Comparing nuclear receptors in worms, flies and humans

Opinion

TRENDS in Pharmacological Sciences Vol.22 No.12 December 2001

Comparing nuclear receptors in worms, flies and humans Eva Enmark and Jan-Åke Gustafsson Complete nucleotide sequences are now available for different species of the animal kingdom: Caenorhabditis elegans – a nematode, Drosophila – an insect, and humans – a mammal. Such information makes it possible to compare the set of nuclear receptors found in these organisms, and to discuss the possible reasons for the differences observed. The human genome sequencing identified few new receptors, which implies that most nuclear receptors have now been found. However, information about polymorphisms and regulating sequences, obtained through genomic sequencing, will be important for understanding receptor function and disease mechanisms. The surprisingly large number of nuclear receptors in C. elegans might have implications for the development of pharmaceuticals and the understanding of the function of these animals. By contrast, Drosophila has few nuclear receptors; however, examination of the unique nuclear receptors provides information about the function of these receptors.

Nuclear receptors constitute a large group of transcription factors that are involved in many important biological processes, including embryonic development, fertility and the regulation of cholesterol metabolism. Approximately 50 members of this protein family are known in mammals and, of these, ~28 possess known ligands or activators1 (Fig. 1). These receptors are important targets in the development of new drugs for the treatment of diseases such as diabetes, cancer and hypercholesterolemia. Recently, complete nucleotide sequences have been determined for different species of the animal kingdom: Caenorhabditis elegans (a nematode), Drosophila (an insect) and humans (a mammal). Thus, comparisons between each set of nuclear receptors found in each organism can be made and the possible reasons for such differences can be discussed. Nuclear receptors in humans: preliminary results of genomic sequencing

Eva Enmark Jan-Åke Gustafsson* Depts of Biosciences and Medical Nutrition, Karolinska Institute, NOVUM, S-14186 Huddinge, Sweden. *e-mail: jan-ake. [email protected]

The human genome is now more or less completely sequenced2,3, which marks a breakthrough in terms of nuclear receptor research. Recently, a preliminary version of the sequence completed by Celera was made available to large parts of the academic community, and a parallel effort has also been conducted at public research centers. From these results, it is clear that few nuclear receptors have evaded detection by conventional cloning methods, as judged from searches based on both DBD http://tips.trends.com

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and LBD homologies. The only new sequences detected to date by the sequencing efforts represent a previously unknown subtype of the farnesoid X receptor (FXR) (NR1H4) and another subtype of RevErbA (NR1D1)4. The available sequences of these two receptors, however, contain several termination codons, and are thus likely to represent pseudogenes. The sequencing results available at present are not yet complete, and there is still a slight possibility that nuclear receptors other than the 50 already identified will be found. At first sight the results of the sequencing efforts might seem a great disappointment. However, although no completely new receptors might remain to be discovered, the new sequence data will be very useful for several reasons. For example, many hitherto unknown splice variants of nuclear receptors have been identified in the new data bank. Furthermore, because all promoter regions of the receptors are now available, their transcriptional regulation can be studied in a systematic manner. Finally, a very important part of Celera’s genomic sequencing efforts is the investigation of naturally occurring variations among individuals, studies made possible by construction of a so-called single nucleotide polymorphism (SNP) database, where a very large amount of data about such individual variations at the DNA level is stored. To date, approximately two million such variations have been identified: that is, several polymorphisms in every existing gene. This information is of great value in identifying links between diseases with genetic components and mutations in individual genes. Only a few studies concerning links between nuclear receptor polymorphisms and human disease have been carried out. The most promising results so far come from studies of the peroxisome proliferator activated receptors (PPARs), and possible links to insulin sensitivity and/or obesity5,6. Some encouraging studies have also shown that the protein short heterodimer partner (SHP) (NR0B2) is probably hypervariable, and is possibly connected to human obesity7. Nuclear receptors in C. elegans and Drosophila

The small nematode C. elegans was the first multicellular organism whose genome was completely sequenced, a task that was completed in December 1998 (Ref. 8). Analysis of the sequence data provided a surprising result regarding the number of nuclear receptors in this organism. Based on similarities in the signature motif of the nuclear receptor superfamily, the DNA-binding domain, the C. elegans genome was shown to contain 270 nuclear receptors, which is more than four times the number identified in any other organism9,10. Subsequent analysis of these genes has shown that the majority is expressed at least at the RNA level11. Several receptor

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Receptor

Ligand hDAX1 hSHP hGCNF hFTF/LRH-1 hSF-1 hERβ hERα hERRα hERRγ hERRβ hAR hPR hMR hGR hRARγ hRARβ hRARα hTHRβ hTHRα hRORγ hRZRβ hRZRα hPPAR-γ hPPAR-δ hPPAR-α hRevErbAβ hRevErbAα hVDR hCAR hPXR hFXR hLXRα hLXRβ hNGFI-B hnor1 rNurr1 hTR4 hTR2 hRXRγ hRXRβ hRXRα hHNF4c hHNF4b hHNF4a hPNR hTLX hEar2 hCOUP-TFII hCOUP-TF

Fig. 1. Phylogenetic tree displaying all known mammalian nuclear receptors and their respective ligands. Receptors that possess known ligands are shown in purple whereas ‘orphan’ receptors are shown in cyan. The tree was constructed using Clustal V analysis, using the program MegAlign of the DNAStar package. For the analysis, all N-terminal domains were excluded because they differ extensively in length and also exhibit very high variability. Abbreviations: AR, androgen receptor; CAR, constitutively active receptor; COUP-TF, chicken ovalbumin upstream promoter transcription factor; DAX1, dosage sensitive sex reversal; Ear2, eel androgen receptor; ER, estrogen receptor; ERR, estrogen receptor-related receptor; FTF, fetoprotein transcription factor; FXR, farnesoid X receptor;

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Estradiol

Testosterone Progesterone Aldosterone Glucocorticoids All-trans retinoic acid Thyroid hormone

Prostaglandins, fatty acids, leukotrienes

Vitamin D3 16-Androstenol Pregnanes Bile acids Hydroxylated cholesterols

9-cis retinoic acid

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GCNF, germ cell nuclear factor; GR, glucocorticoid receptor; h, human; HNF, hepatocyte nuclear factor; LRH-1, liver receptor homolog 1; LXR, liver X receptor; MR, mineralocorticoid receptor; NGFI-B, nerve growth factor induced; nor1, nuclear orphan receptor; Nurr1, Nur-related receptor 1; PNR, photoreceptor cell-specific nuclear receptor; PPAR, peroxisome proliferator activated receptor; PR, progesterone receptor; PXR, pregnane X receptor; r, rat; RAR, retinoic acid receptor; ROR, retinoid-related orphan receptor; RXR, retinoid X receptor; RZR, RAR-related orphan receptor; SF-1, steroidogenic factor 1; SHP, short heterodimer partner; THR, thyroid hormone receptor; TLX, tailless; TR, testis receptor; VDR, vitamin D receptor.

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classes that are present in insects and mammals are also found in C. elegans. However, the majority of these receptors lack known homologs in other types of organisms. Many worms similar to C. elegans cause health problems, mainly in developing countries. For example, the trematode Schistosoma mansoni causes bilharzia (schistosomiasis). Nematodes such as pinworm, threadworm, roundworms and aschelminths, in addition to tapeworms, infect both humans and domestic animals, and are also commonly found in pets such as cats, dogs and horses. The discovery that some of these organisms possess a large number of nuclear receptors that are not found in humans opens up a novel possible strategy for the treatment of diseases caused by these parasites. For example, targeting of the nematode receptors by drugs that interfere with important functions of these receptors might result in malfunctioning and death of the nematode12. In December 1999 the complete genomic sequence of Drosophila was made public13. Drosophila has, for many decades, been a well-characterized model system for genetic analyses, and it was of great interest to compare nuclear receptors in Drosophila with those in C. elegans and humans. To the surprise of many scientists, Drosophila possesses a much smaller number of receptors than that found in C. elegans, with up to a total of 21 unique genes encoding nuclear receptors. Furthermore, all the receptors identified in Drosophila can be placed into one of the previously defined receptor classes (Nuclear Receptors Nomenclature Committee14) (Fig. 2). Of the 21 genes identified, only four were previously unknown. Two of these unknown receptors belong to the subfamily of orphan receptors related to tailless, one is the Drosophila homolog of estrogen receptor-related receptors (ERRs) and one is closely related to the mammalian orphan receptor germ cell nuclear factor (GCNF). ERR

From an evolutionary point of view, the most interesting of the previously unknown receptors in Drosophila is the homolog of ERRs because these receptors might represent evolutionary precursors of the estrogen receptors, and perhaps also of other steroid hormone receptors. One nucleotide sequence similar to that of ERRs has previously been identified in a coral, Renilla sp. (B. Blumberg, pers. commun.), making this receptor evolutionarily much older than has previously been assumed. In mammalian species three subtypes of ERR are known: ERRα, β and γ (NR3B1, 2 and 3). These subtypes have clearly distinct functions in embryonic, in addition to adult, tissues. All three subtypes of ERR in mammals can bind to estrogen response elements (EREs) as homodimers, and to steroidogenic factor 1 (SF-1) response elements (SF-REs) as monomers15. Binding of ERR to SF-REs is of great interest because http://tips.trends.com

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SF-1 (NR5A1) plays an important role in sexual differentiation and in the regulation of steroid hormone synthesis, constituting an essential regulator of the various steroidogenic enzyme genes in the adrenal cortex and gonad. Physiological ligands to these receptors have long evaded identification. However, two recent studies suggest that two well known pharmacological substances, diethylstilbestrol and 4-hydroxytamoxifen, might be activators of ERRβ and ERRγ, respectively16,17. Another study suggests that certain xeno-estrogens might act as antagonists of the constitutively active ERRα (Ref. 18), which indicates that physiological ligand(s) might regulate the activity of this receptor. GCNF

The second of the previously unknown nuclear receptors identified during sequencing of the Drosophila genome represents a homolog of the mammalian GCNF receptor (NR6A1)19. Homologs of this receptor have previously been identified in C. elegans, in addition to insects other than Drosophila. GCNF thus represents yet another ancestral receptor, with an evolutionary origin preceding that of vertebrate receptors. GCNF is unique in that only one representative of its subfamily is known in mammals, whereas all other receptor families have two or more subtypes encoded by paralogous genes. The available data from Drosophila genomic sequencing suggest that two different variants of GCNF exist, which most likely represent two different splicing forms of the gene. The larger isoform encodes a 1506-amino-acid protein, making it one of the largest of the known nuclear receptors. Analysis of this amino acid sequence reveals a sequence motif in the N-terminal domain, a so-called P-loop, characteristic of ATP/GTP binding proteins, which, to our knowledge, is not known in any other receptor.This motif is conserved in all known non-mammalian GCNFs (Drosophila, C. elegans, Bombyx mori and Tenebrio molitor), which indicates that it has an important function in these proteins; the motif is otherwise most common in membrane-bound receptors. Mammalian forms of GCNF bind to DNA in the form of homodimers, whereas the GCNF homolog in B. mori is claimed to bind to DNA as a monomer20. The amino acids that are positioned towards the C-terminus from the DNA-binding domain core – which contributes to the DNA binding specificity in nuclear receptors – are conserved between B. mori and Drosophila GCNF, suggesting that the Drosophila receptor also binds to DNA as a monomer. ‘tailless’ and its relatives

Two of the previously unknown receptors in Drosophila belong to the ‘tailless’ subgroup of orphan receptors. One of these receptors probably represents a homolog of the mammalian orphan receptor called

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dros. TLX hTLX CG16801 hPNR

dros. DSF CG10296

dHNF4 hHNF4a hHNF4g

dros. 7UP dros. 7up2 hCOUP-TF hCOUP-TFII hEar2 hTR2 dros. HR78 hRXRβ hRXRγ hRXRα dros.USP hNurr1 hnor1 dros. DHR38 hNGFI-B hRevErbAα hRevErbAβ dros. E75A dros. E78A dros. Eip78C hPPAR-α hPPAR-δ hPPAR-γ hRZRα hRZRβ hRORγ dros. DHR3 hRARα hRARβ hRARγ hTHRα hTHRβ EG133E12 hGCNF dros. ECR hFXR hLXRβ hLXRα hERRα hERRβ hERRγ CG7404 hERα hERβ hGR hPR hMR hAR hSF-1 hFTF/LRH-1 dros. FTZ dros. DHR39 hVDR hPXR DHR96 hCAR dros. knirps dros. knirl dros. egon

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Fig. 2. All known nuclear receptors in humans and Drosophila. Human receptors are shown in cyan whereas Drosophila receptors are shown in purple. Abbreviations: AR, androgen receptor; CAR, constitutively active receptor; COUP-TF, chicken ovalbumin upstream promoter transcription factor; dros., Drosophila; DSF, dissatisfaction; Ear2, eel androgen receptor; ECR, ecdysteroid receptor; ER, estrogen receptor; ERR, estrogen receptor-related receptor; FTF, fetoprotein transcription factor; FXR, farnesoid X receptor; GCNF, germ cell nuclear factor; GR, glucocorticoid receptor; h, human; HNF, hepatocyte nuclear factor; LRH-1, liver receptor

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homolog 1; LXR, liver X receptor; MR, mineralocorticoid receptor; NGFI-B, nerve growth factor induced; nor1, nuclear orphan receptor; Nurr1, Nur-related receptor 1; PNR, photoreceptor cell-specific nuclear receptor; PPAR, peroxisome proliferator activated receptor; PR, progesterone receptor; PXR, pregnane X receptor; RAR, retinoic acid receptor; ROR, retinoid-related orphan receptor; RXR, retinoid X receptor; RZR, RAR-related orphan receptor; SF-1, steroidogenic factor 1; THR, thyroid hormone receptor; TLX, tailless; USP, ultraspiracle protein; TR, testis receptor; VDR, vitamin D receptor.

Opinion

TRENDS in Pharmacological Sciences Vol.22 No.12 December 2001

photoreceptor specific nuclear receptor (PNR) (NR2E3), whereas the other receptor represents a novel receptor subtype. In both mammals and insects these receptors have important functions in the CNS. The first receptor in this receptor family to be identified was termed ‘tailless’ in Drosophila and ‘TLX’ (NR2E1) in vertebrates, and is of great importance in the development of the CNS, and in the determination of anterior–posterior orientation of the early embryo21. By contrast, PNR is expressed exclusively in the retina22, and in the human this receptor is associated with certain hereditary forms of retinal degeneration23. In Drososphila yet another receptor subtype exists that lacks known homologs in mammals.This receptor is termed ‘dissatisfaction’(DSF) (NR2E4), and is one of the factors that determines sexual preferences in insects24. In evolutionary terms these receptors are also ancient, with several homologs in, for example, C. elegans. Why does C. elegans have so many receptors, and Drosophila so few?

Acknowledgements This work was supported by grants from the Swedish Medical Research Council (No. 13X-2819) and from KaroBio AB.

An obvious question following the mapping of the genomes of one nematode and one insect is why the nuclear receptor gene family has undergone this unparalleled amplification in the nematode C. elegans. To understand the relevance of this observation one must take a closer look at the biology of C. elegans. One property that distinguishes C. elegans from both mammals and insects is the extremely large proportion of the cells of the animal that are nerve cells. The complete organism has 959 cells, and no fewer than a third of these are nerve cells of different types. It is not unlikely that many of the receptors that lack counterparts in other organisms are involved in communication between these nerve cells.

References 1 Gronemeyer, H. and Laudet, V. (1995) Transcription factors 3: nuclear receptors. Protein Profile 2, 1173–1308 2 McPherson, J.D. et al. (2001) A physical map of the human genome. Nature 409, 934–941 3 Venter, J.C. et al. (2001) The sequence of the human genome. Science 291, 1304–1351 4 Maglich, J.M. et al. (2001) Complete nuclear receptor sets from the human, C. elegans and Drosophila genomes. Genome Biol. 2, 0029.1–0029.7 5 Deeb, S.S. et al. (1998) A Pro12Ala substitution in PPARγ2 associated with decreased receptor activity, lower body mass index and improved insulin sensitivity. Nat. Genet. 20, 284–287 6 Swarbrick, M.M. et al. (2001) A Pro12Ala polymorphism in the human peroxisome proliferator-activated receptor γ 2 is associated with combined hyperlipidaemia in obesity. Eur. J. Endocrinol. 144, 277–282 7 Nishigori, H. et al. (2001) Mutations in the small heterodimer partner gene are associated with mild obesity in Japanese subjects. Proc. Natl. Acad. Sci. U. S. A. 98, 575–580 8 C. elegans Sequencing Consortium (1998) Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282, 2012–2018 http://tips.trends.com

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Another obvious difference between C. elegans and other organisms is its highly permeable ‘skin’. This property is used in experiments where the nematode is soaked in solutions containing RNA, whereby the RNA is taken up and causes effects (gene knockouts) in the cells, by so-called RNA interference. The mechanism by which this occurs is still not clear; for example, does the RNA enter through the skin or, in fact, through the digestive tract? Whatever route is employed, the nematode is susceptible to foreign substances in its environment, such as pollutants, heavy metal ions and plant hormones. One possibility is that some of these unique receptors serve as mediators of environmental signals, giving the nematode the capacity to adapt to its surroundings by appropriate adjustment of gene expression patterns. Thus, an interesting task for the future is to investigate the biological function of the nuclear receptors of C. elegans, even if it turns out that they lack homologs in other parts of the animal kingdom. In particular, if it is found that some of the unique nematode receptors possess physiological ligands, this would open up new strategies for the development of drugs directed against certain pathogenic worms. Another interesting question is how widespread in other parts of the animal kingdom are C. elegans type receptors. Nuclear receptors have been isolated from many kinds of animals, including eukaryotes as distant from mammals as corals and schistosomas25. To date, only receptors belonging to the receptor classes represented in Drosophila have been identified in these species, but it is also of interest to investigate in other animals the presence of the receptors hitherto only found in C. elegans.

9 Sluder, A.E. and Maina, C.V. (2001) Nuclear receptors in nematodes: themes and variations. Trends Genet. 17, 206–213 10 Enmark, E. and Gustafsson, J-Å. (2000) Nematode genome sequence dramatically extends the nuclear receptor superfamily. Trends Pharmacol. Sci. 21, 85–87 11 Sluder, A.E. et al. (1999) The nuclear receptor superfamily has undergone extensive proliferation and diversification in nematodes. Genome Res. 9, 103–120 12 Link, E.M. et al. (2000) Therapeutic target discovery using Caenorhabditis elegans. Pharmacogenomics 1, 203–217 13 Adams, M.D. et al. (2000) The genome sequence of Drosophila melanogaster. Science 287, 185–195 14 Nuclear Receptors Committee (1999) A unified nomenclature system for the nuclear receptor subfamily. Cell 97, 1–20 15 Johnston, S.D. et al. (1997) Estrogen-related receptor α 1 functionally binds as a monomer to extended half-site sequences including ones contained within estrogen-response elements. Mol. Endocrinol. 11, 342–352 16 Tremblay, G.B. et al. (2001) Diethylstilbestrol regulates trophoblast stem cell differentiation as a ligand of orphan nuclear receptor ERR β. Genes Dev. 15, 833–838 17 Coward, P. et al. (2001) 4-Hydroxytamoxifen binds to and deactivates the estrogen-related receptor γ. Proc. Natl. Acad. Sci. U. S. A. 98, 8880–8884

18 Yang, C. and Chen, S. (1999) Two organochlorine pesticides, toxaphene and chlordane, are antagonists for estrogen-related receptor α-1 orphan receptor. Cancer Res. 59, 4519–4524 19 Chen, F. et al. (1994) Cloning of a novel orphan receptor (GCNF) expressed during germ cell development. Mol. Endocrinol. 8, 1434–1444 20 Greschik, H. et al. (1999) Characterization of the DNA-binding and dimerization properties of the nuclear orphan receptor germ cell nuclear factor. Mol. Cell Biol. 19, 690–703 21 Pignoni, F. et al. (1990) The Drosophila gene tailless is expressed at the embryonic termini and is a member of the steroid receptor superfamily. Cell 62, 151–163 22 Kobayashi, M. et al. (1999) Identification of a photoreceptor cell-specific nuclear receptor. Proc. Natl. Acad. Sci. U. S. A. 96, 4814–4819 23 Haider, N.B. et al. (2000) Mutation of a nuclear receptor gene, NR2E3, causes enhanced S cone syndrome, a disorder of retinal cell fate. Nat. Genet. 24, 127–131 24 Finley, K.D. et al. (1998) Dissatisfaction encodes a tailless-like nuclear receptor expressed in a subset of CNS neurons controlling Drosophila sexual behavior. Neuron 21, 1363–1374 25 Escriva, H. et al. (2000) Ligand binding and nuclear receptor evolution. BioEssays 22, 717–727