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Drosophila Melanogaster Ecdysone Receptor Isoforms and Ultraspiracle in Saccharomyces Cerevisiae

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Drosophila Melanogaster Ecdysone Receptor Isoforms and Ultraspiracle in Saccharomyces Cerevisiae 183 Transcriptional activity of Drosophila melanogaster ecdysone receptor isoforms and ultraspiracle in Saccharomyces cerevisiae F E Dela Cruz, D R Kirsch and J N Heinrich Molecular Genetic Screen Design, Cyanamid Agricultural Research Center, P.O. Box 400, Princeton, New Jersey 08543–0400, USA (Requests for offprints should be addressed to J N Heinrich; Email: [email protected]) (F E Dela Cruz is now at Bayer Pharmaceutical Division, Bayer Corporation, 400 Morgan Lane, West Haven, Connecticut 06516-4175, USA; D R Kirsch andJNHeinrich are now at Wyeth–Ayerst Research, CN 8000, Princeton, New Jersey 08543-8000, USA) ABSTRACT The Drosophila melanogaster ecdysone receptor (mAR) identified one chimera, composed of the (EcR) is produced in three isoforms, which mediate mAR N-terminus and the remainder from EcR developmental processes such as metamorphosis. (mAR\EcR-CDEF) that was transcriptionally silent These isoforms were expressed in Saccharomyces and inducible by Usp. In contrast, the vertebrate cerevisiae to elucidate aspects of receptor transcrip- homologue, human retinoic acid receptor (RXR), tion activity in a highly defined genetic model showed ligand-independent transcription when system. All three EcR isoforms showed ligand- co-expressed with EcRA/B but not mAR\ independent transcriptional activation of an EcR-CDEF. Therefore, RXR does not require its ecdysone reporter gene and the amount of activation partner to possess an N-terminal domain, yet is correlated with the size of the N-terminal A/B intolerant of a heterologous N-terminus. Similarly, (transactivation) domain present in the isoform: the human vitamin D receptor, which has a short EcR-B1>EcR-A>>EcR-B2. Upon co-expression N-terminal region, showed greater ligand- with ultraspiracle (Usp), transcriptional activation independent transcription in the presence of RXR was further increased with EcR-B1 or EcR-A, but than in the presence of Usp. These results reveal a was unchanged with EcR-B2 or a truncated EcR mechanistic basis for the differential activities lacking the A/B N-terminal domain (EcRA/B). among the EcR isoforms, and between Usp and Thus, the enhanced activity from Usp may depend RXR. Furthermore, they provided the foundation on the presence of an N-terminal domain of EcR. for a genetic screen to identify potential insecticides Co-expression with Usp of several chimeric recep- as well as accessory proteins for Usp and EcR. tors of the EcR and the mouse androgen receptor Journal of Molecular Endocrinology (2000) 24, 183–191 INTRODUCTION (Koeller 1992, Talbot et al. 1993). The EcRs also bind DNA in the absence of ecdysone, suggesting Insect metamorphosis is in part controlled by that unliganded EcRs also regulate target genes ecdysone, a steroid hormone. Ecdysone binds to the (Buszczak & Segraves 1998, Hall & Thummel 1998 ecdysone receptor (EcR), inducing the transcription and references therein). The EcRs are differentially of a hierarchy of genes that harbor ecdysone-specific expressed, with EcR-A being present in cells that response elements (RE) upstream of their pro- differentiate to form adult tissues while EcR-B1 and moters. The Drosophila melanogaster EcR gene EcR-B2 are predominantly expressed in larval cells encodes for three isotypes (-A, -B1 or -B2) that are fated to die. Such observations suggest that cell fate typical steroid/nuclear receptors and share common is, at least in part, controlled by EcR isoform C-terminal DNA-binding domain and ligand- expression (Talbot et al. 1993). binding domain (LBD) sequences, but have The EcR has an obligatory heterodimer partner different N-terminal A/B domain sequences called ultraspiracle (Usp). Usp is a homologue of Journal of Molecular Endocrinology (2000) 24, 183–191 Online version via http://www.endocrinology.org 0952–5041/00/024–183 2000 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 10/01/2021 02:02:13AM via free access 184 and others · Activity of ecdysone receptors in yeast the vertebrate retinoid X receptor alpha (RXR) 1991). Yeast expression plasmids (YEp) and the (Oro et al. 1990), a steroid/nuclear receptor that is a derivative containing a multiple cloning site heterodimeric partner to a growing list of nuclear (YEpc) were used to insert the open reading receptors (reviewed in Mangelsdorf & Evans 1995). frames of the receptor used. Plasmids YEpcUSP Usp and RXR can substitute for one another and YEpEcR-B1 were constructed as described in forming a heterodimeric receptor: RXR can elsewhere (Dela Cruz & Mak 1997). Plasmids heterodimerize with EcR-B1 and Usp with the YEpEcR-A and YEpEcR-B2 were made from human vitamin D receptor (huVDR) (Yao et al. YEpEcR-B1, by excising an AflII-AscI fragment, 1992). However, Usp is distinctly different from and inserting polymerase chain reaction (PCR) RXR in at least two properties. First, it does not fragments containing EcR-A and EcR-B2 specific bind retinoic acid, and is probably an orphan recep- sequences. The YEpEcRA/B plasmid was made tor, although it has been reported that Usp binds by excising from YEpEcR-B1 a BamHI-BspEI juvenile hormone III (Jones & Sharp 1997). fragment and inserting two PCR fragments. Secondly, like RXR, Usp is a global regulator of Plasmids encoding chimeras were constructed using genetic pathways, including some independent of YEpEcR-B1 and YEpmAR5 (Mak et al. 1994b). ecdysone (Segraves 1994, Sutherland et al. 1995, Plasmid YEpEcR\mAR-DE was made by excising a Hall & Thummel 1998 and references therein). SacI-BspEI fragment and replacing it with a PCR However, despite extensive searches for hetero- product derived from YEpmAR5 (mAR being dimeric partners other than EcRs, only the mouse androgen receptor). Plasmid YEpmAR\- Drosophila hormone receptor 38 (Sutherland et al. EcR-DEF was made by inserting a DraIII-BssHII 1995) has been identified. The complex roles of Usp fragment from YEpmAR5 (Mak et al. 1994b) into and EcRs in insect development may include more the SacI-BspEI site of YEpEcR-B. Plasmids diverse mechanisms than those accommodated by YEpmAR\EcR-EF and YEPmAR\EcR-E were simple models of ligand-activated gene transcription. made by cloning PCR fragments derived from Saccharomyces cerevisiae lacks steroid/nuclear EcR-B1 into YEpmAR6 (P Mak, unpublished receptors and their specific co-regulators. As a observations). Plasmid YEpmAR-D\EcR-DEF was result, yeast has been extensively employed by cloned similarly to YEpmAR\EcR-DEF except researchers in basic science and drug discovery to the DraIII-BssHII fragment was cloned into reconstitute steroid/nuclear receptor function YEpmAR6. The plasmid YEpmAR\EcR-CDEF (Garabedian 1993, Butt & Walfish 1996). Faithful was constructed by inserting a PCR product from ligand-activated target gene transcription is com- YEpmAR (Mak et al. 1994b) into YEpEcRA/B. monly observed in yeast cells expressing steroid/ Plasmids YEpmAR5 and YEpRXR are as nuclear receptor(s) through the use of reporter described (McDonnell et al. 1989, Mak et al. 1994a), plasmids with cognate REs upstream of synthetic except the copper-responsive yeast metallothionein promoters. Co-transfecting steroid-specific co- (CUP1) promoter drives expression of Usp and activator proteins and other transcription factor RXR, and the constitutive yeast glyceraldehyde-3- accessory proteins can optimize the activity of some phosphate dehydrogenase (TDH3) promoter drives receptors. Conversely, the absence of co-regulators expression of the EcRs, the chimeras, and huVDR. in yeast provides a powerful in vivo system in which the intrinsic properties of receptors and subtle dif- ferences among receptors can be investigated. Using Construction of reporter plasmids the yeast system, we identified in this study three The YEpEcRRE2-LacZ reporter plasmid (Dela Cruz novel aspects of the EcR and Usp: differences among & Mak 1997) (RE=reporter element; subscript=in EcR isoforms; a Usp specific activity; and a distinc- duplicate) was used to make YEp-UspRE2-LacZ and tion between Usp and RXR. Additionally, we con- YEp-VDRRE2 (vitamin D reporter response ele- structed a yeast strain in which Usp activity is easily ment2). A XhoI fragment from YEpEcRRE2-LacZ monitored. This strain can be used to screen for was excised and replaced with tandem duplicate chemicals with potential insecticide activity, or for enhancer sequences separated by an arbitrary six novel Usp and EcR accessory proteins. bases. For YEp-UspRE2-LacZ, the enhancer was the 21 base pair sequence corresponding to the 64 to 44 region of the chorion s15 promoter (Shea et al. MATERIALS AND METHODS 1990). For YEp-VDRRE2-LacZ, the enhancer was a 25 base pair sequence corresponding to the 262 Construction of yeast expression plasmids to 242 region of the rat 24-hydroxylase distal gene Standard methods for manipulating DNA and yeast (r24-Hlase(d)) (Kephart et al. 1996). YRpA2 has were used (Sambrook et al. 1989, Guthrie & Fink been described previously (Mak et al. 1994b). Journal of Molecular Endocrinology (2000) 24, 183–191 www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/2021 02:02:13AM via free access Activity of ecdysone receptors in yeast · and others 185 S. cerevisiae host strains 195·2 Ci/mmol (New England Nuclear, Boston, MA, USA), in the absence or presence of 1000-fold Plasmid YEpUspRE -LacZ was used in the protease 2 excess of muristerone A (Mur A). After incubation deficient strain BJ2168 which has the following for 1 h at 25 C with constant agitation, the reaction genotype: MATa leu2 trp1 ura3–52 prb1–1122 was passed through an Inotech A21 glass fiber filter pep4–3 prc1–407 gal2. Plasmid YEpEcRRE -LacZ 2 attached to the Inotech Cell Harvester (Inotech and YRpA were used in a canavanine permease 2 Biosystems Int., Rockville, MD, USA) maintained (can1) mutant yeast strain, CGY44(DC45): MAT under vacuum, washed with three volumes of ste11-1 his4–519 leu2 trp1 ura3 can1–101. Trans- TEDG, and subjected to the vacuum for 1 formations of yeast cells were performed according additional min.
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