The C-terminal Subdomain Makes an Important Contribution to the DNA Binding Activity of the Pax-3 Paired Domain
1997; Elsevier BV; Volume: 272; Issue: 45 Linguagem: Inglês
10.1074/jbc.272.45.28289
ISSN1083-351X
Autores Tópico(s)Genomics and Chromatin Dynamics
ResumoThe recognition of DNA targets by Pax-3 is achieved through the coordinate use of two distinct helix-turn-helix-based DNA-binding modules: a paired domain, composed of two structurally independent subdomains joined by a short linker, and a paired-type homeodomain. In mouse, the activity of the Pax-3 paired domain is modulated by an alternative splicing event in the paired domain linker region that generates isoforms (Q+ and Q−) with distinct C-terminal subdomain-mediated DNA-binding properties. In this study, we have used derivatives of a classical high affinity paired domain binding site (CD19-2/A) to derive an improved consensus recognition sequence for the Pax-3 C-terminal subdomain. This new consensus differs at six out of eight positions from the C-terminal subdomain recognition motif present in the parent CD19-2/A sequence, and includes a 5′-TT-3′ dinucleotide at base pairs 15 and 16 that promotes high affinity binding by both Pax-3 isoforms. However, with a less favorable guanine at position 15, only the Q− isoform retains high affinity binding to this sequence, suggesting that this alternative splicing event might serve to stabilize binding to suboptimal recognition sequences. Finally, mutagenic analysis of the linker demonstrates that both the sequence and the spacing in this region contribute to the enhanced DNA-binding properties of the Pax-3/Q− isoform. Altogether, our studies establish a clear role for the Pax-3 C-terminal subdomain in DNA recognition and, thus, provide insights into an important mechanism by which Pax proteins achieve distinct target specificities. The recognition of DNA targets by Pax-3 is achieved through the coordinate use of two distinct helix-turn-helix-based DNA-binding modules: a paired domain, composed of two structurally independent subdomains joined by a short linker, and a paired-type homeodomain. In mouse, the activity of the Pax-3 paired domain is modulated by an alternative splicing event in the paired domain linker region that generates isoforms (Q+ and Q−) with distinct C-terminal subdomain-mediated DNA-binding properties. In this study, we have used derivatives of a classical high affinity paired domain binding site (CD19-2/A) to derive an improved consensus recognition sequence for the Pax-3 C-terminal subdomain. This new consensus differs at six out of eight positions from the C-terminal subdomain recognition motif present in the parent CD19-2/A sequence, and includes a 5′-TT-3′ dinucleotide at base pairs 15 and 16 that promotes high affinity binding by both Pax-3 isoforms. However, with a less favorable guanine at position 15, only the Q− isoform retains high affinity binding to this sequence, suggesting that this alternative splicing event might serve to stabilize binding to suboptimal recognition sequences. Finally, mutagenic analysis of the linker demonstrates that both the sequence and the spacing in this region contribute to the enhanced DNA-binding properties of the Pax-3/Q− isoform. Altogether, our studies establish a clear role for the Pax-3 C-terminal subdomain in DNA recognition and, thus, provide insights into an important mechanism by which Pax proteins achieve distinct target specificities. Pax-3 belongs to a family of transcription factors that regulate a variety of developmental processes in vertebrates and invertebrates (1Noll M. Curr. Opin. Genet. Dev. 1993; 3: 595-605Crossref PubMed Scopus (294) Google Scholar). In the mouse, Pax-3 is expressed in a subset of neuroectodermal and mesodermal lineages (2Goulding M.D. Chalepakis G. Deutsch U. Erselius J.R. Gruss P. EMBO J. 1991; 10: 1135-1147Crossref PubMed Scopus (749) Google Scholar), and is required for proper neural tube closure and neural crest cell migration (3Epstein D.J. Vekemans M. Gros P. Cell. 1991; 67: 767-774Abstract Full Text PDF PubMed Scopus (578) Google Scholar), and for the development of particular skeletal muscle lineages (4Bober E. Franz T. Arnold H.-H. Gruss P. Tremblay P. Development. 1994; 120: 603-612Crossref PubMed Google Scholar,5Tajbakhsh S. Rocancourt D. Cossu G. Buckingham M. Cell. 1997; 89: 127-138Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar). To interact with target genes, Pax-3 contains a paired domain, a conserved 128-amino acid DNA-binding domain (6Chalepakis G. Fritsch R. Fickenscher H. Deutsch U. Goulding M. Gruss P. Cell. 1991; 66: 873-884Abstract Full Text PDF PubMed Scopus (219) Google Scholar, 7Treisman J. Harris E. Desplan C. Genes Dev. 1991; 5: 594-604Crossref PubMed Scopus (288) Google Scholar) composed of two structurally distinct HTH 1The abbreviations used are: HTH, helix-turn-helix; Prd, paired; PCR, polymerase chain reaction; SELEX,in vitro selection of optimal binding sites; bp, base pair(s). 1The abbreviations used are: HTH, helix-turn-helix; Prd, paired; PCR, polymerase chain reaction; SELEX,in vitro selection of optimal binding sites; bp, base pair(s).-based subdomains joined by a short linker region, as revealed by crystallographic studies with a related family member, Drosophila Prd (8Xu W. Rould M.A. Jun S. Desplan C. Pabo C.O. Cell. 1995; 80: 639-650Abstract Full Text PDF PubMed Scopus (307) Google Scholar). In addition to the paired domain with its bipartite structure, Pax-3, like Prd, also contains a second conserved DNA-binding domain, a paired-type homeodomain, which folds into a classical HTH-based structure (9Wilson D.S. Guenther B. Desplan C. Kuriyan J. Cell. 1995; 82: 709-719Abstract Full Text PDF PubMed Scopus (303) Google Scholar) and which binds cooperatively as a dimer to specific palindromic DNA sequences (10Wilson D. Sheng G. Lecuit T,. Dostatni N. Desplan C. Genes Dev. 1993; 7: 2120-2134Crossref PubMed Scopus (328) Google Scholar). To complement the distinct DNA binding activities of the isolated domains, the paired domain and homeodomain of Prd can also cooperate to recognize specific composite binding sites with high affinity (11Jun S. Desplan C. Development. 1996; 122: 2639-2650Crossref PubMed Google Scholar). Consequently, the interaction of Pax-3 with DNA targets has the potential to be quite complex, with up to three distinct HTH-based modules contributing to target site selection.Within Pax proteins, the N-terminal subdomain of the paired domain is highly conserved (12Walther C. Guenet J.-L. Simon D. Deutsch U. Jostes B. Goulding M.D. Plachov D. Balling R. Gruss P. Genomics. 1991; 11: 424-434Crossref PubMed Scopus (356) Google Scholar), and it interacts with a well defined 10–12-bp consensus sequence through a series of base-specific major groove and minor groove contacts (8Xu W. Rould M.A. Jun S. Desplan C. Pabo C.O. Cell. 1995; 80: 639-650Abstract Full Text PDF PubMed Scopus (307) Google Scholar). In contrast, the C-terminal subdomain in these proteins is more variable (12Walther C. Guenet J.-L. Simon D. Deutsch U. Jostes B. Goulding M.D. Plachov D. Balling R. Gruss P. Genomics. 1991; 11: 424-434Crossref PubMed Scopus (356) Google Scholar), and its precise involvement in DNA recognition has not been extensively characterized. In the case of Prd, the C-terminal subdomain does not contact the 15-bp recognition sequence used for crystallization (8Xu W. Rould M.A. Jun S. Desplan C. Pabo C.O. Cell. 1995; 80: 639-650Abstract Full Text PDF PubMed Scopus (307) Google Scholar), and appears to be dispensable both for DNA binding by Prd in vitro (11Jun S. Desplan C. Development. 1996; 122: 2639-2650Crossref PubMed Google Scholar) and for Prd function in vivo (13Bertuccioli C. Fasano L. Jun S. Wang S. Sheng G. Desplan C. Development. 1996; 122: 2673-2685PubMed Google Scholar). However, deletion studies have shown that this subdomain is required for the recognition of a class of naturally occurring target sequences by members of at least three Pax subfamilies (14Czerny T. Schaffner G. Busslinger M. Genes Dev. 1993; 7: 2048-2061Crossref PubMed Scopus (352) Google Scholar), and in the case of human PAX6 (15Azuma N. Nishina S. Yanagisawa H. Okuyama T. Yamada M. Nature Genet. 1996; 13: 141-142Crossref PubMed Scopus (166) Google Scholar, 16Tang H.K. Chao L.-Y. Saunders G.F. Hum. Mol. Genet. 1997; 6: 381-386Crossref PubMed Scopus (68) Google Scholar) and the Caenorhabditis elegans Pax-6 homologuemab-18 (17Chisholm A.D. Horvitz H.R. Nature. 1995; 377: 52-55Crossref PubMed Scopus (145) Google Scholar), point mutations in this subdomain lead to specific developmental disorders, suggesting that the C-terminal subdomain plays an important role in DNA recognition by some Pax proteins in vivo.Thus far, selection experiments in vitro with the Pax-3 paired domain have yielded a consensus recognition sequence which, by analogy with Prd, does not include a C-terminal subdomain recognition motif (18Chalepakis G. Gruss P. Gene (Amst.). 1995; 162: 267-270Crossref PubMed Scopus (69) Google Scholar, 19Epstein J.A. Shapiro D.N. Cheng J. Lam P.Y.P. Maas R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4213-4218Crossref PubMed Scopus (314) Google Scholar), raising the question of whether the C-terminal subdomain in Pax-3 contributes at all to sequence-specific DNA binding. In a preliminary study addressing this question, we recently described the DNA-binding properties of two alternatively spliced isoforms of Pax-3 (called Q+ and Q−), which differ by the presence or absence of a single glutamine residue in the paired domain linker region (20Vogan K.J. Underhill D.A. Gros P. Mol. Cell. Biol. 1996; 16: 6677-6686Crossref PubMed Scopus (89) Google Scholar). This alternative splicing event was found to have no noticeable effect on the DNA-binding properties of the N-terminal subdomain of Pax-3; however, the novel isoform lacking the glutamine was found to exhibit a higher affinity for representative paired domain recognition sequences bearing consensus elements for both subdomains (20Vogan K.J. Underhill D.A. Gros P. Mol. Cell. Biol. 1996; 16: 6677-6686Crossref PubMed Scopus (89) Google Scholar). This finding suggested that, in at least one naturally occurring Pax-3 isoform, the C-terminal subdomain is competent to bind DNA. However, the inability of either isoform of Pax-3 to bind to the Pax6-5a consensus (20Vogan K.J. Underhill D.A. Gros P. Mol. Cell. Biol. 1996; 16: 6677-6686Crossref PubMed Scopus (89) Google Scholar), a C-terminal subdomain recognition sequence derived using an alternatively spliced isoform of Pax6 defective in N-terminal subdomain binding (21Epstein J.A. Glaser T. Cai J. Jepeal L. Walton D.S. Maas R.L. Genes Dev. 1994; 17: 2022-2034Crossref Scopus (317) Google Scholar), suggested that the C-terminal subdomain of Pax-3 may exhibit sequence preferences distinct from other Pax family members, and moreover, that such differences might be important for the discrimination of genomic targets by Pax-3 and other Pax proteins in vivo.In this study, we have used derivatives of CD19-2/A, a classical paired domain recognition sequence bearing consensus elements for both subdomains (14Czerny T. Schaffner G. Busslinger M. Genes Dev. 1993; 7: 2048-2061Crossref PubMed Scopus (352) Google Scholar), to define an improved consensus for the Pax-3 C-terminal subdomain, and to evaluate the contribution of this subdomain to the sequence-specific DNA binding activity of the Pax-3 paired domain. In addition, we have used these derivatives to further define differences in the DNA binding activities of the two alternatively spliced Pax-3 isoforms, and have used site-directed mutagenesis to probe the structural basis for these differences. Altogether, these studies establish a clear role for the Pax-3 C-terminal subdomain in DNA recognition, suggesting that this subdomain may exert an important influence on the selection of DNA targets by Pax-3 within the developing embryo.DISCUSSIONThe ability of tissue-specific transcription factors to recognize specific DNA sequences within the regulatory regions of target genes is an important means of controlling gene expression. Faced with the enormous complexity of the genetic material present in living cells, DNA-binding proteins rely on a number of distinct mechanisms to achieve target specificity in vivo (30Nelson H.C.M. Curr. Opin. Genet. Dev. 1995; 5: 180-189Crossref PubMed Scopus (78) Google Scholar). Among these is the use of complex DNA-binding domains, such as POU domains and zinc fingers, that achieve specificity through the coordinate use of multiple, structurally independent DNA-binding subdomains (30Nelson H.C.M. Curr. Opin. Genet. Dev. 1995; 5: 180-189Crossref PubMed Scopus (78) Google Scholar). The bipartite paired domain, which is composed of two distinct HTH-based subdomains joined by a short linker (8Xu W. Rould M.A. Jun S. Desplan C. Pabo C.O. Cell. 1995; 80: 639-650Abstract Full Text PDF PubMed Scopus (307) Google Scholar), is a well conserved DNA-binding structure present in a family of transcription factors controlling diverse developmental processes in a wide variety of vertebrate and invertebrate species (1Noll M. Curr. Opin. Genet. Dev. 1993; 3: 595-605Crossref PubMed Scopus (294) Google Scholar). The two helical subdomains and the extended linker region allow a single paired domain to interact with up to 20 base pairs of DNA, and together provide a large number of base-specific major groove and minor groove contacts that contribute to the recognition of specific DNA sequences as potential targets for regulation (6Chalepakis G. Fritsch R. Fickenscher H. Deutsch U. Goulding M. Gruss P. Cell. 1991; 66: 873-884Abstract Full Text PDF PubMed Scopus (219) Google Scholar, 8Xu W. Rould M.A. Jun S. Desplan C. Pabo C.O. Cell. 1995; 80: 639-650Abstract Full Text PDF PubMed Scopus (307) Google Scholar, 14Czerny T. Schaffner G. Busslinger M. Genes Dev. 1993; 7: 2048-2061Crossref PubMed Scopus (352) Google Scholar). However, with nine distinct Pax genes in mammals and seven in Drosophila, understanding how individual paired domains achieve distinct target specificities is an important step toward identifying the downstream targets of these transcription factors during development.While the binding specificity of the N-terminal subdomain has been defined for several members of the Pax family (8Xu W. Rould M.A. Jun S. Desplan C. Pabo C.O. Cell. 1995; 80: 639-650Abstract Full Text PDF PubMed Scopus (307) Google Scholar, 11Jun S. Desplan C. Development. 1996; 122: 2639-2650Crossref PubMed Google Scholar, 18Chalepakis G. Gruss P. Gene (Amst.). 1995; 162: 267-270Crossref PubMed Scopus (69) Google Scholar, 19Epstein J.A. Shapiro D.N. Cheng J. Lam P.Y.P. Maas R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4213-4218Crossref PubMed Scopus (314) Google Scholar, 25Epstein J. Cai J. Glaser T. Jepeal L. Maas R. J. Biol. Chem. 1994; 269: 8355-8361Abstract Full Text PDF PubMed Google Scholar), the role of the C-terminal subdomain in DNA recognition has not been characterized as extensively. Significantly, the selection of optimal binding sites with isolated paired domains in vitro, including the Pax-3 paired domain, has often failed to define a consensus that extends far enough to include a recognition motif for the C-terminal subdomain (8Xu W. Rould M.A. Jun S. Desplan C. Pabo C.O. Cell. 1995; 80: 639-650Abstract Full Text PDF PubMed Scopus (307) Google Scholar, 11Jun S. Desplan C. Development. 1996; 122: 2639-2650Crossref PubMed Google Scholar, 18Chalepakis G. Gruss P. Gene (Amst.). 1995; 162: 267-270Crossref PubMed Scopus (69) Google Scholar, 19Epstein J.A. Shapiro D.N. Cheng J. Lam P.Y.P. Maas R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4213-4218Crossref PubMed Scopus (314) Google Scholar, 25Epstein J. Cai J. Glaser T. Jepeal L. Maas R. J. Biol. Chem. 1994; 269: 8355-8361Abstract Full Text PDF PubMed Google Scholar). However, many naturally occurring paired domain recognition sequences identified within candidate target promoters are longer than the consensus sequences defined in vitro, and require a contribution from both subdomains for recognition (14Czerny T. Schaffner G. Busslinger M. Genes Dev. 1993; 7: 2048-2061Crossref PubMed Scopus (352) Google Scholar). A clear example of this is seen with Pax-2, where selection from a pool of random oligonucleotides defined a 13-bp consensus lacking a C-terminal subdomain recognition motif (25Epstein J. Cai J. Glaser T. Jepeal L. Maas R. J. Biol. Chem. 1994; 269: 8355-8361Abstract Full Text PDF PubMed Google Scholar), whereas the purification of Pax-2-bound complexes from native chromatin defined a 23-bp consensus with recognition motifs for both subdomains (31Phelps D.E. Dressler G.R. J. Biol. Chem. 1996; 271: 7978-7985Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Therefore, in some instances, the role of the C-terminal subdomain in DNA recognition by Pax proteins may have been masked by the tendency of the N-terminal subdomain to dominate over the C-terminal subdomain in standard in vitro selection protocols.The recent discovery of an alternatively spliced isoform of Pax-3 with a higher affinity for full-length paired domain recognition sequences provided the first evidence that the C-terminal subdomain might influence the DNA-binding properties of the Pax-3 paired domain (20Vogan K.J. Underhill D.A. Gros P. Mol. Cell. Biol. 1996; 16: 6677-6686Crossref PubMed Scopus (89) Google Scholar). To follow up these observations, we have generated derivatives of CD19-2/A, a well studied paired domain recognition bearing consensus binding motifs for both subdomains (14Czerny T. Schaffner G. Busslinger M. Genes Dev. 1993; 7: 2048-2061Crossref PubMed Scopus (352) Google Scholar), to further evaluate the role of the Pax-3 C-terminal subdomain in sequence discrimination. Since structural studies with Drosophila Prd had shown that the N-terminal subdomain makes contacts with base pairs 4 through 14 of the recognition sequence (8Xu W. Rould M.A. Jun S. Desplan C. Pabo C.O. Cell. 1995; 80: 639-650Abstract Full Text PDF PubMed Scopus (307) Google Scholar), and since the Pax-3 N-terminal subdomain had been shown to select a consensus very similar to Prd in vitro (18Chalepakis G. Gruss P. Gene (Amst.). 1995; 162: 267-270Crossref PubMed Scopus (69) Google Scholar, 19Epstein J.A. Shapiro D.N. Cheng J. Lam P.Y.P. Maas R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4213-4218Crossref PubMed Scopus (314) Google Scholar), we tested the importance of sequences 3′ to this 10–12-bp N-terminal subdomain recognition motif for DNA recognition by Pax-3. Significantly, we find that the affinity of Pax-3 for derivatives of CD19-2/A is sensitive to single base substitutions in the C-terminal subdomain recognition motif. Moreover, a comparison between CD19-2/A (16G), the lowest affinity site identified in these studies (Fig. 2), and P3-C-OPT (15T), a sequence optimized to interact with the Pax-3 C-terminal subdomain (Fig. 3), reveals that the affinity of Pax-3 for these two sequences differs by nearly two orders of magnitude. Since the N-terminal subdomain recognition motif is identical between these two sequences, this result demonstrates that the C-terminal subdomain makes a significant contribution to the DNA binding activity of the Pax-3 paired domain.In addition to establishing a clear DNA-binding function for the Pax-3 C-terminal subdomain, these studies also provide some interesting insights into the role of the C-terminal subdomain in contributing to paired domain DNA-binding specificity. As has been noted previously (14Czerny T. Schaffner G. Busslinger M. Genes Dev. 1993; 7: 2048-2061Crossref PubMed Scopus (352) Google Scholar), the high degree of conservation in the N-terminal subdomain among paired domains leads to the recognition of a very similar core sequence by divergent family members, raising the question of how these proteins are able to achieve distinct DNA-binding specificities in vivo. Significantly, the C-terminal subdomain in these proteins shows a much higher degree of sequence diversity, raising the possibility that this more divergent DNA-binding structure might be important means of discriminating between DNA targets (14Czerny T. Schaffner G. Busslinger M. Genes Dev. 1993; 7: 2048-2061Crossref PubMed Scopus (352) Google Scholar). Indeed, using our panel of singly substituted CD19-2/A derivatives, we do observe clear differences in specificity between the C-terminal subdomains of Pax-3 and Pax6, lending support to this hypothesis. The panel of CD19-2/A derivatives described herein may therefore serve as a useful tool for defining the distinct sequence preferences conferred by the C-terminal subdomain in other Pax family members.A comparison of the behavior of the naturally occurring isoforms of Pax-3 on the CD19-2/A derivatives has also provided us with a better understanding of the effect of this alternative splicing event on Pax-3 DNA recognition. In particular, we find that the presence of a 5′-TT-3′ dinucleotide at base pairs 15 and 16 significantly enhances DNA recognition by both isoforms. While the structural basis for these distinct sequence preferences is not known, we have shown that the recognition of these two base pairs is not affected by the presence of an additional residue at position 75 of the paired domain linker, or by the nature of the amino acid side chain at this position. We note also that in the paired domain crystal structure, the N-terminal portion of the linker is inserted into the minor groove, allowing residues 69–71 to interact with base pairs 12–14 of the recognition sequence (8Xu W. Rould M.A. Jun S. Desplan C. Pabo C.O. Cell. 1995; 80: 639-650Abstract Full Text PDF PubMed Scopus (307) Google Scholar). In the light of our observations, it is possible that residues 72–74 may extend these minor groove contacts and interact with base pairs 15 and 16, contributing to the specificity observed at these positions.In contrast to the similar affinity of the two alternatively spliced isoforms of Pax-3 for derivatives of CD19-2/A bearing a 5′-TT-3′ dinucleotide at base pairs 15 and 16, the Q− isoform of Pax-3 demonstrates a significantly higher affinity for sequences bearing a suboptimal guanine at position 15. In this respect, it is interesting to note that many natural targets of Pax proteins deviate significantly from the optimal recognition sequences defined for these proteins in vitro (14Czerny T. Schaffner G. Busslinger M. Genes Dev. 1993; 7: 2048-2061Crossref PubMed Scopus (352) Google Scholar, 32Zannini M. Francis-Lang H. Plachov D. Di Lauro R. Mol. Cell. Biol. 1992; 12: 4230-4241Crossref PubMed Scopus (271) Google Scholar, 33Holst B.D. Wang Y. Jones F.S. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1465-1470Crossref PubMed Scopus (59) Google Scholar). The enhanced recognition of suboptimal binding sites by the Q− isoform may therefore be an important activity in vivo, enabling this isoform to regulate a broader range of target genes than its Q+counterpart, or making it a more potent activator or repressor of some target genes recognized by both isoforms. However, further studies will be needed to clarify the biological relevance of this alternative splicing event with respect to Pax-3 function in the developing embryo.Despite significant advances in our understanding of the factors governing Pax-3/DNA interactions in vitro, little progress has been made thus far in the identification of actual targets of Pax-3 regulation in vivo. However, the evidence for a significant role for the C-terminal subdomain in DNA recognition, together with the previously established DNA binding activities of the homeodomain and N-terminal subdomain, suggests that the recognition of some genomic targets by Pax-3 may involve the coordinate use of all three HTH-based DNA-binding modules. Indeed, functional interdependence between the paired domain and homeodomain has been demonstrated recently for both Pax-3 (34Underhill D.A. Vogan K.J. Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3692-3696Crossref PubMed Scopus (49) Google Scholar, 35Underhill D.A. Gros P. J. Biol. Chem. 1997; 272: 14175-14182Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 36Fortin A.S. Underhill D.A. Gros P. Hum. Mol. Genet. 1997; 6: 1781-1790Crossref PubMed Scopus (39) Google Scholar) and Prd (11Jun S. Desplan C. Development. 1996; 122: 2639-2650Crossref PubMed Google Scholar, 13Bertuccioli C. Fasano L. Jun S. Wang S. Sheng G. Desplan C. Development. 1996; 122: 2673-2685PubMed Google Scholar, 37Fujioka M. Miskiewicz P. Raj L. Gulledge A.A. Weir M. Goto T. Development. 1996; 122: 2697-2707PubMed Google Scholar). Moreover, there is evidence that cooperative interactions with other homeodomain-containing proteins (10Wilson D. Sheng G. Lecuit T,. Dostatni N. Desplan C. Genes Dev. 1993; 7: 2120-2134Crossref PubMed Scopus (328) Google Scholar) or with members of the Ets family (38Fitzsimmons D. Hodsdon W. Wheat W. Maira S.-M. Wasylyk B. Hagman J. Genes Dev. 1996; 10: 2198-2211Crossref PubMed Scopus (204) Google Scholar) may also contribute to the recognition of some genomic targets by Pax proteins, suggesting an additional mechanism influencing target selection by Pax-3 in vivo. Ultimately, a deeper knowledge of these and other mechanisms by which Pax-3 achieves DNA-binding specificity will be an important step toward defining the genetic pathways functioning downstream of Pax-3, and more broadly, will help us to understand how different Pax proteins are able to achieve unique target specificities in the developing embryo. Pax-3 belongs to a family of transcription factors that regulate a variety of developmental processes in vertebrates and invertebrates (1Noll M. Curr. Opin. Genet. Dev. 1993; 3: 595-605Crossref PubMed Scopus (294) Google Scholar). In the mouse, Pax-3 is expressed in a subset of neuroectodermal and mesodermal lineages (2Goulding M.D. Chalepakis G. Deutsch U. Erselius J.R. Gruss P. EMBO J. 1991; 10: 1135-1147Crossref PubMed Scopus (749) Google Scholar), and is required for proper neural tube closure and neural crest cell migration (3Epstein D.J. Vekemans M. Gros P. Cell. 1991; 67: 767-774Abstract Full Text PDF PubMed Scopus (578) Google Scholar), and for the development of particular skeletal muscle lineages (4Bober E. Franz T. Arnold H.-H. Gruss P. Tremblay P. Development. 1994; 120: 603-612Crossref PubMed Google Scholar,5Tajbakhsh S. Rocancourt D. Cossu G. Buckingham M. Cell. 1997; 89: 127-138Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar). To interact with target genes, Pax-3 contains a paired domain, a conserved 128-amino acid DNA-binding domain (6Chalepakis G. Fritsch R. Fickenscher H. Deutsch U. Goulding M. Gruss P. Cell. 1991; 66: 873-884Abstract Full Text PDF PubMed Scopus (219) Google Scholar, 7Treisman J. Harris E. Desplan C. Genes Dev. 1991; 5: 594-604Crossref PubMed Scopus (288) Google Scholar) composed of two structurally distinct HTH 1The abbreviations used are: HTH, helix-turn-helix; Prd, paired; PCR, polymerase chain reaction; SELEX,in vitro selection of optimal binding sites; bp, base pair(s). 1The abbreviations used are: HTH, helix-turn-helix; Prd, paired; PCR, polymerase chain reaction; SELEX,in vitro selection of optimal binding sites; bp, base pair(s).-based subdomains joined by a short linker region, as revealed by crystallographic studies with a related family member, Drosophila Prd (8Xu W. Rould M.A. Jun S. Desplan C. Pabo C.O. Cell. 1995; 80: 639-650Abstract Full Text PDF PubMed Scopus (307) Google Scholar). In addition to the paired domain with its bipartite structure, Pax-3, like Prd, also contains a second conserved DNA-binding domain, a paired-type homeodomain, which folds into a classical HTH-based structure (9Wilson D.S. Guenther B. Desplan C. Kuriyan J. Cell. 1995; 82: 709-719Abstract Full Text PDF PubMed Scopus (303) Google Scholar) and which binds cooperatively as a dimer to specific palindromic DNA sequences (10Wilson D. Sheng G. Lecuit T,. Dostatni N. Desplan C. Genes Dev. 1993; 7: 2120-2134Crossref PubMed Scopus (328) Google Scholar). To complement the distinct DNA binding activities of the isolated domains, the paired domain and homeodomain of Prd can also cooperate to recognize specific composite binding sites with high affinity (11Jun S. Desplan C. Development. 1996; 122: 2639-2650Crossref PubMed Google Scholar). Consequently, the interaction of Pax-3 with DNA targets has the potential to be quite complex, with up to three distinct HTH-based modules contributing to target site selection. Within Pax proteins, the N-terminal subdomain of the paired domain is highly conserved (12Walther C. Guenet J.-L. Simon D. Deutsch U. Jostes B. Goulding M.D. Plachov D. Balling R. Gruss P. Genomics. 1991; 11: 424-434Crossref PubMed Scopus (356) Google Scholar), and it interacts with a well defined 10–12-bp consensus sequence through a series of base-specific major groove and minor groove contacts (8Xu W. Rould M.A. Jun S. Desplan C. Pabo C.O. Cell. 1995; 80: 639-650Abstract Full Text PDF PubMed Scopus (307) Google Scholar). In contrast, the C-terminal subdomain in these proteins is more variable (12Walther C. Guenet J.-L. Simon D. Deutsch U. Jostes B. Goulding M.D. Plachov D. Balling R. Gruss P. Genomics. 1991; 11: 424-434Crossref PubMed Scopus (356) Google Scholar), and its precise involvement in DNA recognition has not been extensively characterized. In the case of Prd, the C-terminal subdomain does not contact the 15-bp recognition sequence used for crystallization (8Xu W. Rould M.A. Jun S. Desplan C. Pabo C.O. Cell. 1995; 80: 639-650Abstract Full Text PDF PubMed Scopus (307) Google Scholar), and appears to be dispensable both for DNA binding by Prd in vitro (11Jun S. Desplan C. Development. 1996; 122: 2639-2650Crossref PubMed Google Scholar) and for Prd function in vivo (13Bertuccioli C. Fasano L. Jun S. Wang S. Sheng G. Desplan C. Development. 1996; 122: 2673-2685PubMed Google Scholar). However, deletion studies have shown that this subdomain is required for the recognition of a class o
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