Targets of Activity

Ecdysteroid signaling in insects is mediated by the ecdysone receptor complex, which is composed of a heterodimer of the Ecdysone receptor and Ultraspiracle. The DNA binding specificity plays a critical role in defining the repertoire of target genes that respond to the hormone. The determination of the preferred core recognition motif by a binding site selection procedure is described. The consensus sequence consists of a perfect palindrome of the heptameric half-site sequence GAGGTCA that is separated by a single A/T base pair. No binding polarity of the Ecdysone receptor/Ultraspiracle heterodimer to the core recognition motif is observed. This core motif mediates the highest level of ligand-induced transactivation when compared to a series of synthetic ecdysone response elements and to the natural element of the Drosophila hsp27 gene. This is the first report of a palindromic sequence being identified as the highest affinity DNA binding site for a heterodimeric nuclear hormone receptor complex. Evidence is presented that the ligand of the Ecdysone receptor preferentially drives Ultraspiracle from a homodimer into a heterodimer. This mechanism might contribute additionally to a tight control of target gene expression (Vogtli, 1998).

Transcription of the Drosophila Fbp1 gene is induced by the steroid hormone 20-hydroxyecdysone and restricted to the late-third-instar fat body tissue. When a nuclear extract from the fat body is used as a source of binding factors, the proximal promoter of Fbp1 forms seven identifiable nucleoprotein complexes. These complexes are all generated from interaction of USP to the proximal promoter of Fbp1. The promoter structure of Fbp1 bears strong similarity to the hsp27 ecdysone response element (Antoniewski, 1994).

USP binds to the follicular-specific chorion protein s15 proximal promoter (Khoury Christianson, 1992). USP can bind alone to a single hormone response element half site (Since nuclear hormone DNA binding sites usually occur as repeated sequence segments, the term half site refers to a monomeric sequence segment). The equilibrium dissociation constant of USP to its response element on the chorion s15 promoter is equivalent to the binding activities of other nuclear hormone receptors to their cognate elements (Khoury Christianson, 1993).

The Larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster is uniquely expressed in the fat body tissue from the beginning of the third instar to the end of adult life. Accumulation of the larval Lsp-2 transcript is enhanced by 20-hydroxyecdysone. A single functional ecdysone response element (EcRE) has been localized at position -75 relative to the Lsp-2 transcription initiation site. A 27-bp sequence harboring the EcRE binds both the Drosophila Ecdysone receptor and Ultraspiracle in a cooperative manner. The affinity of the Lsp-2 EcRE for the Ecdysone receptor complex is comparable to that of the EcRE of the hsp27 gene and is at least four times greater than that of Fbp1, another fat body-specific Drosophila gene. Structural features of this EcRE form the basis for differential hormone responsiveness within the fat body (Antoniewski, 1995).

BmEcR from the commercial silkmoth, Bombyx mori, is a functional ecdysone receptor. Upon dimerization with BmCF1 (the silkmoth homolog of Drosophila Usp), BmEcR binds the radiolabeled steroid ligand 125I-iodoponasterone A with Kd = 1.1 nM, rendering it indistinguishable from that exhibited by DmEcR/DmUSP. BmEcR/BmCF1 forms a specific complex with an ecdysone response element (EcRE) derived from the Drosophila heat shock protein 27 (hsp27) gene promoter; as with DmEcR/DmUSP, formation of this complex is stimulated by the presence of 20-hydroxyecdysone. BmEcR can substitute for DmEcR in an EcR-deficient Drosophila tissue culture line, stimulating trans-activation of an ecdysone-inducible reporter gene construct. Thus, BmEcR and BmCF1 are the functional counterparts of DmEcR and DmUSP, respectively and, despite considerable sequence divergence between the Drosophila and Bombyx proteins, the counterparts are (at least qualitatively) functionally equivalent (Swevers, 1996).

A fundamental unresolved question in endocrinological research is how systemic signals like pulses of steroid hormones are converted into a variety of tissue- and stage-specific responses. The existence of three different Ecdysone receptor isoforms, which are differentially expressed in larval and imaginal tissues, may provide the first clue for the differential regulation of these responses. Sgs genes are salivary gland secretion protein genes, regulated in Drosophila by the molt cycle. Two regulatory elements were identified in the upstream region of the Drosophila Sgs-3 gene that are both able to bind the Ecdysone receptor (EcR/USP) and the product of the fork head gene. Interestingly, only one of the EcR/USP binding sites is able to recognize in vitro-translated EcR/USP, which provides evidence for the existence of different receptor forms having different DNA binding specificities. Deletions of the elements leads to a reduced accumulation of Sgs-3 mRNA without altering the temporal expression profile of the gene. The data are consistent with the hypothesis that the Ecdysone receptor directly contributes to the transcriptional activation of Sgs-3 by binding to at least one of the two elements. Since the Sgs-4 gene is also controlled by a functional EcR/USP binding site, a direct participation of EcR/USP in the formation of regulatory complexes may be of general importance for the hormonal control of Sgs genes (Lehmann, 1997).

In Drosophila, peaks of the titer of the steroid hormone ecdysone act as molecular signals that trigger all the major developmental transitions occurring along the life cycle. The EcR/USP heterodimer, known to constitute the functional ecdysone receptor, binds with high affinity to specific target sequences. The target sequences, known as ecdysone response elements (EcREs) still remain to be fully characterized at both the molecular and functional levels. In order to investigate the properties of EcREs composed of directly repeated half-sites (DRs), an analysis was carried out of the binding properties of the ng-EcRE, a DR element located within the coding region of ng-1 and ng-2, two highly homologous genes mapping at the ecdysone-regulated 3C intermolt puff. The ng-EcRE contacts the Ecdysone receptor through its directly repeated half-sites spaced by 12 bp, and this element may interact efficiently with at least three Drosophila orphan receptors, namely DHR38, DHR39 and beta FTZ-F1. Interestingly, DHR38 is bound alone or in combination with USP, providing the first evidence that the EcR-USP and DHR38-USP may directly compete for binding to a common response element. These results suggest that EcREs composed of widely spaced DRs may contribute to the establishment of extensive cross-talk between nuclear receptors, thus modulating ng-1 and ng-2 intermolt expression (Crispi, 1998).

The expression of Hormone-receptor-like in 78 in larval salivary glands allowed for the identification of potential regulatory targets by antibody staining of polytene chromosomes. Whereas no binding sites can be detected in polytene chromosomes prepared from Hr78 mutant larvae, multiple stained sites could be detected in polytene chromosomes prepared from wild-type mid-third instar larvae. Hr78 protein can bind to a subset of 20E receptor binding sites in vitro, suggesting that Hr78 might function at the top of the ecdysteroid regulatory hierarchies. In order to determine if Hr78 exhibits a similar binding specificity in vivo, polytene chromosomes were stained with antibodies directed against either Hr78 or Ultraspiracle. The staining pattern of Usp is identical to that of EcR, and thus indicative of sites bound by the 20E receptor. Some sites are bound primarily by the EcR or Hr78, while the majority of sites are bound by both proteins, consistent with an overlap in their binding specificity. In order to map the sites bound by Hr78, salivary glands were dissected from newly formed white prepupae, when Hr78 protein is most abundant, and polytene chromosome preparations were stained with anti-Hr78 antibodies. Over 100 Hr78 binding sites have been identified, many of which correspond to ecdysteroid regulated puff loci (Fisk, 1998).

Response to the insect hormone ecdysone is mediated by a nuclear receptor complex containing Ultraspiracle (Usp) and the Ecdysone Receptor (EcR). Among other phenotypes, loss of functional Usp in Drosophila eye development results in an accelerated morphogenetic furrow, although loss of ecdysone arrests the furrow. Usp both represses and activates a gene affecting furrow movement, the ecdysone-responsive Z1 isoform of Broad-Complex. Using targeted replacement of Usp to rescue usp mutant clones in the eye, various USP functions have been mapped and whether the USP nuclear receptor has an activating as well as a repressive effect on furrow movement has been tested. Furrow movement and related phenotypes are rescued by the presence of Usp in a limited domain near the furrow, while other phenotypes are rescued by Usp expression posterior to the furrow. These data indicate roles for Usp activity at multiple developmental stages and help explain why loss of functional Usp leads to furrow advancement while loss of ecdysone stops furrow movement (Ghbeish, 2002).

These data demonstrate that the usp gene regulates the expression of multiple genes involved in differentiation in developing imaginal discs. Most, but not all, usp mutant phenotypes in developing discs appear to involve premature gene expression in otherwise normal target cells. In the eye disc, normal morphogenetic furrow movement, photoreceptor differentiation and ommatidial organization depend on Usp activity in a limited domain near the furrow while other functions require Usp posterior to the furrow. The usp gene likely affects furrow movement and photoreceptor differentiation in part by regulating the BrC-Z1. Loss of the function of the Usp protein as a repressor of BrC-Z1, and perhaps other genes, in the furrow region is sufficient to account for many of the usp- furrow-associated phenotypes. Conversely, stoppage of the furrow in the absence of ecdysone probably results from continued BrC-Z1 repression by the EcR/Usp complex (Ghbeish, 2002).

Loss of functional Usp affects multiple genes involved in cell determination in the eye such as scabrous, cut and tramtrack as well as neuronal seven-up, elav and spalt. For most of these markers, expression occurs prematurely, although cellular differentiation appears to occur normally. For example, in usp mutant regions, the Cut protein is first expressed toward the posterior of the third instar eye disc, while in wild-type eye discs Cut expression in the same region is seen later during pre-pupal development. In the wing, multiple markers of various stages of bristle complex determination and differentiation, such as Elav, Cut, ttk-lacZ and Cyclin B, are expressed prematurely at the site of the future triple row of bristles. These data suggest that the usp gene behaves as a timer causing cellular differentiation to occur at the proper pace in imaginal disc development (Ghbeish, 2002).

Scabrous protein misexpression posterior to the furrow in usp- clones appears to be an exception to the model of premature but normal differentiation in usp mutant clones. The sca gene is required for spacing and specification of the R8 photoreceptors and expression associated with this function is normal in usp- tissue. In contrast, usp mutant regions show Sca positive cells posterior to the furrow. No Sca expression is seen in these regions in wild-type larval or pre-pupal discs, and no additional roles in eye development have been reported for sca. Thus, this phenotype appears to be true misexpression rather than premature expression. It remains possible that ttk-lacZ expression in usp mutant clones represents ectopic as well as premature expression, suggesting increased numbers of differentiating cone cells. However, examination of Cut expression suggests merely premature differentiation, not extra cone cells (Ghbeish, 2002).

The lack of striking furrow acceleration seen in usp mutant clones using Sca as a marker is of interest. This brings up the possibility that loss of usp does not result in furrow acceleration as previously reported, although premature photoreceptor differentiation is still evident. However, it is clearly seen that furrow acceleration occurs in usp- tissue as judged by dpp-lacZ, which has a more limited expression pattern within the furrow than does Sca. Loss of usp results in a subtle advancement of the furrow, which can only clearly be seen in long usp- clones (Ghbeish, 2002).

usp mutant phenotypes occur at multiple regions of the eye disc. Using targeted Usp protein in usp mutant clones, various usp mutant phenotypes could be rescued and the different regions in which Usp protein is needed have been mapped. The initiation of Usp protein expression in a limited domain near or in the furrow is able to rescue a series of phenotypes, some of which are revealed posterior to the furrow. These include premature furrow advancement and neuronal differentiation, as well as disarrangement of the ommatidial clusters. Usp protein expression initiating posterior to the furrow will not rescue these phenotypes. Other functions, such as maintaining the appropriate number of Cyclin B positive cells, and posterior repression of ttk-lacZ, cut, and sca can be rescued by targeting Usp protein posterior to the furrow (Ghbeish, 2002).

It has been posited that extra cells taking on a cone cell fate, or cells being recruited into clusters ahead of appropriate mitotic divisions, might leave too few cells for later functions, resulting in disarrangement of the ommatidia. The data do not support these models. ttk, a gene expressed in cone cells, is expressed prematurely and perhaps more broadly, in usp- clones. However, this phenotype is rescued by usp transcription beginning posterior to the furrow, even though the ommatidia remain disarranged. Thus, premature cone-cell fate does not cause the abnormal cluster morphology. Similarly, the number of cells expressing Cyclin B returns to normal with post-furrow targeting of the Usp protein, but the ommatidial clusters remain disorganized. Neither precocious recruitment of these Cyclin B-expressing cells into clusters nor a decrease in the number of potential mitotic cells posterior to the furrow causes the ommatidial disarrangement phenotype present in usp mutant clones (Ghbeish, 2002).

This and previous studies clearly show that the ultraspiracle gene functions as a repressor of differentiation in both the eye and wing imaginal discs. Loss of functional Usp results in the premature differentiation of multiple cell types including the photoreceptors and the cone cells in the eye. Furthermore, Usp represses the ectopic expression of at least one gene, scabrous. This repressive function may or may not be due to direct activity of the Usp nuclear receptor on target genes either as part of the EcR complex, alone, or as part of another nuclear receptor complex (Ghbeish, 2002).

Examination of the role of the usp gene in the regulation of BrC-Z1 may be particularly informative. BrC-Z1, a known target for regulation by Usp/EcR protein complexes, is not normally expressed anterior to the furrow, but is upregulated just posterior to the furrow. Loss of Usp nuclear receptor repressive function leads to high level expression of BrC-Z1 protein both anterior and posterior to the furrow. A null allele of usp shows no further post-furrow activation, but the partially functional usp3 allele allows increased posterior expression consistent with Usp-mediated activation as well as repression. Readdition of usp+ in a position specific manner shows that wild-type BrC-Z1 levels return once Usp protein is expressed (Ghbeish et al., 2001).

The expression of BrC-Z1 protein in the presence and absence of usp gene activity allows the analysis of the relative magnitude of the Usp nuclear receptor repression and activation functions in an in vivo context. Surprisingly, the level of post-furrow BrC-Z1 protein in wild-type regions is substantially lower than the level of expression resulting from the complete loss of usp gene activity. Thus, for at least this particular situation, the repression of intrinsic gene expression activity by an RXR-containing complex is far greater than the level of gene activation. This may explain why the eye phenotypes of all three usp alleles are similar, since both null and missense alleles have levels of BrC-Z1 protein well above those seen in wild-type discs (Ghbeish, 2002).

The observations concerning the effects of loss of usp gene function on BrC-Z1 protein expression may resolve a superficial paradox of eye development. usp mutations increase the rate of furrow movement while loss of ecdysone stops the furrow. Additionally, loss of ecdysone decreases the level of BrC-Z1 protein posterior to the furrow and loss of BrC-Z1 results in disruption of neural differentiation and the furrow. This suggests that ecdysone regulation in the eye is mediated in part by the BrC-Z1 gene. It is possible that additional ecdysone-responsive genes are regulated in this dual manner by the EcR/Usp complex during furrow movement. These results are completely consistent with the observations made in this study. In the absence of hormone activity, BrC-Z1 gene activation is repressed. Posterior to the furrow, the consequences of such repression help explain the furrow stoppage phenotype, especially as loss of BrC-Z1 gene function leads to a loss of the critical furrow activator, Hedgehog. By contrast, loss of the fully functional Usp nuclear receptor, far from leading to a loss of BrC-Z1 protein expression, leads to high level expression, both anterior and posterior to the furrow. This high level of BrC-Z1 protein in usp mutant regions may help explain the furrow advancement phenotypes. In the region in or very near the furrow, other regulatory genes (such as hairy, extramacrochaete, atonal, hedgehog, patched and decapentaplegic) function to initiate eye differentiation. High levels of BrC-Z1 protein in this region may induce slightly premature differentiation of photoreceptor cells. This would lead to slightly premature Hedgehog protein expression and possibly incrementally faster furrow movement. Summed across a large usp mutant clone, this would appear as notable furrow advancement (Ghbeish, 2002).

Recent results suggest that the EcR may not play a role in eye development. Similarly, it has been seen that partially functional EcR mutants do not lead to any obvious defects in furrow movement, neuronal differentiation or cluster formation. Since these data are based on partially functional alleles, conditional rescue of a null allele and a limited number of small EcR mutant eye clones, it is possible that the EcR nuclear receptor may play a role in eye development not uncovered by these experiments. Alternatively, a novel EcR and partner for Usp may exist in the eye (Ghbeish, 2002).

In summary, these results show a role for the usp gene in controlling the timing of gene expression and differentiation in developing imaginal discs. In the eye disc some functions are very closely linked to events at the morphogenetic furrow, while others are clearly posterior to the furrow. It is suggested that the Usp nuclear receptor mediates the effects of ecdysone on furrow movement and neuronal differentiation in the eye imaginal disc in part by regulating the BrC-Z1. The specifics of gene repression and gene induction in this interaction account for the differences between loss of the Usp protein and loss of ecdysone on furrow movement. This analysis shows that in vivo the magnitude of repression of the BrC-Z1 gene is far greater than the magnitude of activation (Ghbeish, 2002).

The steroid hormone, 20-hydroxyecdysone (20E), directs Drosophila metamorphosis via a heterodimeric receptor formed by two members of the nuclear hormone receptors superfamily, EcR and Usp. A study on EcR and Usp DNA-binding domains (EcRDBD and UspDBD, respectively) has suggested that UspDBD may act as a specific anchor that preferentially binds the 5' half-site of the pseudo-palindromic response element from the hsp27 gene promoter and thus locates the heterocomplex in the defined orientation. This study analyzes in detail the determinants of the UspDBD interaction with the hsp27 element. The roles of individual amino acids in the putative DNA recognition alpha helix and the roles of the base pairs of the UspDBD target sequence have been probed by site-directed mutagenesis. The results show how the hsp27 element specifies UspDBD binding and thus the polar assembly of the UspDBD/EcRDBD heterocomplex. It is suggested how possible nucleotide deviations within the 5' half-site of the element may be used for the fine-tuning of the 20E-response element specificity and consequently the physiological response (Grad, 2001).

Ligand-dependent de-repression via EcR/USP acts as a gate to coordinate the differentiation of sensory neurons in the Drosophila wing

Loss of function of either the ecdysone receptor (EcR) or Ultraspiracle (USP), the two components of the ecdysone receptor, causes precocious differentiation of the sensory neurons on the wing of Drosophila. It is proposed that the unliganded receptor complex is repressive and that this repression is relieved as the hormone titers increase at the onset of metamorphosis. The point in development where the receptor complex exerts this repression varies for different groups of sensilla. For the chemosensory organ precursors along the wing margin, the block is at the level of senseless expression and is indirect, via the repressive control of broad expression. Misexpressing broad or senseless can circumvent the repression by the unliganded receptor and leads to precocious differentiation of the sensory neurons. This precocious differentiation results in the misguidance of their axons. The sensory precursors of some of the campaniform sensilla on the third longitudinal vein are born prior to the rise in ecdysone. Their differentiation is also repressed by the unliganded EcR/USP complex but the block occurs after senseless expression but before the precursors undertake their first division. It is suggested that in imaginal discs the unliganded EcR/USP complex acts as a ligand-sensitive 'gate' that can be imposed at various points in a developmental pathway, depending on the nature of the cells involved. In this way, the ecdysone signal can function as a developmental timer coordinating development within the imaginal disc (Schubiger, 2005).

The ecdysone signal is transmitted via the ecdysone receptor to activate a number of direct target genes. It has generally been assume that the hormone and its receptor activate a hierarchy by activating early genes that then activate the many late genes. A large body of work based primarily on larval tissues has supported this model. Thus when the ecdysone receptor is non functional, the first step in the cascade fails, the early genes are not activated, and the tissues are unable to undergo a metamorphic response. In imaginal discs it has been shown that loss of function of USP leads to the inability to activate early genes, such as DHR3, EcR and E75B, but also results in precocious differentiation, rather than in a failure to initiate a particular metamorphic response. It has now been demonstrated that loss of EcR function in the wing discs gives similar results (precocious BR-Z1 expression and sensory neuron differentiation) to the ones reported for loss of USP function, and it is concluded that the unliganded EcR/USP heterodimer is the functional repressor. Thus at least some processes at the onset of metamorphosis are not controlled by the ecdysone-induced hierarchy, but rather through the relief of the repressive function of the unliganded EcR/USP complex once the ecdysone titers rise. The importance of this repressive function of the unliganded receptor is further demonstrated by experiments using a dominant negative EcR, which does not bind the hormone, and as a consequence repression cannot be relieved and target genes are not expressed. The repressive role proposed for the unliganded ecdysone receptor complex would also explain why loss-of-function USP clones, in general, result in the differentiation of normal adult bristle organs since loss of receptor function would only control the timing of differentiation. Such an interpretation is supported by early pigmentation of abdominal bristles in usp3 clones that has been observed on occasions. In vivo studies of activation by EcR/USP suggest that activation plays a major role in the metamorphic response of larval tissues but has only a minor role in the development of the imaginal discs. It remains to be seen which processes are activated by the ecdysone hierarchy and which by loss of the repressive actions of the unliganded receptor (Schubiger, 2005).

Adult chemosensory neurons on the wing margin undergo precocious differentiation in loss-of-USP clones. To understand which step is repressed by the EcR/USP complex the early expression patterns of a set of genes involved in neuron differentiation was examined in such mutant clones. In the absence of USP function the early pattern of Achaete (AC) expression in the margin is unaffected. In contrast, both Neur (as visualized by A101) and Sens are expressed in usp mutant cells before they are detected in the surrounding wild-type tissue. In vitro experiments revealed that A101 expression that is already on at the time cultures were set up, remains on through the culture period, but that there is a block by EcR/USP at the level of sens expression that prevents the maturation of the SOPs of the chemosensory neurons in the wing margin. The block is released once the hormone titers rise (Schubiger, 2005).

The repressive function of the unliganded receptor does not act directly on the genes tested. The block of SOP differentiation is controlled through BR-Z1, and br function is required for the activation of sens, a gene necessary and sufficient for sensory organ differentiation. Thus expressing BR-Z1 or Sens early in the margin allows the inhibition from the unliganded ecdysone receptor to be by-passed and the sensory neurons in the margin to differentiate precociously. When Sens is misexpressed it was found that clusters of extra neurons differentiate in the region of high driver expression. This is in agreement with reports that high levels of Sens activate the proneural genes and promote the formation of SOPs. By contrast, when BR-Z1 is misexpressed in the margin, a more normal pattern of sensory neuron arrangement is observed, that is very similar to what is observed in loss-of-function USP or EcR cells. This indicates that BR-Z1 does not induce the formation of SOPs but rather causes the up-regulation of Sens in cells that have already committed to the SOP fate. Occasionally expressing BR-Z1 in the margin leads to the differentiation of a sensory neuron in the posterior margin, normally devoid of neurons. It is possible that in such a situation BR-Z1 misexpression can at times lead to sufficiently high expression of Sens to cause SOP differentiation (Schubiger, 2005).

Loss-of-function of BR demonstrates the requirement for BR to activate the high levels of Sens in the SOPs, as well as the low levels in the posterior margin, but without molecular data it is not known if br is directly activating sens. Since BR-Z1 normally appears later than the initial low expression of Sens, it is proposed that early Sens expression is most probably controlled by BR-Z2. BR-Z2 is expressed shortly after the molt to the third instar, and ectopic BR-Z2 expression induces low levels of Sens. BR-Z3, which also induces Sens when ectopically expressed may induce low levels of Sens as well, but since BR-Z3 is normally expressed at very low levels in the wing disc, it is thought that Br-Z3 plays a minor role. BR-Z1 then is needed for the accumulation of Sens in the mature SOPs (Schubiger, 2005).

In summary the above genetic interactions suggest that unliganded EcR/USP represses BR expression that is required for sens activation and the formation of the mature SOPs in the margin (Schubiger, 2005).

The SOPs are born in a specific temporal sequence in the wing disc. The first SOPs arise in the third instar, 20-30 hours before pupariation; they include GSR, ACV and L3-2 along the third vein. The SOPs of the margin arise later, at 10-12 hours before pupariation, so they are at a very different stage from that of the early born SOPs at the time metamorphosis begins. Since the unliganded receptor is acting as a repressor it is postulated that the block must be occurring at different times during the progression of sensory organ differentiation for these two groups of sensilla. Based on genetic studies, the ecdysone-sensitive arrest for the chemosensory sensilla of the margin occurs in the up-regulation of Sens, since the SOP is undergoing maturation. For the early born sensilla, however, Sens levels are already elevated before the rise in 20E and are not dependent on BR function. For these early born sensilla the ecdysone-sensitive arrest occurs after high Sens expression but prior to the division of the SOP. It is thought that for different sets of sensilla the imposition of an ecdysone-sensitive arrest at different points in development is important to coordinate the differentiation of the sensilla. Such a mechanism would ensure that the outgrowing axons begin to elongate in a choreographed manner leading to the correct axon pathways and to their finding of the correct targets in the CNS according to their physiological function. This idea is supported by the observation that the axons of sensilla forced to differentiate precociously by the absence of a functional ecdysone receptor or by early expression of BR-Z1 or Sens often take abnormal routes (Schubiger, 2005).

Ecdysone is also acting as a timer for the formation of the chordotonal and Johnston's organs as well as for the initiation of the morphogenetic furrow. These structures arise early in the third instar (80 hours after egg laying) and appear to be under the control of the small ecdysone peak at that time. In the case of the leg chordotonal organ, ecdysone appears to be controlling the proneural gene atonal (ato). It is not known yet if this control also occurs via de-repression as is seen for the wing (Schubiger, 2005).

The subsequent progression of the morphogenetic furrow is also dependent on ecdysone. This action of ecdysone has been proposed not to occur via EcR. However, loss of USP leads to an advancement of the furrow and precocious differentiation of the photoreceptors. It has been reported that the progression of the morphogenetic furrow, as well as the timing of differentiation of the chordotonal organs in the leg, are controlled by the insulin receptor (InR)/Tor pathway, with increased InR signaling leading to precocious differentiation. In the wing margin, by contrast, increasing or decreasing InR signaling does not affect the timing of differentiation of the chemosensory neurons. Thus there must be multiple temporal control mechanisms for sensory structures. The current results have demonstrated repression of sensory organs by the unliganded ecdysone receptor at the end of the third instar, but do not rule out additional steps controlled by ecdysone or other factors. It remains to be elucidated which timer(s) is used when, and for which sensory structures (Schubiger, 2005).

In holometabolous insects functional larval tissues are replaced by the differentiating imaginal ones. The endocrine system is acting on larval tissues composed of differentiated cells that are thus in an equivalent state to initiate programs such as cell death and neuronal remodeling. Here EcR/USP's role is activational. For the differentiation of the imaginal tissues the endocrine system faces a varied cellular landscape where some cells may still be dividing while other have begun to differentiate. In these tissues the unliganded receptor acts as a repressor to interrupt the sequence of differentiation at different points in order to coordinate the response to the rising 20E titers. Release of repression by 20E may therefore function as a 'gate' at the onset of metamorphosis and thus would enable development of imaginal tissues to be coordinated and tightly controlled by the rising ecdysone titers. In metamorphosing amphibians a similar situation is seen with functional larval tissues such as the tail and the gills dying and adult limbs and lungs developing in response to thyroid hormone. It would not be surprising to find that the thyroid hormone receptor is activational in the larval tissues but that the forming adult tissues are controlled through de-repression (Schubiger, 2005).

Protein Interactions

USP binds to promoters as a heterodimer with Ecdysone receptor (Yao, 1992). Both EcR and USP co-localize on ecdysone-responsive loci (puffs) in polytene chromosomes (Yao, 1993).

EcR-RXR, the heterodimer of Ecdysone receptor and vertebrate retinoid-X-receptor, DNA-binding activity is stimulated by either ecdysteroid or 9-cis-retinoic acid, demonstrating that hormone can play a role in heterodimer formation (Thomas, 1993).

Seven-up (Svp) is required to prevent photoreceptors R1, R3, R4 and R6 from becoming R7 photoreceptors. Members of the nuclear/steroid receptor superfamily include Seven-up, the Drosophila homolog of the chicken ovalbumin upstream transcription factor (COUP-TF); Ultraspiracle, the Drosophila homolog of the retinoid X receptor; and the ecdysone receptor. SVP, like COUP-TF, can modulate Ultraspiracle-based hormonal signaling both in vitro and in vivo. Seven-up can inhibit ecdysone-dependent transactivation by the ecdysone receptor complex, a heterodimeric complex of USP and ecdysone receptor. This repression depends on the dose of Svp and occurs with two different Drosophila ecdysone response elements. Ectopic expression of Svp in vivo induces lethality during early metamorphosis, the time of maximal ecdysone responsiveness. Concomitant overexpression of usp rescues the larvae from the lethal effects of Svp. Thus Svp-mediated repression can occur by both DNA binding competition and protein-protein interactions (Zelhof, 1995).

Heterodimers of USP-Ecdysone receptor proteins bind to ecdysone response elements (EcREs) and ecdysone to modulate transcription. Drosophila hormone receptor 38 (DHR38) and Bombyx hormone receptor 38 (BHR38) are two insect homologs of rat nerve growth factor-induced protein B (NGFI-B). Although members of the NGFI-B family are thought to function exclusively as monomers, DHR38 and BHR38 in fact interact strongly with USP; this interaction is evolutionarily conserved. DHR38 can compete in vitro against EcR for dimerization with USP and consequently disrupt EcR-USP binding to an EcRE. This suggests that DHR38 plays a role in the ecdysone response and more generally, that NGFI-B type receptors may be able to function as heterodimers in regulating transcription with retinoid X receptor type receptors (Sutherland, 1995).

DHR38 is a member of the steroid receptor superfamily in Drosophila that is homologous to the vertebrate NGFI-B-type orphan receptors. In addition to binding to specific response elements as a monomer, DHR38 interacts with the USP component of the ecdysone receptor complex in vitro, in yeast and in a cell line, suggesting that DHR38 might modulate ecdysone-triggered signals in the fly. It is interesting that DHR38 homologs in mammals, the NGFI-B type receptors, can act as both monomers and heterodimers with the RXR homologs of USP. The molecular structure and expression of the Dhr38 gene has been characterized and an in vivo analysis of its function(s) in development has been initiated. The Dhr38 transcription unit spans more than 40 kb in length, includes four introns, and produces at least four mRNA isoforms differentially expressed in development; two of these are greatly enriched in the pupal stage and encode nested polypeptides. Four alleles of Dhr38 have been characterized: a P-element enchancer trap line, l(2)02306, which shows exclusively epidermal staining in the late larval, pre-pupal and pupal stages, and three EMS-induced alleles. Dhr38 alleles cause localized fragility and rupturing of the adult cuticle, demonstrating that Dhr38 plays an important role in late stages of epidermal metamorphosis (Kozlova, 1998).

The EcR/USP heterodimer can bind in vitro with various affinities to direct repetitions of the motif AGGTCA separated by 0 to 5 nucleotides. Repeats separated by 0 and 3 nucleotides can drive a strong fat body-specific ecdysteroid response of the Fat body protein 1 gene. Directly repeated ECR/USP binding sites are as effective as palindromic EcR elements in vivo, but additional flanking regulatory sequences potentiate the hormonal response mediated by both types of elements (Antoniewski, 1996).

Small hydrophobic hormones like steroids control many tissue-specific physiological responses in higher organisms. Hormone response is characterized by changes in gene expression, but the molecular details connecting target-gene transcription to the physiology of responding cells remain elusive. The salivary glands of Drosophila provide an ideal model system to investigate gaps in knowledge, because exposure to the steroid 20-hydroxyecdysone (20E) leads to a robust regulated secretion of glue granules after a stereotypical pattern of puffs (activated 20E-regulated genes) forms on the polytene chromosomes. A convenient bioassay for glue secretion is described in this study, and this bioassay is used to analyze mutants in components of the puffing hierarchy. 20E mediates secretion through the EcR/USP receptor, and two early-gene products, the rbp+ function of BR-C and the Ca2+-binding protein E63-1, are involved. Furthermore, 20E treatment of salivary glands leads to Ca2+ elevations by a genomic mechanism, and elevated Ca2+ levels are required for ectopically produced E63-1 to drive secretion. The results presented establish a connection between 20E exposure and changes in Ca+ levels that are mediated by Ca2+-effector proteins, and thus establish a mechanistic framework for future studies (Biyasheva, 2001).

SgsDelta3-GFP transgenes were used to monitor glue secretion. SgsDelta3-GFP is expressed in a pattern identical to that of the endogenous Sgs3 gene, and the fusion protein is properly sorted, secreted, and expelled from the salivary gland in a manner identical to that of the endogenous glue mix. The chromophore of this fusion protein is stable for several days and can be used to observe glue synthesis and secretion in living animals and dissected tissues. This allows for the easy performance of genetic screens for mutations that affect exocytosis. Finally, the SgsDelta3-GFP transgenes are important staging tools, allowing one to monitor development of third-instar larvae more precisely than current 'blue food' methods. This is possible because one can select individual larvae with specific patterns of 'green glue' in their salivary glands resulting from a developmentally regulated physiological stimulus. Thus, introducing an SgsDelta3-GFP transgene into the genetic background should allow for a more reliable method of selecting mass quantities of similarly aged animals for physiological and biochemical analyses (Biyasheva, 2001).

Although the majority of data presented here focuses on the secretion of glue granules by the premetamorphic pulse of ecdysteroids, there are reports that speculate that a small ecdysteroid pulse induces a series of developmental events, including glue gene induction during the mid-third instar (90-100 h AEL). However, the idea that 20E acts through the EcR/USP heterodimer to mediate these changes has recently been questioned, and the results presented here substantiate the challenge. l(3)ecd1 mutants, severely compromised but probably not devoid of circulating ecdysteroids, synthesize SgsDelta3-GFP. In addition, animals depleted of Ultraspiracle (Usp), EcR-B1, or EcR-B2 also express the fusion gene. However, since neither the EcR nor the usp mutants tested remove all protein (EcR-A remains intact; Usp is truncated in usp2 mutants, and some leaky expression might occur from the hs-usp construct), the caveat that glue gene induction requires only a small amount of ecdysteroid-EcR/USP signaling must be considered. It is also noted that the studies reporting that BR-C null mutants do not produce glue, are consistent with the observation that an SgsDelta3-GFP transgene is not expressed in npr1 animals (Biyasheva, 2001).

Using classical genetic approaches and RNAi technology, it has been shown that the ecdysteroid receptor, consisting of EcR and Usp, is required for glue secretion. Since limited secretion is detected in EcR-B1 and usp mutants, RNAi was used to establish their absolute requirement. This new technology is a rapid and simple approach to compromise candidate genes, to determine their requirement in secretion, especially when mutants are either not available or die early in development. Using osmotic shock to introduce double-stranded RNA into cultured glands greatly increases the utility of this method (Biyasheva, 2001).

Ultraspiracle interacts with juvenile hormone

The identification of potential endogenous or synthetic ligands for orphan receptors in the steroid receptor superfamily is important both for discerning endogenous regulatory pathways and for designing receptor inhibitors. The insect nuclear receptor Ultraspiracle (USP), an ortholog of vertebrate RXR, has long been treated as an orphan receptor. The fit of terpenoid ligands to the JH III-binding site of monomeric and homo-oligomeric Usp from Drosophila has been tested. Usp specifically binds juvenile hormone III (JH III), but not control farnesol or JH III acid, and also specifically changes in conformation upon binding of JH III in a fluorescence binding assay. Juvenile hormone III binding causes intramolecular changes in receptor conformation, and stabilizes the receptor's dimeric/oligomeric quaternary structure. In both a radiometric competition assay and the fluorescence binding assay the synthetic JH III agonist methoprene specifically competes with JH III for binding to Usp, the first demonstration of specific binding of a biologically active JH III analog to an insect nuclear receptor. The recombinant Usp binds with specificity to a DR12 hormone response element in a gel shift assay. The same DR12 element confers enhanced transcriptional responsiveness of a transfected juvenile hormone esterase core promoter to treatment of transfected cells with JH III, but not to treatment with retinoic acid or T3. The activity of JH III or JH III-like structures, but not structures without JH III biological activity, to bind specifically to Usp and activate its conformational change, provide evidence of a terpenoid endogenous ligand for Ultraspiracle, and offer the prospect that synthetic, terpenoid structures may be discovered that can agonize or antagonize Usp function in vivo (Jones, 1997).

The invertebrate nuclear receptor, Ultraspiracle (Usp), an ortholog of the vertebrate RXR, is typically modelled as an orphan receptor that functions without a ligand-binding activity. The identification of a ligand that can transcriptionally activate Usp would provide heuristic leads to the structure of potentially high affinity activating compounds, with which to detect unknown regulatory pathways in which this nuclear receptor participates. The application of the sesquiterpenoid methyl epoxyfarnesoate (juvenile hormone III) to Sf9 cells is shown to induce transcription from a transfected heterologous core promoter, through a 5'-placed DR12 enhancer to which the receptor Usp binds. Isolated, recombinant Usp from Drosophila specifically binds methyl epoxyfarnesoate, whereupon the receptor homodimerizes and changes tertiary conformation, including the movement of the ligand-binding domain alpha-helix 12. Ligand-binding pocket point mutants of USP that do not bind methyl epoxyfarnesoate act as dominant negative suppressors of methyl epoxyfarnesoate-activation of the reporter promoter, and addition of wild-type Usp rescues this activation. These data establish a paradigm in which the Usp ligand-binding pocket can productively bind ligand with a functional outcome of enhanced promoter activity, the first such demonstration for an invertebrate orphan nuclear receptor. USP thus establishes the precedent that invertebrate orphan receptors are viable targets for development of agonists and antagonists with which to discern and manipulate transcriptional pathways dependent on UspP or other orphan receptors. The demonstration here of these functional capacities of Usp in a transcriptional activation pathway has significant implications for current paradigms of Usp action that do not include a ligand-binding activity for Usp (Jones, 2001).

The invertebrate nuclear receptor, Ultraspiracle, an ortholog of the vertebrate RXR, is typically modelled as an orphan receptor that functions without a ligand-binding activity. The identification of a ligand that can transcriptionally activate Usp would provide heuristic leads to the structure of potentially high affinity activating compounds, with which to detect unknown regulatory pathways in which this nuclear receptor participates. Here, the application of the sesquiterpenoid methyl epoxyfarnesoate (juvenile hormone III) to Sf9 cells induces transcription from a transfected heterologous core promoter, through a 5'-placed DR12 enhancer to which the receptor Usp binds. Isolated, recombinant Usp from Drosophila melanogaster specifically binds methyl epoxyfarnesoate, whereupon the receptor homodimerizes and changes tertiary conformation, including the movement of the ligand-binding domain alpha-helix 12. Ligand-binding pocket point mutants of USP that do not bind methyl epoxyfarnesoate act as dominant negative suppressors of methyl epoxyfarnesoate-activation of the reporter promoter, and addition of wild-type Usp rescues this activation. These data establish a paradigm in which the USP ligand-binding pocket can productively bind ligand with a functional outcome of enhanced promoter activity, the first such demonstration for an invertebrate orphan nuclear receptor. Usp thus establishes the precedent that invertebrate orphan receptors are viable targets for development of agonists and antagonists with which to discern and manipulate transcriptional pathways dependent on Usp or other orphan receptors. The demonstration here of these functional capacities of Usp in a transcriptional activation pathway has significant implications for current paradigms of Usp action that do not include for Usp a ligand-binding activity (Xu, 2002).

Bonus interacts with hormone receptors and inhibits transcription

The Drosophila bonus (bon) gene encodes a homolog of the vertebrate TIF1 transcriptional cofactors. bon is required for male viability, molting, and numerous events in metamorphosis including leg elongation, bristle development, and pigmentation. Most of these processes are associated with genes that have been implicated in the ecdysone pathway, a nuclear hormone receptor pathway required throughout Drosophila development. Bon is associated with sites on the polytene chromosomes and can interact with numerous Drosophila nuclear receptor proteins. Bon binds via an LxxLL motif to the activator function AF-2 domain present in the ligand binding domain of betaFTZ-F1 and behaves as a transcriptional inhibitor in vivo (Beckstead, 2001).

Database searches have revealed that bon encodes the only Drosophila homolog of mammalian TIF1s. Bon exhibits 29% identity with mouse TIF1alpha and mouse TIF1beta, and 26% identity with human TIF1gamma. The overall identity between Bon and TIF1s is similar to the identity observed between the TIF1 members. A higher degree of identity is seen in the N- and C-terminal regions spanning the conserved domains. At the N terminus, a C3HC4 zinc-finger motif or RING finger is followed by two cysteine-rich zinc binding regions (B-boxes) and a coiled coil domain forming a tripartite motif designated RBCC. At the C terminus, a bromodomain is preceded by a C4HC3 zinc-finger motif or PHD finger (Beckstead, 2001 and references therein).

Northern analysis demonstrates that bon produces one predominant 6 kb transcript and two 4 kb transcripts, which each encode a protein of ~140 kDa. The two 4 kb transcripts are only present in 0-3 hr embryos and adult females. It is therefore possible that the 4kb mRNAs are maternal components. bon is expressed throughout embryogenesis and in first instars. Its levels increase in 9-12 hr embryos and are low during the second instar stage. bon is upregulated in late third instar larvae. The upregulation of bon during midembryogenesis and prior to pupariation correlates well with known high titer pulses of ecdysone (Beckstead, 2001).

Immunohistochemical staining of numerous tissues show that Bon is a nuclear protein expressed in most and possibly all cells during embryogenesis, in fat body, imaginal discs, salivary glands, brain, gut, Malpighian tubules, and trachea. Bon is a chromatin-associated protein that localizes to ~10%-15% of the polytene chromosome bands. This pattern is highly reproducible (Beckstead, 2001).

To test whether Bon interacts with betaFTZ-F1 as well as other Drosophila nuclear receptors in vitro, binding assays were performed using purified recombinant proteins. Glutathione-S transferase (GST)-fused betaFTZ-F1, alphaFTZ-F1 (amino acids 154-1029), Seven-up (SVP), DHR3, USP, and EcR were immobilized on glutathione-Sepharose and incubated with purified N-terminally His-tagged Bon (His-Bon). His-Bon binds to GST-betaFTZ-F1, GST-alphaFTZ-F1, GST-DHR3, GST-SVP, GST-USP, and GST-EcR, but not to GST alone. Thus, Bon can bind directly to many members of the nuclear receptor family in vitro (Beckstead, 2001).

To determine whether Bon is able to repress transcription, the coding sequence of Bon was fused to the yeast GAL4 DNA binding domain. The resulting fusion protein was tested for its ability to repress transcription activated by ER(C)-VP16, a chimeric activator containing the DBD of ERalpha fused to VP16. GAL4-Bon and ER(C)-VP16 were transiently transfected into S2 cells with a reporter containing a GAL4 binding site (17M) and an estrogen response element (ERE) in front of a thymidine kinase (tk) promoter-CAT fusion (17M-ERE-tk-CAT). GAL4-Bon efficiently represses transcription in a dose-dependent manner. In contrast, coexpression of Bon without the GAL4 DNA binding domain causes a reproducible increase in CAT activity, indicating that repression by Bon is entirely dependent on DNA binding (Beckstead, 2001).

To map the domain of Bon responsible for transcriptional repression, a set of N- and C-terminally truncated derivatives were assayed for their ability to repress VP16-activated transcription in S2 cells. In the absence of the RBCC motif, the GAL4-Bon fusion protein, GAL4-Bon [471-1133]) fails to repress transcription, indicating that the N-terminal region of Bon is required for repression. However, this region is not sufficient for full repression. Consistent with this, a C-terminal truncation, GAL4-Bon (1-890), is a less potent repressor, indicating that the C-terminal residues of the protein including the PHD finger and the bromodomain also contribute to the repression potential of Bon. However, this domain on its own exhibits little repression. A 3- to 4-fold increase in CAT activity is observed with the central region between the coiled-coil and the PHD finger, suggesting that Bon may also contain a 'masked' activation domain. Note, however, that no significant activation was observed with GAL4-Bon (471-890) tested in the absence of ER(C)-VP16. Taken together, these results indicate that most of the repression activity of Bon resides within the N-terminal RBCC domain (Beckstead, 2001).

Bon and TIF1s contain an N-terminal RBCC (RING finger/B boxes/coiled coil) motif. In the absence of the RBCC motif, the GAL4-Bon protein, unlike the full-length protein, fails to repress transcription. The TIF1beta RBCC domain has been shown to be necessary for the oligomerization of TIF1beta and KRAB binding. Because Bon is able to homodimerize, this domain may be involved in formation of protein complexes (Beckstead, 2001).

The PHD finger and bromodomain are characteristic features of nuclear proteins known to be associated with chromatin and/or to function at the chromatin level. For instance, the chromosomal proteins Trithorax and Polycomb-like contain multiple PHD fingers, while the histone acetyltransferases CBP and GCN5 as well as the chromatin-remodeling factor SWI2/SNF2 are also bromodomain containing proteins. Bromodomains have been shown to bind to acetyl-lysine and specifically interact with the amino-terminal tails of histones H3 and H4, suggesting a chromatin-targeting function for this highly evolutionarily conserved domain. Because Bon is localized to hundreds of chromatin bands on Drosophila polytene chromosomes, it is probably involved in chromatin-mediated regulation of transcription of numerous genes (Beckstead, 2001).

Bon can repress both basal and activated transcription when recruited to the promoter region of a target gene, similar to TIF1alpha, -beta, and -gamma. For TIF1alpha and TIF1beta, a link between silencing and histone modification has been established, and TIF1beta is part of a large multiprotein complex that possesses histone deacetylase activity. Moreover, TIF1beta was also reported to colocalize and interact directly with members of the heterochromatin protein 1 (HP1) family. Similar to TIF1beta, TIF1alpha can bind the HP1 proteins in vitro. However, TIF1alpha-mediated repression in transfected cells does not require the integrity of the HP1 interaction domain, nor is there any significant subnuclear colocalization of HP1alpha and TIF1alpha. No interactions were observed between Bon and HP1 in a yeast two-hybrid assay, nor was any evidence found for genetic interactions. However, in a yeast two-hybrid screen, Bon interacted with members of the Polycomb group, suggesting that Bon may also be part of heterochromatin-like complexes and/or may require some of the members of the Polycomb group genes to repress transcription. This would imply that Bon has a dual role, similar to some members of the Polycomb group family: transcriptional repression and heterochromatin formation. Both of these roles may be required in transcriptional repression (Beckstead, 2001).

The interaction of Bon with nuclear receptors is similar to TIF1alpha but unlike TIF1beta and TIF1gamma. This interaction requires the integrity of the nuclear receptor AF-2 activation domain and is mediated by the Bon/TIF1alpha LxxLL motif. These observations suggest that Drosophila nuclear receptors and Bon have co-evolved to maintain their interaction. It is therefore likely that the biological role of this interaction has been conserved in mammals (Beckstead, 2001).

The Drosophila homolog of the retinoid X receptor, ultraspiracle (Usp), heterodimerizes with the ecdysone receptor (EcR) to form a functional complex that mediates the effects of the steroid molting hormone ecdysone by activating and repressing expression of ecdysone response genes. As with other retinoid X receptor heterodimers, EcR/USP affects gene transcription in a ligand-modulated manner. The functions were analyzed of two usp alleles, usp3 and usp4, which encode stable proteins with defective DNA-binding domains. Usp is able to activate as well as repress the Z1 isoform (BrC-Z1) of the ecdysone-responsive Broad Complex gene. Activation of BrC-Z1 as well as EcR, itself an ecdysone response gene, can be mediated by both the USP3 and USP4 mutant proteins. USP3 and USP4 also activate an ecdysone-responsive element, hsp27EcRE, in cultured cells. These results differ from the protein null allele, usp2, which is unable to mediate activation. BrC-Z1 repression is compromised in all three usp alleles, suggesting that repression involves the association of Usp with DNA. These results distinguish two mechanisms by which Usp modulates the properties of EcR: one that involves the Usp DNA-binding domain and one that can be achieved solely through the ligand-binding domain. These newly revealed properties of Usp might implicate similar properties for retinoid X receptor (Ghbeish, 2001).

These data suggest a separation between the repressive functions of Usp and some of its activating functions, since the Usp DBD is dispensable for the activation of some ecdysone targets. usp3 and usp4 are capable of heterodimerizing with EcR, although they are defective in DNA binding. On some EcREs, USP3 or 4/EcR heterodimers mediate activation. In culture, the Usp LBD alone seems sufficient for the formation of an activating complex with EcR. Thus, for some genes, models explaining USP/EcR gene activation must accommodate the fact that the Usp DNA-binding domain is not necessary, whereas the LBD is. One such model posits that EcR monomers, homodimers, or alternative EcR complexes can bind some EcREs but only activate these response elements if the Usp LBD is present to promote formation of the EcR complex, ligand-binding, and/or interaction with coactivators. This model suggests that USP3, USP4, USPL, and possibly USP+ can activate through a multimeric complex in which the LBDs heterodimerize and DNA binding occurs largely via one or more EcR DBDs. In support of this model, ligand-induced EcR homodimers are able to form on DNA (Ghbeish, 2001).

In contrast to activation, repression of BrC-Z1 clearly requires functional Usp DNA-binding abilities, whereas its post-furrow activation in the eye imaginal disc does not. The apparent differential requirement for DNA binding in repression and activation suggests that, in some situations, the switch between repression and activation regulated by the EcR/Usp heterodimer may involve more than just changes in the LBD in response to ligand. It is also possible that normally the switch from repression to activation occurs without a change in the DNA binding of either EcR or Usp but that on some target sites in the absence of the wild-type complex, an alternative complex can form and allow activation (Ghbeish, 2001).

The ability of added wild-type Usp to restore BrC-Z1 repression in the eye imaginal disc suggests that the Z1 isoform of BrC may be a direct target of Usp regulation. Since it has been shown that Usp has the ability to homodimerize on a DNA element able to mediate repression in cultured Drosophila cells, it is possible that an alternative Usp complex other than EcR/Usp represses BrC-Z1. If Usp is able to repress target genes through a homodimer but requires heterodimerization with EcR to mediate activation, a situation could arise in which gene repression absolutely requires the DNA-binding activity of Usp while this function can be abolished for gene activation (Ghbeish, 2001).

In this study a dual role has been uncovered for Usp in the ecdysone response. Depending on the particular target gene, activation and repression may be more complicated than just a simple ligand-activated switch. This adds potential complexity to the roles that ecdysone, Usp, and EcR play in metamorphosis. This work separates aspects of the Usp component of the ecdysone response into repressive and activating functions, with unique and separable effects attributable to the DNA-binding and ligand-binding domains (Ghbeish, 2001).

Ultraspiracle and RXR interact with Hormone receptor-like in 38, an orphan nuclear receptor that mediates an atypical ecdysteroid signaling pathway

Ecdysteroid pulses trigger the major developmental transitions during the Drosophila life cycle. These hormonal responses are thought to be mediated by the ecdysteroid receptor (EcR) and its heterodimeric partner Ultraspiracle (Usp). Evidence is provided for a second ecdysteroid signaling pathway mediated by Hormone receptor-like in 38 (Hr38), the Drosophila ortholog of the mammalian NGFI-B subfamily of orphan nuclear receptors. Hr38 also heterodimerizes with Usp, and this complex responds to a distinct class of ecdysteroids in a manner that is independent of EcR. This response is unusual in that it does not involve direct binding of ecdysteroids to either Hr38 or Usp. X-ray crystallographic analysis of Hr38 reveals the absence of both a classic ligand binding pocket and coactivator binding site, features that seem to be common to all NGFI-B subfamily members. Taken together, these data reveal the existence of a separate structural class of nuclear receptors that is conserved from fly to humans (Baker, 2003).

To investigate the possibility that Hr38 may function in an ecdysteroid-mediated transcriptional pathway, a screening assay was developed in which Hr38 was heterodimerized with ligand-activated RXR. The feasibility of this approach was based on the finding that RXR can substitute for Usp as a productive heterodimeric partner for EcR. A distinct advantage of substituting RXR for Usp is that although ligands for Usp are not known, several potent RXR ligands (i.e., rexinoids) have been characterized. It was reasoned that assaying Hr38 activity in the presence of rexinoid-activated RXR may be important because previous work has shown that some RXR heterodimers require sensitization with ligand for one receptor before the partner receptor can become ligand responsive (Baker, 2003 and references therein).

To screen for a Hr38 ecdysteroid response, transient cotransfections were performed in the Drosophila SL2 cell line using chimeric GAL4-receptor proteins (hormone receptor proteins with an inserted GAL4 transcriptional activation domain) and a GAL4-responsive luciferase reporter gene (Baker, 2000). In this assay, GAL4-EcR and GAL4-Hr38 were screened in the presence of RXR, the synthetic rexinoid LG268, and the potent plant ecdysteroid muristerone A. The GAL4-EcR/RXR heterodimer responds to 100 nM muristerone A as expected but is not activated significantly by the RXR-specific ligand LG268 alone. Addition of both ligands results in only a modest increase in EcR/RXR activity. In contrast, GAL4-Hr38/RXR, which is known to have a potent basal activity, is not induced by the addition of ecdysteroid alone but, instead, exhibits a strong, dose-dependent response to LG268. Rexinoid activation of the Hr38/RXR heterodimer is consistent with the rexinoid response seen with other NGFI-B family members when paired with RXR. Surprisingly, however, there is a significant, 3- to 4-fold response to muristerone A when it is added together with LG268. A similar response is obtained with the endogenous insect ecdysteroid 20E. Both rexinoid and ecdysteroid responses are abolished when a GAL4-Hr38 construct was utilized that lacks the ligand-dependent activation function-2 (AF-2) domain. Identical results are obtained (i.e., loss of rexinoid and ecdysteroid response) when the AF-2 domain of RXR is also deleted. These data suggest that the Hr38 heterodimeric complex is responsive to ecdysteroid but, like other RXR heterodimers, it requires transactivation of both receptor partners for full agonist activity (Baker, 2003).

The results above reveal the possible existence of two ecdysteroid signaling pathways, one mediated by EcR and the other by Hr38. To begin to delineate the specificity of the Hr38 pathway and show that it functions independently of the EcR pathway, double-stranded RNA (dsRNA) directed against the coding region of the EcR ligand binding domain was used to reduce the expression of endogenous EcR in the SL2 cell assay. Treatment of SL2 cells with increasing amounts of EcR dsRNA completely eliminates muristerone-A-dependent transcription when tested using endogenous EcR/Usp heterodimers on an hsp27-EcRE reporter gene. This RNAi-mediated repression of the EcR-dependent response also completely blocks the activity of exogenously transfected EcR and GAL4-EcR. In contrast, under the same experimental conditions, where the EcR response is abolished, the GAL4-Hr38/RXR heterodimer is fully responsive to ecdysteroid and LG268. These results demonstrate that the Hr38 response to ecdysteroids is independent of EcR (Baker, 2003).

To define the spectrum of potential Hr38 agonists and further delineate the differential ecdysteroidal response of Hr38 and EcR, a panel of naturally occurring Drosophila ecdysteroids, phytoecdysteroids, synthetic ecdysteroids, and a synthetic juvenile hormone (methoprene acid) were tested for activity using the reporter gene assay described above. SL2 cells were transfected with either GAL4-EcR or GAL4-Hr38 plus RXR and tested for agonist activity in the presence of 10 nM LG268. GAL4-EcR responds selectively to the endogenous ecdysteroids 20E and makisterone A and the plant ecdysteroids muristerone A, ponasterone A, and cyasterone. In marked contrast, the GAL4-Hr38/RXR response is promiscuous for several different ecdysteroids when LG268 is included as a coagonist. In addition to the compounds that activated EcR, at least six other ecdysteroids (α-ecdysone, 3-epi-20E, 2-deoxy-20E, 3-dehydromakisterone A, 3-epimakisterone A, and 3-dehydro-20-deoxyponsterone) also exhibit significant Hr38-dependent activity. Dose-response profiles demonstrated that all of these compounds are more potent agonists for Hr38 than for EcR. In fact, 20E, which is believed to be the endogenous hormone agonist for EcR, exhibits a 100-fold greater potency for Hr38-dependent transcription. These data suggest that the Hr38/RXR heterodimer is a potent sensor of a distinct class of physiologically relevant ecdysteroids (Baker, 2003).

An unusual characteristic of the Hr38/RXR heterodimer is that it required transactivation of both receptors to elicit an ecdysteroid response. In particular, the Hr38/RXR heterodimer fails to respond to ecdysteroid in the absence of ligand-activated RXR. Interestingly, Hr38 also fails to respond to ecdysteroid when Usp, the physiologic partner of Hr38, is used instead of RXR. These results raise the intriguing possibility that Usp, like RXR, must also be transactivated (e.g., by ligand) in order to enable the ecdysteroid response. Attempts were made to address this question by using VP16-Usp, a constitutively active form of Usp that circumvents the requirement for Usp ligand by fusing the strong transcriptional activation domain of the herpes simplex viral protein-16 (VP16) to Usp. As expected, in the absence of agonist, the GAL4-Hr38/VP16-Usp heterodimer shows a high constitutive level of basal activity that effectively mimics that of ligand-activated USP. Importantly, the addition of muristerone A to the GAL4-Hr38/VP16-Usp heterodimer elicits a significant increase in reporter gene activity, analogous to the effect seen with LG268-activated GAL4-Hr38/RXR. Similar to the results obtained above with ligand-activated RXR, the GAL4-Hr38/VP16-Usp heterodimer responds to a wide variety of ecdysteroids at comparably low concentrations. These results support the idea that Hr38 mediates a distinct heterodimer-dependent ecdysteroid signaling pathway (Baker, 2003).

To test the prediction that Hr38 is activated by ecdysteroids in Drosophila, transgenic flies were created that carry a heat-inducible hs-GAL4-Hr38 transgene in combination with a GAL4-dependent UAS-nlacZ reporter gene. Since target genes for Hr38 in the fly are unknown, this model permits the assaying of Hr38 transactivation directly in fly tissues. This transgenic fly model has been used to follow the ecdysteroid-dependent activation patterns of the EcR and Usp in Drosophila and has provided data consistent with the known biochemical and genetic activities of the full-length receptors in vivo (Kozlova, 2002). This strategy has also been employed to track ligand-dependent activation of the RAR and RXR ligand binding domains in the mouse central nervous system. To determine if GAL4-Hr38 is activated by ecdysteroids, third instar larval organs from this transgenic line were dissected at ~8 hr before puparium formation and cultured in the presence of either α-ecdysone or 3-epi-20E, two ecdysteroids that activate Hr38, but not EcR in SL2 cells. In the presence of 1 μM α-ecdysone, significant activation above background is seen for GAL4-Hr38. In contrast, these ecdysteroids have no effect on GAL4-EcR, although this same transgenic line shows robust activation by 20E (Kozlova, 2002). GAL4-Hr38 is also activated by 3-epi-20E in both the epidermis and fat body, consistent with the ability of this agonist to selectively activate Hr38 in SL2 cells. These organs contain significant amounts of endogenous Usp, consistent with the interpretation that GAL4-Hr38 ecdysteroid activation is dependent on heterodimerization with a Usp partner. Although similar results were seen in several independent experiments, not all hs-GAL4-Hr38; UAS-nlacZ animals display robust activation, indicating that a specific stage might be competent to respond to the hormone. In agreement with this idea, a complex and dynamic pattern of GAL4-Hr38 activation can be seen in untreated animals. This observation is consistent with the notion that the endogenous Hr38 response may be spatially and temporally regulated by the presence of a number of factors, including ecdysteroids, Hr38/Usp-specific coactivators, and potentially a Usp

Structure of the heterodimeric ecdysone receptor DNA-binding complex

Ecdysteroids initiate molting and metamorphosis in insects via a heterodimeric receptor consisting of the ecdysone receptor (EcR) and ultraspiracle (USP). The EcR-USP heterodimer preferentially mediates transcription through highly degenerate pseudo-palindromic response elements, resembling inverted repeats of 5'-AGGTCA-3' separated by 1 bp (IR-1). The requirement for a heterodimeric arrangement of EcR-USP subunits to bind to a symmetric DNA is unusual within the nuclear receptor superfamily. The 2.24 Å structure is described of the EcR-USP DNA-binding domain (DBD) heterodimer bound to an idealized IR-1 element. EcR and USP use similar surfaces, and rely on the deformed minor groove of the DNA to establish protein-protein contacts. Since retinoid X receptor (RXR) is the mammalian homolog of USP, the 2.60 Å crystal structure of the EcR-RXR DBD heterodimer on IR-1 is also solved; the dimerization and DNA-binding interfaces are the same as in the EcR-USP complex. Sequence alignments indicate that the EcR-RXR heterodimer is an important model for understanding how the FXR-RXR heterodimer binds to IR-1 sites (Devarakonda, 2003).

Nuclear receptor DBDs do not form homo- or hetero-dimers in the absence of DNA. Receptor homo- or heterodimer formation through DBDs is strictly dependent and enhanced by the cognate DNA-binding sites. In the heterodimeric complexes studied in this work, the structures suggest that there are three mechanisms by which the IR-1 appears cooperatively to enhance the dimerization between the EcR and the USP/RXR homologs. First, the same Zn-II regions involved in the formation of the dimer interface are also used extensively for making DNA contacts. In particular, residues Arg51 and Lys52 from EcR and residue Asn51 of USP are simultaneously involved in both dimerization and DNA binding functions. This implies that DNA binding and subunit dimerization are mutually supportive (Devarakonda, 2003).

A second mechanism exists by which the DNA enhances the dimer interactions; the subunit interfaces are in part embedded in the minor groove. A significant minor groove distortion is associated with the spacer AT base pair, this being the convergence point of the protein-protein interactions. Importantly, these minor groove widths represent sharp departures from standard B-DNA values, and are associated with both the EcR-USP and EcR-RXR structures. In particular, there is a 4.3 Å minor groove width in the EcR-USP DNA and a <4.0 Å width in the EcR-RXR DNA. The reliance on minor groove distortions to stabilize dimer binding is reminiscent of the RXR-RAR/DR-1 and the RevErb-RevErb/DR-2 complexes on their cognate DNA targets (Devarakonda, 2003 and references therein).

A third mechanism exists for subunit cooperation; the EcR-DBD footprint on DNA extends well beyond its own AGGTCA site to reach over both its 3'-flanking sequences and a large portion of the USP half-site. In total, the EcR footprint in the USP complex extends over a region totaling 13 bp, and to 12 bp in the RXR complex. This is consistent with mutational studies that have identified base pairs within the USP half-site that are to be critical not for the USP-DBD binding (as a monomer) but for effective heterodimer formation. This extended binding mode exhibited by EcR may contribute to the cooperativity of subunit association, by reducing the conformational flexibility at the USP site and as such pre-paying the entropic costs associated with adjacent site binding by USP. A similar mechanism based on tandem site stabilization has been suggested as the basis for the cooperation between the POU domains of Oct-1 on DNA, as well as the binding of the RXR-RAR heterodimer on DNA (Devarakonda, 2003 and references therein).

rigor mortis encodes a nuclear receptor interacting protein required for ecdysone signaling during Drosophila larval development

Pulses of the steroid hormone ecdysone trigger the major developmental transitions in Drosophila, including molting and puparium formation. The ecdysone signal is transduced by the EcR/USP nuclear receptor heterodimer that binds to specific response elements in the genome and directly regulates target gene transcription. A novel nuclear receptor interacting protein is described, encoded by rigor mortis (rig), that is required for ecdysone responses during larval development. rig mutants display defects in molting, delayed larval development, larval lethality, duplicated mouth parts, and defects in puparium formation -- phenotypes that resemble those seen in EcR, usp, E75A and ßFTZ-F1 mutants. Although the expression of these nuclear receptor genes is essentially normal in rig mutant larvae, the ecdysone-triggered switch in E74 isoform expression is defective. rig encodes a protein with multiple WD-40 repeats and an LXXLL motif, sequences that act as specific protein-protein interaction domains. Consistent with the presence of these elements and the lethal phenotypes of rig mutants, Rig protein interacts with several Drosophila nuclear receptors in GST pull-down experiments, including EcR, USP, DHR3, SVP and ßFTZ-F1. The ligand binding domain of ßFTZ-F1 is sufficient for this interaction, which can occur in an AF-2-independent manner. Antibody stains reveal that Rig protein is present in the brain and imaginal discs of second and third instar larvae, where it is restricted to the cytoplasm. In larval salivary gland and midgut cells, however, Rig shuttles between the cytoplasm and nucleus in a spatially and temporally regulated manner, at times that correlate with the major lethal phase of rig mutants and major switches in ecdysone-regulated gene expression. Taken together, these data indicate that rig exerts essential functions during larval development through gene-specific effects on ecdysone-regulated transcription, most likely as a cofactor for one or more nuclear receptors. Furthermore, the dynamic intracellular redistribution of Rig protein suggests that it may act to refine spatial and temporal responses to ecdysone during development (Gates, 2003).

Mutations in rig result in prolonged second and third instar larval stages, defects in molting, larval lethality and duplicated mouth parts. These phenotypes are characteristic of defects in ecdysone signaling, suggesting a critical role for rig in ecdysone responses during larval development. Two classes of genes produce mutant phenotypes that resemble those seen in rig mutant animals: those required for ecdysone biosynthesis or release -- including ecdysoneless (ecd), dare and itpr -- and those encoding nuclear receptors that mediate the ecdysone signal -- EcR, usp, E75A, and ßFTZ-F1. Unlike ecdysone-deficient mutants, the lethal phenotypes of rig mutants cannot be rescued by feeding 20E, indicating that ecdysone is not limiting in these animals and that rig acts downstream from hormone biosynthesis or release. Rather, it is proposed that Rig is functioning as a nuclear receptor cofactor, based on five lines of evidence. (1) The lethal phenotypes of rig mutants are very similar to those defined for EcR, usp, E75A and ßFTZ-F1, although all of these nuclear receptor genes are expressed in an essentially normal manner in rig mutant larvae. (2) rig mutants display a defect in the ecdysone-triggered switch in E74 isoform expression that is characteristic of reduced ecdysone signaling, indicating that rig is required for the appropriate expression of specific ecdysone-inducible genes. (3) These effects on gene expression are likely to be indirect as the predicted Rig protein sequence contains multiple protein-protein interaction domains and no known DNA-binding motifs. (4) Rig protein can interact physically with several Drosophila nuclear receptors, including EcR, USP and ßFTZ-F1, all of which have mutant phenotypes in common with rig mutants. (5) Rig protein shuttles between the cytoplasm and nucleus of larval cells in a manner similar to the active subcellular redistribution that has been reported for known Drosophila and vertebrate nuclear receptor cofactors (Gates, 2003).

Five Drosophila nuclear receptor cofactors have been identified to date: Alien, SMRTER, MBF1, Taiman and Bonus. Of these, only bonus appears to have activities in common with rig, although relatively limited genetic studies have been undertaken for most of these cofactors. No mutants have been characterized for SMRTER or Alien, which act as co-repressors in tissue culture transfection assays. MBF1 null mutants are viable and display a strong genetic interaction with tdf/apontic mutants: this indicates a role in tracheal and nervous system development. Somatic clones of taiman mutants reveal a role in border cell migration during oogenesis. In contrast, bonus mutants display first instar larval lethality as well as defects in salivary gland cell death and cuticle and bristle development, implicating a role for bonus in ecdysone responses during development. Also like rig, bonus mutations result in gene-specific defects in ecdysone-regulated transcription, and Bonus protein can interact with a range of Drosophila nuclear receptors, including EcR, USP, SVP, DHR3 and FTZ-F1. Bonus, however, interacts with these receptors in an AF-2-dependent manner, unlike Rig. Moreover, the larval lethal phenotypes of rig mutants do not resemble those reported for bonus mutants and, unlike Rig, Bonus protein appears to be exclusively nuclear in both larval and imaginal tissues. Further work is required to determine whether bonus and rig might act together to regulate ecdysone response pathways (Gates, 2003).

Rig is distinct from all known Drosophila nuclear receptor cofactors in that it is not part of an evolutionarily conserved protein family. Alien, SMRTER, MBF1, Taiman and Bonus all have vertebrate homologs, and Taiman and Bonus are the fly orthologs of the well characterized vertebrate nuclear receptor cofactors AIB1 and TIF1, respectively. In contrast, Rig does not contain identifiable enzymatic activities nor the conserved functional domains that define most nuclear receptor cofactors. BLAST searches with the Rig protein sequence did not reveal any closely related sequences in other organisms, although the top hits, which show limited homology in the WD-40 repeats, are in factors known to modify chromatin, including human histone acetyltransferase type B subunit 2 (RBBP-7) and chromatin assembly factor 1 (CAF-1) (Gates, 2003).

The WD-40 repeats that comprise about half of the Rig protein sequence are likely to play an important role in its activity. Consistent with this proposal, an N-terminal fragment of Rig, containing two WD-40 repeats but missing the LXXLL motif (amino acids 1-300), is capable of interacting with GST-DHR3 and GST-USP, suggesting that these repeats are sufficient for Rig-nuclear receptor interactions. WD-40 repeats provide multiple surfaces for protein-protein interactions and have been identified in over 150 proteins that function in a wide range of processes, including cytoskeleton assembly, transcriptional regulation, and pre-mRNA processing. In Drosophila, WD-40 repeats are associated with several transcriptional regulators, including the p85 subunit of TFIID, the Polycomb group protein encoded by extra sex combs, and the Groucho corepressor. In addition, a WD-40 repeat protein, TBL1, has been identified as part of a multiprotein complex with thyroid hormone receptor that contains the SMRT nuclear receptor corepressor and HDAC-3. The presence of these sequences in Rig may thus provide a scaffold for protein-protein interactions that could mediate the formation of multiprotein transcriptional complexes on ecdysone-regulated promoters. Further biochemical studies of Rig should provide insights into the significance of its WD-40 repeats as well as a foundation for understanding how Rig exerts its effects on transcription (Gates, 2003).

It is not clear how Rig expression in the brain, imaginal discs and salivary glands of second and third instar larvae is related to the lethal phenotypes of rig mutants, although neuroendocrine signaling is clearly required for molting, a process that is defective in rig mutant larvae. The subcellular localization of Rig protein at later stages, however, correlates with the distinct fates of larval and imaginal cells during metamorphosis. Rig protein appears to be restricted to the cytoplasm of cells that are fated to form parts of the adult fly, including neuroblasts, imaginal discs, and the imaginal islands of the larval midgut. In contrast, Rig shows dynamic changes in its subcellular distribution in larval salivary gland and midgut cells, both of which undergo steroid-triggered programmed cell death during metamorphosis. It is possible that these differences in subcellular localization could contribute to the distinct fates of these tissues in response to ecdysone signaling (Gates, 2003).

In addition to this spatial correlation, there is also a temporal correlation between the times at which Rig protein shuttles between the cytoplasm and nucleus in larval tissues and the coordinated changes in ecdysone-regulated gene expression that occur during the third instar. The switch from cytoplasmic to nuclear localization in larval salivary glands and midguts occurs at approximately the same time, 24-30 hours after the second-to-third instar larval molt, suggesting that Rig may be responding to a common temporal signal. Cell type-specific factors, however, must also contribute to this regulation as Rig is localized to the nucleus of only a subset of cells in the larval midgut. Interestingly, this protein redistribution correlates with a poorly understood event that is represented by widespread changes in ecdysone-regulated gene expression, called the 'mid-third instar transition.' It is possible that the cytoplasmic-to-nuclear transport of Rig in larval tissues contributes to the regulation of this response, which prepares the animal for metamorphosis one day later. Similarly, Rig returns to the cytoplasm of salivary gland cells at puparium formation, in synchrony with the widespread changes in ecdysone-regulated gene expression associated with the onset of metamorphosis. This translocation, however, is not seen in the larval midgut, where Rig protein remains in the nucleus of some cells. Rig shuttling thus appears to be differentially controlled in both a temporally and spatially restricted manner, correlating with major switches in ecdysone-regulated transcription. The observation that the first of these shifts in subcellular distribution occurs during the major lethal phase of rig mutants -- the mid-third instar -- suggests that these intracellular movements contribute to the critical functions of Rig during development (Gates, 2003).

Interestingly, several recent reports have described the subcellular redistribution of nuclear receptor cofactors in both vertebrate and Drosophila cells. The p/CIP vertebrate nuclear receptor coactivator is differentially distributed within the cells of the mouse female reproductive organs. For example, p/CIP is detected primarily in the nuclei of highly proliferative follicular cells while it is most abundant in the cytoplasm of terminally differentiated cells of the corpus luteum. p/CIP displays active nucleocytoplasmic shuttling in response to growth factors in cell culture, and interacts directly with the microtubule network in the cytoplasm. Similarly, MEK-1 kinase-mediated phosphorylation of the SMRT mammalian corepressor leads to the translocation of this factor from the nucleus to the cytoplasm in cell culture transfection assays. The functional homolog of this protein in flies, SMRTER, also shows active redistribution from the nucleus to the cytoplasm in response to a MAP kinase pathway, in this case mediated by EGFR/Sno/Ebi in the Drosophila eye. In both of these systems, regulated phosphorylation of SMRT/SMRTER results in dissociation of a repressor complex and derepression of target gene transcription (Gates, 2003).

These observations raise the possibility that the subcellular location of Rig could determine its regulatory function in different cell types. For example, by analogy with SMRT/SMRTER, loss of Rig from the nucleus of larval cells might disrupt a corepressor complex on specific promoters, leading to coordinate target gene derepression. This is consistent with the proposal that the ecdysone receptor exerts critical repressive functions during larval development. Alternatively, Rig protein in the cytoplasm may tether one or more nuclear receptors, preventing them from acting on their cognate target genes in the nucleus. This model is not favored, however, because antibody stains reveal an exclusively nuclear localization for EcR, USP and ßFTZ-F1 at the onset of metamorphosis. It is also interesting to note that Rig protein appears to localize to discrete regions within the nuclei of larval midgut cells that do not contain chromosomes while Rig co-localizes with the giant polytene chromosomes in larval salivary gland cells. Rig may thus exert some functions in the nucleus that are independent of chromatin binding. Further biochemical studies of Rig, including the identification of additional proteins that interact with this factor, should provide insights into the significance of the subcellular localization of Rig protein as well as a mechanistic understanding of how Rig contributes to ecdysone responses during Drosophila larval development (Gates, 2003).

Dynamic regulation of Drosophila nuclear receptor activity in vivo

Nuclear receptors are a large family of transcription factors that play major roles in development, metamorphosis, metabolism and disease. To determine how, where and when nuclear receptors are regulated by small chemical ligands and/or protein partners, a `ligand sensor' system was used to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live developing animals. Transgenic lines were established that express the ligand binding domain of each nuclear receptor fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, the fusion proteins show tissue- and stage-specific patterns of activation. These responses accurately reflect the presence of endogenous and exogenously added hormone, and that they can be modulated by nuclear receptor partner proteins. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. Dynamic changes in activation were seen that are indicative of sweeping changes in ligand and/or co-factor production. The screening of a small compound library using this system identified the angular psoralen angelicin and the insect growth regulator fenoxycarb as activators of the Ultraspiracle (USP) ligand-binding domain. These results demonstrate the utility of this system for the functional dissection of nuclear receptor pathways and for the development of new receptor agonists and antagonists that can be used to modulate metabolism and disease and to develop more effective means of insect control (Palanker, 2006).

Nine GAL4-LBD ligand sensor lines described in this study show tissue-specific patterns of activity during development: EcR, USP, ERR, FTZ-F1, HNF4, E78, DHR3, DHR38 and DHR96. These transgenic lines will serve as valuable tools for the genetic and molecular dissection of the receptors they represent, the pathways they regulate and the upstream factors and co-factors that modulate their activity. Specifically, the data reported here show that these lines can be used to: (1) indicate tissues and stages in which the corresponding NRs are likely to function; (2) indicate where endogenous ligands and co-factors are likely to be found; (3) suggest NR biological functions; (4) suggest possible NR-NR interactions, cascades and target genes; (5) evaluate putative co-factors and ligands; (6) screen chemical compound libraries for new agonists and antagonists; and (7) screen genetically for new pathway components. The results of these studies will also provide important insights into the ligands, co-factors and functions of their vertebrate NR homologues (Palanker, 2006).

Hormonal regulation of GAL4-LBD activation in the amnioserosa and yolk

Examination of the nine active ligand sensor lines provided a number of insights into possible relationships between their corresponding NRs. For example, although each of these ligand sensors displays unique temporal and spatial patterns of activity, activation in specific tissues and stages is common to many. These common sites of LBD activity may indicate shared functions, hierarchical or physical interactions, or related ligands. Examples of tissues that represent hotspots for GAL4-LBD activation include the amnioserosa, yolk, midgut and fat body (Palanker, 2006).

Each of these tissues, and the stages at which they score positively, correlates well with the presence of putative ligands. The yolk, for example, is believed to act as a storage site for maternally provided ecdysteroids during embryogenesis. Work with other insects has shown that these ecdysteroids are conjugated in an inactive form to vitellin proteins via phosphate bridges. Around mid-embryogenesis, these yolk proteins and phosphate bonds are cleaved, thereby releasing what are presumed to be the earliest biologically active ecdysteroids in the embryo. Interestingly though, GAL4-EcR activation in the amnioserosa depends on the disembodied (dib) gene, which encodes a cytochrome P450 enzyme required in the penultimate step of Ecdysone (E) biosynthesis, suggesting that the final steps in the linear E biosynthetic pathway are required for EcR function in this tissue and contradicting the prediction that this activity would be dependent on maternal ecdysteroids and independent of the zygotic biosynthetic machinery. The mechanisms by which dib exerts this essential role in providing an EcR ligand, however, remain to be determined (Palanker, 2006).

The response of the EcR and USP ligand sensors in the adjacent amnioserosa tissue shows that active ecdysteroids are not present until the hormone reaches the amnioserosa. A recent study of yolk-amnioserosa interactions has revealed dynamic transient projections that emanate from one tissue and contact the other, suggesting that there may be functional interactions between these two cell types. It is possible that these projections mediate the transfer of lipophilic ligand precursors from the yolk to the amnioserosa. This transfer, in turn, could determine the proper timing of EcR activation in the amnioserosa, thus triggering the major morphogenetic movements that establish the body plan of the first instar larva (Palanker, 2006).

Studies of the DHR38 receptor have demonstrated that it can be activated by a distinct set of ecdysteroids from those that activate EcR, through a novel mechanism that does not involve direct ligand binding. The activation of GAL4-DHR38 that was observed in the embryonic amnioserosa is consistent with this model of DHR38 regulation. First, exogenous 20E can only weakly activate GAL4-DHR38, relative to the strong ectopic activation seen with 20E on the EcR ligand sensor. This correlates with the weak ability of 20E to activate DHR38 in cell culture transfection assays relative to the strong 20E activation of EcR. Second, the DHR38 ligand sensor is activated in the amnioserosa earlier than the EcR construct, suggesting that it is responding to a different signal. It is possible that this signal is an ecdysteroid precursor that can act on DHR38 but not EcR - paralleling the ability of DHR38 to be activated by E, the precursor to 20E, which activates EcR. This putative ecdysteroid must be produced in a manner independent of the conventional ecdysteroid biosynthetic pathway, however, since a zygotic dib mutation has no effect on GAL4-DHR38 activation in the amnioserosa. Rather, this early activation may be due to maternal ecdysteroids that are conjugated and inactive in the yolk and transferred to the amnioserosa. These studies highlight the value of combining mutations in hormone biosynthesis with ligand sensor activation as a powerful means of dissecting hormone signaling pathways. Further studies of DHR38 function and regulation in embryos could help clarify the potential significance of this distinct activation response (Palanker, 2006).

DHR3, DHR38 and HNF4 ligand sensors appear to respond to metabolic signals Interestingly, the midgut continues to be a hotspot for ligand sensor activity long after it has engulfed the yolk during embryogenesis. This seems logical, as the midgut is responsible for most lipid absorption and release, and many vertebrate NRs are involved in fatty acid, cholesterol and sterol metabolism and homeostasis. The observed restriction of ligand sensor activity to a narrow group of cells located at the base of the gastric caeca is of particular interest. This is the site where nutrients in a feeding larva are absorbed into the circulatory system. The activation of DHR3, DHR38 and HNF4 ligand sensors in this region of the gastric caeca suggests that these receptors are activated by one or more small nutrient ligands. Moreover, this suggests that the corresponding receptors may exert crucial metabolic functions by acting as nutrient sensors (Palanker, 2006).

Further evidence of metabolic functions for DHR3, DHR38 and HNF4 arises from their ligand sensor activation patterns in the embryonic yolk and larval fat body. The yolk is the main nutrient source for the developing embryo and represents an abundant source of lipids, correlating with specific activation of DHR3, DHR38 and HNF4 ligand sensors in this cell type during embryogenesis. Upon hatching into a larva, the fat body acts as the main metabolic organ of the animal, functionally equivalent to the mammalian liver. Upon absorption by the gastric caeca, nutrients travel through the circulatory system and are absorbed by the fat body, where they are broken down and stored as triglycerides, glycogen and trehalose. Once again, the efficient activation of the DHR3, DHR38 and HNF4 ligand sensors in the fat body of metabolically active third instar larvae, and lack of sensor activity in non-feeding prepupae, supports the model that the corresponding NRs operate as metabolic sensors. This proposed function is consistent with the roles of their vertebrate orthologs. Mammalian ROR, the ortholog of DHR3, binds cholesterol and plays a crucial role in lipid homeostasis. Similarly, mammalian HNF4 can bind C14-18 fatty acids, is required for proper hepatic lipid metabolic gene regulation and lipid homeostasis, and is associated with human Maturity-Onset Diabetes of the Young (MODY1). The studies described here suggest that DHR3 and HNF4 may perform similar metabolic functions in flies, defining a new genetic model system for characterizing these key NRs (Palanker, 2006).

New insights into the regulation of Drosophila xenobiotic responses

Several vertebrate NRs play a central role in xenobiotic responses by directly binding toxic compounds and inducing the expression of key detoxification enzymes such as cytochrome P450s and glutathione transferases. Ligand sensor activation observed in the gut, epidermis, tracheae or fat body could represent xenobiotic responses insofar as toxic compounds could enter the organism through any of these tissues. Directed screens that test xenobiotic compounds for their ability to activate Drosophila NR ligand sensors will provide a means of identifying potential xenobiotic receptors. Understanding these response systems, in turn, could facilitate the production of insect resistant crops and the development of more effective pesticides (Palanker, 2006).

Like its vertebrate orthologs SXR/PXR and CAR, DHR96 has been recently shown to act in insect xenobiotic responses, providing resistance to the sedative effect of phenobarbital and lethality caused by chronic exposure to DDT (King-Jones et al., 2006). DHR96 is also required for the proper transcriptional response of a subset of phenobarbital-regulated genes. DHR96 can be activated by the CAR-selective agonist CITCO, suggesting that it may be regulated in a manner similar to that of the vertebrate xenobiotic receptors. It is also interesting to note that angelicin was found to activate the USP ligand sensor fusion. Angelicin is an angular furanocoumarin that has the furan ring attached at the 7,8 position of the benz-2-pyrone nucleus. Detailed studies have shown that insects have adapted to the presence of furanocoumarins in their host plants by expressing specific cytochrome P450 enzymes that detoxify these compounds. In the black swallowtail butterfly (Papilio polyxenes), furanocoumarins induce the transcription of P450 genes through an unknown regulatory pathway, thereby aiding in xenobiotic detoxification. The observation that angelicin, and not the linear furanocoumarins 8-methoxypsoralen (xanthotoxin) or 5-methoxypsoralen (bergapten), can activate GAL4-USP suggests that NRs may mediate this detoxification response and may be capable of distinguishing between the linear and angular chemical forms. It is possible that USP may mediate this effect on its own or, more likely, as a heterodimer partner with another NR. Similarly, the activation of GAL4-USP by fenoxycarb may represent a xenobiotic response. This activation, however, is weaker and more variable than the activation observed with angelicin. Identifying other factors that mediate xenobiotic responses in Drosophila would provide a new basis for dissecting the control of detoxification pathways in higher organisms (Palanker, 2006).

ERR activity appears to be regulated by a temporally restricted and widespread signal

GAL4-ERR displays a remarkable switch in activity during mid-embryogenesis, from strong activation in the myoblasts to specific and strong activation in the CNS. The ERR ligand sensor also shows widespread transient activation in the mid-third instar, a time when larval ERR gene expression begins, together with a global switch in gene expression that prepares the animal for entry into metamorphosis 1 day later. This so-called mid-third instar transition includes upregulation of EcR, providing sufficient receptor to transduce the high titer late larval 20E hormone pulse, upregulation of the Broad-Complex, which is required for entry into metamorphosis, and induction of the genes that encode a polypeptide glue used to immobilize the puparium for metamorphosis. The signal and receptor that mediate this global reprogramming of gene expression remain undefined. The widespread activation of GAL4-ERR at this stage raises the interesting possibility that it may play a role in this transition. Moreover, given that the only ligand sensors to display widespread transient activation are EcR and USP, in response to 20E, it is possible that this response reflects a systemic mid-third instar pulse of a ERR hormone. Vertebrate members of the ERR family can bind the synthetic estrogen diethylstilbestrol and the selective ER modulator tamoxifen, as well as its metabolite, 4-hydroxytamoxifen, suppressing their otherwise constitutive activity in cell culture. This is notably different from the highly restricted patterns of ERR ligand sensor activity that was detected in Drosophila, which suggests that it does not function as a constitutive activator in vivo. Rather, it is envisioned that the patterns of ERR activation are precisely modulated by protein co-factors and/or one or more ligands to direct the dynamic shifts in activation that are detect during embryogenesis and third instar larval development. Functional studies of the Drosophila homolog of the ERR receptor family may provide a basis for understanding these dynamic shifts in LBD activation, as well as revealing a natural ligand for this NR (Palanker, 2006).

Alternative sumoylation sites in the Drosophila nuclear receptor Usp

The Ultraspiracle protein (Usp), together with an ecdysone receptor (EcR) forms a heterodimeric ecdysteroid receptor complex, which controls metamorphosis in Drosophila. Although the ecdysteroid receptor is considered to be a source of elements for ecdysteroid inducible gene switches in mammals, nothing is known about posttranslational modifications of the receptor constituents in mammalian cells. Up until now there has been no study about Usp sumoylation. Using Ubc9 fusion-directed sumoylation system, Usp was identified as a new target of SUMO1 and SUMO3 modification. Mutagenesis studies on the fragments of Usp indicated that sumoylation can occur alternatively on several defined Lys residues, i.e., three (Lys16, Lys20, Lys37) in A/B region, one (Lys424) in E region and one (Lys506) in F region. However, sumoylation of one Lys residue within A/B region prevents modification of other residues in this region. This was also observed for Lys residues in carboxyl-terminal fragment of Usp, i.e. comprising E and F regions. Mass spectrometry analysis of the full-length Usp indicated that the main SUMO attachment site is at Lys20. EcR, the heterodimerization partner of Usp, and muristerone A, the EcR ligand, do not influence sumoylation patterns of Usp. Another heterodimerization partner of Usp - HR38 fused with Ubc9 interacts with Usp in HEK293 cells and allows sumoylation of Usp independent of the direct fusion to Ubc9. Taken together, it is proposed that sumoylation of DmUsp can be an important factor in modulating its activity by changing molecular interactions (Bielska, 2012).

The molecular basis of conformational instability of the ecdysone receptor DNA binding domain studied by in silico and in vitro experiments

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, regulates gene expression associated with molting and metamorphosis in insects. The DNA binding domains (DBDs) of the Usp and EcR play an important role in their DNA-dependent heterodimerization. Analysis of the crystal structure of the UspDBD/EcRDBD heterocomplex from Drosophila melanogaster on the hsp27 gene response element, suggested an appreciable similarity between both DBDs. However, the chemical denaturation experiments showed a categorically lower stability for the EcRDBD in contrast to the UspDBD. The aim of this study was an elucidation of the molecular basis of this intriguing instability. Toward this end, the EcRDBD amino acid sequence positions which have an impact on the stability of the EcRDBD were mapped. The computational protein design and in vitro analyses of the EcRDBD mutants indicate that non-conserved residues within the alpha-helix 2, forming the EcRDBD hydrophobic core, represent a specific structural element that contributes to instability. In particular, the L58 appears to be a key residue which differentiates the hydrophobic cores of UspDBD and EcRDBD and is the main reason for the low stability of the EcRDBD. These results might serve as a benchmark for further studies of the intricate nature of the EcR molecule (Szamborska-Gbur, 2014).

UTX coordinates steroid hormone-mediated autophagy and cell death

Correct spatial and temporal induction of numerous cell type-specific genes during development requires regulated removal of the repressive histone H3 lysine 27 trimethylation (H3K27me3) modification. This study shows that the H3K27me3 demethylase dUTX is required for hormone-mediated transcriptional regulation of apoptosis and autophagy genes during ecdysone-regulated programmed cell death of Drosophila salivary glands. dUTX binds to the nuclear hormone receptor complex Ecdysone Receptor/Ultraspiracle, and is recruited to the promoters of key apoptosis and autophagy genes. Salivary gland cell death is delayed in dUTX mutants, with reduced caspase activity and autophagy that coincides with decreased apoptosis and autophagy gene transcripts. It was further shown that salivary gland degradation requires dUTX catalytic activity. These findings provide evidence for an unanticipated role for UTX demethylase activity in regulating hormone-dependent cell death and demonstrate how a single transcriptional regulator can modulate a specific complex functional outcome during animal development (Denton, 2013).

UTX function is known to be critical in mammalian embryonic development and somatic and germ cell reprogramming. This study found a novel role for dUTX in steroid hormone-mediated cell death during development. dUTX, together with nuclear hormone receptor EcR/Usp, is capable of regulating gene expression both spatially and temporally in a hormone-dependent manner. UTX gene mutations are frequently observed in malignancies including lethal castration-resistant prostate cancer, although a role for UTX in androgen receptor-mediated transcription has not yet been identified. This study indicates that UTX is a good candidate to extend the investigation to examine the role of UTX in coordinating nuclear hormone receptor-regulated gene expression, particularly in androgen receptor-mediated transcription during mammalian development and hormone-dependent cancers (Denton, 2013).

The complete degradation of larval salivary glands during metamorphosis utilizes both apoptosis and autophagy and by coordinately controlling the expression of critical genes in these two distinct biological pathways, dUTX ensures timely removal of salivary glands in response to temporal ecdysone pulse. The majority of studies addressing induction of autophagy have focused upon autophagosome formation and protein degradation. The transcriptional regulation of autophagy induction remains poorly understood. Indeed, several Atg genes are transcriptionally upregulated following autophagy induction; however, the molecular pathways are only beginning to be revealed. For example, the master gene controlling lysosomal biogenesis, transcription factor EB, coordinates the expression of both autophagy and lysosomal genes to induce autophagy in response to starvation. Induction of autophagy has been linked to reduced histone H4 lysine 16 acetylation (H4K16ac) through downregulation of the histone acetyltransferase hMOF. Downregulation of H4K16 deacetylation was associated with the downregulation of several Atg genes, whereas antagonizing H4K16ac downregulation upon autophagy induction resulted in cell death. The study indicates that a specific histone modification during autophagy modulates the expression of Atg genes, and is important for survival versus death responses upon autophagy induction. This work now describes dUTX as another regulator of autophagy and cell death in the context of developmental PCD and in concert with the steroid hormone response. Future studies to understand the complex nuclear events regulating both repression and induction of autophagy gene expression in response to particular signals will be important (Denton, 2013).

Despite the opposing roles of H3K27 and H3K4 methylation in transcriptional regulation, UTX has been identified in association with H3K4 methyltransferase and to play demethylase-independent functions. This study suggests that the demethylase activity of dUTX is necessary for hormone-mediated cell death. The nuclear hormone receptor response to ecdysone initiates a hierarchical transcription cascade by induction of transcription factors, including BR-C, E74 and E93. These transcription factors drive expression of downstream genes including cell death genes. The data show that dUTX regulates E93 and suggests that this HDM can regulate cell death both directly, through the transcription of apoptosis and autophagy genes through direct recruitment via EcR/Usp, as well as indirectly through key transcription factor E93. This additional level of regulation through the stage-specific transcription factor E93 may provide temporal control of ecdysone response during metamorphosis (Denton, 2013).

The role of autophagy in cell death is a matter of considerable debate as autophagy is generally a cell survival mechanism in response to cellular stress and nutrient limitations. Studies in Drosophila have provided perhaps some of the strongest evidence for a role of autophagy in developmental cell death in vivo. The data presented in this paper demonstrating coordinate regulation of both key apoptosis and autophagy genes by a single histone-modifying enzyme further provide genetic and molecular evidence linking autophagy and apoptosis in PCD during metamorphosis (Denton, 2013).

Nutritional control of body size through FoxO-Ultraspiracle mediated ecdysone biosynthesis

Despite their fundamental importance for body size regulation, the mechanisms that stop growth are poorly understood. In Drosophila melanogaster, growth ceases in response to a peak of the molting hormone ecdysone that coincides with a nutrition-dependent checkpoint, critical weight. Previous studies indicate that insulin/insulin-like growth factor signaling (IIS)/Target of Rapamycin (TOR) signaling in the prothoracic glands (PGs) regulates ecdysone biosynthesis and critical weight. This study elucidates a mechanism through which this occurs. This study shows that Forkhead Box class O (FoxO), a negative regulator of IIS/TOR, directly interacts with Ultraspiracle (Usp), part of the ecdysone receptor. While overexpressing FoxO in the PGs delays ecdysone biosynthesis and critical weight, disrupting FoxO-Usp binding reduces these delays. Further, feeding ecdysone to larvae eliminates the effects of critical weight. Thus, nutrition controls ecdysone biosynthesis partially via FoxO-Usp prior to critical weight, ensuring that growth only stops once larvae have achieved a target nutritional status (Koyama, 2014).

ultraspiracle: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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