Daxx Mediates the Small Ubiquitin-like Modifier-dependent Transcriptional Repression of Smad4
2005; Elsevier BV; Volume: 280; Issue: 11 Linguagem: Inglês
10.1074/jbc.m409161200
ISSN1083-351X
AutoresChe‐Chang Chang, Ding-Yen Lin, Hsin-I Fang, Ruey‐Hwa Chen, Hsiu-Ming Shih,
Tópico(s)Cancer-related gene regulation
ResumoDaxx has been shown to function as an apoptosis regulator and transcriptional repressor via its interaction with various cytoplasmic and nuclear proteins. Here, we showed that Daxx interacts with Smad4 and represses its transcriptional activity via the C-terminal domain of Daxx. In vitro and in vivo interaction studies indicated that the binding of Smad4 to Daxx depends on Smad4 sumoylation. Substitution of Smad4 SUMO conjugation residue lysine 159, but not 113, to arginine not only disrupted Smad4-Daxx interaction but also relieved Daxx-elicited repression of Smad4 transcriptional activity. Furthermore, chromatin immunoprecipitation analyses revealed the recruitment of Daxx to an endogenous, Smad4-targeted promoter in a Lys159 sumoylation-dependent manner. Finally, down-regulation of Daxx expression by RNA interference enhanced transforming growth factor β-induced transcription of reporter and endogenous genes through a Smad4-dependent, but not K159R-Smad4-dependent, manner. Together, these results indicate that Daxx suppresses Smad4-mediated transcriptional activity by direct interaction with the sumoylated Smad4 and identify a novel role of Daxx in regulating transforming growth factor β signaling. Daxx has been shown to function as an apoptosis regulator and transcriptional repressor via its interaction with various cytoplasmic and nuclear proteins. Here, we showed that Daxx interacts with Smad4 and represses its transcriptional activity via the C-terminal domain of Daxx. In vitro and in vivo interaction studies indicated that the binding of Smad4 to Daxx depends on Smad4 sumoylation. Substitution of Smad4 SUMO conjugation residue lysine 159, but not 113, to arginine not only disrupted Smad4-Daxx interaction but also relieved Daxx-elicited repression of Smad4 transcriptional activity. Furthermore, chromatin immunoprecipitation analyses revealed the recruitment of Daxx to an endogenous, Smad4-targeted promoter in a Lys159 sumoylation-dependent manner. Finally, down-regulation of Daxx expression by RNA interference enhanced transforming growth factor β-induced transcription of reporter and endogenous genes through a Smad4-dependent, but not K159R-Smad4-dependent, manner. Together, these results indicate that Daxx suppresses Smad4-mediated transcriptional activity by direct interaction with the sumoylated Smad4 and identify a novel role of Daxx in regulating transforming growth factor β signaling. Sumoylation, the covalent attachment of ubiquitin-like SUMO 1The abbreviations used are: SUMO, small ubiquitin-like modifier; TGF, transforming growth factor; PML, promyelocytic leukemia protein; HDAC, histone deacetylase; PAI-1, plasminogen activator inhibitor-1; NEM, N-ethylmaleimide; GST, glutathione S-transferase; HA, hemagglutinin; β-Gal, β-galactosidase; SBE, Smad-binding element. to lysine residues, is an important post-translational modification that regulates the functions of proteins involving in many cellular processes (1Seeler J.S. Dejean A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 690-699Crossref PubMed Scopus (578) Google Scholar, 2Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1384) Google Scholar, 3Girdwood D.W. Tatham M.H. Hay R.T. Semin. Cell Dev. Biol. 2004; 15: 201-210Crossref PubMed Scopus (152) Google Scholar). With an increasing number of sumoylated proteins being identified, it has been proposed that SUMO conjugation affects target protein function by two major mechanisms (1Seeler J.S. Dejean A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 690-699Crossref PubMed Scopus (578) Google Scholar, 4Hay R.T. Trends Biochem. Sci. 2001; 26: 332-333Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 5Hochstrasser M. Cell. 2001; 107: 5-8Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 6Kim K.I. Baek S.H. Chung C.H. J. Cell. Physiol. 2002; 191: 257-268Crossref PubMed Scopus (134) Google Scholar). First, sumoylation alters the molecular interaction properties and/or the subcompartmentalization of its targets. For instance, sumoylation of the transcriptional factor Elk-1 not only regulates the nucleo-cytoplasmic shuttling of this protein (7Salinas S. Briancon-Marjollet A. Bossis G. Lopez M.A. Piechaczyk M. Jariel-Encontre I. Debant A. Hipskind R.A. J. Cell Biol. 2004; 165: 767-773Crossref PubMed Scopus (83) Google Scholar) but also results in the recruitment of histone deacetylase HDAC2 to Elk-1-regulated promoters, thereby repressing their transcription (8Yang S.H. Sharrocks A.D. Mol. Cell. 2004; 13: 611-617Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Second, sumoylation antagonizes other post-translational modifications, such as ubiquitination and acetylation, by targeting a common acceptor lysine residue. The prototypical example is that SUMO conjugation of IκBα at lysine 21 stabilizes this protein by blocking ubiquitination at the same site (9Desterro J.M. Rodriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). Because many transcriptional regulatory proteins are subjected to SUMO modification (1Seeler J.S. Dejean A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 690-699Crossref PubMed Scopus (578) Google Scholar, 2Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1384) Google Scholar, 3Girdwood D.W. Tatham M.H. Hay R.T. Semin. Cell Dev. Biol. 2004; 15: 201-210Crossref PubMed Scopus (152) Google Scholar), sumoylation has emerged as an important mechanism in controlling gene expression. Transforming growth factor (TGF) β regulates a wide array of biological activities (for reviews, see Refs. 10Derynck R. Feng X.H. Biochim. Biophys. Acta. 1997; 1333: 105-150Crossref PubMed Scopus (508) Google Scholar and 11Massague J. Nat. Rev. Mol. Cell. Biol. 2000; 1: 169-178Crossref PubMed Scopus (1653) Google Scholar). The cellular effects of TGF-β are mediated by both the type I and type II receptor serine/threonine kinases. Upon ligand binding, the type II receptor phosphorylates the type I receptor, which subsequently phosphorylates Smad2 and Smad3 (receptor-regulated Smads, R-Smad). The activated R-Smads then form complexes with the common-mediator Smad4 (Co-Smad) and translocate into the nucleus to regulate the transcription of target genes that mediate TGF-β-induced cellular processes (12Wrana J.L. Cell. 2000; 100: 189-192Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 13Miyazono K. ten Dijke P. Heldin C.H. Adv. Immunol. 2000; 75: 115-157Crossref PubMed Google Scholar, 14Moustakas A. Souchelnytskyi S. Heldin C.H. J. Cell Sci. 2001; 114: 4359-4369Crossref PubMed Google Scholar, 15Shi Y. Massague J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4832) Google Scholar, 16Derynck R. Zhang Y.E. Nature. 2003; 425: 577-584Crossref PubMed Scopus (4292) Google Scholar). Recently, several groups have demonstrated that Smad4 could be covalently conjugated by SUMO-1 at lysine 113 and 159, and mutation of both sumoylation residues significantly increases Smad4 transcriptional activity, suggesting a negatively regulatory mode of sumoylation on Smad4 activity (17Lin X. Liang M. Liang Y.Y. Brunicardi F.C. Melchior F. Feng X.H. J. Biol. Chem. 2003; 278: 18714-18719Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 18Lee P.S. Chang C. Liu D. Derynck R. J. Biol. Chem. 2003; 278: 27853-27863Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 19Long J. Wang G. He D. Liu F. Biochem. J. 2004; 379: 23-29Crossref PubMed Scopus (92) Google Scholar). Currently, the underlying mechanism as to how the sumoylation modulates Smad4 transcriptional activity has not been completely unraveled. Mutation of Smad4 sumoylation sites does not alter the ability of Smad4 to form a complex with its interacting partners on promoter (17Lin X. Liang M. Liang Y.Y. Brunicardi F.C. Melchior F. Feng X.H. J. Biol. Chem. 2003; 278: 18714-18719Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) but rather modestly increases the stability of Smad4 (18Lee P.S. Chang C. Liu D. Derynck R. J. Biol. Chem. 2003; 278: 27853-27863Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 20Lin X. Liang M. Liang Y.Y. Brunicardi F.C. Feng X.H. J. Biol. Chem. 2003; 278: 31043-31048Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). In addition, sumoylation was found to inhibit Smad4 intrinsic transcriptional activity per se (19Long J. Wang G. He D. Liu F. Biochem. J. 2004; 379: 23-29Crossref PubMed Scopus (92) Google Scholar), implicating a mechanism involving the recruitment of specific transcriptional factors to Smad4-regulated promoters via SUMO-modified Smad4. Daxx is initially identified as a cytoplasmic signaling molecule linking Fas receptor to Jun N-terminal kinase signaling (21Yang X. Khosravi-Far R. Chang H.Y. Baltimore D. Cell. 1997; 89: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (826) Google Scholar). Daxx has also been reported to associate directly with the cytoplasmic domain of the type II TGF-β receptor, thereby mediating TGF-β-induced apoptosis and Jun N-terminal kinase activation (22Perlman R. Schiemann W.P. Brooks M.W. Lodish H.F. Weinberg R.A. Nat. Cell Biol. 2001; 3: 708-714Crossref PubMed Scopus (301) Google Scholar). Besides functioning as a signal transducer in the cytoplasm, Daxx also acts as a transcriptional corepressor in the nuclear compartments. Daxx was found to suppress several transcription factor-responsive reporter activities, including reporters of CRE, E2F1, Sp1, and NF-κB (23Ecsedy J.A. Michaelson J.S. Leder P. Mol. Cell. Biol. 2003; 23: 950-960Crossref PubMed Scopus (79) Google Scholar). Furthermore, through direct protein-protein interactions, Daxx can inhibit the transcriptional potential of several transcription factors, such as ETS1 (24Li R. Pei H. Watson D.K. Papas T.S. Oncogene. 2000; 19: 745-753Crossref PubMed Scopus (158) Google Scholar), Pax3 (25Hollenbach A.D. Sublett J.E. McPherson C.J. Grosveld G. EMBO J. 1999; 18: 3702-3711Crossref PubMed Scopus (185) Google Scholar, 26Lehembre F. Muller S. Pandolfi P.P. Dejean A. Oncogene. 2001; 20: 1-9Crossref PubMed Scopus (93) Google Scholar), glucocorticoid receptor (27Lin D.Y. Shih H.M. J. Biol. Chem. 2002; 277: 25446-25456Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 28Lin D.Y. Lai M.Z. Ann D.K. Shih H.M. J. Biol. Chem. 2003; 278: 15958-15965Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), p53 family proteins (29Kim E.J. Park J.S. Um S.J. Nucleic Acids Res. 2003; 31: 5356-5367Crossref PubMed Scopus (61) Google Scholar), and mineralocorticoid receptor (30Obradovic D. Tirard M. Nemethy Z. Hirsch O. Gronemeyer H. Almeida O.F. Mol. Pharmacol. 2004; 65: 761-769Crossref PubMed Scopus (72) Google Scholar). Whether Daxx is involved in TGF-β-induced Smad4 transcriptional regulation has not been explored. In the present study, we showed that Daxx is capable of repressing the transcriptional activity of Smad4 through its interaction with SUMO-modified Smad4. Mutation of Smad4 sumoylation residue Lys159 but not Lys113 disrupted its association with Daxx, thereby abolishing the inhibitory effect of Daxx on Smad4 transactivation. Furthermore, chromatin immunoprecipitation experiments showed that Daxx forms complexes with the wild-type Smad4 but not with K159R mutant on the promoter of plasminogen activator inhibitor-1 (PAI-1), suggesting that Daxx controls Smad4 transcriptional activation via Lys159 sumoylation. Accordingly, down-regulation of Daxx expression by RNA interference increased Smad4 transactivation and PAI-1 expression regulated by TGF-β. Our results identify an important role for Daxx in mediating sumoylation-dependent modulation of Smad4 transcriptional potential. Yeast Two-hybrid Screen and β-Galactosidase (β-Gal) Assays—Yeast two-hybrid array screen using LexA-Smad4 as bait against preys consisting of 600 human full-length cDNA clones fused to the VP16 transactivation domain. Briefly, AMR70 yeast strain expressing LexA-Smad4 was mated with L40 yeast expressing different preys in 96-well plates. After overnight incubation, the resulting yeast cells were cultured in medium lacking tryptophan and leucine for selection of diploid cells. Diploid cells were further transferred to medium lacking tryptophan, leucine, and histidine for scoring protein-protein interactions. Positive clones were selected and subsequently verified by one-on-one transformation assays. β-Gal liquid assays were performed with the Galacto-light Plus kit (Tropix Inc., Bedford, MA), and three separate liquid cultures for each yeast transformant were assayed. Plasmid Construction—The cDNA fragments for various Smads and Smad4 subdomains were amplified by PCR and subcloned into the pBTM116 in-frame with the LexA to generate pBTM116-Smad1,- Smad2, -Smad3, -Smad4, -MH1, -Linker, and -MH2. The cDNA fragments of Smad1, Ubc9, Daxx, and its deletion mutants were inserted into the pACT2 vector to produce pGalAD-Smad1, -Ubc9, -DaxxFL, -Daxx1–625, -Daxx1–501, and -Daxx570–740. HA-Daxx and its mutant constructs were described (27Lin D.Y. Shih H.M. J. Biol. Chem. 2002; 277: 25446-25456Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 28Lin D.Y. Lai M.Z. Ann D.K. Shih H.M. J. Biol. Chem. 2003; 278: 15958-15965Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). pCMV-Myc-Smad4, pCMV-FLAG-Smad4, and pRK5-HA-Smad4 were constructed by inserting full-length cDNA of Smad4 into the pcDNA3.1-Myc vector (Invitrogen), pCMV-Tag 2 (Stratagene), and pRK5-HA vector, respectively. pEGFP-Daxx and p2XFLAG-Daxx were generated by subcloning full-length cDNA of Daxx into the pEGFP-C1 vector (Clontech) and pCAG-2XFLAG vector. The mammalian and yeast constructs expressing sumoylation site mutants K113R, K159R, and K113/159R were created using a QuikChange site-directed mutagenesis kit (Stratagene) with pCMV-Myc-Smad4 and pBTM116-Smad4 as templates, respectively. For constructing pGST-Smad4-Linker, cDNA fragment coding the linker domain was subcloned into the pGEX-4T-1 vector (Amersham Biosciences). To generate p6XHis-Daxx570–740, the cDNA fragment of Daxx was subcloned into the gateway pENTR3C vector (Invitrogen) followed by a switch to the pDEST17 vector (Invitrogen). pSUPER-Daxx was engineered by inserting an oligonucleotide corresponding to Daxx sequence 46GAT GAA GCA GCT GCT CAG C64 into the pSUPER vector (a generous gift from Dr. Reuven Agami). SBE4-Luc is a gift from Dr. Bert Vogelstein. The sequences of all constructs were confirmed by DNA sequencing analysis. Cell Culture, Transient Transfection, and Luciferase Reporter Assay—COS-1 and MDA-MB-468 cell lines were obtained from the American Type Culture Collection (Manassas, VA). Mv1Lu mink lung cell line is a gift from Dr. Neng-Yao Shih (National Health Research Institutes). All of the cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. To generate stable transfectants of Smad4 and its mutants, MDA-MB-468 cells were transfected with pCMV-Myc-Smad4, pCMV-Myc-Smad4K113R, pCMV-Myc-Smad4K159R, or pCMV-Myc-Smad4K113/159R and then selected in medium containing 1 mg/ml G418. Transient transfections of COS-1 and Mv1Lu mink lung cells were carried out using the Lipofectamine transfection kit (Invitrogen). For coimmunoprecipitation and Western blot analyses, the cells were harvested at 48 h after transfection. For the reporter gene assay, 2 × 105 cells of COS-1, Mv1Lu, MDA-MB-468, or its derivatives were seeded on 6-well plates 24 h prior to transfection. Various expression constructs, together with the β-Gal expression vector (as an indicator for normalization of transfection efficiency), were introduced into these cells by FuGENE 6 (Roche Applied Science). The transfectants were starved for 12 h followed by TGF-β treatment for additional 18 h. The cells were lysed and assayed for relative luciferase activity (firefly luciferase for the reporter and β-Gal activity for the indicator) as manufacture instructed (Packard, Meriden, CT). For reporter gene experiments involving pSUPER-Daxx, transfection and reporter assays were performed as described above, except the cells were harvested at 60 h after transfection. Immunoprecipitation and Western Blot Analysis—The cells were lysed in lysis buffer containing 50 mm Tris (pH 7.8), 0.15 m NaCl, 5 mm EDTA, 0.5% Triton X-100, 0.5% Nonidet P-40, and 0.1% sodium deoxycholate and protease inhibitor mixture (Complete; Roche Applied Science). For assaying Daxx-Smad4 interaction, the lysis buffer was supplemented with 20 mmN-ethylmaleimide (NEM). Lysates containing equal amounts of proteins were subjected to immunoprecipitation and immunoblot analyses as described previously (28Lin D.Y. Lai M.Z. Ann D.K. Shih H.M. J. Biol. Chem. 2003; 278: 15958-15965Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Antibodies to Smad4 (B-8, monoclonal) (G-20, polyclonal), Smad3 (I-20), and SUMO-1 (d-11) were purchased from Santa Cruz. Another anti-SUMO-1 antibody (GMP-1) was purchased from Zymed Laboratories Inc.. Antibodies to Daxx and FLAG were purchased from Sigma. Anti-Myc and anti-HA antibodies were from Covance. Anti-actin antibody was from Chemicon. Immunofluorescence—MDA-MB-468 cells were plated onto coverslips the day before transfection. The expression vectors for EGFP-Daxx and/or HA-Smad4 were transiently transfected into MDA-MB-468 cells. Twenty-four hours after transfection, the cells were incubated in Dulbecco's modified Eagle's medium containing 0.2% fetal bovine serum together with or without 200 pm TGF-β for 2 h. The cells were then fixed in 4% paraformaldehyde in phosphate-buffered saline, permeabilized with 0.4% Triton X-100, and then incubated with the anti-HA antibody for 1 h at room temperature. Following this incubation, the cells were washed three times for 10 min with phosphate-buffered saline at room temperature and the incubated with the Texas Red-conjugated anti-mouse IgG (DAKO) for 1 h at 20 °C. The nuclei were revealed by 4′,6′-diamidino-2-phenylindole staining (10 μg/ml). The coverslips were inverted and mounted on slides. The images were visualized by fluorescent microscopy. In Vitro Sumoylation Assays and Binding Assays for Sumoylated Proteins—The FLAG-tagged Smad4 proteins were purified from transiently transfected COS-1 cells using immunoprecipitation as described above. The precipitated Smad4 proteins were divided equally into two portions for sumoylation reactions in the presence or absence of SUMO-1 proteins. In vitro sumoylation was performed in 20 μl of reaction mixture containing 2 mm ATP, 20 mm HEPES (pH 7.5), 5 mm MgCl2, 15 ng of SUMO E1 recombinant proteins (LAE Biotechnology), 200 ng of Ubc9, 100 ng of SUMO-1, and FLAG-Smad4 proteins bound on beads. The reactions were carried out at 37 °C for 1 h and then washed extensively with phosphate-buffered saline. Half of the resulting sample was examined for sumoylation by Western blot analysis with anti-FLAG antibody. The other half of the sample was further incubated with lysates of COS-1 cells overexpressing HA-Daxx or its mutants at 4 °C for overnight. Beads of samples were collected by centrifugation and washed with phosphate-buffered saline, and bound proteins were analyzed by Western blot with anti-HA antibody. GST Pull-down Assay—The expression and purification of GST-Smad4-Linker fusion protein were performed as described (27Lin D.Y. Shih H.M. J. Biol. Chem. 2002; 277: 25446-25456Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 28Lin D.Y. Lai M.Z. Ann D.K. Shih H.M. J. Biol. Chem. 2003; 278: 15958-15965Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). 2 μg of purified GST fusion proteins bound on glutathione agarose beads were subjected to in vitro sumoylation assay. A fraction of the reaction mixture was analyzed by immunoblotting to indicate the amount of input and SUMO-1-modified GST fusion proteins. The resulting samples were washed, blocked with BSA, and then incubated for 2 h with 0.3 ml of binding mixture containing 10 mm HEPES (pH 7.5), 50 mm NaCl, 0.1% Nonidet P-40, 0.5 mm dithiothreitol, and 0.5 mm EDTA, together with recombinant Daxx570–740 proteins generated from BL21 codon plus strain transformed with p6XHis-Daxx570–740. The bound samples were washed four times and analyzed by immunoblotting with anti-Daxx antibody. Chromatin Immunoprecipitation Analysis—The chromatin immunoprecipitation experiments were performed essentially as described (31Hussein S.M. Duff E.K. Sirard C. J. Biol. Chem. 2003; 278: 48805-48814Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Briefly, ∼8 × 106 cells of each MDA-MB-468 Smad4 stable clone were seeded on four plates (10 cm), cultured for 36 h, and then starved in Dulbecco's modified Eagle's medium with 0.2% fetal bovine serum for additional 12 h. The cells were further stimulated with TGF-β (200 pmol) for 2 h and then subjected to fixation with 1% formaldehyde for 20 min at room temperature. Glycine was added to a final concentration of 125 mm to stop cross-linking. After chromatin extraction, shearing, and preclearing steps, the samples were split for immunoprecipitation with 5 μg of anti-Daxx (Sigma), anti-Smad4 (B-8, Santa Cruz), control rabbit IgG (SC-2027, Santa Cruz) antibodies, or no antibody (as input chromatin control). Bound DNA-protein complexes were washed and then eluted as described (31Hussein S.M. Duff E.K. Sirard C. J. Biol. Chem. 2003; 278: 48805-48814Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), and the cross-links were removed by incubating in 250 mm NaCl at 65 °C for 4 h. The resulting samples were precipitated and resuspended for proteinase K digestion and followed by DNA purification with a PCR purification kit (Qiagen). 5 μl of purified DNA (one-ninth of each sample) was used for PCR amplification (42 cycles). Primers flanking Smad-binding elements (SBEs) in the PAI-1 promoter were 5′-GACAAGGTTGTTGACACAAGAG-3′ (forward) and 5′-GATAACCTCCATCAAAACGTGG-3′ (reverse), which are corresponding to –894/–873 and –614/–593 from the initiation ATG, respectively. PCR products were run on a 2% agarose gel and analyzed by ethidium bromide staining. Quantification of PAI-1 Expression Level in pSUPER-Daxx-transfected MDA-MB-468 Smad4 Stable Cells—MDA-MB-468 Smad4 wild-type and mutant stable cells were cotransfected with pSUPER or pSUPER-Daxx along with pEGFP-C1 vector (in a 10:1 ratio). Transfected cells were cultured for 48 h, and the GFP-positive cells were isolated by FACS Vantage flow cytometer (BD Biosciences). The resulting cells were cultured for additional 24 h and subsequently treated with or without TGF-β for 4 h. Total cellular RNAs from these cells were extracted using the TRIzol reagent (Invitrogen). Five microgram of RNA of each sample was then reverse transcribed using ThermoScript reverse transcription-PCR system (Invitrogen) in 20 μl of reaction mix. A1-μl aliquot of the reverse transcription reaction product was used for semiquantitative PCR analysis with specific PAI-1 primers (forward primer: 5′-CAGACCAAGAGCCTCTCCAC-3′ and reverse primer: 5′-ATCACTTGGCCCATGAAAAG-3′) for an initial denaturation step at 95 °C for 5 min; 35 cycles of 15 s at 95 °C, 15 s at 52 °C, and 30 s at 72 °C; and a final elongation step at 72 °C for 10 min. As an internal control, an aliquot of each sample was analyzed for the level of glyceraldehyde-3-phosphate dehydrogenase RNA by semiquantitative and real time PCR with the forward primer (5′-TGGTATCGTGGAAGGACTCA-3′) and reverse primer (5′-AGTGGGTGTCGCTGTTGAAG-3′). The PCR products were then subjected to electrophoresis on 1% agarose gel containing ethidium bromide. The real time quantitative PCR were performed on the Applied Biosystem PRISM 7700 sequence detector with SYBR Green dye (Applied Biosystems) for detection as described in the manufacturer's guidelines. For each sample, the average threshold (Ct) value was resulted from quadruplicate assays, and the ΔCt value was determined by subtracting the average glyceraldehyde-3-phosphate dehydrogenase Ct value from the average PAI-1 Ct value. Three independent experiments were performed for measuring PAI-1 levels of pSUPER-Daxx-transfected MDA-MB-468 Smad4 stable cells. Daxx Interacts with Smad4 and Suppresses Its Transcriptional Activity—In a search for potential partner of the Smad4, we carried out a yeast two-hybrid array screen using a fusion protein comprised of the full-length human Smad4 and the LexA DNA-binding domain (LexA-Smad4) as bait. Daxx and Ubc9 were recovered from this screen as positive clones. The specificity of the interactions of Daxx, Ubc9, and Smad4 was verified by one-on-one transformation (Fig. 1A, left panel). Introduction of both LexA-Smad4 and GalAD-Daxx or GalAD-Ubc9 (Daxx or Ubc9 fused with Gal4 activation domain, respectively) constructs conferred onto transformants the ability to grow in medium lacking histidine. By contrast, yeast transformed with the control bait, LexA-MST3, or LexA-Smad1, along with GalAD-Daxx or GalAD-Ubc9 failed to do so, indicating that the interactions of Smad4 with Daxx and Ubc9 are specific. We further tested the possibility of Daxx associating with other Smad proteins and demonstrated that neither Smad2 nor Smad3 interacted with Daxx in yeast two-hybrid assays (Fig. 1A, right panel). Because Daxx can act as a transcriptional coregulator, identification of Daxx interacting with Smad4 prompted us to examine whether Daxx is involved in regulating Smad4 transcriptional activity. To this end, expression constructs of Smad4 and Smad3 were cotransfected with increasing amount of HA-Daxx into COS-1 cells along with the 3TP-Lux reporter, which contains TGF-β-responsive elements from the PAI-1 and collagenase promoters (32Wrana 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 (1369) Google Scholar). Consistent with previous reports (33Yingling J.M. Datto M.B. Wong C. Frederick J.P. Liberati N.T. Wang X.F. Mol. Cell. Biol. 1997; 17: 7019-7028Crossref PubMed Google Scholar, 34Zhang Y. Musci T. Derynck R. Curr. Biol. 1997; 7: 270-276Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), overexpression of both Smad3 and Smad4 induced a TGF-β-independent 3TP-Lux reporter activity (Fig. 1B). Introduction of Daxx, however, suppressed the transcriptional activity of Smad3/4 in a dose-dependent manner. To further substantiate the Daxx transcriptional repression on Smad4, MDA-MB-468 breast cancer cells lacking endogenous Smad4 were transfected with FLAG-Smad4 and HA-Daxx along with the reporter 3TP-Lux or SBE4-Luc, with the latter containing four copies of the Smad-binding element CAGA (35Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar), followed by TGF-β treatment. As expected, Daxx also inhibited the TGF-β-induced reporter activities of 3TP-Lux (Fig. 1C) and SBE4-Luc (Fig. 1D) in MDA-MB-468 cells carrying exogenous Smad4. Furthermore, the repressive effect of Daxx on Smad4-mediated reporter activities of 3TP-Lux (Fig. 1E) and SBE4-Luc (Fig. 1F) was also observed in Mv1Lu mink lung cells, another TGF-β-responsive cell line. Together, these results indicate that Daxx could suppress Smad4-mediated transcriptional activity, and this suppression is not a cell type-specific event. To further establish the link between the interaction and regulation of Smad4 by Daxx, we delineated the domain(s) of Daxx required for interacting with Smad4. Several Daxx deletion mutants were generated and then characterized for their interplays with Smad4 by yeast two-hybrid assays and Smad4 reporter experiments. In yeast two-hybrid assays, a truncated mutant of Daxx expressing amino acid residues 570–740 (Daxx570–740) was still able to interact with LexA-Smad4 (Fig. 2A). By contrast, two N-terminal fragments, Daxx1–501 and Daxx1–625, failed to do so. Therefore, the C-terminal domain of Daxx is sufficient for binding Smad4. Consistent with the results from yeast two-hybrid assays, Daxx570–740, instead of Daxx1–625, inhibited the 3TP-Lux reporter gene activity in COS-1 cells cotransfected with a fusion construct consisting of both TGF-β type I and II receptor cytoplasmic domains, R(II-I)C (Fig. 2B) that has been shown to activate TGF-β responses in a ligand-independent manner (36Feng X.H. Derynck R. J. Biol. Chem. 1996; 271: 13123-13129Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Likewise, the repressive effect of Daxx and Daxx570–740, but not Daxx1–625, on Smad4 was also observed in the Smad4-transfected MDA-MB-468 cells (data not shown). Thus, the interaction capabilities of these Daxx mutants are well correlated with their transcriptional repression abilities toward Smad4. Daxx Does Not Alter the Nuclear Translocation of Smad4 — Because Daxx was reported to associate with the TGF-β type II receptor in the cytoplasm (22Perlman R. Schiemann W.P. Brooks M.W. Lodish H.F. Weinberg R.A. Nat. Cell Biol. 2001; 3: 708-714Crossref PubMed Scopus (301) Google Scholar), we next examined whether the repressive effect of Daxx on Smad4 is due to an inhibition of Smad4 nuclear translocation. To test this possibility, MDA-MB-468 cells were transiently transfected with HA-Smad4 and/or EGFP-Daxx followed by TGF-β treatment. Immunofluorescence analysis revealed the translocation of Smad4 from the cytoplasmic to the nuclear compartment upon TGF-β stimulation (Fig. 3, panel a versus panel c). Daxx, however, was mainly distributed in the nucleus, and its localization was not altered by TGF-β treatment (panel e and panel g). Notably, when both Smad4 and Daxx were coexpressed in the same cells, Daxx did not block
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