Ultrabithorax

Targets of Activity

There are 30 common binding sites for the proteins encoded by the Ultrabithorax (Ubx) and abdominal-A (abd-A) genes within a negatively regulated target, the P2 promoter of the Antennapedia gene. In certain neuronal cells, UBX and ABD-A proteins appear to repress by competing for common binding sites with another homeodomain protein, which may be ANTP acting to induce P2 transcription in an autoregulatory manner. In sets of cells that contribute to the tracheal system, UBX and ABD-A repress by counteracting the function of a factor acting at independent sites. The latter mechanism of repression requires only that multiple homeodomain binding sequences be present and is not dependent on any particular binding site (Appel, 1993).

Muscle diversification in the Drosophila embryo is manifest in a stereotyped array of myofibers that exhibit distinct segment-specific patterns. The homeotic genes of the Bithorax complex control the identities of abdominal somatic muscles and their precursors by functioning directly in cells of the mesoderm. Ultrabithorax and abdominal-A have equivalent functions in promoting the formation of particular muscle precursors in the anterior abdominal segments (Michelson, 1994).

In the abdominal cuticle Scr is repressed by UBX, and thereby prevented from inducing prothoracic structures (Andrew, 1994).

Scr, Antp, Ubx and Abd-B repress Dfd both transcriptionally and at the phenotypic level, if their products are available in sufficient amounts (Gonzalez-Reyes, 1992).

unplugged expression occurs in portions of the tracheal system that penetrate the CNS, including the cerebral branch specific to T1. To test the possibility that genes in the BX-C play a role in regulating unpg expression, the distribution of unpg transcript was examined in Ultrabithorax and abdominal-A mutants, and in Ubx, abd-A, Abd-B triple mutants. In Ubx mutant embryos additional unpg expression is observed in cells surrounding the tracheal pits of T2 and T3, indicative of a role for Ubx in repression of unpg in the posterior segments and consistent with homeotic transformation in Ubx mutants of posterior T2 and T3 toward a T1 identity. In abd-A mutants embryos extra patches of unpg-expressing cells around the tracheal pits extend posteriorly to A7, indicating a role for Abd-A in the repression of unpg expression in the abdominal segments. The homeotic gene abd-B probably contributes to the repression of unpg expression in A7, since slightly elevated expression in A7 is observed in the triple mutants (Chiang, 1995b).

Segmental modulation of 18 wheeler expression later in the tracheal system is dependent upon the function of the homeotic genes of the bithorax complex. In Ultrabithorax mutant embryos, a larger, more intense patch of 18w extends to cells surrounding the tracheal pits in T2 and T3, indicative of a role for Ultrabithorax protein in repression of 18w in T2 and T3 tracheal pits and consistent with a homeotic transformation in Ubx mutants of posterior T2 and T3 towards a T1 identity. Expanded 18w similarly extends posteriorly to A6 in flies lacking both Ubx and abd-A functions and to A7 in a triple mutant also deficient in Abd-B, indicating a role for abd-A and Abd-B in the repression of 18w in the posterior abdominal segment. In the triple mutant, loss of intense posterior spiracle staining suggests that Abd-B may also be required in A8 for positive regulation of 18w. It is not known whether BX-C regulation of 18w is direct or indirect (Chiang, 1995a).

In the Drosophila midgut transcription of the dpp gene is activated by UBX and repressed by Abdominal-A. A 45 bp fragment of DNA from within the dpp midgut enhancer correctly responds to both UBX and ABD-A in a largely tissue-specific manner, thus representing the smallest in vivo homeotic response element identified to date (Manek, 1994).

A 674 bp enhancer of dpp controls its expression in the second constriction domain of the visceral mesoderm (parasegment 7). Normal enhancer function requires positive regulation by Ubx and negative regulation by abd-A. This enhancer contains UBX- and ABD-A-binding sites defined in vitro. By generating complementary alterations of the binding sites and the binding specificity of UBX, Capovilla has shown that UBX directly regulates dpp expression (Capovilla, 1994).

An investigation was carried out into the mechanisms by which Hox genes compete for the control of positional identity. Functional dominance is often observed where different Hox genes are co-expressed, and frequently the more posteriorly expressed Hox gene is the one that prevails, a phenomenon known as posterior prevalence. To investigate functional dominance among Hox genes on a molecular basis, dpp674, a visceral mesoderm-specific enhancer of decapentaplegic was used. In the visceral mesoderm, dpp is expressed in parasegment 7 (PS7), where it is required for the formation of the second midgut constriction. Expression of dpp is positively regulated by Ubx in PS7, and negatively regulated by abd-A in PS8-12. Regulation of dpp by Ubx and abd-A takes place through a 680-bp visceral mesoderm-specific enhancer (dpp674). This enhancer contains Ubx/Abd-A protein binding sites defined by DNase I protection assays. Ubx regulation of dpp674 is direct, as shown in experiments in which expression from a mutated enhancer is reconstituted by compensatory changes in the UBX protein that alter its DNA-binding specificity (Capovilla, 1998 and references).

Posterior prevalence is not adequate to describe the regulation of dpp by Hox genes. Instead, abdominal-A dominates over the more posterior Abdominal-B and the more anterior Ultrabithorax. In the context of the dpp674 enhancer, abd-A functions as a repressor whereas Ubx and Abd-B function as activators. Thus, these results suggest that other cases of Hox competition and functional dominance may also be understood in terms of competition for target gene regulation, in which repression dominates over activation (Capovilla, 1998).

The idea that posterior prevalence is the dominance of repression over activation is supported by the observation that abd-A functions as an activator through the 5' portion of the dpp enhancer and as a repressor through its 3' portion. When these portions are fused together in the full enhancer, repression by abd-A prevails over activation. These findings suggest the possibility that other cases of functional dominance may be explained in terms of Hox proteins functioning as repressors and prevailing over Hox proteins functioning as activators. For example, in accordance with posterior prevalence, repression of Distal-less (Dll) by Ubx prevails over Dll activation by genes of the Antennapedia (Antp) complex. Similarly, apterous (ap) repression by Ubx prevails over Antp activation in the central nervous system. In contrast, and in violation of posterior prevalence, repression of centrosomin (cnn) by Antp dominates activation by Ubx in the visceral mesoderm. In another case, a phosphorylation-defective Antp protein (Antp [1,2,3,4]A ) has novel functions in addition to the wild-type Antp functions. At least some of its novel functions are a consequence of the ability of Antp [1,2,3,4]A to misregulate regulatory targets of other Hox genes. Antp [1,2,3,4]A violates posterior prevalence by repressing empty spiracles (ems) expression, which is normally activated by Abd-B. However it is interesting that the novel function of Antp [1,2,3,4]A as a dpp activator cannot overcome repression by abd-A, hence respecting the phenomenon of posterior prevalence. Thus the attractiveness of this model is that it explains the cases of posterior prevalence in which the posterior gene is the repressor, but it also explains other cases of functional dominance in which posterior prevalence is violated (Capovilla, 1998 and references).

Expression patterns of wingless, teashirt and decapentaplegic are altered in the embryonic midgut of embryos lacking extradenticle. EXD acts with UBX to activate dpp expression in parasegment 7 (PS7), via a minimal visceral mesoderm enhancer, and EXD represses dpp expression anterior to PS7. Even when Ubx is ubiquitously expressed at high levels in exd embryos, Ubx is incapable of activating dpp enhancer expression (Rauskolb, 1994).

UBX directly activates DPP expression in parasegment 7 of the embryonic visceral mesoderm. Other factors are also required, including one that appears to act through homeodomain protein binding sites and may be encoded by extradenticle. The EXD protein binds in a highly co-operative manner to regulatory sequences mediating PS7-specific dpp expression, consistent with a genetic requirement for exd function in normal visceral mesoderm expression of dpp (Sun, 1995).

teashirt is necessary for proper formation of anterior and central midgut structures. ANTP activates tsh in anterior midgut mesoderm. Ubx, abd-A, dpp, and wg are required for proper tsh expression in the central midgut mesoderm. The control of tsh by Ubx and abd-A, and probably also by Antp, is mediated by secreted signaling molecules. By responding to signals as well as localized transcription regulators, the TSH transcription factor is produced in a spatial pattern distinct from any of the homeotic genes (Mathies, 1994).

A teashirt gene enhancer is differentially activated by Hox proteins in epidermis and mesoderm. Sites where ANTP and UBX proteins bind in vitro have been mapped within evolutionarily conserved sequences. Although ANTP and UBX bind to identical sites in vitro, ANTP activates the tsh enhancer only in epidermis while UBX activates the tsh enhancer in both epidermis and in somatic mesoderm. The DNA elements driving tissue-specific transcriptional activation by ANTP and UBX are separable (McCormick, 1995).

odd paired is positively regulated by ANTP and ABD-A at the location of the first and third midgut constructions respectively. Between these domains opa is negatively regulated by UBX and DPP (Cimbara, 1995). scabrous is a target for UBX. Parts of the last intron and exon of the scabrous gene contain five ATTA sequences, the core sequence shared by most homeodomain binding sites. Mutation of Ubx results in the ectopic transcription of sca in the first abdominal segment. Transcript localization in several combinations of deficiencies in the Bithorax complex indicates that sca is downregulated by Abdominal-A and Abdominal-B, and suggests it is a common target of the three genes of BX-C (Graba, 1992).

Ubx is sufficient to repress pdm-1 expression in abdominal segments. The effect of Ubx is not direct, but is through interaction of endoderm with Ubx- expressing mesoderm. dpp is not responsible for this inductive process, as it is with endodermal expression of labial (Affolter, 1993).

beta 3 tubulin expression accompanies the specification and differentiation of the Drosophila mesoderm. An enhancer element in the intron leads to expression in a subdomain restricted along the anterior-posterior axis. This pattern is altered in mutants in Ubx, whereas ectopic Ubx expression leads to activity of the enhancer in the entire visceral mesoderm (Hinz, 1992).

A subtractive hybridization procedure has been used to isolate activated target genes of Ultrabithorax. Homeotic genes can regulate target gene expression at the start of gastrulation, suggesting a previously unknown role for the homeotic genes at this early stage. In abdominal segments, the levels of expression for two target genes increase in response to high levels of Ubx, demonstrating that the normal down-regulation of Ubx in these segments is functional. Finally, the DNA sequence of cDNAs for one of these genes, nervy, predicts a protein that is similar to a human proto-oncogene involved in acute myeloid leukemias (Feinstein, 1995).

An immunopurification method has been used to clone target genes of the Antennapedia protein. centrosomin (cnn) is expressed in the developing visceral mesoderm (VM) of the midgut and the central nervous system (CNS). In the VM, Antp and abdominal-A negatively regulate cnn, while Ultrabithorax shows positive regulation. In the CNS, cnn is regulated positively by Antp and negatively by Ubx and abd-A. Evidence suggests that the expression of the cnn gene in the VM correlates with the morphogenetic function of Ubx in that tissue, i.e., the formation of the second midgut construction (Heuer, 1995).

homothorax expression is regulated by genes of the bithorax complex. Starting at germband extension, and throughout the rest of embryonic development, hth expression is modulated in a segment-specific fashion. Most notable is the repression of hth expression in the ectoderm and VNC of abdominal segments during late stages of embryonic development. The genes of the homeotic complexes are the major regulators of segmental identity. hth expression has therefore been analyzed in embryos deficient for the abd-A gene, or the abd-A and Ubx genes together. In the absence of abd-A activity, hth expression is derepressed in the abdominal ectoderm in cells along the segment boundaries. In the absence of both abd-A and Ubx, the derepression is more prominent; hth is expressed in ectodermal cells throughout the segment. In addition, a uniform level of the Hth protein is observed in all the thoracic and abdominal neuromers of Df(3R)Ubx 109 homozygous embryos (Kurant, 1998).

During Drosophila wing development growth and patterning are mediated by signaling from the dorsoventral (D/V) organizer. In the metathorax, wing development is essentially suppressed by the homeotic selector gene Ubx to mediate development of a pair of tiny balancing organs, the halteres. Expression of Ubx in the haltere D/V boundary down-regulates the haltere's D/V organizer signaling compared to that of the wing D/V boundary. Somatic loss of Ubx from the haltere D/V boundary thus results in the formation of a wing-type D/V organizer in the haltere field. Long-distance signaling from this organizer was analyzed by assaying the ability of a Ubx minus clone induced in the haltere D/V boundary to effect homeotic transformation of capitellum cells (the capitellum is the main body of the haltere) away from the boundary. The clonally restored wing D/V organizer in mosaic halteres not only enhances the homeotic transformation of Ubx minus cells in the capitellum but also causes homeotic transformation of even Ubx plus cells in a genetic background known to induce excessive cell proliferation in the imaginal discs. In addition to demonstrating a non-cell-autonomous role for Ubx during haltere development, these results reveal distinct spatial roles for Ubx during maintenance of cell fate and patterning in the halteres. Ubx modulates the expression of wingless and cut in the haltere D/V boundary and represses vestigial in the capitellum, thereby suggesting a mechanism for the Ubx mediated down-regulation of the D/V organizer activity in the haltere. While the repression of wingless and cut expression is cell-autonomous, that of the quadrant vestigial-lacZ is non cell-autonomous: pouch cells farther away from the D/V boundary show more severe reduction in lacZ expression. Given the fact that quadrant vestigial-lacZ repression is dependent on the formation of the D/V boundary, the non-cell-autonomy in quadrant vestigial-lacZ repression by ectopic Ubx would not be surprising if Ubx function is to negatively regulate D/V signaling. Thus all the results provide strong evidence for the negative regulation of D/V organizer signaling by Ubx during haltere specification. It is likely that during haltere development, repression of wing patterning signals results in the specification of cell shape and volume that are unique to the haltere (Shashidhara, 1999).

Connectin is a cell-surface molecule containing leucine-rich repeats. Connectin can mediate cell-cell adhesion, suggesting a direct link between homeotic gene function and processes of cell-cell recognition. A 4 kb restriction enzyme fragment of Connectin, encompassing the 100 base pair clone of the Connectin promoter immunopurified with anti Ultrabithorax antibody, gives a consistent pattern of expression corresponding to a subset of the total Connectin expression pattern. High levels of expression are detected in a small group of cells in the gnathal and thoracic segments and in a posterior segment. This fragment does not produce CNS expression. The reduced levels of expression of the 4 kb fragment suggest a down regulation by Ubx and the abdominal homeotic genes. In Ubx mutants, expression is derepressed in the abdominal segments A1 and A2. Derepression is more dramatic in a Ubx/abd-A double mutant indicating that both genes repress the 4 kb construct. Antp is required for the high levels of expression found in T2 and T3 of wild type embryos. There is an additional pattern of expression in the visceral mesoderm of the midgut, with a smaller 1 kb construct that is not observed with the 4 kb construct. The visceral mesoderm expression corresponds to the developing second midgut constriction and corresponds to a subset of parasegment 7. This is particularly interesting as Ubx is expressed in PS7 in the visceral mesoderm and Ubx function is required for the formation of the second midgut constriction (Gould, 1992).

The beta3 tubulin gene of Drosophila is expressed in the major mesodermal derivatives during their differentiation. The gene is subject to complex stage- and tissue-specific transcriptional control by upstream as well as downstream regions. Analysis of the vm1 enhancer, which is responsible for tissue-specific expression in the visceral mesoderm and is localized in an intron, reveals a complex modular arrangement of regulatory elements. In vitro and in vivo experiments uncovered two binding sites [termed UBX1 and UBX2, for the product of the homeotic gene Ultrabithorax(Ubx)] that are required for high-level expression in pPS6 and PS7. Further analysis of the vm1 enhancer has revealed that deletion of a specific element, termed element 7 (e7), abolishes transcription of the lacZ reporter gene in all parasegments except pPS6/PS7. Gel-retardation and footprint analysis has identified a binding site for the homeodomain protein Tinman, which is essential for the specification of the dorsal mesoderm, within e7. Simultaneous deletion of two further sequence blocks in the vml enhancer, named elements 3 (e3), and 6 (e6), results in a reduction analogous to that caused by removal of e7. The e6 sequence contains conserved motifs also found in the visceral enhancer of the Ubx gene. It is therefore concluded that these elements act in concert with the Tinman binding site to achieve high expression levels. Thus the vm1 enhancer of the beta3 tubulin gene contains a complex array of elements that are involved in transactivation by a combination of tissue- and position-specific factors, including Tinman and UBX (Kremser, 1999b).

Zygotic expression of modifier of variegation modulo depends on the activity of genes that pattern the embryo along dorsoventral and anteroposterior axes and specify diversified morphogenesis. dorsal and the mesoderm-specific genes twist and snail direct modulo expression in the presumptive mesoderm. The homeotic genes Sex combs reduced and Ultrabithorax positively regulate the gene in the ectoderm of parasegment 2 and abdominal mesoderm (Graba, 1994).

UBX isoforms Ia and IVa accumulate in about 100 discrete chromosomal sites. If not all the sites, then certainly most of them are the same for the two UBX isoforms. These sites are all euchromatic, including both bands and interbands. Good candidates for UBX direct regulation include 18-wheeler, nervy, Antp, and Ubx itself. Binding is not detected at the cytological locations of Dll, tsh, centrosomin, Dwnt-4, and scabrous, all good candidates for genes directly regulated by UBX (Botas, 1996).

During embryogenesis Polycomb is found in all tissues, though in later stages it preferentially accumulates in the CNS. Polycomb and Ubx are involved in a feedback-type regulation: Ultrabithorax, a homeotic target gene of Pc in its own domain of expression, down-regulates Polycomb (Paro, 1993).

Fifty-three DNA fragments that can mediate activation by UBX isoform Ia in this test were recovered after screening 15% of the Drosophila genome. Four of these putative target genes are expressed in patterns that suggest roles in the development of regional specializations within mesoderm derivatives; in three cases these expression patterns depend on Ultrabithorax function. Extrapolation from this pilot study indicates that 85-170 candidate target genes can be identified by screening the entire Drosophila genome with UBX isoform Ia (Mastick, 1995).

Signaling by the Epidermal growth factor receptor (EGFR) plays a critical role in the segmental patterning of the ventral larval cuticle in Drosophila: by expressing either a dominant-negative EGFR molecule or Spitz, an activating ligand of EGFR, it is shown that EGFR signaling specifies the anterior denticles in each segment of the larval abdomen. rhomboid, spitz and argos are expressed in denticle rows 2 and 3, just posterior to denticle row 1 in the engrailed expression "posterior" domain of larval ectoderm. High EGFR signalling activity depend on bithorax complex gene function. In mutants lacking abdominal-A and Ultrabithorax, rhomboid expression is very weak. In these mutants, there is very little expression of argos. These homeotic genes account for the main difference in shape between abdominal and thoracic denticle belts (Szuts, 1997).

Diversification of Drosophila segmental morphologies requires the function of Hox transcription factors. However, little information is available that describes pathways through which Hox activities effect the discrete cellular changes that diversify segmental architecture. Serrate is a Hox gene target. Serrate acts in many segments as a component of such pathways. In the embryonic epidermis, Serrate is required for morphogenesis of normal abdominal denticle belts and maxillary mouth hooks, both Hox-dependent structures. The Hox genes Ultrabithorax and abdominal-A are required to activate an early stripe of Serrate transcription in abdominal segments. In the abdominal epidermis, Serrate promotes denticle diversity by precisely localizing a single cell stripe of rhomboid expression, which generates a source of EGF signal that is not produced in thoracic epidermis. In the head, Deformed is required to activate Serrate transcription in the maxillary segment, a region where Serrate is required for normal mouth hook morphogenesis. However, Serrate does not require rhomboid function in the maxillary segment, suggesting that the Hox-Serrate pathway to segment-specific morphogenesis can be linked to more than one downstream function (Wiellette, 1999).

Ser transcripts in the trunk are first detected at the extended germband stage in ventral patches in the middle of abdominal segments A2-A8 and in offset lateral patches. The ventral regions of thoracic segments do not exhibit Ser expression at this stage. As the germband retracts, the abdominal stripes intensify and develop sharp anterior borders. The first abdominal segment (A1) is unique: Ser expression begins later than in the other abdominal segments and forms a narrower stripe after germband retraction. After germband retraction, Ser transcripts can also be detected in the ventral regions of thoracic segments in broad, faint patches. Embryos mutant in all genes of the Bithorax Complex (BX-C), Ubx, abd-A and Abdominal-B (Abd-B), develop thoracic-type denticles throughout the trunk region. Consistent with this transformation, stage 11 and 12 BX-C mutant embryos have no Ser expression in ventral regions. Ventral Ser expression does begin in BX-C mutants after germband retraction, but the location and level of expression matches that of the thoracic segments. As expected, Ubx mutant embryos show a transformation of abdominal- to thoracic-type Ser expression only in A1; abd-A mutants show Ser transcript stripes in A2-A8 that are similar to the wild-type A1 pattern, and Abd-B mutants display no change in A1-A8 ventral Ser transcription. Thus Ubx function is sufficient to activate some Ser expression in the center of each segment, but abd-A function is required for the earlier, broader pattern of Ser transcription in A2-A8, a transcript pattern that correlates with complete diversification of denticle belts. Embryos lacking all trunk Hox functions express Ser at the margins of the anterior part of each trunk segment and at lower levels in the center of this region, a pattern almost the inverse of that seen in wild type. Transcription of Ser in the posterior-most region of each segment, probably corresponding to the posterior compartment, is completely suppressed. The delimitation of Ser expression to reiterated subsegmental stripes in the embryonic metameres suggests that segment polarity genes also regulate the Ser transcript pattern. ptc mutants lack ventral abdominal Ser transcripts, correlating with the loss of denticle diversity and number in ptc denticle belts. Ser transcription in wingless (wg) mutants appears in broad stripes, while hedgehog (hh) and engrailed (en) mutant embryos exhibit Ser transcription throughout almost the entire ventral epidermis of the abdominal segments. Broadened patterns of Ser transcription in these segment polarity mutants correspond to expanded fields of denticles that lack significant diversity of denticle type (Wiellette, 1999).

A model is presented for the roles of Ser, rho and Hox genes in the generation of denticle belt patterns in the thorax and abdomen. Three Hox genes (Antp, Ubx, and abdA) serve to establish the segmentally specific levels of Ser expression in the third abdominal segment and in the first two thoracic segments respectively. Ser is activated at stage 11 in abdominal parasegments by Ubx and abd-A functions but not in thoracic parasegments where Antp is the principal Hox function. Ubx function is required for the A1-type abdominal expression pattern of Ser, which is narrower and fainter than the pattern in other abdominal segments. This pattern correlates with a narrower, less complex denticle pattern in A1 than in more posterior segments. abd-A function is required for the wider, more abundant Ser stripes in A2-A8. Expression of Ser in the embryonic epidermis results in context-dependent responses, including rho expression, denticle belt patterning and normal development of the mouth hooks. These embryonic roles of Ser are apparently different from its roles in wing margin determination and wing outgrowth. One similarity is the short range over which Ser function is exerted, either at the anterior border of its ventral A2-A8 expression pattern, or at the dorsal/ventral margin of its expression boundary in the wing pouch. The spitz-group gene rho can potentiate Egfr activation via the Spitz (Spi) ligand. Egfr activation is required from late stage 11 to early stage 13 for patterning of the denticle belts, and rho, unlike spi and Egfr, has a spatially and temporally regulated expression pattern. Abdomen-specific rho expression is required for patterning of abdominal denticle rows 1 through 4, probably by allowing secretion of Spitz protein from denticle row 2 and 3 cells, which activates Egfr in neighboring cells. Ser function is required for activation of the abdomen-specific posterior row of rho transcription, expression of which is also dependent on Ubx/abd-A. The evidence presented in this paper suggests that Ser provides a critical intermediate that translates broad Hox and segment polarity domains into narrow stripes of rho expression, which then specify diversification at the single cell level (Wiellette, 1999 and references).

Cross-regulation of Homeotic Complex (Hox) genes by ectopic Hox proteins during the embryonic development of Drosophila was examined using Gal4 directed transcriptional regulation. The expression patterns of the endogenous Hox genes were analyzed to identify cross-regulation while ectopic expression patterns and timing were altered using different Gal4 drivers. Evidence is provided for tissue specific interactions between various Hox genes and the induction of endodermal labial (lab) by ectopically expressed Ultrabithorax outside the visceral mesoderm (VMS). Similarly, activation and repression of Hox genes in the VMS from outside tissues seems to be mediated by decapentaplegic gene activation. Additionally, it has been found that proboscipedia (pb) is activated in the epidermis by ectopically driven Sex combs reduced (Scr) and Deformed (Dfd); however, mesodermal pb expression is repressed by ectopic Scr in this tissue. Mutant analyses demonstrate that Scr and Dfd regulate pb in their normal domains of expression during embryogenesis. Ectopic Ultrabithorax and Abdominal-A repress only lab and Scr in the central nervous system (CNS) in a timing dependent manner; otherwise, overlapping expression in the CNS in tolerated. A summary of Hox gene cross-regulation by ectopically driven Hox proteins is tabulated for embryogenesis (Miller, 2001).

The expression of the lab gene is regulated in a time and tissue specific manner by ectopic Ubx accumulation. Lab accumulation in the CNS and epidermis are differentially affected by ectopic Ubx. lab repression in the CNS only occurs in prd=>Ubx (prdGal4;UASUbx driver=>responder) animals even though both the 31-1=>Ubx and 69B=>Ubx demonstrate expression in this tissue. However, 31-1=>Ubx animals do not show significant accumulation of Ubx in the CNS prior to stage 9 while 69B=>Ubx genotypes do. This suggests that timing may be the critical factor in setting up regulatory interactions between these genes. If timing is the difference between the 31-1=>Ubx and 69B=>Ubx repression of lab in the CNS, then after stage 9, the locus becomes immune to Ubx's negative influence. This could be due to the absence of an important cofactor, the masking of a lab cis-regulatory site used by Ubx, or changes in the signaling environment (Miller, 2001).

Driver expression levels in the nearby VMS may be a factor since the prd=>LacZ confocal images shows significantly more LacZ accumulation there. Expanded endodermal lab induction is demonstrated in all three driver=>Ubx combinations. Normally, endodermal lab expression is activated by Ubx in the VMS through a signaling mechanism involving dpp. However, only the prd=>LacZ combination shows significant VMS accumulation which indicates that the VMS may not be the only tissue contributing to this process. It is possible that the observed 31-1 driver expression in the endoderm could autonomously activate the Ubx/Dpp/Lab cascade and provide the observed expansion of Lab accumulation (Miller, 2001).

Additionally, the sparse VMS accumulation seen using the 69B driver could reflect sufficient ectopic Ubx accumulation to activate the Dpp signal and subsequent lab induction in the endoderm. Consistent with this latter possibility is the fact that the dpp gene demonstrates auto-catalytic regulation in this tissue and could amplify low level stimulation by ectopic Ubx. The activation of Ubx in the VMS by 31-1=>Ubx and 69B=>Ubx, suggests that this signaling pathway is involved. Reduced first midgut constrictions are found in a few 69B=>Ubx and 31-1=>Ubx embryos, likely due to high levels of Dpp protein and subsequent Antp repression. Typically, however there is not enough ectopic Ubx expression in the VMS by any driver=>Ubx combination to repress the first midgut constriction. Despite this, expansion of the Lab endodermal domain is seen in these animals indicating that either the threshold response for lab induction is lower than that for Antp repression or that there is a signaling source other than the VMS for the Ubx generated signal. This signal could originate from the CNS for 31-1=>Ubx or the CNS, somatic mesoderm and epidermis for 69B=>Ubx. Additionally, the amnioserosa normally exhibits dpp expression where 31-1=>LacZ and 69B=>LacZ expression is observed. Gal4 activation of the Ubx responder in the amnioserosa could contribute to dpp activation levels and hence the pool of the diffusible agonist through stage 14 (Miller, 2001).

Only two of the Hox genes tested, Scr and lab, are affected in the CNS by expression of more posterior members of the complexes. Additionally, Scr repression occurs only when the prd=>Hox driver combinations are used; this suggests that timing (early expression) is critical for significant down regulation. Persistent expression is probably also important since significant lab CNS repression is seen in both prd=>Ubx and 69B=>Ubx animals but not in the 31-1=>Ubx genotypes, which exhibit high early expression levels that fade by stage 15. Other than these examples, surprisingly few instances of posterior dominance (transcriptional repression) of Hox genes from the ectopically expressed Hox proteins were observed in the CNS during embryogenesis (Miller, 2001).

Ubx is involved in the complex regulation of vestigial in the haltere disc

In the third thoracic segment of Drosophila, wing development is suppressed by the homeotic selector gene Ultrabithorax (Ubx) in order to mediate haltere development. Ubx represses dorsoventral (DV) signaling to specify haltere fate. The mechanism of Ubx-mediated downregulation of DV signaling has been studied. Wingless (Wg) and Vestigial (Vg) are differentially regulated in wing and haltere discs. In wing discs, although Vg expression in non-DV cells is dependent on the DV boundary function of Wg, Vg maintains its expression by autoregulation. Thus, overexpression of Vg in non-DV cells can bypass the requirement for Wg signaling from the DV boundary. Ubx functions on at least two levels to repress Vestigial expression in non-DV cells of haltere discs. At the DV boundary, it functions downstream of Shaggy/GSK3ß to enhance the degradation of Armadillo (Arm), which causes downregulation of Wg signaling. In non-DV cells, Ubx inhibits event(s) downstream of Arm, but upstream of Vg autoregulation. Repression of Vg at multiple levels appears to be crucial for Ubx-mediated specification of the haltere fate. Overexpression of Vg in haltere discs is enough to override Ubx function and cause haltere-to-wing homeotic transformations (Prasad, 2003).

Several experiments were designed to test the current model of Wg and Vg regulation (which is essentially based on studies on wing imaginal discs) in haltere discs. In wing discs, both Wg and Vg are subjected to an elaborate regulatory circuit. Wg and Vg interact to maintain each other's expression at the DV boundary. Vg-mediated activation of Wg is independent of Arm and TCF/pan function, which suggests that Vg may activate Wg either directly or through the N signaling pathway. Vg is capable of specifying wing development, even in the absence of Wg signaling. Overexpression of Vg in a vg¹/vg¹ background (in which no Wg or Vg is expressed) is sufficient to rescue wing phenotypes. This is particularly significant because Vg was expressed in this experiment only in non-DV cells. These results also suggest that Vg cell-autonomously regulates its own expression through its quadrant enhancer. Clonal analysis of arm suggests that Wg is required to activate vg-QE and Arm is not able to activate this enhancer in vg¹ background. Wg signaling might activate Vg either indirectly or by activating some other enhancer of Vg. Once activated, Vg might maintain its expression by autoregulation, which is mediated through its quadrant enhancer. This could ensure the maintenance of Vg expression in non-DV cells, once it is activated by Wg signaling. It might also explain how the Wg gradient is translated into uniformly higher levels of Vg in non-DV cells (Prasad, 2003).

However, the above-mentioned model does not reconcile the observation that Vg, and not Wg, is capable of activating vg-QE in Ser^– background. Since the vg gene is intact in Ser^– background, ectopic expression of Wg using dpp-GAL4 should have activated one of the enhancers to induce Vg expression, which in turn would activate vg-QE. A model that reconciles all the results would, therefore, include a third component, which may act either parallel to or downstream of Wg and Vg at the DV boundary. Although there is no direct evidence for the existence of such a molecule, the fact that N23-GAL4 expression in non-DV cells is dependent on N function and independent of Vg and Wg function suggests such a possibility (Prasad, 2003).

The downregulation of Wg signaling by Ubx occurs at the level of Arm stabilization. Ubx inhibits stabilization of Arm by acting on event(s) downstream of Sgg. Normally, the Arm degradation machinery is very efficient and can degrade even overexpressed Arm. This is evident from the fact that embryos overexpressing Arm (from arm^S2) secrete normal denticle belts. If a downstream component functions with enhanced efficiency (either by direct enhancement of its expression by Ubx or owing to repression of a positive component of Wg signaling), residual activity of Sgg may be sufficient to cause enhanced degradation of Arm. Thus, enhanced degradation of Arm in haltere discs provides a new assay system to identify additional components of Wg signaling. For example, in microarray experiments to identify genes that are differentially expressed in wing and haltere discs, several transcripts of known (e.g., Casein kinase) and putative (e.g., Ubiquitin ligase) negative regulators of Wg signaling are upregulated in haltere discs (Prasad, 2003).

The results suggest that Wg and Vg regulation in haltere discs is different from that in wing discs. Wg is not autoregulated in haltere discs. In addition, Vg expression at the haltere DV boundary is independent of Wg function. However, in both wing and haltere discs, Wg expression at the DV boundary is dependent on Vg. Wg expression at the anterior DV boundary of haltere discs could be redundant because overexpression of DN-TCF at the haltere DV boundary shows no phenotype. However, Vg at the DV boundary appears to have an independent function. vg¹ flies exhibit much smaller halteres than do wild-type flies. Since Wg function (and expression in the posterior compartment) is already repressed in haltere discs, reduction in haltere size in vg¹ flies suggests Wg-independent long-range effects of Vg from the DV boundary. This could be one of the reasons why Ubx does not affect Vg expression at the DV boundary but represses Vg expression in non-DV cells. In wing discs too, Vg may have such a function on cells at a distance (Prasad, 2003).

One way to test the requirement of Ubx in DV and non-DV cells directly is by removing Ubx only from the haltere DV boundary or from non-DV cells. Clonal removal of Ubx solely from the haltere DV boundary does not induce cuticle phenotype in the capitellum. However, it was not possible to ascertain the effect on vg-QE because of haploinsufficiency: Ubx^–-heterozygous haltere discs themselves show activation of lacZ in the entire haltere pouch. The activation of vg-QE in Ubx^–/+ haltere discs could be a result of reduced Ubx function at the DV boundary, or in non-DV cells, or in both. Misexpression of Ubx at the wing disc DV boundary causes non-cell-autonomous reduction in vg-QE expression. The current results suggest that Ubx represses additional event(s) in non-DV cells to downregulate Vg expression. This is consistent with the recent report on cell-autonomous repression of vg-QE by ectopic Ubx in wing discs. It is proposed that Ubx inhibits the activation of Vg in non-DV cells at three different levels: (1) Wg in the posterior compartment; (2) event(s) downstream of Sgg that inhibit the stabilization of Arm, and (3) additional event(s) downstream of Arm in non-DV cells. In wing discs, Wg and a hitherto unknown DV component may function together to activate Vg in non-DV cells. Since Vg-autoregulation is not inhibited in haltere discs, it is possible that Ubx represses Vg activation in non-DV cells by interfering with the Wg-mediated activation of Vg and/or by repressing the activity of the unknown DV-signal molecule in the haltere (Prasad, 2003).

Additional evidence is provided that repression of Vg in non-DV cells by Ubx is crucial for haltere development. Overexpression of Vg in haltere discs causes haltere-to-wing transformations. This is particularly significant considering the fact that haltere-to-wing homeotic transformations are always associated with loss of Ubx, by direct removal of Ubx, by activation of its repressors (e.g., polycomb proteins) or by suppression of its activators (e.g. trithorax proteins). Mitotic clones of Ubx^– alleles in the haltere capitellum normally 'sort out' and often remain as an undifferentiated mass of cells. This is attributed to differential cell-adhesion properties of transformed (Ubx^–) and non-transformed (Ubx⁺) cells. No such sorting out of wing-like trichomes was observed in halteres overexpressing Vg. This implies that cells surrounding the wing-like trichomes are also transformed, at least at the level of cell-adhesion properties. This is consistent with observations that removal of Ubx from the DV boundary or over-growth caused by mutations in the tumor-suppressor gene fat confers wing-like cell-adhesion properties to capitellum cells. Since DV signaling is closely associated with the activation of Vg in non-DV cells and Vg is primarily a growth-promoting gene, it is likely that the cell-sorting behavior of Ubx^– clones is linked to their changed growth properties (Prasad, 2003).

Ubx regulation of Distalless

There are independent enhancers for Distal-less, arrayed over 40 kb of DNA. Head and leg synthesis is independently regulated. One distal 5' enhancer sequence, an early enhancer regulating Dll expression in germ band extended embryos, is subject to repression by Ultrabithorax and abdominal-A (Vachon, 1992).

The 26 bp bx1 element from the regulatory region of Distal-less is capable of imposing control by the homeotic genes Ultrabithorax and abdominal-A on a general epidermal activator in Drosophila. This provides an assay to analyze the sequence requirements for specific repression by these Hox genes. Both the core Hox binding site, 5'-TAAT, and the adjacent Exd 5'-TGAT core site are required for repression by Ultrabithorax and abdominal-A. The Distal-less bx1 site thus fits with the model of Hox protein binding specificity based on the consensus PBX/HOX-family site TGATNNAT[g/t][g/a], where the key elements of binding specificity are proposed to lie in the two base pairs following the TGAT. A single base pair deletion in the bx1 sequence generates a site, bx1:A^-mut, which on the consensus PBX/HOX model would be expected to be regulated by the Deformed Hox gene. It has been observed, however, that the bx1:A^-mut site is regulated predominantly by Sex combs reduced, Ultrabithorax and abdominal-A. The analysis of this site indicates that the specificity of action of Hox proteins may depend not only on selective DNA binding but also on specific post-binding interactions (White, 2000).

A homeotic response element that mediates repression by the Hox genes Ubx and abd-A has been built. The element is based on two short sequence modules; the 21 bp binding site Grainyhead binding site element (Gbe) for the transcription factor GRH and the 26 bp UBX/ ABD-A bx1 footprint site from the Dll regulatory sequences. On its own the Gbe mediates uniform epidermal activation. However, the combination of the Gbe and the bx1 element produces a homeotically modulated response. Specifically in the domain of expression of the Hox genes Ubx and abd-A the epidermal expression is repressed. This homeotic element thus successfully recapitulates the features of endogenous regulatory elements from homeotic target genes giving tissue-specific regulation by multiple homeotic genes (e.g. connectin). However, while endogenous target gene regulatory elements tend to be large (typically covering several kb) and are correspondingly difficult to analyze, this constructed element is appealingly simple and provides a manageable system for analyzing the specificity of the homeotic response (White, 2000).

The bx1 element contains the notable sequences 5' - TAAT (the core Hox binding site) and 5' -TGAT (the core Exd binding site). These two sequences are contained within the 5' -TGATTTAATT which is similar to the proposed PBX/HOX consensus site 5' -TGATNNAT[g/t][g/a]. Mutation of the 5' -TAAT site within bx1 abolishes the abdominal repression conferred by this element, presumably by reducing the affinity of the site for Ubx and Abd-A proteins. Thus, both Ubx and Abd-A appear to bind in vivo to the same site in the bx1 element. Mutation of the 5' -TGAT putative Exd binding site also abolishes the abdominal repression, suggesting that both Ubx and Abd-A require the binding of the cofactor Exd in order to function as repressors on this construct (White, 2000).

Specificity of Distalless repression and limb primordia development by Abdominal Hox proteins

In Drosophila, differences between segments, such as the presence or absence of appendages, are controlled by Hox transcription factors. The Hox protein Ultrabithorax (Ubx) suppresses limb formation in the abdomen by repressing the leg selector gene Distalless, whereas Antennapedia (Antp), a thoracic Hox protein, does not repress Distalless. The Hox cofactors Extradenticle and Homothorax selectively enhance Ubx, but not Antp, binding to a Distalless regulatory sequence. A C-terminal peptide in Ubx stimulates binding to this site. However, DNA binding is not sufficient for Distalless repression. Instead, an additional alternatively spliced domain in Ubx is required for Distalless repression but not DNA binding. Thus, the functional specificities of Hox proteins depend on both DNA binding-dependent and -independent mechanisms (Gebelein, 2002).

This work begins with a characterization of a Ubx binding site in the Dll gene that is critical for Dll repression; both Exd and Hth play a role in Ubx binding and repression. The Dll304 enhancer is sufficient to recapitulate the early expression pattern of Dll in the embryonic leg primordia. In addition to activation functions, Dll304 contains two Hox binding sites, Bx1 and Bx2, that repress enhancer activity in the abdomen and thereby restrict Dll expression to the thorax. Most of the repression activity is conferred by Bx1, a sequence bound by Ubx and Abd-A. In agreement with this result, a Distalless minimal element (DME) that lacks the Bx2 site accurately recapitulates the expression of Dll304 in the embryonic thorax. The DME enhancer also shows no derepression within the abdomen, suggesting that Bx1 is sufficient to fully repress Dll (Gebelein, 2002).

To better understand how Bx1 represses Dll, the presence of Exd and Hth binding sites were sought near the previously characterized Hox binding site. A consensus Exd site and a near consensus Hth site are in close proximity to the Hox site of Bx1. The Hox/Exd site (5'-AAATTAAATCA-3'), however, is unlike other previously characterized Hox/Exd binding sites because it contains an additional base pair in between the Hox and Exd half-sites. The Bx1 region containing this Hox/Exd/Hth site is referred to as the Distalless repression element (DllR). To determine whether DllR is required to repress DME expression in the abdomen, it was deleted from the DME enhancer (DME^act) and its ability to activate a reporter gene was tested in vivo. DME^act drives gene expression in all abdominal segments as well as in the thoracic region. Because the thoracic expression driven by DME^act is similar to that of DME, the DllR region is not required for DME activation but solely functions in the repression of Dll in the abdomen (Gebelein, 2002).

To determine whether Exd and Hth stimulate Hox binding to DllR, electrophoretic mobility shift assays (EMSAs) were performed with purified Ubx, Exd, and Hth proteins. Unless stated otherwise, all of these experiments were performed with UbxIa, the most widely expressed of several Ubx isoforms. By themselves, Ubx or an Exd/Hth heterodimer are capable of weakly interacting with DllR. The combination of all three proteins results in a slower migrating band indicating the formation of a Ubx/Exd/Hth/DNA complex. The formation of this protein/DNA complex is highly cooperative when compared to the amount of binding observed with Ubx or Exd/Hth alone. To test the contribution of each binding site, point mutations were introduced within the individual Hox, Exd, and Hth sites. Mutation of any one of these sites results in a decrease in the formation of the trimeric protein/DNA complex, suggesting that all three are required for optimal binding to DllR (Gebelein, 2002).

To test whether the Hox, Exd, and Hth binding sites are also required for Dll repression in vivo, reporter constructs were created containing the lacZ gene under the control of mutant versions of the DME enhancer. Mutation of the Hox site (DME^Hox) results in a similar level of derepression of reporter gene expression throughout the abdomen, as does the the complete deletion of DllR. Mutation of the Exd (DME^Exd) and Hth (DME^Hth) sites individually also results in derepression, albeit slightly weaker than mutation of the Hox site. However, if both the Exd and Hth sites are mutated together, full derepression is observed. Taken together, these results demonstrate that the efficient formation of a Hox/Exd/Hth trimeric complex on DllR is required for Dll repression within the abdomen (Gebelein, 2002).

These above data support a model in which a Ubx/Exd/Hth complex bound to DllR is necessary for Dll repression. Whether a single copy of DllR is sufficient to repress a heterologous enhancer element was tested. An artificial enhancer, called fkh(250^con), is activated by Scr, Antp, and Ubx (with Exd and Hth), and thus provides a useful heterologous activator to test for DllR function. A reporter construct under the control of both fkh(250^con) and DllR was created. Unlike fkh(250^con), which is expressed in parasegments (PS) 2-6, the composite enhancer (fkh250^con-DllR) is not expressed in PS 6, where Ubx is expressed. Ubx-mediated repression of fkh(250^con)-DllR is more obvious in embryos mutant for abd-A, which derepress Ubx and, consequently, fkh(250^con) throughout the abdomen. In this genetic background, fkh(250^con)-DllR is still active only in PS 2-5. Furthermore, misexpression of Ubx throughout the embryo activates fkh(250^con) but represses fkh(250^con)-DllR. Taken together, these results indicate that DllR is sufficient to confer Ubx-mediated repression of a heterologous enhancer. In addition, these results also illustrate that Ubx/Exd/Hth complexes can mediate repression through DllR in the same cells as it mediates activation through fkh(250^con) (Gebelein, 2002).

A general question for all transcription factors is how they achieve specificity in vivo. For the Hox proteins, a large number of studies have implicated sequences both within and outside the homeodomain as being important for their in vivo specificities. But how do these sequences function? Because DNA binding domains, including homeodomains, can also be protein interaction domains, studies that map the domains necessary for target gene regulation cannot answer this question by themselves. Instead, direct transcriptional targets must be identified and, once binding sites are characterized, DNA binding, in addition to target gene regulation, must be measured. The results allow two steps to be discriminated in the repression of Dll by Ubx. First, Exd and Hth stimulate Ubx, but not Antp, binding to DllR. In contrast, Ubx/Exd/Hth and Antp/Exd/Hth have similar affinities for a different "consensus" binding site (5'-CCATAAATCA-3'), suggesting that subtle differences in the DNA sequence, in addition to differences between Ubx and Antp, contribute to specificity. A C-terminal peptide in Ubx stimulates this cofactor-dependent binding to DllR. DNA binding, however, is not sufficient for Dll repression. Instead, an additional linker domain included in only a subset of Ubx isoforms is required for repression. Thus, a second step, the recruitment of additional factors to the Ubx/Exd/Hth complex bound to DllR, is implied by these data. In addition to the UbxIa linker, this step also requires the specific sequences and conformation imposed on the Ubx/Exd/Hth trimer by DllR (Gebelein, 2002).

Although the Ubx C terminus plays an important role in cofactor-dependent binding to DllR, additional domains contribute to optimal binding. In the presence of Exd and Hth, the AAUU chimera, but not heterologous AAUA or AAAU, binds DllR, suggesting that both the Ubx homeodomain and C terminus are important for optimal DNA binding to this site. The C terminus is not absolutely required for binding because a Ubx protein that lacks this domain (UUU*) is still able to bind well to DllR. Last, the finding that UUU*, but not AAU*, binds DllR suggests that a domain N terminal to the homeodomain also enhances DllR binding. Based on the crystal structures of Hox/Exd/DNA complexes, this difference could be due to the YPWM motif. Taken together, the data suggest that multiple regions of Ubx contribute to binding DllR and that no one domain is sufficient for full binding activity. This finding may be understood in light of the fact that the entire Ubx coding sequence has been constrained over millions of years of insect evolution to maintain leg (and Dll) repression in the abdomen (Gebelein, 2002).

How might the Ubx C terminus and YPWM motifs contribute to DNA binding? It is suggested that these regions could make additional protein-DNA contacts and/or protein-protein interactions that help stabilize the DllR-bound form of the trimeric complex. In support of this idea, the C termini of other homeodomain proteins also contribute to DNA binding. The Exd C terminus, for example, consists of an alpha helix that packs against its homeodomain and contributes to DNA binding. The C terminus of the MATalpha2 protein from yeast forms an alpha helix that contacts the MATa1 homeodomain to stabilize heterodimer formation on DNA. Interestingly, the two Hox proteins that repress Dll expression, Ubx and Abd-A, share sequence homology in their C termini, and are the only Drosophila Hox proteins predicted to form an alpha helix after their homeodomains (Gebelein, 2002).

The Ubx YPWM motif may also help stabilize complex formation on DllR. In the Hox/Exd/DNA crystal structures, this motif, together with flanking amino acids, directly contacts a hydrophobic pocket within the Exd homeodomain. These protein-protein contacts are thought to stabilize protein-DNA contacts made by the complex. The amino acids surrounding the YPWM motifs are different in Ubx and Antp and thus could contribute to DNA binding specificity by such an indirect mechanism (Gebelein, 2002).

The finding that UbxIa, but not UbxIVa, is able to repress Dll suggests that the linker region in UbxIa is required for repression. In addition, these results suggest that alternative splicing has the potential to modulate Ubx's control of gene expression. In support of this view, the expression of Ubx isoforms is temporally and spatially regulated. In addition, misexpression experiments using UbxIa and UbxIVa have shown that while both perform many of the same functions, only UbxIa efficiently transforms the peripheral nervous system. The finding that UbxIa and UbxIVa have different transcriptional regulatory properties provides a possible explanation for their distinct abilities to transform this tissue (Gebelein, 2002).

One argument against the idea that the different Ubx isoforms have distinct functions is that flies containing a genetic inversion that prevents the inclusion of the second microexon are, for the most part, normal. Although this mutation prevents the expression of UbxIa, it is unclear which other Ubx isoforms are expressed in this mutant because the inversion does not include both microexons. Furthermore, the effect that this mutation has on Dll expression has not been examined. A definitive test of the idea that Ubx isoforms have unique functions will require determining whether a Ubx allele in which both microexons are eliminated can provide all Ubx functions in vivo (Gebelein, 2002).

As in Drosophila, Dll expression is a marker for leg primordia in many animal phyla. Animals with appendages on their abdominal segments, such as crustaceans and onychophora, coexpress Ubx with Dll, demonstrating that Ubx is not a repressor of Dll in these species. The ability of Ubx to repress Dll probably arose in a subset of arthropods, the hexapods. Consistent with these findings, two recent studies suggest that one relevant difference between Ubx orthologs that repress Dll (for example, Drosophila Ubx) and Ubx orthologs that do not repress Dll (for example, onychophoran Ubx) maps to the C-terminal regions of these Hox proteins (Galant, 2002; Ronshaugen, 2002). These two groups, however, propose different mechanisms for how these sequences function. Galant suggests that the Drosophila Ubx C terminus actively represses transcription via a polyalanine motif that is present in the Ubx orthologs from all hexapods. Ronshaugen suggests that the Drosophila Ubx C terminus is only permissive for repression. Instead, they argue that crustaceans, which have abdominal legs, evolved a C-terminal sequence that inhibits Dll repression. However, neither study analyzed the binding of these proteins to the relevant binding sites in Dll, leaving open the possibility that the effects they observe could also be due to effects on DNA binding (Gebelein, 2002).

The data provide additional insights into how repression mechanisms may have evolved in these different species. It was found that the Drosophila Ubx C terminus contributes to DllR binding but is not sufficient for Dll repression in vivo. Thus, the positive role, observed by Galant, that the Drosophila sequence plays in Dll repression, could be due to an effect on DNA binding. These experiments also implicate the linker region of UbxIa as important for repression, but not DNA binding. Because some of the onychophora/ Drosophila and crustacean/ Drosophila chimeras lack this linker but are able to repress Dll, the crustacean and onychophoran Ubx orthologs must have repression domains that are different from the one identified in Drosophila Ubx (Gebelein, 2002).

Ronshaugen suggests that the phosphorylation of serine and threonine residues in the crustacean Ubx C terminus is necessary for it to prevent Dll repression (Ronshaugen, 2002). This is an intriguing possibility in light of the fact that phosphorylation of a Hox C terminus can inhibit cooperative DNA binding with Exd. Taken together with the current data that the C terminus of Ubx enhances DNA binding to DllR, it is suggested that the inhibition of Dll repression by the crustacean C terminus may be due to a reduced ability to bind DllR with Exd and Hth. This model accounts for why a Drosophila UbxIa protein containing the crustacean C terminus is unable to repress Dll (Ronshaugen, 2002) and for the inability of onychophora Ubx, which also contains a putative phosphorylation site in its C terminus, to repress Dll. Taken together, it is suggested that the evolution of limb suppression by Hox proteins, and probably many other Hox functions, depended upon the modification of both DNA binding-dependent and -independent mechanisms controlling Hox specificity (Gebelein, 2002).

Although these experiments focused on understanding why Antp is different from Ubx, the results provide some insights into the mechanism of transcriptional repression. The data strongly argue that a DNA-bound Ubx/Exd/Hth complex is necessary, but not sufficient, for repression. First, in addition to repressing Dll, Ubx/Exd/Hth activates fkh(250^con). When both fkh(250^con) and DllR simultaneously regulate the same reporter gene, DllR is able to repress gene expression in the same cells in which fkh(250^con) normally activates gene expression. This result suggests that the repressor proteins required for DllR activity are not cell type specific and are widely expressed in the embryo. Further, these results suggest that differences between the fkh(250^con) and DllR sequences determine whether transcription is activated or repressed. These sequences may recruit additional DNA binding factors that interact with the trimeric complex. These factors, which have not yet been identified, might provide or reveal a latent activation or repression domain within the Hox/Exd/Hth complex. Alternatively, another DNA binding factor may not be needed. Instead, the unique arrangement or spacing of the Hox, Exd, and Hth sites in these two elements may result in distinct conformations of the trimeric complex that recruit different coactivators or corepressors. Such a mechanism has been suggested for the nuclear receptor family of transcription factors and for the POU domain protein Pit-1, where a difference in spacing in a Pit-1 dimer binding site regulates the recruitment of a corepressor. Consistent with such a mechanism, it was found that the DllR^con binding site, which has one less base pair between the Hox and Exd half-sites than the DllR binding site, fails to repress transcription despite having a higher affinity for Ubx/Exd/Hth complexes. In addition, although repression activity for the UbxIa linker and C terminus in S2 cells can be measured, the experiments suggest that their activities are context dependent. The abdominal expression of DME^con-lacZ suggests that the mere presence of these domains is not sufficient for repression. Thus, the data suggest that transcription factor domains have distinct properties when assayed by themselves versus when they are part of a multiprotein complex. Further, it is concluded that the unique architecture of the complex assembled on DllR is necessary for efficient repression (Gebelein, 2002).

Direct integration of Hox and segmentation gene inputs during Drosophila development

During Drosophila embryogenesis, segments, each with an anterior and posterior compartment, are generated by the segmentation genes while the Hox genes provide each segment with a unique identity. These two processes have been thought to occur independently. This study shows tha abdominal Hox proteins work directly with two different segmentation proteins, Sloppy paired and Engrailed, to repress the Hox target gene Distalless in anterior and posterior compartments, respectively. These results suggest that segmentation proteins can function as Hox cofactors and reveal a previously unanticipated use of compartments for gene regulation by Hox proteins. The results suggest that these two classes of proteins may collaborate to directly control gene expression at many downstream target genes (Gebelein, 2004).

The segregation of groups of cells into compartments is fundamental to animal development. Originally defined in Drosophila, compartments are critical for providing cells with their unique positional address. The first compartments to form during Drosophila development are the anterior and posterior compartments and the key step to defining them is the activation of the gene engrailed (en). Expression of en, which encodes a homeodomain transcription factor, results in a posterior compartment fate, and the absence of en expression results in an anterior compartment fate. Once activated by gap and pair-rule genes, en expression and, consequently, the anterior–posterior compartment boundary later become dependent upon the protein Wingless (Wg), which is secreted from adjacent anterior compartment cells. Concurrently with anterior–posterior compartmentalization and segmentation, the expression of the eight Drosophila Hox genes is also initially established by the gap and pair-rule genes. The Hox genes, however, which also encode homeodomain transcription factors, do not contribute to the formation or number of segments but instead specify their unique identities along the anterior–posterior axis (Gebelein, 2004).

This flow of genetic information during Drosophila embryogenesis has led to the idea that anterior–posterior compartmentalization and segment identity specification are independent processes. In contrast to this view, this study shows that these two pathways are interconnected in previously unrecognized ways. Evidence is provided that Hox factors directly interact with segmentation proteins such as En to control gene expression. Moreover, Hox proteins collaborate with two different segmentation proteins in anterior and posterior cell types to regulate the same Hox target gene, revealing a previously unknown use of compartments to control gene expression by Hox proteins (Gebelein, 2004).

Distalless (Dll) is a Hox target gene that is required for leg development in Drosophila. In each thoracic hemisegment, wg, expressed by anterior cells adjacent to the anterior–posterior compartment boundary, activates Dll in a group of cells that straddle this boundary. A cis-regulatory element derived from Dll, called DMX, drives accurate Dll-like expression in the thorax. The abdominal Hox genes Ultrabithorax (Ubx) and abdominalA (abdA) directly repress Dll and DMX-lacZ in both compartments, thereby blocking leg development in the abdomen. DMX is composed of a large activator element (DMXact) and a 57-base-pair (bp) repressor element referred to here as DMX-R. Previous work demonstrated that Ubx and AbdA cooperatively bind to DMX-R with two homeodomain cofactors, Extradenticle (Exd) and Homothorax (Hth). In contrast, the thoracic Hox protein Antennapedia (Antp) does not repress Dll and does not bind DMX-R with high affinity in the presence or absence of Exd and Hth. Thus, repression of Dll in the abdomen depends in part on the ability of these cofactors to selectively enhance the binding of the abdominal Hox proteins to DMX-R (Gebelein, 2004).

Exd and Hth, as well as their vertebrate counterparts, are used as Hox cofactors at many target genes. Moreover, Hox/Exd/Hth complexes are used for both gene activation and repression, raising the question of how the decision to activate or repress is determined. One view posits that these complexes do not directly recruit co-activators or co-repressors, but instead are required for target gene selection. Accordingly, other DNA sequences present at Hox/Exd/Hth-targeted elements would determine whether a target gene is activated or repressed. Consistent with this notion, DMX-R sequences isolated from six Drosophila species show extensive conservation outside the previously identified Hox (referred to here as Hox1) Exd and Hth binding sites, suggesting that they also play a role in Dll regulation (Gebelein, 2004).

To test a role for these conserved sequences, a thorough mutagenesis of DMX-R was performed. Each mutant DMX-R was cloned into an otherwise wild-type, full-length DMX and tested for activity in a standard reporter gene assay in transgenic embryos. Thoracic expression was normal in all cases. However, surprisingly, many of the DMX-R mutations, such as X5, resulted in abdominal de-repression only in En-positive posterior compartment cells, whereas other mutations, such as X2, resulted in abdominal de-repression only in En-negative anterior compartment cells. Single mutations in the Hox1, Exd, or Hth sites also resulted in de-repression predominantly in posterior cells. In contrast, deletion of the entire DMX-R (DMXact-lacZ), or mutations in both the X2 and X5 sites (DMX[X2 + X5]-lacZ), resulted in de-repression in both compartments. These results suggest that distinct repression complexes bind to the DMX-R in the anterior and posterior compartments and that segmentation genes play a role in Dll repression (Gebelein, 2004).

One clue to the identity of the proteins in these repression complexes is that the sequence around the Hth site is nearly identical to a Hth/Hox binding site that had been identified previously by a systematic evolution of ligands by exponential enrichment (SELEX) approach using vertebrate Hox and Meis proteins. This similarity suggested the presence of a second, potentially redundant Hox binding site, Hox2. In agreement with this idea, mutations in both the Hox1 and Hox2 binding sites resulted in de-repression in both the anterior and posterior compartments of the abdominal segments. Similarly, although individual mutations in the Exd and Hth binding sites lead predominantly to de-repression in the posterior compartment, mutation of both sites resulted in de-repression in both compartments. These results suggest that a Hox/Exd/Hth/Hox complex may be used for repression in both compartments. Furthermore, they suggest that although single mutations in these binding sites are sufficient to disrupt the activity of this complex in the posterior compartment, double mutations are required to disrupt its activity in the anterior compartment (Gebelein, 2004).

To provide biochemical evidence for a Hox/Exd/Hth/Hox tetramer, DNA binding experiments were performed using DMX-R probes and proteins expressed and purified from E. coli. Previous experiments demonstrated that a Hox/Exd/Hth trimer cooperatively binds to the Hox1, Exd and Hth sites. The function of the Hox2 site was tested in two ways. First, binding was measured to a probe, DMX-R2, that includes the Exd, Hth and Hox2 sites, but not the Hox1 site. It was found that Exd/Hth/AbdA and Exd/Hth/Ubx trimers cooperatively bind to this probe and that mutations in the Hth, Exd or Hox2 binding sites reduced or eliminated complex formation (Gebelein, 2004).

Second, if both the Hox1 and Hox2 sites are functional, the full-length DMX-R may promote the assembly of Hox/Exd/Hth/Hox tetramers. Using a probe containing all four binding sites (DMX-R1 + 2), the formation of such complexes was observed. Mutation of any of the four binding sites reduced the amount of tetramer binding whereas mutation of both Hox sites or both the Exd and Hth sites eliminated tetramer binding. Furthermore, Antp, which does not repress Dll, formed tetramers with Exd and Hth that were approximately tenfold weaker than with Ubx or AbdA, but bound well to a consensus Hox/Exd/Hth trimer binding site. Because mutation of both Hox sites or both the Exd and Hth sites resulted in de-repression in both compartments, these experiments correlate the binding of a Hox/Exd/Hth/Hox complex on the DMX-R with the ability of this element to mediate repression in both compartments (Gebelein, 2004).

Although binding of a Hox/Exd/Hth/Hox tetramer is sufficient to account for the necessary abdominal Hox-input into Dll repression, it does not explain the compartment-specific de-repression exhibited by some DMX-R mutations. The X2 and X5 mutations, for example, result in abdominal de-repression but do not prevent the formation of the Hox/Exd/Hth/Hox tetramer. Sequence inspection of the DMX-R revealed that the X2 mutation, which resulted in de-repression specifically in the anterior compartment, disrupts two partially overlapping matches to a consensus binding site for Forkhead (Fkh) domain proteins. With this in mind, the expression pattern of Sloppy paired 1 (Slp1), a Fkh domain factor encoded by one of two partially redundant segmentation genes, slp1 and slp2, was examined. The two slp genes are expressed in anterior compartment cells adjacent and anterior to En-expressing posterior compartment cells. In the thorax, cells expressing Dll and DMX-lacZ co-express either Slp or En at the time Dll is initially expressed. In the abdomen, the homologous group of cells, which express DMXact-lacZ (a reporter lacking the DMX-R), co-express either Slp in the anterior compartment or En in the posterior compartment. The expression patterns of Slp and En were compared with Ubx and AbdA. Ubx levels are highest in anterior, Slp-expressing cells whereas AbdA levels are elevated in posterior, En-expressing cells. In contrast, both Exd and Hth are present at similar levels in both compartments throughout the abdomen (Gebelein, 2004).

On the basis of these data, a model is presented for Hox-mediated repression of Dll in both the anterior and posterior compartments of the abdominal segments. In the anterior compartment it is proposed that Slp binds to DMX-R directly with a Ubx/Exd/Hth/Ubx tetramer. In the posterior compartment it is suggested that En binds to DMX-R directly with an AbdA/Exd/Hth/AbdA tetramer. One important feature of this model is that Antp/Exd/Hth/Antp complexes fail to form on this DNA, thereby accounting for the lack of repression in the thorax. Furthermore, the model proposes that Slp and En should, on their own, have only weak affinity for DMX-R sequences because repression does not occur in the thorax, despite the presence of these factors. The Hox/Exd/Hth/Hox complex, perhaps in conjunction with additional factors, is required to recruit or stabilize Slp and En binding to the DMX-R. Both Slp and En are known repressor proteins that directly bind the co-repressor Groucho. Thus, the proposed complexes in both compartments provide a direct link to this co-repressor and, therefore, a mechanism for repression. DNA binding and genetic experiments are presented that test and support this model (Gebelein, 2004).

To test the idea that En is playing a direct role in Dll repression, the ability of En and Hox proteins to bind to DMX-R probes was examined. On its own, En binds to DMX-R very poorly. Surprisingly, it was found that En binds DMX-R with the abdominal Hox proteins Ubx or AbdA in a highly cooperative manner. The thoracic Hox protein Antp does not bind cooperatively with En to this probe. Mutations in the Hox1 or X5 binding sites block AbdA/En binding in vitro, consistent with these mutations showing posterior compartment de-repression in vivo. In contrast, the X6, X7 and Hth mutations do not affect AbdA/En complex formation (Gebelein, 2004).

On the basis of DMX-R's ability to assemble a Hox/Exd/Hth/Hox tetramer, whether En could bind together with an AbdA/Exd/Hth/AbdA complex was tested. Addition of En to reactions containing AbdA, Exd and Hth resulted in the formation of a putative En/AbdA/Exd/Hth/AbdA complex. This complex contains En because its formation is inhibited by an anti-En antibody. A weak antibody-induced supershift is also observed. Moreover, this complex fails to form on the X5 mutant, which causes posterior compartment-specific de-repression. It is noted that En/Exd/Hth complexes also bind to the DMX-R and that it cannot be excluded that an En/Exd/Hth/AbdA complex may be important for Dll repression. The model emphasizes a role for an En/AbdA/Exd/Hth/AbdA complex because it better accommodates the cooperative binding observed between En and AbdA on the DMX-R (Gebelein, 2004).

Repression in the anterior compartments of the abdominal segments requires the sequence defined by the X2 mutation, which is similar to a Fkh domain consensus binding site. The model predicts that this sequence is bound by Slp. Consistent with this view, Slp1 binds weakly to wild type, but not to X2 mutant DMX-R probes. However, in contrast to En, no cooperative binding was observed between Slp and Hox or Hox/Exd/Hth/Hox complexes, suggesting that additional factors may be required to mediate interactions between Slp and the abdominal Hox factors (Gebelein, 2004).

Together, these results suggest that En and Slp play a direct role in DMX-lacZ and Dll repression. However, these experiments do not unambiguously determine the stoichiometry of binding by these factors. Furthermore, in vivo, additional factors may enhance the interaction between these segmentation proteins and Hox complexes, thereby increasing the stability and/or activity of the repression complexes (Gebelein, 2004).

The model for Dll repression is supported by previous genetic experiments that examined the effect of Ubx and abdA mutants on Dll expression in the abdomen. Ubx abdA double mutants de-repress Dll in both compartments of all abdominal segments. In contrast, Ubx mutants de-repress Dll in the anterior compartment of only the first abdominal segment, which lacks AbdA. abdA mutant embryos de-repress Dll in the posterior compartments of all abdominal segments, where Ubx levels are low (Gebelein, 2004).

Several genetic experiments were performed to provide in vivo support for the idea that Slp and En work directly with Ubx and AbdA to repress Dll. The design of these experiments had to take into consideration that the activation of Dll in the thorax depends on wg, and that wg expression depends on both slp and en. Consequently, Dll expression is mostly absent in en or slp mutants, making it impossible to characterize the role that these genes play in Dll repression from examining en or slp loss-of-function mutants. However, some of the mutant DMX-Rs described here provide the opportunity to test the model in alternative ways (Gebelein, 2004).

According to the model, DMX[X5]-lacZ is de-repressed in the posterior compartments of the abdominal segments because it fails to assemble the posterior, En-containing complex. Repression of DMX[X5]-lacZ in the anterior compartments still occurs because it is able to assemble the anterior, Slp-containing complex. According to this model, DMX[X5]-lacZ should be fully repressed if Slp is provided in posterior cells. A negative control for this experiment is that ectopic Slp should be unable to repress DMX[X2]-lacZ because this reporter gene does not have a functional Slp binding site. To mis-express Slp, paired-Gal4 (prd-Gal4), which overlaps both the Slp and En stripes in the odd-numbered abdominal segments, was used. As predicted, ectopic Slp repressed DMX[X5]-lacZ but not DMX[X2]-lacZ, providing strong in vivo support for Slp's direct role in Dll repression in the anterior compartments (Gebelein, 2004).

Conversely, the model posits that DMX[X2]-lacZ is de-repressed in the anterior compartment because it cannot bind Slp, but remains repressed in the posterior compartment because it is able to assemble the En-containing posterior complex. Thus, providing En in the anterior compartment should repress DMX[X2]-lacZ. A complication with this experiment is that En is a repressor of Ubx, which is the predominant abdominal Hox protein in the anterior compartment. It was confirmed that prd-Gal4-driven expression of En represses Ubx and that AbdA levels remain low at the time Dll is activated in the thorax. Consequently, ectopic En expression is not sufficient to repress DMX[X2]-lacZ, consistent with the observation that low levels of abdominal Hox proteins are present. Therefore, to promote the assembly of the posterior complex in anterior cells, En was co-expressed with AbdA using prd-Gal4. As predicted, this combination of factors repressed DMX[X2]-lacZ but not DMX[X5]-lacZ, providing strong in vivo evidence for En playing an essential role in Dll repression in the posterior compartments (Gebelein, 2004).

Several observations provide additional support for the model. First, ectopic expression of AbdA or Ubx in the second thoracic segment (T2) represses DMX[X5]-lacZ in the anterior compartment, but not in the posterior compartment. Conversely, expression of AbdA or Ubx in T2 represses DMX[X2]-lacZ only in posterior compartment cells. Second, co-expression of Slp with Ubx completely represses DMX[X5]-lacZ in T2 but does not repress DMX[X2]-lacZ in T2. Third, in those cases where repression is incomplete (for example, En + AbdA repression of DMX[X2]-lacZ in the abdomen), cells that escape repression have low levels of either an abdominal Hox protein or Slp/En. Together, these data provide additional evidence that the abdominal Hox proteins work together with Slp and En to repress Dll (Gebelein, 2004).

The segregation of cells into anterior and posterior compartments during Drosophila embryogenesis is essential for many aspects of fly development. The results presented in this study reveal an unanticipated intersection between anterior–posterior compartmentalization by segmentation genes and segment identity specification by Hox genes. Specifically, it is suggested that the abdominal Hox proteins collaborate with two different segmentation proteins, Slp and En, to mediate repression of a Hox target gene (Dll) in the anterior and posterior compartments of the abdomen, respectively. This mechanism of transcriptional repression suggests a previously unknown use of compartments in Drosophila development. The mechanism proposed here contrasts with the alternative and simpler hypothesis in which the abdominal Hox proteins would have used the same set of cofactors to repress Dll in all abdominal cells, regardless of their compartmental origin (Gebelein, 2004).

These results provide further support for the view that Hox/Exd/Hth complexes do not directly bind co-activators or co-repressors but instead indirectly recruit them to regulatory elements. Consistent with previous analyses, it is suggested that Hox/Exd/Hth complexes are important for the Hox specificity of target gene selection. Additional factors, such as Slp or En in the case of Dll repression, are required to determine whether the target gene will be repressed or activated. In the future, it will be important to dissect in similar detail other Hox-regulated elements, to assess the generality of this mechanism (Gebelein, 2004).

These results also broaden the spectrum of cofactors used by Hox proteins to regulate gene expression. Although the analysis of Exd/Hth in Drosophila and Pbx/Meis in vertebrates has provided some insights into how Hox specificity is achieved, there are examples of tissues in which these proteins are not available to be Hox cofactors and of Hox targets in which Exd and Hth seem not to play a direct role. This study shows that En, a homeodomain segmentation protein, is used as a Hox cofactor to repress Dll in the abdomen. Although the complex defined at the DMX-R includes Exd and Hth, the DNA binding studies demonstrate that Hox and En proteins can bind cooperatively to DNA in the absence of Exd and Hth. These findings suggest that En may function with Ubx and/or AbdA to regulate target genes other than Dll, and perhaps independently of Exd and Hth. Consistent with this idea are genetic experiments showing that, in the absence of Exd, En can repress slp and this repression requires abdominal Hox activity. Although these experiments were unable to distinguish whether the Hox input was direct or indirect, the results suggest that En may bind directly with Ubx and AbdA to repress slp, and perhaps other target genes (Gebelein, 2004).

Finally, these results raise the question of why a compartment-specific mechanism is used by Hox factors to repress Dll. The activation of Dll at the compartment boundary by wg may be important for accurately positioning the leg primordia within each thoracic hemisegment, but this mode of activation requires that Dll is repressed in both compartments in each abdominal segment. The utilization of segmentation proteins such as En and Slp may be the simplest solution to this problem. Compartment-specific mechanisms may also provide additional flexibility in the regulation of target genes by Hox proteins by allowing them to turn genes on or off specifically in anterior or posterior cell types. For these reasons, compartment-dependent mechanisms of gene regulation may turn out to be the general rule instead of the exception (Gebelein, 2004).

Direct regulation of knot gene expression by Ultrabithorax

The regulation of development by Hox proteins is important in the evolution of animal morphology, but how the regulatory sequences of Hox-regulated target genes function and evolve is unclear. To understand the regulatory organization and evolution of a Hox target gene, a wing-specific cis-regulatory element was identified controlling the knot gene, which is expressed in the developing Drosophila wing but not the haltere. This regulatory element contains a single binding site that is crucial for activation by the transcription factor Cubitus interruptus (Ci), and a cluster of binding sites for repression by the Hox protein Ultrabithorax (Ubx). The negative and positive control regions are physically separable, demonstrating that Ubx does not repress by competing for occupancy of Ci-binding sites. Although knot expression is conserved among Drosophila species, this cluster of Ubx binding sites is not. The knot wing cis-regulatory element was isolated from D. pseudoobscura, which contains a cluster of UBX-binding sites that is not homologous to the functionally defined D. melanogaster cluster. It is, however, homologous to a second D. melanogaster region containing a cluster of UBX sites that can also function as a repressor element. Thus, the knot regulatory region in D. melanogaster has two apparently functionally redundant blocks of sequences for repression by UBX, both of which are widely separated from activator sequences. This redundancy suggests that the complete evolutionary unit of regulatory control is larger than the minimal experimentally defined control element. The span of regulatory sequences upon which selection acts may, in general, be more expansive and less modular than functional studies of these elements have previously indicated (Hersh, 2005).

The knot gene is expressed in the developing Drosophila wing imaginal disc at the anteroposterior compartment boundary, but is not expressed in the haltere imaginal disc. Furthermore, knot expression is genetically downstream of Ubx; overexpression of Ubx in clones in the wing causes cell-autonomous loss of knot expression. Because these features make knot a candidate for direct regulation by Ubx, attempts were made to identify the regulatory element that controls knot expression in the wing (Hersh, 2005).

One regulatory element of knot has been identified that drives reporter gene expression in the embryonic head and mesoderm. This element extends ~5 kb from the transcriptional start site of knot. When a knot cDNA was placed under control of this element, embryonic lethality was rescued, but wing vein defects were not, indicating that the wing regulatory element is located elsewhere. The lesion underlying the wing-specific knot^SA2 allele, which is a complex translocation with a breakpoint 10-20 kb 5' of the knot-coding region, suggested the location of a wing-specific regulatory element (Hersh, 2005).

Based on the location of the knot^SA2 lesion, reporter constructs were generated with genomic DNA from the region 5-20 kb 5' of knot. A 6.8 kb region of DNA ~15 kb 5' of knot was identified that drives expression of lacZ in a stripe at the anteroposterior compartment boundary in the wing imaginal disc, consistent with the expression pattern of the Knot protein. No expression of lacZ was observed in the haltere, demonstrating that this large region accurately recapitulates the expression and regulation of the endogenous knot gene. To determine if both wing and haltere regulation was confined to a single region within this 6.8 kb, the activity was further narrowed to a 2.3 kb region that drives appropriate reporter gene expression. All subsequent numbering of constructs is in reference to this 2.3 kb region, kn^Mel1-2330 (Hersh, 2005).

Sequence conservation has been successfully employed in the identification of regulatory elements. Attempts were made to use conservation to direct the further dissection of kn^Mel1-2330 to define a minimal regulatory element. Based on several scattered blocks of sequence conservation between D. melanogaster and D. pseudoobscura, PCR primers were designed to amplify sequence from a more distantly related fruit fly, D. virilis. Three conserved blocks were shared between these three flies, and kn^Mel1-2330 was split into two overlapping constructs, each containing two of the conserved blocks. Though both constructs included the central conserved block that contained several potential sites for regulators, only the 1.3 kb kn^Mel701-1991 construct was capable of driving expression in a stripe in the wing, whereas the kn^Mel224-1426 construct was only weakly expressed in a single small spot at the intersection of the DV and AP axes in the wing. Therefore, the 1.3 kb region accurately recapitulates the knot expression pattern in both the wing and haltere, and must contain binding sites for the regulators that generate this pattern (Hersh, 2005).

Expression of the knot gene is dependent on Hedgehog (Hh) activity, and overexpression of Hh can trigger ectopic knot expression in the wing. The transcriptional effector of Hh signaling is the Cubitus interruptus (Ci) protein. Ci is a zinc-finger transcription factor of the Gli family, and binds a 9 bp consensus sequence TGGG(T/A)GGTC. In the 1.3 kb kn^Mel701-1991 fragment, three potential Ci-binding sites were identified that matched at least seven out of nine consensus residues and that are conserved in D. pseudoobscura. Two additional potential sites were present, but were not conserved in D. pseudoobscura. The three conserved binding sites were independently mutagenized, converting a crucial guanine to an adenine, and the mutagenized element was re-introduced into flies. Changes at two of the three candidate sites had no effect on reporter gene expression, whereas the mutation of site Ci1680 almost completely abolishes reporter expression. Mutation of all three sites did not have a more severe effect than mutation of Ci1680 alone. These results indicate that activation of the wing-specific enhancer element by Hh signaling is dependent primarily on a single Ci-binding site at position 1680 in the kn^Mel701-1991 element (Hersh, 2005).

Because the kn^Mel701-1991 element drives expression in the wing, but not the haltere, it was postulated that this sequence integrates information from Hh signaling and the homeotic regulator, Ubx. Therefore, attempts were made to identify possible binding sites for Ubx within this element. Isolated Ubx homeodomain binds optimally in vitro to the sequence TTAATGG, but binding sites in characterized Ubx-responsive regulatory elements often are not exact matches to this optimal sequence. Therefore, the TAAT core sequence commonly bound by homeodomain proteins was sought. The kn^Mel701-1991 fragment contains clusters of TAAT core sequences near both its 5' and 3' limits that might mediate knot repression in the haltere. In addition, there is a single TAAT core sequence located within 10 bp of the crucial Ci-binding site and in a conserved block of sequence, suggesting that it may be important for repression by Ubx (Hersh, 2005).

To determine which TAAT sequences might be functionally important for Ubx repression, sequences were removed from each end of kn^Mel701-1991 and the effect on reporter gene expression in vivo was observed. Removal of the 5' end, with its small cluster of four core sequences, had no effect on expression. By contrast, removal of 156 bp from the 3' end, including nine putative Ubx-binding sites (kn^Mel701-1835), caused the reporter to be expressed at the AP compartment boundary in both the wing and the haltere. Therefore, kn^Mel701-1991 does appear to be directly negatively regulated by Ubx in the haltere, and removal of Ubx-binding sites relieves repression in the haltere. In addition to the ectopic activation of expression in the haltere, it was noted that the expression level in the wing is also elevated compared with kn^Mel701-1991, suggesting that additional repressor binding sites important for appropriate wing expression may have been removed in kn^Mel701-1835. Importantly, the response to local spatial information within the wing field (encompassing both the wing and haltere) was maintained; expression was appropriately observed at the AP compartment boundary in both tissues. Because the single deletion preserved the response to spatial information within the dorsal appendage wing field but altered the response to spatial information along the anteroposterior axis, it is suggested that activation by Ci and repression by Ubx are mediated through physically separable sites within knot cis-regulatory sequences (Hersh, 2005).

To identify which potential binding sites could be occupied by Ubx in vitro, DNaseI footprinting was performed on a 392 bp fragment (kn^Mel1599-1991) that includes the functional Ci site and the 156 bp required for repression in the haltere. This fragment is itself capable of driving expression in the wing, although at a significantly lower level than that driven by the full kn^Mel701-1991, and is repressed in the haltere. Four regions protected from DNaseI digestion by binding of Ubx were identified. These four regions include all TAAT core sequences present in the 392 bp fragment (10 in total) (Hersh, 2005).

Although Ubx site 1 is located only 4 bp from the Ci-binding site, it is still present in the kn^Mel701-1835 construct that is derepressed in the haltere, so this site alone is not sufficient to mediate repression by Ubx. To determine whether this site is necessary for repression by Ubx, Ubx site 1 alone (kn^Mel701-1991Ubx1KO) was mutated and no derepression of reporter gene expression in the haltere was observed. Therefore, Ubx Site 1, unlike individual Ubx-binding sites in the spalt enhancer, does not appear to contribute significantly to repression of this element by Ubx. Of the other regions protected by Ubx, the largest spans six TAAT core sequences and ~24 bp of sequence, and is located ~250 bp from the Ci binding site. Therefore, the DNA sequences necessary for repression in the haltere appear to be comprised of multiple, functional Ubx-binding sites that do not overlap with the activating Ci-binding site. This organization suggests that Ubx does not repress knot in the haltere by competing for activator binding sites (Hersh, 2005).

Individual Ubx-binding sites can additively contribute to repression in the haltere of the sal wing-specific regulatory element. To determine how individual Ubx binding sites in the knot element contribute to repression in the haltere, TAAT core sequences in Ubx site 1, and Ubx site 4 were independently mutated in kn^Mel701-1991 and these mutated constructs were introduced into flies. Elimination of these individual sites had no detectable effect on reporter gene expression in the haltere, so all 10 TAAT core sequences were mutated in kn^Mel701-1991, and this construct (kn^Mel701-1991KO) was introduced into flies. Elimination of all Ubx sequences results in de-repression in the haltere, demonstrating that some combination of these sites is required for repression in vivo. However, it is noted that the level of expression of this construct is lower than that observed in the deletion construct, kn^Mel701-1835. This difference was not expected and suggests the presence of additional regulatory sequences that contribute to repression in the haltere (Hersh, 2005).

To determine where additional potential regulatory sequences are located, sequence 3' of the kn^Mel701-1835 construct was restored. Addition of 43 bp (kn^Mel701-1878) was sufficient to partially restore repression in the haltere, suggesting the additional regulatory information was contained within this region. Deletion of this block of sequence (kn^Mel701-1991Delta) resulted in very weak, inconsistent de-repression in the haltere. By contrast, point mutations introduced at positions 1834-1837 (kn^Mel701-1991mut), the boundary of the derepressed kn^Mel701-1835 construct, resulted in consistent, partial, de-repression. Since this position is not a Ubx site, this result suggests that at least one transcription factor acts in addition to Ubx to repress knot in the haltere through this regulatory element. Mutation of both positions 1834-1837 and all Ubx TAAT core sequences (kn^Mel701-1991KOmut) resulted in full de-repression in the haltere, suggesting that Ubx and another repressor act together to reduce expression in the haltere through sequences located between kn^Mel1835-1991. The DNA sequence at kn^Mel1834-1837 does not clearly match any binding sites archived in transcription factor databases, and as the identity of the factor that may act with Ubx to repress knot in the haltere is not known (Hersh, 2005).

To understand how Ubx-regulated target gene networks evolve, it is crucial to determine how Ubx regulation of individual target genes evolves. Dissection of the knot wing regulatory element was combined with comparative genomics within Drosophila to establish how Ubx-responsive regulatory sequences in knot have evolved. The 156 bp knot repressor element from D. melanogaster was compared to D. pseudoobscura sequence, and neither significant sequence conservation nor a comparable cluster of potential Ubx-binding sites was observed in D. pseudoobscura. Because the expression pattern of knot is the same between these two species, these significant sequence differences suggest that regulation by Ubx is mediated through different regulatory sequences in D. pseudoobscura. Therefore, attempts were made to identify a functional regulatory element from D. pseudoobscura that could regulate reporter expression in the appropriate pattern (Hersh, 2005).

Using blocks of sequence identity as relational anchor points, a fragment from D. pseudoobscura (kn^Pse1-1935) that roughly corresponded to the kn^Mel1-2330 D. melanogaster fragment was amplified. This fragment was introduced into D. melanogaster and it was found that it could properly drive expression in the wing while repressing expression in the haltere. The kn^Pse1-1935 construct contained at its 3' end a cluster of 12 TAAT Ubx core binding sites. To determine if this region is important for repression by Ubx in D. pseudoobscura, a truncation of kn^Pse1-1935 was generated that eliminated the TAAT core sequences. This kn^Pse1-1643 construct appropriately drove expression in the wing, but now also drove haltere expression. Therefore, the region containing these putative Ubx-binding sites acts as a repressor element in the haltere (Hersh, 2005).

Interestingly, this functional cluster of Ubx-binding sites is conserved between D. pseudoobscura and D. melanogaster, and is located ~500 bp 3' of the kn^Mel1835-1991 sequence necessary for repression, just 3' of the limit of the 6.8 kb fragment originally isolated that contains the functional D. melanogaster knot regulatory element. Therefore, the knot regulatory region in D. melanogaster could potentially contain two sets of functional repressor input sites. To determine whether this second, conserved block can also function to repress the D. melanogaster knot regulatory element, the D. melanogaster sequence was attached to the de-repressed kn^Mel701-1835 construct. Addition of 222 nucleotides (kn^Mel2499-2722), homologous to the D. pseudoobscura sequence necessary for repression, to kn^Mel701-1835 (to generate kn^Melcomposite) restored repression in the haltere. Therefore, D. pseudoobscura has a single element (located between kn^Pse1643-1935) that represses expression of knot in the haltere, and this element is shared with D. melanogaster. However, D. melanogaster possesses a second element (located between kn^Mel1835-1991), not shared with D. pseudoobscura, that also functions to repress expression in the haltere (Hersh, 2005).

Next, attempts were made to determine whether Ubx-binding sites in the kn^Mel2499-2722 conserved element are sufficient to repress reporter expression, or whether this element also requires the action of a collaborating repressor. All Ubx core binding sites were mutated in this sequence and the mutated kn^Mel2499-2722KO sequence was attached to the de-repressed kn^Mel701-1835 (generating kn^MelcompositeKO). Whereas mutation of Ubx sites alone in kn^Mel701-1991KO did not fully de-repress in the haltere, mutation of Ubx sites in kn^MelcompositeKO was sufficient for complete de-repression in the haltere. Thus, the kn^Mel2499-2722 and kn^Mel1835-1991 repressor elements appear to be organized differently -- the former with input only from Ubx, and the latter with input from Ubx and an additional trans-acting factor (Hersh, 2005).

Does the presence of two elements in D. melanogaster indicate the acquisition of a new element in this lineage or the loss of an element in D. pseudoobscura? To analyze the distribution of these two regulatory elements in other drosophilids, the knot regulatory region was amplified from three additional Drosophila species – D. mauritiana, D. biarmipes and D. malerkotliana – phylogenetically intermediate between D. melanogaster and D. pseudoobscura. All three species have sequence similar to kn^Pse1643-1935, but also possess sequence similar to kn^Mel1835-1991 in varying degrees. For example, the core TAAT of Ubx site 3 is shared by all three additional species (though sequence surrounding the core is non-identical), whereas Ubx site 2 is found only in D. mauritiana. The most interesting pattern is observed for Ubx site 4. D. malerkotliana has only a single core Ubx sequence conserved with D. melanogaster; D. biarmipes has two conserved core sequences and two additional core sequences that are unique, and D. mauritiana has five of the six core sequences present in D. melanogaster. Therefore, in this sample of five drosophilid species, the pattern of an apparent accretion of Ubx-binding sites in this region is observed in the evolution of the D. melanogaster lineage (Hersh, 2005).

Thus, a wing-specific cis-regulatory element was identified for the gene knot. This regulatory element is activated in the wing by direct input from Ci and is repressed in the haltere by direct input from Ubx. The regulatory sequences governing activation and repression are physically separable, and the repression element was found not to be shared with D. pseudoobscura. A distinct functional repression element was identified in D. pseudoobscura that is shared with D. melanogaster, indicating that the entire knot wing regulatory region in D. melanogaster contains two apparently redundant repressor elements. One element appears to have been acquired in the course of the evolution of the D. melanogaster lineage. These results suggest that complete functional cis-regulatory elements, the units of function that selection is operating upon, may be larger and more diffuse than the minimal functional sequences typically defined by molecular dissection (Hersh, 2005).

Hox control of morphogen mobility and organ development through regulation of glypican expression

Animal bodies are composed of structures that vary in size and shape within and between species. Selector genes generate these differences by altering the expression of effector genes whose identities are largely unknown. Prime candidates for such effector genes are components of morphogen signaling pathways, which control growth and patterning during development. This study shows that in Drosophila the Hox selector gene Ultrabithorax (Ubx) modulates morphogen signaling in the haltere through transcriptional regulation of the glypican dally. Ubx, in combination with the posterior selector gene engrailed (en), represses dally expression in the posterior (P) compartment of the haltere. Compared with the serially homologous wing, where Ubx is not expressed, low levels of posterior dally in the haltere contribute to a reduced P compartment size and an overall smaller appendage size. One molecular consequence of dally repression in the posterior haltere is to reduce Dpp diffusion into and through the P compartment. These results suggest that Dpp mobility is biased towards cells with higher levels of Dally and that selector genes modulate organ development by regulating glypican levels (Crickmore, 2007).

Upon comparing Dpp signaling readouts in the wing and haltere, it was noticed that, in addition to a general narrowing of Dpp pathway activity, Dpp signaling was also asymmetric relative to its source (the AP organizer) in the haltere. Specifically, the P-Mad signal was stronger anterior to the AP organizer (roughly demarcated by the domain of peak P-Mad staining) than it was posterior to the organizer. To test if this asymmetry is due to asymmetric ligand distribution or differences in signal transduction, an extracellular staining protocol was used to examine the distribution of a Dpp::GFP fusion protein following its expression in AP organizer cells. In wing cells, Dpp::GFP was detected in a broad gradient on both sides of the AP organizer. In the haltere, the distribution of Dpp::GFP is limited in both directions owing to high tkv expression levels, but this restriction is stronger in the P direction. Dpp::GFP spread was abruptly halted a few cell diameters posterior to the haltere AP compartment boundary, contrasting with a tapering signal seen in the anterior direction. By contrast, the Gal4 driver used to express Dpp::GFP (ptc-Gal4) drove nearly symmetrical expression of a UAS-GFP transgene, demonstrating that the distribution of Dpp::GFP in the haltere is not due to asymmetric activity of the ptc-Gal4 driver. In both the wing and haltere, the pattern of extracellular Dpp::GFP was very similar to the P-Mad pattern, suggesting that Ubx does not affect Dpp signal transduction downstream of ligand binding, at least as detected with the anti-P-Mad antibody. Furthermore, in both the wing and haltere, a similar coincidence of extracellular Dpp::GFP and P-Mad patterns was observed when Dpp::GFP was expressed in clones. The correlation between the P-Mad and extracellular Dpp::GFP patterns in both the wing and haltere allows inference of extracelluar Dpp ligand distribution by visualizing P-Mad in the proceeding experiments (Crickmore, 2007).

In the wing, Dpp::GFP distribution and P-Mad staining were also asymmetric, owing to slightly higher levels of Tkv in the P compartment, which impedes diffusion. By contrast, because Tkv levels are similar on both sides of the AP boundary of the haltere, Tkv levels are unlikely to account for the Dpp signaling asymmetry in this appendage. This idea directly by providing uniform levels of UAS-tkv to both the haltere and wing. Under these conditions, P-Mad staining became symmetric in the wing, but remained asymmetric in the haltere. These results suggest that the more-restricted P-Mad staining in the P compartment of the wild-type haltere is due to a tkv-independent and haltere-specific anterior bias in the diffusion of Dpp (Crickmore, 2007).

In previous work, it was showed how the upregulation of the Dpp receptor, thickveins, in the haltere causes an overall decrease in Dpp mobility as compared with the wing, and consequently contributes to the small size of the haltere. This study shows that the HSPG dally is repressed in the P compartment of the haltere and that this regulation decreases the P:A ratio and overall size of the haltere. Posterior dally repression causes Dpp diffusion to be biased away from P cells, generating an AP asymmetry in Dpp signaling. The findings reported here therefore provide another instance wherein Ubx controls the extracellular signaling environment of the developing haltere and thereby distinguishes it from the wing (Crickmore, 2007).

The movement of most or all signaling molecules through tissues is regulated by HSPGs, including glypicans such as dally. In contrast to receptors, HSPGs control the distribution of multiple signaling molecules. Regulation of HSPG expression and activity by selector genes is therefore a potentially very powerful mechanism for shaping signaling pathway activation profiles and molding organ shapes and sizes. However, the promiscuity of HSPGs also makes it difficult to assign the morphological consequences of their expression patterns to the alteration of individual signaling pathways. Indeed, it is likely that the altered dally expression pattern in the haltere has implications for Hh, Wg and Dpp signaling, all of which control growth and patterning. This study has focused on the relationship between dally expression and Dpp signaling (Crickmore, 2007).

Dpp signaling is increased in dally⁺ clones and decreased in dally^- clones. These and other findings have suggested that Dally participates in the control of Dpp mobility. The current results add to these earlier observations by suggesting that variations in the levels of Dally between the cells of a tissue influence the direction and extent of Dpp diffusion. Specifically, it is proposed that in addition to simply being promoted by Dally, Dpp mobility is biased towards cells with higher Dally levels. This idea derives mainly from the observation that Dally can influence Dpp movement in a cell-non-autonomous manner. For example, when Dally levels are increased in the haltere P compartment, there is a shift in Dpp signaling from the A to the P compartments, as visualized by the levels of P-Mad. Similarly, knocking down Dally levels in the P compartment of the wing influences the extent and levels of P-Mad in the A compartment. If discontinuities in Dally levels can non-autonomously influence Dpp signaling across compartment borders, it follows that differences in Dally levels between cells within a compartment can also shape the Dpp signaling landscape. This might be important for wild-type wing development, where graded Dpp signaling represses dally, resulting in an inverse dally gradient that increases towards the lateral edge of the disc. It is suggested that this inverse dally gradient helps to attract Dpp to more lateral regions of the disc. Accordingly, in a dally-mutant wing disc, the Dpp gradient is less broad than in a wild-type wing disc. It is possible that other HSPGs control the mobility of signaling molecules in a similar manner (Crickmore, 2007).

Altering dally levels in either the A or P compartment changes relative compartment size, but only P compartment dally levels are relevant for total organ size. Two possible explanations are considered that link the P-specific dally repression seen in the haltere to a reduction in final organ size. Both of these scenarios (which are not mutually exclusive) focus on the role of P cells in producing Hh, which diffuses into A cells to instruct Dpp production and, consequently, controls final organ size. Importantly for both models, it was found that there is in fact less Hh detected in the P compartment of the wild-type haltere as compared with the wing. In the first model, the repression of dally reduces overall Hh production simply by reducing the size of the P compartment, which is a consequence of reduced Dpp signaling. In this scenario, fewer Hh-producing P cells result in less total Hh production from the P compartment, and therefore less Dpp produced in the A compartment. The logic of this potential mode of size regulation is interesting: a selector gene (Ubx) restricts growth factors (Wg and Dpp) from the pool of cells (the P compartment) that produces another growth factor (Hh). In the second scenario, dally repression may directly reduce the amount of Hh in the P compartment that can be transported into the A compartment. In support of this idea, Hh staining was found to be reduced in clones of cells where Dally levels are reduced through UAS-dallyRNAi (Crickmore, 2007).

Together, dally and dlp influence the mobility of all known morphogens in Drosophila. In addition to the compartmental regulation of dally, it is also noted that Dlp levels are generally lower throughout the haltere as compared with the wing. The haltere also lacks the domain of dlp repression seen at the DV boundary of the wing. Finally, it was also noticed that the expression of Notum-lacZ, an enhancer trap into a gene that encodes an HSPG-modifying enzyme, is different between the wing and haltere. The combined alteration of dally, dlp and Notum levels in the haltere is likely to have consequences for any signaling molecule that uses HSPGs for transport. When these observations are combined with those of earlier work showing that the levels of both Dpp and its receptor are regulated differently in the haltere and wing, and the observation that wg is repressed in the posterior haltere, a picture emerges in which selector genes alter the expression of multiple components of multiple signaling pathways to change morphogen signaling landscapes between tissues and thereby modify organ shapes and sizes. It is hypothesized that the summation of all signaling pathway changes may be sufficient to understand the size and shape differences between fundamentally similar epithelia such as the wing and haltere imaginal discs (Crickmore, 2007).

Evolutionarily conserved domains required for activation and repression functions of the Drosophila Hox protein Ultrabithorax

While testing the functions of deletion mutants in the Hox protein Ultrabithorax (Ubx), it was found that the embryonic repression function of Ubx on Distal-less transcription in limb primordia is highly concentration dependent. The steep sigmoidal relationship between in vivo Ubx concentration and Distal-less repression is dependent on the Ubx YPWM motif. This suggests that Ubx cooperatively assembles a multi-protein repression complex on Distal-less regulatory DNA with the YPWM motif as a key protein-protein interface in this complex. Deletion mutants also provide evidence for a transcriptional activation domain in the N-terminal 19 amino acids of Ubx. This proposed activation domain contains a variant of the SSYF motif that is found at the N termini of many Hox proteins, and is conserved in the activation domain of another Hox protein, Sex combs reduced. These results suggest that the N-terminal region containing the SSYF motif has been conserved in many Hox proteins for its role in transcriptional activation (Tour, 2005).

These results suggest that many Hox N-terminal regions possess a conserved transcriptional activation domain that includes an evolutionarily conserved SSYF motif. This region is required for the Drosophila Ubx and Scr proteins to activate four different downstream target genes with differing tissue-specific expression patterns. In Ubx, this domain is not just required for general functional activity; the deletion of Ubx N-terminal sequences dramatically reduces transcriptional activation function, but has no influence on repression function. In fact, the deletion of the region containing the Ubx variant of the SSYF motif (NSYF) appears to convert it from an activator to a repressor of dpp transcription (Tour, 2005).

The most relevant previous work on Hox N-terminal function in Drosophila embryos involved tests of mouse HoxA5 deletion mutants. Multiple regions N-terminal to the homeodomain are required for HoxA5 to activate a forkhead promoter-reporter gene. One of the required regions included amino acid residues 2-39, and it has been proposed that this region might be required for activation function or co-factor specificity. Similarity of Hox protein N-terminal sequences in Drosophila and mammals has been long noted, and is a characteristic of Hox proteins from a wide variety of animal species. In both mammal and Drosophila Hox proteins, the core conserved motif in this N-terminal region is a Ser-Ser-Tyr-Phe (SSYF) amino acid sequence (Tour, 2005).

The mechanism through which the Hox SSYF activation domain operates is not know: it may interact with DNA-binding transcription factors dedicated to transcriptional activation or with co-activator protein complexes. One possible SSYF interactor is the histone acetyltransferase CBP (CREB-binding protein). Mutations in the Drosophila CBP gene are dose-sensitive modifiers of Deformed and Ubx biological function. In addition, CBP increases the transactivation activity of human HOXB7 protein in breast cancer cells and interacts with the N-terminal region of HOXB7 in GST pull-down assays, in a manner that required the presence of the first 18 N-terminal amino acids of HOXB7. Mammalian CBP interacts with the first 141 N-terminal amino acids of human HOXD4 in co-immunoprecipitation assays, and increases transactivation activity of HOXD4-PBX complexes on a synthetic element containing five HOX/PBX sites in cultured human embryonic kidney cells. Another possibility is that the N terminus interacts with the IkappaBalpha protein, which binds to the N-terminal regions of human HOXB7, a region of HOXB7 that is required for normal function in a murine myelomonocytic cell line (Tour, 2005).

A detailed analysis of Ubx domains required for transactivation function in Drosophila cultured S2 cells, which are derived from embryonic hemocytes, has been carried out. In these assays, the N-terminal 67 amino acid residues were not required for Ubx-dependent transcriptional activation. The disparity between the current results and the previous results might be explained by the different assay systems (cultured S2 cells versus embryos), the different target elements, and/or the exact size and extent of the deletion mutants that were tested (Tour, 2005).

The current results indicate that at least for its limb and Dll repression functions, Ubx contributes to a cooperative on/off switch over a small concentration range. When Dll repression is plotted as a function of Ubx concentration, the best-fit curve has a Hill slope of 4.9±2.2. These results suggest a highly cooperative assembly of a multiprotein repression complex containing Ubx on Dll regulatory DNA. Although the repression dose-response curves cannot be extrapolated into the number of cooperative protein-protein interactions within a repression complex, they are a surprisingly good fit to a model in which the Ubx-mediated repression of a Dll limb enhancer requires at least five clustered DNA sites that cooperatively bind two molecules of Ubx, Extradenticle (Exd) and Homothorax, while the fifth site binds the Sloppy paired 1 protein. The high sensitivity of Ubx phenotypes to concentration may explain why previous experiments using ectopic expression of Ubx have come to different conclusions, and illustrates why the validity of conclusions from ectopic expression studies should be interpreted with caution, unless great care is taken to achieve near-normal physiological levels (Tour, 2005).

Why is the Ubx repressive effect on Dll so concentration sensitive? It is instructive to look at other biological systems with similar concentration-dependent transcriptional switches. For example, the steep concentration dependence of the lambda transcriptional repressor allows prophages in E. coli cells to switch, at crucial levels of cellular distress, from one stable state to another, lysogenic to lytic. For Ubx, one likely reason for the highly concentration-dependent effects on Dll expression and limb development is to ensure that all the cells in a limb field are stably programmed to adopt either the limb state, or body wall fate. At least in extant Drosophila, a mosaic appendage that developed from a mixed field of limb and body wall cells would presumably be little benefit to the animal that carried it, and thus selected against during evolution (Tour, 2005).

Tests of mutant Hox proteins in Drosophila and in mice have demonstrated the importance of the YPWM motif for Hox function in vivo, although both loss- and gain-of-function phenotypes were observed. In vitro, the YPWM region has been shown to mediate Hox interactions with the PBC family of homeodomain proteins. The PBC proteins (Exd protein in Drosophila, Pbx proteins in mammals) bind cooperatively with Hox proteins on composite DNA sites, and are important co-factors in the regulation of many Hox target genes (Tour, 2005).

A Ubx protein with a YAAA substitution for YPWM exhibits reduced cooperative binding with Exd on a consensus composite Ubx-Exd DNA-binding site. Reduced affinity between UbxDeltaYPWM and Exd might compromise the assembly of the entire repression complex, resulting in an inefficient transcriptional repression of Dll in the anterior segmental compartments (Tour, 2005).

The in vivo results are also consistent with models in which the YPWM region contributes in other ways to repression cooperativity. For example, the YPWM region appears to influence Hox activation and repression functions in a manner that is independent of its role in enhancing the affinity of Hox/PBC protein complexes for binding sites. In vitro, Ubx is also known to bind cooperatively to DNA in homomeric complexes, and the YPWM motif might be required for the formation of such complexes on Dll regulatory sequences (Tour, 2005).

No single deletion abolishes the Ubx repression function, although some regions are required for robust repression. Hox protein repression function appears to be quite complex. Embryonic tests of the deletion mutants, suggest that Ubx contains multiple regions that additively contribute to repression. In additi