The palindromic DNA-bound USP/EcR nuclear receptor adopts an asymmetric organization with allosteric domain positioning.

Nuclear receptors (NRs) regulate gene expression through DNA- and ligand-binding and thus represent crucial therapeutic targets. The ultraspiracle protein/ecdysone receptor (USP/EcR) complex binds to half-sites with a one base pair spaced inverted repeat (IR1), a palindromic DNA response element (RE) reminiscent of IRs observed for vertebrate steroid hormone receptors. Here we present the cryo electron microscopy structure of the USP/EcR complex bound to an IR1 RE which provides the first description of a full IR-bound NR complex. The structure reveals that even though the DNA is almost symmetric, the complex adopts a highly asymmetric architecture in which the ligand-binding domains (LBDs) are positioned 5' off-centred. Additional interactions of the USP LBD with the 5'-flanking sequence trigger transcription activity as monitored by transfection assays. The comparison with DR-bound NR complexes suggests that DNA is the major allosteric driver in inversely positioning the LBDs, which serve as the main binding-site for transcriptional regulators.

Quantitative cell-based reporter gene assays using droplet-based microfluidics.

We used a droplet-based microfluidic system to perform a quantitative cell-based reporter gene assay for a nuclear receptor ligand. Single Bombyx mori cells are compartmentalized in nanoliter droplets which function as microreactors with a >1000-fold smaller volume than a microtiter-plate well, together with eight or ten discrete concentrations of 20-hydroxyecdysone, generated by on-chip dilution over 3 decades and encoded by a fluorescent label. The simultaneous measurement of the expression of green fluorescent protein by the reporter gene and of the fluorescent label allows construction of the dose-response profile of the hormone at the single-cell level. Screening approximately 7500 cells per concentration provides statistically relevant data that allow precise measurement of the EC(50) (70 nM +/- 12%, alpha = 0.05), in agreement with standard methods as well as with literature data.

The ligand-binding domains of the three RXR-USP nuclear receptor types support distinct tissue and ligand specific hormonal responses in transgenic Drosophila.

In insects, 20-hydroxyecdysone acts by binding on a heterodimer constituted by the ecdysone receptor (EcR) and Ultraspiracle (USP), the homolog to the vertebrate retinoid X receptor (RXR). Two types of USP have been characterized based on their structure and function, Mecopterida USP (Diptera/Lepidoptera USP), in particular the fruitfly Drosophila melanogaster USP (DmUSP) and non Mecopterida USP, exemplified by the beetle Tribolium castaneum USP (TcUSP) both showing structural differences from the vertebrate RXR. Here, by combining in vivo and organ culture observations in Drosophila transgenic animals, we show that ectopic expression of GAL4-DmUSP, GAL4-TcUSP or GAL4-HsRXR results in tissue- and ligand-dependent activities. In parallel, we show that neither juvenile hormone (JH) nor the related methyl farnesoate has an effect on GAL4-USP activation although JH induces the expression of a factor inhibiting the receptor transcriptional activity in the presence of EcR or RXR agonists. This study suggests that not only is USP important for hormonal regulation, via heterodimer formation, but that tissue-specific expression of cofactors may represent a higher level of control of this regulation. This in vivo approach should lead to a better understanding of the modes of action of USP and the identification of transcriptional cofactors essential for its function.

Structural and functional characterization of a novel type of ligand-independent RXR-USP receptor.

Retinoid X receptor (RXR) and Ultraspiracle (USP) play a central role as ubiquitous heterodimerization partners of many nuclear receptors. While it has long been accepted that a wide range of ligands can activate vertebrate/mollusc RXRs, the existence and necessity of specific endogenous ligands activating RXR-USP in vivo is still matter of intense debate. Here we report the existence of a novel type of RXR-USP with a ligand-independent functional conformation. Our studies involved Tribolium USP (TcUSP) as representative of most arthropod RXR-USPs, with high sequence homology to vertebrate/mollusc RXRs. The crystal structure of the ligand-binding domain of TcUSP was solved in the context of the functional heterodimer with the ecdysone receptor (EcR). While EcR exhibits a canonical ligand-bound conformation, USP adopts an original apo structure. Our functional data demonstrate that TcUSP is a constitutively silent partner of EcR, and that none of the RXR ligands can bind and activate TcUSP. These findings together with a phylogenetic analysis suggest that RXR-USPs have undergone remarkable functional shifts during evolution and give insight into receptor-ligand binding evolution and dynamics.

Roles of Drosophila Kruppel-homolog 1 in neuronal morphogenesis.

The molecular mechanisms underlying remodeling of neural networks remain largely unknown. In Drosophila, widespread neural remodeling occurs during metamorphosis, and is regulated by ecdysone. Kruppel-homolog 1 (Kr-h1) is a zinc finger transcription factor known to play a role in orchestrating ecdysone-regulated transcriptional pathways and, furthermore, implicated in governing axon morphogenesis. Interestingly, in honey bee workers, neural expression of the Apis mellifera homolog of Kr-h1 is enhanced during their transition to foraging behavior when there is increased neurite outgrowth, branching, and synapse formation. Here, we assessed the role(s) of KR-H1 in Drosophila neuronal remodeling and morphology. We characterized the effect of Kr-h1 expression on neuronal morphology through Drosophila larval, pupal, and adult stages. Increased expression of Kr-h1 led to reduced branching in individual neurons and gross morphological changes in the mushroom bodies (MBs), while knocking down Kr-h1 did not produce any obvious changes in neural morphology. Drosophila Kr-h1 is normally expressed when MB neurons do not undergo active morphogenesis, suggesting that it may play a role in inhibiting morphogenesis. Further, loss of endogenous KR-H1 enhanced the neuronal morphogenesis that is otherwise delayed due to defective TGF-beta signaling. However, loss of KR-H1 alone did not affect neuronal morphogenesis. In addition, Kr-h1 expression remains strongly linked to ecdysone-regulated pathways: Kr-h1 expression is regulated by usp, which dimerizes to the ecdysone receptor, and Kr-h1 expression is essential for proper patterning of the ecdysone receptor isoforms in the late larval central nervous system. Thus, although KR-H1 has a potential for modulating neuronal morphogenesis, it appears physiologically involved in coordinating general ecdysone signaling.

Dynamic localisation of KR-H during an ecdysone response in Drosophila.

The propagation of a hormonal response following an increase in titre involves intensive cross-talk between the products of the key regulatory genes. The Kr-h gene of Drosophila is a modulator of both the embryonic and metamorphic hierarchies of ecdysone responsive genes, but its mode of action is puzzling as mutants have both quantitative and qualitative (timing) effects on the ecdysone responses. We have used an antibody against KR-H to follow its distribution in larval tissues as they prepare for metamorphosis. While in most tissues protein levels remain stable, its distribution within salivary gland cells changes throughout the late larval ecdysone response and the ensuing prepupal period. We show that, at the chromosomal level, KR-H localisation is dynamic and that the protein is recruited to, and released from, loci harbouring an important subset of the known regulatory genes as the response advances. Such behaviour is most likely a conserved characteristic of hormonal responses.

Krüppel-homolog is essential for the coordination of regulatory gene hierarchies in early Drosophila development.

Drosophila development is marked by two major morphogenetic processes: embryogenesis and metamorphosis. While insect metamorphosis is known to be controlled by the steroid hormone ecdysone, relatively little is known concerning the hormonal control of embryogenesis. Here we show that many ecdysone-regulated transcripts of metamorphosis are also expressed in a wavelike manner during embryogenesis, suggesting that these genes also participate in an embryonic ecdysone response. At metamorphosis, the Krüppel-homolog (Kr-h) gene, coding for a zinc finger protein, is required during the prepupal ecdysone response. Kr-h mutants die at the prepupal-pupal transition. In these mutants, the expression of several ecdysone-regulated genes is disrupted and we concluded that Kr-h was a key modulator of the hormonal response [Dev. Biol. 221 (2000) 53]. While Kr-h is expressed in many tissues at metamorphosis, in embryos expression is restricted to neurons. Here, we investigate its role during early Drosophila development using new alleles with an earlier lethality than those previously described. Although we detect only minor morphological defects in these mutants, we show that Kr-h expression is necessary for the early development of Drosophila and that, during metamorphosis, Kr-h acts as a modulator of the expression of many of these ecdysone-regulated genes.