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A leucine zipper domain of the suppressor of Hairy-wing protein mediates its repressive effect on enhancer function.

The suppressor of Hairy-wing [su(Hw)] protein mediates the mutagenic effect of the gypsy retrotransposon by repressing the function of transcriptional enhancers controlling the expression of the mutant gene. A structural and functional analysis of su(Hw) was carried out to identify domains of the protein responsible for its negative effect on enhancer action. Sequence comparison among the su(Hw) proteins from three different species allows the identification of evolutionarily conserved domains with possible functional significance. An acidic domain located in the carboxy-terminal end of the Drosophila melanogaster protein is not present in su(Hw) from other species, suggesting a nonessential role for this part of the protein. A second acidic domain located in the amino-terminal region of su(Hw) is present in all species analyzed. This domain is dispensable in the D. melanogaster protein when the carboxy-terminal acidic domain is present, but the protein is nonfunctional when both regions are simultaneously deleted. Mutations in the zinc fingers result in su(Hw) protein unable to interact with DNA in vivo, indicating a functional role for this region of the protein in DNA binding. Finally, a region of su(Hw) homologous to the leucine zipper motif is necessary for the negative effect of this protein on enhancer function, suggesting that su(Hw) might exert this effect by interacting, directly or indirectly, with transcription factors bound to these enhancers.

su(Hw) protein can also interfere with regulatory sequences located in genes for which gypsy-induced mutations have not been found. For example, the insertion of the su(Hw)-binding region in the 5' end of the hsp70 gene, between the heat shock element and the promoter, interferes with proper heat shock induction of transcription (Holdridge and Dorsett 1991).
These results indicate that the su(Hw)-mediated mutagenic effect of gypsy is attributable to an inhibitory effect of the su(Hw) protein on enhancer elements that control the expression of the mutated gene. The lack of specificity in the nature of the affected enhancers and the directionality of the inhibitory effect suggest a few altematives to explain the mechanism underlying this phenomenon. One possibility is that binding of the su(Hw) protein causes changes in the conformation of the adjacent chromatin that spread distally with respect to the promoter and interfere with the binding of transcription factors to distal enhancers. The requirement for a directional (away from the promoter) spreading of chromatin changes can be explained if binding of su (Hw) to DNA creates boundaries between higher order domains of gene activity (Roseman et al. 1993). A second possibility is that the inhibitory effect of su(Hw) on enhancer function is attributable to the interaction of this protein with enhancer-bound transcription factors as they try to reach the transcription complex by tracking along the DNA or looping the intervening sequences (Geyer and Corces 1992). This model explains the directionality of the su(Hw) effect better than that based on chromatin changes, but the direct contact of su (Hw) with transcription factors might require a degree of specificity in the interaction that is at odds with the apparent universal effect of su(Hw) on all enhancers tested.
To further the understanding of the mechanism by which su(Hw) affects enhancer function, we have sought to determine whether specific domains of su(Hw) are involved in the negative effect of this protein on tissuespecific transcription. Here, we report a functional study of the su(Hw) protein carried out by analysis of extant su(Hw) mutants, as well as new mutations induced in vitro and introduced into the fly by P element-mediated transformation. These studies suggest that an extended amphipathic s-helix of the su(Hw) protein with structural characteristics similar to the leucine zipper motif might be responsible for the inhibitory effect of su(Hw) on enhancer-promoter interactions, whereas two acidic domains located in the amino-and carboxy-terminal ends of the su(Hw) protein might play auxiliary functions in this process.

Mutations in su(Hw) only affect oogenesis
To gain insights into the normal role of the su(Hw) protein and the mechanism by which su(Hw) mediates the mutant effect of gypsy, we have analyzed in detail the phenotypic consequences of the lack of the su(Hw) gene product. The only phenotypic effect of null mutations in the su(Hw) gene is female sterility. Females carrying the su(Hw) v null allele (Harrison et al. 1992) are unable to lay eggs because the egg chambers degenerate before completion of oogenesis. Egg chambers from su(Hw) v females appear less regular in shape and spaced more closely on the ovarioles than wild-type chambers (data not shown). In stages 7, 8, and 9, nurse cells of su (Hw) mutants can often be seen to shrink away from one another, leaving gaps in the anterior portions of the egg chambers. In addition, wild-type oocytes begin accumulating yolk at stage 8. The stage 8 and 9 egg chambers of su(Hw) mutant females show a considerable reduction in deposition of yolk in the oocyte. Egg chambers of the mutant cease to grow and eventually degenerate before stage 10. DAPI staining shows that wild-type ovaries contain nurse cells with homogeneously staining chromosomes within the nucleus after very early stages of oogenesis (King et al. 1956). The DNA appears to be decondensed and spread throughout the nucleus (Fig. 1A).
In su(Hw) mutant nurse cells, the chromosomes look normal until stage 3 or 4, but afterward become aggregated and condensed (Fig. 1B). The bulbous nature of the chromosomes from su(Hw) mutants is reminiscent of that seen fleetingly in normal egg chambers only at stage 4. It seems that su(Hw) v nurse cell chromosomes generally retain the bulbous morphology throughout later stages of oogenesis, suggesting that the su(Hw) gene product might be required for decondensation of the nurse cell chromosomes. As su(Hw) is a DNA-binding protein capable of interacting with many sites throughout the genome (Spana et al. 1988), it could be hypothesized that the binding of su(Hw) directly to these bulbous chromosomes is responsible for decondensation. The chromosomes of follicle cells appear to be unaffected in the mutant.
An additional defect seen in some of the su(Hw) alleles is that many egg chambers contain more than the normal 15 nurse cells. This defect is likely to be the result of the fusion of egg chambers rather than the overproliferation of nurse cells, because most of the aberrant chambers contain -30 nurse cells, twice the normal number. In addition, the anterior nurse cells are often smaller than the posterior, as might be expected because the younger chambers are located more anteriorly in the ovariole. Perhaps the strongest evidence is the presence of some chambers that appear to be in the process of fusing. These chambers are pushed together, and material from degenerating follicle cells can be seen between the cysts (Fig. 1C). These fused chambers continue to develop until stage 9 of oogenesis, arrest, and eventually degenerate similar to nonfused su(Hw) egg chambers. These results suggest that the su(Hw) protein is not essential in males and is only required in females during oogenesis.

su(Hw) is a ubiquitous nuclear protein
To understand the apparent discrepancy between su(Hw) RNA expression in all tissues of the fly and the restric- tive mutant phenotype, and to test whether the enhancer-specific effects of su(Hw) on the expression of the yellow gene are attributable to a restriction in the spatial expression of this protein, we have determined the tissue distribution of su(Hw) protein during Drosophila development. Frozen sections through adults, pupae, and larvae were immunoreacted with anti-su(Hw) antibodies [Spana et al. 1988), incubated with a rhodamine-conjugated secondary antibody, and viewed under epifluorescence ( Fig. 1). su(Hw) protein was detected in the nuclei of cells from all stages of development; therefore, su(Hw) is a ubiquitous nuclear protein. Also, staining was detected in males as well as females, contrary to what might be expected from the ovary-specific phenotype of su(Hw) mutations. Sections of flies from the su(Hw) v null mutation immunoreacted with these antibodies failed to stain above background levels (data not shownl, indicating that the antibodies recognize only the su(Hw) protein in situ.
These results indicate that su(Hw) protein is present in the nuclei of all Drosophila cells. The role of this protein in tissues other than the nurse cells is dispensable or the function of su(Hw) can be replaced by other proteins with similar properties. These results also clarify the role of the su(Hw) protein in mediating the mutagenic effect of gypsy on the yellow gene. The specificity of the inhibitory effect of su(Hw) on enhancers located distal to the su(Hw)-binding site with respect to the promoter could not be attributable to the presence of the protein in the tissues where yellow expression is controlled by these enhancers, whereas nonaffected tissues lack su(Hw) protein. Rather, the specificity must depend on the particular mechanism underlying the inhibitory effect. To further understand the molecular basis of this effect we have determined the nature of putative conserved structural domains in the su(Hw) protein that could have functional significance in its interaction with enhancers in the yellow gene.

Structural conservation of the su(Hw) protein in different Drosophila species
As a first step in determining the nature of structural domains of functional importance for the role of su(Hw), both during oogenesis and in the generation of gypsyinduced phenotypes, we have determined the amino acid sequence of this protein in several Drosophila species. The su(Hw) gene was cloned from Drosophila ananassae and D. virilis by cross-hybridization with the gene from D. melanogaster. Figure 2 shows the deduced amino acid sequence for all three proteins.
The amino-terminal region of the protein, including the first 218 amino acids, is poorly conserved, with only 20% identity and 46% similarity. This region of the protein contains a highly acidic domain located between residues 154 and 202 in D. melanogaster. In spite of the low overall sequence conservation in this domain, the acidic characteristic is well preserved: 23 of 48 amino acid residues are aspartic acid or glutamic acid in D. has been more conserved during evolution than the amino-terminal region. Comparison of this region among all three species indicates 80% amino acid identity and 95% similarity when chemically similar amino acids are taken into account {Fig. 2). This region contains 12 copies of the zinc finger motif, which are presumably involved in the interaction of su(Hw) with gypsy DNA.
The high degree of conservation not only extends to the zinc fingers but also to some of the interfinger regions This indicates that these alleles are not affected at the level of transcript accumulation and are suitable candidates for further characterization. The production of   Figure 4, although longer exposure of the film allows the visualization of low amounts of a protein of wild type size (Harrison 1991). This mutation is caused by the insertion of the jockey transposable element into the first intron of the su(Hw) gene (Fig. 5). This insertion results in very low levels of su(Hw) RNA (Parkhurst et al. 1988) owing to premature termination of transcription in the polyadenylation site located in the jockey element (Harrison 1991). The su(Hw) t3 mutation is fertile and suppresses the y2 phenotype weakly (see below), suggesting that very low levels of su(Hw) protein can still affect the function of transcriptional enhancers in the yellow gene.
This mutant has been included in the Westem analysis to establish a correlation between levels of wild-type protein and the severity of the su(Hw) phenotype.
Genomic DNA was isolated from each of the mutants and subjected to PCR using three pairs of nested primers covering the entire transcribed region of the su(Hw) locus. The PCR products were cloned into pUC18, and several clones were sequenced to confirm the lesion in these su(Hw) mutants. Results of this analysis are described below and are summarized in Figure 5. Also summarized in Figure 5 is the structure of several su(Hw) alleles analyzed but not used in these studies. Mutations in the zinc fingers affect the binding of su(Hw) to gypsy DNA Two of the mutations that accumulate protein of normal size and abundance have been identified as amino acid substitutions in the seventh zinc finger motif, su(Hw) ~a is a G--~ A transition at nucleotide 2116 [see Parkhurst et al. (1988) for a description of nucleotide numbering in the su(Hw) gene], resulting in the replacement of an arginine by a histidine residue. This change occurs in the region of the zinc finger base, between the two histidine residues that coordinate the zinc ion (Fig. 3B). The su(Hw) ~8 allele is a C---> T transition at base 2109 (Parkhurst et al. 1988), resulting in the replacement of a histidine with a tyrosine residue. The lost histidine would have been one of the amino acids that coordinates zinc in the finger (Fig. 3B). These two amino acid replacements occur only two residues apart, yet the phenotypes of these mutants are extremely different (Fig. 6).
su(Hw) ~8 ranks among the most severe of su(Hw) alleles, with strong suppression and complete female sterility. Presumably, the mutant protein is rendered incapable of binding its normal recognition site. The loss of one of the ligand-binding residues has been shown to be deleterious to the function of zinc finger proteins (Blumberg et al. 1987;Redemann et al. 1988). Inability of the seventh finger to fully participate in the interaction with the recognition site may destabilize binding of the entire protein, suggesting that this finger plays a central role in the interaction of su(Hw) with gypsy DNA. Evidence to support this conclusion is presented below. On the other hand, su(Hw) e2 is among the mildest of the known mutations in the locus. Flies are suppressed only partially for the scored gypsy-induced mutations, and the females show no detectable reduction in fertility, suggesting that the lesion in the su(Hw) ee mutation causes only a subtle alteration in protein function. A third mutation studied that affects the zinc finger region is su(Hw) f. Sequence analysis shows that this mutation is caused by a nucleotide change from G to A at position 2377. This results in a change of a cysteine for a tyrosine residue in the tenth finger of the su(Hw) protein (Fig. 3B). The cysteine residue is involved in coordination with the zinc ion; therefore, the mutation should result in a change in the conformation of the finger that would severely affect its ability to interact with DNA. Nevertheless, the su(Hw) f mutation does not affect female fertility and suppresses the y2 phenotype only partially (Fig. 6), suggesting that the tenth finger does not play an essential role in the interaction between su(Hw) protein and gypsy DNA.
To test whether mutations in the zinc fingers affect the binding of su(Hw) to DNA, we have used the immu-nolocalization of su(Hw) to polytene chromosomes as an assay for the ability of the mutant protein to bind DNA in vivo. When larval salivary glands are prepared from animals of the genotype su(Hw)ES/su(Hw) v [the latter produces no su(Hw) protein; see Fig. 4], no binding to the chromosomes is detected (Fig. 7). Whole-mount preparations of the mutant and wild-type salivary glands indicate that su(Hw) protein is present within the nucleus of both genotypes (data not shown); thus, the lack of binding is not attributable to underproduction or improper localization of the protein. A similar analysis was carried out with the su(Hw) e2 and su(Hw) f mutations, which are the result of alterations in the zinc finger region that result in only weak mutant phenotypes. In agreement with this observation, immunolocalization of su(Hw) protein can be detected on polytene chromosomes from these two strains (Fig. 7). These two mutations show approximately wild-type levels of su(Hw) protein on Western blots (Fig. 4). The intensity of antibody reaction on polytene chromosomes is close to normal in su(Hw) ~2 (Fig. 7), suggesting that the ability of the protein to interact with DNA is not affected severely. On the contrary, the intensity of antibody staining in su(Hw) f polytene chromosomes is lower compared with that of wild type (Fig. 7), suggesting that although the su(Hw) f protein can still bind to DNA, its affinity is reduced. The difference in binding between su(Hw) ee and su(Hw) f correlates with the respective abilities of these mutations to suppress the 312 phenotype, as the su(Hw) f allele reverses  the coloration of the wing and body cuticle to a greater extent than su(Hw) ~2 (Fig. 6).

Deletion of the carboxy-terminal region affects su(Hw) function
Flies carrying the su(Hw) I or su(Hw) ~z mutations produce su(Hw) protein smaller than the expected size (Fig.  4). The precise location of each mutation was determined by cloning and sequencing these alleles. Both contain nonsense mutations resulting in the premature termination of the protein product (Fig. 2). These mutants are able to accumulate protein in significant amounts; su(Hw) I produces higher than normal amounts, and su(Hw) ~z produces about twofold less protein than wild type (Fig. 4). The mutation in su(Hw) I is a G ~ T transversion at nucleotide 3318 (Parkhurst et al. 1988) and results in the loss of the terminal 149 amino acids of the protein (Figs. 2 and 5). su(Hw) ~z is a C ~ T transition at base 3096 and lacks the last 223 amino acids (Figs. 2 and 5). These two mutations are female fertile but differ in their phenotypic effect on y2. The degree of female fer-tility of these and other su(Hw) alleles was quantitated by measuring the number of eggs laid by 100 females over a period of 4 days and comparing this number to the amount of eggs laid by the bx 3*e parental stock. The su(Hw) I mutation has no effect on fertility, as females carrying this mutation lay the same number of eggs as the parental strain. The fertility of su(Hw) ez could not be determined precisely, as this allele is homozygous lethal, probably because of the presence of other mutations in the same chromosome. Nevertheless, females of the genotype su(Hw)eZ/su(Hw) v have clearly reduced fertility with respect to wild type. The difference in the effect of these two mutations on female fertility correlates with their effect on the phenotype of the y2 mutation, su(Hw) l is a mild mutation only capable of weakly suppressing gypsy-induced mutations (Fig. 6), whereas su(Hw)eZ/ su(Hw) v shows a stronger phenotype (data not shown). Deletion of carboxy-terminal su(Hw) sequences in either of these two mutants does not interfere with the binding of the altered protein to polytene chromosomes (data not shown, but identical to su(Hw) Al~176 in Fig. 7).
From the lesion in su(Hw) 1 it is apparent that there are proteins on polytene chromosomes. Salivary glands were dissected from third-instar larvae, carrying the su(Hw) mutations indicated in each panel, and fixed. Samples were then incubated with su(Hw) affinitypurified polyclonal antibodies, followed by rhodamine-conjugated secondary antibody, and detection by epifluorescence. In each panel, the photograph at left was taken under phase contrast; the photograph at right shows the same field under fluorescence.
elements in the protein within the last 149 amino acids that are necessary for complete function of the protein, but these are not vital for critical aspects of su(Hw) function. The most striking structural feature within this region is a charged acidic domain, which is 33% aspartic acid or glutamic acid (Fig. 2). The su(Hw) proteins from D. ananassae and D. virilis lack most of this domain, suggesting that it might not play an essential role in the function of su(Hw). The su(Hw) I mutation is not strong, perhaps indicating that the charged region plays some auxiliary role in the activity of su(Hw). The acidic domain in the amino end of the protein may be able to partially compensate for the loss of the charged region at the carboxyl terminus. An alternative and more likely possibility is that the subtle alteration in su(Hw) activity detected in su(Hw) 1 is attributable to the lack of amino acid residues 794-860, as this region of the protein is highly conserved among the Drosophila species analyzed (Fig. 2). An additional 84 amino acids are lost in su(Hw) ez beyond those deleted in su(Hw) 1. Whereas the amount of detectable protein is somewhat reduced in su(Hw) ~z mutants (Fig. 4), protein levels are not likely to be the cause of the phenotype. In the very weak su(Hw) ~3 allele, there is much less protein than that seen in su(Hw) ez (Fig. 4). Despite greater levels of protein in su(Hw) ez, it has a stronger mutant phenotype than su(Hw) ~3. This would support the idea that a qualitative change in the protein is responsible for the severe phenotype associated with the su(Hw) ~z mutation. Within the extra 84 deleted amino acids between su(Hw) ~z and su(Hw) 1 is an extended amphipathic c~-helical region that shows homol- ogy to the helix 2-coiled-coil domain of bHLH-Zip proteins (Fig. 3A), suggesting that the phenotype of the su-(Hw) ~z allele is derived from a loss of this domain. To discriminate between these two possibilities, we have analyzed the effect of site-directed deletions and single amino acid substitutions in the extended leucine zipper region on the ability of the su(Hw) protein to reverse the mutant phenotype of y2.

Deletion of the amino-terminal acidic domain does not affect the mutagenic effect of su(Hw)
To further study the domains of the su(Hw) protein required for its function, a deletion of a small region of the su(Hw) gene was generated and tested in vivo. su(Hw) aI~176 is a deletion of 144 bp from bases 1074 to 1217 resulting in an in-frame deletion of the 48-aminoacid region defined as the amino-terminal acidic domain shown in Fig. 2. A similar acidic region is present in other transcriptional regulators and has been found to be essential to the transcriptional activation function in the proteins studied (Ma and Ptashne 1987;Hope et al. 1988;Berger et al. 1990). The su(Hw) a~~176 construct was introduced into flies by P element-mediated germ-line transformation. Four independently transformed lines were recovered and tested for ability to complement the su-(Hw) v amorphic allele. Surprisingly, all four lines were capable of rescuing the phenotypic suppression of gypsyinduced mutations associated with the su(Hw) v allele (Fig. 6). The su(Hw) mutant genotype of the flies from two of these lines was confirmed by subjecting the transformed flies to Westem blot analysis. Only the protein encoded by the su(Hw) a~~176 construct is present at wildtype levels, as determined by its slightly smaller size (Fig. 4). This mutant protein binds to polytene chromosomes with the same affinity as wild type (Fig. 7). Nevertheless, in spite of their normal behavior in the phenotypic suppression assay, females carrying the su(Hw) A~~176 mutation lay only 31% of the eggs deposited by the parental strain. We conclude that the amino-terminal acidic domain of su(Hw) is not essential to mediate the mutagenic effect of gypsy, but it may play some role during oogenesis. Perhaps the charged region in the carboxyl terminus of the protein is functionally redundant with the major acidic domain, explaining why deletion of either domain alone results in no effect or only a subtle phenotype.

Deletion of both acidic domains affects su(Hw) function
To examine the question of the role of the acidic domains in the function of the su(Hw) protein we have constructed in vitro a su(Hw) gene encoding a protein lacking both charged regions [i.e., a protein containing the deletions of both su(Hw) 1 and su(Hw)al~176 This altered gene was designated su(Hw) N~ and was inserted into the pCaSper P-element transformation vector. The resulting plasmid was then injected into preblastoderm embryos. Transformants homozygous for su(Hw) v and the transformed su(Hl4r) NOAD gene express higher than wild-type levels of a protein of the expected size (Fig. 4), and this protein accumulates on polytene chromosomes at normal levels (Fig. 7). The transformed strains were then examined for their phenotypic effect on female fertility and the expression of the y2 gene. These flies are weakly fertile (16% of the parental strain) and show a wild-type yellow phenotype (Fig. 6), indicating that the altered su(Hw) protein, lacking the amino-terminal acidic domain and the last 150 amino acid residues in the carboxy-terminal end of the protein, is able to suppress the gypsy-induced phenotype of y~. Therefore, simultaneous deletion of these two regions of su(Hw) results in a protein that is not functional in mediating gypsy-induced phenotypes. The lack of functionality in the su(Hw) N~ protein might be attributable to the simultaneous deletion of both acidic domains or to deletion of sequences adjacent to the leucine zipper domain compounded with alterations in other parts of the protein.

The Ieucine zipper region of su(Hw) is required for the mutagenic effect of gypsy
To show that the increased severity of the su(Hw) e7 allele with respect to su(Hw) I is derived from a loss of the extended leucine zipper domain, we have generated a site-directed deletion of part of this motif. A deletion named su(Hw) a283 was made in vitro, removing 57 bp that encodes 19 amino acids (base pairs 3213--3269) in the region identified as a possible leucine zipper domain in the carboxy-terminal third of the protein (Fig. 2). The deletion was generated by use of an oligonucleotide that includes 15 bases on either side of the deleted sequence. This construct, containing the su(Hw) a283 gene, was then introduced into flies by P element-mediated transformation. Ten independently transformed lines of the su(Hw) a283 construct were recovered. Flies transformed with su(Hw) a283 and homozygous for the su(Hw) v null allele accumulate normal amounts of su(Hw) protein that is able to bind to polytene chromosomes with the same affinity as wild type (Fig. 7). Nevertheless, these flies are weakly fertile, laying only 2% of the number of eggs deposited by the parental strain, and reverse the y2 phenotype completely (Fig. 6). Only the leucine zipper region is disrupted by the 19-amino-acid deletion in su(Hw) a283, yet the effect is similar in severity to the null phenotype of su(Hw) v. These results suggest that the putative extended leucine zipper domain is a region of functional importance within the su(Hw) protein.
To further establish that the mutant phenotype of the su(Hw) a2s3 protein is attributable to a lack in the functionality of the leucine zipper domain, we analyzed the effect of specific point mutations affecting this region. Several amino acids are conserved in the same relative positions among su(Hw) and bHLH-Zip proteins, interspersed with less conserved sequences (Fig. 3A). It is expected that ff the sequence preservation is attributable to a maintenance in the function of the putative leucine zipper motif, changes that affect conserved amino acids would have an effect on su(Hw) function, whereas mu-tations in other residues would not have a phenotypic effect. To test this hypothesis we made two different mutations in vitro affecting one conserved and one nonconserved amino acid, respectively. The first mutation is a change in nucleotides 3256-3258 from CTT to AAA. This results in a change in leucine at position 775 (marked with an asterisk in Fig. 3A) to lysine. This construct was named su(Hw) L7zSK and was introduced into the Drosophila germ line by P element-mediated transformation. Flies homozygous for su(Hw)v and any one of four different su(Hw) rzzSK transformants accumulate normal levels of the su(Hw) r7751~ protein {data not shown}. These flies show the same level of fertility as the parental strain and contain normal levels of su(Hw) protein but strongly suppress the y2 phenotype in a manner indistinguishable from su(Hw) a283 [data not shown; the cuticle phenotype is identical to that of su(Hw) a283 in Fig. 6]. The second mutation analyzed consists of an alteration in nucleotide 3226 from a G to an A. This changes the aspartate residue at position 765 to asparagine (marked with an asterisk in Fig. 3A). This construct was named su(Hw) Dz65N and introduced into the germ line as described above. Transformants homozygous for the su(Hw) v mutation and carrying this insertion are unable to suppress the y2 phenotype (data not shown; the phenotype is identical to that of y2 in Fig. 6) and therefore show wild-type su(Hw) function, indicating that the change from aspartate to asparagine does not affect the functionality of su(Hw). These results suggest that the region of su(Hw) affected by the su(Hw) a283 and su(Hw) rzzsz': mutations is structurally and functionally equivalent in mediating protein interactions to the leucine zipper motif and that this region plays a crucial role in su(Hw) function.

Discussion
The repressive effect of the su(Hw) protein on enhancer function shows an interesting directionality: Only enhancers located distally from the promoter with respect to the position of the su(Hw)-binding region are affected by the presence of this protein (Corces and Geyer 1991;Jack et al. 1991;Geyer and Corces 1992}. This directional effect offers some clues as to the mechanism by which su(Hw) represses enhancer action, suggesting that su(Hw) acts either by interfering with DNA looping that allows transcription factors bound to enhancers to interact with the transcription complex, by interfering with the tracking process of these factors towards the promoter, or by establishing chromatin domains of independent gene activity that insulate DNA sequences within a domain from neighboring regions {Geyer Roseman et al. 1993}. Several structural domains of the su(Hw) protein seem to have important roles in eliciting gypsy-induced mutant phenotypes and during oogenesis. In general, there is a direct correlation between the degree of female sterility of a particular su(Hw) allele and its ability to suppress gypsy-induced mutations. The zinc fingers are necessary for DNA binding, suggesting that the interaction of su(Hw) with gypsy DNA is also a prerequisite for its effect on upstream enhancers. It is interesting to note that the three mutations analyzed affecting zinc finger structure are clustered in a specific subset of zinc fingers, suggesting that not all 12 fingers of the su(Hw) protein are involved in DNA sequence recognition. This is supported by the varying degree of sequence conservation of the different fingers among the su(Hw) protein of three different Drosophila species; fingers 6, 7, 8, and 9 show 100% identity or similarity in all three species examined, whereas the rest of the fingers contain several nonconserved amino acids. This relative degree of conservation correlates with the importance of the various fingers in DNA binding as judged by the nature of the zinc fingers affected by random mutagenesis of the su(Hw) gene.
A second structural domain identified as functionally important in this study is composed of the two acidic regions located in the amino-and carboxy-terminal ends of the su(Hw) protein. Several results suggest that these domains might act in conjunction and that they are important for su(Hw) function. Deletion of the amino-terminal acidic domain from the su(Hw) protein of D. melanogaster has no major consequence on the mutagenic effect of this protein. In addition, analysis of the sequence of the su(Hw) protein in various Drosophila species indicates that the carboxy-terminal acidic domain is missing in D. ananassae and is shortened considerably in D. virilis; the weak effects observed in the su(Hw) I mutation, which is missing this region of the protein, are probably attributable to additional sequences that affect the integrity of the leucine zipper region. These two lines of evidence suggest a nonessential role for the acidic domains of the su(Hw) protein. Nevertheless, deletion of both regions simultaneously renders the protein nonfunctional, suggesting that the acidic domains have a functional role, but each one can substitute for the other in mediating the yellow mutant phenotype induced by the insertion of the gypsy element. The functional interchangeability of the acidic domains also suggests that they might act synergistically and therefore might be located in close proximity to each other in the mature su(Hw) protein. The normal role of the acidic domains in su(Hw) function has not been determined, but it might be related to the process of transcriptional activation characteristic of transcription factors such as GAL4, GCN4, and VP16 (Gill and Ptashne 1988;Hope et al. 1988;Cress and Triezenberg 1991}. A third functional domain identified in the su(Hw) protein is defined by the su(Hw) ae83 allele. This mutation deletes 19 amino acid residues in a region that shows homology to the leucine zipper motif characteristic of some DNA-binding proteins (Landschulz et al. 1988}. The homology is higher to proteins of the bHLH-Zip family of transcription factors such as Myc, Max, AP-4, and FIP (Murre et al. 1989;Carr and Sharp 1990;Blackwood and Eisenman 1991;Blanar and Putter 1992}. These proteins typically contain a basic region that binds DNA followed by a HLH motif and, immediately adjacent, a coiled-coil region similar to the leucine zipper sequence of C/EBP (Vinson and Garcia 1992}. The Cold Spring Harbor Laboratory Press on July 19, 2018 -Published by genesdev.cshlp.org Downloaded from su(Hw) protein has considerable homology to the second helix and the coiled-coil region of these proteins (Fig. 3), showing perfect conservation of residues in the d position, with the sole exception of the second leucine in the coiled-coil region that is changed to an aspartate. This position is also variable in other members of the family; for example, the leucine has been substituted by a methionine in 1-Myc and a histidine in Max. In addition to the conserved amino acids in Fig. 3, the su(Hw) protein shows a high degree of homology to other members of the family in other residues of the bHLH-Zip motif. For example, helix 2 contains conserved hydrophobic amino acids in the e, g, and a positions that are located on four contiguous spokes of an a-helical wheel projection, creating a side of the helix that is highly hydrophobic and might be important for dimerization (Vinson and Garcia 1992). In addition, the e and g positions of the coiled-coil region contain conserved hydrophilic amino acids that have been implicated in regulating the specificity of dimerization in basic leucine zipper (bZIP) proteins by interhelical interactions (Cohen and Parry 1990;O'Shea et al. 1991). The functionality of the s u(Hw) leucine zipper region is not only supported by the high degree of homology to members of the bHLH-Zip family but also by results of the mutational analysis carried out in this region of the protein. In particular, substitution of a conserved leucine for a lysine in the last d position renders the su(Hw) protein nonfunctional, whereas a mutation affecting an aspartate residue in a nonconserved a position of the coiled-coil region has no effect on su(Hw) function (Fig. 3). These results suggest that the essential role of this region of su(Hw) in eliciting a yellow phenotype is attributable to its function as a leucine zipper domain. This domain might not be involved in the dimerization of su(Hw), an event that is necessary in bHLH-Zip proteins to bind DNA, as su(Hw) contains zinc fingers that interact with the DNA instead of the basic region of bHLH-Zip proteins. In support of this contention, the su(Hw) protein migrates as a monomer in gel filtration columns (D. Gdula and V. Corces, unpubl.), and mutations affecting the leucine zipper region of su(Hw) do not affect DNA binding. Instead, this region might be involved in interactions with other proteins that cooperate with su(Hw) in the transcription of gypsy and other cellular genes.
The question then arises as to the specific role of this region in mediating gypsy-induced phenotypes. The repressive effect of su(Hw) on enhancer function might involve a direct interaction between the su(Hw) protein and transcription factors present in enhancers of the yellow gene, resulting in the inability of these transcription factors to interact with the promoter. If this is the case, the results discussed above suggest that this interaction is mediated by the acidic and/or leucine zipper domains of the su(Hw) protein, as deletion of either domain results in the reversion of the yellow mutant phenotype.
But as a role for the leucine zipper region in mediating su(Hw)-induced phenotypes, one has to assume that the affected transcription factors also contain leucine zipper motifs. Although this could be possible for yellow and the few other Drosophila genes affected by gypsy insertions, it could not explain that the su(Hw)-binding region protects white expression from chromosomal position effects, independent of the chromosomal location of the white gene (Roseman et al. 1993). This lack of specificity suggests that the leucine zipper region might interact with a second protein that in turn mediates the negative effect of su(Hw) on enhancer function. An alternative explanation for the repressive effect of su(Hw) on gene expression could be that the binding of su (Hw) to gypsy DNA causes directional changes in chromatin structure by establishing boundaries between higher order domains of gene activity (Geyer and Corces 1992;Roseman et al. 1993). The need for acidic and leucine zipper domains for this function is not clear, but one can postulate that these regions of su(Hw) interact with other chromosomal proteins necessary for the formation of chromatin domains.

Isolation and enzymology of nucleic acids
Isolation of plasmid DNA, construction and screening of ?t libraries, labeling of DNA, and enzymology of nucleic acids were carried out by standard procedures (Maniatis et al. 1989). Genomic DNA from Drosophila adults was prepared as described by Parkhurst and Cortes (1985). Total RNA for Northern analysis was isolated by homogenization in 10 mM Tris-HC1 (pH 7.4), 0.1 M NaC1, 1 mM EDTA, and 0.5% SDS, followed by phenol extraction and ethanol precipitation. Poly(A) + RNA was selected by chromatography on oligo{dT)-cellulose. Southern and Northern analyses were done as described by Parkhurst and Corces (1985). DNA sequence analysis was performed by dideoxy chain-termination methodology (Sanger et al. 1977). P element-mediated transformation was carried out as described by Rubin and Spradling {1982), using the white gene as a selectable marker in the CaSpeR vector (Pirrotta et al. 1985).

DNA amplification by PCR and site-specific in vitro mutagenesis
Genomic DNAs were subjected to the PCR to amplify sequences from the su(Hw) region (Saiki et al. 1985;Mullis and Faloona 1987). Three pairs of primers containing restriction sites were used to amplify overlapping fragments covering the entire transcribed region of su(Hw). Amplified DNA was digested and cloned by standard techniques (Maniatis et al. 1989).
Small deletions within the coding region of su(Hw) were directed in vitro by a 30-base oligonucleotide containing the sequences flanking the desired deletion using the Amersham in vitro mutagenesis system based on methods described in Sayers et al. (1988). Both the su(Hw) A~83 and su(Hw) al~176 plasmids were constructed using this approach. Generated deletions were used to replace fragments of CaSpeR 5.2, a P-element construct containing a minimum of sequence necessary for the complete phenotypic rescue of su(Hw) mutants. The su(Hw) al~176 plasmid was generated by replacing a 541-bp Bsu36I fragment (from base 678 to 1219) with the fragment deleting the acidic domain (missing from base 1074 to 1217). The su(Hw) A~83 plasmid was generated by replacing a 1.95-kb XbaI-BamHI fragment (1325-3272) by the 1.6-kb eDNA fragment containing a deletion in the bHLH-Zip region (from nucleotide 3213 to 3269). The su(Hw) Lz7sK mutation was constructed as follows. Two oligonucleotides

Functional domains of su(I-Iw)
were used for mutagenesis by PCR. The first oligonucleotide, GTGCGTGAATTCCTCGTGGACGAAG, contains an EcoRI site 5' to the leucine zipper domain; the second one, GGTG-GATCCACTAGCTATACAATTTTTTACCCACA, contains a BamHI site 3' to the domain and three nucleotide changes that result in a codon change of leucine to lysine. The resulting PCR fragment was digested with EcoRI and BamHI and ligated into the EcoRI-BamHI sites of pBSsuHw, which contains a fulllength su(Hw) cDNA. This plasmid was then digested with BamHI and XbaI, and the 1.6-kb fragment containing the mutation was used to replace the homologous wild-type fragment from plasmid pCaSpeR 5.2. The presence of the mutation was confirmed by DNA sequencing. The su(Hw) D765N mutation was constructed using a similar strategy, but the 3' primer GGTG-GATCCACTAGCATATCAATAAGTTTACCCACATTCTC-TTTGAGCTTTATGTTCTCCTCAGTTAG was used for PCR. This primer contains one nucleotide change with respect to the wild-type DNA that results in a substitution of aspartic acid with asparagine. Both plasmids were injected into preblastoderm embryos as described above.