Ecdysone receptor
Ultraspiracle, a Drosophila RXR homolog, can substitute for RXR in stimulating the DNA binding of receptors for retinoic acid, T3, vitamin D, and peroxisome proliferator activators. These observations led to the search and ultimate identification of the Ecdysone receptor as a Drosophila partner of USP. Together, USP and EcR bind DNA in a highly cooperative fashion. Cotransfection of both EcR and usp expression vectors is required to render cultured mammalian cells ecdysone responsive. By demonstrating that receptor heterodimer formation precedes the divergence of vertebrate and invertebrate lineages, these data underscore a central role for RXR and its homolog USP in the evolution and control of the nuclear receptor-based endocrine system (Yao, 1992). Native EcR and USP are co-localized on ecdysone-responsive loci of polytene chromosomes. Natural ecdysones selectively promote physical association between EcR and USP, and high-affinity hormone binding requires both EcR and USP. Replacement of USP with retinoid X receptor produces heterodimers with distinct pharmacological and functional properties (Yao, 1993). DNA-binding activity of EcR-RXR, the heterodimer of Ecdysone receptor and vertebrate retinoid-X-receptor, is stimulated by either ecdysteroid or 9-cis-retinoic acid, demonstrating that hormone can play a role in heterodimer formation (Thomas, 1993). 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. Such interactions are evolutionarily conserved. DHR38 can compete in vitro against EcR for dimerization with USP and consequently disrupt EcR-USP binding to an Ecdysone receptor response element. This suggests that DHR38 plays a role in the ecdysone response and that more generally NGFI-B type receptors may be able to function as heterodimers with retinoid X receptor type receptors in regulating transcription (Sutherland,1995). 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). Pulses of the steroid hormone ecdysone function as key
temporal signals during insect development, coordinating
the major postembryonic developmental transitions,
including molting and metamorphosis. In vitro studies have
demonstrated that the Ecdysone receptor (EcR) requires an
RXR heterodimer partner for its activity, encoded by the
ultraspiracle locus. usp exerts no
apparent function in mid-third instar larvae, when a
regulatory hierarchy prepares the animal for the onset of
metamorphosis. Rather, usp is required in late third instar
larvae for appropriate developmental and transcriptional
responses to the ecdysone pulse that triggers puparium
formation. The imaginal discs in usp mutants begin to evert
but do not elongate or differentiate; the larval midgut and
salivary glands fail to undergo programmed cell death, and
the adult midgut fails to form. Consistent with these
developmental phenotypes, usp mutants show pleiotropic
defects in ecdysone-regulated gene expression at the larval-prepupal
transition. usp mutants also recapitulate aspects
of a larval molt at puparium formation, forming a
supernumerary cuticle. These observations indicate that
usp is required for ecdysone receptor activity in vivo. They
demonstrate that the EcR/Usp heterodimer functions in a
stage-specific manner during the onset of metamorphosis
and implicate a role for usp in the decision to molt or
pupariate in response to ecdysone pulses during larval
development (Hall, 1998).
In the early stages of Drosophila metamorphosis DHR3 represses the ecdysone induction of early genes turned on by the pulse of ecdysone that triggers metamorphosis. DHR is shown to interact directly with the Ecdysone receptor. The mechanism of DHR3 repression may involve an interaction between the DHR3 and Ecdysone receptor ligand binding domains. DHR also induces ßFTZF1, an orphan nuclear receptor that is essential for the appropriate response to the subsequent prepupal pulse of ecdysone. The DNA binding domain of DHR3, and perhaps sequences NH2-terminal to it, are necessary for the activating function of DHR3. The E75B receptor, which lacks a complete DNA binding domain, inhibits this inductive function by forming a complex with DHR3 on the ßFTZF1 promoter, thereby providing a timing mechanism for ßFTZF1 induction that is dependent on the disappearance of E75B. DHR3 has two high-affinity binding sites approximately 300 base pairs apart, that lie downstream of the transcription start site of ßFTZF1. DHR3 appears to bind as a monomer to these sites, since sequencing and footprinting analysis have uncovered single consensus DHR3 sites at each of these DNA sites. E75B fails to bind DNA in the absence of DHR3. Thus E75B acts like a co-repressor with DHR3, rather than as a competitor with DHR3 for DNA binding; the restricted temporal expression of E75B apparently acts as a precise timer for the onset of ßFTZF1 expression (White, 1997).
A Drosophila corepressor mediates transcriptional silencing of the Ecdysone receptor:Ultraspiracle heterodimer. SMRT-related ecdysone receptor-interacting factor (Smrter), formally known as SANT domain protein , is a large nuclear protein that, surprisingly, shows only limited homology to the vertebrate corepressors SMRT and N-CoR. Nevertheless, the fact that EcR:USP associates with Smrter and Smrter associates with murine Sin3A and Drosophila Sin3A, co-repressors known to form a complex
with the histone deacetylase Rpd3/HDAC (see Drosophila Rpd3), indicates a conserved mechanism underlying transcriptional
repression by vertebrate and invertebrate nuclear receptors. Given the genetic and biochemical evidence that Sin3A associates with Rpd3/HDAC in both yeast and
mammalian cells, and the likelyhood for a similar association in Drosophila, it is expected that Smrter also recruits a
histone deacetylase complex to EcR. The linkage of EcR to Rpd3 is a potential explaination for the role of histone deacetylase in triggering the regression of chromosome
puffs. Yet the presence of Smrter in puffed loci of polytene chromosomes indicates that a complete dissociation of Smrter complex may not be a prerequisite
step for the formation of chromosomal puffs. Rather, other factors, such as coactivators with histone acetyltransferase activity, may play a significant role in triggering
the formation of chromosome puffs (Tsai, 1999 and references).
Using Gal4-DBD fusions, a series of Smrter deletion and truncation constructs were generated to map receptor interaction domains. Their interaction
with the EcR complex was measured in mammalian two-hybrid assays with EcR-vp16 and USP. The EcR harbors a VP16 activation domain, so that association
with Smrter results in activation of a Gal4-responsive luciferase gene. Two independent ecdysone receptor-interacting domains (the ERID1 and the ERID2) were
identified with this assay. ERID1, which maps to aa 1698-1924 of Smrter, confers a 17-fold induction of a reporter gene in the presence of EcR-vp16 and USP. ERID2 maps to aa 2951-3038 and, along with EcR-vp16 and USP, produces an 8-fold induction of the reporter gene. ERID2, but not ERID1, is
located within the original E52 clone. The inclusion of regions flanking ERID1 (aa 1698-2063) and ERID2 (aa 2094-3040) or ERID2 (aa 2929-3181) increases the
reporter activities by several-fold, although these additional regions possess no autonomous EcR-interacting activity. Both ERID1 and ERID2 display a dramatic
preference to bind the EcR:USP heterodimer and to dissociate from the EcR:USP when ligand is added. Interestingly, vertebrate retinoic X receptor (RXR), the
mammalian homolog of USP, fails to substitute for USP in potentiating the interaction of EcR with Smrter. This result further strengthens the
notion that EcR:USP is a preferred binding complex for Smrter, since the EcR:RXR complex requires ligand to be stabilized while the formation of EcR:USP
dimer is independent of ligand binding (Tsai, 1999).
Since cell culture results leave open the question of whether the interaction between Smrter and the EcR complex is direct, pull-down experiments were
conducted with GST fusions of ERID1 and ERID2, mixed with either 35S-labeled EcR or 35S-labeled USP. GST-ERID1 (aa 1698-2063) and GST-ERID2 (aa 2951-3038), but not GST alone, both pull down labeled EcR, whereas little interaction is found
between USP and any of the three GST proteins. In addition, the pull-down complex is disrupted by the addition of hormone when USP is present.
These in vitro results establish that Smrter and EcR may interact directly. In vivo interference assays were used to assess whether ERID1 and ERID2 as well as SMRT bind similar regions within the EcR. In this experiment, Gal4 fusions
encoding either ERID1 or ERID2 were transfected along with plasmids encoding EcR:USP heterodimers. A test was then carried out to see whether coexpression of excess
nuclear-targeted ERID1, or ERID2, or c-SMRT interferes with or reduces reporter activity. Interaction between each Gal4-ERID fusion
and EcR-vp16:USP is significantly decreased by both ERIDs and c-SMRT. Interestingly, a more prominent effect is observed in experiments when
Gal4-ERID1 (aa 1698-2063) is challenged by ERID2, and, conversely, a more efficient competition is achieved by ERID1 to Gal4-ERID2 (aa 2094-3181).
Together, these results suggest that ERID1, ERID2, and c-SMRT may bind similar or overlapping surface(s) in EcR (Tsai, 1999).
Experiments indicating that EcR A483T disrupts the interaction with SMRT, led to experiments to see whether this mutation also severs its association
with ERID1 and 2. Gal4 fusions harboring either ERID1 or ERID2 were examined for their interaction with wild-type EcR or
with EcR A483T in the presence of vp16-USP. In both cases, no significant induction of reporter was observed in cells transfected with the EcR mutation, A483T,
in either the presence or absence of ligand, confirming that A483 of EcR represents a common target for corepressor binding (Tsai, 1999).
The steroid hormone 20-hydroxyecdysone coordinates the stages of Drosophila development by activating a nuclear receptor heterodimer consisting of the
ecdysone receptor, EcR, and the Drosophila RXR receptor, USP. EcR/USP DNA binding activity requires activation by a chaperone heterocomplex
like that required for activation of the vertebrate steroid receptors, but not previously shown to be required for activation of RXR heterodimers. Six proteins normally
present in the chaperone complex were individually purified and shown to be sufficient for this activation. Two of the six (Hsp90 and Hsc70) are
required in vivo for ecdysone receptor activity, and EcR is shown to be the primary target of the chaperone complex (Arbeitman, 2000).
The results show that the generation of a fully functional ecdysone receptor is a multistep process. The ability to bind ligand is not inherent to EcR or USP alone but
arises by an interaction between these two proteins that does not require accessory factors. Such a naive heterodimer formed from purified receptors is
different from the purified steroid receptors for progesterone (PR) or glucocorticoid (GR), which can not bind ligand or DNA prior to chaperone activation. This naive heterodimer was transformed to the fully functional EcR/USP heterodimer (defined here by its ability to bind both
ligand and EcRE DNA) by exposure to six purified chaperones and an ATP regenerating system. This is an artificial system in that four of the six purified
chaperones (Hip, Hop, FKBP52, and p23) are from humans rather than Drosophila. It is also redundant, in that removal of any one of the six decreases the yield but
does not eliminate activation. The important point at this stage is not what combination of these chaperones is most efficient for the in vitro transformation of the naive
heterodimer to the fully active state; rather, it is that such a transformation requires only chaperones and an ATP regenerating system. The data do indicate that the two Drosophila chaperones Hsp90 and Hsc70 are required for activation of the EcR/USP heterodimer in vivo (Arbeitman, 2000).
Two additional characteristics of the EcR/USP activation reaction deserve emphasis. One is that activation to the fully functional state can occur in the absence of
ecdysone response element DNA since such a fully functional heterodimer has been produced and purified under this condition. The second is the finding that
EcR is the target of the molecular chaperone-containing heterocomplex.
Given that almost all the data presented in this paper concerns events that occur after the synthesis of all the polypeptides involved in the formation of the fully
functional ecdysone receptor, the following model is limited to events after that completion. It is assumed that the USP polypeptide folds appropriately into a relatively
stable configuration that is not further stabilized by chaperones. By contrast, the EcR polypeptide folds into an unstable configuration easily subject to irreversible
unfolding or protease degradation. This postulated difference in the behavior of the two polypeptides derives in large part from the observation that overexpression
of each in cultured cells yields much more USP than EcR. Similar observations have been reported for RAR and RXR in E. coli, where
RAR is the unstable entity. It is assumed that the unstable EcR interacts with appropriate Drosophila chaperones including Hsp90 and Hsc70, that stabilize EcR in a configuration
appropriate for formation of EcR/USP heterodimers capable of binding EcRE DNA sequences. It is postulated that formation of the active heterodimer is
accompanied by dissociation of the chaperones from EcR, accounting for the isolation of an ecdysone receptor response element (EcRE) DNA-binding form of the EcR/USP heterodimer. USP binding is
postulated to stabilize EcR in a DNA binding configuration, which is enhanced by chromosomal EcRE binding, and can be further enhanced by the binding of EcR to
other chromatin binding proteins (Arbeitman, 2000).
Some members of nuclear hormone receptors, such as the thyroid hormone receptor (TR), silence gene expression in the absence of the
hormone. Corepressors, which bind to the receptor's silencing domain, are involved in this repression. Hormone binding leads to
dissociation of corepressors and binding of coactivators, which in turn mediate gene activation.
Alien (Drosophila homolog: Alien) is a novel corepressor. Alien interacts with TR only in the absence of hormone. Addition of thyroid hormone leads to dissociation of
Alien from the receptor, as shown by the yeast two-hybrid system, glutathione S-transferase pull-down, and coimmunoprecipitation
experiments. Reporter assays indicate that Alien increases receptor-mediated silencing and that it harbors an autonomous silencing function. Immune staining shows
that Alien is localized in the cell nucleus. Alien is a highly conserved, showing 90% identity between human and Drosophila proteins. Drosophila Alien shows similar
activities in that it interacts in a hormone-sensitive manner with TR and harbors an autonomous silencing function. Specific interaction of Alien is seen with Drosophila nuclear hormone receptors, such as the ecdysone receptor and Seven-up, the Drosophila homolog of COUP-TF1, but not with retinoic acid receptor, RXR/USP, DHR 3, DHR 38, DHR 78, or DHR 96. These properties, taken together, show that Alien has the characteristics of a corepressor. Thus, Alien represents a member of a novel class of corepressors specific for selected members of the nuclear hormone receptor superfamily (Dressel, 1999).
To gain insight into the mechanism of Alien-mediated gene repression, tests were performed to see whether the silencing function of Alien
is based on complex formation with the known corepressors SMRT and N-CoR or with SIN3A. The ability of full-length human Alien (h-Alien) to interact with
either full-length SMRT, the C terminus of N-CoR, or mouse SIN3A was tested in the yeast two-hybrid system. No interaction of h-Alien with the
SMRT/N-CoR class of corepressors was detected. Interestingly, a strong interaction of Alien with SIN3A, a protein shown to be part of a deacetylase
complex, was detected. A specific Alien-SIN3A interaction was observed in GST pull-down experiments with GST-h-Alien and in vitro-translated SIN3A. To verify this interaction, coimmunoprecipitations were performed. The anti-Alien antibody immunoprecipitates SIN3A from HeLa extracts. This indicates that Alien is mediating silencing, at least in part, by recruiting a factor known to be involved in deacetylase activity (Dressel, 1999).
To further investigate the association of Alien with a deacetylase activity, trichostatin A (TSA), a specific inhibitor of histone deacetylases, was used. CV1 cells were transfected with Gal-h-Alien or Gal-N-CoR and treated with TSA for 8 h. Addition of TSA reduces silencing by both Alien and N-CoR, a protein known to repress transcription by recruitment of deacetylase activity. Taken together, this supports a role for deacetylase activity in Alien-mediated silencing. Since the SMRT/N-CoR class of corepressor also interacts with SIN3A, the observed TR-Alien-SIN3A interaction may strengthen the recruitment of a deacetylase complex to genes regulated by selected NHRs. Thus, these data indicate that one mechanism by which Alien confers silencing may, at least to some extent, be based on recruitment of deacetylase activity via interaction with SIN3A (Dressel, 1999).
Steroid hormones fulfil important functions in animal development. In Drosophila, ecdysone triggers molting and metamorphosis through its effects on gene expression. Ecdysone works by binding to a nuclear receptor, EcR, which heterodimerizes with the retinoid X receptor homolog Ultraspiracle. Both partners are required for binding to ligand or DNA. Like most DNA-binding transcription factors, nuclear receptors activate or repress gene expression by recruiting co-regulators, some of which function as chromatin-modifying complexes. For example, p160 class coactivators associate with histone acetyltransferases and arginine histone methyltransferases. The Trithorax-related gene of Drosophila encodes the SET domain protein TRR. TRR is a histone methyltransferase capable of trimethylating lysine 4 of histone H3 (H3-K4). trr acts upstream of hedgehog (hh) in progression of the morphogenetic furrow, and is required for retinal differentiation. Mutations in trr interact in eye development with EcR, and EcR and TRR can be co-immunoprecipitated on ecdysone treatment. TRR, EcR and trimethylated H3-K4 are detected at the ecdysone-inducible promoters of hh and BR-C in cultured cells, and H3-K4 trimethylation at these promoters is decreased in embryos lacking a functional copy of trr. It is proposed that TRR functions as a coactivator of EcR by altering the chromatin structure at ecdysone-responsive promoters (Sedkov, 2003).
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 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 bon21B/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 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).
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 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).
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).
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).
Drosophila NURF is an ISWI-containing ATP-dependent chromatin remodeling complex that regulates transcription by catalyzing nucleosome sliding. To determine in vivo gene targets of NURF, whole genome expression analysis was performed on mutants lacking the NURF-specific subunit NURF301. Strikingly, a large set of ecdysone-responsive targets is included among several hundred NURF-regulated genes. Null Nurf301 mutants do not undergo larval to pupal metamorphosis, and also enhance dominant-negative mutations in ecdysone receptor. Moreover, purified NURF binds EcR in an ecdysone-dependent manner, suggesting it is a direct effector of nuclear receptor activity. The conservation of NURF in mammals has broad implications for steroid signaling (Badenhorst, 2005).
The ligand dependency of the NURF-EcR interaction implies that NURF functions as a coactivator for the ecdysone receptor. Like other NRs, EcR has two transcriptional activation function (AF) domains, the conserved AF2 located within the ligand-binding domain and isoform-specific AF1s at the N terminus. NURF is able to pull down, in a ligand-dependent manner, a minimal construct that contains the entire AF2 domain. However, inactivation of AF2 either by C-terminal truncation, or by mutation of F645 (a conserved residue critical for interaction of mammalian NRs with coactivators) blocks interaction with NURF. These results extend the repertoire of transcription factors shown to interact with NURF. These data indicate that purified NURF is a coactivator that binds to the AF2 region of EcR, in addition to previously demonstrated interactions with the GAGA factor, GAL4-VP16, and HSF (Badenhorst, 2005).
To confirm further that NURF functions in ecdysone signaling in vivo, genetic interactions between EcR and components of NURF were assayed. A dominant-negative EcR mutant (EcR-F645A) was expressed in follicle cells in the developing egg chamber of female flies. EcR-F645A is defective in transcriptional activation and interferes with ecdysone signaling, leading to a number of embryo defects, including malformed dorsal appendages. Decreasing the titer of a coactivator has been shown to enhance this phenotype. Mutation of a single copy of any of three NURF subunits increases the frequency and severity of these aberrations, consistent with NURF functioning as a coactivator for the ecdysone receptor (Badenhorst, 2005).
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