Archives of Insect Biochemistry and Physiology 65:125–133 (2007) Nuclear Localization and DNA Binding of Ecdysone Receptor and Ultraspiracle M.V. Cronauer, S. Braun, Ch. Tremmel, K.-D. Kröncke, and M. Spindler-Barth* The Ecdysone receptor (EcR) is distributed between cytoplasm and nucleus in CHO cells. Nuclear localization is increased by the ligand Muristerone A. The most important heterodimerization partner Ultraspiracle (Usp) is localized predominantly in the nucleus. We used the diethylentriamine nitric oxide adduct DETA/NO, which releases NO and destroys the zinc-finger structure of nuclear receptors, to investigate whether nuclear EcR and Usp interact with DNA. If expressed separately, Usp and EcR in the absence of hormone do not interact with DNA. The hormone-induced increase in nuclear EcR is due to enhanced DNA binding. In the presence of Usp, EcR is shifted nearly quantitatively into the nucleus. Only a fraction (approximately 30%) of the heterodimer is sensitive to DETA/NO. Interaction of the heterodimer with DNA is mediated mainly by the C-domain of EcR. Deletion of the DNA-binding domain of Usp only slightly reduces nuclear localization of EcR/Usp, although the nuclear localization signal of Usp is not present anymore. The results indicate that EcR and Usp can enter the nucleus independently, but cotransport of both receptors mediated by dimerization via the ligand binding domains is possible even in the absence of hormone. Arch. Insect Biochem. Physiol. 65:125–133, 2007. © 2007 Wiley-Liss, Inc. KEYWORDS: dimerization; insect; molting hormone; NO; nuclear receptors; Zn-finger INTRODUCTION The functional ecdysone receptor is considered as a heterodimer of the ecdysteroid receptor EcR and Ultraspiracle (Usp). A prerequisite for transcriptional regulation of ecdysone-dependent genes is nuclear localization of EcR/Usp and interaction with specific DNA sequences, the hormone response elements. DNA binding is mediated by the C-domain of nuclear receptors encompassing two zinc fingers, which interact with the major groove of the DNA-helix. Intracellular localization is the result of continuous nucleocytoplasmatic shuttling (Shank et al., 2005), which may be shifted in favour of the nucleus by interaction with DNA, since proteins bound to DNA may not be fully available for the export machinery (Sackey et al., 1996). To study the influence of DNA binding on nuclear localization of EcR and Usp, we selectively destroyed the 3-D structure of the C-domain with nitric oxide (NO) known to remove zinc from the cysteine bonds in a reversible manner, without affecting the amino acid sequence (Kröncke and Carlberg, 2000; Garban et al., 2005; Hongo et al., 2005). NO inhibits DNA-binding activity in a reversible manner in different members of the nuclear steroid hormone receptor superfamily, e.g.. the vitamin D3 receptor (VDR), the retinoic X receptor (RXR), the glucocorticoid receptor (GR), the estrogen receptor α (ERα), and the androgen receptor (AR), as well as the transcription factor YY1 (Kröncke and Carlberg, 2000; Galigniana et al., 1999; Garban et al., 2005; Cronauer et al., 2006; Hongo et al., 2005). Although NO inhibits DNAbinding activity of the nuclear steroid hormone receptors, it does not affect intracellular receptor protein levels or its nuclear import as recently shown for the AR (Cronauer et al., 2006). Institute of General Zoology and Endocrinology, University of Ulm, Ulm, Germany *Correspondence to: Prof. Dr. M. Spindler-Barth, Institute of General Zoology and Endocrinology, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany. E-mail: [email protected] © 2007 Wiley-Liss, Inc. DOI: 10.1002/arch.20184 Published online in Wiley InterScience (www.interscience.wiley.com) 126 Cronauer et al. MATERIAL AND METHODS Plasmids The following plasmids were used for transfection studies: pEYFP-EcR-B1 (obtained from Dr. Ozyhar, Wrozlaw, Poland). The plasmid CFP-VP16Usp I and III was generated from DNA fragments containing the coding sequences (cds) for wild type sequences of Usp from Drosophila melanogaster. A plasmid containing the cds for VP16-Usp I and III (provided by Dr. VC Henrich, Greensboro, NC; Beatty et al., 2006) was digested with BglII and HindIII. The resulting DNA fragment was subcloned in the pECFP-C1 vector (Clontech, Takara Bio Clontech, Saint-Germain-en-Laye, France). The original AB domain of Usp was replaced by the activation domain of VP16 to overcome the inhibitory effect of the AB domain of Usp on transactivation of reporter genes in vertebrate cells (Henrich, 2005). Heterologeous Expression of EcR and USP CHO-K1 cells were cultured under standard conditions in Ham F12 medium. Cells were seeded in six-well plates (Nunc, Wiesbaden, Germany) with 3 × 105 cells per well the day prior to transfection. Transfection was performed with lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. Each well received 0.25 µg of expression plasmid. Empty vector was added when only one expression plasmid was used. The cells were incubated with plasmid DNA for 1 hr. Then the NO donor DETA/NO ((Z)-1-[N-(2aminoethyl)-N-(2-ammonioethyl)amino]diazen-1ium-1,2-diolate), which releases NO during several hours (t½ = 8 h), or denitrosylated DETA (Sigma, Deisenhofen, Germany,) which served as a control, was added (final concentration 800 µM), dissolved in DMSO. Seven hours after transfection, intracellular localization was evaluated. Microscopy Intracellular distribution of fluorescence was evaluated with an inverted microscope (Zeiss Axio- vert, Jena, Germany). To ensure a randomised approach, various samples from different regions were analysed with a total of about 50 cells. Three independent transfection experiments were performed. Western Blots Cell extracts were prepared as described previously (Nieva et al., 2005). After SDS gel electrophoresis (Laemmli, 1970) Western blots were performed according to Khyse-Andersen (1984). Gels were electroblotted on nitrocellulose membranes (BA 85, 45 µm pore size, Schleicher and Schuell, Dassel, Germany). Membranes were stained with Ponceau as a loading control and to check transblotting efficiency (data not shown). The membranes were soaked in blocking buffer (Tris-HCl, 137 mM NaCl, 0.1% Tween 20, pH 7.6, 0.02% Thimerosal) containing 3% milk powder (low fat <1%) and probed with a monoclonal anti-GFP (Clontech, Heidelberg, Germany) diluted in the same buffer 1:100. For detection of specific Western signals, peroxidase conjugated secondary antibodies (Anti-mouse IgG; no. A-5906 Sigma, Deisenhofen, Germany) diluted 1:1,000 in TBS (0.1% Tween 20) was used. After visualization with an ECL detection kit (Amersham, Freiburg, Germany) according to the instructions of the supplier, specific signals were scanned (Scanner JX-325, Sharp, 600 dpi, software ViceVersa Scan 1.2. Krystec EDV, Norderstedt, Germany) and analysed with an image analysis system. Toxicity Test The activity of mitochondrial dehydrogenases was used as a measure for toxicity (Mossman et al., 1983) of DETA and DETA/NO. Cells were seeded in 96-well plates (12,000 cells suspended in 200 µl cell culture medium/well) and allowed to adhere for 8 h. Subsequently, medium was changed and cells were cultured for 24 h in 200 µl of medium with increasing concentrations of DETA/NO. Following DETA/NO treatment, 20 µl of MTT (3-(4,5-Dimethylthiazol-2yl)-2,5 diphenylArchives of Insect Biochemistry and Physiology July 2007 doi: 10.1002/arch. Intracellular Localization of EcR and Usp tetrazoliumbromid (Sigma, Deisenhofen, Germany; 5 mg/ml in phosphate buffered saline) was added, mixed carefully and incubated for 60 min at 37°C. The medium was removed and the cells lysed with DMSO-SDS-solution (99.4 ml DMSO, 0.6 ml acetic acid, 10 g SDS). After 10 min OD560 was measured. 127 of NO (Figs. 2 and 3). Nuclear localization of YFPEcR–B1 is considerably lower and is also not affected by NO (Figs. 4 and 5). These results confirm that both Usp and EcR can enter the nucleus independently and show that neither receptor interact with DNA to a significant extent in the absence of hormone. RESULTS DNA Binding Is Not Important for Nuclear Localization of EcR and Usp The concentration of DETA/NO used does not cause significant toxicity problems during the incubation time chosen; 83 ± 3.2 % of the cells were still viable after incubation with 800 µM DETA/ NO for 6 h as determined by mitochondrial hydroxylase activity (Mossman et al., 1983). No degradation products of EcR and Usp are observed by Western blots in the presence of DETA/NO although receptor concentrations were diminished after incubation for 24 h (Fig. 1) presumably due to toxic effects after prolonged incubation times. Nuclear localization of CFP-Usp I, which is present to about 80% in the nucleus 6 h after transfection, is not significantly reduced in the presence Fig. 1. Influence of DETA/NO on EcR and Usp. Extracts from CHO-K1 cells were separated on SDS-PAGE and nuclear receptors detected with antibodies against YFP. * = CFP-Usp (calculated MW = 83.6 KD); ** = YFP-EcR (calculated MW = 122 KD). Archives of Insect Biochemistry and Physiology July 2007 doi: 10.1002/arch. Fig. 2. Intracellular localization of Usp fused with a fluorescent protein. DETA/NO or DETA were added 1 h after transfection. Localization of Usp was evaluated 6 h after addition of DETA/NO. a: Effect of DETA/NO on CFP-UspI. b: Intracellular localization of CFP-UspIII, where the Cdomain was deleted. c: Influence EcR on intranuclear localization of CFP-UspIII. E = EcR-B1, UI = CFP-UspI, UIII = CFP-UspIII; DNO = DETA/NO; H= 1 µM Muristerone A (n = 3, mean ± SD). 128 Cronauer et al. Fig. 3. Microphotographs of CHO cells showing the intracellular localization of Usp fused to a fluorescent protein. a: CFP-UspI. b: CFP-UspI+DETA/NO. c: CFP-UspI + DETA. d: CFP-UspIII. e: CFP-UspIII in the presence of EcR. f: CFP-UspIII with EcR and 1 µM Muristerone A. g: CFP- UspIII + DETA/NO. h: CFP-UspIII with EcR and DETA/ NO. i: CFP-UspIII with EcR, DETA/NO, and 1 µM Muristerone A. Experimental conditions are the same as described in Figure 2. Ligand and Heterodimerization With Usp Promote Interaction of EcR With DNA C-domain was deleted (Beatty et al., 2006). CFPUsp III is localized predominately in the cytoplasm (Figs. 2 and 3). Coexpression with EcR increases nuclear localization of CFP-Usp III (Figs. 2 and 3) and also of EcR compared to separate expression of each receptor protein (Figs. 4 and 5) as shown for YFP-EcR and CFP-Usp III in the presence of the corresponding receptor without a fused YFP-tag. We, therefore, conclude that EcR can heterodimerize with Usp in the absence of the C-domain via the ligand binding domains and that heterodimerization with EcR can compensate the missing nuclear localization signal of Usp III. It shows also that nuclear localization is not dependent on the DNAbinding ability of Usp. The decrease in nuclear localization of CFP-UspIII after addition of DETA/NO of about 40% (Fig. 2) indicates that a considerable Nuclear localization of EcR is increased in the presence of ligand (Fig. 4). This shows that ligand binding to EcR is of functional importance. The enhanced nuclear localization of EcR in the presence of Muristerone A is abolished by NO, demonstrating that hormone binding is necessary for interaction of EcR with DNA (Fig. 4). About 30% of the EcR is sensitive to DETA/NO (Fig. 4). The C-Domain of Usp Is Not Essential for Nuclear Localization of EcR. To investigate further whether DNA-binding capacity of Usp is necessary for nuclear localization of EcR, we used a Usp variant (Usp III), where the Archives of Insect Biochemistry and Physiology July 2007 doi: 10.1002/arch. Intracellular Localization of EcR and Usp 129 nitrosative stress caused by NO. The inhibitory effect of NO on the DNA-binding of zinc-finger proteins is most likely due to a reversible S-nitrosation of cysteine residues of the two cys4-type zinc fingers, which are essential for DNA-binding (Garban et al., 2005). Although NO inhibits DNA-binding activity of the nuclear steroid hormone receptors, it does not affect intracellular receptor protein levels or its nuclear import as recently shown for the AR (Cronauer et al., 2006). The nuclear localization signal of EcR located in the CD-domain is functional in the absence of zinc-fingers (Gwozdz et al., 2007), which confirms that NO does not change nuclear import. In the following study, the diethylenetriamine nitric oxide adduct Deta/NO, which spontaneously liberates NO in aqueous solutions, was used as a tool to selectively inhibit DNA binding activity of EcR and Usp. Nuclear Localization of EcR and Usp Fig. 4. Intracellular localization of YFP-EcR. DETA/NO was added 1 h after transfection. Intracellular localization was evaluated 6 h after addition of DETA/NO. a: YFPEcR. b: YFP-EcR + UspI. c: YFP-EcR + UspIII. UI = UspI; UIII = UspIII; E= YFP-EcR-B1; DNO = DETA/NO; H= 1 µM Muristerone A (n = 3, mean ± SD). fraction seems to be bound to DNA. although Usp III cannot directly interact with DNA itself. DISCUSSION NO as a Tool to Study Intranuclear Localization of Nuclear Receptors Zinc finger transcription factors are redox-sensitive and, therefore, molecular targets during Archives of Insect Biochemistry and Physiology July 2007 doi: 10.1002/arch. EcR is heterogeneously distributed between cytoplasm and nucleus (Nieva et al., 2005). The dynamic equilibrium between different intracellular localizations is influenced by various import signals, which are identified in the AB, C, and E domains and an export signal present in the E-domain of EcR (Nieva et al., 2005; Gwozdz et al., 2007), which allows leptomycin B sensitive transport of EcR into the cytoplasm (Betanska et al., 2007). The effective nuclear export seems to be responsible for the low nuclear concentration of EcR, since nuclear import is quite effective. The high nuclear content of EcR decreases with time as shown by Nieva et al. (unpublished observation). No export signal could be identified for Usp so far (Gwozdz et al., 2007), which may be responsible for the constantly high Usp concentration in nuclei. A reasonable explanation for the enhanced nuclear localization of EcR in the presence of Usp is that heterodimerization renders the nuclear export signal in the ligand-binding domain of EcR inaccessible (Betanska et al., 2007). Although both receptors can enter the nuclei independently, the heterodimerization partners mutually enhance nuclear localization. This is most 130 Cronauer et al. Fig. 5. Microphotographs of CHO cells showing the intracellular localization of YFP-EcR. a: YFP-EcR. b: YFPEcR with 1 µM Muristerone A. c: YFP-EcR with DETA/ NO. d: YFP-EcR with DETA/NO and 1 µM Muristerone A. e: YFP-EcR with UspI. f: YFP-Ecr with UspI and 1 µM Muristerone A. g: YFP-EcR with UspI and DETA/NO. h: YFP-EcR with UspI, DETA/NO, and 1 µM Muristerone A. i: YFP-EcR with UspIII (C-domain of Usp deleted). j: YFPEcR with UspIII and 1 µM Muristerone A. k: YFP-EcR with UspIII and DETA/NO. l: YFP-EcR with UspIII, DETA/ NO, and 1 µM Muristerone A. Experimental conditions are the same as described in Figure 2. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com] Archives of Insect Biochemistry and Physiology July 2007 doi: 10.1002/arch. Intracellular Localization of EcR and Usp obvious for EcR and can be demonstrated for Usp only, if the nuclear localization signal in the DNAbinding domain of Usp is deleted. In the absence of DNA, dimerization of nuclear receptors in cytoplasm is mediated by the ligandbinding domains, which apparently occurs already in the absence of hormone to a sufficient degree, although ligand binding increases dimerization of LBDs of EcR and Usp significantly (Grebe et al., 2003; Greb-Markiewicz et al., 2005). Within the nucleus, receptors are present in different compartments. Interaction of nuclear receptors with DNA seems to regulate interaction with comodulators, which depends also on the relative concentrations of the proteins involved (Voss et al., 2005). As pointed out by Voss et al. (2005), subnuclear distribution of receptor proteins provides an additional mechanism for regulation of gene expression. Generally, it is assumed that DNA binding enhances nuclear localization of receptors by reducing nuclear export as reported for several vertebrate nuclear receptors, e.g., the glucocorticoid receptor (Sackey et al., 1996). Initially, both nuclear receptors do not interact with DNA. As shown previously, ecdysteroids bind to EcR already in the absence of a heterodimerization partner (Grebe et al., 2003, 2004) leading to increased nuclear localization of EcR in the presence of ligand. Only the increased nuclear fraction of EcR observed after hormone treatment is NO sensitive, indicating association with DNA. Therefore, we conclude that liganded EcR can bind to DNA even in the absence of a dimerization partner, since the concentration of RXR, the ortholog of Usp, in CHO-K1 cells is extremely low and cotransfection with RXR has only a very weak influence on nuclear localization of EcR (Nieva et al., personal communication), This is confirmed by EMSA with EcR containing cell extracts and reporter gene assays in the absence of Usp resp RXR (Braun and Tremmel, unpublished results). As reported by Nieva et al. (2005) and Gwozdz et al. (2007), nuclear localization of EcR is greatly enhanced in the presence of the heterodimerization partner Usp, although Usp alone present predominantly in the nucleus does not interact with DNA. Archives of Insect Biochemistry and Physiology July 2007 doi: 10.1002/arch. 131 Heterodimerization with EcR seems to be required for efficient interaction with DNA. Mutational analysis of the ligand-binding domain (LBD) of EcR (Grebe et al., 2003, 2004) revealed that heterodimerization can rescue to a certain extent the deleterious effects of some mutations in the LBD of EcR, which indicates an altered three-dimensional structure of the heterodimer. An altered conformation of the LBD of EcR may be required for interaction with DNA. This view is confirmed by Nieva et al. (unpublished results), who showed that the LBD of EcR enhances DNA binding of the heterodimer. Since the only nuclear localization signal present in Usp (Gwozdz et al., 2007) is situated in the Cdomain, UspIII is localized predominantly in the cytoplasm. Interestingly, nuclear localization of EcR and UspIII is only modestly reduced, although Usp III lacks a DNA-binding domain and NiedzielaMaijka et al. (2000) report that the DNA-binding domain of Usp plays an important role in directing EcR to the hormone response element. From our results, we conclude that EcR can heterdimerize with Usp in the absence of the C-domain via the ligandbinding domains and that heterodimerization with EcR can compensate for the missing nuclear localization signal of Usp III. The reduction in nuclear localization of CFP-Usp III after addition of DETA/ NO of about 40% (Fig. 2) indicates that a considerable fraction seems to be bound to DNA mediated by the C-domain of EcR. It shows also that dimerization via the ligand-binding domains of EcR and Usp occurs already in the absence of ligand and stimulates DNA binding of the heterodimer, although UspIII itself can not directly interact with DNA. According to Gbheish et al. (2001), some actions of EcR/Usp require DNA binding of both transcription factors, whereas for other effects only DNA binding of the heterodimeric complex mediated only by the DBD of EcR is sufficient. Is NO a Functional Regulator of Nuclear Receptor Activity In Vivo? NO was used in this study as an experimental tool only. Its presence and functional importance, 132 Cronauer et al. e.g., during development in Drosophila melanogaster, raises the question of whether regulation of DNA binding of Zn-finger proteins by NO is also a physiological mechanism. It is well known that NO mediates its effects by modulating the activity of guanylyl cyclase, e.g., dilatation of blood vessels in vertebrates, but NO plays essential roles as a second messenger also in insects (Regulski et al., 2004). An influence on transcriptional regulation by the nuclear receptor E75 was shown by Reinking et al. (2005). However, in this case a heme group is present in the ligand-binding domain, which can bind NO in addition to S-nitrosylation of the cyteines of the zinc-fingers. The tight control of NO synthase activity in Drosophila underlines the importance of NO-controlled pathways. NO regulates cell proliferation in Drosophila (Kuzin et al., 1996, 2000) and in the optic lobe of Manduca sexta in an ecdysteroid hormone–controlled manner (Champlin and Truman, 2000). Nitric oxide (NO) has been shown to play an important role in a variety of physiological functions in vertebrates and invertebrates. In vertebrates, genes for three isoformes of NO synthase (NOS) have been cloned. In Drosophila, only one NOS was identified so far. The reversible regulation of protein function by S-nitrosation has led to the proposal that posttranslational modification of proteins by NO might be of physiological importance, although the role of nitrosation in vivo remains unclear. Kröncke and Carlberg (2000) proposed a functional role of NO for regulation of Zn-finger containing transcription factors activity. This would provide an additional level of complexity for regulation of gene induction by nuclear receptors. The effect of NO on DNA binding is most pronounced on receptor molecules before binding to DNA and is attenuated by hormones. This may indicate that the impact of NO is part of a regulatory network used for fine tuning of the hormone response. ACKNOWLEDGMENTS YFP-EcR was a kind gift from Dr. A. Ozyhar (Wrozlaw, Poland). Usp I and Usp-III were pro- vided by Dr. V.C. Henrich (University of Greensboro, NC). The skilful technical assistance of N. Möbius and M. Burret is gratefully acknowledged. LITERATURE CITED Beatty J, Fauth T, Callender JL, Spindler-Barth M, Henrich VC. 2006. Analysis of transcriptional activity mediated by Drosophila melanogaster ecdysone receptor isoforms in a heterologeous cell culture system. Insect Mol Biol 15:785–795. Betanska K, Nieva C, Spindler-Barth M, Spindler KD. 2007. Nucleocytoplasmic shuttling of EcR and Usp from Drosophila melanogaster: Energy requirement and interaction with exportin. Arch Insect Physiol Biochem 65:134–142 (this issue). Champlin DT, Truman JW. 2000. Ecdysteroid coordinates optic lobe neurogenesis via a nitric oxide signaling pathway. Development 127:3543–3551. Cronauer MV, Ince Y, Engers R, Rinnab L, Weidemann W, Sushek CV, Burchardt M, Kleinert H, Wiedemann J, Sies H, Ackermann R, Kröncke KD. 2006. Nitric oxide-mediated inhibition of androgen receptor activity: Possible implications for prostate cancer progression. Oncogene: in press, online publication; doi:10.1038/sj. Onc. 120 9984. Galigniana MD, Piwien-Pilipuk G, Assreuy J. 1999. Inhibition of glucocorticoid receptor binding by nitric oxide. . Mol Pharmacol 55:317–323. Garban HJ, Marquez-Garban DC, Pietras RJ, Ignarro LJ. 2005. Rapid nitric oxide-mediated S-nitrosylation of estrogen receptor: regulation of estrogen-dependent gene transcription. Proc Natl Acad Sci USA 102:2632–2636. Ghbeish N, Tsai CC, Schubiger M, Zhou JY, Evans RM, McKeown M. 2001. The dual role of ultraspiracle, the Drosophila retinoid X receptor, in the ecdysone response. Proc Natl Acad Sci USA 98:3867–3872. Greb-Markiewicz B, Fauth T, Spindler-Barth M. 2005. Ligand binding is without effect on complex formation of the ligand binding domain of the ecdysone receptor (EcR). Arch Insect Physiol Biochem 59:1–11. Grebe M, Przibilla S, Henrich VC, Spindler-Barth M. 2003. Characterization of the ligand-binding domain of the Archives of Insect Biochemistry and Physiology July 2007 doi: 10.1002/arch. Intracellular Localization of EcR and Usp ecdysteroid receptor from Drosophila melanogaster. Biol Chem 384:105–116. Grebe M, Fauth T, Spindler-Barth M. 2004. Dynamic of ligand binding to Drosophila melanogaster ecdysteroid receptor. Insect Biochem Mol Biol 34:981–989. Gwozdz T, Dutko-Gwozdz J, Nieva C, Betanska K, Orlowski M, Kowalska A, Dobrucki J, Spindler-Barth M, Spindler KD, Ozyhar A. 2007. EcR and Usp, components of the ecdysteroid nuclear receptor complex, exhibit differential distribution of molecular determinants directing subcellular trafficking. Cell Signal 19:490–503. 133 Laemmli UK.1970. Cleavage of structural proteins during the assembly of the head of baceriophage T4.Nature 227:680– 685. Mossman T. 1983. Rapid colorimetric assay for cell growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63. Niedziela-Majka A, Kochman M, Ozyhar A. 2000. Polarity of the ecdysone receptor complex interaction with the palindromic response element form the hsp27 gene promoter. Eur J Biochem 267:507–519 Henrich VC. 2005. The ecdysteroid receptor. In: Gilbert LI, Jatrou K, Gill SS, editors. Comprehensive molecular insect science vol. 3. Oxford: Elsevier, Pergamon Press. p 243–282. Nieva C, Gwozdz T, Dutko-Gwozdz J, Wiedenmann J, Spindler-Barth M, Wieczorek E, Dobrucki J, Dus D, Henrich V, Ozyhar A, Spindler KD. 2005. Ultraspiracle promotes the nuclear localization of ecdysteroid receptor in mammalian cells. Biol Chem 386:463–470. Hongo F, Garban H, Huerta-Yepez S, Vega M, Jazirehi AR, Mizutani Y, Miki T, Bonavida B. 2005. Inhibition of the transcription factor Yin Yang 1 activity by S-nitrosation. Biochem Biophys Res Commun 336:692–701. Regulski M, Stasiv Y, Tully T, Enikolopov G. 2004,Essential function of nitric oxide synthase in Drosophila. Curr Biol 14:881–882. Khyse-Andersen J. 1984. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods 10:203–209. Kröncke KD, Carlberg C. 2000. Inactivation of zinc finger transcription factors provides a mechanism for a gene regulatory role of nitric oxide. FASEB J 14:166–173. Reinking J, Lam MM, Pardee K, Sampson HM, Liu S, Yang P, Williams S, White W, Lajoie G, Edwards A, Krause HM. 2005. The drosophila nuclear receptor E75 contains heme and is gas responsive. Cell 122:195–207. Sackey, FNA., Haché, Reich T, Kwast-Welfeld, Leberve, YA. 1996. Determinants of subcellular distribution of the glucocorticoid receptor. Mol Endocrinol 10:1191–1205. Kuzin B, Roberts I, Peunova N, Enikolopov G. 1996. Nitric oxide regulates cell proliferation during Drosophila development. Cell 87:639–649. Shank, LC, Paschal, BM. 2005. Nuclear transport of steroid hormone response. Crit Rev Eukar Gene Express 15:49– 73. Kuzin B, Regulski M, Stasiv Y, Scheinker V, Tully T, Enikolopov G. 2000. Nitric oxide interacts with the retinoblastoma pathway to control eye development in Drosophila. Curr Biol 10:4594–4562. Voss TC, Cemarco IA, Booker CF, Day RN. 2005. Corepressor subnuclear organization is regulated by estrogen receptor via a mechanism that requires the DNA-binding domain. Mol Cell Endocrinol 231:33–47. Archives of Insect Biochemistry and Physiology July 2007 doi: 10.1002/arch.