Transcriptional Activators of TGF-β Responses: Smads
1998; Cell Press; Volume: 95; Issue: 6 Linguagem: Inglês
10.1016/s0092-8674(00)81696-7
ISSN1097-4172
AutoresRik Derynck, Ying E. Zhang, Xin‐Hua Feng,
Tópico(s)Genetic factors in colorectal cancer
ResumoSmads are a class of proteins that function as intracellular signaling effectors for the TGF-β superfamily of secreted polypeptides. TGF-β-related factors regulate cell proliferation and differentiation in organisms ranging from insects and worms to mammals. Although only the receptors for TGF-βs, activins, and BMPs have been characterized, all TGF-β-related factors, with the exception of the distantly related GDNF, are thought to act through a cell surface complex of two types of transmembrane serine/threonine kinase receptors. Most receptor complexes bind several ligands, and several type I receptors form combinatorial interactions with type II receptors, thus creating signaling diversity. Following ligand binding, the type II receptor kinases phosphorylate and thereby activate the type I receptor cytoplasmic domains. The Smads then act as type I receptor–activated signaling effectors, which regulate transcription of select genes in response to ligand (7Derynck R Feng X.-H Biochim. Biophys. Acta: Reviews on Cancer. 1997; 1333: F105-F150Crossref Scopus (504) Google Scholar; 10Heldin C.-H Miyazono K ten Dijke P Nature. 1997; 390: 465-471Crossref PubMed Scopus (3244) Google Scholar; 17Massagué J Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3893) Google Scholar; 24Whitman M Genes Dev. 1998; 12: 2445-2462Crossref PubMed Scopus (437) Google Scholar). The Smads received their name as a contraction of the names of the C. elegans Sma and Drosophila Mad, the first identified members of this class of signaling effectors. Their sequence alignments show two large conserved domains, the MH1 or N domain, and the MH2 or C domain, separated by a less conserved linker (L) segment (Figure 1A). The model of how Smads transfer information from the activated receptor complex to regulate transcription is illustrated in Figure 1B. The “receptor-activated” Smads interact transiently with the activated receptor complex and are C-terminally phosphorylated by the type I receptor. Thus, Smad2 and -3 are phosphorylated and activated by activin and TGF-β receptors, whereas Smad1, -5, and -8 are activated by BMP receptors and, in Drosophila, Mad is activated by receptors for the BMP-related Dpp. Following dissociation, the phosphorylated, receptor-activated Smads form complexes with Smad4 (or Medea in Drosophila), which are then translocated into the nucleus, where they regulate transcription. This model shows conceptual similarity with the Jak/STAT signal transduction pathway from activated cytokine receptors, whereby receptor activation results in phosphorylation of STATs by receptor-associated Jak kinases, and the phosphorylated STATs translocate into the nucleus to act as transcription factors (4Darnell Jr., J.E Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3282) Google Scholar). Over the last ten years, several TGF-β- and activin-responsive elements have been identified in various promoters, but no consensus sequences were apparent. Instead, binding sites for known transcription factors, such as AP-1, Sp1, and CTF/NF-1, were often shown to be required for ligand-induced transcription. Since coexpression of a receptor-activated Smad with Smad4 activates transcription and dominant-negative interference with Smad signaling inhibits the ligand-induced response, Smads are considered as effectors for the ligand-induced transcriptional responses. Recent progress provides insight into the mechanisms through which Smads regulate transcription and explains the sequence heterogeneity of the TGF-β/activin-responsive promoter sites. Thus, Smads have been shown to act as transcription factors through their ability to directly bind DNA, and to induce transcriptional responses through cooperativity with other transcription factors. Following nuclear translocation, Smads regulate transcription through physical interaction of the heteromeric Smad complex with a ligand-responsive promoter sequence. For example, the Smad2/4 complex interacts in response to activin with the Mix.2 promoter of Xenopus through FAST-1, a winged-helix transcription factor that binds to an activin-response element (1Chen X Rubock M.J Whitman M Nature. 1996; 383: 691-696Crossref Scopus (620) Google Scholar, 2Chen X Weisberg E Fridmacher V Watanabe M Naco G Whitman M Nature. 1997; 389: 85-89Crossref Scopus (486) Google Scholar). Whereas Smad2 interacts directly with FAST-1, Smad4 participates in the Smad2/FAST-1 complex primarily through its association with Smad2 (2Chen X Weisberg E Fridmacher V Watanabe M Naco G Whitman M Nature. 1997; 389: 85-89Crossref Scopus (486) Google Scholar; 16Liu F Pouponnot C Massagué J Genes Dev. 1997; 11: 3157-3167Crossref PubMed Scopus (394) Google Scholar). At the goosecoid promoter, the Smad2/4 complex interacts similarly with an activin-response sequence, but this interaction occurs through FAST-2, which is structurally related to FAST-1 and binds to a sequence similar to the FAST-1 binding sequence (15Labbé E Silvestri C Hoodless P.A Wrana J.L Attisano L Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). In an analogous way, the Smad3/4 complex interacts in response to TGF-β with responsive sequences in the PAI-1 (6Dennler S Itoh S Vivien D ten Dijke P Huet S Gauthier J.-M EMBO J. 1998; 11: 3091-3100Crossref Scopus (1536) Google Scholar; 8Feng X.-H Zhang Y Wu R.-Y Derynck R Genes Dev. 1998; 12: 2153-2163Crossref Scopus (443) Google Scholar; 11Hua X Liu X Ansari D.O Lodish H.F Genes Dev. 1998; 12: 3084-3095Crossref Scopus (251) Google Scholar), JunB (13Jonk L Itoh S Heldin C.-H ten Dijke P Kruijer W J. Biol. Chem. 1998; 273: 21145-21152Crossref PubMed Scopus (505) Google Scholar), or collagenase I (28Zhang Y Feng X.-H Derynck R Nature. 1998; 394: 909-912Crossref PubMed Scopus (666) Google Scholar) promoters. At these promoters, Smads interact directly with defined DNA sequences. This may have come as a surprise because the first report on the Smad2 interaction with the Mix.2 promoter emphasized an indirect interaction through FAST-1 (1Chen X Rubock M.J Whitman M Nature. 1996; 383: 691-696Crossref Scopus (620) Google Scholar). However, the DNA binding of Smads in various promoters is now well documented. Thus, Drosophila Mad interacts directly with a GC-rich sequence in the vestigial promoter. Whereas full-length Mad does not bind, removal of the C domain allows its binding (14Kim J Johnson K Chen H Carroll S Laughon A Nature. 1997; 388: 304-308Crossref PubMed Scopus (444) Google Scholar). The inhibition of the DNA binding of the N–L segment by the C domain is consistent with the notion that the N and C domains interact with each other, and that receptor activation results in exposure of the N and C domain sequences (9Hata A Lo R.S Wotton D Lagna G Massagué J Nature. 1997; 388: 82-87Crossref PubMed Scopus (290) Google Scholar). Similarly, Medea interacts through its N domain with several GC-rich sequences in the tinman promoter (25Xu X Yin Z Hudson J.B Ferguson E.L Frasch M Genes Dev. 1998; 12: 2354-2370Crossref PubMed Scopus (218) Google Scholar). A PCR-based selection has led to the definition of an optimal DNA sequence for Smad3 and -4 binding. This consensus “Smad-binding element” is GTCTAGAC, a palindromic sequence with two copies of GTCT and its reverse complement AGAC in the opposite DNA strand. Tandem repeats of this sequence confer TGF-β-inducible transcriptional activation (27Zawel L Dai J Buckhaults P Zhou S Kinzler K Vogelstein B Kern S Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar). Smad3 and -4 also bind to TGAGTCAGAC, i.e. an AP-1 binding site TGAGTCA that overlaps with an AGAC-containing Smad-binding sequence (26Yingling J Datto M Wong C Frederick J Liberati N Wang X.-F Mol. Cell. Biol. 1997; 17: 7019-7028Crossref Google Scholar; 28Zhang Y Feng X.-H Derynck R Nature. 1998; 394: 909-912Crossref PubMed Scopus (666) Google Scholar). Finally, Smad3 and -4 bind directly to CAGA-like sequences in the PAI-1 and JunB promoters (6Dennler S Itoh S Vivien D ten Dijke P Huet S Gauthier J.-M EMBO J. 1998; 11: 3091-3100Crossref Scopus (1536) Google Scholar; 13Jonk L Itoh S Heldin C.-H ten Dijke P Kruijer W J. Biol. Chem. 1998; 273: 21145-21152Crossref PubMed Scopus (505) Google Scholar) and, as with the “Smad-binding element,” concatemerization of these sequences confers TGF-β and Smad3/4 responsiveness. These sequences again contain tandem repeats of AGAC or its reverse complement GTCT. Consistent with the direct DNA binding properties of Mad, deletion of the C domain strongly increases the affinity of the Smad for these sequences (6Dennler S Itoh S Vivien D ten Dijke P Huet S Gauthier J.-M EMBO J. 1998; 11: 3091-3100Crossref Scopus (1536) Google Scholar; 27Zawel L Dai J Buckhaults P Zhou S Kinzler K Vogelstein B Kern S Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar; 28Zhang Y Feng X.-H Derynck R Nature. 1998; 394: 909-912Crossref PubMed Scopus (666) Google Scholar). In contrast, Smad4 interacts with an unrelated GC-rich, bipartite sequence in the goosecoid promoter (15Labbé E Silvestri C Hoodless P.A Wrana J.L Attisano L Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar), possibly reminiscent of the Mad/Medea-binding sequences. The direct interaction of the N domain of Smad3 with the Smad-binding element of 27Zawel L Dai J Buckhaults P Zhou S Kinzler K Vogelstein B Kern S Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar has been characterized through crystallographic analysis (20Shi Y Wang Y.-F Jayaraman L Yang H Massagué J Pavletich N.P Cell. 1998; 94: 585-594Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). The results reveal that a base-specific DNA interaction is provided by an 11–amino acid β hairpin in the N domain, which is embedded in the major groove of the DNA and contacts the GTCT sequence. Three residues in the β hairpin make five hydrogen bonds to three bases in the major groove and three additional contacts to DNA backbone phosphates. This observation, together with the reduced DNA binding affinity following single base mutations in the GTCT sequence (27Zawel L Dai J Buckhaults P Zhou S Kinzler K Vogelstein B Kern S Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar), suggests that the GTCT (or AGAC) sequence is optimal for Smad3 binding. The second base of the GTCT sequence tolerates single base substitutions with only a minor reduction in DNA binding affinity (6Dennler S Itoh S Vivien D ten Dijke P Huet S Gauthier J.-M EMBO J. 1998; 11: 3091-3100Crossref Scopus (1536) Google Scholar; 27Zawel L Dai J Buckhaults P Zhou S Kinzler K Vogelstein B Kern S Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar; 28Zhang Y Feng X.-H Derynck R Nature. 1998; 394: 909-912Crossref PubMed Scopus (666) Google Scholar), which is consistent with the lack of direct contact with the N domain of Smad3 (20Shi Y Wang Y.-F Jayaraman L Yang H Massagué J Pavletich N.P Cell. 1998; 94: 585-594Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). Remarkably, Smad2 does not bind these DNA sequences (6Dennler S Itoh S Vivien D ten Dijke P Huet S Gauthier J.-M EMBO J. 1998; 11: 3091-3100Crossref Scopus (1536) Google Scholar; 27Zawel L Dai J Buckhaults P Zhou S Kinzler K Vogelstein B Kern S Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar), demonstrating that, in spite of their 92% sequence identity, Smad2 and -3 are not functionally equivalent. This difference in DNA binding is likely due to a sequence insert in the N domain of Smad2, immediately before the DNA binding β hairpin, which may interfere with DNA recognition (20Shi Y Wang Y.-F Jayaraman L Yang H Massagué J Pavletich N.P Cell. 1998; 94: 585-594Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). The difference in DNA binding properties of Smad2 and -3 may explain why Smad3 can not replace Smad2 in activating transcription from the goosecoid promoter (15Labbé E Silvestri C Hoodless P.A Wrana J.L Attisano L Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). The ability of Smads to activate transcription in response to ligand results from a functional cooperativity with other transcription factors in a nucleoprotein complex (Figure 2). Thus, the interactions of the C domains of Smad2 with FAST-1 or -2 are essential for activin- and Smad2/4-mediated transcription from the Mix.2 or goosecoid promoters, respectively (2Chen X Weisberg E Fridmacher V Watanabe M Naco G Whitman M Nature. 1997; 389: 85-89Crossref Scopus (486) Google Scholar; 16Liu F Pouponnot C Massagué J Genes Dev. 1997; 11: 3157-3167Crossref PubMed Scopus (394) Google Scholar; 15Labbé E Silvestri C Hoodless P.A Wrana J.L Attisano L Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). Smad2, but not Smad1, associates with FAST-1, and this specificity is determined by a sequence difference in helix 2 of their C domains (3Chen Y.G Hata A Lo R.S Wotton D Shi Y Pavletich N Massagué J Genes Dev. 1998; 12: 2144-2152Crossref Scopus (298) Google Scholar). At TGF-β-responsive AP-1-binding sites, Smad3 interacts through its N–L segment with c-Jun and through its C domain with c-Fos, and thus forms a multiprotein complex. The cooperativity between Smad3/4 and c-Jun/c-Fos then results in TGF-β-induced transcription from these promoter sequences (28Zhang Y Feng X.-H Derynck R Nature. 1998; 394: 909-912Crossref PubMed Scopus (666) Google Scholar). In a conceptually similar way, Smad3/4 cooperates with the basic helix-loop-helix protein TFE3 to induce transcription from the PAI-1 promoter in response to TGF-β. Although no physical association between TFE3 and Smad3/4 has been shown, all three proteins interact with a promoter segment, which contains both a TFE3-binding E box sequence and a Smad-binding site (11Hua X Liu X Ansari D.O Lodish H.F Genes Dev. 1998; 12: 3084-3095Crossref Scopus (251) Google Scholar). Finally, the responsiveness of the promoters for the p15 and p21 Cdk inhibitors to TGF-β has been localized to Sp1-binding sites, suggesting that Sp1 is required for their transcriptional activation by Smads in response to TGF-β (5Datto M.B Yu Y Wang X.-F J. Biol. Chem. 1995; 270: 28623-28628Crossref PubMed Scopus (398) Google Scholar). The associations of Smad3 with c-Jun (28Zhang Y Feng X.-H Derynck R Nature. 1998; 394: 909-912Crossref PubMed Scopus (666) Google Scholar), and Smad2 with FAST-1 (2Chen X Weisberg E Fridmacher V Watanabe M Naco G Whitman M Nature. 1997; 389: 85-89Crossref Scopus (486) Google Scholar) or FAST-2 (15Labbé E Silvestri C Hoodless P.A Wrana J.L Attisano L Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar), all depend on ligand stimulated receptor activation, consistent with the ligand-induced Smad activation and nuclear translocation of the heteromeric Smad complex. In Drosophila, analyses of the transcriptional activation of the tinman and Ubx promoters in response to Dpp further support this model of transcriptional cooperativity. The mesoderm-specific induction of tinman requires both Medea/Mad- and Tinman-binding sites in the promoter; thus, the Dpp-induced Mad/Medea complex may cooperate with the homeobox protein Tinman to induce tinman transcription (25Xu X Yin Z Hudson J.B Ferguson E.L Frasch M Genes Dev. 1998; 12: 2354-2370Crossref PubMed Scopus (218) Google Scholar). Similarly, the Ubx promoter contains a Mad-binding sequence and a predicted cAMP-response element (CRE), which are both required for transcriptional induction by Dpp, suggesting that the Mad/Medea complex cooperates with CREB to induce Ubx (22Szuts D Eresh S Bienz M Genes Dev. 1998; 12: 2022-2035Crossref Scopus (88) Google Scholar). The cooperativity of Smads with other transcription factors is likely a consequence of ligand-induced associations, although direct physical interactions of Smads have only been described for FAST-1 and -2 and for c-Jun/c-Fos. As a result of this interaction, Smad3 enhances the activity of c-Jun at the AP-1-binding site (28Zhang Y Feng X.-H Derynck R Nature. 1998; 394: 909-912Crossref PubMed Scopus (666) Google Scholar) and of Sp1 at its binding site (18Moustakas A Kardassis D Proc. Natl. Acad. Sci. USA. 1998; 95: 6733-6738Crossref Scopus (319) Google Scholar). It is as-yet unclear whether FAST-1 and FAST-2 have an intrinsic transcription activity, with which Smad2/4 can cooperate, or whether they merely function as DNA binding adaptors to allow transcription by Smad2/4. It also remains to be determined whether Smad3/4 enhances the intrinsic transcriptional activity of TFE3. Although Smads have been shown to cooperate with several unrelated transcription factors, the diversity of transcription factors with which Smads can interact, and the structural bases for these multiple interactions remain to be defined. Nevertheless, these observations suggest that functional cooperation with a select set of transcription factors is at the basis of the ability of Smads to activate transcription. The transcriptional activity of Smads also depends on interactions with coactivators within the complex. Thus, Smad2 or -3 increase the transcription activity of Smad3, suggesting that oligomerization of receptor-activated Smads regulates the transcriptional activity (8Feng X.-H Zhang Y Wu R.-Y Derynck R Genes Dev. 1998; 12: 2153-2163Crossref Scopus (443) Google Scholar). Smad4, which does not have transcriptional activity on its own, is presumably an essential coactivator in the Smad complex, which may result from two possible functions of Smad4. In the Smad2/4 complex with FAST-1, Smad4 promotes DNA binding of the complex to the promoter DNA (16Liu F Pouponnot C Massagué J Genes Dev. 1997; 11: 3157-3167Crossref PubMed Scopus (394) Google Scholar), and in the Smad2/4 complex with FAST-2 (and presumably also with FAST-1), Smad4 binding to DNA is required for efficient transcription (15Labbé E Silvestri C Hoodless P.A Wrana J.L Attisano L Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). In addition, Smad4 may act as a coactivator through its ability to stabilize the Smad-activator complex and its interactions with the general transcription machinery (16Liu F Pouponnot C Massagué J Genes Dev. 1997; 11: 3157-3167Crossref PubMed Scopus (394) Google Scholar; 8Feng X.-H Zhang Y Wu R.-Y Derynck R Genes Dev. 1998; 12: 2153-2163Crossref Scopus (443) Google Scholar). This may explain why Smad4 strongly increases the transcriptional activity of Smad1 in response to BMP-4 (16Liu F Pouponnot C Massagué J Genes Dev. 1997; 11: 3157-3167Crossref PubMed Scopus (394) Google Scholar) and of Smad2 or -3 in response to TGF-β (16Liu F Pouponnot C Massagué J Genes Dev. 1997; 11: 3157-3167Crossref PubMed Scopus (394) Google Scholar; 8Feng X.-H Zhang Y Wu R.-Y Derynck R Genes Dev. 1998; 12: 2153-2163Crossref Scopus (443) Google Scholar). The closely related CBP and p300 proteins are also important coactivators for Smad activity. CBP and p300 act as coactivators of several transcription factors by bringing the sequence-specific activators within proximity of the general transcription machinery and by modifying the chromatin structure through histone acetylation. Accordingly, CBP/p300 acts as coactivator of Smad2 or -3 through direct physical interactions with their C domains, the transcriptional effector domains (8Feng X.-H Zhang Y Wu R.-Y Derynck R Genes Dev. 1998; 12: 2153-2163Crossref Scopus (443) Google Scholar; 12Janknecht R Wells N.J Hunter T Genes Dev. 1998; 12: 2114-2119Crossref PubMed Scopus (426) Google Scholar; 19Pouponnot C Jayaraman L Massagué J J. Biol. Chem. 1998; 273: 22865-22868Crossref PubMed Scopus (285) Google Scholar; 23Topper J.N diChiara M.R Brown J.D Williams A.J Falb D Collins T Gimbrone M.A Proc. Natl. Acad. Sci. USA. 1998; 95: 9506-9511Crossref PubMed Scopus (154) Google Scholar). In response to TGF-β, Smad3 associates with CBP/p300 and TGF-β-induced C-terminal phosphorylation of Smad3 promotes this association. This association with CBP/p300 is likely to be essential for transcriptional activity of Smad3. Thus, C-terminal deletions of Smad3, that abolish CBP association, also inactivate transcription, and interference with CBP/p300 function also inhibits Smad3/4- and TGF-β-induced transcription. Accordingly, increased expression of CBP/p300 enhances the transactivation activity of Smad3 and the transcriptional induction by TGF-β and Smad3/4. Importantly, CBP can not act as coactivator unless Smad4 is present, presumably because Smad4 stabilizes the Smad3/CBP complex (8Feng X.-H Zhang Y Wu R.-Y Derynck R Genes Dev. 1998; 12: 2153-2163Crossref Scopus (443) Google Scholar). Other coactivators may also play important roles. A candidate for an additional coactivator is MSG-1, which can interact with the C domain of Smad4 in vitro and enhances the transcriptional activity induced by transfected Smad4 (21Shioda T Lechleider R.J Dunwood S.L Li H Yahata T de Caestecker M.P Fenner M.H Roberts A.B Isselbacher K.J Proc. Natl. Acad. Sci. USA. 1998; 95: 9785-9790Crossref PubMed Scopus (100) Google Scholar). Based on the observations summarized above, the mechanism of transcriptional activation by Smads is most likely defined by the combined requirements for interactions with other transcription factors and with promoter DNA sequences. Consequently, Smad-responsive promoters have a double DNA sequence requirement. One sequence confers the specificity to bind the transcription factors that cooperate with the Smad complex. Another adjacent sequence is required for direct Smad binding and confers Smad selectivity to the first sequence. Thus, only a subset of promoter sequences that bind these cooperating transcription factors are targets for Smad signaling, and this selectivity is provided by flanking or partially overlapping sequences that allow Smad binding. For example, Smad3/4 cooperates with c-Jun to induce transcription from the collagenase I promoter in response to TGF-β; thus, the AP-1-binding site, which binds c-Jun/c-Fos, and the partially overlapping Smad-binding sequence AGAC mediate responsiveness to TGF-β and Smad3/4 (26Yingling J Datto M Wong C Frederick J Liberati N Wang X.-F Mol. Cell. Biol. 1997; 17: 7019-7028Crossref Google Scholar; 28Zhang Y Feng X.-H Derynck R Nature. 1998; 394: 909-912Crossref PubMed Scopus (666) Google Scholar). However, the AP-1-binding site without a flanking GAC sequence does not allow inducibility by TGF-β or Smad3/4 (6Dennler S Itoh S Vivien D ten Dijke P Huet S Gauthier J.-M EMBO J. 1998; 11: 3091-3100Crossref Scopus (1536) Google Scholar). Similarly, in the PAI-1 promoter, the TFE3-binding E-box sequence and the flanking sequence that binds the Smad3/4 complex are both required for TGF-β-induced transcription, which is mediated by the cooperation of Smad3/4 and TFE3 (11Hua X Liu X Ansari D.O Lodish H.F Genes Dev. 1998; 12: 3084-3095Crossref Scopus (251) Google Scholar). The dual sequence requirement is also exhibited by an activin-response element, which requires adjacent FAST-1- and Smad-binding sequences for full ligand-inducible transcription (29Zhou S Zawel L Lengauer C Kinzler K.W Vogelstein B Mol. Cell. 1998; 2: 121-127Abstract Full Text Full Text PDF Scopus (208) Google Scholar). Therefore, at the Mix.2 promoter, Smad2/4 may not only interact with FAST-1 (2Chen X Weisberg E Fridmacher V Watanabe M Naco G Whitman M Nature. 1997; 389: 85-89Crossref Scopus (486) Google Scholar), but presumably also with an adjacent sequence, most likely through Smad4 (since Smad2 does not bind DNA). Accordingly, Smad2/4 interacts with FAST-2 at the goosecoid promoter and Smad4 interacts with an adjacent GC-rich bipartite sequence. Again, the FAST-2- and Smad4-binding sequences are both required for full inducibility of transcription by ligand or Smad2/4 (15Labbé E Silvestri C Hoodless P.A Wrana J.L Attisano L Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). Finally, this model may also explain why a CRE sequence and a Mad-binding sequence were independently proposed as sequences required for transcriptional induction of the Ubx promoter by Dpp in Drosophila. Mutational analysis suggests that these overlapping sequences are both required for Dpp-mediated induction (22Szuts D Eresh S Bienz M Genes Dev. 1998; 12: 2022-2035Crossref Scopus (88) Google Scholar). The required interactions of Smads with other DNA-binding transcription factors explains in retrospect why no consensus sequences for TGF-β and activin-response elements were found in different promoters, and why previously known transcription factors were implicated as essential factors for ligand-induced transcription. Given this cooperativity with other transcription factors, it may be surprising that the Smad3/4-binding elements themselves allow transcription in response to TGF-β and Smad3/4 (6Dennler S Itoh S Vivien D ten Dijke P Huet S Gauthier J.-M EMBO J. 1998; 11: 3091-3100Crossref Scopus (1536) Google Scholar; 13Jonk L Itoh S Heldin C.-H ten Dijke P Kruijer W J. Biol. Chem. 1998; 273: 21145-21152Crossref PubMed Scopus (505) Google Scholar; 27Zawel L Dai J Buckhaults P Zhou S Kinzler K Vogelstein B Kern S Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar). However, since a single Smad-binding sequence by itself does not allow transcriptional activation by ligand or Smad3/4, perhaps the multimerization of these sequences in the reporter assays allows recruitment of cooperating transcription factors. Alternatively, multimerization of Smad-binding sites may synergistically amplify the low transcriptional activity of Smad3, which is apparent in yeast or in the absence of cooperating transcription factors (28Zhang Y Feng X.-H Derynck R Nature. 1998; 394: 909-912Crossref PubMed Scopus (666) Google Scholar). This transcriptional cooperativity model also provides a mechanism for integration of two signaling pathways at the promoter sequence. Thus, transcription by c-Jun/c-Fos is induced by mitogenic stimuli and stress or UV irradiation, whereas Smad3/4 activation is induced by TGF-β. The required cooperation of Smad3/4 with c-Jun/c-Fos at select AP-1-binding sites thus illustrates that a convergence of both signaling pathways is at the basis of Smad3/4-induced transcription from these promoters (28Zhang Y Feng X.-H Derynck R Nature. 1998; 394: 909-912Crossref PubMed Scopus (666) Google Scholar). A similar type of cross-talk is also required for Dpp-induced transcription from the Ubx promoter. Thus, CREB binding to the promoter results from EGF receptor activation, whereas Mad binding is induced by Dpp receptor activation (22Szuts D Eresh S Bienz M Genes Dev. 1998; 12: 2022-2035Crossref Scopus (88) Google Scholar), and Mad is likely to cooperate with CREB for Dpp-induced transcription from this promoter. Based on these examples, a variety of Smad responses may depend on activation of other signaling pathways and modifications in the cooperating transcription factors may regulate Smad responses.
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