INTRODUCTION

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Cell fate determinations often depend on the ability to recognize and respond to subtle differences in the concentrations of regulatory proteins. The quantitative nature of the problem is particularly clear in Drosophila melanogaster sex determination, for which a twofold difference in the collective concentration of four X-linked gene products ultimately signals sexual fate. Central to the sex determination mechanism are several proteins of the basic helix-loop-helix (bHLH) family. bHLH proteins play important roles in a variety of cellular process, including cell proliferation, blood and muscle development, and neurogenesis (37, 39). These proteins share a bipartite DNA binding and dimerization motif, which consists of a basic -helix that mediates sequence-specific binding to the consensus E-box sequence CANNTG, and two amphipathic -helices, which are separated by a loop of variable length, controlling protein dimerization. Although DNA binding by homodimers is not uncommon, most bHLH proteins appear to function as heterodimers. Based on their evolutionary, structural, and DNA-binding characteristics, most bHLH proteins can be grouped into two classes (1, 2). Class A bHLH proteins, including the MyoD family, E12/E47, and the achaete-scute-related proteins, favor E-box sites containing a central GC pair (CAGCTG), while class B proteins, including Myc, Max, and the Hairy-related proteins, prefer E boxes with a central CG pair (CACGTG) (5, 6, 16, 40).

In Drosophila, the class A bHLH protein encoded by the X-linked sisterlessB gene (sisB, also called scute or T4) participates in several highly dose-sensitive transcriptional processes. During sex determination, SISB functions as a direct indicator of X-chromosome number and, along with SISA, is largely responsible for the ability of the fly to discriminate between the XY male and XX female signals (13, 20, 42, 50). During dorsal-ventral fate determination, SISB, and its close relatives in the achaete-scute complex, function to interpret the axis-defining gradient of the dorsal morphogen (23). Later, during neurogenesis, the concentration of SISB (as Scute) determines the capacity of cells to form neural precursors (31, 32). During all these processes, SISB interacts with the ubiquitous product of the daughterless (da) gene to form DNA binding heterodimers (B/Da) that activate the appropriate target genes (8, 15, 17).

The target of the X-chromosome sex determination signal is the regulatory switch gene Sex-lethal (Sxl). Sxl lies at the top of the sex determination and dosage compensation hierarchies and ultimately controls all aspects of somatic sexual development (reviewed in reference 14). In precellular female (XX) embryos, the diplo-X dose of the sisA, sisB, sisC, and runt gene products activates the establishment promoter Sxl_Pe, creating a pulse of Sxl mRNA and protein synthesis that initiates the female developmental program (12, 13, 18, 35, 36, 46). In male (XY) embryos, the haplo-X dose of the four X-counting genes generates too few sisterless and runt proteins to activate Sxl_Pe, and male development follows by default.

While sisB and the other X-linked elements are the true determinants of X-chromosome dose, their action on Sxl_Pe requires a variety of other proteins. These include Da, the maternally supplied dimerization partner of SISB, as well as the positively acting hermaphrodite (her) and Stat92E gene products (13, 14, 34, 45, 46). In addition to these maternal activators, several maternally or zygotically expressed negative regulators, including Groucho, Emc, and Deadpan (Dpn), are needed for proper sex-specific regulation (3, 43, 53).

With the exception of sisC, which encodes a ligand for the JAK-STAT pathway (46), all of the sex signal elements encode transcription factors. SISB and Da, as well as the negative regulator Dpn, are bHLH proteins, while SISA, Her, Stat92E, and Runt are members of the basic-leucine zipper, Zn finger, STAT, and Runt domain families, respectively. How does this diverse collection of transcription factors mediate the on-or-off regulation of Sxl_Pe in response to a twofold difference in SIS and Runt concentrations? While all available evidence suggests that the action of these proteins on Sxl_Pe is direct, little is known about the specific sequences that mediate sex signal element binding. Given the central importance of SISB and Da to sex determination and other dose-sensitive processes, a logical place to begin is with the interactions between B/Da and Sxl_Pe.

Because B/Da is a prototypical bHLH molecule known to bind E boxes in vivo and in vitro, a straightforward prediction would be that the distribution of E-box sites at Sxl_Pe would be well correlated with the major enhancer activities. In fact, only 3 of the 13 class A E-box sequences present in the 3.7-kb region upstream of Sxl_Pe map to regions known to be important for promoter function, and only one maps in the minimal 400-bp segment needed for sex-specific expression (21). We have resolved this paradox in the work reported here. We show that the predominant binding sites for B/Da in the critical region of Sxl_Pe are noncanonical and that many are of relatively low affinity. We show these non-E-box sites play an essential role in Sxl activation, thus providing the first direct evidence that non-E-box sites play important roles in the in vivo functions of class A bHLH proteins. Furthermore, our analysis of binding site affinities along with the deletion analysis of Estes et al. (21) suggests that threshold binding to proximal low-affinity B/Da sites may be critical for the sex-specific response of Sxl_Pe. We propose that once Sxl_Pe is active, higher-affinity distal sites amplify the response, producing the high-level Sxl expression needed to initiate female development.

	MATERIALS AND METHODS

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Plasmids. The His₆-Da plasmid was based on pRSET-B and encoded C-terminal amino acids 362 to 710. The glutathione S-transferase (GST)-SISB and His₆-SISB plasmids carried the C-terminal portion of sisB (residues 82 to 345) in pGEX-2TK and pRSET-B, respectively. Most promoter fusions were derived from an Sxl segment extending from 1.45 kb to +44 bp. This region was PCR amplified from a sequenced genomic plasmid clone with 5' primer cgaattcgatATCTCTTTTCGCAGCTTCGTA and 3' primer ggggtaccCAAGATCTCTGAACACAAGTTG, cut with BglII and EcoRI, and inserted into BamHI and EcoRI sites of pGEM-11zf(+) to create pG01 (underlined nucleotides are restriction sites, and lowercase nucleotides are nontemplate bases). Repair-proficient DNA polymerase was used for all PCR experiments. Sxl_Pe-Fluc deletion reporters were made by cloning PCR-amplified Sxl_Pe DNA into SmaI and HindIII cut pGL3-Basic (Promega). Forward primers began at the indicated positions, and the common backward primer extended from 9 to +6 and carried a HindIII site at its 5' end. Construct 94 bp was made using a forward primer containing 15-base extension GAAAGATCTGAATTC abutting nucleotides 95 to 78 and the common backward primer. The 4X-B/Da site reporters have four copies of the following sequences between the XhoI and EcoRI sites of the 95 bp Sxl_Pe-Fluc plasmid: TGCAGCCGGCA, CGCACCTTGCC; and AACATCTGCCT (underlined nucleotides are B/Da sites). Cell culture sisB and da expression plasmids carried the entire coding regions plus a Kozak sequence in pAct5CPPA (27). Point mutant plasmids were derived from pG01 by site-directed oligonucleotide mutagenesis. All mutations were confirmed by sequencing. The changes were as follows: site 1, GCgaCTTGC; site 2, GCtaGCGGG, site 4, TCtCgagG; sites 5 and 6, GatGCTTGCG CAttaTGCCACGTTCatCC (underlined nucleotides are B/Da sites, and lowercase nucleotides are mismatches). For P-element transformation, the 1.5-kb EcoRI to NotI fragments from pG01 and its [1 2], [1 4], [1 5 6], [2 5 6], and [4 5 6] derivatives were cloned into the P-element vector pCaSpeR-AUG-gal with a modified polylinker. Upstream sequences were removed as a 1.1-kb EcoRI to SwaI fragment to create the 390 to +44 bp Sxl_Pe-lacZ transformation vectors. P-element vectors carrying the site 1, 3, 4, and [5 6] mutations were made by cloning DraI to BglII (390 to +44 bp) Sxl_Pe restriction fragments into pCaSpeR-AUG-gal. The mutated sequences were as follows: site 1, GggCCcTGC; site 3, ACgTCgaC; site 4, TtACgTagC; sites 5 and 6, GgAGCTCGC GaAtaTTGCCgACGTcCCA.

Protein expression and purification. To produce GST-SISB protein, BL21(DE3) cells carrying the expression vector were grown in Luria broth at 21°C to an optical density at 600 nm of 0.3 and induced with 0.5 mM IPTG (isopropyl--D-thiogalactopyranoside) for 1 to 3 h. Cell pellets were suspended in a 1/40 culture volume of 20 mM HEPES-0.6 M NaCl-0.5 mM EDTA-1% (vol/vol) NP-40-2 mM dithiothreitol (DTT) (pH 7.9) and were sonicated. After a 10-min centrifugation at 10,000 × g, the supernatant was diluted with 1 volume of 20 mM HEPES (pH 7.9), and GST-SISB was purified to homogeneity using glutathione-agarose beads. For His₆-tagged proteins, similar procedures were followed except that the lysis buffer contained 1.0 M NaCl and no EDTA or DTT, and a Ni²⁺-nitrilotriacetic acid affinity resin was used.

DNase I footprinting and electrophoretic mobility shifts. Binding reaction mixtures (20 µl) contained 15 mM HEPES, 200 mM KCl, 1 mM EDTA, 5 mM DTT, 7.5% (vol/vol) glycerol, 0.1% (vol/vol) NP-40, 1 µg of poly(dI)-poly(dC), 5 µg of bovine serum albumin (BSA) (pH 7.9), and the indicated amounts of premixed SISB and Da proteins. One B/Da unit equaled 0.3 pmol each of GST-SISB (15 ng) and His₆-Da (12 ng) proteins. In some experiments with the proximal promoter, His₆-SISB was used instead of GST-SISB, with indistinguishable results. For DNase I footprinting, probes were made by PCR amplification with one ³²P-end-labeled primer and were gel purified. Between 10⁴ and 10⁵ cpm of probe was incubated with or without B/Da as previously described for electrophoretic mobility shift assays (EMSA). After 30 min at 21°C, 0.05 U of DNase I (Epicentre) was added. Reactions were stopped after 2 min by addition of 80 µl of 0.1 M EDTA-1.0 M NaCl. Samples were phenol-CHCl₃ extracted, ethanol precipitated, and dissolved in 80% formamide-0.01 N NaOH-1 mM EDTA, heated to 90°C for 5 min, and loaded on 6% polyacrylamide-8 M urea gels. MspI-cut ³²P-labeled pBR322 fragments served as size standards. DNAs shown in Fig. 1 extended (from top to bottom) from 749 to 1013, 229 to 373, and 229 to 16. For EMSA, reaction mixtures were preincubated at 21°C for 10 min before addition of ~5× 10⁴ cpm of ³²P-labeled probe. For competition experiments, unlabeled probes were added immediately after labeled probes. After 30 min, samples were electrophoresed on prerun 0.25× Tris-borate-EDTA-4% polyacrylamide gels at 21°C. Double-stranded probes for gel shifts were prepared from oligonucleotides carrying four extra 5' bases (GATC) Annealed oligonucleotides were 5' end labeled with polynucleotide kinase, and then the 5' overhangs were filled in using Klenow and unlabeled deoxynucleoside triphosphates. Competitor oligonucleotides were filled in but not labeled. Probes used for the experiments in Table 2 but not shown in Table 1 extended from positions 932 to 911, 691 to 673, 613 to 593, 330 to 313, 321 to 291, 271 to 252, 190 to 169, 177 to 150, 131 to 99, 83 to 69, and 70 to 43.

View larger version (70K): [in this window] [in a new window]   FIG. 1.   B/Da heterodimers bind multiple sites in Sxlpe. DNase I footprinting assays were done at increasing concentrations of GST-tagged SISB and His6-tagged Da proteins. Numbers indicate B/Da footprinting units. One unit equaled 0.3 pmol (15 nM) of each protein. Panels (left to right) illustrate protection from the distal to the proximal region within the 1.4-kb Sxlpe promoter. Site 6 is not shown. Locations of the 11 regions protected by B/Da are summarized in the schematic. Shading represents estimated relative B/Da binding affinity. High-affinity site 7 is black, moderate-affinity sites are gray, low-affinity sites are striped, and putative weak-binding sites are white. Protection of putative sites 2a and 2b was apparent only at 540 nM B/Da (not shown).

Cell culture, P-element transformation, and -galactosidase staining. Cultivation, transfection, and assay of Schneider L2 cells were performed according to procedures described by Han et al. (27). One microgram of DNA was used per plate and included 0.1 µg of Fluc reporter, 0.05 µg each of sisB and/or da expression constructs, 0.1 µg of simian virus 40 (SV40) or cytomegalovirus (CMV)-Renilla luciferase reporters to control for transfection efficiency (pRL-SV40 and pRL-CMV; Promega), and carrier DNA. Luciferase activity was determined using a Dual-Luciferase assay kit (Promega) and a Berthold Lumat LB9501 luminometer. P-element transformants were obtained from w¹¹¹⁸ flies with a heterozygous 2-3 transposase source. Four- to seven-hour-old embryos were stained for -galactosidase activity as described previously (21). Transcription of wild-type 390-bp Sxl_Pe-lacZ fusions occurs during the normal precellular period, as measured by the production of nascent lacZ nuclear transcripts (19); however, there was a lag in the accumulation of active enzyme, necessitating a later assay.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

DNase I footprinting of B/Da binding sites in Sxl_Pe. Deletion analysis has established that a 1.4-kb region upstream of Sxl_Pe is sufficient to drive uniform high-level female-specific expression of Sxl_Pe-lacZ fusion in a B/Da-dependent manner (21). To identify the B/Da binding sites in Sxl_Pe, we expressed soluble GST-tagged SISB and His₆-tagged Da fusion proteins in Escherichia coli and used the purified proteins to systematically footprint the 1.4-kb proximal promoter. In total, we observed 11 protected regions distributed in two clusters. Regions 1 to 6 were located in the proximal 390 bp while regions 7 to 11 mapped between 0.8 and 1.1 kb (Fig. 1). Protected regions 3, 7, and 8 were centered on three type A E boxes, but the sequences in the other eight protected areas lack even the minimal CANNTG E-box consensus (Fig. 2), suggesting that B/Da can bind to non-E-box sites at Sxl_Pe.

View larger version (50K): [in this window] [in a new window]   FIG. 2.   Sequence of proximal 1.4-kb Sxlpe. The DNA sequence from 1400 to +1 is shown. Sequence blocks that are identical in D. melanogaster and D. subobscura are shown in white in black boxes. B/Da sites 1 to 9 and 11 are identified by name and number with a shaded block marking each core binding site. The sequence of D. melanogaster B/Da site 9 is shown above the corresponding D. subobscura sequence. Putative sites 2a, 2b, 5a, and 10 are identified by number. Shaded blocks mark the closest matches to the B/Da consensus. The locations of the tandem Dpn binding sites (28), putative STAT sites (34, 46), and TATA box are marked. Schematic is like that in Fig. 1. The numbering scheme differs from those previously published, as in vitro expression and evolutionary analysis place the +1 position 6 bp downstream of that proposed by Keyes et al. (35).

CA(G/C)CTTG and CA(G/C)CCG are B/Da binding core sites. To identify the binding core sequences in the B/Da protected regions, we performed gel mobility shift assays using overlapping oligonucleotide probes from each protected segment. We found that probes from windows 1, 4, and 11 carrying the common sequence CACCTTG were efficiently bound by B/Da in gel mobility shift experiments but that mutations within the common sequence prevented B/Da binding (Fig. 3; Table 1). Windows 5 and 6 carry a common sequence, CAGCTTG, that has previously been suggested as a possible B/Da binding site (28). We found that wild-type, but not mutant, region 5 and 6 oligonucleotides were bound by B/Da in gel mobility shift assays (Fig. 3; Table 1). Taken together, the footprinting and gel mobility shift data suggest that B/Da can bind to a 7-bp core sequence, CA(G/C)CTTG, which is related to the CA(G/C)CTG E box, by the insertion of an internal T residue.

View larger version (67K): [in this window] [in a new window]   FIG. 3.   EMSA of B/Da DNA binding. (A) B/Da binding to Sxlpe oligonucleotides. B/Da samples (0.1 U) were incubated with 32P-labeled oligonucleotides containing a CAATTG E box or sites 7, 2, 3, 8, 6, 4, and 6m1 (Table 1), and the protein-DNA complexes were resolved on a gel. (B) Binding competition experiments. Complexes were formed between B/Da (0.1 U) and 32P-labeled site 7 and challenged with 10-, 30-, and 100-fold molar excesses of the indicated unlabeled competitor oligonucleotides (Table 1). The site 7 oligonucleotide sequence was GATCAAGGCAGCTGCTATTGGATC.

Relative affinities of consensus and nonconsensus B/Da binding sites. To test further the relative affinity of the B/Da sites in Sxl_Pe, we performed binding competition experiments using the gel mobility shift assay. The B/Da heterodimer was incubated with a radiolabeled probe carrying the symmetrical site 7 E box CAGCTG, and increasing concentrations of unlabeled oligonucleotides were added to compete for binding. We found that B/Da could be competed off the consensus site by sites 2, 3, 4, and 7, but not by a mutant site 2 sequence (Fig. 3B and data not shown). The site 7 E box was the strongest binding site, but the nonconsensus site 2 and E-box site 3 oligonucleotides were also reasonably effective competitors. Site 4 CACCTTG was least effective in competing for B/Da binding, consistent with the relative affinities observed in DNase I footprinting experiments (Fig. 1 and data not shown). Based on the relative binding affinities observed in the sum of our footprinting and gel mobility shift experiments, we classify the B/Da sites as being of relatively high (site 7), moderate (sites 2, 3, and 8), or low (sites 1, 4, 5, 6, 9, and 11) DNA binding affinity.

Site distribution and evolutionary conservation of Sxl_Pe structure. The deletion analysis of Estes et al. (21) suggested that two subsegments within the 1.4-kb promoter account for most Sxl_Pe enhancer activity. An upstream segment (1.4 to 0.8 kb) contributes to the strength of the promoter but is not essential for sex specificity. A proximal segment, including the start site and 390 upstream base pairs, drives a low-level, nonuniform female-specific expression. The sequence conservation between Sxl_Pe in D. melanogaster and Drosophila subobscura (44) correlates well with the functional analysis (Fig. 2). There is extensive sequence identity in the proximal region, with more limited matches in the distal segment, and no detectable similarity in the similarly sized central spacer segment. Within the proximal 390 bp, the sequences of all six B/Da binding sites are perfectly conserved. In the distal region, E-box sites 7 and 8 are conserved. Interestingly, while the sequence of site 9 is not conserved, another low-affinity B/Da site, CAGCTTG, is present in the equivalent position in D. subobscura (Fig. 2).

Nonconsensus sites can support B/Da-activated transcription in cultured cells. To determine whether the binding sites we identified in vitro can be recognized in vivo, we asked if multimerized sites could support B/Da-mediated transcription in Drosophila Schneider L2 cells. Full-length SISB and Da were expressed under control of the Actin5C promoter and the expression plasmids were introduced into cells along with firefly luciferase (Fluc) reporter plasmids carrying four tandem copies of various B/Da binding sites fused to an otherwise inactive promoter. We found that four copies of the noncanonical CACCTTG or CAGCCG core supported levels of B/Da-activated transcription indistinguishable from those produced by four copies of the canonical site 3 E-box CATCTG (Fig. 4, bottom). It has been shown previously that multimerized fragments containing the sequence CAGCTTG can support B/Da-activated transcription in Kc cells (28), suggesting that this noncanonical core sequence is also recognized by B/Da in vivo.

View larger version (30K): [in this window] [in a new window]   FIG. 4.   SISB and Da can activate transcription through noncanonical sites in SL2 cell culture. (Top) Activity of a Sxlpe deletion series. Schematics show the structures of the Sxlpe-firefly luciferase plasmids. Only B/Da binding sites in the proximal promoter are shown. The Sxlpe-Fluc plasmids were introduced with full-length sisB and da expression vectors and a CMV-Renilla luciferase (Rluc) plasmid to normalize for transfection efficiency. Normalized F-luc activities are shown in comparison to the 100% active 3.7 kb Sxlpe fusion. Data are the averages of duplicates differing by 10%. (Bottom) Activity of multimerized B/Da binding sites. Four copies each (top to bottom) of the site 2, sites 1, 4, and 11, and site 3 E-box core sequences were joined to the 95 bp Sxlpe-Fluc plasmid. The 4 × B/Da site-Fluc plasmids were cotransfected with sisB and da expression vectors and an SV40-Rluc control. Data are the average ratios of experimental Fluc to control Rluc activities. Neither sisB nor da expression vectors alone significantly stimulated Sxlpe or the multimerized site constructs (not shown).

Mutational analysis of B/Da sites in P-element transgenes. To determine if the sex-specific response of Sxl_Pe to X-chromosome dose in flies depends on the B/Da sites we identified in vitro, we engineered a variety of inactivating point mutations in the proximal consensus and nonconsensus B/Da binding sites and analyzed their effects in P-element-transformed embryos. We examined the effect of the mutations in the context of the minimal 390 bp promoter because such constructs are expressed sex specifically and are extremely sensitive to changes in the dose of the X-counting elements (21). Accordingly, wild-type and mutant Sxl_Pe promoters were inserted into the P-element transformation vector pCaSpeR-AUG-gal and injected into flies to create mutant 390 bp Sxl_Pe-lacZ transgenes for in vivo analysis.

View larger version (119K): [in this window] [in a new window]   FIG. 5.   Noncanonical B/Da sites are needed for Sxlpe expression in P-element-transformed embryos. (A) Female and male embryos carrying wild-type 390 bp Sxlpe-lacZ fusions. Representative 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal)-stained XX embryos from strongly, moderately, and weakly expressing transformant lines are shown. XY embryos do not express Sxlpe. (B) Wild-type (wt) and mutant Sxlpe-lacZ transgenes stained for -galactosidase activity. Numbers refer to the B/Da site mutations carried by the transgenes. None of the [1 5 6], [2 5 6], [4 5 6], [1 2], or [1 4] lines expressed detectable -galactosidase activity. Other mutant and wild-type lines exhibited a range of expression levels, but all were active. Representatives of moderate strength lines are shown.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Successful dissection of a transcriptional response requires a detailed analysis of the cis elements at promoter targets. A weakness of the Drosophila sex determination system is that the analysis of the Sxl establishment promoter has lagged behind the discovery of its regulators. The identities of most of the important proteins are known, yet with few exceptions, their binding sites at Sxl_Pe are not. In this study, we present evidence that the bHLH heterodimer formed by the sex signal elements SISB and Da activates transcription of Sxl by binding to numerous noncanonical binding sites in Sxl_Pe. Using P-element transformation, we have shown that the noncanonical B/Da sites are required for the activation of Sxl_Pe in embryos. These results suggest that Sxl_Pe utilizes cis elements with different locations and binding affinities to sense the twofold male-female difference in collective SIS protein concentration.

Noncanonical bHLH binding sites. As measured by footprinting and gel mobility shift assays, the B/Da protein binds to canonical CA(G/C/T)CTG E boxes as well as to noncanonical sites with 6-bp CA(G/C)CCG and 7-bp CA(G/C)CTTG core sequences. These noncanonical sequences can be bound directly by B/Da in vivo, as evidenced by their ability to drive B/Da-dependent transcription from multimerized sites and from truncated versions of Sxl_Pe in cell culture assays. While examples have been found of bHLH proteins that prefer non-E-box sites, such as the Hairy-type repressors (40, 51) and the class C bHLH-PAS proteins (47), the overwhelming majority of bHLH molecules are thought to bind with a strong, if not exclusive, preference for E-box sequences (37, 39). Although so far there have been no other reports of class A proteins binding to non-E-box sites in vivo, there is evidence that noncanonical sequences may be important target sites for the class B Myc-Max heterodimer (24, 25). While the highest-affinity Myc-Max core site is CACGTG, random site selection experiments have demonstrated that the protein also binds efficiently to a number of noncanonical sequences, including CAYGCG, CACGAG, and CACGTTG (4). The importance of this relaxed DNA binding specificity was highlighted by the surprising finding that non-E-box sequences accounted for the majority of genomic Myc-Max sites in immunopurified chromatin (24, 25). Interestingly, if one accounts for the class B preference for CG in the central positions, the 6-bp CANNCG and 7-bp CANNTTG sequences bound by Myc and Max are identical to those we identified as the predominant B/Da sites at Sxl_Pe. This striking similarity in noncanonical DNA sequence recognition by long-diverged representatives of the major bHLH classes (2) suggests that many, if not all, subgroups of bHLH proteins will be found to regulate gene expression through noncanonical sequence elements.

Distribution of B/Da sites at Sxl_Peimplications for the X-chromosome counting mechanism. A critical question for sex determination is as follows: how can Sxl_Pe sense the twofold difference in male and female SIS and Runt concentrations and translate that into a strong all-or-nothing response? At some level, Sxl_Pe expression must be related to sex-specific differences in binding site occupancy. This is true whether dose sensitivity arises from cooperative DNA binding, competition with negative regulators, or from the sum of multiple independent interactions between the sex signal elements and the transcription machinery (10).

View larger version (29K): [in this window] [in a new window]   FIG. 6.   A model for Sxlpe function. (A) Distribution of identified and predicted B/Da binding sites from 3.7 kb to +1 in Sxlpe. Note the distal to proximal gradation in relative binding affinities. Predicted high-affinity sites all contain a canonical CAGCTG E box. Of the predicted moderate-affinity sites, the most distal are CATCTG and the third CAGCCG. (B) "Reservoir" model for Sxlpe. Sxlpe is activated in XX embryos as a result of the SIS-directed assembly of a proximal enhancer complex at the XCR. B/Da and other proteins bound to upstream sites function synergistically to augment Sxl transcription when the XCR complex is formed. Sxlpe is left inactive in XY embryos as haplo-X SIS and Runt concentrations are insufficient to drive assembly of the XCR complex. Occupied distal sites cannot stimulate transcription in the absence of a proximal complex. The analogy is to a filled reservoir awaiting release. In XY embryos, the reservoir remains full. In XX embryos, the opening of the proximal promoter causes a flood of Sxl expression. (C) Model for snail regulation in response to dorsal morphogen gradient (30). Dl binds to one high-affinity and nine low-affinity sites in the distal PLA and VA elements. These sites are occupied only in regions of high Dl concentration. The proximal AE contains sites for Twist and other proteins. High-level snail expression requires synergism between the dose sensitivity and augmentation elements.

Structure and function at the Sxl establishment and snail promoters. The model for Sxl_Pe shares important similarities with that proposed for snail, a target of the dorsal (dl) morphogen (30) (Fig. 6B and C). First, the dose-sensitive control elements are composed of low-affinity sites. For snail, these consist of nine low-affinity D1 binding sites that promote transcription only when D1 concentration is high (Fig. 6C). Second, the dose-sensing regions are not sufficient to promote high-level transcription: separate augmentation elements are required. In the snail promoter, the augmentation element contains two Twist binding sites and functions to increase transcription and refine the boundaries of snail expression (30). Third, the proximal element is necessary for detectable levels of transcription, indicating that the upstream sites function, in a formal genetic sense, through the proximal ones. Thus, both genes may exploit binding site affinity to set the activation threshold and use synergism between distal and proximal regulatory elements to achieve full expression.

Sex specificity and role of negative regulators. Our model provides an explanation for how the initial decision to turn on may be amplified to generate a strong response, but it does not answer the question of how the critical on or off decision is actually made. While the affinity of the B/Da sites in the XCR may well play an important role, we envision that activators, such as SISA and Runt, as well as maternally provided Stat92E and Her, also contribute to the assembly and function of the XCR complex, as all of the proteins have been shown by genetic means to work through the proximal promoter (21, 34, 36; H. Lu, unpublished data). Intriguingly, no specific Runt binding sites have been identified at Sxl_Pe (36; D. Yang, unpublished data), suggesting that Runt may also bind to sequences of low intrinsic affinity. By analogy, we predict that several low-affinity binding sites for SISA will be present in the XCR and that cooperative binding between various signal proteins will play an important role in determining site occupancy. Direct precedent for these types of protein-protein interactions comes from the rhomboid promoter, where cooperative interactions between ASC/Da and Dl proteins facilitate Dl binding (29, 33, 48). The idea that a large protein complex forms at the XCR is consistent with the number of proteins involved as well as with the extent and relative order of the conserved sequence blocks in the promoter (Fig. 2). Interestingly, the ordering may even extend to the different subunits of heterodimers, as the asymmetric B/Da sites all have their noncanonical half-sites directed proximally. Such local directionality can be important for cooperative binding (7) and is a characteristic of highly structured enhancer complexes, such as those formed at the beta interferon and T-cell receptor  promoters (22, 26, 49).