Smad4 and β-Catenin Co-activators Functionally Interact with Lymphoid-enhancing Factor to Regulate Graded Expression of Msx2
2003; Elsevier BV; Volume: 278; Issue: 49 Linguagem: Inglês
10.1074/jbc.m305472200
ISSN1083-351X
AutoresSamer M. I. Hussein, Eleanor K. Duff, Christian Sirard,
Tópico(s)Developmental Biology and Gene Regulation
ResumoRecent in vivo evidence suggests that Wnt signaling plays a central role in determining the fate of stem cells in the ectoderm and in the neural crest by modulating bone morphogenetic protein (BMP) levels, which, in turn, influence Msx gene expression. However, the molecular mechanism regulating the expression of the Msx genes as key regulators of cell fate has not been elucidated. Here we show in murine embryonic stem cells that BMP-dependent activation of Msx2 is mediated via the cooperative binding of Smad4 at two Smad binding elements and of lymphoid enhancing factor (Lef1) at two Lef1/TCF binding sites. Lef1 can synergize with Smad4 and Smad1 to activate Msx2 promoter, and this transcriptional complex is assembled on the endogenous promoter in response to BMP2. The Wnt/β-catenin signaling pathway can activate Msx2 via the binding of Lef1 to its promoter and synergizes with BMP2 to activate Msx2 expression, possibly via enhanced recruitment of the p300/cAMP-response element-binding protein-binding protein co-factor. Interestingly, the Wnt/β-catenin-dependent activation of Msx2 was defective in Smad4-deficient embryonic stem cells or when Smad binding elements were mutated but persisted in the presence of various BMP antagonists, indicating that Smad4 was involved in transducing the Wnt/β-catenin signals in the absence of a BMP autocrine loop. A chromatin immunoprecipitation analysis revealed that endogenous Smad4, but not Smad1, was part of the Lef1 transcriptional complex in response to β-catenin activation, dismissing any implication of BMP signaling in this response. We propose that Wnt signaling pathway could dictate cell fate not only by modulating BMP levels but also by directly regulating cooperatively BMP-target genes. Recent in vivo evidence suggests that Wnt signaling plays a central role in determining the fate of stem cells in the ectoderm and in the neural crest by modulating bone morphogenetic protein (BMP) levels, which, in turn, influence Msx gene expression. However, the molecular mechanism regulating the expression of the Msx genes as key regulators of cell fate has not been elucidated. Here we show in murine embryonic stem cells that BMP-dependent activation of Msx2 is mediated via the cooperative binding of Smad4 at two Smad binding elements and of lymphoid enhancing factor (Lef1) at two Lef1/TCF binding sites. Lef1 can synergize with Smad4 and Smad1 to activate Msx2 promoter, and this transcriptional complex is assembled on the endogenous promoter in response to BMP2. The Wnt/β-catenin signaling pathway can activate Msx2 via the binding of Lef1 to its promoter and synergizes with BMP2 to activate Msx2 expression, possibly via enhanced recruitment of the p300/cAMP-response element-binding protein-binding protein co-factor. Interestingly, the Wnt/β-catenin-dependent activation of Msx2 was defective in Smad4-deficient embryonic stem cells or when Smad binding elements were mutated but persisted in the presence of various BMP antagonists, indicating that Smad4 was involved in transducing the Wnt/β-catenin signals in the absence of a BMP autocrine loop. A chromatin immunoprecipitation analysis revealed that endogenous Smad4, but not Smad1, was part of the Lef1 transcriptional complex in response to β-catenin activation, dismissing any implication of BMP signaling in this response. We propose that Wnt signaling pathway could dictate cell fate not only by modulating BMP levels but also by directly regulating cooperatively BMP-target genes. The bone morphogenetic protein 2/4 (BMP2/4) 1The abbreviations used are: BMPbone morphogenetic proteinSBESmad binding elementLef1lymphoid enhancing factorTCFT-cell factorCBPcAMP-response element-binding protein-binding proteinEMSAelectrophoretic mobility shift assayChIPchromatin immunoprecipitationESembryonic stemrrhombomeresRTreverse transcriptaseGSTglutathione S-transferase. signaling pathway is activated by the binding of the ligand to a family of type I and type II serine-threonine kinase receptors (1.Massague J. Cell. 1996; 85: 947-950Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 2.Wrana J.L. Miner Electrolyte Metab. 1998; 24: 120-130Crossref PubMed Scopus (83) Google Scholar). This activated receptor complex recruits and phosphorylates receptor Smads (Smad1, Smad5, or Smad8), which, in turn, associate with Smad4. This complex then translocates to the nucleus where it participates in transcriptional regulation. The N-terminal, Mad homology-1 domain of Smad4 binds DNA via a Smad binding element (SBE) identified as AGAC using a random pool of oligonucleotides (3.Zawel 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 (902) Google Scholar) or as CAGAC based on the crystal structure of the Mad homology-1 domain bound to the SBE (4.Shi Y. Wang Y.F. Jayaraman L. Yang H. Massague J. Pavletich N.P. Cell. 1998; 94: 585-594Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar). The Smad complex can act either as a co-repressor or co-activator, depending on the co-factor with which it associates. For example, it can recruit the p300/CBP histone acetyltransferase (5.Pouponnot C. Jayaraman L. Massague J. J. Biol. Chem. 1998; 273: 22865-22868Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 6.Janknecht R. Wells N.J. Hunter T. 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Of interest, Smad4 was shown to functionally interact with the high mobility group member, lymphoid enhancing factor (Lef1), to regulate the expression of Xtwn in Xenopus (11.Nishita M. Hashimoto M.K. Ogata S. Laurent M.N. Ueno N. Shibuya H. Cho K.W. Nature. 2000; 403: 781-785Crossref PubMed Scopus (404) Google Scholar, 12.Labbe E. Letamendia A. Attisano L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8358-8363Crossref PubMed Scopus (387) Google Scholar). Lef1 is a transcriptional mediator of the Wnt signaling pathway activated by the binding of β-catenin (13.Miller J.R. Genome Biol. 2002; 3: 3001.1-300115Google Scholar). In unstimulated cells, β-catenin is constantly degraded by ubiquitination via phosphorylation by glucose synthase kinase-3β. Upon Wnt signaling, the constitutive kinase activity of glucose synthase kinase-3β is inhibited and allows the accumulation of β-catenin in the nucleus (14.Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1758) Google Scholar). β-Catenin binds and activates Lef1 by recruiting p300/CBP protein to the complex (15.Takemaru K.I. Moon R.T. J. Cell Biol. 2000; 149: 249-254Crossref PubMed Scopus (412) Google Scholar, 16.Hecht A. Vleminckx K. Stemmler M.P. van Roy F. Kemler R. EMBO J. 2000; 19: 1839-1850Crossref PubMed Google Scholar). bone morphogenetic protein Smad binding element lymphoid enhancing factor T-cell factor cAMP-response element-binding protein-binding protein electrophoretic mobility shift assay chromatin immunoprecipitation embryonic stem rhombomeres reverse transcriptase glutathione S-transferase. Some of the genes known to be induced by BMP2/4 include the homeobox containing genes Msx-1 and -2 (17.Davidson D. Trends Genet. 1995; 11: 405-411Abstract Full Text PDF PubMed Scopus (311) Google Scholar). In chick embryos, BMP2/4 is secreted from the neuroepithelium along the roof of the neural tube (18.Maden M. Graham A. Gale E. Rollinson C. Zile M. Development. 1997; 124: 2799-2805PubMed Google Scholar). Concomitant to the Bmp expression, Msx genes are expressed in the neural crest cells of the dorsal region of the neural tube to the hindbrain (17.Davidson D. Trends Genet. 1995; 11: 405-411Abstract Full Text PDF PubMed Scopus (311) Google Scholar, 19.Hogan B.L. Curr. Opin. Genet. Dev. 1996; 6: 432-438Crossref PubMed Scopus (665) Google Scholar). Recent studies in the avian system indicate that Msx2 expression inhibits chondrogenic differentiation of the migratory cranial neural crest cells (20.Takahashi K. Nuckolls G.H. Takahashi I. Nonaka K. Nagata M. Ikura T. Slavkin H.C. Shum L. Dev. Dyn. 2001; 222: 252-262Crossref PubMed Scopus (77) Google Scholar). The role of Msx genes in maintaining undifferentiated cells has also been well established in myoblasts (21.Thompson-Jaeger S. Raghow R. Mol. Cell. Biochem. 2000; 208: 63-69Crossref PubMed Google Scholar, 22.Woloshin P. Song K. Degnin C. Killary A.M. Goldhamer D.J. Sassoon D. Thayer M.J. Cell. 1995; 82: 611-620Abstract Full Text PDF PubMed Scopus (132) Google Scholar), and overexpression of Msx-1 in myotube cells triggers their de-differentiation into multipotent stem cells (23.Odelberg S.J. Kollhoff A. Keating M.T. Cell. 2000; 103: 1099-1109Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). Consistent with their role in modulating cell fate, BMP2/4 are potent neural inhibitors. In Xenopus, the default fate of the ectodermal cells is to acquire a neural phenotype. Secretion of BMP4 from the dorsal neural tube prevents neural determination and allow the cells to take on an epidermal fate (24.Sasai Y. Lu B. Steinbeisser H. De Robertis E.M. Nature. 1995; 376: 333-336Crossref PubMed Scopus (542) Google Scholar). The Msx genes appear to mediate this response, because overexpression of Msx1 in the ectoderm of Xenopus embryos inhibits neural and promotes epidermal differentiation. Similar default mechanisms for neural differentiation is inhibited by BMP/Smad4 signaling in mouse embryonic stem (ES) cells (25.Tropepe V. Hitoshi S. Sirard C. Rossant J. Mak W.T. van der Kooy D. Neuron. 2001; 30: 65-78Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). An additional role of the Msx genes is to mediate BMP-induced apoptosis. In the avian system, increased expression of BMP2/4 in rhombomeres (r) 3 and 5 leads to increased Msx2 expression and to the apoptotic elimination of their neural crests cells (26.Graham A. Francis-West P. Brickell P. Lumsden A. Nature. 1994; 372: 684-686Crossref PubMed Scopus (451) Google Scholar). Consequently, misexpression of Msx2 in r2 and r4 leads to apoptosis of its neural crest cells, which would otherwise migrate and contribute to the formation of craniofacial structures (27.Takahashi K. Nuckolls G.H. Tanaka O. Semba I. Takahashi I. Dashner R. Shum L. Slavkin H.C. Development. 1998; 125: 1627-1635PubMed Google Scholar). Taken together, these results indicate that graded expression of Msx2 can result in different cell fate, emphasizing the importance of tight regulatory mechanisms. Although the Msx genes are sufficient to induce these biological responses, recent studies in avian embryos suggest that it is the Wnt signaling pathway that dictates these BMP-dependent responses. The Wnt-induced neural inhibition is achieved by interfering with fibroblast growth factor-induced inhibition of Bmp4 expression (28.Wilson S.I. Rydstrom A. Trimborn T. Willert K. Nusse R. Jessell T.M. Edlund T. Nature. 2001; 411: 325-330Crossref PubMed Scopus (234) Google Scholar). The ectodermal cells close to the region expressing Wnt (lateral zone) take on an epidermal fate whereas the marginal ectoderm, with low Wnt expression, acquire a neural fate. Similarly, exogenous Wnt, but not BMP4, can provoke induction of neural crest cells from the neural plate (29.Garcia-Castro M.I. Marcelle C. Bronner-Fraser M. Science. 2002; 297: 848-851PubMed Google Scholar), and increased levels of Wnt are required for the apoptotic elimination of neural crest cells in r3 and r5 (30.Ellies D.L. Church V. Francis-West P. Lumsden A. Development. 2000; 127: 5285-5295PubMed Google Scholar). In this latter system, Wnt activity is enhanced by decreasing the levels of the Wnt antagonist, cSFRP2, in r3 and r5 relative to the others. Thus, it can be deduced from these studies that the levels of Wnt signaling determines cell fate, in part, by modulating BMP production, which, in turn, could influence the activation of Msx gene expression. However, the molecular mechanisms regulating the graded expression of Msx2 are unclear, because BMP-induced activation of Msx genes appears insufficient to induce most of these biological responses (29.Garcia-Castro M.I. Marcelle C. Bronner-Fraser M. Science. 2002; 297: 848-851PubMed Google Scholar, 30.Ellies D.L. Church V. Francis-West P. Lumsden A. Development. 2000; 127: 5285-5295PubMed Google Scholar). In this study we show that BMP-induced activation of Msx2 is regulated by two canonical SBEs and two Lef1/TCF binding sites. The Smad4/1 co-activators functionally cooperate with the transcription factor Lef1 and together form a complex on the endogenous Msx2 promoter in response to BMP2. Activation of the Wnt pathway induces Msx2 expression via these same Lef1/TCF binding sites, and this response is partially dependent on the presence of Smad4 protein and its ability to form a complex with Lef1, indicating that Smad4 can act as a transcriptional co-activator for Wnt signaling. Thus, Smad4 integrates signals from the Wnt or the BMP pathways either by respectively interacting with the Lef1/β-catenin complex or by recruiting Smad1 to the DNA-bound Lef1. We proposed that in the presence of both signals, the levels or affinity of p300/CBP is enhanced via its independent interaction with β-catenin or Smad1 (7.Feng X.H. Zhang Y. Wu R.Y. Derynck R. Genes Dev. 1998; 12: 2153-2163Crossref PubMed Scopus (457) Google Scholar, 16.Hecht A. Vleminckx K. Stemmler M.P. van Roy F. Kemler R. EMBO J. 2000; 19: 1839-1850Crossref PubMed Google Scholar, 31.Pearson K.L. Hunter T. Janknecht R. Biochim. Biophys. Acta. 1999; 1489: 354-364Crossref PubMed Scopus (47) Google Scholar) and facilitate the recruitment of transcriptional machinery to the Msx2 promoter. These findings reveal a molecular mechanism explaining how Wnt signaling can act as a determinant factor influencing cell fate by directly modulating the expression of BMP-target genes. Cell Culture and Transfections—The wild-type ES cells and Smad4–/– ES cells (clone F9–2A2) (32.Sirard C. de la Pompa J.L. Elia A. Itie A. Mirtsos C. Cheung A. Hahn S. Wakeham A. Schwartz L. Kern S.E. Rossant J. Mak T.W. Genes Dev. 1998; 12: 107-119Crossref PubMed Scopus (418) Google Scholar) were maintained on 0.1% gelatin-treated tissue culture dishes in Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum (Hyclone), 0.1 μg/ml leukemia inhibitory factor, 1 mm sodium pyruvate, 2 mm l-glutamine, and β-mercaptoethanol (10–4m). All transfections were performed using wild-type ES cells (unless otherwise specified). They were prepared in triplicates in 96-well dishes containing 5000 to 8000 cells seeded the previous day, and LipofectAMINE plus transfection reagent (Invitrogen) was used as recommended by the manufacturers. Equal amounts of DNA (total of 110 ng/well) were co-transfected for all experimental constructs except for Smad1-expressing construct, which was transfected at110 of the total amount of DNA to reduce background activity. All transfections were normalized to Renilla Luciferase (pRL-CMV) expression vector (10 ng) or to protein content measured at A570. The normalized luciferase values were expressed relative to the specified reporter. BMP2 was added at 25 ng/ml the following day of transfection for 10 to 24 h depending on cell density of seeding. All transfections were performed in triplicate and repeated several times. Luciferase assays were carried out using the Dual-Luciferase Reporter Assay System (Promega). Plasmid Construction—Msx2-lux was derived by cloning a 1.7-kb BamHI genomic fragment ∼3 kb upstream of the transcriptional start site of Msx2, into pGL3-promoter vector (Promega) (33.Sirard C. Kim S. Mirtsos C. Tadich P. Hoodless P.A. Itié A. Maxson R. Wrana J.L. Mak T.W. J. Biol. Chem. 2000; 275: 2063-2070Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Mutations of the Smad and Lef1/TCF binding elements were done by a multi-step PCR system using Msx2-lux as a template. The first PCR reaction generated Fragment A using a common forward primer, 5′-GGAGCGGCCGCAATAAAATA-3′, in combination with one of the following reverse primers: 5′-ACCTCCAACTGGGCgagtAAATCCGAAGGG-3′, 5′-ATCAGAAGCGGGCgagtAAATGGGCGCCA-3′, 5′-CGGTTCTGAACCTTcaTAGACCTGTTCTCA-3′, or 5′-ACTCAACAGTCCTTcaTTCCTGGGCCTCT-3′ containing mutations (lowercase) in potential Smad and Lef1/TCF consensus binding sites at –766, –600, –1210, and –1060, respectively. In the second PCR, a common reverse primer, 5′-AAAAATTAGTCAGCGATGGGGC-3′, was used in conjunction with the reverse complement of the above mentioned primers containing mutations in the SBEs and Lef1/TCF consensus sites to obtain Fragment B. 100 ng of gel-purified Fragments A and B were mixed together and used as template for a third PCR reaction using a second set of nested forward and reverse primers, 5′-CAAGTGCAGGTGCCCGAACATTT-3′ and 5′-GCGGGATCCTGGTTGCTGACTAATTGAG-3′, to amplify 1.7 kb fragment with a terminal BamHI site (underlined) at the 3′ end. The amplified fragment was then subcloned in the MluI and BglII sites of pGL3 promoter and sequenced. The same approach was used to obtain constructs with double mutations using the specified single mutation constructs as templates. cDNA of full-length Smad4 protein was cloned in BamHI sites of pGex-4T2 (Amersham Biosciences). Reverse Transcriptase (RT)-PCR Analysis—ES cells were plated on 10-cm tissue culture dishes (BD Biosciences) at 5 × 105 cells/dish for 24 h. Cells were then starved in serum-free medium for 3–4 h, and RNA was extracted at different time points after addition of BMP2 (25 ng/ml) and 1% fetal bovine serum. Extracted RNA was reverse-transcribed with the Advantage RT-PCR kit (Clontech) and amplified for 30 cycles at an annealing temperature of 58 °C using the following primers (sense:antisense): Msx-2, 5′-CCGGGCCTCTCGTCAAAG-3′:5′-CGCCGTATATGGATGCTGCTT-3′. To control for the amount of RNA in different samples, expression of glucose-6-phosphate dehydrogenase was analyzed using oligonucleotides described elsewhere (33.Sirard C. Kim S. Mirtsos C. Tadich P. Hoodless P.A. Itié A. Maxson R. Wrana J.L. Mak T.W. J. Biol. Chem. 2000; 275: 2063-2070Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Total Cell Extracts and GST Fusion Proteins—Wild-type and Smad4-deficient ES cells were plated on 15-cm tissue culture dishes (BD Biosciences) at 2.5 × 106 cells/dish for 24 h. Cells were then starved in serum-free medium for 3–4 h, and total protein was extracted after addition of BMP2 (25 ng/ml) for 3 h. The cells were homogenized in a buffer containing 20 mm Hepes, pH 7.9, 100 mm KCl, 1 mm dithiothreitol, 25% glycerol, and supplemented with protease and phosphatase inhibitors. Expression of GST-Smad4 fusion protein was induced in BL21 strain of Escherichia coli by addition of 0.3 mm isopropyl-1-thio-β-d-galactopyranoside and incubated at 30 °C for 4 h. GST-Smad4 was purified using glutathione-Sepharose beads and following the Amersham Biosciences Bulk-protein purification protocol. Electrophoretic Mobility Shift Assays (EMSA)—Reactions were performed with 1 μg of purified GST-Smad4 or 5 μg of total protein from ES cell extract in a final volume of 50 μl in 20 mm HEPES, pH 7.9, 25 mm KCl, 2.5 mm MgCl2 or 7.5 mm for total protein, 1 mm EDTA, pH 8.0, 1 mm dithiothreitol, 10% glycerol. Five micrograms of bovine serum albumin was included in the reaction mixture. Proteins from ES cell extracts were preincubated with 1 μg of poly(dI·dC) for 5 min at room temperature, whereas bacterially expressed proteins were preincubated with 50 ng of poly(dI·dC). Double-stranded SBE-600 and SBE-766 probes were generated by EcoRI/XmaI or SacI digestion of Msx2-lux construct, respectively, and double-stranded probe containing both SBEs was generated by Bsu36I/XmaI digestion. The probes were end-labeled with [γ-32P]ATP (50,000 cpm, ∼1 pmol). Upon addition of probes, samples were further incubated for 30 min at 30 °C. For competition assays, 30-fold molar excess of the unlabeled probes were added 10 min prior to the addition of the radiolabeled probe. Samples were loaded on a 4% polyacrylamide gel (80:1 acrylamide/bisacrylamide), and complexes were separated at 75 V for 12 h using 24-cm-long glass plates in Tris-glycine high ionic strength buffer. Supershifting of the complex was performed by adding 1 μl of Smad4 polyclonal antibody (33.Sirard C. Kim S. Mirtsos C. Tadich P. Hoodless P.A. Itié A. Maxson R. Wrana J.L. Mak T.W. J. Biol. Chem. 2000; 275: 2063-2070Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) together with the probe. Gels were dried and scanned using a phosphorimager (Bio-Rad). Chromatin Immunoprecipitation Analysis—The ChIP analysis was performed as described elsewhere (34.Boyd K.E. Farnham P.J. Mol. Cell. Biol. 1999; 19: 8393-8399Crossref PubMed Scopus (147) Google Scholar) with the following modifications. The wild-type ES cells were grown in 6-well plates at 1.0 × 105 cells/ well for 24 h and then transfected with equal amounts of either an empty vector (pCDNA3.0) (Invitrogen) or Msx2-lux and ΔN89-β-catenin constructs to give a total of 1 μg of DNA/well. The next day, the cells were treated with BMP2 (25 ng/ml) for 10 h. The cells were then cross-linked with 1% formaldehyde for 25 min at room temperature, and glycine (125 mm) was added for 10 min at room temperature to quench the formaldehyde. The cells were washed with cold phosphate-buffered saline and lysed in 1 ml of lysis buffer (5 mm PIPES, pH 8.0, 85 mm KCl, 0.5% Nonidet P-40, protease inhibitors) for 30 min on ice. Nuclei were centrifuged (5000 rpm) for 10 min at 4 °C and resuspended in 0.4 ml of nuclear lysis buffer (50 mm Tris, pH 8.0, 10 mm EDTA, 1% SDS, protease inhibitors). 0.1 g of glass beads (212–300 μm; Sigma) was added, and samples were vortexed for 30 min at 4 °C and sonicated four times for 10 s using a Brason 450 Sonifier (output control 5 and 60% duty cycle). The average DNA fragments ranged between 300 and 1000 bp. The lysates were then clarified by centrifugation (13,000 rpm) for 10 min at 4 °C and diluted 5-fold in ChIP dilution buffer (15 mm Tris, pH 8.0, 1% Triton X-100, 0.01% SDS, 1 mm EDTA, 150 mm NaCl, protease inhibitors). The sonicated samples were precleared using Protein G-Sepharose beads (Amersham Biosciences) for 1 h at 4 °C, and 1% of the lysate was used for Input control. Samples were incubated with 5 μg of anti-Lef1 (Santa Cruz Biotechnology), anti-Smad4 (Santa Cruz Biotechnology), or anti-Smad1 (Upstate Biotechnology) or no antibody at 4 °C for 12 h. Immunoprecipitation was carried out using Protein G-Sepharose beads for 1 h at 4 °C. Immune complexes were washed consecutively for 5 min with each of the following solutions: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris, pH 8.0, 150 mm NaCl), high salt wash buffer (same as low salt wash solution but 500 mm NaCl), LiCl wash buffer (0.25 m LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mm EDTA, 10 mm Tris, pH 8.0) and twice with TE. Complexes were then eluted twice at 65 °C for 10 min in elution buffer (50 mm Tris, pH 8.0, 10 mm EDTA, 1% SDS). Immunoprecipitated DNA was reverse cross-linked at 65 °C overnight and purified using a PCR purification kit (Qiagen). 2 μl of purified DNA from cells transfected with the reporter construct was used for PCR amplification (30 cycles), and 15 μl of purified DNA from non-transfected cells was used and amplified for 55 cycles. All reactions were done under an annealing temperature of 63 °C. The set of primers for amplifying the Lef1/TCF binding sites (292 bp) are as follows: forward, 5′-GAAACCGAGAAGGCAGCAGTGTC-3′; reverse, 5′-CAGTGGAAGTTGAGGGGCAGAAGA-3′; and for the SBEs (301 bp) they are as follows: forward, 5′-CCGAGTATCTACCTAAATTCCCTGCTG-3′; reverse, 5′-TTCCTTCTAATGGGCCGCTTGTT-3′. A sonication control PCR (734 bp) was done using the forward Lef1/TCF primer and the reverse SBE primer. Identification of SBE on the Msx2 Promoter—The ability of BMP2/4 to induce the expression of Msx genes has been established in several biological systems, including the avian and Xenopus embryos. To determine whether BMP2 can induce endogenous expression of Msx2 in murine ES cells, kinetic analyses were performed by RT-PCR. Total RNA was obtained from wild-type ES cells at various time points after BMP2 stimulation. As early as 15 min after the addition of BMP2, expression of Msx2 was induced and reached maximal activation after 4 h (Fig. 1A). This BMP response was preserved in the presence of the protein synthesis inhibitor, cyclohexamide (Fig. 1B), indicating that Msx2 activation was an immediate early response following BMP2 stimulation. Our previous study demonstrated that a potential enhancer element present in a 1.7-kb fragment of the Msx2 locus, 3 kb upstream of the initiation start site, required Smad4 and synergistically interacted with Smad1 for its activation (33.Sirard C. Kim S. Mirtsos C. Tadich P. Hoodless P.A. Itié A. Maxson R. Wrana J.L. Mak T.W. J. Biol. Chem. 2000; 275: 2063-2070Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Sequence analysis of this region revealed two potential SBEs (GTCTG), at –600 and –766 (Fig. 1C). Interestingly, the homology between these two sites extended to seven additional nucleotides (ATTTgtctgCCC) and was similar to functional SBEs found in the several other promoters (35.Jonk L.J. Itoh S. Heldin C.H. ten Dijke P. Kruijer W. J. Biol. Chem. 1998; 273: 21145-21152Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar, 36.Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1611) Google Scholar, 37.Yeo C.Y. Chen X. Whitman M. J. Biol. Chem. 1999; 274: 26584-26590Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), suggesting that they could contribute to the efficient binding of Smad proteins. To determine whether these sites played a functional role in the Smad4-dependent activation of Msx2, we performed a PCR-based site-directed mutagenesis of the –600 and –766 CAGA boxes either separately or in combination (Fig. 1C). When transfected into wild-type ES cells, inactivation of either the –600 or the –766 SBE reduced the BMP-dependent activation by 2-fold relative to the wild-type Msx2-lux reporter construct (Fig. 1D). The simultaneous inactivation of both SBEs resulted in a further 2-fold reduction, bringing the luciferase expression to basal levels. These results indicate that both SBEs were required for the transcriptional activation of Msx2. The activation of the wild-type reporter remained relatively elevated at low levels of BMP2 concentrations (0.25 ng/ml) and was not significantly enhanced over a 50-fold increase in BMP2 (Fig. 1D), suggesting that increasing BMP2 levels might not be sufficient to enhance Msx2 expression. Smad4 Specifically Binds Both SBEs—To confirm Smad4 binding to the SBE, an EMSA was performed with 100 bp end-labeled oligonucleotide probes containing either one of the two SBEs (Fig 2A). The band shift was initially performed with recombinant GST-Smad4 fusion protein. A DNA-protein complex was obtained with the radiolabeled Probe A containing the wild-type –600 SBE, but not with the probe containing nucleotide substitutions at the core sequence of the SBE (Fig. 2B, lanes 1–4), indicating that the SBE was responsible for the binding of Smad4. Binding of Smad4 to the radiolabeled wild-type probe was competed with as little as 30-fold molar excess of unlabeled wild-type probe but not with unlabeled probe mutated at the –600 SBE (Fig. 2B, lanes 5–8). These results indicated that Smad4 binding to this SBE was specific. Similar results were obtained for the –766 SBE, whereby the radiolabeled Probe B containing the wild-type SBE, but not the one containing nucleotide substitutions at the SBE, formed a DNA-protein complex with Smad4 protein (Fig. 2B, lanes 9–12). Moreover, competition assays with the unlabeled –766 SBE revealed that only the fragment containing the wild-type SBE, but not the one containing a mutated SBE, could prevent Smad4 protein from binding to the wild-type radiolabeled probe (Fig. 2B, lanes 13–16). These results indicate that Smad4 binds to both SBEs with similar efficiency. To examine whether Smad4 was part of an endogenous transcriptional complex binding to the SBE, nuclear extracts from wild-type and Smad4-deficient ES cells, treated or not with BPM2, were subjected to EMSA. A specific DNA-protein complex was formed with Probe C, encompassing both SBEs, with cellular extract derived from wild-type but not Smad4-deficient ES cells, and this complex was greatly enhanced in response to BMP2 (Fig. 2B, lanes 18–21). The presence of Smad4 in this DNA-protein complex was confirmed by a supershift of the radiolabeled probe with anti-Smad4 antibody (Fig. 2B, lanes 23 and 24). These results indicate that under physiological conditions endogenous Smad4 is assembled at the Msx2 promoter in response to BMP signaling. Smad4 Interacts with the LEF1 Transcription Factor on the Msx2 Promoter—The requirement for Smad4 to associate with tran
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