ftz-f1

Protein interactions

The orphan nuclear receptor alpha Ftz-F1, which is deposited in the egg during oogenesis, is an obligatory cofactor for Fushi tarazu. Mutation of Ftz-F1 causes a pair-rule phenotype even though the maternal alphaFTZ-F1 gene product is uniformly distributed through the embryo. Surprisingly, patterns of FTZ mRNA and protein expression in the alphaFTZ-F1 mutants are indistinguishable from wild type. In Ftz-F1 mutant embryos, as in ftz mutant embryos, Ftz-dependent engrailed stripes fail to be expressed, and wingless stripes expand. Thus alphaFtz-F1 is required for all Ftz activities tested except that for which it was first identified: regulation of the ftz promoter. Given this result, a test was made for direct interaction between Ftz protein and alphaFtz-F1. The two proteins interact specifically and directly, both in vitro and in vivo, through a conserved domain in the Ftz polypeptide. The conserved motif is independent of the Ftz homeodomain and is located in the central portion of the protein, flanked by prolines. Deletion of this motif disrupts all but one of the the Ftz activities described above, that is, it is still capable of broadening endogeneous ftz expression stripes. Thus removal of the alphaFtz-F1 interaction domain from the Ftz polypeptide results in the same loss of Ftz activities as removal of alphaFtz-F1. The Ftz-mediated repression of wingless requires both Paired and alphaFtz-F1. This interaction could involve either simultaneous or competitive interactions among the three proteins, as Prd also contacts residues 101-150 of Ftz. Paired may be a cofactor of Ftz or Ftz-F1 that is required for target genes that are repressed by Ftz, because Prd is required for Ftz-dependent wingless repression, but not for Ftz-dependent activation of engrailed or ftz auto-regulation (Guichet, 1997).

A native binding site for the homeodomain protein Fushi tarazu, the upstream regulatory region of the ftz gene itself, was used to isolate Ftz-F1, a protein of the nuclear hormone-receptor superfamily and a new Ftz cofactor. Ftz and Ftz-F1 are present in a complex in Drosophila embryos. The upstream regulatory region contains five Ftz binding sites and three Ftz-F1 binding sites. One Ftz-F1 site is adjacent to a medium-affinity Ftz binding site. Ftz-F1 facilitates the binding of Ftz to DNA, allowing interactions with weak-affinity sites at concentrations of Ftz that alone would bind only high-affinity sites. By virtue of mutual binding to the same regulatory region, Ftz-f1 and Ftz are both implicated in Ftz autoregulation. Embryos lacking Ftz-F1 display ftz-like pair-rule cuticular defects, and engrailed transcription sites are not present in Ftz-f1 mutant embryos. The pair-rule phenotype is a result of abnormal ftz function, because ftz is expressed but fails to activate downstream genes (Yu, 1997).

One Drosophila Tbp interaction with transcription factors involves a coactivator of the transcription factor FTZ-F1. The coactivator, Multiprotein bridging factor 1 (MBF1), makes possible a connection, or bridges, the TATA box-binding protein (Tbp) and the nuclear hormone receptor FTZ-F1. MBF1 is functional in interactions with Tbp and a positive cofactor MBF2. MBF1 makes a direct contact with FTZ-F1 through the C-terminal region of the FTZ-F1 DNA-binding domain and stimulates the binding of FTZ-F1 to its recognition site. The central region of MBF1 (residues 35-113) is essential for the binding of FTZ-F1, MBF2, and Tbp. MBF1, in the presence of MBF2, and FTZ622 bearing the FTZ-F1 DNA-binding domain, support selective transcriptional activation of the fushi tarazu gene. Mutations that disrupt the binding of FTZ622 to DNA or MBF1, or an MBF2 mutation that disrupts the binding to MBF1, all abolish the selective activation of transcription. These results suggest that tethering the positive cofactor MBF2 to a FTZ-F1-binding site through FTZ-F1 and MBF1 is essential for the binding site-dependent activation of transcription (Takemaru, 1997).

To activate transcription, most nuclear receptor proteins require coactivators that bind to their ligand-binding domains (LBDs). The Drosophila FTZ-Factor1 (Ftz-f1) protein is a conserved member of the nuclear receptor superfamily, but has been thought to lack an AF2 motif, a motif that is required for ligand and coactivator binding. It is shown here that Ftz-f1 does in fact have an AF2 motif and that it is required to bind a coactivator, the homeodomain-containing protein Fushi tarazu (Ftz). Ftz contains an AF2-interacting nuclear receptor box, the first to be found in a homeodomain protein. Both interaction motifs are shown to be necessary for physical interactions in vitro and for functional interactions in developing embryos. These unexpected findings have important implications for the conserved homologs of the two proteins (Schwartz, 2001).

Ftz-f1 deletion constructs were sequenced to ensure that no errors had been introduced during the course of PCR and cloning procedures. These analyses revealed a discrepancy between the sequences in this study and those of the published Ftz-f1 sequence. An 8 bp repeat beginning at position 3006 of the alphaFtz-f1 sequence was contained in the sequences obtained in this study. The predicted coding region of the revised sequence diverges at amino acid 1003 and encodes an additional 24 amino acids. This revised sequence contains an AF2 consensus that is highly similar to the sequences of vertebrate Ftz-f1 homologs. Confirmation of this revised sequence has recently been provided by the Drosophila genome sequencing project. The sequence of the Ftz-f1 protein of B. mori has also been reported to lack an AF2 motif. A brief examination of the DNA coding sequence reveals that the addition of a single nucleotide in an appropriate location would change the reading frame and yield an AF2 nearly identical to that of the Drosophila protein. Hence, a Bombyx Ftz-f1 cDNA was obtained and sequenced. An additional adenosine nucleotide was found that follows the thymidine at position 1659. The shifted reading frame generates a new C-terminus with 27 of the 34 predicted amino acids identical to the Drosophila sequence. An additional four residues are conservative substitutions. The C-terminal AF2s of the two insect proteins are identical at 15 of 16 positions. It is concluded that both insect proteins contain highly conserved AF2 motifs (Schwartz, 2001).

It is not yet known how Ftz and Ftz-f1 regulate their target genes. Interestingly, Ftz and Ftz-f1 are known to function as both transcriptional activators and repressors. Thus, Ftz may be able to act as both a coactivator and corepressor for Ftz-f1 function. Alternatively, these differential activities may depend on the actions of other cofactors, some of which may be target gene specific. The fact that Ftz-f1 utilizes Ftz as a coregulator of target genes, and that Ftz contains a nuclear receptor box, suggests the possibility that, like other nuclear receptor cofactors, Ftz may help recruit histone acetyltransferase or deacetylase complexes. Alternatively, Ftz may act via regulatory factors or complexes that are quite different from these. For example, Ftz target genes such as engrailed and homeotic genes of the Antennapedia and bithorax gene clusters are regulated by chromatin-organizing Polycomb and Trithorax group protein complexes. The Ftz-Ftz-f1 complex may play a role in recruiting these larger protein complexes (Schwartz, 2001).

Does Ftz-f1 have a ligand? There are several observations that argue both for and against the existence of a Ftz-f1 ligand. Motifs and residues required for ligand binding by other receptors are conserved in Ftz-f1. These include residues in the AF2 helix that, based on other receptor structures, are expected to contact the ligand-binding pocket. Ftz-f1 also contains a conserved residue in the third helix of the LBD (N840) that has been shown in other receptors to be a key ligand-contacting residue. Although a study with the Ftz-f1 homolog SF-1 suggests that its transcriptional activity could indeed be enhanced in the presence of certain oxysterols, subsequent studies have been unable to validate this finding. A more general observation in favor of a ligand is that while there are a large number of orphan nuclear receptors whose ligands have not been identified, there are currently no clear examples of receptors that are fully functional as activators in the absence of ligand (Schwartz, 2001 and references therein).

Observations that argue for the absence of a Ftz-f1 ligand include the avid binding of Ftz to Ftz-f1 observed in vitro in the absence of ligand. For most nuclear receptors with known ligands, the ligand is required to position the overlying AF2 in the proper orientation for coactivator binding. The ability of Ftz to bind in the absence of ligand suggests that coactivator binding to Ftz-f1 may be constitutive, and that temporal and spatial regulation of Ftz-f1 activity is controlled by the presence or absence of cofactors such as Ftz rather than by ligands. Alternatively, conditions in the cell may be more stringent, making the Ftz-Ftz-f1 interaction ligand dependent (Schwartz, 2001).

The Drosophila homeodomain protein Fushi Tarazu (Ftz) and its partner, the orphan receptor Ftz-F1, are members of two distinct families of DNA binding transcriptional regulators. Ftz and Ftz-F1 form a novel partnership in vivo as a Hox/orphan receptor heterodimer. The murine Ftz-F1 ortholog SF-1 functionally substitutes for Ftz-F1 in vivo, rescuing the defects of ftz-f1 mutants. This finding identified evolutionarily conserved domains of Ftz-F1 as critical for activity of this receptor in vivo. These domains function, at least in part, by mediating direct protein interactions with Ftz. The Ftz-F1 DNA binding domain interacts strongly with Ftz and dramatically facilitates the binding of Ftz to target DNA. This interaction is augmented by a second interaction between the AF-2 domain of Ftz-F1 and the N-terminus of Ftz via an LRALL sequence in Ftz that is reminiscent of LXXLL motifs in nuclear receptor coactivators. It is proposed that Ftz-F1 serves as a cofactor for Ftz by facilitating the selection of target sites in the genome that contain Ftz/Ftz-F1 composite binding sites. Ftz, in contrast, influences Ftz-F1 activity by interacting with the Ftz-F1 AF-2 domain in a manner that mimics a nuclear receptor coactivator (Yussa, 2001).

The orphan receptor Ftz-F1 and homeodomain (HD) protein Ftz cooperate to promote the development of alternate body segments, presumably by activating expression of downstream target genes such as engrailed. Ftz and Ftz-F1 are each sequence specific DNA binding proteins with strong transcriptional activation domains. However, neither is apparently able to function in Drosophila embryos to regulate target gene expression in the absence of its partner, since mutation of either protein results in lethality accompanied by identical pair-rule defects. Each protein is necessary but neither Ftz nor Ftz-F1 alone is sufficient to select and activate target gene transcription in the embryo. Why do Ftz and Ftz-F1 require partners in vivo to regulate gene expression? By analogy to the function of beta ftz-f1 as a competence factor for the ecdysone response, alpha ftz-f1 can be seen as a competence factor for a 'pulse' of Ftz expression in seven stripes in the blastoderm embryo (Yussa, 2001).

Why does Ftz-F1 fail to activate Ftz-F1/Ftz (F1F) targets in the absence of Ftz protein? Ftz-F1 proteins are strong transcriptional activators in a variety of cell systems. In Drosophila, ftz-f1 is maternally deposited and Ftz-F1 is found in all somatic nuclei before Ftz is expressed in seven stripes. Yet, Ftz-F1 does not detectably activate transcription in the absence of Ftz either temporally (before Ftz is expressed zygotically) or spatially (in regions of the embryo outside the seven Ftz stripes). Thus the presence of Ftz functions as an apparent 'on' versus 'off' switch to enable Ftz-F1 to activate transcription. Two mechanisms are proposed that may contribute Ftz-F1's requirement for Ftz to activate transcription. (1) Ftz-F1 might require Ftz for stable DNA binding. In vitro, Ftz-F1 activates transcription of the ftz proximal enhancer (323-fPE) and F1F reporter constructs more strongly than does Ftz, consistent with the in vitro DNA binding properties of the two proteins. In fact, the order of magnitude of synergy of transcriptional activation of Ftz-F1 compared to Ftz-F1 + Ftz (5-10 fold) is similar to the enhancement of DNA binding conferred by Ftz on Ftz-F1. While this mechanism likely contributes to Ftz-F1 activation, the enhancement seen in vitro may not translate into the apparent 'on' vs. 'off' state of Ftz-F1 in Ftz-expressing vs. Ftz-non-expressing cells in vivo (Yussa, 2001). Therefore, (2) a model in which Ftz-F1 is actively repressed in the absence of Ftz, even if it is bound to cognate DNA target sites, is favored. Ftz-F1 is present in all somatic nuclei before Ftz is expressed and may bind cognate DNA sequences, but remains quiescent until it is activated by interaction with Ftz protein. It is proposed that interaction of Ftz with Ftz-F1 through its LXXLL motif displaces corepressor molecules, allowing productive transcription complexes to form. The candidates for repressor molecules that keep Ftz-F1 in an 'off' state are the corepresssors that inhibit activity of other nuclear receptor family members. The corepressors identified in Drosophila that are expressed throughout the blastoderm embryo include Alien, which interacts with Ftz-F1 in GST-pulldown assays and SMRTER. A corepressor might bind directly to Ftz-F1. It is also possible that another partner of Ftz-F1 recruits a corepressor to the complex, as has been shown for the murine ortholog of Ftz-F1 (SF-1) which is regulated by interaction with the nuclear receptor Dax-1 that recruits corepressors and inhibits SF-1 activity (Yussa, 2001).

Why does Ftz require Ftz-F1 for target gene regulation in vivo? Ftz and Ftz-F1 bind cooperatively to DNA to select specific target sites that are composite binding sites for the two proteins. The binding of Ftz and Ftz-F1 to composite sites is stabilized by protein-protein interactions mediated by at least two regions of each partner protein. The composite nature of the binding site raises the selectivity of Ftz binding by requiring a heterodimeric site for productive interaction. The affinity of Ftz for composite sites is dramatically increased by Ftz-F1. Ftz protein can bind to monomeric 'ATTA' core sites in vitro and it appears to associate with a wide array of sites in the genome, as determined by UV cross-linking experiments. However, it is proposed that in vivo, binding of Ftz to monomeric 'ATTA' sites is transient, during the time that Ftz scans the genome for cognate DNA binding sites. This relatively unstable binding does not allow Ftz to effectively activate transcription on its own, consistent with the finding that concatamerized Ftz binding sites do not mediate a Ftz-dependent pattern of gene expression in vivo. Ftz binds productively to composite heterodimeric sites, where it is stabilized by protein-protein interactions with Ftz-F1. This notion is also consistent with reports that fusion of Ftz to a strong VP16 activator does not alter target gene regulation in vivo. Thus, specificity of gene regulation by Ftz protein is achieved at the level of DNA binding/target site selection, as a result of interaction with DNA binding cofactors such as Ftz-F1 (Yussa, 2001).

Future studies are required to elucidate the detailed mechanism whereby transcription is activated by the Ftz/ Ftz-F1 complex. One candidate mechanism is direct contact of TFIIB by Ftz and/or Ftz-F1. Ftz was shown to directly contact TFIIB, activating transcription via a C-terminal region that does not appear to be involved in contacting Ftz-F1. Similarly, SF-1 interacts with TFIIB via the Ftz-F1 box and adjacent proline-rich region, neither of which appear to be necessary for interaction with Ftz. Thus, these regions of the proteins could be available in the DNA bound ternary complex to directly contact the basal transcription machinery (Yussa, 2001).

Does Ftz interaction obviate a requirement for a Ftz-F1 coactivator? The novel possibility is suggested that Ftz substitutes for coactivator function for Drosophila Ftz-F1. For mammalian SF-1, standard coactivator interactions have been demonstrated in vitro and in cell culture, suggesting that SRC and CBP/p300 family proteins are partners of SF-1. Drosophila CBP has been well characterized and one p160/SRC-type coactivator, Taiman, was recently identified. Like Ftz, Drosophila CBP has one LXXLL motif while Tai has four such motifs, as is typical for mammalian coactivators. However, it is unlikely that interaction with either of these coactivators is sufficient to activate Ftz-F1 since both dCBP and Tai are expressed ubiquitously in the embryo, including the cells where Ftz-F1 is apparently inactive. One interesting possibility is that dCBP acts as a corepressor in the context of Ftz-F1, as it has been shown to do for TCF. Thus dCBP might silence Ftz-F1 by interacting with its AF-2 domain via an LXXLL motif. This interaction could be displaced by Ftz because of an intrinsically higher affinity of its LXXLL motif as compared to that of dCBP. Note that peptides with variations of the LXXLL motif have different affinities for AF-2 domains of nuclear receptors. Alternatively, Ftz may interact preferentially with Ftz-F1 because of the additional protein-protein and protein-DNA interactions that bring high levels of Ftz protein in close proximity to Ftz-F1, driving the interaction of its LXXLL motif with Ftz-F1 (Yussa, 2001).

In addition to its role in DNA binding, the Ftz HD is involved in direct protein-protein interactions with its cofactor Ftz-F1. These findings underscore the importance of the HD, which is absolutely required for the wild type function of Ftz, as it is for other Hox proteins. Some years ago, a 'HD-independent' activity of Ftz was described. These studies made use of a protein carrying a deletion within the HD (DHD) that removes helix 2 and portions of helices 1 and 3. It was shown that this protein is able to cause 'anti-ftz' phenotypes that result from mis-expression of Ftz with a heat inducible promoter and it was suggested that this protein can rescue ftz-dependent cuticle when similarly overexpressed. In contrast to these results, Ftz DHD was unable to rescue ftz mutants when the protein was expressed under control of endogenous ftz regulatory elements. In addition, even subtle mutations within this region of the HD abolish rescue potential. Finally, FtzDHD was unable to rescue any cuticular defects associated with ftz mutations when expressed using native ftz regulatory elements. Thus it is likely that under conditions of overexpression, interactions with Ftz-F1 through the AF-2/LXXLL domains of the proteins can to some extent overcome the endogenous requirement for the HD by positioning Ftz on the DNA of some target elements, allowing for gene activation (Yussa, 2001).

Given the highly conserved nature of the HD, the finding that it is involved in the Ftz/Ftz-F1 interaction led the authors to ask if other Hox proteins could interact with Ftz-F1. Preliminary results indicate that a number of Hox proteins coordinately activate transcription in conjunction with Ftz-F1 in cells. Experiments are underway to determine whether these interactions -- which are less potent than Ftz -- are strong enough to support interactions between Ftz-F1 and Hox proteins during Drosophila development. Such interactions would have been masked in previous genetic studies by the fact that the earliest function of Ftz-F1 in segmentation results in a phenotype which precludes analysis of Ftz-F1 function at slightly later stages when Hox genes such as Ubx, Antp and Scr are active. An additional question is whether conserved Hox proteins are partners of mammalian Ftz-F1 proteins. Co-expression of and interaction between Hox proteins and Ftz-F1 have not been investigated in mammals. Several partners of SF-1 have been characterized, including the nuclear receptor Dax-1, the zinc finger transcription factor WT-1, and the Bcd-family HD protein Ptx-1. For Ptx-1, the interaction domain with SF-1 maps outside of the HD. However, interactions between other nuclear receptors and the HDs of other proteins have been reported. Since ftz-f1 but not ftz genes are found in the vertebrate lineage, an intriguing possibility consistent with the results discussed above is that vertebrate Ftz-F1 proteins interact with HDs of Hox proteins that are conserved throughout evolution, to execute unique functions as nuclear receptor/Hox protein heterodimers (Yussa, 2001).

Orphan receptors for whom cognate ligands have not yet been identified form a large subclass within the nuclear receptor superfamily. To address one aspect of how they might regulate transcription, the mode of interaction between the Drosophila orphan receptor FTZ-F1 (NR5A3) and a segmentation gene product, Fushi tarazu (FTZ), was investigated. Strong interaction between these two factors was detected by use of the mammalian one- and two-hybrid interaction assays without addition of ligand. This interaction requires the AF-2 core and putative ligand-binding domain of FTZ-F1 and the LXXLL motif of FTZ. The requirement of these elements has been further confirmed by examination of their target gene expression in Drosophila embryos and observation of a cuticle phenotype in transgenic fly lines that express mutated factors (Suzuki, 2001).

In Drosophila cultured cells, FTZ is required for FTZ-F1 activation of a FTZ-F1 reporter gene. These results reveal a resemblance in the mode of interaction between FTZ-F1 and FTZ and that of nuclear receptor-coactivator and indicate that direct interaction is required for regulation of gene expression by FTZ-F1. Thus, it is proposed that FTZ may represent a category of LXXLL motif-dependent coactivators for nuclear receptors (Suzuki, 2001).

The general structure of the LBD of nuclear receptor superfamily members is composed mainly of 12 helices. Interaction with ligand induces allosteric changes in conformation, especially in the configuration of helix 12 at the C terminus of the LBD, leading to transcriptional activation or repression. Helix 12 is often referred to as the AF-2 core (or AF-2 activation domain, tauc or tau4) that serves in some receptors as a conserved domain essential for ligand-dependent transcriptional activation. Transcriptional coactivators such as CBP/p300, TRAP220, and p160 family factors, SRC-1/NcoA-1, TIF2/GRIP1/NcoA-2, and p/CIP/ACTR/AIB1, have been shown to mediate activating signals through binding to nuclear receptors in a ligand-dependent manner. For this receptor-coactivator interaction, conserved sequences containing a short signature motif of LXXLL (where L is leucine and X can be any amino acid) have been implicated. The conserved leucines in these so-called LXXLL motifs, or NR boxes, appear indispensable for interaction with nuclear receptors. In nuclear receptors, the importance of helices 3, 5, and 12 (AF-2 core) in the LBD has been demonstrated, and computational modeling studies have predicted that helices 3, 5, and an appropriately realigned helix 12 form an interacting surface for the LXXLL. The majority of nuclear receptors, however, are 'orphans', for which cognate ligands have not yet been identified and the molecular mechanisms of their transcriptional regulation remain unclear. From an evolutionary aspect, the extension of the structural conservation to domains including the LBD strongly suggests a functional significance, raising a naive question. Despite large-scale ligand screenings that have been undertaken by many groups, why are there still so many 'orphans' remaining? One answer may be that the structural conservation in the LBD implicates an importance for interactions with various intracellular factors other than small lipophilic molecules (Suzuki, 2001).

Ubiquitous expression of FTZ in early embryos under control of the heat shock promoter broadens even-numbered engrailed (en) stripes, represses alternate wg stripes, and results in a so-called anti-ftz cuticle phenotype, in which roughly reciprocal segments are missing compared with the ftz larval cuticle phenotype. However, ectopic en induction or wg repression was not observed by expressing a construct (mutFTZ) containing substitutions in the tandem leucines, indicating that the LXXLL motif in FTZ is necessary for the ectopic expression of en and the repression of wg. FTZ-dependent en induction and wg repression were also observed by expression of both FTZ-F1 and FTZ under control of the heat shock promoter in the ftz-f1 mutant embryo but not when FTZ-F1DeltaAF2C was used instead of FTZ-F1. An anti-ftz cuticle phenotype was produced by forced expression of wild-type FTZ but not by expression of mutFTZ. In ftz-f1 mutant embryos, anti-ftz phenotypes were obtained when FTZ-F1 and FTZ were coexpressed under heat shock control but not upon replacement of FTZ-F1 with FTZ-F1DeltaAF2C. These observations indicate that interaction through the LXXLL motif in FTZ and AF-2 core in FTZ-F1 is necessary for producing an anti-ftz phenotype and further support the results of the one- and two-hybrid assay (Suzuki, 2001).

Thus, results using embryos strongly suggest that FTZ-F1 activates engrailed in vivo through the AF-2-LXXLL-dependent direct interaction. In early fly embryos, FTZ-F1 seems to function as an activator for engrailed only in regions where FTZ is also present despite the uniform expression of FTZ-F1. Such situations mimic that of the requirement for a ligand by a nuclear receptor in controlling its function and specificity in gene expression. The characteristic cooperation of FTZ-F1 and FTZ provides a novel example of transcriptional regulation by a nuclear receptor, which may be an alternative pathway to the conventional one using lipophilic ligands. From an evolutionary aspect, it has been proposed that the ancestral nuclear receptor had no ligand and the ability to bind a ligand was acquired by a subset of descendent receptors later in evolution. It has also been presumed that FTZ-F1 is one of the most ancient receptors based on its distribution among species. It is believed that transcriptional activation by FTZ-F1 through binding to FTZ might represent a primitive style of regulation by nuclear receptors before the acquisition of ligand-binding ability. The existence is presumbed of yet unidentified corresponding factors for other orphan receptors as well as for ligand-responsive receptors, which may form a new group of nuclear receptor coactivators and play critical roles for development and metabolism (Suzuki, 2001).

Bonus interacts with FTZ-F1 and inhibits FTZ-F1-dependent 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).

bon was isolated in a screen for mutations affecting embryonic peripheral nervous system (PNS) development. Three independent P-element alleles, bonS024108 (bon²⁴¹), bonS024912 (bon²⁴⁹), and bonS048706 (bon⁴⁸⁷), which mapped to 92E8-14, fail to complement each other. An additional bon allele, bon^21B, was generated by imprecise excision of bon²⁴¹. This allele fails to complement all bon alleles and a deficiency, Df(3R)H^B79, which removes chromosomal region 92E (Beckstead, 2001).

To establish the strength of each allele, complementation tests were performed and the lethal phase associated with each allelic combination was defined. Df(3R)H^B79/bon^21B animals exhibit the earliest stage of lethality, while homozygous bon²⁴¹/bon²⁴¹ animals display the least severe phenotype, with 34% of the expected animals surviving to pharate adults. In all genetic combinations, some first instar larvae survive up to a week and fail to molt into second instar larvae. No male third instar larvae, pharate adults or adults survived in any bon genetic background, indicating that loss of bon has a more deleterious effect on males than females. Based on the complementation data, the bon alleles were ordered as follows: Df(3R)H^B79 > bon^21B > bon⁴⁸⁷ > bon²⁴¹ = bon²⁴⁹ (Beckstead, 2001).

To pinpoint the phenotypes associated with bon mutations, morphological defects associated with different allelic combinations were analyzed. Df(3R)H^B79/bon^21B mutant embryos fully develop. However, many are unable to hatch from their egg case, and both Df(3R)H^B79/bon^21B embryos and first instar larvae exhibit disrupted fluid-filled trachea. The embryonic/first instar lethality probably corresponds to the zygotic null or a severe loss of function phenotype as there is very little maternal protein remaining in mature Df(3R)H^B79/bon^21B embryos (Beckstead, 2001).

Less severe loss of Bon function results in pupal defects. The majority of bon⁴⁸⁷/bon^21B mutant pupae display an almost complete lack of pigmentation. They initiate but fail to complete development of legs, wings, head, eyes, and cuticle. Salivary glands, which normally undergo apoptosis at 12 hr postpupariation, are present in 4 days post-pupariation bon⁴⁸⁷/bon^21B pupae and are similar in size to third instar larval glands. In bon²⁴¹/bon⁴⁸⁷ animals, defects in cuticle and bristle development are observed. The abdominal cuticle of these flies appears immature and the tergite and sternal bristles are severely reduced or absent. Bristles of the anterior wing margin of bon²⁴¹/bon⁴⁸⁷ pharate adults are almost entirely lacking. Finally, there is a dramatic reduction in pigmentation in the mutant wing cuticle. This data indicates that bon is required for numerous developmental processes, including control of larval molting, cuticle deposition and pigmentation, bristle development, and elimination of salivary glands by cell death (Beckstead, 2001).

To determine the effect of complete loss of bon on the development of adult tissues, the FLP/FRT system was used to create mutant clones in the developing eye imaginal disc with the eyeless enhancer driving FLP. Flies heterozygous for FRT bon^21B and a cell lethal gene marked with w⁺ (FRT w⁺ cl3R) expressing FLP in the eye disc generate clones of bon^21B/bon^21B. Loss of bon in the eye causes a loss of all mutant photoreceptors. A small patch of red photoreceptors remains because of a limited number of bon^21B/cl3R cells. This indicates that bon is required for cell viability or proliferation of photoreceptors. In addition, much of the head cuticle is missing, indicating that most or all cells of the eye disc that produce cuticle are also lacking. In addition, no adult mutant bon clones were observed in FLP/FRT experiments using heat shock-FLP; FRT82 bon^21B animals, even though numerous wild-type twin spots were observed. Hence, early and complete loss of bon is lethal to cells or disrupts proliferation (Beckstead, 2001).

To clone bon, genomic DNA flanking bon²⁴¹ was isolated and used to identify cDNAs. The bon cDNA (AF210315) permitted isolation of genomic phages and determination of the structure of the locus. Flanking sequences from bon²⁴¹, bon²⁴⁹, and bon⁴⁸⁷ were used to map the P-elements. Sequencing of bon^21B revealed a deletion of most of exon 1 and the 5' end of intron 1 (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 determine whether the defects seen in bon mutants are due to disruptions in the ecdysone-regulated pathway, the expression of several ecdysone-regulated genes were examined in y w and bon²⁴¹/bon²⁴¹ larvae, prepupae, and pupae. In bon²⁴¹/bon²⁴¹ animals, levels of betaFTZ-F1, EcR-A, EcR-B, E74A, E74B, and BR-C are reduced. It appears that each gene is upregulated in response to the ecdysone pulse, but is unable to maintain expression in the bon mutants. However, DHR3 transcripts are prematurely expressed and the overall level of expression is elevated in bon²⁴¹/bon²⁴¹ animals when compared to y w control animals. In addition, the EcR-A transcript levels appear slightly reduced in bon²⁴¹/bon²⁴¹ animals, while the EcR-B transcript levels are severely reduced when compared to controls. Similar observations were made for all of the above genes in bon^21B/bon⁴⁸⁷ animals, except that DHR3 transcript levels are also reduced. Based on these effects on gene expression, defects in larval molting and metamorphosis, and the temporal expression pattern of Bon, it is proposed that Bon plays an important role in the regulation of genes in the ecdysone response pathway (Beckstead, 2001).

To better characterize the function of Bon, interacting proteins were sought. A Drosophila embryonic cDNA library was screened using Bon as bait. Isolated cDNAs were classified as positive when retested in another version of the two-hybrid system using the DNA binding domain of the estrogen receptor fused to Bon (DBD-Bon) and an ERE-URA3 reporter gene. One positive clone encoded the 488 C-terminal residues of betaFTZ-F1 (amino acids 315-802). Coexpression of DBD-Bon with AAD-betaFTZ-F1(315-802) transactivates the URA3 reporter. Hence, Bon is able to interact with betaFTZ-F1(315-802) in yeast cells (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 define the domain(s) of betaFTZ-F1 responsible for Bon interaction, a deletion analysis of betaFTZ-F1 was performed using the yeast two-hybrid system. Various segments of betaFTZ-F1 were fused to the VP16 AAD and assayed for DBD-Bon interaction. No increase in reporter activity was observed with the N-terminal A/B region or with a fusion protein containing residues 270-631, which include the DNA binding domain and the hinge region of the receptor. In contrast, a 7-fold activation was detected in the presence of AAD-betaFTZ-F1(555-802), indicating that the E region encompassing the putative ligand binding domain (LBD) is sufficient for interaction with Bon (Beckstead, 2001).

Sequence analysis of the E region of betaFTZ-F1 has revealed a conserved transcriptional activation domain 2 core motif (AF-2 AD core) between residues 791 and 797. To investigate its activity, an expression vector encoding the E region of betaFTZ-F1 fused to the yeast GAL4 DNA binding domain was cotransfected into Drosophila Schneider (S2) cells together with a GAL4 reporter plasmid. An increase in reporter gene activity was observed, whereas no transactivation was detected with a GAL4-betaFTZ-F1 construct lacking the AF-2 AD core. Deletion of the AF-2 AD core also abolished Bon interaction with the betaFTZ-F1 E region in yeast. Thus, the LBD of betaFTZ-F1 contains an AF-2 activation domain, whose integrity is required for Bon interaction (Beckstead, 2001).

To determine which domain of Bon interacts with betaFTZ-F1, a series of DBD-Bon deletion constructs were generated and assayed for interaction with the E region of betaFTZ-F1. No significant increase in reporter activity was observed when fusion proteins of the RBCC motif (1-450) and the PHD/bromodomain (891-1133) of Bon were coexpressed with AAD-betaFTZ-F1 E region. In contrast, a 15-fold enhancement was observed in the presence of DBD-Bon (527-700) domain. Analysis of this region has revealed a predicted alpha-helical segment extending from residues 561 to 570. This domain contains an LxxLL consensus sequence, originally identified in the nuclear receptor-interacting domain of TIF1alpha and subsequently found in many other AF-2 mediators. In the presence of AAD-betaFTZ-F1 (E) wild-type, but not AAD-betaFTZ-F1(E)DeltaAF-2 AD-core, residues 561 to 570 of Bon fused to the ERalpha DBD activate the reporter gene ~8-fold above the level of unfused AAD. Thus, Bon contains an LxxLL motif that is sufficient to interact with the LBD of betaFTZ-F1 in an AF-2-integrity-dependent manner. To investigate whether Bon actually binds betaFTZ-F1 through this LxxLL motif, mutations in Bon were generated that eliminate the conserved leucine residues at positions 566 and 567. The replacement of these leucines by alanine residues abolishes the interaction with the LBD of betaFTZ-F1 in yeast. Hence, Bon interacts with the AF-2 of betaFTZ-F1 through an LxxLL motif (Beckstead, 2001).

betaFTZ-F1 plays an important role in the stage-specific response to the prepupal ecdysone pulse by positively regulating the expression of E74A, E75B, BR-C, EDG84A, and E93, and negatively regulating its own expression. Mutant betaFTZ-F1 animals display variable defects in early pupal events such as adult head eversion, leg elongation, and salivary gland cell death. Similar phenotypes are observed in bon mutant pupae (Beckstead, 2001).

The phenotypes associated with betaFTZ-F1^ex17/Df(3L)Cat^Dh104 mutants have been categorized into three lethal pupal classes: 38% die as pharate adults with short malformed legs; 45% undergo head eversion, but arrest early in pupal development; and 17% fail to undergo head eversion, but continue developing into cryptocephalic pharate adults. All betaFTZ-F1 mutants have deformed legs.

Because Bon is able to interact with betaFTZ-F1 in vitro, attempts were made to establish whether Bon interacts with betaFTZ-F1 in vivo. Flies were generated with either bon²⁴¹ or bon⁴⁸⁷ in the Df(3L)Cat^Dh104/betaFTZ-F1^ex17 or betaFTZ-F1^ex17/betaFTZ-F1^ex17 mutant backgrounds and assessed for their effect on betaFTZ-F1 phenotypes. Loss of one copy of bon is able to suppress the phenotypes associated with loss of betaFTZ-F1. In the Df(3L)Cat^Dh104/betaFTZ-F1^ex17 background, partial loss of Bon rescues the majority of mutant animals to pharate adult stages: 87% for bon⁴⁸⁷ and 70% for bon²⁴¹. In the betaFTZ-F1^ex17 homozygotes, partial loss of Bon dramatically increases the number of adult escapers: 72% for bon⁴⁷⁸ and 60% for bon²⁴¹, compared to 31% in a wild-type background. In addition, one mutant copy of bon also strongly suppresses the leg phenotypes associated with loss of betaFTZ-F1. In summary, these data indicate that partial loss of Bon suppresses the phenotypes associated with a partial loss of betaFTZ-F1 (Beckstead, 2001).

betaFTZ-F1^ex17 has been shown to be a hypomorphic allele that is the result of a deletion of a positive regulatory element. Northern analysis has demonstrated that betaFTZ-F1^ex17/betaFTZ-F1^ex17 animals exhibit low levels of betaFTZ-F1 transcripts. It was therefore hypothesized that bon suppression of the betaFTZ-F1^ex17 phenotypes may be the result of betaFTZ-F1 upregulation. To test this hypothesis, Northern analysis was performed on betaFTZ-F1^ex17/+, betaFTZ-F1^ex17/betaFTZ-F1^ex17, and betaFTZ-F1^ex17 bon⁴⁸⁷/betaFTZ-F1¹⁷ staged prepupae and the levels of betaFTZ-F1 expression was estimated. One mutant copy of bon⁴⁸⁷ results in a 1.8- and a 1.9-fold upregulation of betaFTZ-F1 in betaFTZ-F1^ex17 mutants. These results suggest that suppression by bon is at least partially due to the upregulation of betaFTZ-F1 and that Bon seems to play a direct role in repressing betaFTZ-F1 expression. These data appear in contrast to, but are not inconsistent with the general loss of Bon function that affects the transcription of most nuclear receptors negatively. Because betaFTZ-F1 is a downstream effector in the ecdysone pathway, the specificity of the interaction between betaFTZ-F1 and Bon is probably masked in a severe loss of function bon animal (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).

To investigate functional consequences of the Bon-betaFTZ-F1 interaction, the transcriptional activity of betaFTZ-F1 AF-2 was assayed alone or in combination with overexpressed Bon in transiently transfected cells. Bon and the GAL4-betaFTZ-F1(E) derivative were cotransfected into S2 cells together with the GAL4-responsive reporter, 17M-ERE-tk-CAT. GAL4-betaFTZ-F1(E) exerts a trans-stimulation activity that is repressed by the addition of Bon. Taken together, these results provide support for the hypothesis that Bon plays a role in downregulating betaFTZ-F1-dependent transcription (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).

Upon ecdysone binding, the EcR/USP complex upregulates the expression of a group of transcription factors, many of which are nuclear receptors. During this ecdysone regulatory cascade, both induction and repression of transcription are required to regulate the timing and the response to the ecdysone signal. Bon is able to interact with many members of the nuclear receptor family, suggesting it may have a role in multiple steps during metamorphosis and affect expression of many ecdysone regulated genes. For example, DHR3, a key component of the ecdysone response, is required for patterning and integrity of the adult cuticle, and DHR3 mutant clones exhibit a loss of pigmentation, cuticle defects, and missing bristles, similar to a partial loss of Bon. In addition, mutations in betaFTZ-F1, E74B, and BR-C exhibit malformed legs, which are a result of failure in the ecdysone response pathway. Again, very similar defects are observed in bon mutants. Salivary glands in betaFTZ-F1, BR-C, and bon mutant pupae also fail to undergo apoptosis. The ability of bon mutations to cause phenotypes that resemble defects associated with mutations with multiple members of the pathway suggests that Bon is interacting with several members of the pathway at several stages, in agreement with the biochemical observations (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 data provide genetic evidence for the biological relevance of the interaction between Bon and the nuclear receptor betaFTZ-F1. Reduction in the level of Bon, but not the complete loss of Bon, which affects the entire pathway, suppresses the phenotypes associated with a regulatory loss of function mutation of betaFTZ-F1. This suppression is likely to be the result of an increase in the transcription of betaFTZ-F1, suggesting that Bon plays a role in the repression of betaFTZ-F1. Because betaFTZ-F1 represses its own transcription, it is likely that a protein complex containing betaFTZ-F1 and Bon is required for this repression. Removal of a copy of Bon may therefore lead to an up-regulation of betaFTZ-F1 transcription. Although these data appear to contrast the loss of betaFTZ-F1 transcription in bon²⁴¹/bon²⁴¹ mutants, they are not inconsistent. In the bon²⁴¹/bon²⁴¹ mutant background, loss of two copies of bon severely affects the entire ecdysone pathway. This is clearly not the case when one copy of bon is mutated. Therefore, removal of one copy of bon in the betaFTZ-F1 mutant background allows for the detection of protein:protein interactions between betaFTZ-F1 and Bon. Thus the phenotypic suppression and the S2 cell transcription data are in agreement with Bon functioning as a negative regulator of betaFTZ-F1-dependent transcription. It is therefore tempting to speculate by analogy that TIF1alpha may also interact with and inhibit transactivation by nuclear receptors in mammals. A model is favored in which Bon (or TIF1alpha), once recruited to particular regions of chromatin containing acetylated histones via Bon's bromodomain, interacts via Bon's bromodomain LxxLL motif with the AF-2 domain of DNA-bound nuclear receptors. This complex then represses transcription from cognate target genes, possibly via an effect on chromatin structure (Beckstead, 2001).

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).

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.