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, 1999).

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 addition, other results suggest that the homeodomain also contributes directly to transcriptional repression function in a manner that is independent of its DNA-binding function (Tour, 2005).

The deletion of the Ubx YPWM region has little detectable effect on the transcriptional activation of the dpp and tsh genes. Because exd genetic function is required for normal levels of dpp and tsh activation in Ubx-expressing cells, this result is difficult to reconcile with a simple model in which the YPWM motif is required for Exd recruitment to activation target sites in dpp and tsh enhancers. However, it is consistent with studies that tested the effect of YPWM mutations on the activation abilities of the Labial and Abd-A Hox proteins in embryos. A YPWM to AAAA mutant of Labial is a more potent activator than wild-type Labial protein of a sequence derived from the Hoxb1 autoregulatory region, whereas a YPWM-to-AAAA mutant of Abd-A converted this protein from a repressor into an activator of dpp transcription. In addition, this YPWM mutation has no effect on the activation function of Abd-A on wingless. The ability of Labial and Abd-A YPWM mutants to retain their transactivation functions is correlated with their ability to bind Exd in vitro in a YPWM-independent fashion. The YPWM-independent interactions between Hox proteins and Exd can be mediated by Hox homeodomains and the C-terminal regions (Tour, 2005).

Since the Ubx-responsive elements from dpp and tsh loci possess a mixture of Ubx monomer and Ubx-Exd heterodimer-binding sites, possible reasons for the ability of the Ubx YMPM deletion mutant to activate these downstream target genes are: (1) Hox activation of target genes often involves a mixture of Exd-dependent and Exd-independent functions; (2) removal of the YPWM motif does not completely abolish Exd-Ubx binding interactions, and (3) the YPWM apparently serves other functions besides binding Exd in the context of developing embryos (Tour, 2005).

scylla and charybde are transcriptionally regulated targets of Dpp/Zen-mediated signal transduction and appear more generally to be downstream targets of homeobox regulation

Robotic methods and the whole-genome sequence of Drosophila melanogaster were used to facilitate a large-scale expression screen for spatially restricted transcripts in Drosophila embryos. In this screen, scylla (scyl) and charybde (chrb), which code for dorsal transcripts in early Drosophila embryos and are homologous to the human apoptotic gene RTP801, were identified. In Drosophila, both gene products are transcriptionally regulated targets of Dpp/Zen-mediated signal transduction and appear more generally to be downstream targets of homeobox regulation. Gene disruption studies revealed the functional redundancy of scyl and chrb, as well as their requirement for embryonic head involution. From the perspective of functional genomics, these studies demonstrate that global surveys of gene expression can complement traditional genetic screening methods for the identification of genes essential for development: beginning from their spatio-temporal expression profiles and extending to their downstream placement relative to dpp and zen, these studies reveal roles for the scyl and chrb gene products as links between patterning and cell death (Scuderi, 2006).

In addition to the dorsal field of scyl and chrb expression in early embryos, segmental expression along the anteroposterior axis suggests that scyl and chrb may also be sensitive to regulation by homeobox genes other than zen. Both scyl and chrb transcripts are localized in anteroposteriorly segmented patterns in ventral regions of the blastoderm and in the three thoracic segments of stage 13 embryos. In stage 13 embryos, genes of the bithorax complex (BX-C) repress expression of target genes in abdominal segments, restricting their expression to the thorax. The expression of scyl and chrb was examined in BX-C mutant embryos: expansion was observed of thoracic expression into abdominal segments of mutant embryos, thereby placing scyl and chrb downstream of the BX-C homeobox transcription factors, Ubx, Abd-A and/or Abd-B. Consistent with this observation is the finding (Chauvet, 2000) that Ubx binds to regulatory regions of both scyl and chrb (Scuderi, 2006).

Hox control of organ size by regulation of morphogen production and mobility

Selector genes modify developmental pathways to sculpt animal body parts. Although body parts differ in size, the ways in which selector genes create size differences are unknown. This study investigated how the Drosophila Hox gene Ultrabithorax (Ubx) limits the size of the haltere, which, by the end of larval development, has ~fivefold fewer cells than the wing. It was found that Ubx controls haltere size by restricting both the transcription and the mobility of the morphogen Decapentaplegic (Dpp). Ubx restricts Dpp's distribution in the haltere by increasing the amounts of the Dpp receptor, thickveins. Because morphogens control tissue growth in many contexts, these findings provide a potentially general mechanism for how selector genes modify organ sizes (Crickmore, 2006).

Changes in body part sizes have been critical for diversification and specialization of animal species during evolution. The beaks of Darwin's finches provide a famous example for how adaptation can produce variations in size and shape that allowed these birds to take advantage of specialized ecological niches and food supplies. Sizes also vary between homologous structures within an individual. For example, vertebrate digits and ribs vary in size, likely due to the activities of selector genes such as the Hox genes. Although the control of organ growth by selector genes is likely to be common in animal development, little is known about the mechanisms underlying this control (Crickmore, 2006).

The two flight appendages of Drosophila, the wing and the haltere, provide a classic example of serially homologous structures of different sizes. Halteres, appendages used for balance during flight, are thought to have been modified from full-sized hindwings during the evolution of two-winged flies from their four-winged ancestors. All aspects of haltere development that distinguish it from a wing, including its reduced size, are under the control of the Hox gene Ultra-bithorax (Ubx), which is expressed in all haltere imaginal disc cells but not in wing imaginal disc cells. At all stages of development, haltere and wing primordia (imaginal discs) are different sizes. In the embryo, the wing primordium has about twice as many cells as the haltere primordium. By the end of larval development, the wing disc has ~five times more cells (~50,000) than the haltere disc (~10,000). The wing and haltere appendages will form from the pouch region of these mature discs. The final step that contributes to wing and haltere size differences occurs during metamorphosis, when wing, but not haltere, cells flatten, thus increasing the surface area of the final appendage (Crickmore, 2006).

To confirm that Ubx has a postembryonic role in limiting the size of the haltere disc, Ubx^– clones were generated midway through larval development. Haltere discs–bearing large Ubx^– clones generated at this time become much larger than wild-type discs. Ubx could limit haltere size cell-autonomously by, for example, slowing the cell cycle of haltere cells relative to wing cells. This was tested by comparing the sizes of isolated Ubx^– clones in the haltere with those of their simultaneously generated wild-type twin clones. Contrary to the prediction of a cell-autonomous function for Ubx in size control, Ubx mutant clones did not grow larger than their twins, a result that is consistent with earlier experiments suggesting that wing and haltere cells have similar mitotic rates during development. Hence, Ubx limits the size of the haltere during larval development by modifying pathways that control organ growth cell-nonautonomously (Crickmore, 2006).

In the fly wing, Decapentaplegic (Dpp) [a long-range morphogen of the bone morphogenetic protein (BMP) family] has been shown to promote growth. In both the wing and the haltere, Dpp is produced and secreted from a specialized stripe of cells called the AP organizer, which is induced by the juxtaposition of anterior (A) and posterior (P) compartments, two groups of cells that have separate cell lineages. The AP organizer is a stripe of A cells that are instructed to synthesize Dpp by the short-range morphogen Hedgehog (Hh) secreted from adjacent P compartment cells. Dpp has a positive role in appendage growth. When more Dpp is supplied to the wing disc, either ectopically or within the AP organizer, more cells are incorporated into the developing wing field. Conversely, mutations that reduce the amount of Dpp lead to smaller wings (Crickmore, 2006).

A comparison of the expression patterns of Dpp pathway components in the wing and the haltere demonstrates that Ubx is modifying this pathway. Compared with the wing, the stripe of dpp expression in the haltere was reduced in both its width and intensity, as reported by a lacZ insertion into the dpp locus (dpp-lacZ). There was also a difference in the profile of Dpp pathway activation, as visualized by an antibody that detects P-Mad, the activated form of the Dpp pathway transcription factor Mothers against Dpp (Mad). In the wing, P-Mad staining was low in the cells that transcribe dpp. Immediately anterior and posterior to this activity trough, P-Mad labeling peaked in intensity and then gradually decayed further from the Dpp source, revealing a bimodal activity gradient. In contrast, in the haltere intense P-Mad staining was detected only in a single stripe of cells that overlaps with Dpp-producing cells of the AP organizer (Crickmore, 2006).

Because of the coincidence between dpp transcription and peak P-Mad staining in the haltere, it was hypothesized that Dpp might be less able to move from haltere cells that secrete this ligand. This idea was tested by generating clones of cells in both wing and haltere discs in which the actin5c promoter drove the expression of a green fluorescent protein (GFP)–tagged version of Dpp (Dpp:GFP). By using an extracellular staining protocol to analyze simultaneously generated clones, Dpp:GFP and P-Mad were observed much further from producing cells in the wing than in the haltere. These observations strongly suggest that, compared with the wing, Dpp's mobility—and consequently the range of Dpp pathway activation—is reduced in the haltere (Crickmore, 2006).

Whether the decreased production of Dpp in the haltere contributes to the different pattern of pathway activation observed in this tissue compared with the wing was tested. This is unlikely because, even in haltere discs that overexpress Dpp in its normal expression domain, peak P-Mad staining was still observed close to Dpp-expressing cells. Despite increased dpp expression, no P-Mad activity trough was observed in these haltere discs. Further, although they become larger, these discs remained smaller than wild-type wing discs. It is concluded that the decreased Dpp production in the haltere contributes to its reduced growth, but there must be mechanisms that also limit the extent of Dpp pathway activation, even in the presence of increased Dpp production (Crickmore, 2006).

One way in which Dpp's activation profile can be modified is by varying the production of the type I Dpp receptor, Thick veins (Tkv). In the wing, tkv expression is low within and around the source of Dpp, resulting in low Dpp signal transduction in these cells and robust Dpp diffusion. Low tkv expression in the medial wing is due to repression by both Hh and Dpp. Accordingly, tkv expression is highest in lateral regions of the wing disc, where Hh and Dpp signaling are low. In contrast to the wing, tkv transcription and protein levels were high in all cells of the haltere. Thus, the more restricted Dpp mobility and P-Mad pattern in the haltere may result from a failure to repress tkv medially. To test this idea, all cells of the wing disc were supplied with uniform UAS-tkv⁺ expression, to mimic the haltere pattern. The resulting P-Mad pattern in these wing discs was very similar to that found in the wild-type haltere: The P-Mad trough was gone, and the activity gradient was compacted into a single stripe that coincides with Dpp-producing cells. Conversely, lowering the amount of Tkv in the haltere by expressing an RNA interference (RNAi) hairpin construct directed against tkv (UAS-tkvRNAi) in Dpp-producing cells induced a bimodal pattern of P-Mad staining similar to that of the wild-type wing disc. Thus, different amounts of Tkv result in qualitative differences in the P-Mad profiles of the wing and the haltere (Crickmore, 2006).

It was hypothesized that the more limited pathway activation in the haltere might contribute to its smaller size. If correct, increasing tkv expression in the wing should reduce its size. Adult wings from flies expressing uniform UAS-tkv⁺ were ~30% smaller than control wings; however, wing cell size remained the same. Similar results were seen in staged imaginal discs and when UAS-tkv⁺ expression was limited to the wing and the haltere. Conversely, reducing Tkv amounts by uniformly expressing UAS-tkvRNAi in wings and halteres increased haltere size by 30 to 60%. In a complementary experiment, tkv transcription was reduced in the haltere by expressing a known tkv repressor, master of thickveins (mtv). In this experiment, haltere discs were measured instead of the adult appendage; it was consistently found that the appendage-generating region of these discs increased in size by ~40%. Thus, different amounts of Tkv not only affect Dpp pathway activation but also affect organ size. The fact that manipulating only Tkv production does not fully transform the sizes of these appendages suggests that additional mechanisms, such as the reduced amounts of dpp transcription and the modulation of other morphogen pathways by Ubx, also contribute to size regulation. Consistently, when Dpp production is decreased in wing discs that uniformly express UAS-tkv⁺, wing size was reduced more than it was by either single manipulation (Crickmore, 2006).

Next, how Ubx up-regulates tkv in the haltere was addressed. tkvlacZ expression and amounts of Tkv protein were cell-autonomously reduced in medial Ubx^– clones, whereas lateral Ubx mutant tissue retained high amounts of Tkv. Because tkv is repressed by Dpp and Hh signaling in the wing, these results suggest that, in the haltere, these signals are not able to repress tkv. Consistently, activation of the Dpp pathway by expressing a constitutively active form of Tkv (Tkv^QD) resulted in cell-autonomous tkv-lacZ repression in the wing pouch, whereas repression is not observed in the corresponding region of the haltere disc (Crickmore, 2006).

In Ubx mosaic haltere discs, it was also found that medial Ubx⁺ tissue showed stronger P-Mad staining than Ubx^– tissue at the same distance from the Dpp source. This observation is interpreted as evidence that Ubx⁺ tissue is more effective at trapping and transducing Dpp than Ubx^– tissue because of higher Tkv production in Ubx⁺ cells (Crickmore, 2006).

To further understand the control of tkv by Ubx, the known tkv repressor, mtv, was examined. In medial wing disc cells, mtv expression is approximately complementary to tkv expression, and mtv^– clones in this region of the wing disc cell autonomously derepressed tkv. In the haltere, very low mtv-lacZ expression was detected in the cells that stained strongly for P-Mad, suggesting that mtv is repressed by Dpp in this appendage. Accordingly, strong repression of mtv-lacZ was seen in UAS-tkv^QD-expressing haltere pouch clones, whereas weak or no repression was seen in analogous wing clones. It was also found that, as expected, Ubx^– clones in the medial haltere cell autonomously derepressed mtv-lacZ (Crickmore, 2006).

In the wing, Dpp and mtv are mandatory repressors of tkv: In the absence of either, tkv expression is high. In the haltere in the presence of Ubx, Dpp is a repressor of mtv. Consequently, high levels of these obligate tkv repressors (Dpp signaling and mtv) do not coexist in the haltere, resulting in tkv derepression. Consistent with this model, when mtv expression was forced in the medial haltere, where it coexists with Dpp signaling, it repressed tkv-lacZ. It is noted, however, that Ubx is likely to control tkv through additional means, because mtv mutant wing clones did not derepress tkv-lacZ expression to haltere levels, and ectopic mtv in the haltere did not repress tkv-lacZ expression to the extent seen in the medial wing (Crickmore, 2006).

Because of high Tkv production in the wild-type haltere disc, peak Dpp signal transduction occurs in the AP organizer, the same cells that transduce the Hh signal. Thus, in the haltere, the activity profiles for these two signal transduction pathways coincide with each other. In contrast, low tkv expression in the wing AP organizer results in two peaks of Dpp signaling that are on either side of Hh-transducing cells. This difference will have important consequences for the expression of genes that are targets of both pathways. For example, dpp is activated by Hh and repressed by Dpp signaling. In the haltere, these two conflicting inputs occur in the same cells, possibly contributing to reduced dpp expression compared with the wing. Ubx^– clones cell-autonomously up-regulated dpp-lacZ in the haltere. To test whether Ubx lowers dpp transcription in part by aligning Dpp and Hh signaling, uniform UAS-tkv⁺ was expressed in the dorsal half of the wing disc. As a result, in this region of the wing disc both signals peaked in the same cells, and dpp-lacZ expression was reduced compared with the ventral half of these wing discs. Conversely, expressing tkvRNAi in dorsal haltere cells increased dpplacZ expression. Thus, Ubx reduces dpp transcription in part by changing where peak Dpp signaling occurs in the disc. Ubx is likely to reduce dpp expression in additional ways, because increasing tkv expression does not lower dpplacZ expression to that observed in wild-type haltere. Nevertheless, varying the relative spatial relationships between signal transduction pathways is a potentially powerful mechanism for modifying the outputs from commonly used pathways. It is suggested that selector genes may work through molecules that control ligand distribution to vary the spatial relationships between these and other signal transduction pathways in diverse contexts during development (Crickmore, 2006).

The finding that increased tkv expression results in decreased dpp transcription reveals an unexpected link between Dpp mobility and Dpp production. Because of this link, the above experiments do not discriminate between growth effects due to differences in Dpp mobility per se as opposed to secondary consequences on Dpp production. To distinguish between these scenarios, use was made of a compartment-specific Ubx regulatory allele, posterior bithorax (pbx), that lacks detectable Ubx in the P compartment when paired with a Ubx null allele but still has normal Ubx expression in the A compartment. Consequently, in pbx/Ubx^– haltere discs, the P compartment increased in size such that the P:A size ratio was 1.45; the P:A ratio of +/Ubx^– haltere discs was ~0.35. It is suggested that Dpp more readily diffuses into and through the P compartments of pbx/Ubx^– discs because of the wing-like expression pattern of tkv and that this wing-like diffusion results in its robust growth (Crickmore, 2006).

To test whether differences in Tkv-regulated Dpp diffusion affect tissue growth independently of an effect on Dpp production, the consequences of expressing UAS-tkv⁺ uniformly in pbx/Ubx^– haltere discs were examined. If Tkv's effect on growth is mediated only by lowering Dpp production, both compartments should be reduced in size and thus maintain the same size ratio. However, if reducing Dpp mobility directly affects growth, the P compartment should be reduced in size more than the A compartment, which, in pbx/Ubx^– discs, already has high tkv expression. It was found that expressing uniform tkv⁺ in pbx/Ubx^– discs decreased the size of the P compartment more than the A compartment, resulting in a P:A ratio of 0.83. Because uniform tkv⁺ returned the P:A ratio back to the wild-type ratio by ~56%, these results suggest that this single variable is sufficient to provide ~50% rescue of the size of an otherwise Ubx mutant P compartment (Crickmore, 2006).

This study has investigated the mechanism underlying a classic yet poorly understood phenomenon in biology: how size variations are genetically programmed in animal development. Many experiments show that organ size is not governed by counting cell divisions but instead depends on disc-intrinsic yet cell-nonautonomous mechanisms, possibly relying on morphogen signaling. The results support this idea by showing that alterations in a morphogen gradient contribute to size differences between appendages. In the example investigated here, Ubx limits the size of the haltere by reducing both Dpp production and Dpp mobility. Moreover, both of these effects are due, in part, to higher tkv expression in the medial haltere. In many morphogen systems, the receptors themselves have been shown to control the distribution of the ligand and, consequently, pathway activation. This study shows that a selector gene exploits this phenomenon to modify organ size (Crickmore, 2006).

Although the mechanism by which Dpp controls proliferation is not fully understood, recent results argue that, in the medial wing disc, cells may compare the amount of Dpp transduction with their neighbors, whereas lateral cells proliferate in response to absolute Dpp levels. The results suggest several ways in which the altered Dpp gradient in the haltere could limit its growth. First, proliferation of lateral haltere cells may be limited because they perceive less Dpp. Second, the narrower Dpp gradient results in fewer cells exposed to the gradient in the medial haltere. Another notable difference is that, because there are two peaks of Dpp signaling in the wing but only one in the haltere, the wing has four distinct slopes whereas the haltere has only two. The less complex Dpp activity landscape of the haltere may also contribute to its reduced growth (Crickmore, 2006).

On the basis of these results, it is suggested that altering the shape and intensity of morphogen gradients may be a general mechanism by which selector genes affect tissue sizes in animal development. Consistent with this view, wingless (wg), another long-range morphogen in the wing, is partially repressed in the haltere. Intriguingly, some of the size and shape differences in the beaks of Darwin's finches are controlled by alterations in the production of the Dpp ortholog BMP4. The results suggest that differences in the diffusion of this ligand may also contribute to the range of beak morphologies that have evolved in these species (Crickmore, 2006).

Negative regulation of Egfr/Ras pathway by Ultrabithorax during haltere development in Drosophila

In Drosophila, wings and halteres are the dorsal appendages of the second and third thoracic segments, respectively. In the third thoracic segment, homeotic selector gene Ultrabithorax (Ubx) suppresses wing development to mediate haltere development. Halteres lack stout sensory bristles of the wing margin and veins that reticulate the wing blade. Furthermore, wing and haltere epithelia differ in the size, shape, spacing and number of cuticular hairs. The differential development of wing and haltere, thus, constitutes a good genetic system to study cell fate determination. Down-regulation of Egfr/Ras pathway is critical for haltere fate specification: over-expression of positive components of this pathway causes significant haltere-to-wing transformations. RNA in situ, immunohistochemistry, and epistasis genetic experiments suggest that Ubx negatively regulates the expression of the ligand vein as well as the receptor Egf-r to down-regulate the signaling pathway. Electromobility shift assays further suggest that Egf-r is a potential direct target of Ubx. These results and other recent findings suggest that homeotic genes may regulate cell fate determination by directly regulating few steps at the top of the hierarchy of selected signal transduction pathways (Pallavi, 2006).

To identify potential targets of Ubx and thereby mechanism of its function, a gain-of-function genetics strategy was employed. Ubx-GAL4 driver, is expressed in the entire anterior compartment of the haltere imaginal disc. Ubx-GAL4 is also a null allele of Ubx and exhibits characteristic dominant phenotype; the presence of wing-type sensory bristles in the capitellum of the haltere. This GAL4 driver provides a fortuitous sensitive background to carry out large-scale screens for identifying the suppressors and enhancers of Ubx function, which otherwise may be less efficient in a wild type background. Indeed, over-expression of Vestigial (Vg), a pro-wing gene and a target of Ubx function, in the developing haltere results in very high degree of haltere-to-wing homeotic transformations, A candidate gene screen was employed to identify downstream targets of Ubx, in which various genes known to be involved in wing development were ectopically expressed in the developing haltere using the Ubx-GAL4 driver. Criterion for defining haltere-to-wing transformation in this study was the presence of wing-type sensory bristles, although increase in haltere size and enhanced pigmentation was frequently observed. For comparison between different genotypes, the degree of transformation was estimated by counting the number of sensory bristles on the haltere capitellum. UAS stocks for a large number of such wing patterning genes were crossed to Ubx-GAL4 driver and were scored for enhancement and suppression of the dominant phenotype of heterozygous Ubx. Progeny for many of the crosses resulted in early embryonic or early larval lethality, reflecting the fact that the GAL4 driver expresses at early stages during embryonic development. Nevertheless, strong haltere-to-wing transformations were observed upon over-expression/mis-expression of most of the positive components of the Egfr/Ras pathway. For example, the bristle number in the capitellum of the Ubx-GAL4 haltere increased when positive components such as Vn or Egf-r were over-expressed and over-expression of negative components such as Aos completely suppressed the heterozygous Ubx phenotype (Pallavi, 2006).

A significant finding of this study is the down-regulation of Egfr/Ras pathway in haltere discs by Ubx. Earlier reports suggest that a short-range signal originating from the D/V boundary activates Egfr/Ras pathway in a zone of cells on the edges of the D/V boundary and that this activation is essential for vg transcription (Nagaraj, 1999). Egfr/Ras pathway has also been implicated in the developmental events along the A/P axis: in wing vein specification. Consistent with the down-regulation of both A/P and D/V signaling events in haltere discs, expression of most of the Egfr/Ras pathway components is repressed in the entire haltere pouch. Observations on the strengths of haltere-to-wing transformations (at the margin bristle level) in different genetic backgrounds establish the specificity of genetic interactions between Egfr/Ras pathway and Ubx during haltere development (Pallavi, 2006).

The abovementioned results on the down-regulation of Egfr/Ras pathway in haltere discs by Ubx are consistent with the previously reported genetic screen for the modifiers of homeotic genes, which indicates that Ras1 activity modulates functions of the homeotic loci Sex combs reduced (Scr) and Ubx. For example, haploinsufficient (haltere-to-wing) phenotype of Ubx¹⁰⁹/+ is significantly enhanced in Gap1⁻Ubx¹⁰⁹/Gap1⁻ adults (Gap1 is a negative regulator of the Egfr/Ras pathway). This effect of Gap1 is reversed in Gap1⁻Ubx¹⁰⁹/Gap1⁻Ras^e1b individuals due to the antagonistic roles of Gap1 and Ras1 in the Egfr/Ras pathway (Pallavi, 2006).

All the components of the Egfr/Ras pathway tested so far are differentially expressed between wing and haltere discs. This suggests the utility of developing wings and halteres as assay systems to identify novel components of Egfr/Ras pathway. Indeed, enhancer-trap screens and microarray analyses to identify genes that are differentially expressed between wing and haltere discs have resulted in the identification of CG32062 (Drosophila homologue of human ataxin-2 binding protein) and Mapmodulin (Drosophila homologue of human Inhibitor-1 of protein phosphatase-2A) as potential modulators of Egfr/Ras pathway (Pallavi, 2006).

The activation of dpERK1/ERK2 and aos in the haltere pouch by the ectopic expression of vn or Egf-r suggests that Ubx regulates Egfr/Ras pathway at ligand as well as receptor levels. Clonal analysis of Ubx function (loss of Ubx in haltere discs and gain in wing discs) demonstrates that Ubx controls vn expression in a cell-autonomous manner. Inability of ectopic Hh to activate vn expression in haltere discs suggests that Ubx functions downstream of Hh to repress vn expression. Putative Ubx-binding sites are present in the cis-regulatory regions of both vn and Egf-r, further suggesting their direct regulation by Ubx. Indeed, electromobility shift experiments suggest that, at least, Egf-r is probably a direct target of Ubx function. Thus, it is likely that Ubx independently down-regulates both vn and Egf-r. Ubx-mediated down-regulation of vn and Egf-r appears to be critical; over-expression of normal or the activated form of Egf-r induced stronger phenotypes when expressed in aos heterozygous background than in Ubx heterozygous background. However, Ubx may exert some influence on the pathway downstream of the receptor, since the strength of the phenotypes induced by the over-expression of Vn or Egf-r was stronger in Ubx heterozygous background. At dpERK1/ERK2 level too, the activation was stronger when Egf-r was over-expressed in Ubx heterozygous genetic background than in the wild type background (Pallavi, 2006).

It is interesting to note that Ubx regulates Egfr/Ras pathway at the level of the receptor itself. Although Egfr/Ras pathway is auto-regulated at several levels including the transcription of Egf-r itself, external factors regulating Egfr/Ras pathway during various developmental events mostly act at the level of the ligand/s or downstream effectors such as MAPK or transcription factors Yan, Pointed, etc. This study has found that the receptor itself is a direct target of Ubx indicating a novel mode of regulation of this pathway (Pallavi, 2006).

Specification of the larval oenocyte has been shown to be dependent on the regulation of just one principal target Rho by the homeotic gene abdominal A. Similarly, Hox proteins AbdA and AbdB specify the lineage of the embryonic NB6-4 neuroblast in abdominal segments by down-regulating CycE. Differential expression of CycE is both required and sufficient to generate segmental differences in NB6-4 lineage. This study reports that down-regulation of Vn and Egf-r is critical for Ubx-mediated suppression of wing margin bristles in the haltere. These results suggest that one common mechanism by which homeotic genes may regulate cell fate determination is by directly regulating few steps at the top of the hierarchy of selected signal transduction pathways. In contrast, Wingless and Decapentaplegic signaling pathways, which regulate more complex traits such as wing growth and shape, are regulated by Ubx at multiple levels in the hierarchy of those pathways (Pallavi, 2006 and references therein).

Although absence of veins in the haltere could be attributed to down-regulation of Egfr/Ras pathway, activation of sensory bristle development in Ubx⁺/Ubx⁺ halteres over-expressing positive components of Egfr/Ras pathway suggests a role for this pathway in cell fate specification in the wing margin. So far, no direct role for Egfr/Ras pathway has been assigned in the specification of sensory bristles of the wing margin, although it is known to specify macrochaete of the notum. Indeed, preliminary investigations suggest that Wg pathway induces EGFR/Ras pathway expression in cells immediately adjacent to the D/V boundary, and the latter pathway is required and sufficient to specify sensory organs of the wing margin (Pallavi, 2006).

The bristle development in the transformed halteres appears to be organized in two parallel rows when various components of Egfr/Ras pathway are over-expressed in Ubx heterozygous background, while the bristles are positioned in a disorganized way when phenotypes are induced in wild type background. This could be due to partial de-repression of D/V signaling in Ubx heterozygous background, which may allow appropriate positioning of the zone of margin bristle development (Pallavi, 2006).

Sex combs reduced and Ultrabithorax establish differential expression of the proneural gene achaete by modifying expression of the achaete prepattern regulator Delta in Drosophila legs

Many studies have shown that morphological diversity among homologous animal structures is generated by the homeotic (Hox) genes. However, the mechanisms through which Hox genes specify particular morphological features are not fully understood. This issue was addressed by investigating how diverse sensory organ patterns are formed among the legs of the Drosophila adult. The Drosophila adult has one pair of legs on each of its three thoracic segments (the T1-T3 segments). Although homologous, legs from different segments have distinct morphological features. Focus was placed is on the formation of diverse patterns of small mechanosensory bristles or microchaetae (mCs) among the legs. On T2 legs, the mCs are organized into a series of longitudinal rows (L-rows) precisely positioned along the leg circumference. The L-rows are observed on all three pairs of legs, but additional and novel pattern elements are found on T1 and T3 legs. For example, at specific positions on T1 and T3 legs, some mCs are organized into transverse rows (T-rows). The T-rows on T1 and T3 legs are established as a result of Hox gene modulation of the pathway for patterning the L-row mC bristles. The findings suggest that the Hox genes, Sex combs reduced (Scr) and Ultrabithorax (Ubx), establish differential expression of the proneural gene achaete (ac) by modifying expression of the ac prepattern regulator, Delta (Dl), in T1 and T3 legs, respectively. This study identifies Dl as a potential link between Hox genes and the sensory organ patterning hierarchy, providing insight into the connection between Hox gene function and the formation of specific morphological features (Shroff, 2007).

It is proposed that T-rows are formed on T1 and T3 legs in response to Scr or Ubx alteration of the L-row prepattern via repression of Dl expression. Dl is expressed in narrow longitudinal stripes that correspond to the L-row primordia. Dl-expressing cells in the L-row primordia signal to adjacent cells to activate N signaling and repress ac expression in the hairy-OFF interstripes and in one hairy-ON interstripe, between the L-row 1 and 8 proneural fields. It is suggested that in T1 and T3 legs, reduction of Dl expression in cells with high-levels of Scr or Ubx establishes a zone where there is no repressive influence on ac expression, resulting in expression of Ac in broad domains from which the T-row precursors will be selected. Cells in the center of T-row primordia are presumably out of range of the Dl signaling that takes place at the interface of Dl-expressing and Dl-non-expressing cells. The anterior and posterior boundaries of Ac expression in the T-row primordia of T1 prepupal legs are likely established by Dl/N signaling. In T3 legs, in contrast, it appears that Hairy rather than Dl/N signaling establishes the boundaries of ac on either side of the T-row primordia. Reduced Dl expression in the T-row primordia of T3 legs, however, is likely required to establish a broader domain of Ac expression than would be observed in the corresponding domain of T2 legs (Shroff, 2007).

A key feature of the model for mC patterning is that position-specific expression of ac expression in the mC proneural fields is established mainly by repression and that differential mC patterns are generated by altering expression or function of the repressive factors, Hairy and/or Dl. It is suggested that altered Dl expression is required in order to reduce N signaling, which allows proneural gene expression within the T-row primordia. An alternative hypothesis is that Dl function in during leg mC development is limited to selection of SOPs via lateral inhibition and that regulation of Dl by Scr/Ubx alters lateral inhibition within the T-row proneural fields. However, the hypothesis that Scr/Ubx regulation of Dl alters the proneural prepattern is supported by several observations. A prepattern function for Dl in mC patterning has been previously demonstrated in the notum. Similarly, it has been observed that in prepupal legs with reduced Dl function, proneural Ac expression expands along the leg circumference and is excluded only from Hairy-expressing cells. Furthermore, in prepupal legs, proneural Ac expression fills the center of large clones lacking Dl function. It is also observed that N signaling is activated only in narrow stripes on either side, but not within the T-row proneural fields. The genetic observations are substantiated by analysis of an enhancer that directs ac expression in both the L-row and T-row proneural fields. This enhancer consists of an activation element that directs uniform expression of ac along the leg circumference and two associated repression elements, one that is N-responsive and another that is Hairy-responsive. This is consistent with genetic studies suggesting that in the absence of repressive influences from Hairy and Dl, proneural ac expression would be uniformly along the leg circumference. Combined, these observations suggest that the mC patterning pathway is modified upstream of proneural gene expression by establishment of differential Dl expression in legs from different thoracic segments (Shroff, 2007).

The finding that Dl expression is down-regulated in the T-row primordia, does not necessarily imply that Dl expression is incompatible with mC formation. That this is not the case is suggested by the observation that Dl is expressed in the L-row primordia. Previous studies in a number of tissues have shown that high-level N-ligand expression renders cells non-responsive to N signaling. Hence, it appears that Dl/N signaling at the boundary of Dl-expressing and Dl-non-expressing cells, not Dl expression per se, is incompatible with mC development (Shroff, 2007).

Many studies have made clear the importance of establishing spatially defined proneural gene expression, largely via transcriptional regulation, for patterning of both the vertebrate and invertebrate nervous system. For example, it has been shown that ectopic proneural gene expression causes disruption of the sense organ pattern in adults. In the leg, compromised hairy function results in ectopic proneural ac expression and disorganization of the adult mC pattern, including formation of extranumerary mCs. However, other studies have implicated post-transcriptional regulation of proneural gene function in neural patterning. This was suggested by a study that showed that generalized and transient sc expression in a background devoid of ac and sc function results in an almost normal sense organ pattern in adult flies. Studies in the notum have provided an explanation for this observation by identification of the Extra macrochaetae (Emc) protein as a post-transcriptional regulator of proneural gene function. Emc, an HLH protein that lacks a basic DNA binding domain, binds proneural bHLH proteins, such as Ac, and inhibits their activity. In the notum, emc is expressed in a complex pattern that partially overlaps proneural gene expression, and it appears that SOPs are selected from cells with the lowest levels of Emc. This would suggest that on the notum, competence to acquire a neural fate depends on the balance of proneural protein to Emc levels. It is probable that similar mechanisms function in leg mC patterning as well, since largely normal sense organ patterns are found in legs ubiquitously expressing Sc. These observations indicate that sense organ patterning is a complex process that involves regulation of both proneural gene expression and function. Hence, it would be of interest to assess the relative contribution of post-transcriptional regulation of proneural gene function on leg mC patterning (Shroff, 2007).

It is proposed that T-row mCs are selected from domains of up-regulated Scr or Ubx expression and that one essential function for Scr and Ubx in T-row development is repression of Dl expression. This proposal is supported by several lines of evidence. The requirement of Scr and Ubx in T-row formation was suggested by prior reports that loss of Scr or Ubx function results in transformation of T1 or T3 legs, respectively, toward a T2 fate. This study shows that adult legs heterozygous for reduced function alleles of Scr (Scr^EdK6/Scr^EfW22) exhibit almost complete loss of T-rows in the adult. Moreover, ectopic expression of Scr or Ubx induces T-row formation on T2 legs, on which T-rows are never normally observed. The domains of elevated Scr and Ubx expression in T1 and T3 prepupal legs correspond to the respective positions of T-rows in adult T1 and T3 legs. Furthermore, comparison of Scr expression to that of an SOP marker, sca-Gal4, shows that T-row mCs are selected from groups of cells that express high-level Scr on T1 legs (Shroff, 2007).

Strong evidence is provided that Scr and Ubx repress Dl expression in the T-row primordia. First, a correlation is observed between up-regulated Scr and Ubx expression and domains of reduced Dl expression. Second, loss and gain of function studies indicate that Scr and Ubx negatively regulate Dl expression. In Scr^EdK6/Scr^EfW22, prepupal legs, Dl is expressed in two longitudinal stripes overlapping the region of high-level Scr expression, whereas in wild type legs Dl stripes flank but do not overlap this domain. In addition, Dl is ectopically expressed in either Scr or Ubx loss of function clones within the T-row primordia. Consistent with loss of function results, it was found that ectopic high-level expression of Scr or Ubx results in repression of Dl expression (Shroff, 2007).

Taken together, these observations suggest a function for Scr and Ubx in specification of a T-row fate. However, the finding that the formation of T-rows in response to ectopic Scr or Ubx is confined to ventro-lateral regions along the circumference implies that there may be other positional cues, in addition to elevated Scr or Ubx expression, that are required for T-row specification. Hence, it is plausible that these genes function combinatorially with other factors to induce T-row formation. Wg, for example, is a good candidate since it is expressed in ventro-lateral regions of the leg. In addition, ectopic Wg expression results in expansion of T-row bristles in T1 legs. It is also plausible that, in addition to or instead of T-row promoting factors in ventro-lateral leg regions, there are factors outside these domains that inhibit T-row formation (Shroff, 2007).

These studies have elucidated a general pathway for leg mC patterning in which an early event is establishment of position-specific expression of the prepattern genes hairy and Delta, presumably in response to the global regulators of limb patterning. The spatial regulation of hairy expression during leg development has been investigated ant it has been determined that hairy expression is controlled by modular enhancer elements that integrate patterning information provided by the signaling molecules known to pattern the leg along its circumference, Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg). Periodic Dl expression is partially established by Hairy. However, it is likely that Dl expression in the mC proneural fields is also regulated, like hairy, by genes that pattern the leg along the circumference (circumferential patterning genes), such as hh, dpp and wg (Shroff, 2007).

Up-regulated expression of Scr and Ubx at specific positions along the circumference and P/D axis of T1 and T3 prepupal legs is key to generating the T-row pattern, raising the question of how Scr and Ubx expression in the T-row primordia is regulated. It is proposed that Scr and Ubx expression is controlled by the circumferential patterning genes and genes that pattern the leg along the P/D axis (P/D patterning genes). For example, Scr and/or Ubx expression might be regulated by Wg, which is known to specify ventral leg identity and patterns the ventral leg along the A/P axis in a concentration dependent manner. Along the P/D axis, a number of genes, such as Distal-less and dachshund, are expressed in defined and partially overlapping domains and might function to define the extent of up-regulated Scr and/or Ubx expression. This model would suggest that circumferential and P/D patterning information is integrated by Scr and Ubx, implying that these Hox genes link the global regulators of leg development to local acting genes, such as ac, that specify a neural fate (Shroff, 2007).

These studies indicate that Scr and Ubx function early in T-row development to repress Dl expression, which allows formation of the T-row proneural fields. It will be of interest to determine whether Dl is a direct target of these Hox genes, especially since few direct Hox-gene targets have been identified to date. However, it is probable that Scr and Ubx have other functions in T-row development. Establishment of ac expression in the T-row primordia is an early and essential step of T-row development, but, while ac specifies a neural fate, it does not specify sensory organ type. Hence, it is likely that Scr and Ubx function, in conjunction with other factors, to specify 'T-row-type' mCs. For example, since the T-row mCs are less pigmented than L-row mCs, a potential role of Scr and Ubx in T-row development is to regulate genes involved bristle pigmentation. A second potential function for these Hox genes is in controlling growth in the regions of the legs where T-rows are formed. In T1 legs, for example, the region between L-rows 7 and 8, in which the T-rows are found, is larger than the corresponding region on T2 legs, implying that there is additional growth in this domain. Inconsistent with this hypothesis, however, is the observation that posterior compartment clones lacking Ubx function in the T3 basitarsus did not have a significant effect on basitarsal width (Shroff, 2007).

Another potential role for Scr and Ubx in patterning T-row bristles is to implement a mechanism for selection and organization of T-row mCs into transverse rows. The mechanisms through which the L-row and T-row bristles are selected and organized within their respective proneural fields are likely to differ substantially. The regular spacing of L-row mCs suggests that the L-row SOPs send inhibitory signals in all directions to establish their proper spacing. This is also suggested by the observation that in hairy mutant legs, Ac is expressed in four broad domains, similar to the broad T-row proneural fields, and the supernumerary mCs that are formed on hairy mutant legs are well spaced along the leg circumference. This would suggest that the lateral inhibitory signals are sent along both the leg circumference and P/D axis. Unlike the L-row bristles, the T-rows mCs are positioned directly adjacent to one another in straight regularly spaced transverse rows. How the T-row precursors are selected from a broad field of ac-expressing cells and are arranged in tandem in straight rows is not understood. Previous studies have implicated N and EGFR signaling in formation of organized T-rows. Although the current studies indicate that N-signaling is down-regulated in the T-row primordia, it is conceivable that N functions at later stages of T-row development to pattern the T-row bristles. For example, N might function to set the register and spacing of the T-rows. Also of interest is how the T-rows are aligned in tandem within the rows. It has been suggested that homophilic adhesion between mC SOPs might be involved in organizing T-row bristles. Hence, it is plausible that Hox genes regulate expression of genes involved in adhesion, N signaling and/or EGFR signaling. Investigation of the mechanisms of T-row SOP selection and organization will provide an opportunity to uncover a potential connection between Hox gene function and morphogenesis (Shroff, 2007).

The proposed function for Scr and Ubx in T-row patterning bears some similarity to that described for Ubx in generation of diverse trichome patterns among the T2 legs of various Drosophila species. It has been shown that late pupal expression of Ubx in the T2 femur primordia correlates with lack of trichome formation in different Drosophila species, implying that Ubx inhibits formation of these structures. This role for Ubx, which has been termed a 'micromanaging role' is analogous to the function described here for Scr and Ubx in directing formation of T-rows in specific domains of T1 and T3 legs. Hence, micromanaging functions for Hox genes in generating complex and detailed morphologies may be a general phenomenon (Shroff, 2007).

Another common theme that has emerged from studies of the mechanisms through which Hox genes generate morphological diversity is that, in many cases, Hox genes function to suppress specific developmental pathways. For example, in legs, Antennapedia functions to repress expression of genes that promote antennal development, and Ubx functions to prevent development of specific macrochaete bristles on T3 legs. Furthermore, Ubx is known to act at several levels of the wing patterning hierarchy to suppress wing development in the haltere disc and as mentioned, Ubx functions late in leg development to suppress trichome formation. Less well understood, in contrast, is how and whether Hox genes act positively to direct the formation of morphological novelties among homologous structures, e.g., the T-row bristles on T1 and T3 legs. Further analysis of the mechanisms involved in T-row specification and morphogenesis is likely to provide insight into this question (Shroff, 2007).

Collaboration between Smads and a Hox protein in target gene repression

Hox proteins control the differentiation of serially iterated structures in arthropods and chordates by differentially regulating many target genes. It is yet unclear to what extent Hox target gene selection is dependent upon other regulatory factors and how these interactions might affect target gene activation or repression. Two Smad proteins, effectors of the Drosophila Dpp/TGF-ß pathway, that are genetically required for the activation of the spalt (sal) gene in the wing, collaborate with the Hox protein Ultrabithorax (Ubx) to directly repress sal in the haltere. The repression of sal is integrated by a cis-regulatory element (CRE) through a remarkably conserved set of Smad binding sites flanked by Ubx binding sites. If the Ubx binding sites are relocated at a distance from the Smad binding sites, the proteins no longer collaborate to repress gene expression. These results support an emerging view of Hox proteins acting in collaboration with a much more diverse set of transcription factors than has generally been appreciated (Walsh, 2007).

The activation of sal in the wing and its repression in the haltere are regulated by a 1.1 kb CRE, sal1.1 (Galant, 2002). Previous studies have shown that sal1.1 is directly repressed by Ubx in the haltere (Galant, 2002). In order to test whether Mad/Med binds to and directly represses the activity of the sal1.1 CRE in the haltere, candidate Mad/Med binding sites were sought in the sal1.1 CRE. One candidate Mad/Med binding site, M1 (5'-AGACGGGCAC-3'), was identified that lies between Ubx binding sites 5 and 6 in sal1.1, using binding site prediction and electrophoretic mobility shift assays (EMSAs). The sequence of M1 deviates somewhat from published Mad/Med silencer consensus binding sites (5'-AGAC-5 bp-GNCGYC-3') (Gao, 2005; Pyrowolakis, 2004), and Mad and Med bound with >10-fold and >25-fold lower affinities, respectively, to the M1 site than to the bam (Gao, 2005) and brk (Pyrowolakis, 2004) silencer elements (Walsh, 2007).

In order to test whether Mad/Med bound specifically to the M1 site, a series of point mutations were introduced within the M1 site, and their effect on protein binding was examined in vitro. Of four point mutations to the M1 site, the single mutation at position 808 reduced the binding of a Med fusion protein (GST-MedMH1) to M1 as compared with the wild-type sequence. The remaining three point mutations did not affect the affinity of GST-MedMH1 for the probe. These results suggest that Med might contact the sequence 5'-AGAC-3' in sal1.1. By contrast, the four individual point mutations each decreased, but did not abolish, binding of a Mad fusion protein (GST-MadN) in vitro, with the point mutation at bp 814 having the strongest effect. The weaker effect of the individual point mutations in M1 on Mad binding affinity in vitro is likely to be due to the affinity of MadN for both 5'-AGAC-3' Smad sites and GC-rich sequence. Combining these four mutations (sal798-824 kM1) had the greatest effect on GST-MadN binding to the probe. This analysis of individual point mutations indicates a putative orientation for a Mad/Med compound-binding site in the sal1.1 CRE (Walsh, 2007).

Most importantly, in transgenic flies, each point mutation of M1 introduced into an otherwise wild-type sal1.1 reporter construct caused derepression of the reporter gene lacZ in the haltere imaginal disc. The strength of derepression correlates with the decreased affinity of Mad for its binding site with the pm814 mutation, the strongest point mutation in vitro, showing the strongest level of derepression in vivo. Full derepression was observed when all four point mutations were combined into a sal1.1 reporter construct. No effect of mutations in M1 were observed on sal1.1-driven reporter gene expression in the wing as compared with the wild-type sal1.1 element or with endogenous sal expression, indicating that this site is not required for gene activation in the wing or haltere disc. Together, the biochemical, reporter gene and genetic evidence indicate that Mad/Med/Shn are directly required for sal repression in the haltere imaginal disc (Walsh, 2007).

This study demonstrates that Mad/Med and Ubx bind to adjacent sites in the sal1.1 CRE and that each protein is required for the direct repression of sal expression in the haltere. Furthermore, the sequence and spacing of Ubx and Smad binding sites are highly conserved and their proximity is required for target gene repression in the haltere. Because no evidence was found that these proteins interact directly, it is suggested this is an example of 'collaboration' or target gene co-regulation without direct cooperative interaction. These results have general implications for understanding how Hox proteins regulate diverse sets of target genes in animal development (Walsh, 2007).

The direct role for Smads in the repression of sal in the haltere is surprising in the light of previous genetic and molecular studies that had indicated that the Dpp pathway and Mad/Med were involved in sal activation in the wing. No direct evidence was found that this is the case and the fact that sal is activated in Mad and Med clones in the haltere indicates that sal is activated independently of Mad/Med in the flight appendages. The requirement for Mad/Med/Shn in shaping the pattern of sal expression in the wing appears to be indirect -- the protein complex represses the expression of brk, a repressor of sal, in cells in the central region of the developing wing and thereby permits sal expression (Walsh, 2007).

The Mad-Med-Shn complex is also active within cells in the central region of the haltere as a consequence of Dpp signaling. However, whereas sal is expressed and the sal1.1CRE is active in the wing, sal and the sal1.1 CRE are repressed in the haltere. These observations raise the question of how the Mad-Med-Shn complex selectively represses sal in the haltere but not in the wing disc? The results suggest that there are two key determinants in the selective repression of sal in the haltere. The first is collaboration with Ubx, which is expressed in the haltere and not in the wing disc. The second key determinant might be the affinity of Mad/Med binding to the sal CRE (Walsh, 2007).

The different responses of the brk and sal genes to Mad/Med/Shn suggests how the different affinities of proteins for binding sites might determine how available transcriptional regulatory inputs are integrated by CREs. Mad/Med binding to the brk CRE is of high affinity (Pyrowolakis, 2004) and apparently sufficient to impart repression, whereas that to the sal CRE is of much lower affinity and insufficient to impart repression in the wing. In the haltere, although Mad-Med-Shn or Ubx binding are alone insufficient, they act together either via simultaneous or sequential occupancy of their binding sites to repress sal (Walsh, 2007).

The requirement for two or more regulators to act together to control gene expression, i.e. combinatorial regulation, is fundamental to the generation of the great diversity of gene expression patterns by a finite set of transcription factors. Several previous studies have revealed the dual requirement for Hox and Smad functions for the activation of a target gene. Studies have suggested a general combinatorial mechanism for gene activation in which apparently separate transcriptional inputs act synergistically in gene activation and, in at least one case, the Hox response element and Dpp response element are separable. In this study, however, a requirement was observed for strict evolutionary conservation of the close topology of Hox and Smad binding sites in the sal CRE. It is suggested that collaboration is a distinct mode of combinatorial regulation in which two or more regulatory proteins must bind to nearby sites, but not necessarily to each other (Walsh, 2007).

The integration of Hox and Smad inputs could work through a number of possible mechanisms in the absence of direct physical interaction. One appealing possibility that might explain the requirement for the close proximity of binding sites is that Ubx and Mad-Med-Shn might interact with, and could therefore cooperatively recruit, the same co-repressor(s) for the repression of sal. Alternatively, if Mad-Med-Shn and Ubx bind sequentially to sal1.1, they might recruit different co-repressors and thereby orchestrate the assembly of a co-repressor complex. A third possibility is that because the Ubx and Mad/Med sites are embedded within a larger block of conserved regulatory DNA sequence in the sal1.1 CRE, the binding of other interacting transcription factors might also be involved in the repression of sal by Ubx and Mad-Med-Shn (Walsh, 2007).

These and recent results raise the question of whether collaboration is a general feature of target gene selection by Hox proteins. It is suggested that collaboration might be a widespread requirement for Hox function in vivo. This proposal is prompted by three observations: (1) Hox proteins alone have low DNA-binding specificity; (2) some, and perhaps all, Hox proteins might act as both repressors and activators; (3) Hox proteins regulate a great diversity of target genes that are also regulated by other transcription factors. In order to be such versatile regulators, it would be too great a constraint to require that Hox proteins always interact cooperatively with the diverse repertoire of transcription factors with which they act. Indeed, it may be argued that too much weight has been ascribed to the cooperative binding of Hox proteins and co-factors to DNA (Walsh, 2007).

Previously, much attention has focused on Exd and Hth, which interact with Hox proteins and bind cooperatively to DNA, thereby increasing Hox DNA-binding selectivity. However, it was only recently shown that the binding of these complexes alone was not sufficient to regulate target gene expression. Rather, Hox-Exd-Hth collaborate with and require the segmentation proteins Slp and En to repress the target gene Dll. This study has shown that the Exd- and Hth-independent target gene repression of sal requires collaboration between Ubx and Mad-Med-Shn. Although still a tiny sample of target genes, cases of transcription factors of various structural types acting as collaborators with Hox proteins are now available. The picture of Hox proteins relying on dedicated interacting co-factors such as Exd and Hth is expanding to a larger pool of collaborating transcription factors that modulate target gene selection (Walsh, 2007).

Indeed, collaboration might be the key to another unresolved mystery of the Hox proteins - the regulation of Hox protein activity. Some Hox proteins appear to act in both gene activation and repression; this is certainly the case for Ubx. This versatility would appear to be crucial to their role as sculptors of major features of body patterns, but how does the same transcription factor act positively in some contexts but negatively in others? There is evidence to suggest that the identity of the collaborating proteins and/or CRE sequences determines the 'sign' of Hox action (Walsh, 2007).

For instance, there is no evidence that the mere binding of Hox-Exd-Hth to a site determines the sign of Hox activity. These co-factors are involved in both Hox target gene activation (e.g., dpp in the midgut) and target repression (e.g.,Dll in the embryonic abdomen). But, in the latter case, En and Slp, two proteins that each harbor motifs for interaction with the co-repressor Groucho, are required collaborators for Dll repression. The roles of En and Slp in this instance might not be so much a matter of facilitating Hox target selection, but rather in regulating the sign of the output of the collaboration (Walsh, 2007).

Similar to the Hox proteins, the Smads can either activate or repress target genes. Furthermore, it has been demonstrated that the topology of Smad binding sites on DNA appears to be critical for determining whether a target gene is activated or repressed. In Drosophila, the topology of Mad and Med binding sites is critical for the recruitment of the co-repressor Shn. The recruitment of Shn was shown here to be necessary for sal repression. These two examples suggest that the positive or negative regulatory activity of a Hox protein depends on the context of surrounding binding sites and how they influence the activity of collaborating factors (Walsh, 2007).

The dependence of Hox proteins upon co-factors and collaborators indicates that, at the molecular level, Hox proteins are not 'master' regulatory proteins that dictate how target genes behave. Rather, they exert their great influence by virtue of their simple binding specificity, broad domains of expression and versatile, collaborative properties (Walsh, 2007).

Multi-step control of muscle diversity by Hox proteins in the Drosophila embryo

Hox transcription factors control many aspects of animal morphogenetic diversity. The segmental pattern of Drosophila larval muscles shows stereotyped variations along the anteroposterior body axis. Each muscle is seeded by a founder cell and the properties specific to each muscle reflect the expression by each founder cell of a specific combination of 'identity' transcription factors. Founder cells originate from asymmetric division of progenitor cells specified at fixed positions. Using the dorsal DA3 muscle lineage as a paradigm, this study shows that Hox proteins play a decisive role in establishing the pattern of Drosophila muscles by controlling the expression of identity transcription factors, such as Nautilus and Collier (Col), at the progenitor stage. High-resolution analysis, using newly designed intron-containing reporter genes to detect primary transcripts, shows that the progenitor stage is the key step at which segment-specific information carried by Hox proteins is superimposed on intrasegmental positional information. Differential control of col transcription by the Antennapedia and Ultrabithorax/Abdominal-A paralogs is mediated by separate cis-regulatory modules (CRMs). Hox proteins also control the segment-specific number of myoblasts allocated to the DA3 muscle. It is concluded that Hox proteins both regulate and contribute to the combinatorial code of transcription factors that specify muscle identity and act at several steps during the muscle-specification process to generate muscle diversity (Enriquez, 2010).

Eve expression in the DA1 muscle lineage provided the first paradigm for studying the early steps of muscle specification. Detailed characterization of an eve muscle CRM showed that positional and tissue-specific information were directly integrated at the level of CRMs via the binding of multiple transcription factors, including dTCF, Mad, Pnt, Tin and Twi. Based on this transcription factor code and using the ModuleFinder computational approach, this study has identified a CRM, CRM276, that precisely reproduces the early phase of col transcription. This CRM also drove expression in cells of the lymph gland, another organ that is issued from the dorsal mesoderm where col is expressed. Parallel to this study, two col genomic fragments were selectively retrieved in chromatin immunoprecipitation (ChIP-on-chip) experiments designed to identify in vivo binding sites for Twi, Tin or Mef2 in early embryos. One fragment overlaps with CRM276. Based on this overlap and interspecies sequence conservation, a 1.4 kb subfragment of CRM276 that retained most of the transcription factor binding sites identified by ModuleFinder was tested, and it was found to specifically reproduced promuscular col expression. This in vivo validation shows that intersecting computational predictions and ChIP-on-chip data should provide a very efficient approach to identify functional CRMs on a genome-wide scale (Enriquez, 2010).

The eve and col early mesodermal CRMs are activated at distinct A/P and D/V positions. It is now possible to undertake a comparison of these two CRMs, in terms of the number and relative spacing of common activator and repressor sites and their expanded combinatorial code, in order to understand how different mesodermal cis elements perform a specific interpretation of positional information (Enriquez, 2010).

A progenitor is selected from the Col promuscular cluster in T2 and T3 but not T1. One cell issued from the Col-expressing promuscular cluster in T1 nevertheless shows transiently enhanced Col expression, suggesting that the generic process of progenitor selection is correctly initiated in T1. This process aborts, however, in the absence of a Hox input, as shown by the loss of progenitor Col expression and DA3 muscle in specific segments in Hox mutants. The similar changes in Nau and Col expression observed under Hox gain-of-function conditions leads to the conclusion that the expression of 'identity' transcription factor iTFs is regulated by Hox factors at the progenitor stage. The superimposition of Hox information onto the intrasegmental information thereby implements the iTF code in a segment-specific manner and establishes the final muscle pattern. Unlike DA3, a number of specific muscles are found in both T1 and T2-A7, such as the Eve-expressing DA1 muscle; other muscles form in either abdominal or thoracic segments, as illustrated by the pattern of Nau expression in stage 16 embryos. This diversity in segment-specific patterns indicates that Hox regulation of iTF expression is iTF and/or progenitor specific (Enriquez, 2010).

As early as 1994, Hox proteins were proposed to regulate the segment-specific expression of iTFs. Seven years later, an apterous mesodermal enhancer (apME680) active in the LT1-4 muscles was characterized and itwas proposed that regulation by Antp was direct. However, mutation of the predicted Antp binding sites present in apME680 abolished its activity also in A segments, suggesting that some of the same sites were bound by Ubx/AbdA. Evidence is now available that the regulation of col expression by Ubx/AbdA in muscle progenitors is direct and involves a single Hox binding site. However, regulation by Antp does require other cis elements. It remains to be seen whether regulation by Antp is also direct. Since Antp, Ubx and AbdA display indistinguishable DNA-binding preferences in vitro, the modular regulation of col expression by different Hox paralogs suggests that other cis elements and/or Hox collaborators contribute to Hox specificity. Direct regulation of col by Ubx has previously been documented in another cellular context, that of the larval imaginal haltere disc, via a wing-specific enhancer. In this case, Ubx directly represses col expression by binding to several sites, contrasting with col-positive regulation via a single site in muscle progenitors. This is the second example, in addition to CG13222 regulation in the haltere disc, of direct positive regulation by Ubx via a single binding site. Hox 'selector' proteins collaborate on some cis elements with 'effector' transcription factors that are downstream of cell-cell signaling pathways. In the DA3 lineage, it seems that Dpp, Wg and Ras signaling act on one col cis element and the Hox proteins on others. The regulation of col expression by Hox proteins in different tissues via different CRMs provides a new paradigm to decipher how different Hox paralogs cooperate and/or collaborate with tissue- and lineage-specific factors to specify cellular identity (Enriquez, 2010).

The DA3 muscle displays fewer nuclei in T2 and T3 than in A1-A7, an opposite situation to that described for an aggregate of the four LT1-4 muscles. It has been proposed that the variation in the number of LT1-4 nuclei was controlled by Hox proteins. These studies of the DA3 muscle extend this conclusion by showing that the variations due to Hox control are specific to each muscle and are exerted at the level of FCs. Since the number of nuclei is both muscle- and segment-specific, Hox proteins must cooperate and/or collaborate with various iTFs to differentially regulate the nucleus-counting process. As such, Hox proteins contribute to the combinatorial code of muscle identity. Identifying the nature of the cellular events and genes that act downstream of the iTF/Hox combinatorial code and that are involved in the nucleus-counting process represents a new challenge (Enriquez, 2010).

Drosophila castor is regulated negatively by the Ubx and abdA genes, but positively by the AbdB gene

The ventral nerve cord (VNC) of Drosophila exhibits significant segmental-specific characteristics during embryonic development. Homeotic genes are expressed over long periods of time and confer identity to the different segments. castor (cas) is one of the genes which are expressed in neuroblasts along the VNC. However, at late embryonic stages, cas transcripts are found only in head and thoracic segments and terminal abdominal segments, while Cas protein lasts longer in all segments. This study investigated the regulation of temporal and spatial expression of cas by the bithorax complex genes. In the loss-of-function mutants of Ultrabithorax (Ubx) and abdominal-A (abdA), cas transcripts were ectopically expressed in abdominal segments at late embryonic stage. However, unlike in Ubx and abdA mutants, in Abdominal-B (AbdB) loss-of-function mutant embryos, cas disappeared in the terminal region. Ectopic Ubx and abdA suppressed cas expression, but ectopic AbdB activated cas expression in most abdominal segments. Moreover, cas was co-expressed in the cells in which AbdB was normally expressed, and overexpressed in the ectopically expressed AbdB embryos. These results suggest that the expression of cas is segment-specifically regulated negatively by Ubx and abdA genes, but positively by the AbdB gene (Ahn, 2010).

cas is transiently expressed in a subset of neuroblasts in their cell lineage. Its transcripts are present with homologous patterns in thorax and abdomen at early embryonic stages. At late embryonic stages, cas transcripts are found in only a few cells per hemisegment in thoracic and posterior abdominal segments, but not in other abdominal segments. This indicates that cas is expressed in segment-specific mode during late embryonic stages. This study investigated how this regional diversity was produced (Ahn, 2010).

The segment-specific expression of cas was regulated by the homeotic genes. Ubx or abdA mutation caused the homeotic transformation of abdominal cuticle belts to the more anterior ones. These transformation patterns were also observed in cas expression. Mutations in Ubx or abdA genes caused ectopic cas expression in abdominal segments, which was normally observed in the thoracic segments at stage 15 of wild-type embryo, suggesting transformation of the thoracic pattern to an abdominal pattern at that stage. This transformation was synergistically enhanced in the Ubx and abdA double mutants (Ahn, 2010).

cas was ectopically expressed in A1 segment in Ubx mutant embryos. This result is coincident with the function of Ubx to specify the posterior thorax and a portion of A1 segment. cas was also ectopically expressed in A1 to A4 segments in abdA mutant embryos, which coincide with the function of abdA. In Ubx abdA double mutant embryos, cas was expressed in virtually all abdominal segments and in more cells than in each single mutant embryo. The roles of Ubx and abdA on cas expression in the abdominal segments were confirmed in the ectopically expressed Ubx and abdA mutant embryos. For this experiment, proper embryonic stages were very important because cas expression changed dramatically in the abdominal segments between stages 14 and 15. Whether ectopic Ubx or abdA repressed cas expression in the abdominal segments was tested at this stage. The GAL4-mediated induction of Ubx or abdA suppressed cas expression in the abdominal segments (Ahn, 2010).

However, in contrast to the Ubx and abdA mutations, AbdB mutation caused reverse effects on cas expression in the abdominal segments, which have never been reported. Loss-of AbdB function caused lack of cas expression, while ectopic ABDB activated cas expression in the abdominal segments. This phenotype was also observed in Polycomb mutant embryos. Although Polycomb mutation induced ectopic expression of Ubx, abdA and AbdB at the same time, cas was ectopically expressed in the abdominal segments of stage15 embryos, suggesting that ABDB dominated the effects of ectopic UBX or ABDA. The co-localization of cas and ABDB is found in a few cells in the posterior abdominal segments, supporting the positive regulation of cas by ABDB (Ahn, 2010).

This idea was further intensified by the appearance of the ectopic cas mRNA in the numerous abdominal cells with the ectopic AbdB expression. Real-time PCR experiment showed the overexpession of cas mRNA in the ectopically expressed AbdB embryos, also supporting the positive regulation of cas by ABDB. Furthermore, seven AbdB DNA binding sites were found within 5kb upstream from the cas transcription start site enhancing the possibility that ABDB directly regulates the cas expression. ABDB binds preferentially to a sequence with an unusual 5'-TTAT-3' core (Ahn, 2010).

One of questions was why all the cells with the AbdB expression does not show cas mRNA expression. In wild-type embryos, all AbdB-expressing cells does not show cas mRNA. Only a few cells among AbdB-expressing cells could maintain the expression of cas and the other cells lost it. This might be that the homoetic proteins carry out their function by interacting with other cofactors to regulate distinct sets of downstream genes (Ahn, 2010).

Accumulating evidence shows that the bithorax complex genes are involved at different steps in the segment-specific divergence of the CNS. Ubx or abdA activity is required for the abdominal pathway of the NB1-1 lineage. Both ectopic induction of Ubx- or abdA expression until several hours after gastrulation and homeotic de-repression in Polycomb mutants, override thoracic determination of NB1-1. The abdominal NB6-4 lineage is also specified by the abdA and AbdB. abdA is expressed in the NB6-4 lineage of abdominal segments A1-A6, whereas AbdB is expressed in the NB6-4 lineage of segments A7-A8. They specify the abdominal NB6-4 lineage by down-regulating levels of G1 Cyclin (CycE) (Ahn, 2010).

In summary, UBX and ABDA suppress cas expression in abdominal segments, so that mutation in both genes causes ectopic expression of cas in abdominal segments at late embryonic stage. However, ABDB activates cas expression, which is supported by co-localization of cas and ABDB in cells ectopically expressing AbdB, and real-time PCR in ectopically expressed AbdB embryos (Ahn, 2010).

Sequence-specific interaction between ABD-B homeodomain and castor gene in Drosophila

The effects were analyzed of bithorax complex genes on the expression of castor gene. During the embryonic stages 12-15, both Ultrabithorax and abdominal-A regulate the castor expression negatively, whereas Abdominal-B showed a positive correlation with the castor gene expression according to real-time PCR. To investigate whether ABD-B protein directly interacts with the castor gene, electrophoretic mobility shift assays were performed using the recombinant ABD-B homeodomain and oligonucleotides, which are located within the region 10 kb upstream of the castor gene. The results show that ABD-B protein directly binds to the castor gene specifically. ABD-B binds more strongly to oligonucleotides containing two 5'-TTAT-3' canonical core motifs than the probe containing the 5'-TTAC-3' motif. In addition, the sequences flanking the core motif are also involved in the protein-DNA interaction. The results demonstrate the importance of HD for direct binding to target sequences to regulate the expression level of the target genes (Kim 2013).

The cis-regulatory code of Hox function in Drosophila

Precise gene expression is a fundamental aspect of organismal function and depends on the combinatorial interplay of transcription factors (TFs) with cis-regulatory DNA elements. While much is known about TF function in general, understanding of their cell type-specific activities is still poor. To address how widely expressed transcriptional regulators modulate downstream gene activity with high cellular specificity, binding regions were identified for the Hox TF Deformed (Dfd) in the Drosophila genome. This analysis of architectural features within Hox cis-regulatory response elements (HREs) shows that HRE structure is essential for cell type-specific gene expression. It was also found that Dfd and Ultrabithorax (Ubx), another Hox TF specifying different morphological traits, interact with non-overlapping regions in vivo, despite their similar DNA binding preferences. While Dfd and Ubx HREs exhibit comparable design principles, their motif compositions and motif-pair associations are distinct, explaining the highly selective interaction of these Hox proteins with the regulatory environment. Thus, these results uncover the regulatory code imprinted in Hox enhancers and elucidate the mechanisms underlying functional specificity of TFs in vivo (Sorge, 2012).

In order to quantitatively identify genomic regions bound by the Hox TF Dfd in Drosophila, two complementing approaches were employed: ChIP-seq, which has been successfully applied previously to identify stage- and tissue-specific enhancer activities, and computational detection of clusters of TF binding sequences, which allows the identification of cis-regulatory modules irrespective of temporal and spatial context. To generate genome-wide maps of Dfd binding in vivo, ChIP was performed using stage 10-12 Drosophila embryos and a Dfd-specific antibod. Stage-independent in silico Dfd-specific Hox response elements (HREs) were identified by searching for clusters of conserved Dfd binding motifs, as defined by a position weight matrix (PWM), in the non-coding regions of the genomes of 12 distinct Drosophila species. By applying both approaches, 4526 genomic regions containing clusters of Dfd binding sites and 1079 Dfd ChIP-seq enrichment peaks were identified, including two out of the three well-characterized Dfd-HREs, namely rpr-4S3 and Dfd-EAE. To study the regulatory capacity of novel in silico and ChIP-seq detected HREs, cell culture-based enhancer assays were performed for 11 randomly selected HREs, and it was found that reporter expression driven by the identified genomic regions was in all cases dependent on Dfd binding. In vivo activity was tested of 21 arbitrarily selected enhancers in transgenic reporter lines, revealing that 7 out of 11 ChIP-identified and 5 out of 10 in silico-predicted Dfd-HREs recapitulate the spatio-temporal expression of adjacent genes). Most importantly, it was possible to demonstrate Dfd-dependent regulation of both transgenic reporter expression and endogenous gene expression, suggesting that they are bona fide direct Dfd target genes. Thus, the identified Dfd-HREs represent a data set of biologically relevant regulatory regions and an excellent resource to unravel sequence features within Hox responsive enhancers that might be essential for the highly selective Hox target gene regulation (Sorge, 2012).

Transcriptional regulation in many cases relies on the assembly of regulatory protein complexes mediated by closely spaced TF binding sites within a cis-regulatory module and previous studies have shown that Hox proteins employ this mechanism to control target gene activity in small subsets of cells. The novel HREs were systematically scanned for TF binding motifs appearing in close proximity to Dfd binding sites. Using a statistical test for pair-wise distance distributions, w11 overrepresented DNA motifs for known TFs were found adjoining to Dfd binding sites with 5 of the motifs occurring in both the ChIP-seq and in silico-identified Dfd-HREs. When the expression patterns of six of these transcriptional regulators known to bind to the 11 motifs that were identified were examined, colocalization with Dfd was found in different sub-populations of cells in all cases. Colocalization was already known for two TFs, whose binding sites were coupled to Dfd motifs, including Extradenticle (Exd) , which is known to cooperatively bind with Hox proteins to DNA and thereby increase Hox DNA-binding selectivity. It was next asked whether the short-distance arrangements in Dfd-HREs are of biological relevance and translated into the regulation of similar classes of target genes. To this end, the overrepresentation was statistically tested of expression and biological terms of genes associated with HREs harbouring specific combinations of Dfd and close-by motifs. This analysis revealed that only those Dfd-HREs with short distance intervals between the Dfd and adjacent motifs were coupled to similar gene classes, while random distance intervals did not show any correlation. Strikingly, genes associated with specific short-distance HREs had similar expression and functional annotations as the TFs interacting with the Hox adjoining motifs, suggesting that time and place of Hox action is dictated by spatio-temporally restricted co-regulators. Support for this hypothesis stems from the observation that one of the close-distance partners, Optix, regulates similar processes as Dfd, since Dfd and Optix mutants displayed comparable morphological defects in the head region, such as the absence of mouth hooks, a maxillary segment-derived structure known to be specified by Dfd. In addition, one of the genes associated with a Dfd-Optix HRE, the known Dfd target gene reaper (rpr), is expressed in the ventral epidermis primordium as predicted by its HRE architecture, and regulated by Dfd and Optix in ventral-maxillary cells, which also express these factors. A cell-culture assay using the well-established Dfd responsive module responsible for rpr expression in a few anterior-maxillary cells, the rpr-4S3 Dfd-HRE, with wild-type or mutated Dfd binding sites or reduction of Dfd levels by RNAi confirmed the requirement for simultaneous activity of Dfd and Optix on the rpr-4S3 Dfd-HRE for strong reporter gene induction. Optix binding to the rpr-4S3 Dfd-HRE was additionally confirmed by electrophoretic mobility shift assay (EMSA) experiments. Furthermore, transgenic reporter expression induced by the rpr-4S3 Dfd-HRE was lost in Optix mutant embryos or when the Optix binding sites were mutated. These results demonstrate that Optix, one of the newly identified factors, is a Dfd co-regulator required for proper regulation of the important Hox target gene rpr (Sorge, 2012).

Whether The precise spacing between Hox and adjacent binding sites plays a role for enhancer activity was explored. The rpr-4S3 Dfd HRE, which induces gene expression in a few anterior-maxillary cells, has previously been shown to be under the control of Dfd and Glial cells missing (Gcm), a Dfd co-regulator also identified in this study. Dfd and Gcm as well as Optix binding sites within the rpr-4S3 HRE are directly adjacent to each other, thus a 5- and 10-bp spacer was introduced to interfere with potential interactions of the proteins on the enhancer. In all cases, reporter gene expression was strongly reduced or completely abolished, showing that the close-distance arrangements between Dfd and Gcm as well as Dfd and Optix are required for the in vivo activity of the rpr-4S3 enhancer (Sorge, 2012).

While the results regarding the close-distance arrangement of Dfd and Gcm binding sites suggested the formation of a Dfd-Gcm protein complex, like in the case of Dfd and Exd, only independent binding of the two proteins to the rpr-4S3 enhancer was observed in EMSA experiments , supporting the idea of Hox proteins collaborating with other TFs on target HREs in the absence of physical contact. It has been shown before that Hox proteins together with other TFs that bind in the immediate vicinity recruit non-DNA binding cofactors to HREs. To test if such factors could interact with Dfd and the newly identified short distance binding TFs, the modENCODE data set was scanned and it was found that dCBP/Nej, a member of the CBP/p300 family of transcriptional co-activators bearing acetyltransferase activity, binds to the rpr-4S3 enhancer in vivo. As nej has been previously reported to genetically interact with Dfd, its function was examined in Dfd/Gcm-mediated transcriptional activation. Both factors, Dfd and Gcm, are required for transcriptional activation, since expression of Gcm in Drosophila D.Mel-2 cells, which have basal levels of Dfd activity, resulted in strong induction of reporter gene expression, while abolishing Dfd binding to the rpr-4S3 HRE by mutating all Dfd binding sites or by reducing Dfd protein levels in D.Mel-2 cells using RNAi, strongly reduced reporter gene expression in the presence of Gcm. Strikingly, Dfd- and Gcm-mediated reporter gene expression was strongly reduced in nej dsRNA-treated cells, whereas inhibition of protein deacetylation by Trichostatin A (TSA0) restored reporter gene expression. Consistently, rpr expression was abolished in nej mutant embryos. These results demonstrate that dCBP/Nej-mediated protein acetylation/histone modification is important for the combined activity of Dfd and Gcm on the rpr-4S3 HRE. While it was not possible to demonstrate that nej physically interacts with Dfd protein using various assays, EMSA experiments show that nej interacts with Gcm. Furthermore, acetylation of transiently transfected Gcm was detected in cultured Drosophila cells. Acetylation of Gcm is dependent on Nej, as it was reduced upon RNAi-mediated downregulation of nej. These results are consistent with published work demonstrating that in human cells CBP interacts with Gcma, resulting in its acetylation and stimulation of its transcriptional activity. Since about 10% of all Dfd and nej in vivo genomic binding events during embryonic stages 10-12 overlap, the functional interaction of Dfd and nej observed at the rpr locus does not seem an exception. This finding suggests that the interaction of co-activators (and co-repressors) with Hox proteins and close distance binding TFs on enhancer modules could be a commonly used mechanism to achieve highly specific spatio-temporal control of target gene activity. In this scenario, Hox proteins would control downstream genes by direct transcriptional and/or epigenetic regulation depending on HRE composition and thus cofactor identity and recruitment (Sorge, 2012)

Despite very similar DNA binding behaviour in vitro, Hox proteins regulate distinct morphological features along the anterior-posterior body axis in animal systems. To elucidate the mechanistic basis for the differences in their regulatory properties, Dfd-HREs identified in this study were compared to genomic regions bound by the Hox TF Ultrabithorax (Ubx) at identical developmental stages, as identified by the modENCODE consortium. Searching for overrepresented DNA motifs in both enriched ChIP regions, it was found that Dfd and Ubx bind to identical DNA sequences in vivo, reminiscent to in vitro systems. However, individual binding motifs seem to play only a minor role for Hox binding site selection in vivo, since this analysis revealed that Dfd and Ubx exclusively interact with non-overlapping genomic regions in embryonic stages 9-12. Consequently, Dfd- and Ubx-HREs were found to be associated with distinct classes of genes, revealing that genes with roles in the epidermis are primarily under the control of Dfd at the analysed embryonic stages while genes with mesoderm-related functions are predominantly regulated by Ubx. Consistently, it was found that the expression of tartan (trn), one of the genes associated with a Dfd-HRE, is regulated exclusively by Dfd, but not by Ubx, in epidermal cells, while parcas (pcs), one of the genes linked to a Ubx-HRE is under the selective control of Ubx in mesodermal cells. Furthermore, only Ubx-HREs were found to substantially overlap with cis-regulatory elements stage specifically bound by the mesoderm-specifying TFs Myocyte enhancer factor 2 (Mef2), Twist and Tinman. In contrast, the common ability of both Dfd and Ubx to regulate genes involved in nervous system development was underlined by comparable representations of binding motifs for the neuronal-specifying TFs Asense, Deadpan and Snail in Dfd- and Ubx-HREs (Sorge, 2012).

Strikingly, the basic design principles of Dfd- and Ubx-HREs were found to be similar: like in Dfd-HREs, six binding motifs for known TFs were located adjacent to Ubx binding sites and colocalization studies showed that they are expressed in subsets of Ubx-positive cells. Again, Ubx binding sites and motifs for potential co-regulators occurred most frequently in specific short intervals and only those Ubx-HREs with the preferred distance were associated with specific gene classes. This analysis also revealed that four of the six short-distance motifs were specific for Ubx-HREs, which is consistent with the data showing that Hox proteins interact with different and spatially restricted co-regulators to control target gene expression in selected cells. Importantly, in the cases of the close-distance motifs detected in both HREs, namely the binding sites for the TFs Ladybird early (Lbe) and Cut (Ct), the associated target genes were also expressed in non-overlapping tissues. This raised the question of how different Hox proteins can act on distinct target genes, even when their target HREs exhibit similar binding site compositions including short-distance arrangements. Since Lbe is active in both mesodermal and epidermal cells, one Dfd-Lbe and one Ubx-Lbe HRE was exemplarily analysed, and binding of Lbe protein was confirmed to both HREs by EMSAs. As predicted by the presence of Lbe binding sequences. Complex formation between the Hox protein and Lbe was observed in the case of Ubx and Lbe while Dfd and Lbe interact independently with the Dfd-Lbe HRE, indicating that the two Hox proteins employ different mechanisms for binding to the selected HREs. Lbe interaction with the Dfd-Lbe and Ubx-Lbe HREs is essential for in vivo activity, since in both cases ectopic reporter gene expression was observed when Lbe binding sites were mutated. Even more important, reporter gene expression was specifically changed only in segments in which either Dfd or Ubx is active, meaning in the case of the Dfd-Lbe HRE in maxillary cells and in the case of the Ubx-Lbe HRE in abdominal segments A1-A7. Taken together, these results demonstrate that the combined activity of Lbe and the Hox proteins Dfd or Ubx on selected HREs is critical for the precise spatiotemporal and segment-specific control of HRE activity. It was next asked whether additional (DNA- and non-DNA-binding) factors contribute to the predicted cell type-specific expression of the Dfd-Lbe and Ubx-Lbe HREs. Using the Drosophila Interactions Database (DroID; Murali, 2011) and published genome-wide DNA binding studies a search was carried out for unique Dfd-lbe and Ubx-lbe interactors. It was discovered that almost 20% of all Ubx-Lbe HREs but none of the Dfd-Lbe HREs were found to interact with the mesoderm-specifying factor Mef2 in vivo, while H3K9me3 histone marks, which are mediated by one of the unique Dfd-lbe interactors, Enhancer of zeste E(z), are enriched only within Dfd-Lbe HREs. Interestingly, E(z) modifies chromatin also by trimethylating H3K27 residues, a histone mark highly enriched at the genomic region spanning the ChIP-detected Dfd-Lbe HRE. Consistent with the repressive function of this histone modification, loss of Lbe binding to the Dfd-Lbe HRE results in ectopic reporter gene expression, suggesting that Lbe (and Dfd) recruits E(z) to the Dfd-Lbe HRE for cell type-specific target gene repression (Sorge, 2012).

Taken together, these results demonstrate that Hox proteins interact with different regulatory proteins on HREs, which allows them to differentially regulate their target genes despite their similar DNA binding properties. The fact that these interactions occur only in a few cells for a short period of time is very likely one of the major reasons why the identification of factors conferring regulatory precision and specificity to Hox function has met with little success so far (Sorge, 2012).

This study, has identified crucial features of HREs, which are essential for cell type-specific regulation of Hox target genes in vivo. In addition to motif composition the exact spatial arrangement of TF binding elements is critical to translate Dfd function into transcriptional regulation in vivo. These architectural features of Dfd-HREs alone accurately predict target gene function and expression patterns. Furthermore, it was found that epigenetic regulators bind to HREs on a genome-wide scale, suggesting that they generally collaborate with Hox proteins to achieve stable target gene regulation. This is in line with recent findings showing that chromatin modifications at enhancers strongly correlate with functional enhancer activity and tissue specificity. By comparing HREs regulated by Dfd and Ubx, two different Hox proteins with different embryonic regulatory specificities, this study shows that while similar design principles apply, specificity is encoded by distinct sets of co-occurring DNA motifs. Due to the highly dynamic regulatory output of Hox TFs in space and time, cell type-specific approaches are required in future to elucidate all relevant aspects of Hox-chromatin and Hox-cofactor interactions (Sorge, 2012).

A common set of DNA regulatory elements shapes Drosophila appendages

Animals have body parts made of similar cell types located at different axial positions, such as limbs. The identity and distinct morphology of each structure is often specified by the activity of different 'master regulator' transcription factors. Although similarities in gene expression have been observed between body parts made of similar cell types, how regulatory information in the genome is differentially utilized to create morphologically diverse structures in development is not known. This study used genome-wide open chromatin profiling to show that among the Drosophila appendages, the same DNA regulatory modules are accessible throughout the genome at a given stage of development, except at the loci encoding the master regulators themselves. In addition, open chromatin profiles change over developmental time, and these changes are coordinated between different appendages. It is proposed that master regulators create morphologically distinct structures by differentially influencing the function of the same set of DNA regulatory modules (McKay, 2013).

This paper addresses a long-standing question in developmental biology: how does a single genome give rise to a diversity of structures? The results indicate that the combination of transcription factors expressed in each thoracic appendage acts upon a shared set of enhancers to create different morphological outputs, rather than operating on a set of enhancers that is specific to each tissue. This conclusion is based upon the surprising observation that the open chromatin profiles of the developing appendages are nearly identical at a given developmental stage. Therefore, rather than each master regulator operating on a set of enhancers that is specific to each tissue, the master regulators instead have access to the same set of enhancers in different tissues, which they differentially regulate. It was also found that tissues composed of similar combinations of cell types have very similar open chromatin profiles, suggesting that a limited number of distinct open chromatin profiles may exist at a given stage of development, dependent on cell-type identity (McKay, 2013).

Different tissues were dissected from developing flies to compare their open chromatin profiles. These tissues are composed of different cell types, each with its own gene expression profile. Formaldehyde-assisted isolation of regulatory elements (FAIRE) data thus represent the average signal across all cells present in a sample. However, data from embryos and imaginal discs indicate that FAIRE is a very sensitive detector of functional DNA regulatory elements. For example, the Dll⁰¹ enhancer is active in 2–4 neurons of the leg imaginal disc; yet, the FAIRE signal at Dll⁰¹ is as strong as the Dll⁰⁴ enhancer, which is active in hundreds of cells of the wing pouch. Thus, FAIRE may detect nearly all of the DNA regulatory elements that are in use among the cells of an imaginal disc. This study does not rule out the existence of DNA regulatory elements that are not marked by open chromatin or are otherwise not detected by FAIRE (McKay, 2013).

Despite this sensitivity, the approach of this study does not identify which cells within the tissue have a particular open chromatin profile. For a given locus, it is possible that all cells in the tissue share a single open chromatin profile or that the FAIRE signal originates from only a subset of cells in which a given enhancer is active. Comparisons between eye-antennal discs, larval CNS, and thoracic discs suggest that the latter scenario is most likely, with open chromatin profiles among cells within a tissue shared by cells with similar identities at a given developmental stage (McKay, 2013).

The observation that halteres and wings share open chromatin profiles demonstrates that Hox proteins like Ubx can differentially interpret the DNA sequence within the same subset of enhancers to modify one structure into another. This is consistent with the idea that morphological differences are largely dependent on the precise location, duration, and magnitude of expression of similar genes, and it is further supported by the similarity in gene expression profiles observed between Drosophila appendages and observed between vertebrate limbs. However, that such dramatic differences in morphology could be achieved by using the same subset of DNA regulatory modules in different tissues genome-wide was not known. The current findings provide a molecular framework to support the hypothesis that Hox factors function as 'versatile generalists,' rather than stable binary switches. The similarity in open chromatin profiles between wings and legs suggests that this framework also extends to other classes of master regulators beyond the Hox genes. It is also noted that, like the Drosophila appendages, vertebrate limbs are composed of similar combinations of cell types that differ in their pattern of organization. Moreover, the Drosophila appendage master regulators share a common evolutionary origin with the master regulators of vertebrate limb development, suggesting that the concept of shared open chromatin profiles may also apply to human development (McKay, 2013).

The data suggest that open chromatin profiles vary both over time for a given lineage and between cell types at a given stage of development. Given the dramatic differences in the FAIRE landscape observed during embryogenesis and between the CNS and the appendage imaginal discs during larval stages, it appears as though the alteration of the chromatin landscape is especially important for specifying different cell types from a single genome. After cell-type specification, open chromatin profiles in the appendages continued to change as they proceeded toward terminal differentiation, suggesting that stage-specific functions require significant opening of new sites or the closing of existing sites. These findings contrast with those investigating hormone-induced changes in chromatin accessibility, in which the majority of open chromatin sites did not change after hormone treatment, including sites of de novo hormone-receptor binding. Thus, it may be that genome-wide remodeling of chromatin accessibility is reserved for the longer timescales and eventual permanence of developmental processes rather than the shorter timescales and transience of environmental responses (McKay, 2013).

Different combinations of 'master regulator' transcription factors, often termed selector genes, are expressed in the developing appendages. Selectors are thought to specify the identity of distinct regions of developing animals by regulating the expression of transcription factors, signaling pathway components, and other genes that act as effectors of identity. One property attributed to selectors to explain their unique power to specify identity during development is the ability to act as pioneer transcription factors. In such models, selectors are the first factors to bind target genes; once bound, selectors then create a permissive chromatin environment for other transcription factors to bind. The finding that the same set of enhancers are accessible for use in all three appendages, with the exception of the enhancers that control expression of the selector genes themselves and other primary determinants of appendage identity, suggests that the selectors expressed in each appendage do not absolutely control the chromatin accessibility profile; otherwise, the haltere chromatin profile (for example) would differ from that of the wing because of the expression of Ubx (McKay, 2013).

What then determines the appendage open chromatin profiles? Because open chromatin is likely a consequence of transcription factor binding, two nonexclusive models are possible. First, different combinations of transcription factors could specify the same open chromatin profiles. In this scenario, each appendage's selectors would bind to the same enhancers across the genome. For example, the wing selector Vg, with its DNA binding partner Sd, would bind the same enhancers in the wing as Dll and Sp1 bind in the leg. In the second model, transcription factors other than the selectors could specify the appendage open chromatin profiles. Selector genes are a small fraction of the total number of transcription factors expressed in the appendages. Many of the non-selector transcription factors are expressed at similar levels in each appendage, and thermodynamic models would predict them to bind the same enhancers. This model could also help to explain how the appendage open chromatin profiles coordinately change over developmental time despite the steady expression of the appendage selector genes during this same period. It is possible that stage-specific transcription factors determine which enhancers are accessible at a given stage of development. This would help to explain the temporal specificity of target genes observed for selectors such as Ubx. Recent work supports the role of hormone-dependent transcription factors in specifying the temporal identity of target genes in the developing appendages (Mou, 2012). Further experiments, including ChIP of the selectors from each of the appendages, will be required to determine the extent to which either of these models is correct (McKay, 2013).

Binding of Ubx results in differential activity of enhancers in the haltere imaginal disc relative to the wing, despite equivalent accessibility of the enhancers in both discs, indicating that master regulators control morphogenesis by differentially regulating the activity of the same set of enhancers. It is likely that functional specificity of enhancers is achieved through multiple mechanisms. These include differential recruitment of coactivators and corepressors, modulation of binding specificity through interactions with cofactors, differential utilization of binding sites within a single enhancer, or regulation of binding dynamics through an altered chromatin context. This last mechanism would allow for epigenetic modifications early in development to affect subsequent gene regulatory events. For example, the activity of Ubx enhancers in the early embryo may control recruitment of Trithorax or Polycomb complexes to the PREs within the Ubx locus, which then maintain Ubx in the ON or OFF state at subsequent stages of development. Consistent with this model, Ubx enhancers active in the early embryo are only accessible in the 2-4 hr time point, whereas the accessibility of Ubx PREs varies little across developmental time or between tissues at a given developmental stage (McKay, 2013).

The current results also have implications for the evolution of morphological diversity. Halteres and wings are considered to have a common evolutionary origin, but the relationship between insect wings and legs is unresolved. The observation that wings and legs share open chromatin profiles supports the hypothesis that wings and legs also share a common evolutionary origin in flies. Because legs appear in the fossil record before wings, the similarity in their open chromatin profiles suggests that the existing leg cis-regulatory network was co-opted for use in creation of dorsal appendages during insect evolution (McKay, 2013).

Hox proteins mediate developmental and environmental control of autophagy

Hox genes encode evolutionarily conserved transcription factors, providing positional information used for differential morphogenesis along the anteroposterior axis. This study shows that Drosophila Hox proteins are potent repressors of the autophagic process. In inhibiting autophagy, Hox proteins display no apparent paralog specificity and do not provide positional information. Instead, they impose temporality on developmental autophagy and act as effectors of environmental signals in starvation-induced autophagy. Further characterization establishes that temporality is controlled by Pontin, a facultative component of the Brahma chromatin remodeling complex, and that Hox proteins impact on autophagy by repressing the expression of core components of the autophagy machinery. Finally, the potential of central and posterior mouse Hox proteins to inhibit autophagy in Drosophila and in vertebrate COS-7 cells indicates that regulation of autophagy is an evolutionary conserved feature of Hox proteins (Banreti, 2013).

Autophagy is a cellular process whose induction or inhibition involves multiple levels of regulation, including developmental signals conveyed by the steroid hormone ecdysone, and environmental signals, sensed in the case of amino acid starvation by the InR/dTOR pathways. These regulatory paths do not act independently but seem rather to be interconnected as illustrated by developmentally induced ecdysone-mediated autophagy that acts by repressing the inhibitory function of the InR pathway. This indicates that whereas upstream control is distinct, downstream control may be common (Banreti, 2013).

This study shows that Drosophila Hox proteins are potent inhibitors of autophagy, with a potent and equivalent impact on both developmental and starvation-induced autophagy, and establish that both converge in the regulation of Hox gene expression. This highlights Hox genes as central regulators of autophagy, acting as a node for mediating autophagy inhibition. In regulating autophagy, Hox proteins act at least through regulation of Atg genes and other autophagy genes. Consistent with a direct transcriptional effect of Hox proteins in controlling Atg genes, Ubx DNA binding was found to be essential for autophagy inhibition, whereas have previously shown that Ubx associates to genomic regions immediately adjacent to Atg5 and Atg7 genes (Banreti, 2013).

A key aspect underlying Hox-mediated autophagy control is the regulation of Hox gene expression, where Hox downregulation induces autophagy. This aspect is true for both developmental- and starvation-induced autophagy, where the dynamics of Hox proteins respond to ecdysone (developmental autophagy) and to InR/dTOR (starvation) signaling. Signals mediating changes in Hox gene expression result from changes in the expression of Pont, a facultative component of the Brm complex known to act as a global and positive regulator of Hox genes. Although not establishing changes in Brm complex composition at the L3 feeding/L3 wandering transition, the dynamics of Pont expression suggest that a Pont-depleted Brm complex loses its ability to maintain the expression of Hox genes, resulting in the release of Hox-mediated inhibition of autophagy (Banreti, 2013).

Hox proteins are widely described as providing spatial information required for differential morphogenesis along the A-P axis, within which they largely display paralog-specific activities. However, in regulating autophagy, Hox function is distinct. First, it appears to be generic, with all Hox proteins tested providing inhibitory activity. The need to alleviate global Hox gene function (achieved in this study by impairing the activity of the Brm complex) in order to induce autophagy, further supports their redundant function in inhibiting autophagy. Second, they provide temporal, instead of spatial, information, mediating the temporality of developmental autophagy downstream of ecdysone signaling. Third, in the case of starvation-induced autophagy, Hox genes respond to the InR/dTOR pathways, acting as environmental effectors (Banreti, 2013).

Investigating the evolutionary conservation of Hox-mediated inhibition of autophagy by exploring the activity of mouse Hox proteins in Drosophila fat body cells as well as in vertebrate COS-7 cells indicates that vertebrate Hox proteins also act as potent autophagy inhibitors. Further studies in vertebrate cells should frame their activity to the multiple physiological and pathological situations that involve autophagy and allow for deciphering the molecular modalities of their regulatory roles (Banreti, 2013).

In summary, these findings broaden the framework of Hox protein functions, showing that besides providing spatial information during development, they also coordinate temporal processes and, more surprisingly, act as mediators of environmental signals for autophagy regulation (Banreti, 2013).

Low affinity binding site clusters confer hox specificity and regulatory robustness

In animals, Hox transcription factors define regional identity in distinct anatomical domains. How Hox genes encode this specificity is a paradox, because different Hox proteins bind with high affinity in vitro to similar DNA sequences.This study demonstrates that the Hox protein Ultrabithorax (Ubx) in complex with its cofactor Extradenticle (Exd) binds specifically to clusters of very low affinity sites in enhancers of the shavenbaby gene of Drosophila. These low affinity sites conferred specificity for Ubx binding in vivo, but multiple clustered sites were required for robust expression when embryos developed in variable environments. Although most individual Ubx binding sites are not evolutionarily conserved, the overall enhancer architecture-clusters of low affinity binding sites-is maintained and required for enhancer function. Natural selection therefore works at the level of the enhancer, requiring a particular density of low affinity Ubx sites to confer both specific and robust expression (Crocker, 2015).

This study has demonstrated that the Hox protein Ubx regulates separate enhancers of the svb gene by binding, with its cofactors Exd and Hth, to clusters of low affinity binding sites. Combining in vitro and in vivo assays, experimental demonstration is provided of an affinity-specificity tradeoff for Hox proteins, such that enhancers that integrate Hox inputs to drive regionalized expression are unlikely to utilize high affinity Hox binding sites. Forced to utilize low affinity sites, enhancers have evolved to contain multiple binding sites to ensure regulatory robustness to genetic and environmental variations. Most individual Ubx-Exd sites have evolved rapidly, but evolution has conserved overall enhancer architecture, with clusters of low affinity sites (Crocker, 2015).

Homotypic clusters of transcription factor binding sites are pervasive in animal genomes and several models have been proposed to explain their existence. The current results provide experimental evidence that homotypic clusters of Hox binding sites can confer robustness to enhancers. This may reflect a more widespread phenomenon. Although many enhancers contain homotypic clusters with low affinity sites, previous studies have rarely detected changes in expression by deleting individual binding sites. However, these mutated enhancers have not been tested in variable environments. It is possible that many of these clustered sites confer regulatory robustness (Crocker, 2015).

It is useful to compare these results with previous studies that have demonstrated specific regulatory functions for homotypic clusters. For example, clustered binding sites in an enhancer of the Drosophila hunchback gene mediate cooperative DNA binding by Bicoid, which provides threshold-dependent enhancer activity. In other cases, clusters of homotypic binding sites act in a noncooperative manner to allow enhancers to respond in a graded fashion, for example to determine expression levels in response to transcription factor concentrations. It is worth noting that in these cases, where homotypic clusters mediate specific linear or nonlinear outputs, enhancers are bound by transcription factors that belong to small paralogous families: e.g., two paralogs for Msn2; three for p53; two for Dorsal; and five for NFκB. In contrast, there are 84 homeodomain-containing proteins encoded in the Drosophila genome, many with overlapping specificities. Therefore, in previously described examples of homotypic clusters, binding affinity may not be a strong constraint on specificity (Crocker, 2015).

For the Hox regulated svb enhancers, low affinity Ubx/AbdA-Exd binding sites enable specificity, while the clustering of low affinity sites confers phenotypic robustness. This is a fundamentally different constraint on clustered binding sites than observed in all previous examples. The affinity-specificity tradeoff, initially supported by computational analysis of in vitro data, was confirmed in vivo by progressively increasing the affinity of the Ubx-Exd binding sites. While replacement of low affinity sites with higher affinity sites always quantitatively altered enhancer activity, either positively or negatively, most higher affinity sites generated strong ectopic expression. This ectopic expression is driven, at least in part, by gaining the binding of additional Hox proteins, which are normally not involved in the regulation of these enhancers. Other studies have performed replacement of low affinity sites with higher affinity sites and, in some cases, they have also observed ectopic expression. These altered patterns of expression may reflect increased sensitivity of enhancers to the same transcription factor that binds to the wild-type enhancer. This study observed a similar effect for Ubx and AbdA-dependent upregulation of svb enhancers in the cells in which they are normally expressed. In addition, however, it was found that sites with higher affinity resulted in a reduced specificity, due to the binding of additional homeodomain proteins, such as Scr, to svb enhancers. Computational analyses suggest that this affinity-specificity tradeoff is a fundamental property of Hox proteins and would therefore influence the architecture of enhancers that must generate specific outputs in response to Hox factors. It is suggested that transcription factors that belong to other large paralog groups may exhibit a similar affinity-specificity tradeoff and that enhancers regulated by these factors may also exploit clusters of low affinity sites (Crocker, 2015).

The results help to explain previous difficulties with bioinformatic prediction of functional Hox binding sites, because low affinity sites are difficult to detect reliably. Indeed, the low affinity sites that implement Hox regulation within svb enhancers share little similarity with canonical Hox or Hox-Exd binding sites. Consequently, a very large number of seemingly disparate DNA sequences can confer low affinity binding for Hox proteins. If Hox-Exd sites are often clustered in the genome, then signals from genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) will reflect binding to the entire cluster (as was observed) and the signals associated with individual low affinity sites may be difficult to discern from noise. Identification of important low affinity sites will require a change in computational approaches to analyzing genome-wide data. Currently, it is de rigueur to apply an arbitrary threshold to genome-wide data and then to analyze only signals above this threshold. This approach is likely to bias detection toward high affinity sites, whose functions may be distinct from those of clusters of low affinity sites (Crocker, 2015).

These findings provide insight into how different Hox proteins regulate specific target genes to generate phenotypic diversity across the anterior-posterior axis. One unanswered question is how the many low affinity DNA sequences, which appear to share little in common, are bound by the same Hox-Exd complex with apparently similar affinity. It is possible that variations in DNA shape (deviations from the structure of canonical B-DNA) influence Hox-Exd binding to low affinity sites. It remains unclear if very different sequences can adopt similar shapes, or whether instead the Hox-Exd complex can recognize a range of shapes. Resolution of this question will require structural studies of Hox-Exd complexes bound to a range of low affinity DNA sequences and quantitative analysis of their binding dynamics in vivo (Crocker, 2015).

Shlyueva, D., Meireles-Filho, A. C. A., Pagani, M. and Stark, A. (2015). Genome-wide Ultrabithorax binding analysis reveals highly targeted genomic loci at developmental regulators and a potential connection to Polycomb-mediated regulation. bioRxiv

Genome-wide Ultrabithorax binding analysis reveals highly targeted genomic loci at developmental regulators and a potential connection to Polycomb-mediated regulation

Hox homeodomain transcription factors are key regulators of animal development. They specify the identity of segments along the anterior-posterior body axis in metazoans by controlling the expression of diverse downstream targets, including transcription factors and signaling pathway components. The Drosophila melanogaster Hox factor Ultrabithorax (Ubx) directs the development of thoracic and abdominal segments and appendages, and loss of Ubx function can lead for example to the transformation of third thoracic segment appendages (e.g. halters) into second thoracic segment appendages (e.g. wings), resulting in a characteristic four-wing phenotype. This study presents a Drosophila melanogaster strain with a V5-epitope tagged Ubx allele, which was employed to obtain a high quality genome-wide map of Ubx binding sites using ChIP-seq. The sensitivity of the V5 ChIP-seq was confirmed by recovering 7/8 of well-studied Ubx-dependent cis-regulatory regions. Ubx binding was shown to be predictive of enhancer activity as suggested by comparison with a genome-scale resource of in vivo tested enhancer candidates. Densely clustered Ubx binding sites were identified at 12 extended genomic loci that included ANTP-C, BX-C, Polycomb complex genes, and other regulators and the clustered binding sites were frequently active enhancers. Furthermore, Ubx binding was detected at known Polycomb response elements (PREs) and was associated with significant enrichments of Pc and Pho ChIP signals in contrast to binding sites of other developmental TFs. Together, these results show that Ubx targets developmental regulators via strongly clustered binding sites and allow the authors to hypothesize that regulation by Ubx might involve Polycomb group proteins to maintain specific regulatory states in cooperative or mutually exclusive fashion, an attractive model that combines two groups of proteins with prominent gene regulatory roles during animal development (Shlyueva, 2015).

This study presents a high-quality ChIP-seq dataset that allowed the identification of individual Ubx binding sites genome-wide. These binding sites frequently overlap with active enhancers and Ubx binding is predictive of enhancer activity, especially outside HOT regions. Importantly, Ubx binds extensively to highly targeted genomic loci (HTGLs), which often overlap the gene loci of developmental regulators and genes that are regulated by the Polycomb complex and the majority of these binding sites are functioning as enhancers during embryogenesis (Shlyueva, 2015).

The observation that Ubx binds to known PREs/TREs and that Ubx binding sites also show a significant Pc and Pho ChIP signal is suggestive of a model in which Ubx could be upstream of Pc targeting and involved in mediating or antagonizing Pc and Pho recruitment to their genomic binding sites. The data are consistent with two scenarios: Ubx and Pc/Trx binding might occur predominantly in the same cells and Ubx could be involved in recruiting Pc/Trx to their binding sites. Alternatively, Ubx and Pc/Trx might occur predominantly in mutually exclusive spatial domains or at different stages in the developing embryo and Ubx could potentially counteract Pc binding (Shlyueva, 2015).

The first hypothesis is consistent with known Polycomb-dependent Ubx repression by high transient levels of Ubx in haltere and the known repression of bxd in Ubx-expressing cells, which involved components of the Trx complex. The finding that Ubx was bound at bxd locus suggests that this repression could be direct and mediated by the Hox factor (Shlyueva, 2015).

In contrast, Ubx binding has not been observed at the Abd-A and abd-B loci in haltere, a tissue in which Abd-A and abd-B are repressed by Pc. Similarly, sites that are bound by Ubx in embryos have high levels of Pc and H3K27me3 in S2 cells that do not express Ubx. Therefore, while Ubx could be involved transiently during initial steps of Pc recruitment, it does not seem to be required for repression and Pc might even restrict TF access to these loci. Moreover, as Pc is typically associated with repression, the strong enrichment of active enhancers at Ubx binding sites suggests that Ubx could counteract Pc, potentially through enhancer activation. In other cells, Pc would then bind to and silence the same regions thereby counteracting Ubx function, leading for example to the high levels of H3K27me3 observed in ChIP experiments from entire embryos (Shlyueva, 2015).

The prediction that Ubx might be involved in specifying or counteracting the recruitment of Polycomb to specific genomic loci is attractive as it links Hox genes, which are involved in the definition of segment identity with Polycomb, which has been implicated in the maintenance of transcriptional regulatory states throughout development. While it was found that several TFs co-localize with Pc/Pho binding sites in ChIP from entire embryos, Ubx had the most prominent effect. Given the attractiveness and potential importance of this link between Hox genes and Polycomb, this observation is shared here with the broader scientific community (Shlyueva, 2015).

Roles of cofactors and chromatin accessibility in Hox protein target specificity

The regulation of specific target genes by transcription factors is central to understanding of gene network control in developmental and physiological processes, yet how target specificity is achieved is still poorly understood. This is well illustrated by the Hox family of transcription factors as their limited in vitro DNA-binding specificity contrasts with their clear in vivo functional specificity. This study generated genome-wide binding profiles for three Hox proteins, Ubx, Abd-A and Abd-B, following transient expression in Drosophila Kc167 cells, revealing clear target specificity and a striking influence of chromatin accessibility. In the absence of the TALE class homeodomain cofactors Exd and Hth, Ubx and Abd-A bind at a very similar set of target sites in accessible chromatin, whereas Abd-B binds at an additional specific set of targets. Provision of Hox cofactors Exd and Hth considerably modifies the Ubx genome-wide binding profile enabling Ubx to bind at an additional novel set of targets. Both the Abd-B specific targets and the cofactor-dependent Ubx targets are in chromatin that is relatively DNase1 inaccessible prior to the expression of Hox proteins/Hox cofactors. It is concluded that there is a strong role for chromatin accessibility in Hox protein binding, and the results suggest that Hox protein competition with nucleosomes has a major role in Hox protein target specificity in vivo (Beh, 2016).

A comparative genomic analysis of targets of Hox protein Ultrabithorax amongst distant insect species

In the fruitfly Drosophila melanogaster, the differential development of wing and haltere is dependent on the function of the Hox protein Ultrabithorax (Ubx). This study compare Ubx-mediated regulation of wing patterning genes between the honeybee, Apis mellifera, the silkmoth, Bombyx mori and Drosophila. Orthologues of Ubx are expressed in the third thoracic segment of Apis and Bombyx, although they make functional hindwings. When over-expressed in transgenic Drosophila, Ubx derived from Apis or Bombyx could suppress wing development, suggesting evolutionary changes at the level of co-factors and/or targets of Ubx. To gain further insights into such events, direct targets of Ubx from Apis and Bombyx were identified by ChIP-seq and compared with those of Drosophila. While majority of the putative targets of Ubx are species-specific, a considerable number of wing-patterning genes are retained, over the past 300 millions years, as targets in all the three species. Interestingly, many of these are differentially expressed only between wing and haltere in Drosophila but not between forewing and hindwing in Apis or Bombyx. Detailed bioinformatics and experimental validation of enhancer sequences suggest that, perhaps along with other factors, changes in the cis-regulatory sequences of earlier targets contribute to diversity in Ubx function (Prasad, 2016).

Ubx dynamically regulates Dpp signaling by repressing Dad expression during copper cell regeneration in the adult Drosophila midgut

The gastrointestinal (GI) tract of metazoans is lined by a series of regionally distinct epithelia. To maintain structure and function of the GI tract, regionally diversified differentiation of somatic stem cell (SC) lineages is critical. The adult Drosophila midgut provides an accessible model to study SC regulation and specification in a regionally defined manner. SCs of the posterior midgut (PM) have been studied extensively, but the control of SCs in the middle midgut (MM) is less well understood. The MM contains a stomach-like copper cell region (CCR) that is regenerated by gastric stem cells (GSSCs) and contains acid-secreting copper cells (CCs). Bmp-like Decapentaplegic (Dpp) signaling determines the identity of GSSCs, and is required for CC regeneration, yet the precise control of Dpp signaling activity in this lineage remains to be fully established. This study shows that Dad, a negative feedback regulator of Dpp signaling, is dynamically regulated in the GSSC lineage to allow CC differentiation. Dad is highly expressed in GSSCs and their first daughter cells, the gastroblasts (GBs), but has to be repressed in differentiating CCs to allow Dpp-mediated differentiation into CCs. WThe Hox gene Ultrabithorax (Ubx) is required for this regulation. Loss of Ubx prevents Dad repression in the CCR, resulting in defective CC regeneration. This study highlights the need for dynamic control of Dpp signaling activity in the differentiation of the GSSC lineage and identifies Ubx as a critical regulator of this process (Li, 2016).

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