BIOLOGICAL OVERVIEW

The Drosophila gene Hormone receptor-like in 46 (Hr46), previously known as DHR3, is an orphan nuclear receptor. The designation "orphan" refers to the fact that unlike other nuclear receptors whose ligands are known, the ligand activating Hr46 is not known, nor is it even certain that a ligand for this protein even exists. Hr46 is homologous to the mammalian orphan nuclear receptor RORalpha (Giguere, 1995).

Before delving into the biology of Hr46 function, a few words about Drosophila nuclear receptors are in order. One might ask, why should biologists interested in human development study Drosophila molting hormones? After all, humans do not molt, leaving Drosophila molting hormones an audience limited to those investigating amphibian or reptilian molting, for example. Studies in these areas might look to the Drosophila molting heirarchy as a model system, but why would anyone else bother? This narrow viewpoint is fallacious as well, when examined in terms of current understanding of the roles played by nuclear hormones in mammalian development.

Perhaps the most well documented involvement of nuclear hormone receptors in mammalian development has to do with the regulation of Hox cluster genes. Hoxa-1 and Hoxa-2 are homologs of Drosophila genes labial and proboscipedia, respectively. In both mouse and Drosophila, these genes have been shown to play a critical role in head development. One enhancer regulating Hoxa-1 and Hoxa-2 expression contains a retinoic acid response element. Point mutations within the retinoic acid response element abolish expression in neuroepithelium caudal to rhombomere 4, supporting a natural role for retinoid responsive nuclear receptors in patterning of the hindbrain and spinal cord. Analysis of the murine Hoxa-2 rhombomere 2-specific enhancer in Drosophila embryos reveals a distinct expression domain within the fly head segments, which parallels the expression domain of proboscipedia. These results suggest an evolutionary conservation between HOM-C/Hox family members, including a conservation of certain DNA regulatory elements and possible regulatory cascades involving nuclear hormone receptors (Frasch, 1995).

Thus it is clear that aspects of a molting hierarchy, at least as far as nuclear hormone receptors, are conserved in mammals. What is this pathway and how does it function in development? The answer to this question is unexpected, and not by any means complete. In Drosophila, the source of molting hormone is the prothoracic gland. In humans, although the genes regulate growth as though there were a central source of the hormone retinoic acid, such a source might not function in hindbrain and spinal cord segmentation. Retinaldehyde dehydrogenase type 2, a major retinoic acid generating enzyme in the early embryo is expressed in mesoderm in the entire posterior part of the embryo up to the base of the headfolds, while there is no more rostral (towards the head) expression (Niederreither, 1997). Perhaps, Hox genes are regulated by nuclear hormone receptors based on intracellular signals modulated as if there were a central source of hormone. The nuclear receptors follow a similar dynamic to that found in flies and frogs, but the response may be cell autonomous and not regulated by exogenously supplied hormone. It could be that the pathway has been evolutionarily conserved, but that the external regulation has been lost. A definitive confirmation of this conclusion awaits more detailed examination of the distribution of retinoids in mammals.

The retinoid responsive nuclear receptors in mammals involved in Hox cluster regulation are only distantly related to Drosophila Ecdysone receptor. EcR is most closely related to the vertebrate Farnesoid X receptor (Mangelsdorf, 1995). Ultraspiracle, a closer homolog of mammalian retinoic acid RXR receptor, functions in Drosophila as a dimerization partner with Ecdysone receptor, the central regulator of the molting process. Perhaps a better analogy than the one given above involving molting would be the mammalian response to thyroid hormone. In this case the response is to an exogenous hormone (thyroxin). The transcription factor components are similar; thyroxin receptor (TR) plays a homologous role to the Ecdysone receptor, and RXR, the partner of TR, plays a homologous role to Ultraspiracle (Collingwood, 1997).

Hr46 represents a second tier regulator, one that acts negatively on Ecdysone receptor. Thus Hr46 plays a direct role in regulating the nuclear receptor hormones involved in Drosophila molting and whose cell autonomous regulation in mammals remains somewhat a mystery. Hr46 acts negatively on Ecdysone receptor and postively on genes expressed subsequently in the molting hierarchy. Perhaps by understanding the gene and protein interactions in Drosophila molting, clues can be discovered as to the roles of nuclear receptors in mammalian development (White, 1997).

Hr46 is termed a early-late gene. This means that it is expressed after early genes such as Ecdysone receptor and before the late hormones involved in metamorphosis. In the early stages of Drosophila metamorphosis, during the formation of pupa (the process of pupariation), prior to metamorphosis into the adult, Hr46 represses the ecdysone induction of early genes turned on by the pulse of ecdysone that triggers pupariation. Hr46 is shown to interact directly with the Ecdysone receptor. The mechanism of Hr46 repression may involve an interaction between the Hr46 and Ecdysone receptor ligand binding domains. Thus the repressive function of Hr46 does not involve binding to DNA but instead involves physical interaction with the Ecdysone receptor (White, 1997).

Hr46 also induces ßFTZF1, an orphan nuclear receptor that is essential for the appropriate response to the subsequent prepupal pulse of ecdysone. This induction requires binding of Hr46 to DNA. The DNA binding domain of Hr46 is necessary for the activating function of Hr46. Another nuclear receptor, the E75B receptor, classified as an early gene, regulates Hr46. E75B lacks a complete DNA binding domain, and inhibits the inductive function of Hr46 by forming a complex with Hr46 on the ßFTZF1 promoter, thereby providing a timing mechanism for ßFTZF1 induction that is dependent on the disappearance of E75B. Hr46 appears to bind the ßFTZF1 promoter as a monomer, since sequencing and footprinting analysis have uncovered single consensus Hr46 sites at each of these DNA sites. E75B fails to bind DNA in the absence of Hr46. Thus E75B acts like a co-repressor with Hr46, rather than as a competitor with Hr46 for DNA binding. The restricted temporal expression of E75B apparently acts as a precise timer for the onset of ßFTZF1 expression (White, 1997).

Hr46 targets a number of proteins besides EcR and ßFTZF1. Hr46 is sufficient to repress BR-C, E74A, E75A and E78B transcription. BR-C, E74A, E75A and E78B are considered early genes that are induced by ecdysone. In the repressive function Hr46 is likely to act through the Ecdysone receptor. In addition, however, direct interaction with the promoters of these genes is likely, as Hr46 is found associated with their salivary gland puffs (Lam, 1997). Hr46 thus appears to function as a switch that defines the larval-prepupal transition by arresting the early regulatory response to ecdysone at puparium formation and facilitating the induction of the betaFTZ-F1 competence factor in mid-prepupae (Lam, 1997).

GENE STRUCTURE

At least three Hr46 transcripts of approximately 5.5, 7, and 9 kb are detected, of which the 9-kb transcript is observed only during pupal development (Koelle, 1992).

Bases in 5' UTR - 227

Exons - 9

Bases in 3' UTR - 2.5 kb

PROTEIN STRUCTURE

Amino Acids - 487

Structural Domains

Hr46 contains two conserved domains characteristic of steroid receptor superfamily members. The more N-terminal and the more C-terminal of these conserved domains are referred to as the C and E regions respectively. The C regions is a 67 amino acid sequence that has been shown to function as a Zn finger DNA binding domain. The E region is an 225 amino acid domain that functions as a hormone binding domain in vertebrate receptors. Knirps, Knirps-related and Egon proteins show homology to the C but not the E region (Koelle, 1992).

ROR alpha isoforms bind to response elements consisting of a single copy of the core recognition sequence AGGTCA preceded by a 6-bp A/T-rich sequence; the distinct amino-terminal domains of each isoform influence DNA-binding specificity. ROR alpha 1 presumably binds along one face of the DNA helix as a monomer. By analogy to previous studies of the orphan receptors NGFI-B and FTZ-F1, extensive mutational analysis of the ROR alpha 1 protein shows that a domain extending from the carboxy-terminal end of the second conserved zinc-binding motif is required for specific DNA recognition. However, point mutations and domain swap experiments between ROR alpha 1 and NGFI-B demonstrate that sequence-specific recognition dictated by the carboxy-terminal extension is determined by distinct subdomains in the two receptors. These results demonstrate that monomeric nuclear receptors utilize diverse mechanisms to achieve high-affinity and specific DNA binding and that ROR alpha 1 represents the prototype for a distinct subfamily of monomeric orphan nuclear receptors (Giguere, 1995).

ROR alpha 1 and ROR alpha 2 bind as monomers to a DNA recognition sequence composed of two distinct moieties, a 3' nuclear receptor core half-site AGGTCA preceded by a 5' AT-rich sequence. Recognition of this bipartite hormone response element (RORE) requires both the zinc-binding motifs and a group of amino acid residues located at the carboxy-terminal end of the DNA-binding domain (DBD) which is referred to here as the carboxy-terminal extension. Binding of ROR alpha 1 and ROR alpha 2 to the RORE induces a large DNA bend of approximately 130 degrees that may be important for receptor function. The overall direction of the DNA bend is towards the major groove at the center of the 3' AGGTCA half-site. The presence of the nonconserved hinge region, located between the DBD and the putative ligand-binding domain (LBD) of ROR alpha, is required for maximal DNA bending. Deletion of a large portion of the amino-terminal domain (NTD) of the ROR alpha protein does not alter the DNA bend angle but shifts the DNA bend center 5' relative to the bend induced by intact ROR alpha. Methylation interference studies using the NTD-deleted ROR alpha 1 mutant indicate that some DNA contacts in the 5' AT-rich half of the RORE are also shifted 5', while those in the 3' AGGTCA half-site are unaffected. These results are consistent with a model in which the ROR alpha NTD and the nonconserved hinge region orient the zinc-binding motifs and the carboxy-terminal extension of the ROR alpha DBD relative to each other in order to achieve proper interactions with the two halves of its recognition site. Transactivation studies suggest that both protein-induced DNA bending and protein-protein interactions are important for receptor function (McBroom, 1995).

date revised: 22 March 2022  

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