Ecdysone receptor


Protein Interactions

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).

Bonus interacts with hormone receptors and inhibits transcription

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

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

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

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

To 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).

Structure of the heterodimeric ecdysone receptor DNA-binding complex

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

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

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

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

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

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

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

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

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

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

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

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

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

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

NURF301 interacts with the Ecdysone receptor

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).

To characterize the physiological function of Drosophila NURF, a series of EMS-induced lesions were generated in the gene encoding the largest NURF subunit, Nurf301. Focus was placed on NURF301, since the large subunit is the only NURF-specific subunit and is obligatory for the assembly of NURF. Twelve EMS-induced lesions were isolated in Nurf301, all of which encode truncated NURF301 products. In addition to the previously reported male X-chromosome and melanotic tumor phenotypes, null Nurf301 mutants that truncate before the putative WAKZ motif displayed a non-pupariating phenotype. Mutants exhibit a slight developmental delay relative to heterozygous siblings, but larvae do not form pupae and can continue to survive in culture for up to 2 wk. In the few cases where white prepupae form, these retain an elongated larval form and fail to evert the anterior spiracles completely. Null Nurf301 mutants appear to undergo the larval molts normally. However, embryos contain a large dowry of maternally loaded Nurf301 mRNA, suggesting that Nurf301 mutants have sufficient NURF301 protein to support initial larval development, including the molt from L2 to L3 (Badenhorst, 2005).

In contrast to null Nurf301 alleles, Nurf301 mutations that truncate NURF301 after the WAKZ motif (Nurf3014, Nurf30110, Nurf30111, and Nurf30112) do pupariate, indicating that N-terminal fragments of NURF301 that extend beyond the WAKZ domain support pupariation. This agrees with in vitro data showing that these fragments contain sites of interaction with the other three NURF subunits and may be able to coordinate the assembly of a NURF complex. Heteroallelic combinations of these alleles survive to adult stages. However, flies exhibit developmental abnormalities and are sterile indicating that the C-terminal regions of NURF301, while dispensable for pupariation, have functions (Badenhorst, 2005).

To define gene targets of NURF that are required for pupariation, whole genome expression profiles of null Nurf301 mutant (Nurf3012/Nurf3018) third instar larvae were compared with those of wild-type larvae. A set of 477 genes was identified for which there was a statistically significant change in expression between mutant and wild-type samples. Of these, 274 genes were decreased at least threefold in Nurf301 mutants, while 203 exhibited at least threefold elevated levels of expression in the mutant samples, suggesting that NURF may function both as an activator and repressor of transcription (Badenhorst, 2005).

Classification according to gene ontology of the 274 genes that require NURF301 for expression revealed that a sizeable number correspond to ecdysone target genes. An additional 30 of the 274 genes have no gene ontology classifications, but are known to be highly expressed at the larval/pupal transition and may be additional ecdysone targets. Taken together, this indicates that NURF is required for expression of targets of the ecdysone receptor. Similar classifications of the genes that show increased expression in Nurf301 mutants indicated that many are immune-related genes (Badenhorst, 2005).

An involvement of NURF in EcR signaling agrees well with the pupariation defects observed in Nurf301 mutants, which resemble the phenotypes of mutants in key downstream regulatory targets of the ecdysone receptor. To further confirm that NURF is required for ecdysone signaling, the expression in Nurf301 mutants was examined of all known ecdysone targets that had been annotated and included in the Affymetrix microarrays. In Nurf301 mutants, expression of a significant majority of these genes was reduced by greater than fivefold. The few genes for which there were no relative differences in expression are genes that are predominantly expressed at the prepupal/pupal transition (after the point sampled; for example, Edg84A and Eip63F-1 or those whose induction would be difficult to detect in whole animals because of a high background of tissues in which expression is constitutive or even repressed by ecdysone -- Eip71CD (Eip28/29) and Eip55E (Eip40) (Badenhorst, 2005).

Remarkably, although approximately equal numbers of genes exhibit elevated or reduced expression in Nurf301 mutants, among known ecdysone-responsive genes, Nurf301 mutation produced only decreases in expression. This suggests that NURF functions specifically as a coactivator in the ecdysone response. Finally, microarray analysis shows that transcript levels of known ecdysone synthetic enzymes are unchanged in Nurf301 mutants, indicating that NURF does not indirectly influence expression of responsive genes by affecting ecdysone levels (Badenhorst, 2005).

Next, the altered expression of selected ecdysone targets was validated by analyzing transcript levels using Northern analysis and semiquantitative RT-PCR. Northern blotting confirms that Sgs1, Sgs3, and Eig71Ee are not expressed in null Nurf301 mutants (Nurf3012/Nurf3013 allelic combination) or Iswi mutants that lack the catalytic subunit of NURF. Similarly, semiquantitative RT-PCR confirms that expression of Eig71Ea, ImpE2, and Fbp1 is reduced in null Nurf301 or Iswi mutants. In contrast, transcript levels of EcR and usp are unchanged in Nurf301 mutants, indicating that the failure to express ecdysone target genes is not an indirect effect of reduced levels of the ecdysone receptor. As expected, allelic combinations with Nurf301 mutants that truncate after the putative WAKZ motif, and that are able to pupariate, do express ecdysone target genes (Badenhorst, 2005).

Lastly, as an additional demonstration that NURF is required for Sgs3 transcription, expression was examined of an Sgs3-GFP reporter transgene in null Nurf301 mutant animals. Sgs3-GFP is expressed in the salivary glands of Nurf3012/+ heterozygous animals but is not expressed in homozygous mutant Nurf3012 animals (Badenhorst, 2005).

The failure of ecdysone-responsive genes to be expressed in NURF mutants indicates that NURF is a coactivator of the Drosophila ecdysone receptor (EcR). Thus, whether NURF could physically interact with EcR was tested. A pull-down assay, in vitro-translated EcR isoforms, EcR-A and EcR-B2, interacted with Flag-tagged recombinant NURF. These interactions were dependent on the presence of added ecdysone. No pull-down was observed in the absence of 20-hydroxyecdysone (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. It was observed that 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 assessed. 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. It was observed that 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).

These results provide one of the first demonstrations of a biological requirement for an ISWI-containing chromatin remodeling enzyme (NURF) in steroid hormone-dependent transcriptional activation. To date, most studies of ATP-dependent chromatin remodeling during NR transactivation have focused on the SWI/SNF family of chromatin remodeling complexes. It has been shown that SWI/SNF enzymes are required for activation by the retinoid receptor heterodimer (RAR/RXR), glucocorticoid receptor, and estrogen receptor. Moreover, interaction studies reveal that SWI/SNF remodeling complexes can be targeted to NRs through interactions with the noncore subunits BAF250, BAF57, and BAF60a (Badenhorst, 2005).

However, there are many families of ATP-dependent chromatin remodeling complexes, including those based on the SWI2/SNF2, ISWI, INO80, and CHD1 catalytic subunits. Each group of remodeling enzymes has distinct mechanisms of operation. For example, SWI/SNF enzymes increase chromatin accessibility by DNA looping, whereas ISWI enzymes induce nucleosome sliding and the SWR1 category catalyzes histone exchange. It is important to determine whether NRs exclusively employ the SWI2/SNF2 branch or use a much wider repertoire of remodeling enzymes to exert their functions (Badenhorst, 2005).

This study provides evidence that the ISWI-containing chromatin remodeling enzyme NURF is a coactivator of a Drosophila NR, the ecdysone receptor. In addition to previous demonstrations of direct interactions between NURF and the GAGA factor and HSF, this study shows that purified NURF binds to EcR in an ecdysone-dependent manner, suggesting it is a direct effector of NR activity. These conclusions are broadly consistent with two previous studies that indicated that ISWI complexes are required for NR-dependent transactivation in vitro (Badenhorst, 2005).

Inspection of the NURF301 coding sequence, and of the corresponding rat and human homologs, reveals that NURF301 contains two conserved NR (LxxLL) boxes. These motifs can mediate interaction between NRs and transcriptional coactivators and suggest a mechanism by which NURF can interact with, and be recruited by, EcR. Despite numerous efforts, no suitable antibody has been isolated that allows direct visualization of NURF recruitment to ecdysone-responsive promoters. However, chromatin immunoprecipitation (ChIP) using anti-ISWI antibodies shows that an ISWI-containing complex binds to the ecdysone response element of the hsp27 promoter. This ISWI ChIP signal is lost in Nurf301 mutants, suggesting that it is due to NURF recruitment (Badenhorst, 2005).

Analyses of Drosophila Iswi mutants have provided critical insights into the functions of ISWI-containing chromatin remodeling enzymes. These studies provided the first demonstration that ISWI chromatin remodeling complexes are required to maintain male X-chromosome morphology, for homeotic gene expression and metamorphosis. However, Drosophila ISWI is the catalytic ATPase subunit of at least three complexes: ACF, CHRAC, and NURF. As such, analysis of Iswi mutants alone does not allow the relative functions of these complexes to be discriminated. By focusing investigations on the NURF-specific subunit NURF301, it was possible to define specific functions of NURF. The pupariation defects seen in Nurf301 mutants, and the reduced ecdysone target gene expression noted in Nurf301 mutants, highlights the critical function of NURF in EcR-dependent activation. As expected, defects seen in Nurf301 mutants are also observed in Iswi mutants. However, mutations in subunits specific to ISWI complexes other than NURF, for example, the ACF1 subunit of ACF and CHRAC, do not affect EcR function. Acf1 null mutants are semi-lethal but are able to produce viable adults (Badenhorst, 2005).

These studies on Drosophila NURF have important implications for NR function in mammals. Homologs of ISWI and the large subunit NURF301 exist in mouse and humans. Moreover, human NURF has been purified and exhibits identical biochemical properties as Drosophila NURF. The human homolog of a Drosophila NURF target, engrailed, is also a target of human NURF. Given this conservation of function, it will be of interest to determine if gene targets of mammalian NRs related to Drosophila EcR also require NURF for expression (Badenhorst, 2005).

dDOR is an EcR coactivator that forms a feed-forward loop connecting insulin and ecdysone signaling

Mammalian DOR (for 'Diabetes- and Obesity Regulated') was discovered as a gene whose expression is misregulated in muscle of Zucker diabetic rats. Because no DOR loss-of-function mammalian models are available, this study analyzed the in vivo function of DOR by studying flies mutant for Drosophila DOR (dDOR). dDOR is a novel coactivator of ecdysone receptor (EcR) that is needed during metamorphosis. dDOR binds EcR and is required for maximal EcR transcriptional activity. In the absence of dDOR, flies display a number of ecdysone loss-of-function phenotypes such as impaired spiracle eversion, impaired salivary gland degradation, and pupal lethality. Furthermore, dDOR knockout flies are lean. dDOR expression is inhibited by insulin signaling via FOXO. This work uncovers dDOR as a novel EcR coactivator. It also establishes a mutual antagonistic relationship between ecdysone and insulin signaling in the fly fat body. Furthermore, because ecdysone signaling inhibits insulin signaling in the fat body, this also uncovers a feed-forward mechanism whereby ecdysone potentiates its own signaling via dDOR (Francis, 2010).

Thyroid hormone receptor (TR) is an important regulator of development and metabolism in animals. TR is a type II nuclear hormone receptor (NR). It resides in the nucleus and binds DNA regardless of ligand binding, and it heterodimerizes with retinoid X receptor (RXR). In the absence of ligand, TR is complexed with corepressors to inhibit transcription, whereas in the presence of ligand, it binds coactivators and activates transcription. One recently discovered TR coactivator is DOR. DOR was first identified as a gene that is downregulated in muscle of diabetic rats. DOR was then shown to have two functions. It acts as a coactivator of thyroid hormone receptor TRα1, binding TRα1 and impacting its transcriptional activity. Furthermore, DOR has a second life outside the nucleus, as a regulator of autophagy. Together, these data implicate DOR as a regulator of NR function and of metabolism. However, no DOR mutant animals have yet been reported, and the in vivo function of DOR remains to be studied (Francis, 2010).

Drosophila has 18 nuclear receptors, including ecdysone receptor (EcR). EcR shares many commonalities with type II NRs, in that it heterodimerizes with the fly RXR homolog USP, binds DNA constitutively, complexes with either coactivators or corepressors depending on its state of ligand binding, and can form a functional complex with mammalian RXR. The EcR/USP complex senses and responds to the hormone 20-hydroxyecdysone (20E) to regulate developmental timing and metabolism. 20E triggers all developmental transitions, such as the molts from one larval stage to the next, and many events occurring during metamorphosis. These include termination of larval feeding, apoptosis, and elimination of larval salivary glands and larval fat body, as well as many morphological changes in tissues that will give rise to the adult fly. Several EcR corepressors and coactivators have been identified and characterized, including Alien, SMRTER, bonus, Trithorax-related gene (TRR), Taiman, and rigor mortis. However the coactivator(s) of EcR required for proper pupal development and metamorphosis remain to be described (Francis, 2010).

Interestingly, crosstalk has recently come to light between ecdysone signaling and insulin signaling, which regulates the growth and metabolism of animals. Ecdysone regulates insulin signaling and vice versa. In particular, in the fat body of the fly, ecdysone signaling inhibits PI3K activity and thereby insulin signaling, suggesting an antagonistic relationship between these two hormonal signaling pathways. The molecular mechanisms underlying these regulatory events, however, are not fully understood (Francis, 2010).

In order to study the function of DOR in an in vivo animal model, the Drosophila genome was searched for homologs of human DOR (hDOR). A BLAST search through all predicted Drosophila proteins with the sequence of hDOR yielded CG11347 as the top hit, which was rename Drosophila DOR (dDOR) (Francis, 2010).

The dDOR locus is predicted to encode six different transcripts, giving rise to three different polypeptides. The -RA, -RB, -RD, and -RE isoforms encode a 387 amino acid protein hereafter referred to as DORlong, whereas the -RC isoform encodes a shorter protein, of 273 amino acids, referred to as DORshort. The -RF isoform encodes an even shorter protein similar to DORshort but lacking 44 amino acids at the N terminus. While performing RT-PCR with oligonucleotides specific for the long isoform, the presence of two differently sized PCR products was detected. Sequencing revealed that one of the products corresponded to the predicted 'long' isoform. The second product corresponded to an unannotated isoform consisting of the 'long' isoform plus a 90 bp extension of the third exon, resulting from use of an alternate splice donor. As a result, 30 amino acids are inserted in the middle of the dDORlong protein. Contained in these 30 amino acids is the sequence FENLL, which is similar to the LXXLL nuclear-receptor-interacting motif found in nuclear receptor coactivators. This FENLL sequence aligns to the transactivation domain motif of human DOR (LEDLL) when the two proteins are aligned to each other. This isoform is referred to as dDORFENLL. The domain of dDORFENLL surrounding the FENLL sequence has 75% identity and 85% homology to human DOR. Three isoforms of dDOR were studied in this work (Francis, 2010).

In order to measure the relative abundance of the three isoforms in vivo, quantitative RT-PCR was performed with isoform-specific primers on RNA extracted from animals of various stages of development. The most abundant isoform is the long one, followed by the FENLL isoform (roughly half the level of the long isoform), whereas the short isoform is expressed at comparatively low levels. This relative expression of the three isoforms is also observed in fat body of wandering third-instar larvae whereas in fat body of early pupae the FENLL isoform strongly predominates. Indeed, the FENLL isoform is highly enriched in fat body of early pupae when compared to the rest of the body (Francis, 2010).

Because expression of human DOR is misregulated in rats, via an unknown mechanism, upon development of diabetes, it was asked whether expression of Drosophila DOR is also regulated by nutritional conditions. Given that the FENLL isoform of dDOR is responsible for the metabolic defects of dDOR mutants, attention was focused on the FENLL isoform. Third-instar larvae were either fasted or fed for 18 hr and then assayed dDORFENLL mRNA levels in fat body by quantitative RT-PCR. When control larvae were fasted, dDORFENLL expression in fat body increased > 2-fold. One important signaling pathway that is inhibited upon fasting is insulin. It was therefore asked whether dDORFENLL expression is inhibited by insulin, because this would explain its upregulation upon fasting. Explanted fat bodies were tested in the presence or absence of 5 μg/ml insulin and dDORFENLL expression levels were assayed by quantitative RT-PCR. In the presence of insulin, dDORFENLL expression decreased by 73%. dDORFENLL expression levels also decreased by 59% in S2 cells treated with 1 μM insulin for 2 hr (Francis, 2010).

One transcription factor mediating much of the transcriptional output of the insulin pathway is FOXO. FOXO activity is suppressed by insulin signaling. Whether regulation of dDORFENLL expression is mediated by FOXO was tested by studying animals containing the FOXO21/25 null allele combination. FOXO21/25 mutants were starved and it was found that the fasting-induced upregulation of dDORFENLL expression in fat body was strongly impaired, indicating that this transcriptional regulation is FOXO dependent. The transcriptional regulation of dDORFENLL is analogous to that of a canonical FOXO target gene, 4E-BP. 4E-BP expression is suppressed by insulin in vivo in fat bodies and increases in vivo in fat body upon fasting of wild-type animals but does not increase upon fasting of FOXO mutant animals. It was therefore asked whether dDOR is also a direct transcriptional target of FOXO. In Drosophila, FOXO targets sites are preferentially located within 1 kb of the target promoter. The dDOR promoter region was screened and a perfect consensus FOXO binding site (GTAAACAA) was found 230 nt upstream of the transcription start site of the –RA and –RB transcripts. To test whether FOXO binds this site in vivo, chromatin immunoprecipitation (ChIP) of endogenous FOXO from third-instar larvae was performed. Two negative controls were performed: a mock ChIP using preimmune serum from wild-type animals, and a ChIP using anti-FOXO antibody from FOXO21/25 null mutant animals. Quantitative PCR (qPCR) on the immunoprecipitated material revealed that the promoter region of 4E-BP, an established direct target of FOXO, was strongly enriched in the FOXO ChIP from wild-type animals, but not in the negative controls. Strikingly, the promoter region of dDOR-RA/B was also strongly enriched in the FOXO ChIP but not in the negative controls, indicating that FOXO binds the dDOR promoter in vivo. As a negative control, the genomic region of mir-278 was not enriched in the FOXO ChIP. Together, these data indicate that expression of dDORFENLL is inhibited by insulin signaling as a direct target of FOXO, and identify a molecular mechanism by which insulin signaling inhibits ecdysone signaling in the fat body. Because dDOR is involved in linking nutrient signaling to EcR signaling, whether dDOR mutants have impaired fitness upon nutrient deprivation was tested. Upon removal of food (but not water), dDOR knockout animals died more rapidly than controls (Francis, 2010).

Thus dDOR functions as a novel coactivator of the ecdysone receptor that plays an important role during metamorphosis. Clearly not all EcR functions are impaired in DOR mutants. For instance, very little lethality is seen during larval stages of development, indicating that larval molts are occurring properly. It is possible that different EcR coactivators are important for different aspects of EcR signaling, for instance with rigor mortis plays an important role in the regulation of larval molts. Alternatively, because induction of EcR target genes is reduced but not completely eliminated in dDOR knockout animals, this could reflect the differential sensitivity of various biological processes to the degree of EcR activation. Future work may shed more light on this issue. Interesting in this context is that it was possible to rescue the lethality of DOR knockouts by feeding 20E. This suggests that either DOR knockouts also have low ecdysone titers due to impaired expression of E75A, which is involved in an ecdysone feed-forward production pathway, or because the elevated ecdysone titers achieved by supplying exogenous 20E allow other coactivators to compensate for DOR loss of function (Francis, 2010).

This work identifies a new link between ecdysone signaling and insulin signaling. It was previously known that ecdysone signaling inhibits insulin signaling in the fat body. This study shows, conversely, that insulin signaling also inhibits ecdysone signaling. When insulin signaling is high, FOXO activation is low and dDOR expression is low. Conversely, when insulin signaling drops, this allows FOXO to become active, resulting in elevated levels of dDOR expression and maximal activation of EcR target genes. In sum, this study found that there is a mutual antagonistic relationship between insulin signaling and ecdysone signaling in the fat body, possibly creating a system with two equilibrium states -- high ecdysone/low insulin and low ecdysone/high insulin. This makes biological sense because insulin plays an anabolic role in the fat body, whereas ecdysone plays a catabolic role, encouraging lipid mobilization and autophagy. By identifying dDOR as a direct FOXO target, this study has shed light on the molecular mechanism by which part of this antagonistic relationship is achieved (Francis, 2010).

A second consequence of the regulation of dDOR by FOXO is the creation of a feed-forward regulatory mechanism. When ecdysone signaling is activated, it inhibits insulin signaling and activates FOXO, causing increased expression of dDOR. This results in potentiation of the ecdysone signal. This type of mechanism may be important for the dramatic activation of the ecdysone pathway at the end of larval development. Indeed, ecdysone signaling has several autoregulatory positive feedback loops, including EcR-dependent transcription of the EcR gene and downregulation of a microRNA, miR-14, which inhibits EcR expression (Francis, 2010).

DOR was first identified as a gene whose expression is aberrant in Zucker diabetic rats. Until DOR knockout mice are analyzed, it is possible that this aberrant regulation is either a cause or a consequence of the diabetes. Because dDOR knockout flies have reduced triglyceride and elevated glycogen stores, it is tempting to speculate that aberrant DOR expression in mammals might actually cause metabolic defects and not simply be a consequence of them. Although DOR expression was downregulated in muscle of diabetic rats, this study found a 2-fold increase in hDOR expression in adipose tissue of type 2 diabetic patients. This indicates that regulation of DOR expression -- and hence the effect on metabolism -- in conditions of metabolic disease in mammals is likely to be tissue specific and complex. The reduction in triglycerides in dDOR knockout flies is also interesting in light of the antagonistic relationship between ecdysone signaling and insulin signaling in the fly. Previous work has shown that flies with systemically reduced insulin signaling have elevated triglyceride levels. Therefore, the leanness of dDOR knockouts would be consistent with increased systemic insulin signaling in dDOR knockout animals (Francis, 2010).

Intriguingly, dDOR shares a number of features with its mammalian homolog. Like hDOR, dDOR functions as a nuclear hormone coactivator. Whereas hDOR binds TRα1, dDOR binds EcR. TRα1 and EcR are similar in that they both form heterodimeric complexes with RXR/USP. In fact, EcR can form a functional complex with the human USP homolog RXR in mammalian cells. Furthermore, EcR and TRα1 both play catabolic roles in some contexts. For instance, ecdysone signaling induces autophagy and lipid mobilization in the fat body and programmed cell death in salivary glands during metamorphosis. Likewise, thyroid hormones increase basal metabolic rates, induce fat mobilization, and enhance fatty acid oxidation. A second similarity between dDOR and hDOR is that both are transcriptionally regulated by nutritional inputs. DOR expression is misregulated in diabetic rats, whereas dDOR expression changes depending on whether the animals are feeding or fasting. Because this study found that regulation of dDOR expression is insulin and FOXO dependent, this raises the possibility that the transcriptional effect on DOR in diabetic rats may also be insulin dependent. A third similarity is that both hDOR and dDOR have two separable functions -- as a nuclear hormone receptor coactivator, and as a regulator of autophagy. This makes particular biological sense within the context of the fat body, where ecdysone signaling induces autophagy during metamorphosis. Therefore, the dual functions of dDOR work in parallel, both by potentiating ecdysone signaling and by interacting with the autophagy proteins Atg8a/b (Francis, 2010).

In sum, this work discovers dDOR as a novel EcR coactivator required during fly metamorphosis. Furthermore, it identifies dDOR as a novel component of a gene regulatory network integrating ecdysone and insulin signaling to regulate fly development and metabolism (Francis, 2010).

Histone recognition and nuclear receptor co-activator functions of Drosophila Cara Mitad, a homolog of the N-terminal portion of mammalian MLL2 and MLL3

MLL2 and MLL3 histone lysine methyltransferases are conserved components of COMPASS-like co-activator complexes. In vertebrates, the paralogous MLL2 and MLL3 contain multiple domains required for epigenetic reading and writing of the histone code involved in hormone-stimulated gene programming, including receptor-binding motifs, SET methyltransferase, HMG and PHD domains. The genes encoding MLL2 and MLL3 arose from a common ancestor. Phylogenetic analyses reveal that the ancestral gene underwent a fission event in some Brachycera dipterans, including Drosophila species, creating two independent genes corresponding to the N- and C-terminal portions. In Drosophila, the C-terminal SET domain is encoded by trithorax-related (trr), which is required for hormone-dependent gene activation. This study identified the cara mitad (cmi) gene (FlyBase name: Lost PHDs of trr), which encodes the previously undiscovered N-terminal region consisting of PHD and HMG domains and receptor-binding motifs. The cmi gene is essential and its functions are dosage sensitive. CMI associates with TRR, as well as the EcR-USP receptor, and is required for hormone-dependent transcription. Unexpectedly, although the CMI and MLL2 PHDf3 domains could bind histone H3, neither showed preference for trimethylated lysine 4. Genetic tests reveal that cmi is required for proper global trimethylation of H3K4 and that hormone-stimulated transcription requires chromatin binding by CMI, methylation of H3K4 by TRR and demethylation of H3K27 by the demethylase UTX. The evolutionary split of MLL2 into two distinct genes in Drosophila provides important insight into distinct epigenetic functions of conserved readers and writers of the histone code (Chauhan, 2012).

Nuclear receptors (NRs) function as transcription factors that respond to cellular signals to initiate new gene expression programs and have essential roles in embryonic development, growth and differentiation. NRs collaborate with greater than 300 co-factors that provide important enzymatic and regulatory functions. Co-factors can be activators or repressors and are typically recruited to gene promoters through associations with receptors. Some co-factors direct changes in the epigenetic environment of target genes by direct covalent chromatin modification or nucleosome remodeling. Co-activators are recruited in a ligand-dependent manner, whereas unliganded receptors often associate with co-repressors. Co-activators exist in large complexes required for the transcription of genes that are regulated by at least 48 vertebrate NRs, including retinoic acid receptor (RAR), liver-X-receptor (LXR), farnesoid-X-receptor (FXR), as well as a co-activator for p53. Disruptions of both NRs and their co-regulators have been linked to many cancers and developmental disorders (Chauhan, 2012).

Hormone signaling pathways in Drosophila melanogaster rely on two primary hormones, the steroid hormone 20-hydroxyecdysone (20HE) and sesquiterpenoid juvenile hormone (JH), and 18 receptors representing all major conserved nuclear receptor subfamilies. Drosophila Ecdysone Receptor (EcR) is an FXR/LXR ortholog, whereas its heterodimeric partner Ultraspiracle (USP) is an RXR ortholog (Chauhan, 2012).

Drosophila Trithorax-related (TRR) is a co-activator of EcR-USP. TRR is a histone lysine methyltransferase (HMT) that trimethylates histone 3 on lysine 4 (H3K4me3) and TRR functions are essential for activating ecdysone-regulated genes. TRR is closely related to another Drosophila protein, Trithorax (TRX), which regulates homeotic (Hox) gene expression through similar methyltransferase activity. The mammalian counterparts of TRR are MLL2 (also known as ALR or MLL4) and MLL3 (also known as HALR). MLL2 and MLL3 are enormous (5537 aa and 4911 aa, respectively), with multiple conserved domains, including histone methyltransferase (SET domain), five plant homeodomain (PHD) zinc fingers, an HMG-I binding motif, LXXLL NR binding motifs and FY-rich regions. Through the SET domain, both MLL2 and MLL3 directly methylate histone H3 to mediate transcription activation (Chauhan, 2012).

MLL2 and MLL3 are components of large SET1/COMPASS-like co-activator complexes that are required for NR-directed gene regulation. These complexes have important human disease connections, including developmental disorders and cancers. MLL2 and MLL3 are mutated in many Kabuki syndrome patients. MLL2 is frequently mutated in childhood medulloblastomas (14%), follicular lymphoma (89%) and diffuse large B-cell lymphoma (32%) (the two most common forms of non-Hodgkin lymphoma), suggesting that MLL2 and MLL3 COMPASS-like complex activities have important epigenetic gene regulatory roles that normally function to inhibit cancer progression (Chauhan, 2012).

Proteins that co-purify with the MLL2 include ASH2, RBBP5 (RBQ3), DPY30, WDR5, adaptor protein ASC2, PTIP, PA1 and histone demethylase UTX. Recently, TRR was found in Drosophila COMPASS-like complexes (Mohan, 2011). Despite functional similarities, TRR is much smaller than MLL2 or MLL3 with homology limited to the C-terminal SET domain portion. TRR lacks the N-terminal PHD and HMG domains that might contribute to chromatin binding. MLL2-related family members are always encoded by large single genes in species other than Brachycera dipterans. To further studies on epigenetic regulation of ecdysone target genes, Drosophila genes were sought that could encode a protein highly related to the N-terminal half of MLL2, and a single open reading frame (CG5591) was identified. The gene was named cara mitad (cmi; translated as 'dear half'). Although cmi is unlinked to trr in the genome, genetic studies using null mutants, in vivo depletion and overexpression revealed functions for cmi as a nuclear receptor co-factor necessary for hormone-regulated gene expression. Unexpectedly, the CMI type 3 PHD finger (PHDf3) was found to accommodate non-methylated, mono- and dimethylated H3K4, rather than trimethylated H3K4. Moreover, CMI-dependent activation also required demethylation functions of UTX, suggesting that NR-stimulated transcription involved at least three steps: binding of H3K4me1/2 by CMI, trimethylation of H3K4 by TRR and demethylation of H3K27 by UTX. The intriguing possibility that COMPASS-like functions in NR-directed transcription are associated with two independent proteins in flies suggests that recognition and binding to modified histones is a distinct step, separate from the epigenetic modification associated with other enzymes in the complex. This presents a unique opportunity to examine functions of histone recognition/binding and covalent histone tail lysine modifications as separate and essential features of NR-directed activation (Chauhan, 2012).

Although the precise roles of proteins directly participating in nuclear receptor signaling remain largely speculative, many are thought to regulate transcription through effects on chromatin. The MLL2 and MLL3 co-activators function to epigenetically decode or modify histone lysine residues and provide activation functions for NR signaling at target genes. In Drosophila, CMI and TRR together have a single MLL family homolog. This is the first example of an evolutionary 'splitting' of an epigenetic regulator involved in nuclear receptor signaling, whereby the essential gene regulatory functions of one protein have been parsed into two distinct proteins. CMI forms complexes with TRR, associates directly with hormone receptors and interacts with other putative COMPASS-like components, suggesting that Drosophila contains a functional counterpart to the mammalian ASCOM-MLL2 nuclear receptor co-activator complex (Chauhan, 2012).

The MLL histone lysine methyltransferases (KMTs) can be divided into two conserved groups, the MLL1-MLL4(2) and MLL2(4)/ALR-MLL3/HALR subfamilies. Each MLL member is capable of forming related discrete complexes with several common components. The MLL-based complexes activate transcription in part through methyltransferase activity on histone H3 Lys4 residues within promoter-associated nucleosomes. There might be partial functional overlap between MLL2 and MLL3; however, they are not redundant with the MLL1-MLL4 subfamily. The SET-domain methyltransferase activity of the MLL proteins is essential for transcription activation through histone lysine methylation, but the precise biological role of PHD fingers remains somewhat elusive. Closely related PHDf3 fingers bind H3K4me3/2, the product of the methyltransferase activity. Within the context of a single protein, such as MLL1, the PHDf3 recognition and binding of H3K4me3 is required for transcription activation of target genes (Chauhan, 2012).

The findings that CMI and TRR function coordinately in a COMPASS-like complex suggest that cmi and trr probably split from a common ancestor. Gene-protein fusions are four times more common than fissions, perhaps reflecting a simpler genetic event. In cases in which fissions occur, it has been suggested that many involve subunits of multimeric complexes in which the two independent proteins interact physically. The process of splitting into two independent genes might involve gene duplication with subsequent partial degeneration, as has been observed in the monkey king (mkg) gene family in Drosophila (Chauhan, 2012).

The notion that a large protein contains domains that function both together and independently is not without precedent. TRX and MLL1 are cleaved by a specific protease, taspase-1. The two 'halves' interact with each other in a functional complex, but there is evidence that the N-terminal TRX peptide (TRX-N) binds chromatin without its TRX-C partner in transcribed regions of Hox genes. Transcription factor TFIIA and herpes simplex virus host cell factor (HCF1) are cleaved during maturation, with both halves necessary for a functional product. There is presently no evidence that MLL2 or MLL3 are cleaved or processed (Chauhan, 2012).

An important question is whether both the chromatin-binding and methyltransferase functions of the MLL family are required for transcription activation. The data indicate that depletion of trr can suppress the effects of overexpressing cmi, suggesting that the activation potential of CMI depends on TRR methyltransferase activity. Similarly, simultaneous depletion of cmi and trr produces stronger phenotypes than depletion of either alone, indicative of cooperation on similar gene targets. Moreover, in vivo depletion of cmi results in reduced global H3 trimethylation, despite a functional trr gene (Chauhan, 2012).

Phenotypes associated with changes in CMI levels reveal important functions in hormone-regulated development. The larval defects in molting, morphogenetic furrow progression and necrosis associated with a cmi null allele, similar to trr, are consistent with impaired hormone signaling. Similarly, depletion of MLL2 in HeLa cells using siRNA led to reduced expression of genes known to be important for development and trimethylation of H3K4 was reduced at some promoters. Knockdown of MLL2 in MCF-7 cells impaired estrogen receptor (ERα) transcription activity and inhibited estrogen-dependent growth. Inactivation of the murine Mll3 resulted in stunted growth and reduced PPARgamma-dependent adipogenesis with increased insulin sensitivity. Perhaps reflecting synonymous functions in Drosophila, cmi/CG5591 was found to be important for regulating muscle triglyceride levels, suggesting conserved adipogenic functions. Furthermore, CG5591 (cmi) is involved in phagocytosis and regulation of caspase functions in response to cellular stress, implicating cmi in immune-cell regulation. The increased hemocyte number associated with elevated CMI suggests functions in hemocyte development, perhaps as an effector of chromatin remodeling or signaling (JAK/STAT, Hedgehog, Notch) pathways. It was previously shown that trr was important for Hedgehog (HH)-dependent signaling during eye development and cmi overexpression and depletion data are consistent with that possibility. However, the dosage-dependent cmi wing phenotypes are not consistent with changes in HH signaling, raising the possibility that cmi and trr are important for other growth and signaling pathways in wing development, including Decapentaplegic (DPP/TGFβ) and Wingless (WG/WNT) pathways (Chauhan, 2012).

Several steps are involved in activation of hormone-responsive target genes, including methylation of H3K4 by the MLL2-MLL3 COMPASS-like complex and displacement of demethylases. Reduced cmi function resulted in lower hormone-responsive enhancer activation and genetic interactions between cmi, trr and Utx revealed that chromatin binding by CMI was important for gene activation in vivo. Furthermore, RNAi depletion of Utx suppressed HA-cmi overexpression wing phenotypes, suggesting that demethylation of H3K27 is a pre-requisite for activation of some hormone target genes. This is supported by genetic evidence from C. elegans that indicated both histone H3K4 methylation by SET-16 (MLL2/MLL3 ortholog) and H3K27 demethylation by UTX-1 were required for attenuation of RAS signaling in the vulva and MLL2-MLL3 complex-related components were required for proper germ line development. Genetic epistasis data reveals that Utx, trr and cmi functions are all required for activation in Drosophila (Chauhan, 2012).

Unexpectedly, the CMI PHDf3.b showed binding to mono- and dimethylated H3K4, rather than trimethylated H3K4. Although CMI contains two PHDf3 domains in two clusters similar to MLL3, MLL2 contains one PHDf3 most closely related to the CMI and MLL3 PHDf3.b domains. The second cluster appears in all isoforms of MLL3, whereas the N-terminal 'a' cluster is optional. Additionally, the 'b' cluster is more closely related to the PHD cluster found in other MLL family proteins. PHD modules are thought to bind histones and present tail residues to the modifying enzyme subunits or stabilize those enzymes with their substrates. Recently, RNAi knockdown of trr in S2 cells was shown to affect H3K4 mono-, di-, and trimethylation, revealing widespread functions in regulating methylation in vivo and suggesting that loss of TRR might destabilize the co-activator complex leading to de-protection of H3K4 methylation. One possibility is that CMI binds mono- and dimethylated H3K4 to prevent demethylation and stabilize TRR to allow for hormone-stimulated methylation and gene activation. CMI might disengage to allow for removal of methylation marks as hormone levels decrease and gene transcription is reduced. In contrast to MLL1-TRX function in maintenance of active gene transcription, CMI and TRR might be required for NR-targeted gene activation in response to temporally restricted hormone-dependent genome reprogramming (Chauhan, 2012).

Cryptocephal, the Drosophila melanogaster ATF4, is a specific coactivator for ecdysone receptor isoform B2

The ecdysone receptor is a heterodimer of two nuclear receptors, the Ecdysone receptor (EcR) and Ultraspiracle (USP). In Drosophila melanogaster, three EcR isoforms share common DNA and ligand-binding domains, but these proteins differ in their most N-terminal regions and, consequently, in the activation domains (AF1s) contained therein. The transcriptional coactivators for these domains, which impart unique transcriptional regulatory properties to the EcR isoforms, are unknown. Activating transcription factor 4 (ATF4) is a basic-leucine zipper transcription factor that plays a central role in the stress response of mammals. Here Cryptocephal (CRC), the Drosophila homolog of ATF4, is shown to be an ecdysone receptor coactivator that is specific for isoform B2. CRC interacts with EcR-B2 to promote ecdysone-dependent expression of ecdysis-triggering hormone (ETH), an essential regulator of insect molting behavior. It is proposed that this interaction explains some of the differences in transcriptional properties that are displayed by the EcR isoforms, and similar interactions may underlie the differential activities of other nuclear receptors with distinct AF1-coactivators (Gauthier, 2012).

The experiments suggest that the 17-residue B2-specific N-terminus binds to the bZIP region of CRC, that an ionic interaction between EcR-B2-E9 and CRC-R361 plays some role in the binding, and that the interaction of the two proteins plays a crucial role in those tissues where EcR-B2 is essential. These tissues include the endocrine Inka cells, which display ecdysone-dependent upregulation of ETH transcripts and which require EcR-B2 and CRC for full ETH expression. Taken together, these findings implicate CRC as an isoform-specific transcriptional activator for EcR-B2 (Gauthier, 2012).

In diverse systems, bZIP proteins interact with dyadic or palindromic promoter sequences as homodimers or heterodimers with other bZIP partners. Dimerization involves regularly spaced hydrophobic amino acids that form a coiled-coil between two leucine zipper domains. Other bZIP transcription factors are known to interact with nuclear receptors, modulating the activities of either AF1 or AF2, but in the cases reported previously, bZIP proteins bind either to the DNA-binding domain or to the hinge domain of the nuclear receptor. By contrast, CRC (through its bZIP domain) appears to bind directly to the EcR-B2 AF1 region, and its interaction is specific to one EcR isoform (Gauthier, 2012).

ATF4, the mammalian homolog of CRC, plays a central role in stress responses. The role of CRC in ecdysone signaling suggests the possibility of interesting and unexpected connections between stress responses and the control of developmental timing and metamorphosis (Gauthier, 2012).

The ETH promoter contains sequences matching the consensus half-sites for binding of ATF4 and EcR to DNA. These half-sites are separated by 4 nucleotides, and they are located within a highly conserved sequence (comparing D. melanogaster to several other Drosophila species) that is 138-171 nucleotides upstream of the ETH transcriptional start site. Since bZIP proteins may bind first sequentially as monomers and then dimerize while bound to DNA, these observations suggest a model in which CRC participates in the stabilization of EcR-B2 binding to the ETH promoter. This interaction provides a basis for understanding some of the differences in transcriptional properties that are displayed by the EcR isoforms and perhaps other nuclear receptors with distinct AF1-coactivators (Gauthier, 2012).

Drosophila Kdm4 demethylases in histone H3 lysine 9 demethylation and ecdysteroid signaling

The dynamic regulation of chromatin structure by histone post-translational modification is an essential regulatory mechanism that controls global gene transcription. The Kdm4 family of H3K9me2,3 and H3K36me2,3 dual specific histone )emethylases has been implicated in development and tumorigenesis. This study shows that Drosophila Kdm4A and Kdm4B, both members of the JHDM3 histone demethylase family are together essential for mediating ecdysteroid hormone signaling during larval development. Loss of Kdm4 genes leads to globally elevated levels of the heterochromatin marker H3K9me2,3 and impedes transcriptional activation of ecdysone response genes, resulting in developmental arrest. It was further shown that Kdm4A interacts with the Ecdysone Receptor (EcR) and colocalizes with EcR at its target gene promoter. These studies suggest that Kdm4A may function as a transcriptional co-activator by removing the repressive histone mark H3K9me2,3 from cognate promoters (Raueumi, 2013).

This study have discovered a role for Kdm4 in the transcriptional regulation of a subset of ecdysone pathway components. Furthermore, an interaction was demonstrated between Kdm4A and EcR in vivo, providing evidence that Kdm4 demethylases may act as co-activators of EcR. A genetic approach has allowed facilitated the detection of a previously uncharacterized, but essential, role of Kdm4 in development, and has identified a direct Kdm4 target gene in euchromatin. Interestingly, Human Kdm4 members interact with the nuclear hormone receptors, Androgen Receptor (AR) and Estrogen Receptor α (ERα), and has been proposed to serve as co-activators, suggesting a molecular mechanism by which Kdm4 can act as an oncogene in prostate and breast cancers. Kdm4B was shown to be a direct target gene of ERα, yielding a feed-forward loop for an augmented hormonal response. The results indicate that a similar epigenetic mechanism exists in Drosophila, where a nuclear hormone receptor requires the Kdm4 family of demethylases to remove H3K9 methylation at the promoter of a target gene. Taken together, the Kdm4 family of demethylases may function as transcriptional co-factors required for transcriptional activation by nuclear hormone receptors (Raueumi, 2013).

Previous studies have shown that the Trithorax-related (Trr) H3K4 methyltransferase, the Nurf nucleosome remodeling complex component, Nurf301, the Brahma (Brm)-containing chromatin remodeler, and the histone acetyltransferase CREB-binding protein (CBP) are also co-activators of EcR, indicating that activation of ecdysone pathway genes requires substantial regulation of the chromatin environment. Since H3K4 hyper-methylation at promoters is a marker of active transcription, and since H3K9 hypo-methylation also promotes upregulation of gene expression, it is feasible that synchronizing these two events would lead to more robust target gene activation. The mammalian Kdm4B (JMJD2B) forms a complex with the mixed-lineage leukemia (MLL) 2 H3K4 methyltransferase and serves as a co-activator of Estrogen Receptor. The complex couples H3K9 demethylation with H3K4 methylation in order to facilitate ERα target gene activation. Similar functional cross-talk between H3K9 demethylation and H3K4 methylation has been described in S. pombe, where the Lsd1 H3K9 demethylase and the Set1 H3K4 methyltransferase were found in a complex. Since, in Drosophila, the Nurf301 subunit, Brm and CBP were also found to interact with EcR, nucleosome remodeling may cooperate as well in the rapid and dynamic activation of ecdysone regulated genes (Raueumi, 2013).

These studies of the Kdm4A and Kdm4B homozygous double mutants demonstrate a requirement for these genes in the ecdysone pathway. This observation is similar to results obtained with mutant alleles of Nurf301 and trr, two seemingly ubiquitous chromatin regulators, where specific downregulation of ecdysone signaling genes has been detected. Additionally, this study is consistent with the reports that adult male Kdm4A mutants display abnormal courtship behavior and concomitant downregulated fru, a gene speculated to be a direct downstream target of EcR (Beckstead, 2005; Dalton, 2009). The specific defects in ecdysone signaling, rather than general transcription, exhibited by the double mutants indicate that either Kdm4 may not be essential for regulating all genes, or that the aberrant expression of ecdysone responsive genes is the earliest manifestation of loss of Kdm4. However, this study does not rule out the possibility that Kdm4 proteins regulate other crucial transcription factors that in turn regulate ecdysone pathway components by secondary effects. Further molecular and genomic studies are required to resolve this issue (Raueumi, 2013).

H3K9 demethylation-dependent transcriptional activation of BR-C was demonstrated. It is possible however, that H3K36 demethylation also contributes to ecdysone pathway component regulation. Previous studies have shown that HP1a is recruited to developmental puffs in polytene chromosomes and that it stimulates H3K36 demethylation by Kdm4A. Perhaps H3K36 demethylation in the gene body and subsequent displacement of the HDAC complex is important for transcriptional elongation or for the activation of downstream nested promoters of ecdysone pathway components. Moreover, H3K36 plays a role in exon splice choice and thus ecdysone pathway genes that produce multiple splice variants may require Kdm4 regulation. However, immunostaining experiments show that HP1a and Kdm4A signals are mostly non-overlapping. Thus, it seems that HP1a's involvement in the demethylase activities of Kdm4 toward H3K9 or H3K36 would have to be transient and dynamic (Raueumi, 2013).

In summary, this study has shown that double homozygous mutants of the two Kdm4 genes in Drosophila display developmental delays and lethality, with compromised activation of ecdysone related genes. Furthermore, it was found that BR-C may be a direct target of H3K9 demethylation, and that the interaction between Kdm4A and EcR may be important in transcriptional activation of BR-C. These results provide insight into the physiological functions and mechanistic roles of Kdm4 in vivo. The interaction between Kdm4 and EcR awaits further investigation. It is conceivable that EcR directs the recruitment of Kdm4A to the promoter of its target genes, or alternatively, that EcR allosterically regulates the demethylase activity of Kdm4A, allowing removal of H3K9m2,3 only upon hormone signaling (Raueumi, 2013).

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

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

UTX coordinates steroid hormone-mediated autophagy and cell death

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

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

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

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

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

CDK8-Cyclin C mediates nutritional regulation of developmental transitions through the Ecdysone receptor in Drosophila

EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval-pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval-pupal transition. Taken together, these results suggest that CDK8-CycC links nutrient intake to developmental transitions (EcR activity) and fat metabolism (SREBP activity) during the larval-pupal transition (Xie, 2015).

In animals, the amount of juvenile growth is controlled by the coordinated timing of maturation and growth rate, which are strongly influenced by the environmental factors such as nutrient availability. This is particularly evident in arthropods, such as insects, arachnids and crustaceans, which account for over 80% of all described animal species on earth. Characterized by their jointed limbs and exoskeletons, juvenile arthropods have to replace their rigid cuticles periodically by molting. In insects, the larval-larval and larval-pupal transitions are controlled by the interplay between juvenile hormone (JH) and steroid hormone ecdysone. Drosophila has been a powerful system for deciphering the conserved mechanisms that regulate hormone signaling, sugar and lipid homeostasis, and the molecular mechanisms underlying the nutritional regulation of development. In Drosophila, all growth occurs during the larval stage when larvae constantly feed, and as a result their body mass increases approximately 200-fold within 4 d, largely due to de novo lipogenesis. At the end of the third instar, pulses of ecdysone, combined with a low level of JH, trigger the larval-pupal transition and metamorphosis. During this transition, feeding is inhibited, and after pupariation, feeding is impossible, thus the larval-pupal transition marks when energy metabolism is switched from energy storage by lipogenesis in larvae to energy utilization by lipolysis in pupae (Xie, 2015).

The molecular mechanisms of ecdysone-regulated metamorphosis and developmental timing have been studied extensively in Drosophila. Ecdysone binds to the Ecdysone Receptor (EcR), which heterodimerizes with Ultraspiracle (USP), an ortholog of the vertebrate Retinoid X Receptor (RXR). By activating the expression of genes whose products are required for metamorphosis, ecdysone and EcR-USP are essential for the reorganization of flies' body plans before emerging from pupal cases as adults. Despite the tremendous progress in understanding of the physiological and developmental effects of EcR-USP signaling, the molecular mechanism of how the EcR-USP transcription factor interacts with the general transcription machinery of RNA polymerase II (Pol II) and stimulates its target gene expression remains mysterious. EcR is colocalized with Pol II in Bradysia hygida and Chironomus tentans. Although a number of proteins, such as Alien, Bonus, Diabetes and Obesity Regulated (dDOR), dDEK, Hsc70, Hsp90, Rigor mortis (Rig), Smrter (Smr), Taiman, and Trithorax-related (TRR), have been identified as regulators or cofactors of EcR-mediated gene expression, it is unknown how these proteins communicate with the general transcription machinery and whether additional cofactors are involved in EcR-mediated gene expression. In addition, it remains poorly understood how EcR activates transcription correctly after integrating nutritional and developmental cues (Xie, 2015).

The multisubunit Mediator complex serves as a molecular bridge between transcriptional factors and the core transcriptional machinery, and is thought to regulate most (if not all) of Pol II-dependent transcription. Biochemical analyses have identified two major forms of the Mediator complexes: the large and the small Mediator complexes. In addition to a separable 'CDK8 submodule', the large Mediator complex contains all but one (MED26) of the subunits of the small Mediator complex. The CDK8 submodule is composed of MED12, MED13, CDK8, and CycC. CDK8 is the only enzymatic subunit of the Mediator complex, and CDK8 can both activate and repress transcription depending on the transcription factors with which it interacts. Amplification and mutation of genes encoding CDK8, CycC, and other subunits of Mediator complex have been identified in a variety of human cancers, however, the function and regulation of CDK8-CycC in non-disease conditions remain poorly understood. CDK8 and CycC are highly conserved in eukaryotes, thus analysis of the functional regulation of CDK8-CycC in Drosophila is a viable approach to understand their activities (Xie, 2015).

Previous, work has shown that CDK8-CycC negatively regulates the stability of sterol regulatory element-binding proteins (SREBPs) by directly phosphorylating a conserved threonine residue. This study now reports that CDK8-CycC also regulates developmental timing in Drosophila by linking nutrient intake with EcR-activated gene expression. Homozygous cdk8 or cycC mutants resemble EcR mutants in both pupal morphology and retarded developmental transitions. Despite the elevation of both EcR and USP proteins in cdk8 or cycC mutants, genome-wide gene expression profiling analyses reveal systematic down-regulation of EcR-target genes, suggesting the CDK8-CycC defect lies between the receptor complex and transcriptional activation. CDK8-CycC is required for EcR-USP transcription factor binding to EcR target genes. Mass spectrometry analysis for proteins that co-immunoprecipitate with EcR and USP has identified multiple Mediator subunits, including CDK8 and CycC, and yeast two-hybrid assays have revealed that CDK8 and Med14 can directly interact with the EcR-AF1 domain. Furthermore, the dynamic changes of CDK8, EcR, USP, and SREBP correlated with the fundamental roles of SREBP in regulating lipogenesis and EcR-USP in regulating metamorphosis during the larval–pupal transition. Importantly, it was shown that starving the early third instar larvae causes precocious increase of CDK8, EcR and USP proteins, as well as premature inactivation of SREBP; whereas refeeding of the starved larvae reduces CDK8, EcR, and USP proteins, but potently stimulates SREBP activity. These results suggest a dual role of CDK8-CycC, linking nutrient intake to de novo lipogenesis (by inhibiting SREBP) and developmental signaling (by regulating EcR-dependent transcription) during the larval–pupal transition (Xie, 2015).

Through EcR-USP, ecdysone plays pivotal roles in controlling developmental timing in Drosophila. This study shows that cdk8 or cycC mutants resemble EcR-B1 mutants and CDK8-CycC is required for proper activation of EcR-target genes. Molecular and biochemical analyses suggest that CDK8-CycC and the Mediator complexes are directly involved in EcR-dependent gene activation. In addition, the protein levels of CDK8 and CycC are up-regulated at the onset of the wandering stage, closely correlated with the activation of EcR-USP and down-regulation of SREBP-dependent lipogenesis during the larval–pupal transition. Remarkably, starvation of the feeding larvae leads to premature up-regulation of CDK8 and EcR-USP, and precocious down-regulation of SREBP, while refeeding of the starved larvae results in opposite effects on the CDK8-SREBP/EcR network. Thus, it is proposed that CDK8-CycC serves as a key mediator linking food consumption and nutrient intake to EcR-dependent developmental timing and SREBP-dependent lipogenesis during the larval–pupal transition (Xie, 2015).

The Mediator complex is composed of up to 30 different subunits, and biochemical analyses of the Mediator have identified the small Mediator complex and the large Mediator complex, with the CDK8 submodule being the major difference between the two complexes. Several reports link EcR and certain subunits of the Mediator complex. For example, Med12 and Med24 were shown to be required for ecdysone-triggered apoptosis in Drosophila salivary glands. It was recently reported that ecdysone and multiple Mediator subunits could regulate cell-cycle exit in neuronal stem cells by changing energy metabolism in Drosophila, and specifically, EcR was shown to co-immunoprecipitate with Med27. However, exactly how Mediator complexes are involved in regulating EcR-dependent transcription remains unknown. The current data suggest that CDK8 and CycC are required for EcR-activated gene expression. Loss of either CDK8 or CycC reduced USP binding to EcR target promoters, diminished EcR target gene expression, and delayed developmental transition, which are reminiscent of EcR-B1 mutants. Importantly, mass spectrometry analysis for proteins that co-immunoprecipitate with EcR or USP has identified multiple Mediator subunits, including all four subunits of the CDK8 submodule (Xie, 2015).

Taken together, previous works and the present work highlight a critical role of the Mediator complexes including CDK8-CycC in regulating EcR-dependent transcription. How does CDK8-CycC regulate EcR-activated gene expression? Biochemical analyses show that CDK8 can interact with EcR and USP in vivo and that CDK8 can directly interact with EcR-AF1. These observations, together with the current understanding of how nuclear receptors and Mediator coordinately regulate transcription, suggest that CDK8-CycC may positively and directly regulate EcR-dependent transcription. Yeast two-hybrid analysis indicates that the recruitment of CDK8-CycC to EcR-USP can occur via interactions between CDK8 and the AF1 domain of EcR. Interestingly, this assay also revealed a direct interaction between EcR-AF1 and a fragment of Med14 that contains the LXXLL motif. In future work, it will be interesting to determine whether CDK8 and Med14 compete with each other in binding with the EcR-AF1, whether they interact with EcR-AF1 sequentially in activating EcR-dependent transcription, and how the Mediator complexes coordinate with other known EcR cofactors in regulating EcR-dependent gene expression (Xie, 2015).

In cdk8 or cycC mutants, the binding of USP to the promoters of the EcR target genes is significantly compromised, even though nuclear protein levels of both EcR and USP are increased. It is unclear how CDK8-CycC positively regulates EcR-USP binding to EcREs near promoters. CDK8 can directly phosphorylate a number of transcription factors, such as Notch intracellular domain, E2F1, SMADs, SREBP, STAT1, and p53. Interestingly, the endogenous EcR and USP are phosphorylated at multiple serine residues, and treatment with 20E enhances the phosphorylation of USP. Protein kinase C has also been proposed to phosphorylate USP. It will be interesting to determine whether CDK8 can also directly phosphorylate either EcR or USP, thereby potentiating expression of EcR target genes and integrating signals from multiple signaling pathways (Xie, 2015).

Although a direct role for CDK8-CycC to regulate EcR-USP activated gene expression is favored, it was not possible to exclude the potential contribution of impaired biosynthesis of 20E to the developmental defects in cdk8 or cycC mutants. For example, the expression of genes involved in synthesis of 20E, such as sad and spok, is significantly reduced in cdk8 or cycC mutant larvae. Indeed, the ecdysteroid titer is significantly lower in cdk8 mutants than control from the early L3 to the WPP stages, and feeding the cdk8 mutant larvae with 20E can partially reduce the retardation in pupariation. Nevertheless, impaired ecdysone biosynthesis alone cannot explain developmental defects that were characterized in this report for the following reasons. First, feeding cdk8 or cycC mutants with 20E cannot rescue the defects in pupal morphology, developmental delay, and the onset of pupariation. Second, the expression of EcRE-lacZ reporter in cdk8 or cycC mutant salivary glands cannot be as effectively stimulated by 20E treatment as in control. Third, knocking down of either cdk8 or cycC in PG did not lead to obvious defects in developmental timing. Therefore, the most likely scenario is that the cdk8 or cycC mutants are impaired not only in 20E biosynthesis in the PG, but also in EcR-activated gene expression in peripheral tissues. Defects in either ecdysone biosynthesis or EcR transcriptional activity will generate the same outcome: diminished expression of the EcR target genes, thereby delayed onset of pupariation (Xie, 2015).

How CDK8-CycC regulates biosynthesis of ecdysone in PG remains unknown. Several signaling pathways have been proposed to regulate ecdysone biosynthesis in Drosophila PG, including PTTH and Drosophila insulin-like peptides (dILPs)-activated receptor tyrosine kinase pathway and Activins/TGFβ signaling pathway. Interestingly, CDK8 has been reported to regulate the transcriptional activity of SMADs, transcription factors downstream of the TGFβ signaling pathway, in both Drosophila and mammalian cells. Thus, it is conceivable that the effect of cdk8 or cycC mutation on ecdysone biosynthesis may due to dysregulated TGFβ signaling in the PG (Xie, 2015).

An effort to explore the potential role of food consumption and nutrient intake on CDK8-CycC has resulted an unexpected observation that the protein level of CDK8 is strongly influenced by starvation and refeeding: starvation potently increased CDK8 level, while refeeding has opposite effect, and both occur post-transcriptionally. The importance of this observation is highlighted in two aspects. First, considering the generally repressive role of CDK8 on Pol II-dependent gene expression, up-regulation of CDK8 may provide an efficient way to quickly tune down most of the Pol II-dependent transcription in response to starvation; while down-regulation of CDK8 in response to refeeding may allow many genes to express when nutrients are abundant. Second, it will be necessary to test whether the effects of nutrient intake on CDK8-CycC is conserved in mammals. If so, considering that both CDK8 and CycC are dysregulated in a variety of human cancers, the effects of nutrient intake on CDK8 may have important implications in not only understanding of the effects of nutrients on tumorigenesis, but also providing nutritional guidance for patients with cancer (Xie, 2015).

Major dietary components including carbohydrates, lipids, and proteins, can strongly influence the developmental timing in Drosophila. Excessive dietary carbohydrates repress growth and potently retard the onset of pupariation. One elegant model proposed to explain how high sugar diet delays developmental timing is that high sugar diet reduces the activity of the Target of Rapamycin (TOR) in the PG, thereby reducing the secretion of ecdysone and delaying the developmental transition. Previously, it was reported that insulin signaling could down-regulate CDK8-CycC, and that ectopic expression of CycC could antagonize the effect of insulin stimulation in mammalian cells, as well as the effect of refeeding on the expression of dFAS in Drosophila (Zhao, 2012). Although the mRNA levels of TOR and insulin receptor (InR) are not significantly affected in cdk8 or cycC mutants, it is necessary to further study whether and how different dietary components may regulate CDK8-CycC in the future (Xie, 2015).

Developmental genetic analyses of the cdk8 and cycC mutants have revealed major defects in fat metabolism and developmental timing. De novo lipogenesis, which is stimulated by insulin signaling, contributes significantly to the rapid increase of body mass during the constant feeding larval stage. This process is terminated by pulses of ecdysone that trigger the wandering behavior at the end of the L3 stage, followed by the onset of the pupariation. Insulin and ecdysone signaling are known to antagonize each other, and together determine body size of Drosophila. The genetic interaction is established, but the detailed molecular mechanisms are not. The SREBP family of transcription factors controls the expression of lipogenic enzymes in metazoans and the expression of cholesterogenic enzymes in vertebrates. Previous work shows that CDK8 directly phosphorylates the nuclear SREBP proteins on a conserved threonine residue and promotes the degradation of nuclear SREBP proteins. Consistent with the lipogenic role of SREBP and the inhibitory role of insulin to CDK8-CycC, the transcriptional activity of SREBP is high while the levels of CDK8-CycC and EcR-USP are low prior to the onset of wandering stage. Subsequently during the wandering and non-mobile, non-feeding pupal stage, the transcriptional activity of SREBP is dramatically reduced, accompanied by the significant accumulation of CDK8-CycC and EcR-USP (Xie, 2015).

The causal relationship of these phenomena was further tested by starvation and refeeding experiments. On the one hand, it was observed that the levels of CDK8, EcR and USP are potently induced by starvation, while the mature SREBP level and the transcriptional activity of SREBP are reduced by starvation. Starvation of larvae prior to the two nutritional checkpoints in early L3, known as minimum viable weight and critical weight, which are reached almost simultaneously in Drosophila, will lead to larval lethality; while starvation after larvae reach the critical weight will lead to early onset of pupariation and formation of small pupae. Thus, this nutritional checkpoint ensures the larvae have accumulated sufficient growth before metamorphosis initiation. If the status with high CDK8, EcR, and USP is regarded as an older or later stage, these results indicate that starvation shifts the regulatory network precociously (see Model for the CDK8-SREBP/EcR regulatory network). On the other hand, the current analyses of refed larvae show that refeeding potently reduced the levels of CDK8, EcR and USP. If the status with low CDK8, EcR, and USP is considered as a younger or earlier stage, these results indicate that refeeding delays the activation of this network, which is consistent with the model and delayed pupariation as observed. Taken together, these results based on starved and refed larvae suggest that CDK8-CycC is a key regulatory node linking nutritional cues with de novo lipogenesis and developmental timing (Xie, 2015).

The larval-pupal transition is complex and dynamic. Although the expression of SREBP target genes fit well with the predicted effects of starvation and refeeding, the expression of EcR targets during the stage that was analyzed in this study does not reflect the changes in the protein levels of EcR and USP. It is reasonable to consider that CDK8-CycC and EcR-USP are necessary, but not sufficient, for the activation of EcR target genes. One possibility is that there is a delay on synthesis of 20E or other cofactors that are required for EcR-activated gene expression in response to starvation. Indeed, the 20E levels were measured during the first 16 hr of starvation, and no significant difference was observed between fed and starved larvae. It will be necessary to further analyze the effect of starvation on 20E synthesis at later time points in the future (Xie, 2015).

Taken together, a model is proposed whereby CDK8-CycC functions as a regulatory node that coordinates de novo lipogenesis during larval stage and EcR-dependent pupariation in response to nutritional cues. It is likely that pulses of 20E synthesized in the PG, and subsequent behavioral change from feeding to wandering, ultimately trigger the transition from SREBP-dependent lipogenesis to EcR-dependent pupariation. The opposite effects of CDK8-CycC on SREBP- and EcR-dependent gene expression suggest that the role of CDK8 on transcription is context-dependent (Xie, 2015).

In conclusion, this study illustrates how CDK8-CycC regulates EcR-USP-dependent gene expression, and the results suggest that CDK8-CycC may function as a regulatory node linking fat metabolism and developmental timing with nutritional cues during Drosophila development (Xie, 2015).

Interactive Fly, Drosophila Ecdysone receptor: Biological Overview | Evolutionary homologs | Transcriptional Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

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