Howard Hughes Medical Institute and Laboratory of Molecular Biology, University of Wisconsin, 1525 Linden Drive, Madison,WI 53706, USA
* Present address: The University of Texas, MD Anderson Cancer Center, Biochemistry and Molecular Biology, 1515 Holcombe Boulevard, Box 117, Houston, TX 77030-4095, USA
Authors for correspondence (e-mail: sbcarrol{at}facstaff.wisc.edu or ghalder{at}odin.mdacc.tmc.edu)
Accepted June 4, 2001
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SUMMARY |
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Key words: Selector gene, Co-factor, Transcription, Wing formation network, Drosophila
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INTRODUCTION |
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The known selector proteins are transcription factors that exert their prominent effects by regulating presumably large but specific sets of target genes. However, the DNA-binding domains of selector proteins often show promiscuous DNA-binding specificity in vitro. For example, recognition sequences for the homeodomains of Hox proteins are typically only 6 bp long (Biggin and McGinnis, 1997; Ekker et al., 1994; Gehring et al., 1994; Mann, 1995; Mann and Affolter, 1998). Similarly, the consensus sequence bound by the TEA domain of the Scalloped (Sd) protein is 8 bp long but degenerate (reviewed by Jacquemin and Davidson, 1997). Potential binding sites for these proteins are predicted to occur once every 2-4 kb in a random sequence, and therefore may be found in cis-regulatory regions of virtually every gene. However, selector proteins presumably do not regulate all genes in a genome. Furthermore, the activity of many selector proteins, particularly Hox proteins, is not restricted to a single field, but may be required during the development of several structures to regulate distinct sets of target genes (Azpiazu and Frasch, 1993; Bodmer, 1993; Halder et al., 1995; Morata and Sanchez-Herrero, 1999) reviewed in (Mann and Morata, 2000). Understanding how the target selectivity of selector proteins is determined in vivo is thus fundamental to understanding how they control gene expression and pattern formation.
Interactions with specific co-factors may be a major determinant of selector protein target selectivity. The DNA-binding specificity of transcription factors is often increased by cooperative interactions with specific co-factors that are also DNA-binding proteins. The Hox proteins and their PBC (Pbx, ceh-20, Extradenticle (Exd)) and MEIS (Homothorax (Hth), Meis, Prep) co-factors provide a prominent example (Mann, 1995; Mann and Affolter, 1998; Mann and Chan, 1996; Mann and Morata, 2000; Wilson and Desplan, 1999). Like the Hox genes, the PBC genes encode homeodomain proteins. They bind cooperatively with Hox proteins to a bipartite DNA sequence. Importantly, they selectively form heterodimers with different Hox proteins, depending on differences within the sequence of the DNA-binding site (Knoepfler et al., 1996; Ryoo and Mann, 1999). The Hth/Meis and Prep1 homeodomain proteins appear to form ternary complexes with Hox and PBC proteins (Berthelsen et al., 1993; Ferretti et al., 2000; Ryoo et al., 1999). Unlike Hox proteins themselves, these complexes bind DNA with higher specificity, which thereby increases the discrimination between target and non-target cis-regulatory elements (Mann, 1995; Mann and Affolter, 1998; Mann and Morata, 2000). Post-translational modifications can also modify DNA-binding and the interactions of Hox proteins with co-factors (Berry and Gehring, 2000; Jaffe et al., 1997). These observations suggest that interactions with and activity regulation by co-factors may be a major determinant of Hox protein selectivity. Little is known, however, about the mechanisms that mediate the target specificity of field-specific selector proteins.
The Scalloped protein (Burglin, 1991; Campbell et al., 1992) controls wing development by directly regulating the expression of a network of genes in the imaginal wing disc (Guss et al., 2001; Halder et al., 1998). Sd binds to essential sites in numerous wing-specific cis-regulatory elements of its target genes (Campbell et al., 1992; Inamdar et al., 1993). Sd is the Drosophila homolog of the vertebrate transcription enhancer factor (TEF) family of transcription factors that contain a TEA DNA-binding domain (Burglin, 1991; Campbell et al., 1992; Jacquemin and Davidson, 1997) and the Sd and TEF-1 proteins possess similar DNA-binding specificities in vitro (Halder et al., 1998). In developing wing cells, Sd forms a complex with Vestigial (Vg) (Paumard-Rigal et al., 1998; Simmonds et al., 1998), a protein with no informative homologies (Williams et al., 1991). This complex is wing specific, because Vg and Sd are not co-expressed in other tissues. The Vg-Sd complex acts as a selector for wing development (Halder et al., 1998; Paumard-Rigal et al., 1998; Simmonds et al., 1998; Bray, 1999; de Celis, 1999). The wing field fails to develop in vg or sd loss-of-function mutants (Campbell et al., 1992; Williams et al., 1991; Williams et al., 1993) and targeted expression of Vg to regions where Sd is also active induces wing-like outgrowths on other structures (Halder et al., 1998; Kim et al., 1996). Sd and Vg physically interact in solution (Paumard-Rigal et al., 1998; Simmonds et al., 1998), but it is not known if they form a complex on DNA. Vg activates transcription in yeast one-hybrid experiments and it has been postulated that Vg acts as a transcriptional activator that is recruited by Sd (Vaudin et al., 1999).
We have examined whether interaction with Vg affects Sd DNA-binding and target gene specificity. We found that Vg and Sd formed a complex on DNA that had a different DNA-binding specificity than Sd alone. We also show that Vg-Sd complex formation on DNA requires protein domains of Vg that are not required for Sd binding in solution. The Vg-Sd complex on DNA appears to be a heterotetramer, and Vg exerts its effect without contacting bases outside the Sd-binding sites. Vg interaction thus switches the DNA target selectivity of Sd, so that Sd and the Vg-Sd complex bind to different sets of binding sites. The presence or absence of Vg in a particular cell is therefore a key determinant of the set of cis-regulatory elements, which are bound and regulated by Sd. The tissue-specific modification of selector protein DNA-binding specificity by co-factors may be a general mechanism for increasing their target selectivity in vivo.
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MATERIALS AND METHODS |
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DNA probes
DNA probes for EMSAs were labeled with 32P-ATP by fill in reaction of double T overhangs at both ends of double-stranded oligonucleotides using the Klenow fragment of DNA polymerase I. Single strand oligonucleotides were annealed at concentrations of 10 µM in 10 mM Tris pH 7.5 and 50 mM NaCl. Labeled probes were purified over Sephadex G50 columns (Princeton Separations). Different probes were diluted to the same specific activities with cold labeled oligonucleotides. Sequences of the upper strand oligonucleotides were 5' to 3': 2xGT (TTCGATACACTTGTGGAATGTGTGGAATGTGTTAGCCCCG), 1xGT (TTCGATACACTTGTGGAATGTGTTTGATTTGTTAGCCCCG), GTspaceGT (TTCGATACACTTGTGGAATGTTATGATCGAAGTGGAATGTGTTAGCCCC), cut-564 (TTGTCAATGTAATTCGAAAAATGTCGTCAG), cut-341 (TTGGCGGCAGATAAAATTATTGAAATTACATTGGCAAGAC); sal-750 (TTTGCTTTCTCTAATCAGACTAATGAGGATT); sal-862 (TTGTTCGCATAACTTATTAAAAA); kni-268 (K. Guss and E. Bier, personal communication; TTCCCCTCTTACATTTGTCGCATAGTTCCCATCTTGGCCA); DSRF (CGATACACTTAAACTATGCCAGGAATTTCTTAGCCCCG); cTNT (TTCCCAGAGAGGAATGCAACACTTGT); and
MHC (TTGCAGGCACGTGGAATGAGCTAT).
Electrophoretic mobility shift assays (EMSAs)
EMSA reactions with TNT produced proteins were carried out in 20 µl binding buffer (8% glycerol, 15 mM Hepes pH 7.9, 150 mM KCl, 1 mM EDTA, 100 µg/ml bovine serum albumin (BSA)) containing 0.7 µl TNT reaction, 0.3 µg dIdC and 3 fmol DNA probe. Equimolar amounts of 35S-labeled proteins were added by diluting the TNT reactions accordingly with unprogrammed TNT extract. Binding reactions were incubated for 15 minutes at room temperature and complexes were separated on 5% polyacrylamide gels and standard 0.5x TBE buffer. Gels were run at 15 V/cm at room temperature. Run gels were dried and exposed with intensifying screens at -70°C overnight. EMSAs with purified TEA domain protein were carried out in essentially the same way, except that 1 fmol probe was used and the buffer contained 100 mM KCl, 2 mM MgCl2 and no dIdC. TEA domain shifts were run on 6% polyacrylamide gels. For supershifts, 100 ng antibody was added to the binding reaction.
Co-immunoprecipitations
To preclear, 10 µl of TNT product were incubated with 400 µl IP buffer (15 mM Hepes pH 7.9, 150 mM KCl, 1 mM EDTA, 1% Triton) and 20 µl of protein A-Sepharose suspension (Amersham Pharmacia Biotech) at 4°C for 20 minutes shaking. Reactions were centrifuged for 2 minutes and 1 µg of antibody (mAB -Myc, mAB
-HA, both from Babco) was added to the supernatant, which was then incubated on a shaker at 4°C for 60 minutes. 20 µl of protein A-Sepharose were added and the reaction was incubated on a shaker at 4°C for 60 minutes. Agarose beads were pelleted by centrifugation at 1500 g for 2 minutes. Supernatant was removed and beads were washed four times with 700 µl IP buffer. Bound proteins were eluted and denatured in 40 µl SDS sample buffer (with 200 mM DTT) by incubation at 68°C for 15 minutes. Proteins were separated by standard 12% and 18% SDS-PAGE. Gels were dried and exposed to BiomaxMR film (Kodak). The IP-buffer differs from the binding buffer used for EMSA only in that it contained 1%Triton X-100 and no BSA. The presence of 1% Triton X-100 had no effect on the EMSA results.
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RESULTS |
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Co-translation of Sd with Vg produced a Vg-Sd complex that bound to these other sites (referred to as B-sites). In contrast to Sd alone, complexes containing Sd and Vg bound strongly to the cut, sal and kni elements (Fig. 1, lanes 3, 7, 11). Quantification of the bound complexes showed that Vg increased Sd binding to these doublet sites by about 10-fold. In addition to enabling binding to B-sites, interaction with Vg reduced Sd binding to the single site templates by at least fivefold (Fig. 1). Importantly, we have not observed binding of Vg alone to any of the binding sites described in this report or to any other DNA templates tested (Fig. 1, data not shown). Therefore, Vg binding to Sd switches the DNA target preference of Sd from the single A-sites to the doublet B-sites.
Only two of the eleven templates that we tested were bound by both Sd and Vg-Sd and thus possessed A- and B-site properties. These were the synthetic 2xGT (see below) and the DSRF probes (data not shown). Thus, while most native templates have either A- or B-site character, sites with both A- and B-site properties also occur.
Two binding sites but not cooperativity of Sd binding are required for Vg-Sd complex formation on DNA
The observation that the DNA templates that were bound by the Vg-Sd complexes (B-sites) contained two binding sites arranged in tandem to which the TEA domain bound cooperatively, raised the possibility that cooperative binding and the presence of two binding sites are required for the Vg-Sd complexes to form on DNA. To test this, we analyzed binding to a series of probes derived from the GT-IIC high-affinity TEF-1 site identified in the SV40 enhancer (Davidson et al., 1988). We selected this probe because templates composed of two GT-IIC-binding sites arranged in tandem (referred to as 2xGT) are bound cooperatively and with high affinity by the TEA domain, full-length Sd and TEF-1, as well as by the Vg-Sd complex (Fig. 2A) (Davidson et al., 1988; Halder et al., 1998; Jacquemin et al., 1996; Xiao et al., 1991). We also designed derivatives of the 2xGT probe that either had only a single Sd binding site (1xGT, resembling the native GTII-C-binding site, Table 1) or that had a 10 bp spacer between the two Sd-binding sites (GTspaceGT) that abolishes cooperative binding (Fig. 2; Davidson et al., 1988; Jiang et al., 2000).
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The Sd-interaction domain of Vg is sufficient for binding to Sd but not for complex formation on DNA
To identify domains within Vg that may be important for Sd interaction and complex formation on DNA, we first searched for conserved domains in Vg homologs and other proteins. A Vg homolog from the mosquito (Jim Williams and S. B. C., unpublished) shows strong conservation of the first 79 amino acids and of the region beginning with the previously identified Sd-interaction domain (SID) to the very C terminus (Fig. 3A,B). The region from position 80 to 280 in the Drosophila protein shows no or only moderate similarity to mosquito Vg. A Vg homolog from vertebrates, Tondu (Tdu; Vaudin et al., 1999), shares the first part of the SID but no other domains (Fig. 3A,B). We have identified a novel vertebrate Vg homolog, Fondue (Fdu) (G. H. and S. B. C., unpublished). The similarity of the SID in Fdu to the SID in Vg is more extensive than in Tdu and spans two exons. In fact the splice site in Vg and Fdu occur at nearly identical positions (Fig. 3B, arrowheads). We now define the extent of the SID by the region that is conserved between insect Vg and Fdu (Fig. 3B). In addition to the SID, Fdu and Vg share two other domains: the N-terminal 66 amino acids (green domain in Fig. 3A,B) and a domain rich in histidine and alanine residues C-terminal to the SID (orange domain in Fig. 3A,B). Therefore, the newly identified Fdu protein is more similar to Vg than is the Tdu protein.
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We also created four deletion mutants to test whether the non-conserved residues between the green domain and the SID have essential functions. Internal deletions of residues 73-176 and 73-274 had essentially no effect on Vg activity in the DNA-binding assay. However, in combination with the C-terminal deletion, they abolished complex formation (Fig. 4A, lanes 10-14). Thus, the internal region may be required to correctly position the green domain, so that it can interact with C-terminal residues to form the Vg-Sd-DNA complex.
Taken together, these data identify three regions in Vg that are required for complex formation with Sd on DNA: the Sd interaction domain (SID), which is sufficient to mediate binding to Sd in solution, the N-terminal 66 residues and the region C-terminal to the SID, both of which are required specifically for forming a Vg-Sd complex on DNA. All three domains show conservation between insect Vg and vertebrate Fdu.
Vg and Sd form a heterodimer in solution but a heterotetramer on target DNA
The observation that the Vg-Sd complex did not bind to the 1xGT probe was surprising given that it bound to the 2xGT probe, which contains two tandemly arranged 1xGT binding sites. This raises the question of why the Vg-Sd complex requires doublet sites but does not recognize single binding sites. One possibility is that DNA binding by the Vg-Sd complex requires more than one Sd and Vg molecule in the complex. For example, a single molecule of Vg could bridge two Sd molecules to enable DNA binding or it may be that Vg-Sd dimers interact to increase the affinity or stability of the complex on DNA.
To determine how many Vg molecules are present in the respective complexes, we examined the mobility of complexes formed when two Vg molecules of different sizes were present. The design of this experiment was to test for the formation of heteromeric complexes of intermediate size that would indicate the presence of two (or more) Vg molecules in the complexes. We made use of two internal deletion mutants that showed nearly normal activity in the DNA-binding and Sd interaction assays (Fig. 4). The Vg deletion (73-274) formed complexes with Sd on DNA that migrate faster on EMSA gels than complexes containing full-length Vg, owing to the smaller size of the mutant protein (Fig. 5A, lanes 1,3). Because we wanted to use the same proteins for the EMSA and the Co-IPs described below, we used an HA-tagged Vg protein (VgHA), which gave the same results as native Vg (not shown). When full length VgHA was co-expressed with Vg
(73-274) and Sd, complexes of intermediate mobility formed (Fig. 5A, lane 2 arrowhead). Complexes migrating at the position of the complexes formed with VgHA were still present (open arrowhead). However, little (if any) complexes with the Vg deletion were observed, which may be due to competition by VgHA, which binds with higher affinity than
(73-274) (Fig. 5A, compare lane 1 with lane 3). We interpret the intermediate complexes as hetero-complexes formed between Sd, a mixture of VgHA and Vg
(73-274), and DNA. Results using the Vg
(73-176) deletion were identical (not shown). Thus, more than one Vg molecule is present in the shifted complexes. More precisely, as one extra complex of intermediate size appeared, it most probably contains one of each of the two Vg forms and is a heterotetramer.
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Vg-Sd complex formation on target DNA does not require bases outside the Sd-binding site
The observation that the Vg-Sd complex has increased affinity for B-sites compared with the Sd protein alone raises the possibility that Vg makes DNA contacts outside the region that is contacted by Sd, which could enlarge the DNA interaction surface and thereby increase the affinity of the complex. It has not been possible to produce sufficient quantities of active Vg-Sd complexes for chemical interference and DNAseI footprinting assays that could localize exactly the region contacted by the Vg-Sd complex. Bacterially produced Sd and Vg are insoluble and do not form active complexes upon renaturation, either refolded together or refolded separately and then mixed together. As an alternative to a chemical interference assay, we designed a series of 2xGT probes with increasingly truncated ends to test whether bases outside of the Sd binding sites are required for Vg-Sd complex formation (Fig. 6A). We chose the 2xGT probe because this template allows observation of Sd and Vg-Sd binding. Our rationale was that, if Vg contacts DNA outside the Sd-binding sites, then the Vg-Sd complex may not form on shorter probes that are nevertheless bound by Sd alone. However, if Vg does not contact DNA, the minimal template length requirement should be similar for Vg-Sd and for Sd binding.
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DISCUSSION |
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Vg binding switches the DNA target selectivity of Sd
We propose a model in which Vg binding to Sd switches the DNA target selectivity of Sd (Fig. 7). We found that the Sd protein alone binds to sites with a particular composition, termed A-sites, which exist singly or as doublets. In the latter case, Sd may bind cooperatively if the two sites are arranged in tandem. When Vg is also present, Vg and Sd interact and form a dimer in solution (Fig. 5B). This complex has two distinct properties. First, the Vg-Sd dimer has a greatly reduced affinity for A-sites (Fig. 1). Vg may either induce a conformational change in Sd that inhibits the TEA domain from interacting with DNA, or Vg could directly mask the TEA domain. Second, the dimer forms a higher order complex on a different set of binding sites, termed B-sites (Fig. 1). These two activities of Vg are distinguished by their structural requirements. While the SID domain of Vg is sufficient to inhibit Sd DNA-binding to A-sites, additional domains N- and C-terminal to the SID are required for complex formation on B-sites (Fig. 4). Importantly, B-sites are poorly bound by Sd in the absence of Vg. Thus, Vg binding to Sd inhibits binding to A-sites while enabling binding to B-sites, that is, Vg switches the DNA-binding preference from A-sites to B-sites.
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Alternatively, Vg interaction may specifically enhance binding to doublet B-sites. We favor this model because we found that Vg-Sd had a similar affinity for several B-sites such as those in cut and 2xGT, even though 2xGT is a much better Sd binding site. The affinities of Sd for these sites therefore do not translate directly into the relative affinities observed for Sd-Vg binding, as would be expected if Vg only enhanced cooperativity. In addition, we found that the TEA domain binds several A- and B-sites with high affinity, but that full-length Sd has a strong preference for A-sites over B-sites. Thus, in the absence of any co-factor, Sd is in a conformation in which a domain of Sd separate from the TEA domain inhibits the TEA domain from binding to B-sites specifically (Fig. 1). In vitro, Vg interaction appears to be able to alleviate this inhibition because Vg-Sd complexes bind strongly to B-sites. This alleviation only occurs when complexes form on doublet sites, as Vg-Sd complexes do not bind to DNA as a dimers. We suggest that some sort of conformational change is associated with binding to doublet B-sites (Fig. 7). Our model is supported by the finding that the region of Sd that binds to the SID of Vg is homologous to a region of the vertebrate TEF-1 that negatively affects DNA binding (Hwang et al., 1993; Simmonds et al., 1998). This model is analogous in part to the role of Exd overcoming the inhibitory effect of the YKWM motif in the Labial Hox protein (Chan et al., 1996).
We have argued here that Sd and the Vg-Sd complex differentiate between A- and B-sites. What then are the distinguishing features of these sites? The sequences of the A- and B-sites are quite diverse and their alignment does not reveal different consensus sequence motifs. However, Sd clearly prefers binding to A-sites, and the inability of Sd to bind strongly to B-sites, such as that in the cut element, must therefore be due to the sequence of the template site. Vg-Sd complexes bind with high affinity to only two sites when arranged in tandem, and do not form on single A- or B-sites. Thus, Sd discriminates between A- and B-sites based on sequence, while the binding of Vg-Sd complex depends both on sequence and the arrangement of the sites. We have identified two sites (DSRF and 2xGT) that have A- as well as B-site properties, so these properties are not mutually exclusive. However, many sites exist that are bound well by Sd or Vg-Sd, but not by both. Most of the essential sites for Vg-Sd regulation in vivo have mainly B-site character and are bound poorly by Sd. The identification of the exact sequence requirements that distinguish native essential Sd sites from the known Vg-Sd target sites will require some knowledge of Sd-regulated target genes in other tissues (see below).
Vg as a determinant of the specificity of Sd action in vivo
Vg binding and its effect on the DNA target selectivity of Sd plays a major role in distinguishing the biological specificity of Sd action in the developing wing from Sd function in other tissues. Sd is required for the development of tissues other than the wing, for example, the eye and the PNS, where it is not co-expressed with Vg (Campbell et al., 1992; Inamdar et al., 1993). Based on our results, we postulate that Sd selects a different set of target genes there, at least in part because its DNA-binding specificity is different in the absence of Vg.
No direct target genes for Sd in these other tissues have been identified. However, many target genes for the vertebrate Sd homolog TEF-1 are known (reviewed by Jacquemin and Davidson, 1997). Sd and TEF-1 may function very similarly, as their TEA domains are 99% identical (Campbell et al., 1992) and have indistinguishable DNA-binding properties in vitro (Halder et al., 1998), and TEF-1 can substitute for Sd in Drosophila (Deshpande et al., 1997). In mammals, TEF-1 directly regulates many genes expressed during muscle differentiation by binding to A-sites containing the so-called m-CAT motif (CATTCCT) (Cooper and Ordahl, 1985; Farrance et al., 1992; Mar and Ordahl, 1990; Nikovits et al., 1986). Importantly, this motif is bound by a single TEF-1 molecule (Farrance et al., 1992). We tested two of these m-CAT sites for Sd binding and found that, as for other single A-sites, Sd alone bound well, but the presence of Vg inhibited Sd binding and did not result in complex formation on DNA. Because these sites are in vivo targets of TEF-1, this suggests that TEF-1 and Sd may directly regulate gene expression by binding to single A-sites alone or in complexes with other factors, but not in complexes containing the Vg/Fdu proteins. Interestingly, it has been found that vertebrate TEF-1 forms a complex with the bHLH protein Max in vivo, and that Max, or another bHLH protein, may be an obligatory co-factor for TEF-1 function during muscle differentiation (Gupta et al., 1997). Because Max contacts DNA sequence specifically, it increases the target selectivity of TEF-1 in muscle cells. The association of TEF-1 with Max may present another example of a tissue-specific co-factor that differentiates the DNA-target selectivity of a TEF transcription factor family member between different tissues.
Implications for the elucidation of regulatory networks on a genomic scale
One of the major aims of genome sequence analysis is to decipher genetic regulatory sequences involved in development and differentiation. One critical challenge in achieving this goal is the ability to correctly predict the in vivo target genes of transcription factors. Several types of data may be considered for such predictions, including the presence or absence of transcription factor binding sites in potential regulatory regions, gene expression profiles and detailed protein function studies. Searching genomic sequences for binding sites is obviously important; however, binding site consensus sequences are often short and degenerate, so that potential binding sites are predicted to occur in regulatory regions of virtually any gene. This also holds true for Sd. The consensus binding site of the TEA domain (T/A A/G A/G T/A AT G/T T) is found once about every 2 kb, on average. However, we have argued that many, if not all, Vg-Sd-regulated target genes possess a doublet of Sd-binding sites. Requiring a second binding site in tandem decreases the frequency of potential biologically relevant Vg-Sd binding sites by a factor of 2000. The fact that most of the Vg-Sd sites would not have been found using full-length Sd protein in footprint assays and that the Sd DNA-binding domain alone binds promiscuously is therefore a note of caution. Understanding the role of tissue-specific co-factors may be imperative to deciphering transcription factor-regulated networks on a genome-wide scale. Efforts are under way, using these new insights into the selectivity of the Vg-Sd complex, towards defining the network of Vg-Sd-regulated genes in the developing wing.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Azpiazu, N. and Frasch, M. (1993). tinman and bagpipe: two homeo box genes that determine cell fates in the dorsal mesoderm of Drosophila. Genes Dev. 7, 1325-1340.[Abstract]
Berry, M. and Gehring, W. (2000). Phosphorylation status of the SCR homeodomain determines the functional activity: essential role for protein phosphatase 2A, B. EMBO J. 19, 2946-2957.
Berthelsen, J., Zappavigna, V., Ferretti, E., Mavillo, F. and Blasi, F. (1993). The novel homeoprotein Prep1 modulates Pbx-Hox protein cooperativity. Genes Dev. 17, 1434-1445.
Biggin, M. D. and McGinnis, W. (1997). Regulation of segmentation and segmental identity by Drosophila homeoproteins: the role of DNA binding in functional activity and specificity. Development 124, 4425-4433.
Bodmer, R. (1993). The gene tinman is required for specification of the heart and visceral muscles in Drosophila. Development 118, 719-729.
Bray, S. (1999). Drosophila development: Scalloped and Vestigial take wing. Curr. Biol. 9, R245-R247.[Medline]
Burglin, T. (1991). The TEA domain: A novel, highly conserved DNA-binding motif. Cell 66, 11-12.[Medline]
Campbell, S., Inamdar, M., Rodrigues, V., Raghavan, V., Palazzolo, M. and Chovnick, A. (1992). The scalloped gene encodes a novel, evolutionarily conserved transcription factor required for sensory organ differentiation in Drosophila. Genes Dev. 6, 367-379.[Abstract]
Carroll, S., Weatherbee, S. and Langeland, J. (1995). Homeotic genes and the regulation and evolution of insect wing number. Nature 375, 58-61.[Medline]
Carroll, S. B., Grenier, J. K. and Weatherbee, S. D. (2001). From DNA to Diversity: Molecular Genetics and the Evolution of Animal Design. Malden: Blackwell Scientific.
Chan, S. K., Popperl, H., Krumlauf, R. and Mann, R. S. (1996). An extradenticle-induced conformational change in a HOX protein overcomes an inhibitory function of the conserved hexapeptide motif. EMBO J. 15, 2476-2487.[Abstract]
Cohen, S., Bronner, M., Kuttner, F., Jurgens, G. and Jackle, H. (1989). Distal-less encodes a homeodomain protein required for limb formation in Drosophila. Nature 338, 432-434.[Medline]
Cooper, T. A. and Ordahl, C. P. (1985). A single cardiac troponin T gene generates embryonic and adult isoforms via developmentally regulated alternate splicing. J. Biol. Chem. 260, 11140-11148.
Dalton, S. and Treisman, R. (1992). Characterization of SAP-1, a protein recruited by serum response factor to the C-fos serum response element. Cell 68, 597.[Medline]
Davidson, I., Xiao, J., Rosales, R., Staub, A. and Chambon, P. (1988). The HeLa cell protein TEF-1 binds specifically and cooperatively to two SV40 enhancer motifs of unrelated sequence. Cell 54, 931-942.[Medline]
de Celis, J. F. (1999). The function of vestigial in Drosophila wing development: how are tissue-specific responses to signalling pathways specified? BioEssays 21, 542-545.[Medline]
Deshpande, N., Chopra, A., Rangarajan, A., Shashidhara, L., Rodrigues, V. and Krishna, S. (1997). The human transcription enhancer factor-1, TEF-1, can substitute for Drosophila scalloped during wingblade development. J. Biol. Chem. 272, 10664-10668.
Ekker, S. C., Jackson, D. G., von Kessler, D. P., Sun, B. I., Young, K. E. and Beachy, M. D. (1994). The degree of variation in DNA sequence recognition among four Drosophila homeotic proteins. EMBO J. 13, 3551-3560.[Abstract]
Farrance, I. K., Mar, J. H. and Ordahl, C. P. (1992). M-CAT binding factor is related to the SV40 enhancer binding factor, TEF-1. J. Biol. Chem. 267, 17234-17240.
Ferretti, E., Marshall, H., Popperl, H., Macoachle, M., Krumlauf, R. and Blasi, F. (2000). Segmental expression of Hoxb2 in r4 requires two separate sites that integrate cooperative interactions between Prep1, Pbx and Hox proteins. Development 127, 155-166.
Frasch, M. (1999). Intersecting signalling and transcriptional pathways in Drosophila heart specification. Semin. Cell Dev. Biol. 10, 61-71.[Medline]
Gehring, W. J., Qian, Y. Q., Billeter, M., Furukubo-Tokunaga, K., Schier, A. F., Resendez-Perez, D., Affolter, M., Otting, C. and Wuthrich, K. (1994). Homeodomain DNA recognition. Cell 78, 211-223.[Medline]
Gorfinkiel, N., Morata, G. and Guerrero, I. (1997). The homeobox gene Distal-less induces ventral appendage development in Drosophila. Genes Dev. 11, 2259-2271.
Gupta, M., Amin, C., Gupta, M., Hay, N. and Zak, R. (1997). Transcription enhancer factor 1 interacts with a basic helix-loop-helix zipper protein, Max, for positive regulation of cardiac -Myosin heavy-chain gene expression. Mol. Cell. Biol. 17, 3924-3936.[Abstract]
Guss, K. A., Nelson, C. E., Hudson, A., Kraus, M. E. and Carroll, S. B. (2001). Control of a genetic regulatory network by a selector gene. Science 292, 1164-1167.
Halder, G., Callaerts, P. and Gehring, W. (1995). Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science 267, 1788-1792.[Medline]
Halder, G., Polaczyk, P., Kraus, M. E., Hudson, A., Kim, J., Laughon, A. and Carroll, S. B. (1998). The Vestigial and Scalloped proteins act together to directly regulate wing-specific gene expression in response to signaling proteins. Genes Dev. 12, 3900-3909.
Hwang, J.-J., Chambon, P. and Davidson, I. (1993). Characterization of the transcription activation function and the DNA binding domain of transcriptional enhancer factor-1. EMBO J. 12, 2337-2348.[Abstract]
Inamdar, M., Vijayraghavan, K. and Rodrigues, V. (1993). The Drosophila homolog of the human transcription factor TEF-1, scalloped, is essential for normal taste behavior. J. Neurogenet. 9, 123-139.[Medline]
Jacquemin, P. and Davidson, I. (1997). The role of the TEF transcription factors in cardiogenesis and other developmental processes. Trends Cardiovasc. Med. 7, 192-197.
Jacquemin, P., Hwang, J.-J., Martials, J., Dollé, P. and Davidson, I. (1996). A novel family of developmentally regulated mammalian transcription factors containing the TEA/ATTS DNA binding domain. J. Biol. Chem. 271, 21775-21785.
Jaffe, L., Ryoo, H. D. and Mann, R. S. (1997). A role for phosphorylation by casein kinase II in modulating Antennapedia activity in Drosophila. Genes Dev. 11, 1327-1340.[Abstract]
Jiang, S. W., Desai, D., Khan, S. and Eberhardt, N. L. (2000). Cooperative binding of TEF-1 to repeated GGAATG-related consensus elements with restricted spatial separation and orientation. DNA Cell Biol. 19, 507-514.[Medline]
Kim, J., Sebring, A., Esch, J., Kraus, M., Vorwerk, K., Magee, J. and Carroll, S. (1996). Integration of positional signals and regulation of wing formation and identity by Drosophila vestigial gene. Nature 382, 133-138.[Medline]
Knoepfler, P. S., Lu, Q. and Kamps, M. P. (1996). Pbx-1 Hox heterodimers bind DNA on inseparable half-sites that permit intrinsic DNA binding specificity of the Hox partner at nucleotides 3' to a TAAT motif. Nucleic Acids Res. 24, 2288-2294.
Lawrence, P. and Morata, G. (1994). Homeobox genes: their function in Drosophila segmentation and pattern formation. Cell 78, 181-189.[Medline]
Lewis, E. B. (1978). A gene complex controlling segmentation in Drosophila. Nature 276, 565-570.[Medline]
Manak, J. R. and Scott, M. P. (1994). A class act: conservation of homeodomain protein functions. Development 120 Suppl., 61-77.
Mann, R. (1995). The specificity of homeotic gene function. BioEssays 17, 855-863.[Medline]
Mann, R. S. and Affolter, M. (1998). Hox proteins meet more partners. Curr. Opin. Genet. Dev. 8, 423-429.[Medline]
Mann, R. S. and Chan, S. K. (1996). Extra specificity from extradenticle: the partnership between HOX and PBX/EXD homeodomain proteins. Trends Genet. 12, 328.
Mann, R. S. and Morata, G. (2000). The developmental and molecular biology of genes that subdivide the body of Drosophila. Annu. Rev. Cell Dev. Biol. 16, 243-271.[Medline]
Mar, J. H. and Ordahl, C. P. (1990). M-CAT binding factor, a novel trans-acting factor governing muscle-specific transcription. Mol. Cell. Biol. 10, 4271-4283.[Medline]
McGinnis, W. and Krumlauf, R. (1992). Homeobox genes and axial patterning. Cell 68, 283-302.[Medline]
Morata, G. and Sanchez-Herrero, E. (1999). Patterning mechanisms in the body trunk and the appendages of Drosophila. Development 126, 2823-2828.
Nikovits, W., Kuncio, G. and Ordahl, C. P. (1986). The chicken fast skeletal troponin I gene: exon organization and sequence. Nucleic Acids Res. 14, 3377-3390.[Abstract]
Paumard-Rigal, S., Zider, A., Vaudin, P. and Silber, J. (1998). Specific interactions between vestigial and scalloped are required to promote wing tissue proliferation in Drosophila melanogaster. Dev. Genes Evol. 208, 440-446.[Medline]
Quiring, R., Walldorf, U., Kloter, U. and Gehring, W. J. (1994). Homology of the eyeless gene of Drosophila to the Small eye gene in mice and Aniridia in humans. Science 265, 785-789.[Medline]
Ryoo, H. D. and Mann, R. S. (1999). The control of trunk Hox specificity and activity by Extradenticle. Genes Dev. 13, 1704-1716.
Ryoo, H. D., Marty, T., Casares, F., Affolter, M. and Mann, R. S. (1999). Regulation of Hox target genes by a DNA bound Homothorax/Hox/Extradenticle complex. Development 126, 5137-5148.
Simmonds, A., Liu, X., Soanes, K., Krause, H., Irvine, K. and Bell, J. (1998). Molecular interactions between Vestigial and Scalloped to promote wing formation in Drosophila. Genes Dev. 12, 381-390.
Vaudin, P., Delanoue, R., Davidson, I., Silber, J. and Zider, A. (1999). TONDU (TDU) a novel human protein related to the product of vestigial (vg) gene of Drosophila melanogaster interacts with vertebrate TEF factors and substitutes for Vg function in wing formation. Development 126, 4807-4816.
Williams, J. A., Bell, J. and Carroll, S. B. (1991). Control of Drosophila wing and haltere development by the nuclear vestigial gene product. Genes Dev. 5, 2481-2495.[Abstract]
Williams, J. A., Paddock, S. W. and Carroll, S. B. (1993). Pattern formation in a secondary field: A hierarchy of regulatory genes subdivides the developing Drosophila wing disc into discrete sub-regions. Development 117, 571-584.
Wilson, D. S. and Desplan, C. (1999). Structural basis of Hox specificity. Nat. Struct. Biol. 6, 297-300.[Medline]
Xiao, J. H., Davidson, I., Matthes, H., Garnier, J. M. and Chambon, P. (1991). Cloning, expression and transcriptional properties of the human enhancer factor TEF-1. Cell 65, 551-568.[Medline]