Identification of a Critical Sp1 Site within the Endoglin Promoter and Its Involvement in the Transforming Growth Factor-β Stimulation
2001; Elsevier BV; Volume: 276; Issue: 37 Linguagem: Inglês
10.1074/jbc.m011611200
ISSN1083-351X
AutoresLuisa M. Botella, Tilman Sanchez‐Elsner, Carlos Rı́us, Ángel L. Corbí, Carmelo Bernabéu,
Tópico(s)Tracheal and airway disorders
ResumoEndoglin, a component of the transforming growth factor-β (TGF-β) receptor complex expressed on endothelial cells, is involved in cardiovascular morphogenesis and vascular remodeling, as exemplified by the fact that the endoglin gene is the target for the autosomal dominant disorder known as hereditary hemorrhagic telangiectasia type 1. Since haploinsufficiency is the underlying mechanism for hereditary hemorrhagic telangiectasia type 1, understanding the regulation of endoglin gene expression appears to be a crucial step to correct the disease. In this study we have identified an Sp1 site at −37 as a critical element for the basal transcription of the endoglin TATA-less promoter. Since endoglin promoter activity is stimulated by TGF-β and this stimulation is located at the Sp1-containing proximal region, we have investigated the possible involvement of Sp1 in the TGF-β-mediated induction. Mutation of the Sp1-binding sequence, or addition of the Sp1 inhibitor WP631, abolished both the basal transcription activity and the TGF-β responsiveness of the endoglin promoter. Binding of Sp1 and Smad3 to the proximal promoter region −50/−29 was evidenced by electrophoretic mobility shift assays and DNA affinity precipitation studies. Furthermore, synergistic cooperation on the promoter activity between Sp1 and TGF-β or Smad3 could be demonstrated by co-transfection experiments of reporter promoter constructs. The molecular mechanism underlying this cooperation appears to involve a direct physical interaction between Sp1 and Smad3/Smad4. Endoglin, a component of the transforming growth factor-β (TGF-β) receptor complex expressed on endothelial cells, is involved in cardiovascular morphogenesis and vascular remodeling, as exemplified by the fact that the endoglin gene is the target for the autosomal dominant disorder known as hereditary hemorrhagic telangiectasia type 1. Since haploinsufficiency is the underlying mechanism for hereditary hemorrhagic telangiectasia type 1, understanding the regulation of endoglin gene expression appears to be a crucial step to correct the disease. In this study we have identified an Sp1 site at −37 as a critical element for the basal transcription of the endoglin TATA-less promoter. Since endoglin promoter activity is stimulated by TGF-β and this stimulation is located at the Sp1-containing proximal region, we have investigated the possible involvement of Sp1 in the TGF-β-mediated induction. Mutation of the Sp1-binding sequence, or addition of the Sp1 inhibitor WP631, abolished both the basal transcription activity and the TGF-β responsiveness of the endoglin promoter. Binding of Sp1 and Smad3 to the proximal promoter region −50/−29 was evidenced by electrophoretic mobility shift assays and DNA affinity precipitation studies. Furthermore, synergistic cooperation on the promoter activity between Sp1 and TGF-β or Smad3 could be demonstrated by co-transfection experiments of reporter promoter constructs. The molecular mechanism underlying this cooperation appears to involve a direct physical interaction between Sp1 and Smad3/Smad4. transforming growth factor-β electrophoretic mobility shift assay polyacrylamide gel electrophoresis hereditary hemorrhagic telangiectasia Smad binding element glutathioneS-transferase hemagglutinin protein position identification with nuclease tail bone morphogenetic protein Endoglin is a component of the TGF-β1 receptor complex strongly expressed at the surface of human endothelial cells. It binds TGF-β1, TGF-β3, activin-A, BMP-2, and BMP-7 in the presence of the signaling receptor types I and II (1Cheifetz S. Bellón T. Calés C. Vera S. Bernabéu C. Massagué J. Letarte M. J. Biol. Chem. 1992; 267: 19027-19030Abstract Full Text PDF PubMed Google Scholar, 2Letamendı́a A. Lastres P. 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HHT1 is a vascular disorder, which shows a prevalence of up to 1 in 8,000, exhibiting age-dependent penetrance and variable expressivity (Online Mendelian Inheritance in Man, OMIM 187300). The most common clinical manifestations involve the development of vascular abnormalities seen as telangiectases on skin and lesions in nasal mucosa with bleeding. The lesions are the result of direct arteriovenous connections that can lead to large arteriovenous malformations in brain, lung, and liver. Many of the mutations reported to date in endoglin include deletions, insertion, and point mutations in the coding region, leading to premature stop codons and frameshifts that result in predicted truncated proteins that are neither expressed at the cell surface nor secreted. This and the existence of null allele mutations support haploinsufficiency as the underlying mechanism for HHT1 (12Pece N. Vera S. Cymerman U. White R.J. Wrana J.L. Letarte M. J. Clin. 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The primary analysis of endoglin promoter reveals that it is a TATA- and CAAT-less promoter, but with GC-rich tracts, including an Sp1 consensus site at −37, in the vicinity of the transcription initiation nucleus. These features are typical of TATA-less promoters with multiple transcription start sites (16Pugh B.F. Tjian R. Cell. 1990; 61: 1187-1197Abstract Full Text PDF PubMed Scopus (736) Google Scholar) and are also present in other components of the mammalian TGF-β system, including human TGF-β1 (17Kim S.J. Glick A. Sporn M.B. Roberts A.B. J. Biol. Chem. 1989; 264: 402-408Abstract Full Text PDF PubMed Google Scholar), mouse inhibin/activin βC gene (18Schmitt J. Hotten G. Jenkins N.A. Gilbert D.J. Copeland N.G. Pohl J. Schrewe H. Genomics. 1996; 32: 358-366Crossref PubMed Scopus (52) Google Scholar), and TGF-β receptor type II (19Bae H.W. Geiser A.G. Kim D.H. Chung M.T. Burmester J.K. Sporn M.B. Roberts A.B. Kim S.J. J. Biol. Chem. 1995; 270: 29460-29468Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 20Humphries D.E. Bloom B.B. Fine A. Goldstein R.H. Biochem. Biophys. Res. Commun. 1994; 203: 1020-1027Crossref PubMed Scopus (30) Google Scholar). We have shown previously that endoglin expression is stimulated by TGF-β1, at protein and mRNA levels (4Lastres P. Letamendı́a A. Zhang H. Rı́us C. Almendro N. Raab U. López L.A. Langa C. Fabra A. Letarte M. Bernabéu C. J. Cell Biol. 1996; 133: 1109-1121Crossref PubMed Scopus (283) Google Scholar), and that TGF-β1 is also able to induce the transcriptional activity of the endoglin promoter (15Rı́us C. Smith J.D. Almendro N. Langa C. Botella L.M. Marchuk D.A. Vary C.P.H. Bernabéu C. Blood. 1998; 92: 4677-4690Crossref PubMed Google Scholar). The stimulation by TGF-β could be detected in different endoglin promoter constructs, representing successive deletions from −400 to −141 base pairs, but the putative TGF-β responsive elements within this fragment have not been identified yet. The transcription modulatory effect of TGF-β is exerted by binding to type I and type II serine-threonine kinase receptors, which propagate intracellular signaling by phosphorylation of members of the Smad family of proteins (21ten Dijke P. Miyazono K. Heldin C.H. Trends Biochem. Sci. 2000; 25: 64-70Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 22Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3999) Google Scholar). Smads may be classified into three different subclasses: receptor-regulated or R-Smads, as Smad2, and Smad3 (activated by TGF-β), or Smad1, Smad5, and Smad8 (activated by BMP); the common partner or Co-Smad, Smad4; and finally the inhibitory Smads, I-Smads, Smad6, and Smad7. R-Smads are activated by phosphorylation at their C-terminal SS(M/V)S-motifs, whereafter they heteroligomerize with the common partner, Smad4. The Smad complexes are then translocated to the nucleus, where the transcriptional activities of different genes are affected. Within the nucleus, Smads bind to their target genes at the motifs called Smad binding elements (SBE) (23Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1588) Google Scholar, 24Jonk L. Itoh S. Heldin C.H. ten Dijke P. Kruijer W. J. Biol. Chem. 1998; 273: 21145-21152Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar, 25Yingling 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, 26Zawel 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 (892) Google Scholar). However, Smads bind with low affinity to their target sequences, requiring interaction with additional transcription factors in order to exert its function (27Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (481) Google Scholar, 28Iavarone A. Massagué J. Nature. 1997; 387: 417-422Crossref PubMed Scopus (330) Google Scholar). Several transcription factors and transcriptional co-activators have been identified to cooperate with Smad in transcriptional activation such as CBP/p300, FAST, TFE-3, PEBP-2/CBF, ATF-2, OAZ, and AP-1 (21ten Dijke P. Miyazono K. Heldin C.H. Trends Biochem. Sci. 2000; 25: 64-70Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 27Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (481) Google Scholar, 29Massagué J. Wotton D. EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar). The mechanisms whereby different transcription factors participate cooperatively in TGF-β induced transcription involve the stabilization of protein-DNA complexes, bridging Smads with the basal transcription machinery, or stimulating the acetyltransferase activity in promoter regions (21ten Dijke P. Miyazono K. Heldin C.H. Trends Biochem. Sci. 2000; 25: 64-70Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar,27Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (481) Google Scholar). In the proximal endoglin promoter, there are G/C-rich motifs, which are putative binding sites for the Sp1 transcription factor. Sp1 is an ubiquitously expressed protein with a zinc finger DNA-binding domain (16Pugh B.F. Tjian R. Cell. 1990; 61: 1187-1197Abstract Full Text PDF PubMed Scopus (736) Google Scholar, 30Berg J.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11109-11110Crossref PubMed Scopus (165) Google Scholar, 31Lania L. Majello B. De Luca P. 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Human hepatoma HepG2 cells, human epithelioid carcinoma HeLa, and monkey kidney COS cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with penicillin-streptomycin. The human monocytic line U-937 was cultured in RPMI medium supplemented with penicillin-streptomycin. The Drosophila Schneider S-2 cells were grown at room temperature in 100-ml culture bottles containing S-2 medium (Sigma) supplemented with 100 IU/ml gentamicin. The reporter vectors pCD105(−250/+350), pCD105(−150/+350), and pCD105(−50/+350) were derived from the human endoglin promoter (15Rı́us C. Smith J.D. Almendro N. Langa C. Botella L.M. Marchuk D.A. Vary C.P.H. Bernabéu C. Blood. 1998; 92: 4677-4690Crossref PubMed Google Scholar). Polymerase chain reaction was carried out in the presence of sequence-specific primers surrounded byHindIII/XhoI sites for directional cloning and the resulting −250/+350, −150/+350, and −50/+350 fragments were inserted into the reporter luciferase vector pXP2 previously digested with HindIII/XhoI. Mutagenesis of Sp1 site at −37 was performed by recombinant polymerase chain reaction, using mutated primers −50/−24 D (GCA GGC GGC CTG GG ttt AGC CCC TTCTC) and −50/−24 R (GAGGG GGG GCT aaa CCC AGG CCG CCT GC), where the mutated nucleotides appear in lowercase (CCC in the original sequence was changed by TTT). Using the pCD105(−50/+350) construct as template, mutant oligonucleotides −50/−24 D and −50/−24 R, together with flanking primers of the pXP2 vector, allowed the amplification of the mutant −50/+350 fragment, that was inserted into theHindIII/XhoI site of the pXP2 vector leading to the pCD105(−50/+350)Sp1Mut construct. The altered construct was confirmed by automated sequencing (ABI Prism 310 genetic analyzer). The reporter plasmid pRL-LUC, containing the prolactin promoter with a minimal TATA box, was kindly provided by Dr. Mercedes Rincón (Yale University School of Medicine, New Haven, CT). Transfections were performed with 1 μg of reporter vector, the indicated amounts of expression vectors, and Superfect reagent (Qiagen), following the instructions of manufacturer for each type of cell line. Luciferase activity was determined with the Luciferase assay reagent (Promega, Madison, WI) using a TD-20/20 luminometer (Promega). Luciferase activity values were corrected by internal normalization using cotransfection of the test plasmids with β-galactosidase, yielding the relative luciferase units as a measurement of the promoter activity. Luciferase assays were carried out in duplicate, and each experiment of transfection was repeated at least three times with similar results. Data plotted in the figures represent the mean ± standard deviations of intra assay values from representative experiments. When required, after transfection, cells were treated for 20 h with 10 ng/ml TGF-β1 (R&D Systems). The increase of the promoter activity by different stimuli was measured by -fold induction values using as a reference the activity of untreated samples whose arbitrary value is 1. For inhibition by bisanthracycline (WP631) of Sp1 binding to DNA, cells were incubated with different concentrations (0.1–1 μm) of WP631 (Tropix) 3 h after transfection and kept for 20 h. The expression vectors pCMV5-Flag-Smad3, and pCMV5-Smad4-HA, encoding human Smad members Flag- or hemagglutinin (HA) epitope-tagged, and pCMV5-TβRI encoding a constitutively activated form of the TGF-β receptor type I (ALK-5) have been described previously (39Labbé E. Silvestri C. Hoodless P.A. Wrana J.L. Attisano L. Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). EMSAs were carried out as described (15Rı́us C. Smith J.D. Almendro N. Langa C. Botella L.M. Marchuk D.A. Vary C.P.H. Bernabéu C. Blood. 1998; 92: 4677-4690Crossref PubMed Google Scholar, 40Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3918) Google Scholar). To obtain nuclear extracts, subconfluent cell cultures, either untreated or treated with TGF-β1 (10 ng/ml), were washed, collected in cold phosphate-buffered saline, and resuspended in appropriate buffers to collect the nuclear fraction and nuclear extracts. Complementary oligonucleotides, constituting the different binding motifs, were annealed followed by 5′ end labeling using [γ-32P]ATP and T4 polynucleotide kinase. Approximately 5 ng (100,000 cpm) of the respective probe was incubated with 5–10 μg of nuclear extract and 2 μg/reaction of poly(dI-dC) for 30 min on ice. Oligonucleotides used as probes were −50/−24 WT (GCAGGCG GCCTGGG CCCAGCC CCTTCTC), and −50/−24 Sp1-Mut (GCAGGCG GCCTGGG TTTAGCC CCTTCTC). For competition experiments, an excess of 100- or 200-fold excess of unlabeled double-stranded oligonucleotide was added. When required, EMSAs were carried out in the presence of recombinant Smad or Sp1 proteins. In experiments with antibody, protein extract and 1 μg of commercial antibody were preincubated for 60 min on ice prior to the addition of the remaining components of the binding reaction. Rabbit polyclonal antibodies anti-Sp1, anti-AP-2, and anti-Smad3 were from Santa Cruz Biotechnology (Santa Cruz, CA). For specific inhibition of Sp1 binding to DNA, nuclear extracts were incubated with the indicated concentrations of WP631 (Tropix). Binding reactions were separated by nondenaturing 6% PAGE in Tris-Borate-EDTA buffer at 4 °C, dried, and visualized by autoradiography. EMSAs were repeated at least three times with similar results, and representative experiments are shown in the corresponding figures. The PINPOINT technique is an alternative, in vivo method to the in vitro footprinting and allows the identification and positioning of a given transcription factor on the promoter (41Lee J.S. Lee C.H. Chung J.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 969-974Crossref PubMed Scopus (20) Google Scholar). The sense +42/+24 (CAG CAG GGA GCT CCC GGC) and antisense −73/−56 (GTG GCA CTT CCT CTA CCC) endoglin primers, as well as the Sp1 pointer (construction p588, kindly provided by Dr. Chung), were used. COS cells were cotransfected with pCD105(−150/+350) as reporter, and the p588 Sp1 pointer vector, which contains the Sp1 domains derived from pPacSp1 fused to the nuclease domain of restriction enzyme FokI. Because the nuclease domain ofFokI lacks sequence specificity, the position and the probability of cleavage by the Sp1 pointer is determined by Sp1 binding to DNA. The cleavage site was detected by primer extension. Two primers were used: an antisense primer, complementary to +46/+24 positions of the endoglin promoter; and the sense primer −73/−56. Total DNA from transfected cells was collected and subjected to primer extension with the above-mentioned 5′-kinased oligonucleotides. Reactions were loaded in a 6% polyacrylamide: 7.5 m urea sequencing gels, in the presence of a G+A sequencing lane. Gels were dried and autoradiographed. PINPOINT experiments were repeated at least four times with similar results, and a representative experiment is shown in the corresponding figure. The glutathioneS-transferase (GST) fusion proteins, GST-Smad3, and GST-Smad4, kindly provided by Dr. Liliana Attisano (University of Toronto, Toronto, Ontario, Canada), were expressed in Escherichia coli strain DH5α and purified as described (42Kardassis D. Papakosta P. Pardali K. Moustakas A. J. Biol. Chem. 1999; 274: 29572-29581Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). In vitro transcribed and translated Sp1 protein was obtained using a TnT kit (TnT® T7 coupled reticulocyte lysate system, Promega) in the presence of 1 μg of pCiNeoSp1 vector generously provided by Dr. Scott L. Friedman (Mount Sinai Medical Center, New York, NY), and one tenth of the reaction was used for each DNA binding experiment. Forty hours after transfection of COS-7 cells, total extracts were prepared by lysing the cells in 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 10 mm aprotinin, and 2 mm Pefabloc at 4 °C. The cell extracts were centrifuged at 10,000 rpm at 4 °C and incubated with anti-Sp1 or anti-Smad3 (Santa Cruz Biotechnology) for 2 h at 4 °C. Afterward, immunocomplexes were precipitated with protein G-Sepharose, washed with lysis buffer twice, and dissolved in Laemmli-SDS-polyacrylamide gel electrophoresis loading buffer. After 8% SDS-PAGE, the resolved proteins were transferred to Hybond-C extra nitrocellulose (Amersham Pharmacia Biotech) and the antigens were detected by incubation with the specific antibodies (anti-Sp1 or anti Smad3), followed by incubation with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Amersham Pharmacia Biotech), and developing with the Super Signal reagent from Pierce for enhanced chemiluminescence on x-ray film. Experiments were repeated at least five times with similar results, and a representative experiment is shown in the corresponding figure. COS-7 cells were transfected with the expression vectors pCMV5-Flag-Smad3, pCMV5-Smad4-HA, and pCMV5-TβRI using Superfect (Qiagen). After 48 h, cells were resuspended in a solution containing 50 mm HEPES, pH 7.4, 50 mm NaCl, 0.1% Tween 20, and 10% glycerol supplemented with protease and phosphatase inhibitors, and lysed by sonication. The lysates were precleared in the presence of ImmunoPure streptavidin-agarose (Pierce), followed by incubation with 200 ng of biotinylated −50/−24 oligonucleotide and 2 μg of poly(dI-dC). Endogenous Sp1 bound to the endoglin probe was then captured by streptavidin-agarose, washed in the same lysis buffer, fractioned on SDS-PAGE (8%), transferred onto nitrocellulose membranes, and developed as indicated in the previous section. Experiments were repeated at least five times with similar results, and a representative experiment is shown in the corresponding figure. We have shown previously that TGF-β1 stimulates the activity of the endoglin promoter (15Rı́us C. Smith J.D. Almendro N. Langa C. Botella L.M. Marchuk D.A. Vary C.P.H. Bernabéu C. Blood. 1998; 92: 4677-4690Crossref PubMed Google Scholar). Since TGF-β signaling is exerted through Smads, we assessed whether Smad proteins could reproduce the TGF-β-mediated stimulation of the endoglin promoter. Thus, HepG2 cells were transiently cotransfected with the reporter constructs pCD105(−350/+350), pCD105(−250/+350), and pCD105(−50/+350), derived from the proximal endoglin promoter (Fig. 1), together with an expression vector encoding Smad3. These constructs were transactivated by Smad3, and significant induction values were obtained with the −350/+350 (3-fold), −250/+350 (2.5-fold) and −50/+350 (2.5-fold) promoter fragments, whereas the promoterless pXP2 vector was unresponsive. The fact that the pCD105(−50/+350) construct, containing only 50 base pairs upstream of the transcription start site, displayed a level of stimulation by Smad3 similar to the longer constructs, suggested that a TGF-β/Smad-responsive element was located in this proximal region of the endoglin promoter. Interestingly, within this region, three putative Smad binding elements and one Sp1 motif were identified (Fig.1). To determine the sequences responsible for the TGF-β/Smad responsiveness of the endoglin promoter, DNase I footprinting experiments with TGF-β treated versusuntreated cells were performed. In different cell lines (U-937, L6E9, and HMEC-1), a protection area spanning the region −50/−20 was revealed (data not shown). Within this sequence, there is a Sp1 consensus site at −37/−29 (CCCAGCCCC) partially overlapping with a preceding G/C-rich region at −50/−35. To test whether the protection of the Sp1 motif was meaningful in vivo, the PINPOINT technique (41Lee J.S. Lee C.H. Chung J.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 969-974Crossref PubMed Scopus (20) Google Scholar) was applied. COS cells were cotransfected with pCD105(−150/+350) as reporter, together with the expression Sp1 pointer, and the cleavage site was detected by primer extension. Specific extended bands were obtained with both sense and antisense primers (Fig. 2 A). Although the antisense primer gave a fragment of around 75 nucleotides, corresponding to a cut point by the nuclease around the sequence GCT from −33 to −35, the sense primer was extended in a series of discrete bands from −35 to −46. Taking into account the flexibility of the nuclease cutting point (around 4 base pairs from the Sp1 binding site in the DNA; Ref. 41Lee J.S. Lee C.H. Chung J.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 969-974Crossref PubMed Scopus (20) Google Scholar), it can be concluded that Sp1 binds the endoglin promoter at around −33/−46. To confirm the Sp1 binding to the endoglin proximal promoter region, a synthetic oligonucleotide encompassing this region (−50/−24) was used as a probe in EMSA experiments. A slow migrating DNA-protein band, which was significantly increased after TGF-β treatment, was detected in both U937 and HepG2 cells (Fig. 2 B). This band was specific because it was competed with a 100-fold excess of cold oligonucleotide. Sp1 was present in the DNA complex, as the band decreased significantly (more than 5 times) using a Sp1-specific antibody, but was not affected by an anti-AP-2 antibody (Fig.2 B). The involvement of Sp1 was also demonstrated by the absence of a retarded complex when using a probe mutated at the Sp1 site (Fig. 2 C, lanes 3 and4). These results support the participation of Sp1 in the DNA-protein complex. Smad3 contains DNA binding activity with affinity toward the so-called SBE (GNCN), whereas Smad4 has affinity toward both the SBE and the G/C-rich motifs (39Labbé E. Silvestri C. Hoodless P.A. Wrana J.L. Attisano L. Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, 43Shi Y. Wang Y.F. Jayaraman L. Yang H. Massagué J. Pavletich N.P. Ce
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