REVIEWS NUCLEAR RECEPTORS — A PERSPECTIVE FROM DROSOPHILA Kirst King-Jones and Carl S. Thummel Abstract | Nuclear receptors are ancient ligand-regulated transcription factors that control key metabolic and developmental pathways. The fruitfly Drosophila melanogaster has only 18 nuclear-receptor genes — far fewer than any other genetic model organism and representing all 6 subfamilies of vertebrate receptors. These unique attributes establish the fly as an ideal system for studying the regulation and function of nuclear receptors during development. Here, we review recent breakthroughs in our understanding of D. melanogaster nuclear receptors, and interpret these results in light of findings from their evolutionarily conserved vertebrate homologues. CLADE Classical signal-transduction pathways originate with a A wide range of small, lipophilic molecules function as A group of organisms that membrane-bound receptor. On binding its cognate nuclear-receptor ligands, including steroids, thyroid includes common ancestor and ligand, the receptor initiates a cascade of events in the hormone, retinoic acid and vitamin D. Not all nuclear all of its descendants, cytoplasm, eventually affecting specific transcription receptors, however, have a known natural ligand, and at representing a distinct branch on a phylogenetic tree. factors in the nucleus. In contrast to these often complex least some of these so-called orphan receptors can and convoluted pathways, members of the nuclear- function in a ligand-independent fashion3,4. receptor superfamily use an ingenious shortcut to trans- Extensive studies over the past two decades have pro- duce their signals — a strategy that is due to their vided a detailed understanding of the molecular mecha- unique structure. Nuclear receptors harbour a receptor nisms by which nuclear receptors convert a hormonal function, DNA-binding capacity and a transcriptional signal into a transcriptional response3–7.Steroid hor- activation function all within the same molecule, allow- mone receptors, such as GR, reside in the cytoplasm in ing a one-step response to a signal that results in direct complexes with chaperone proteins. On binding to hor- effects on expression of the target gene. mones, these receptors translocate to the nucleus, where Members of the nuclear-receptor superfamily are they function as homodimers to regulate the transcrip- defined by the presence of a highly conserved DNA- tion of target genes. Other receptors, such as TR and binding domain (DBD) and a less conserved C-terminal RAR, function as heterodimers with RXR. Some of these ligand-binding and dimerization domain (LBD) (BOX 1). receptors function as repressors in the absence of a ligand, Nuclear receptors are ancient proteins that have been maintaining the silence of target genes through the found in CLADES as diverse as sponges, echinoderms, recruitment of co-repressor complexes (FIG. 1a).On bind- Howard Hughes Medical tunicates, arthropods and vertebrates, and are there- ing to hormones, these receptors recruit co-activators, Institute, Department of fore believed to be present throughout the Metazoa1,2. displace the co-repressors, and thereby induce tran- Human Genetics, University Phylogenetic analysis of nuclear-receptor genes has scription of the target gene (FIG. 1a).The ability to readily of Utah School of Medicine, revealed six distinct subfamilies, defined by clusters of switch the functional state of nuclear receptors by the 15 North 2030 East, Room 5100, Salt Lake City, receptors that share significant sequence conservation simple addition of small lipophilic compounds has put Utah 84112-5331, USA. between their respective DBDs and LBDs (BOX 2).Well- these factors in the vanguard of transcriptional regula- Correpondence to known vertebrate receptors that are members of some tion studies, providing key insights into the control of K.K.-J. or C.S.T. of these subfamilies include the steroid receptors, such eukaryotic gene expression. e-mails: kirst@genetics. utah.edu; carl.thummel@ as the oestrogen and glucocorticoid receptors (ER and Nuclear receptors function as powerful regulators of genetics. utah.edu GR), the thyroid hormone receptor (TR), the retinoic diverse biological processes, including lipid and glucose doi:10.1038/nrg1581 acid receptor (RAR), and the retinoid X receptor (RXR). homeostasis, detoxification, cellular differentiation and NATURE REVIEWS | GENETICS VOLUME 6 | APRIL 2005 | 311 © 2005 Nature Publishing Group REVIEWS embryonic development4,5.Consistent with these crucial This review focuses on recent genetic studies of the regulatory roles, mutations in nuclear receptors are nuclear-receptor superfamily of D. melanogaster in the associated with many common and lethal human disor- context of their respective mammalian homologues, ders, including cancer, diabetes and heart disease4,5.As a and identifies common themes and divergent functions. consequence, extensive efforts by the pharmaceutical We discuss how maturation is controlled by hormones industry have focused on modulating nuclear-receptor and nuclear receptors in insects and vertebrates, and function by developing new and specific agonists and describe how studies of 20E signalling in D. melanogaster antagonists, resulting in the production of leading drugs have provided a molecular framework for understand- that are currently on the market. ing the genetic regulation of maturation. We discuss the Although studies in vertebrate systems have provided developmental roles of fly nuclear receptors, presenting insights into the molecular mechanisms by which examples of remarkable functional conservation with nuclear receptors regulate transcription of target genes, their vertebrate counterparts. Recent structural studies much remains to be learned about their biological roles of LBDs and DBDs in D. melanogaster are discussed in during development. Several unique advantages have the context of their roles during development. Finally, we pushed the fruitfly, Drosophila melanogaster,to the fore- survey fly orphan receptors that are likely to have a ligand front of these studies. In contrast to the complexity of and describe our current understanding of their possible vertebrate hormone signalling pathways, D. melanogaster regulatory functions. These studies show how genetic has only two known physiologically active lipophilic hor- analysis of nuclear receptors in D. melanogaster provides a mones, the steroid hormone 20-hydroxyecdysone (20E) valuable developmental context for understanding the and the sesquiterpinoid juvenile hormone (JH). In addi- regulation and function of their vertebrate orthologues. tion, the fly genome contains only 18 nuclear-receptor In addition, we identify directions for future research that genes, as opposed to the 48 genes found in humans, with hold the promise of providing new insights into develop- these fly receptors representing all 6 of the main nuclear- ment, metabolism, longevity and physiology. receptor subfamilies (BOX 2).Taken together with the well-established genetic and genomic tools for studying The 20-hydroxyecdysone regulatory cascades the biology of D. melanogaster, this distilled set of fly One of the most remarkable hormonally triggered nuclear-receptor genes and the apparent relative simplic- developmental responses in higher organisms is the ity of insect hormone-signalling pathways define this maturation of a non-reproductive juvenile to a mature insect as an ideal model system for characterizing adult form. In humans, this is manifested by marked nuclear-receptor function and regulation. changes that occur during puberty and adolescence, Box 1 | Nuclear receptors: clues from their structure Nuclear receptors are a Dimerization Dimerization defined by the presence (weak) (strong) of common structural elements: a highly conserved DNA-binding Ligand-binding domain DNA-binding domain AF-1domain AF-2 (DBD) and a ligand-binding Hinge domain (LBD) are joined by Zinc finger 1 a flexible hinge region (panel Zinc finger 2 a). The DBD comprises two C zinc fingers. The first 4 b zinc finger provides DNA- binding specificity through a stretch of five amino acids AGGTCA AGGTCA Gene known as the P-box143.The second zinc finger harbours a relatively weak dimerization interface that allows DBDs to dimerize, but only in the presence of a target DNA molecule144.The less conserved LBD is located C-terminal to the DBD, and constitutes the principal dimerization interface of this protein family. The LBD allows different receptors to dimerize, thereby vastly expanding the repertoire of potential DNA target sequences and regulatory functions. The LBD comprises 11–13 α-helices that generally form a hydrophobic pocket for the binding of small lipophilic molecules. The activation function 1 (AF-1) domain can act in a ligand-independent fashion and is located at the N-terminus, whereas the AF-2 activation domain is located at the C-terminal end of the LBD and is often ligand-dependent. Detailed structural and molecular studies have shown that, on hormone binding, the LBD switches into an active state by rotating helix 12 into a configuration in which it can recruit transcriptional co-activators through its AF-2 domain6.Each nuclear receptor binds to a half site that is derived from, or identical to, the archetypal AGGTCA sequence (panel b). Some nuclear receptors, such as vertebrate steroidogenic factor 1 (SF-1), can function as monomers through a single response element (FIG. 1b).Other nuclear receptors function as homodimers