Site-specific Heterodimerization by Paired Class Homeodomain Proteins Mediates Selective Transcriptional Responses
1999; Elsevier BV; Volume: 274; Issue: 45 Linguagem: Inglês
10.1074/jbc.274.45.32325
ISSN1083-351X
Autores Tópico(s)RNA Research and Splicing
ResumoAlx4 is a paired class homeodomain protein involved in defining anterior/posterior polarity in the developing limb bud. The paired class of homeodomain proteins cooperatively bind palindromic DNA elements as homodimers or as heterodimers with other paired homeodomain proteins. Previous characterization demonstrates that the strength of the cooperativity as well as the preference for targets is dictated largely by the identity of amino acid 50 of the homeodomain. Here we compare and contrast the DNA binding properties of a glutamine 50 paired homeodomain protein, Alx4, and a lysine 50 paired homeodomain protein, Goosecoid. We demonstrate that Alx4 homodimers, Gsc homodimers, and Alx4/Gsc heterodimers each have distinct DNA binding properties, and each can discriminate between highly related palindromic elements. Using reporter gene assays, we show that Alx4 activates transcription in a site-specific manner, and that Gsc is capable of antagonizing Alx4-mediated activation only from promoter elements that support heterodimer formation. These data demonstrate that paired homeodomain proteins with different DNA binding properties are able to form heterodimeric complexes with unique DNA binding and transcriptional activities. Thus, heterodimerization regulates the DNA binding specificity of these transcription factors and may partially explain how paired homeodomain proteins direct specific developmental functions. Alx4 is a paired class homeodomain protein involved in defining anterior/posterior polarity in the developing limb bud. The paired class of homeodomain proteins cooperatively bind palindromic DNA elements as homodimers or as heterodimers with other paired homeodomain proteins. Previous characterization demonstrates that the strength of the cooperativity as well as the preference for targets is dictated largely by the identity of amino acid 50 of the homeodomain. Here we compare and contrast the DNA binding properties of a glutamine 50 paired homeodomain protein, Alx4, and a lysine 50 paired homeodomain protein, Goosecoid. We demonstrate that Alx4 homodimers, Gsc homodimers, and Alx4/Gsc heterodimers each have distinct DNA binding properties, and each can discriminate between highly related palindromic elements. Using reporter gene assays, we show that Alx4 activates transcription in a site-specific manner, and that Gsc is capable of antagonizing Alx4-mediated activation only from promoter elements that support heterodimer formation. These data demonstrate that paired homeodomain proteins with different DNA binding properties are able to form heterodimeric complexes with unique DNA binding and transcriptional activities. Thus, heterodimerization regulates the DNA binding specificity of these transcription factors and may partially explain how paired homeodomain proteins direct specific developmental functions. zone of polarizing activity anterior-posterior chloramphenicol acetyltransferase homeodomain The mechanisms involved in vertebrate limb patterning serve as a model for understanding pattern formation throughout the body plan. Classical developmental studies in avian systems have provided a foundation for understanding this process by defining three major signaling centers within the developing limb bud: 1) the apical ectodermal ridge, a region of specialized epithelial cells at the distal margin of the limb bud; 2) the dorsal ectoderm; and 3) the zone of polarizing activity (ZPA),1 a group of mesenchymal cells in the posterior aspect of the limb bud. These signaling centers are derived from both epithelial and mesenchymal cells and form a series of interdependent feedback loops such that this system also serves as a model for understanding epithelial-mesenchymal interactions (for reviews, see Refs. 1Tickle C. Eichele G. Annu. Rev. Cell Biol. 1994; 10: 121-152Crossref PubMed Scopus (168) Google Scholar and 2Johnson R.L. Tabin C.J. Cell. 1997; 90: 979-990Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar). The establishment of anterior-posterior (A/P) polarity in the limb bud is controlled largely by the ZPA. The ZPA was initially defined by studies in which explants of cells from various regions of a donor limb bud were grafted to the anterior margin of a recipient limb bud. When posterior mesenchymal cells of the donor limb bud are grafted onto the anterior margin of the recipient limb bud, the resulting limb displays A/P axis duplications (3Saunders J.W. Gassling M.T. Fleischmajor R. Billingham R.F. Epithelial-Mesenchymal Interactions. Williams and Wilkins, Baltimore1968Google Scholar). These duplications are marked by the appearance of additional anterior digits with posterior characteristics (preaxial polydactyly). This is a dose-dependent phenomenon; more ZPA cells grafted to the anterior margin result in a greater number of ectopic digits. The ZPA does not contribute tissue to the digits, but instead serves as an organizer that patterns surrounding tissue. Two lines of evidence suggest that the key molecule mediating the polarizing activity of the ZPA is Sonic hedgehog (Shh). 1) Shh expression coincides with cells that have polarizing activity, both in the limb and in other tissues; and 2) direct application of Shh to the anterior margin of a limb bud results in digit duplications in a dose-dependent fashion (4Riddle R.D. Johnson R.L. Laufer E. Tabin C. Cell. 1993; 75: 1401-1416Abstract Full Text PDF PubMed Scopus (1930) Google Scholar, 5Chang D.T. Lopez A. von Kessler D.P. Chiang C. Simandl B.K. Zhao R. Seldin M.F. Fallon J.F. Beachy P.A. Development. 1994; 120: 3339-3353Crossref PubMed Google Scholar). We have previously identified and characterized a novel homeodomain protein, aristaless-like 4 (Alx4) (6Qu S. Li L. Wisdom R. Gene ( Amst. ). 1997; 203: 217-223Crossref PubMed Scopus (46) Google Scholar, 7Qu S. Niswender K.D. Ji Q. van der Meer R. Keeney D. Magnuson M.A. Wisdom R. Development. 1997; 124: 3999-4008PubMed Google Scholar). In wild-type embryos, Alx4 is expressed in the anterior mesenchyme of the limb bud. Mice that are homozygous for a null Alx4 allele exhibit preaxial polydactyly that is associated with ectopic Shh expression along the anterior margin of the limb bud during development. Genes previously demonstrated to be downstream targets of the Shh signaling pathway in the limb bud, such as patched and HoxD13, are misexpressed in the anterior mesenchyme of the limb bud as well (7Qu S. Niswender K.D. Ji Q. van der Meer R. Keeney D. Magnuson M.A. Wisdom R. Development. 1997; 124: 3999-4008PubMed Google Scholar). These results demonstrate that although Alx4 is not required for normal ZPA formation, it is required to prevent an ectopic anterior ZPA from forming. Although several polydactylous mouse mutants have been shown to misexpress Shh at the anterior margin of the limb bud, the genes responsible have been identified in only two cases. Alx4mutations are known to be responsible for the defects inStrong's luxoid, and Gli3 is disrupted in the mutant Extra toes (8Hui C.C. Joyner A.L. Nat. Genet. 1993; 3: 241-246Crossref PubMed Scopus (600) Google Scholar, 9Vortkamp A. Gessler M. Grzeschik K.H. Nature. 1991; 352: 539-540Crossref PubMed Scopus (472) Google Scholar). To understand the molecular mechanism by which Alx4 regulates A/P polarity in the limb, a detailed characterization of its biochemical and molecular properties is required. The most outstanding structural feature of Alx4 is the presence of a homeodomain, an evolutionarily conserved DNA binding motif shared by a large family of eukaryotic transcription factors. Genetic experiments demonstrate that these proteins play fundamental roles rooted in the establishment of cell identity or position. Furthermore, these experiments indicate that, in most cases, the homeodomain is necessary for function and biologic specificity (10Duboule D. Tooze S.A. Guidebook to the Homeobox Genes. Oxford University Press, New York1994: 3-10Google Scholar, 11Duncan I. Bioessays. 1996; 18: 91-94Crossref PubMed Scopus (21) Google Scholar, 12Miskiewicz P. Morrissey D. Lan Y. Raj L. Kessler S. Fujioka M. Goto T. Weir M. Development. 1996; 122: 2709-2718PubMed Google Scholar). Despite the diverse roles that homeodomain proteins play biologically, early molecular characterizations indicated that most bind a similar 5–6-base pair A-T-rich DNA element (13Hoey T. Levine M. Nature. 1988; 332: 858-861Crossref PubMed Scopus (307) Google Scholar). It seems implausible that this mode of DNA binding can accommodate the diverse functional specificity implied by the genetic data (for a review, see Ref. 14Scott M.P. Tamkun J.W. Hartzell III, G.W. Biochim. Biophys. Acta. 1989; 989: 25-48Crossref PubMed Scopus (799) Google Scholar; for an alternative view, see Ref. 15Biggin M.D. McGinnis W. Development. 1997; 124: 4425-4433PubMed Google Scholar). More recently, it has been shown that several classes of homeodomain proteins are able to generate target site specificity by forming heterodimeric DNA binding complexes with other proteins (16Ryan A.K. Rosenfeld M.G. Genes Dev. 1997; 11: 1207-1225Crossref PubMed Scopus (437) Google Scholar, 17Lu Q. Kamps M.P. Mol. Cell. Biol. 1996; 16: 1632-1640Crossref PubMed Scopus (100) Google Scholar, 18Chan S.K. Jaffe L. Capovilla M. Botas J. Mann R.S. 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One mechanism by which prd HD proteins generate target site specificity is by binding DNA as cooperative homodimers. The target elements consist of palindromic repeats of the sequence 5′-TAAT-3′ (P elements) separated by a variable number of nucleotides. Physical and biochemical experiments have shown that the prd HD is both necessary and sufficient for this activity. Furthermore, these studies demonstrate that a major determinant of the DNA binding properties exhibited by prd HD proteins is the identity of residue 50 within the homeodomain (22Wilson D. Sheng G. Lecuit T. Dostatni N. Desplan C. Genes Dev. 1993; 7: 2120-2134Crossref PubMed Scopus (328) Google Scholar). Amino acid 50 resides within the DNA recognition helix of the homeodomain, and crystallographic studies reveal that this residue mediates contacts with the nucleotide bases immediately 3′ to the 5′-TAAT-3′ target site (25Wilson D.S. Guenther B. Desplan C. Kuriyan J. Cell. 1995; 82: 709-719Abstract Full Text PDF PubMed Scopus (302) Google Scholar). Members of the prd class of HDs have either a serine (Ser-50), lysine (Lys-50) or glutamine (Gln-50) at position 50, and each subclass has unique DNA binding properties (22Wilson D. Sheng G. Lecuit T. Dostatni N. Desplan C. Genes Dev. 1993; 7: 2120-2134Crossref PubMed Scopus (328) Google Scholar). The Ser-50 subclass of prd HD proteins preferentially dimerizes on P elements in which the palindromic half-sites are separated by two intervening nucleotides (5′-TAAT NN ATTA-3′; P2 sites). Representative members of this subclass are encoded by the paired(prd) gene from Drosophila (26Bopp D. Burri M. Baumgartner S. Frigerio G. Noll M. Cell. 1986; 47: 1033-1040Abstract Full Text PDF PubMed Scopus (366) Google Scholar) and the vertebrate Pax genes (27Gruss P. Walther C. Cell. 1992; 69: 719-722Abstract Full Text PDF PubMed Scopus (350) Google Scholar). In addition to the homeodomain, this subclass is characterized by the presence of a second DNA binding motif, the paired domain, which expands the repertoire of DNA target elements with which these proteins can interact (28Czerny T. Schaffner G. Busslinger M. Genes Dev. 1993; 7: 2048-2061Crossref PubMed Scopus (349) Google Scholar). The Lys-50 subclass of prd HDs selectively forms dimers on P elements separated by three intervening nucleotides (P3 elements), with a strong preference for cytosines 3′ to each core half-site, i.e. 5′-TAAT CCG ATTA-3′ (P3C). The half-site comprising this element (5′-TAAT CC-3′) is also recognized by Lys-50 homeodomain monomers from other classes, such as bicoid (28Czerny T. Schaffner G. Busslinger M. Genes Dev. 1993; 7: 2048-2061Crossref PubMed Scopus (349) Google Scholar, 29Hanes S.D. Brent R. Science. 1991; 251: 426-430Crossref PubMed Scopus (180) Google Scholar). The Gln-50 subclass preferentially binds P3 sites as well, but has a less stringent requirement for particular residues 3′ to each TAAT half-site. In addition to the differences in target site specificity, the Lys-50 and Gln-50 prd HDs differ in one other aspect; the Gln-50 subclass binds approximately 15-fold more cooperatively to palindromic P3 elements than does the Lys-50 subclass. Cooperativity is defined as the extent to which binding to the first half-site enhances binding to the second half-site, and can be represented by the cooperativity coefficient, τ. Previous τ measurements indicate that for the Lys-50 subclass, τ ≈ 20, while for Gln-50 prd HD proteins, τ ≈ 300 (22Wilson D. Sheng G. Lecuit T. Dostatni N. Desplan C. Genes Dev. 1993; 7: 2120-2134Crossref PubMed Scopus (328) Google Scholar). Many of the vertebrate genes encoding prd HD proteins show overlapping expression patterns; in particular, expression of a large number of these genes, including Alx4, Alx3, Cart1, Prx1, Prx2, and Gsc, has been detected in mesenchymal cells of both the limb bud and the first branchial arch (6Qu S. Li L. Wisdom R. Gene ( Amst. ). 1997; 203: 217-223Crossref PubMed Scopus (46) Google Scholar, 30Driever W. Nusslein-Volhard C. Nature. 1989; 337: 138-143Crossref PubMed Scopus (463) Google Scholar, 31ten Berge D. Brouwer A. el Bahi S. Guenet J.L. Robert B. Meijlink F. Dev. Biol. 1998; 199: 11-25Crossref PubMed Scopus (71) Google Scholar, 32Zhao G.Q. Eberspaecher H. Seldin M.F. de Crombrugghe B. Mech. Dev. 1994; 48: 245-254Crossref PubMed Scopus (79) Google Scholar, 33Luessink B. Brouwer A. El Khattabi M. Poelman R.E. Gittenberger-de Groot A.C. Meijlink F. Mech. Dev. 1995; 52: 51-64Crossref PubMed Scopus (110) Google Scholar, 34Gaunt S.J. Blum M. De Robertis E.M. Development. 1993; 117: 769-778PubMed Google Scholar). Given the previously described DNA binding properties of the prd HD, as well as the ability of prd HD proteins to form heterodimeric complexes with one other, functional and genetic redundancy is possible. Consistent with this model, we have previously shown that the Cart1 gene, which encodes the closest identified relative of Alx4, functions as a dose-dependent enhancer of the polydactyly observed in Alx4 mutant mice (36Qu S. Tucker S.C. Zhao Q. deCrombrugghe B. Wisdom R. Development. 1999; 126: 359-369PubMed Google Scholar). In addition, analysis of double mutant animals revealed a role for both Alx4 and Cart1 in patterning structures derived from the first branchial arch, a result that is consistent with the high levels of expression of both genes at this site during development (6Qu S. Li L. Wisdom R. Gene ( Amst. ). 1997; 203: 217-223Crossref PubMed Scopus (46) Google Scholar, 34Gaunt S.J. Blum M. De Robertis E.M. Development. 1993; 117: 769-778PubMed Google Scholar, 35Zhao G.Q. Zhou X. Eberspaecher H. Solursh M. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8633-8637Crossref PubMed Scopus (98) Google Scholar). Thus, genetic evidence suggests that an understanding of the molecular basis of Alx4 function will need to account for the activities of other prd HD proteins that are co-expressed during development. Although previous studies demonstrated that the ability to bind palindromic response elements is a property of many, if not all, prd HD proteins, there is also evidence for functional specificity. As described above, the Gln-50 and Lys-50 prd HD proteins have been shown to have similar, yet distinct, DNA binding preferences. The fact that over 30 vertebrate proteins belong to the prd HD family suggests that, in addition to differential expression patterns, other mechanisms for providing functional specificity within the class may exist. One unexplored possibility is that members of separate prd HD subclasses form heterodimeric complexes with distinct DNA binding and transcriptional properties. The presence of both activators and repressors within the prd HD class could add further complexity to such a mechanism. The Lys-50 prd HD protein Goosecoid (Gsc) is expressed in mesenchymal cells of the limb bud and first branchial arch in domains that broadly overlap those of Alx4 (34Gaunt S.J. Blum M. De Robertis E.M. Development. 1993; 117: 769-778PubMed Google Scholar). 2S. C. Tucker and R. Wisdom, unpublished observations.2S. C. Tucker and R. Wisdom, unpublished observations.This protein is of particular interest for two reasons. First, the gsc protein of Drosophila has been shown to function as a transcriptional repressor and antagonist of the Lys-50 activator orthodenticle (otd) (37Mailhos C. Andre S. Mollereau B. Goriely A. Hemmati-Brivanlou A. Desplan C. Development. 1998; 125: 937-947Crossref PubMed Google Scholar). Second, mice homozygous for a targeted loss of function allele of Gsc display defects in structures that are also affected by Alx4 mutations, including the skull, mandible, and limb (37Mailhos C. Andre S. Mollereau B. Goriely A. Hemmati-Brivanlou A. Desplan C. Development. 1998; 125: 937-947Crossref PubMed Google Scholar, 38Rivera-Perez J.A. Mallo M. Gendron-Maguire M. Gridley T. Behringer R.R. Development. 1995; 121: 3005-3012PubMed Google Scholar). Here we present a characterization of the DNA binding and transcriptional regulatory properties of Alx4 and Gsc. We find that Alx4 homodimers, Gsc homodimers, and Alx4/Gsc heterodimers all display unique DNA binding properties. Each of these complexes differentiate between P3 elements based on the nucleotides separating the palindromic half-sites. Thus, the selective homo- and heterodimerization of prd HD proteins converts the generic P3 element into a family of related elements, each capable of providing unique transcriptional responses in the presence of Gln-50 and Lys-50 prd HD proteins. These results have implications for understanding the developmental specificity demonstrated by prd HD proteins. Peptides containing the homeodomains of Alx4 (residues 185–265), Gsc (residues 159–219), Msx1 (residues 163–226), and Cart1 (128–195) were expressed as (His)6-tagged peptides in Escherichia coli BL21 and purified to homogeneity by chromatography on Ni2+-nitrilotriacetic acid-agarose as described previously (40Qu S. Tucker S.C. Ehrlich J.S. Levorse J.M. Flaherty L.A. Wisdom R. Vogt T.F. Development. 1998; 125: 2711-2721PubMed Google Scholar). Unc4 protein was prepared similarly and was a generous gift of David Miller and Kim Liptig. Gel shift reactions contained 15 mm Tris, pH 7.5, 75 mm NaCl, 1.5 mmEDTA, 0.3% Nonidet P-40, 0.8 μg of dI-dC, 4 mmspermidine, 4 mm spermine, 1.5 mmdithiothreitol, and 7.5% glycerol. Where indicated (Figs. 3 and 4), low dose gel shifts contained 5 nm protein and high dose gel shifts 20 nm protein. After incubation on ice for 10 min, 32P-labeled probe was added and the mixture was incubated at room temperature for 15 min before separation on 7% polyacrylamide gels that contained 0.5× TBE. In competition experiments, a 20-fold molar excess of the competing oligonucleotide was added during the preincubation phase. In preliminary experiments, DNA binding was shown to be dependent on protein concentration. The sequence of the gel shift probes is shown (top strand with TAAT repeats in bold): P1/2C, 5′-CCTGAGAATAATCTGAGGACTGTACA-3′; P2C, 5′-CCTGAGAATAATCGATTACTGTACA-3′; P3C, 5′-CCTGAGAATAATCCGATTACTGTACA-3′; P4C, 5′-CCTGAGAATAATCCGGATTACTGTACA-3′; P5C, 5′-CCTGAGAATAATCCTGGATTACTGTACA-3′; P3Cmut, 5′-CCTGAGAATGGTCCGAGGACTGTACA-3′; P3A, 5′-CCTGAGAATAATAGTATTACTGTACA-3′; P3G, 5′-CCTGAGAATAATGGCATTACTGTACA-3′; P3T, 5′-CCTGAGAATAATTGAATTACTGTACA-3′; P3TTC, 5′-CCTGAGAATAATTTCATTACTGTACA-3′; P3TGG, 5′-CCTGAGAATAATTGGATTACTGTACA-3′.Figure 4Gsc preferentially binds P3C. A, gel shifts using the P3 half-site probes shown in Fig.3 A were performed with the recombinant Gsc HD peptide fragment. Each reaction contained the indicated 32P-labeled probe and 20 nm peptide. B, using the idealized P3 elements shown in Fig. 3 B as gel shift probes, gel shift assays were performed with the indicated 32P-labeled probe and 20 nm Gsc peptide.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cooperativity measurements were made using a Molecular Dynamics PhosphorImager. Dried gels were scanned and the relevant bands quantitated. By definition, τ =Kd2/Kd1, whereKd1 and Kd2 are functions of the following binding reactions.P+D→Kd1PDREACTION 1 PD+P→Kd2P2DREACTION 2 P is free protein, D is DNA, PD is monomerically bound DNA, and P2D is dimerically bound DNA. Using the PhosphorImager to quantitate bands representing the relevant complexes, τ can be calculated via Equation 1.τ=Kd2/Kd1=4[P2D][D]/[PD]2(Eq. 1) τ measurements were made from lanes in which approximately 50% of the probe was shifted and represent an average from three independent experiments with a deviation of less than 15% (for details, see Ref. 22Wilson D. Sheng G. Lecuit T. Dostatni N. Desplan C. Genes Dev. 1993; 7: 2120-2134Crossref PubMed Scopus (328) Google Scholar). Three copies of the relevant sequences were cloned upstream of a basal promoter (from the adenovirus E1b gene) and chloramphenicol acetyltransferase (CAT) gene in the plasmid pE1b-CAT to generate the indicated reporters (P3C-CAT, P3TTC-CAT, etc.). The Alx4 expression plasmid pCMX-Alx4 has previously been described (40Qu S. Tucker S.C. Ehrlich J.S. Levorse J.M. Flaherty L.A. Wisdom R. Vogt T.F. Development. 1998; 125: 2711-2721PubMed Google Scholar). The mouse Gsc cDNA with a hemagglutinin tag was cloned into pCMX (41Umesono K. Murakami K.K. Thompson C.C. Evans R.M. Cell. 1991; 65: 1255-1266Abstract Full Text PDF PubMed Scopus (1478) Google Scholar) to generate the expression plasmid pCMX-Gsc. Transient transfections utilized 0.1 μg (Fig. 2 A) or 0.5 μg of the indicated reporter construct, 0.5 μg of the β-galactosidase expression plasmid pCH110 (Amersham Pharmacia Biotech), and the indicated amounts of expression plasmids; the total amount of expression plasmid DNA was held constant by the addition of empty vector. DNA samples were transfected into 293 (human embryonic kidney) cells using LipofectAMINE (Life Technologies, Inc.); in preliminary experiments, it was shown that 293 cells do not express either Alx4 or Gsc, do not harbor P3 DNA binding activity, and do not efficiently express any of the indicated reporter constructs. Forty-eight hours after transfection, lysates were made and CAT and β-galactosidase activities were determined by previously described methods and represent an average of at least three independent experiments that deviate by less that 15%. Alx4 is a member of the prd class of homeobox genes. It has been previously demonstrated that prd class HD proteins bind preferentially and with high affinity as cooperative homodimers to palindromic repeats of the sequence 5′-TAAT-3′. The preferred spacing of the palindromes is dictated in large part by residue 50 of the homeodomain, such that prd HDs bearing a glutamine or a lysine at position 50 (Gln-50 or Lys-50) prefer P3 elements (5′-TAAT NNN ATTA-3′), while Ser-50 prd HDs prefer P2 elements (5′-TAAT NN ATTA-3′) (22Wilson D. Sheng G. Lecuit T. Dostatni N. Desplan C. Genes Dev. 1993; 7: 2120-2134Crossref PubMed Scopus (328) Google Scholar). Alx4 contains a Gln-50 HD and therefore is predicted to bind P3 elements. To test this prediction, gel shift assays were performed using probes containing TAAT repeats separated by a variable number of nucleotides (Fig. 1 B). In these experiments, we used a recombinant Alx4 peptide fragment containing the HD that was expressed in bacteria and purified to homogeneity via nickel agarose chromatography (Fig. 1 A). Alx4 bound all of the indicated probes to some extent (Fig.1 C). Migration of monomeric protein-DNA complexes is defined by binding to P1/2C, which contains a single half-site (Fig.1 C, lanes 2–4), and the more slowly migrating species on other probes represents dimeric binding. The prd HD is known to exist as a monomer in solution and interact with DNA in a stepwise fashion resulting in dimeric protein-DNA complexes (22Wilson D. Sheng G. Lecuit T. Dostatni N. Desplan C. Genes Dev. 1993; 7: 2120-2134Crossref PubMed Scopus (328) Google Scholar). Thus, the overall binding affinity is a function of the affinity of peptide monomers for the first half-site and the enhanced affinity for a monomeric peptide binding the second half-site. The -fold increase for the second binding event is defined as the cooperativity coefficient, τ (see "Experimental Procedures"). Measurements of τ in this and similar experiments indicated that cooperativity is greatest on the P3C site where τ ≈ 300; on P2C and P4C sites, τ ≈ 20; and binding was noncooperative on P5C. These results are consistent with values previously reported for other Gln-50 prd HD proteins (22Wilson D. Sheng G. Lecuit T. Dostatni N. Desplan C. Genes Dev. 1993; 7: 2120-2134Crossref PubMed Scopus (328) Google Scholar). The preference for P3 elements was further demonstrated by a competition experiment in which binding of Alx4 to P3C was competed by a 20-fold excess of cold P3C probe (Fig. 1 D, lane 3), but not P1/2C, P2C, P4C, or P5C (Fig.1 D, lanes 4–7). To ensure that the DNA binding properties observed for the recombinant peptide fragment are shared by full-length Alx4, nuclear extracts were made from 293 cells transiently transfected with either pCMX-Alx4 or empty vector. These extracts were then used in gel shift assays along with the same series of P element probes. An Alx4-specific gel shift was detected only on the P3C probe (Fig. 1 E, lane 9). Because of the decreased mobility of protein-DNA complexes with full-length Alx4, dimeric and monomeric complexes could not be resolved. The P3C-specific binding observed with the full-length protein is in contrast to the previous series of gel shifts in which the recombinant Alx4 peptide bound all sites tested. This difference is most likely due to the fact that lower levels of Alx4 protein are present in the nuclear extracts. Consistent with this idea, gel shifts of the various P element probes at intermediate doses of recombinant peptide indicate that dimeric binding was detected only on the P3C site (Fig. 1 C, compare lane 11 tolanes 3, 7, 15, and19). This suggests that the binding activity detected on sites that lack the P3 spacing require amounts of protein greater than that expressed in transfected 293 cells, and therefore high affinity cooperative dimerization on P3 sites may be the only binding event that occurs in vivo. To test the possibility that the DNA binding activity described in Fig. 1 can result in transactivation, a series of synthetic reporter constructs were utilized. The various P elements used in DNA binding assays (see Fig. 1 B) were cloned in triplicate upstream of a basal E1b promoter driving (CAT expression. Transient cotransfections were performed in 293 cells using the P element reporters with or without pCMX-Alx4. Reporter gene activity was detected only on the P3C reporter (P3C-CAT) (Fig. 2 A and data not shown). This activation was dependent on the dose of pCMX-Alx4 to a maximal activation observed at 100 ng of input plasmid. At higher doses, the level of activation declined, probably due to squelching effects. These results suggest that high affinity, cooperative DNA binding is required for transcriptional activation and that the non-cooperative and weakly cooperative binding detected in vitro are not sufficient to mediate a biologic response. The P3 elements were previously identified as binding sites for prd HD proteins using a polymerase chain reaction-based site selection technique, which over several rounds of selection and enrichment generates a consensus site (48Gehring W.J. Gene ( Amst. ). 1993; 135: 215-221Crossref PubMed Scopus (92) Google Scholar). However, this method may fail to identify potential target elements with a lower binding affinity, or become biased against some high affinity sites. Since the ability of Alx4 to bind different P3 elements as homodimers is determined, in part, by the intrinsic affinity for different half-sites, we defined the half-site preference for Alx4 by varying the nucleotide immediately 3′ to the core TAAT half-site. Using double-stranded oligonucleotides containing P1/2 sites as probes (Fig.3 A, top), we performed gel shifts with two doses of Alx4 peptide. At lower doses (Fig. 3 A, lanes 1, 3,5, and 7), Alx4 bound to P1/2C and P1/2T only. A 4-fold higher concentration of peptide (Fig. 3 A, lanes 2, 4, 6, and8) resulted in binding to all sites, with binding to P1/2C and P1/2T greater than that of P1/2A and P1/2G. Therefore, the preferred half-site can be represented as 5′-TAAT Py-3′. We went on to compare Alx4 binding to a series of idealized P3 elements in which the nucleotides separa
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