) Google Scholar). It has been proposed that Dach1 and Dach2 are partially functionally redundant, since Dach1–/– mice survive to birth but exhibit postnatal lethality associated with a failure to suckle, cyanosis, and respiratory distress (19Davis R.J. Shen W. Sandler Y.I. Amoui M. Purcell P. Maas R. Ou C.N. Vogel H. Beaudet A.L. Mardon G. Mol. Cell. Biol. 2001; 21: 1484-1490Crossref PubMed Scopus (73) Google Scholar). Dach1 is a direct target gene for fibroblast growth factor signaling during limb skeletal development (23Horner A. Shum L. Ayres J.A. Nonaka K. Nuckolls G.H. Dev. Dyn. 2002; 225: 35-45Crossref PubMed Scopus (26) Google Scholar). Muscle development is regulated by a synergistic interaction of Dach2, Pax3, Eya2, and Six1 (18Heanue T.A. Reshef R. Davis R.J. Mardon G. Oliver G. Tomarev S. Lassar A.B. Tabin C.J. Gene Dev. 1999; 13: 3231-3243Crossref PubMed Scopus (302) Google Scholar). This group of genes is composed of vertebrate genes structurally and functionally related to the Drosophila genes dac, ey, eya, and so, respectively. Dach1 in combination with Six6 regulates proliferation of retinal and pituitary precursor cells by repressing cyclin-dependent kinase inhibitors including p27Kip1 (24Li X. Perrisi V. Liu F. Rose D.W. Rosenfeld M.G. Science. 2002; 297: 1180-1183PubMed Google Scholar). The current studies examined the role of DACH1 in TGF-β signaling. DACH1 was expressed in breast cancer cell lines and inhibited TGF-β-induced apoptosis. DACH1 regulated endogenous TGF-β responsive genes and repressed TGF-β induction of AP-1 and Smad signaling. The conserved DS domain of DACH1 was required for binding to endogenous NCoR and for repression of TGF-β signaling by DACH1. These studies thus identify DACH1 as a regulator of TGF-β signaling that may contribute a better understanding of this complex pathway.MATERIALS AND METHODSPlasmid Construction—The full-length DACH1, DACH1 DS domain alone (DS), or DACH1 DS domain deleted (ΔDS) were cloned to pKW10 vector containing N-terminal FLAG peptide. The FLAG-tagged DACH1 cDNA was subcloned into the pIND vector (Clontech) to produce pIND-FLAG-DACH1. The DACH1 cDNA was also subcloned into the vectors to form HA-DACH1. CS2-FLAG Smad2 was a gift from Dr. J. Massague, CMV2-FLAG Smad3 was from Dr. Chang (25Kang H.-Y. Lin H.-K. Hu Y.-C. Huang K.-E. Chang C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3018-3023Crossref PubMed Scopus (134) Google Scholar), pCMV5-HA-Smad4 was from Dr. Bottinger (26von Gersdorff G. Susztak K. Rezvani F. Bitzer M. Liang D. Bottinger E.P. J. Biol. Chem. 2000; 275: 11320-11326Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), and the FLAG-tagged NCoR expression vector was from Dr. Rosenfeld (24Li X. Perrisi V. Liu F. Rose D.W. Rosenfeld M.G. Science. 2002; 297: 1180-1183PubMed Google Scholar). The reporter plasmids 3TP Lux and SBE-4 Luc were previously described (27Watanabe G. Pena P. Albanese C. Wilsbacher L.D. Young J.B. Pestell R.G. J. Biol. Chem. 1997; 272: 20063-20069Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Ski and Sno cDNAs were obtained from Dr. Ishii (28Normura T. Khan M. Kaul S.C. Dong H. Wadhwa R. Colmenares C. Kohno I. Ishii S. Genes Dev. 1999; 13: 412-423Crossref PubMed Scopus (251) Google Scholar) and subcloned into 3× FLAG-CMV7.1 vector (Sigma).Western Blotting and Immunohistochemistry—Western blot analysis was conducted as previously described with minor changes (29Albanese C. Wu K. D'Amico M. Jarrett C. Joyce D. Hughes J. Hulit J. Sakamaki T. Fu M. Ben-Ze'ev A. Bromberg J.F. Lamberti C. Verma U. Gaynor R.B. Byers S.W. Pestell R.G. Mol. Biol. Cell. 2003; 14: 585-599Crossref PubMed Scopus (132) Google Scholar). Proteins were separated by electrophoresis in 6–10% graded polyacrylamide gel and transferred to nitrocellulose filters, immunoblotted with anti-Smad4, anti-Smad2/3, anti-NCoR, anti-mSin3A antibody (Santa Cruz Biotechnology), anti-phosphorylated Smad2/3 (Upstate Biotechnology, Inc., Lake Placid, NY), or anti-FLAG M2 antibody (Sigma). The bands were detected using the enhanced chemiluminescence detection system (Amersham Biosciences). Guanine nucleotide dissociation inhibitor antibody, a generous gift from Dr. Perry Bickel (Washington University, St. Louis, MO) (30Lee R.J. Albanese C. Fu M. D'Amico M. Lin B. Watanabe G. Haines III, G.K. Siegel P.M. Hung M.C. Yarden Y. Horowitz J.M. Muller W.J. Pestell R.G. Mol. Cell. Biol. 2000; 20: 672-683Crossref PubMed Scopus (307) Google Scholar) was used as an internal control for protein abundance. We generated anti-DACH1 antibody by hyperimmunizing rabbits with purified DACH1 DS domain peptide. For purification of DACH1 DS domain antibody, 2 ml of immune serum diluted to 40 ml was bound to 2 ml of protein-A agarose beads (Sigma) as a column overnight. The bound immunoglobulin was eluted with a high salt method using 3 m NaCl, and the immunoglobulin concentration was estimated using the spectrophotometer. This antibody was used for Western blotting. Immunostaining of the mouse embryo tissues was performed in a manner similar to a previous method (31D'Amico M. Wu K. Di Vizio D. Reutens AT. Stahl M. Fu M. Albanese C. Russell R.G. Muller W.J. White M. Negassa A. Lee H.W. DePinho R.A. Pestell R.G. Cancer Res. 2003; 63: 3395-3402PubMed Google Scholar) using a polyclonal DACH1 antibody that was a gift from Dr. G. Nuckolls (23Horner A. Shum L. Ayres J.A. Nonaka K. Nuckolls G.H. Dev. Dyn. 2002; 225: 35-45Crossref PubMed Scopus (26) Google Scholar).Cell Culture, Luciferase Reporter Assays, and Fluorescence-activated Cell Sorting—HaCaT, MDA-MB-231, MCF10A, Panc-1, HTB-134, and 293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin/streptomycin. The cells were maintained in a humidified atmosphere with 5% CO2 at 37 °C. Human recombinant TGF-β1 is from Calbiochem. Transfections were performed using Superfect transfection reagent (Qiagen, Valencia, CA) according to the manufacturer's protocol. Stable MDA-MB-231 cell lines were generated expressing the VgEcR/RXRα plasmid and the (EGRE)3 FLAG DACH1 plasmid. Colonies selected with Zeocin (400 μg/ml) and G418 (500 μg/ml) were analyzed for low basal and robust inducibility by Western blot analysis of the FLAG epitope.For reporter gene assays, cells were transiently transfected with an appropriate combination of the reporter, expression plasmids, and control vector. In some experiments, cells were serum-starved for 36 h and stimulated with or without TGF-β for 12 h before collecting cells for luciferase assay. The transfection efficiency was normalized by cotransfection with 0.2 μg of pRL-CMV plasmid (Promega, Madison, WI) and was measured with the Promega dual-luciferase reporter assay system according to the manufacturer's protocol. Luciferase assays were performed at room temperature using an Autolumat LB 953 (EG&G Berthold) (32Watanabe G. Albanese C. Lee R.J. Reutens A. Vairo G. Henglein B. Pestell R.G. Mol. Cell. Biol. 1998; 18: 3212-3222Crossref PubMed Scopus (145) Google Scholar). Statistical analyses were performed using Student's t test, and significant differences were established as p < 0.05. FACS analysis was used to determine the proportion of cells in the subG1 or apoptotic phase as previously described (33Ashton A.W. Watanabe G. Albanese C. Harrington E.O. Ware J.A. Pestell R.G. J. Biol. Chem. 1999; 274: 20805-20811Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar).Microarray Analysis—mRNA was prepared from MDA-MB-231 pIND-DACH1 stable lines, treated with either vehicle or ponasterone A (2 μg/ml) for serial time points using Trizol reagent (Invitrogen). Following DNase I treatment (Takara Bio Inc., Japan) according to the manufacturer's instructions, total RNA was amplified according to the Eberwine procedure (34Van Gelder R.N. von Zastrow M.E. Yool A. Dement W.C. Barchas J.D. Eberwine J.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1663-1667Crossref PubMed Scopus (1032) Google Scholar) using the Ambion MessageAmp™ kit (Ambion). During in vitro transcription, biotin-11-CTP and biotin-16-UTP (Enzo Diagnostics, Farmingdale, NY) were incorporated. 20 μg of the biotinylated cRNA product was fragmented at 94 °C for 35 min. The sample was used for each hybridization reaction. Hybridization to a set of two Affymetrix U133A GeneChips (representing ∼22,000 open reading frames and at least 17,000 genes) was performed overnight, followed by staining and washing as per the manufacturer's instructions. The clean processed chips were then scanned using Agilent GeneArray scanner. Grid alignment and raw data generation were performed using Affymetrix GeneChip 5.0 software. For quality control, oligonucleotide B2 was hybridized to analyze the checkerboard pattern in each corner of the chip bioB, bioC, and bioD probes were added to each sample with varying concentrations to standardize hybridization; and staining and washing procedures were performed. Raw expression values, representing the average difference in hybridization intensity between oligonucleotides that perfectly match the transcript sequence and oligonucleotides containing single base pair mismatches, were measured. A noise value (Q) based on the variance of low intensity probe cells was used to calculate a minimum expression threshold (2.1 × Q) for each chipGene Selection—Data generated after scanning were subjected to comparison analysis to select change calls at 100% increase or decrease compared with vehicle control at each time point. The data selected after comparison analysis were further filtered based on absolute analysis using the Mann-Whitney test and detection calls, and 422 genes were selected for multidimensional scaling and hierarchical clustering.Multidimensional Scaling and Cluster Analysis—Multidimensional scaling (Matlab) was used to visualize the differences between control and treated samples selected above (see "Gene Selection"). To measure distance, the Pearson correlation coefficient was applied to each pair of control (•) and treated (♦, ▴) samples. To visualize expressions of genes, those that were selected above (see "Gene Selection") and intra- and intersample pairs, hierarchical clustering was performed using Cluster 3.0 (Stanford University). A gene list corresponding to clusters was generated using the data mining tool from Affymetrix.Immunoprecipitation and Immunoblotting—293T cells and HaCaT cells were used for the detection of protein-protein interaction in vivo. Cells were transfected with the expression plasmids, cultured for 2 days, washed, scraped, and lysed in a buffer containing 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, and proteinase inhibitor mixture (Sigma). Lysates were cleared by centrifugation at 4 °C for 15 min. The protein concentration was measured by the Bio-Rad assay. 500 μg of total protein was incubated with anti-FLAG M2 antibody (Sigma) or anti-Smad4 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by incubation with protein G-Sepharose beads. The beads were washed five times with buffer containing 0.5% Tween 20 instead of 1% Triton X-100. The immunoprecipitates were eluted by boiling for 5 min in SDS sample buffer (100 mm Tris-HCl, 10 mm dithiothreitol, 4% SDS) and subjected to SDS-gel electrophoresis.Protein Purification and Immunodepletion—500 μg of anti-FLAG M2 gel (Sigma) was equilibrated in TBS (50 mm Tris, pH 7.4, 0.15 m NaCl). 1 ml of whole cell extract derived from 293T cells, transfected with expression vector-encoding FLAG-DACH1, was incubated with the gel in TBS/Ca2+ binding buffer (TBS containing 1 mm CaCl2) at 4 °C, rotating overnight. The supernatant was saved as immunoprecipitated DACH1 whole cell extract for further Western blot and EMSA. The gel was washed five times with 1 ml of TBS/Ca2+, followed by a 30-min incubation of TBS/EDTA (TBS containing 2 mm EDTA) at room temperature. FLAG-tagged DACH1 was incubated and eluted by five sequential 50-μl TBS/EDTA elutions. 10 μl of FLAG-DACH1-transfected 293T whole cell extract, 10 or 20 μl of immunoprecipitated DACH1 whole cell extract, and 5, 10, or 20 μl of purified FLAG-DACH was loaded on 4–15% precast protein gel (Bio-Rad). Electrophoresis was conducted at 150 V for 1.5 h at 4 °C, and samples were transferred to the 0.2-μm nitrocellulose membrane (Bio-Rad) at 100 V for 2 h at 4 °C. Blocking with 5% nonfat milk (Bio-Rad) at room temperature for 2 h and a blot with 1:1000 diluted anti-FLAG M2 monoclonal antibody (Sigma) were performed. Membranes were washed and blotted with anti-mouse antibody for 1 h at room temperature. The fluorescence signal was detected after SuperSignal treatment (Pierce).Electrophoretic Mobility Shift Assays—2 μl of 293T whole cell extracts prepared from cells transfected with Smad4, 1 μl of whole cell extract transfected with DACH1, 1 μl of immunoprecipitated DACH1, and 1 μl of purified DACH1 were incubated with 1 μg of poly(dI-dC) in the presence or absence of specific self-oligo competitor on ice for 10 min. The FAST-1/SBE oligonucleotide (5′-CTGCCCTAAAATGTGTATTCCATGGAAATGTCTGCCCTTCTCTCCAG-3′) (35Yeo C.-Y. Chen X. Whitman M. J. Biol. Chem. 1999; 274: 26584-26590Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) was end-labeled with polynuclotide kinase using [γ-32P]ATP and incubated with whole cell extracts of 293T cells at room temperature for 10 min. Protein-DNA complexes were separated by 5% native polyacrylamide gels, running at room temperature in 0.5 TBE. Gels were dried and visualized by autoradiography.In Vitro Expression of Protein—In vitro [35S]methionine-labeled protein was prepared by coupled transcription-translation with a Promega TNT coupled reticulocyte lysate kit (Promega, Madison, WI) using 1 μg of Smad4 expression plasmid DNA in a total of 50 μl. GST, GST-DACH1 DS/EYAD, GST-DACH1 DS, and GST-DACH1 EYAD/C-end were expressed in E. coli BL21 DE3 and purified using glutathione-Sepharose 4B. In vitro protein-protein interactions were performed as described (36Fu M. Wang C. Reutens A.T. Angelletti R. Siconolfi-Baez L. Ogryzko V. Avantaggiati M.L. Pestell R.G. J. Biol. Chem. 2000; 275: 20853-20860Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). The in vitro translated protein (45 μl of Smad4) and 5 μg of purified GST protein were incubated with glutathione-Sepharose 4B beads in binding buffer at 4 °C for 6 h and then washed five times; 50 μl of binding buffer and 10 μl of 6× SDS loading buffer were added after the final wash; and the samples were denatured at 95 °C and subjected to electrophoresis on 8% SDS-polyacrymide gel. The gel was fixed and incubated with Amplifier (Amersham Biosciences) for 30 min to enhance the signal and dried for 1 h at 80 °C, and autoradiography was performed at –80 °C.Subcellular Localization of DACH1 and NCoR—Subcellular localization of DACH1 and NCoR was essentially examined as described (28Normura T. Khan M. Kaul S.C. Dong H. Wadhwa R. Colmenares C. Kohno I. Ishii S. Genes Dev. 1999; 13: 412-423Crossref PubMed Scopus (251) Google Scholar). A mixture of 1.5 μg of FLAG-NCoR expression plasmid and 1 μg of the plasmid encoding DACH1 was transfected into HaCaT cells. Forty hours after transfection, cells were fixed and stained with anti-FLAG polyclonal antibodies (Sigma) and the anti-HA monoclonal antibody (Santa Cruz Biotechnology). The DACH1 and NCoR signals were visualized by Cy3- and FITC-conjugated donkey anti-secondary antibodies (Jackson Immunoresearch), respectively, and analyzed by confocal microscopy.RESULTSDACH1 Represses TGF-β Signaling—The amino acid sequence of Dachshund has significant homology with the Ski and Sno family of proto-oncogenes, greatest in the region of DACH box-N (DS domain) (28% sequence identity with the vertebrate Ski and Sno proteins) (Fig. 1A). This domain of Ski has been implicated in transformation and the induction of myogenesis. Additional weak homology is found between DACH box-C and the C-terminal domain of the Ski/Sno proteins, which are believed to share an α-helical structure capable of forming coiled-coil structures upon homodimerization (Fig. 1A) (14Hammond K.L. Hanson I.M. Brown A.G. Lettice L.A. Hill R.E. Mech. Dev. 1998; 74: 121-131Crossref PubMed Scopus (120) Google Scholar). To examine the role of the DACH1 DS domain in DACH1 function, expression constructions encoding full-length DACH1, a DS domain-deleted mutant (ΔDS), and the DS domain alone (DS) were assessed in cultured cells. Western blot analysis demonstrated that all three constructs were expressed well using either the anti-FLAG antibody or an antibody to the DACH1 DS domain. The DACH1 antibody (see "Materials and Methods") showed no cross-immunoreactivity with Ski (Fig. 1C). Immunohistochemical study was performed of the murine 15.5-day embryo. Consistent with previous observations (20Ayres J.A. Shum L. Akarsu A.N. Dashner R. Takahashi K. Ikura T. Slavkin H.C. Nuckolls G.H. Genomics. 2001; 77: 18-26Crossref PubMed Scopus (27) Google Scholar), DACH1 immunoreactivity was identified within cells of the cochlea duct and retinal epithelial cells with some staining of the overlying ectoderm (Fig. 1D). Western blot analysis was conducted with the DACH1 antibody of several TGF-β-responsive cell lines, including MCF10A, MDA-MB-231, and Panc-1 cells. A 97-kDa band corresponding to DACH1 was identified in each cell line. 293T cells transfected with the DACH1 expression vector demonstrated a band of identical molecular weight (Fig. 1E). Thus, DACH1 is expressed in human breast cancer and a pancreatic cancer cell line.Since the Sno and Ski proteins regulate TGF-β signaling (10Xu W. Angelis K. Danielpour D. Haddad M.M. Bischof O. Campisi J. Stavnezer E. Medrano E.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5924-5929Crossref PubMed Scopus (180) Google Scholar), we assessed the role for DACH1 in this pathway. TGF-β increased activity of both the AP-1-responsive reporter 3TP Lux (38Wrana J.L. Attisano L. Carcamo J. Zentella A. Doody J. Laiho M. Wang X.F. Massague J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1366) Google Scholar) and an artificial promoter containing the Smad-binding elements (SBE-4 Luc). TGF-β induced reporter activity in the human keratinocyte cell line HaCaT (Fig. 2A). Coexpression of Ski or Sno repressed TGF-β-induced activity of both reporters (Fig. 2B), consistent with previous studies (8Sun Y. Liu X. Eaton E.N. Lane W.S. Lodish H.F. Weinberg R.A. Mol. Cell. 1999; 4: 499-509Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 9Stroschein S.L. Wang W. Zhou S. Zhou Q. Luo K. Science. 1999; 286: 771-774Crossref PubMed Scopus (436) Google Scholar, 10Xu W. Angelis K. Danielpour D. Haddad M.M. Bischof O. Campisi J. Stavnezer E. Medrano E.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5924-5929Crossref PubMed Scopus (180) Google Scholar). To investigate the role of DACH1 in TGF-β signaling, DACH1 expression constructs were assessed for activity on the 3TP Lux and SBE-4 Luc reporter genes. DACH1 repressed both reporters in HaCaT cells. Deletio
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