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Hox Transcription Factor Ultrabithorax Ib Physically and Genetically Interacts with Disconnected Interacting Protein 1, a Double-stranded RNA-binding Protein

2004; Elsevier BV; Volume: 279; Issue: 25 Linguagem: Inglês

10.1074/jbc.m312842200

1083-351X

Sarah E. Bondos, Daniel J. Catanese, Xin-Xing Tan, Alicia A. Bicknell, Likun Li, Kathleen S. Matthews,

RNA Research and Splicing

The Hox protein family consists of homeodomain-containing transcription factors that are primary determinants of cell fate during animal development. Specific Hox function appears to rely on protein-protein interactions; however, the partners involved in these interactions and their function are largely unknown. Disconnected Interacting Protein 1 (DIP1) was isolated in a yeast two-hybrid screen of a 0–12-h Drosophila embryo library designed to identify proteins that interact with Ultrabithorax (Ubx), a Drosophila Hox protein. The Ubx ·DIP1 physical interaction was confirmed using phage display, immunoprecipitation, pull-down assays, and gel retardation analysis. Ectopic expression of DIP1 in wing and haltere imaginal discs malforms the adult structures and enhances a decreased Ubx expression phenotype, establishing a genetic interaction. Ubx can generate a ternary complex by simultaneously binding its target DNA and DIP1. A large region of Ubx, including the repression domain, is required for interaction with DIP1. These more variable sequences may be key to the differential Hox function observed in vivo. The Ubx ·DIP1 interaction prevents transcriptional activation by Ubx in a modified yeast one-hybrid assay, suggesting that DIP1 may modulate transcriptional regulation by Ubx. The DIP1 sequence contains two dsRNA-binding domains, and DIP1 binds double-stranded RNA with a 1000-fold higher affinity than either single-stranded RNA or double-stranded DNA. The strong interaction of Ubx with an RNA-binding protein suggests a wider range of proteins may influence Ubx function than previously appreciated. The Hox protein family consists of homeodomain-containing transcription factors that are primary determinants of cell fate during animal development. Specific Hox function appears to rely on protein-protein interactions; however, the partners involved in these interactions and their function are largely unknown. Disconnected Interacting Protein 1 (DIP1) was isolated in a yeast two-hybrid screen of a 0–12-h Drosophila embryo library designed to identify proteins that interact with Ultrabithorax (Ubx), a Drosophila Hox protein. The Ubx ·DIP1 physical interaction was confirmed using phage display, immunoprecipitation, pull-down assays, and gel retardation analysis. Ectopic expression of DIP1 in wing and haltere imaginal discs malforms the adult structures and enhances a decreased Ubx expression phenotype, establishing a genetic interaction. Ubx can generate a ternary complex by simultaneously binding its target DNA and DIP1. A large region of Ubx, including the repression domain, is required for interaction with DIP1. These more variable sequences may be key to the differential Hox function observed in vivo. The Ubx ·DIP1 interaction prevents transcriptional activation by Ubx in a modified yeast one-hybrid assay, suggesting that DIP1 may modulate transcriptional regulation by Ubx. The DIP1 sequence contains two dsRNA-binding domains, and DIP1 binds double-stranded RNA with a 1000-fold higher affinity than either single-stranded RNA or double-stranded DNA. The strong interaction of Ubx with an RNA-binding protein suggests a wider range of proteins may influence Ubx function than previously appreciated. During animal development, differential transcription regulation is key to subdivision and specification of a diverse array of tissues. Position along the anterior-posterior axis in developing metazoans is specified by the Hox transcription factor family (1Biggin M.D. McGinnis W. Development. 1997; 124: 4425-4433PubMed Google Scholar, 2Manak J.R. Scott M.P. Development. 1994; 120: 61-71Google Scholar, 3Beachy P.A. Krasnow M.A. Gavis E.R. Hogness D.S. Cell. 1988; 55: 1069-1081Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 4Vigano M.A. Rocco G.D. Zappavigna V. Mavilio F. Mol. Cell. Biol. 1998; 18: 6201-6212Crossref PubMed Scopus (37) Google Scholar, 5Weatherbee S.D. Halder G. Kim J. Hudson A. Carroll S. Genes Dev. 1998; 12: 1474-1482Crossref PubMed Scopus (266) Google Scholar, 6Bondos S.E. Tan X.-X. Crit. Rev. Eukaryotic Gene Expression. 2001; 11: 145-171Crossref PubMed Google Scholar, 7Wolberger C. Curr. Opin. Genet. Dev. 1998; 8: 552-559Crossref PubMed Scopus (47) Google Scholar). Alterations in expression patterns of Hox transcription factors can rearrange appendages, a dramatic example of their capacity to instigate developmental programs (1Biggin M.D. McGinnis W. Development. 1997; 124: 4425-4433PubMed Google Scholar). However, Hox transcription factors bind short, partially degenerate DNA binding sites via the highly conserved homeodomain, suggesting that DNA recognition solely by the homeodomain is insufficient to achieve the precise regulatory control required for Hox function in vivo (2Manak J.R. Scott M.P. Development. 1994; 120: 61-71Google Scholar). Further, members of the Hox family can activate or repress transcription, often of the same gene, depending on the cellular context (3Beachy P.A. Krasnow M.A. Gavis E.R. Hogness D.S. Cell. 1988; 55: 1069-1081Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 4Vigano M.A. Rocco G.D. Zappavigna V. Mavilio F. Mol. Cell. Biol. 1998; 18: 6201-6212Crossref PubMed Scopus (37) Google Scholar). Despite this variability, genetic and molecular studies demonstrate that Hox proteins specifically regulate a large number of genes throughout the genetic hierarchy (1Biggin M.D. McGinnis W. Development. 1997; 124: 4425-4433PubMed Google Scholar, 5Weatherbee S.D. Halder G. Kim J. Hudson A. Carroll S. Genes Dev. 1998; 12: 1474-1482Crossref PubMed Scopus (266) Google Scholar). Thus, Hox proteins require a mechanism to assimilate external information to produce differential DNA binding and transcription regulation functions. The function of many transcription factor families relies on a complex web of protein interactions to generate the requisite specificity, diversity, and reliability of transcriptional outcomes (6Bondos S.E. Tan X.-X. Crit. Rev. Eukaryotic Gene Expression. 2001; 11: 145-171Crossref PubMed Google Scholar, 7Wolberger C. Curr. Opin. Genet. Dev. 1998; 8: 552-559Crossref PubMed Scopus (47) Google Scholar, 8Massagué J. Wotton D. EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar). Indeed, the transcriptional activity of Hox proteins is influenced by homomeric and heteromeric cooperative DNA binding (9Piper D.E. Batchelor A.H. Chang C.-P. Cleary M.L. Wolberger C. Cell. 1999; 96: 587-597Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 10Chan S.-K. Pöpperl H. Krumlauf R. Mann R.S. EMBO J. 1996; 15: 2476-2487Crossref PubMed Scopus (107) Google Scholar, 11Beachy P.A. Varkey J. Young K.E. von Kessler D.P. Sun B.I. Ekker S.C. Mol. Cell. Biol. 1993; 13: 6941-6956Crossref PubMed Scopus (67) Google Scholar, 12van Dijk M.A. Murre C. Cell. 1994; 78: 617-624Abstract Full Text PDF PubMed Scopus (235) Google Scholar, 13Passner J.M. Ryoo H.D. Shen L. Mann R.S. Aggarwal A.K. Nature. 1999; 397: 714-719Crossref PubMed Scopus (273) Google Scholar) and by specific interactions with components of the transcription apparatus (14Zhu A. Kuziora M.A. J. Biol. Chem. 1996; 271: 20993-20996Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Hox proteins bind cooperatively to DNA with Extradenticle (Exd) 1The abbreviations used are: Exd, Extradenticle; Pbx, vertebrate homolog of Exd; Ubx, ultrabithorax; Ubx Ib, longest Ubx isoform; DIP1, Disconnected Interacting Protein 1; dsRBD, double-stranded RNA-binding domain; DIP1-c, longest DIP1 isoform; Disco, disconnected; Su(var)3–9, suppressor of variegation 3–9; RED1, RNA editase 1; BSA, bovine serum albumin; RNAP, RNA polymerase; GST, glutathione S-transferase; dsRNA, double-stranded RNA; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; HIV, human immunodeficiency virus. in arthropods or with Pbx in vertebrates via their YPWM motifs (10Chan S.-K. Pöpperl H. Krumlauf R. Mann R.S. EMBO J. 1996; 15: 2476-2487Crossref PubMed Scopus (107) Google Scholar, 13Passner J.M. Ryoo H.D. Shen L. Mann R.S. Aggarwal A.K. Nature. 1999; 397: 714-719Crossref PubMed Scopus (273) Google Scholar). Residues flanking this YPWM motif stabilize the protein complex and also make contacts with the DNA (13Passner J.M. Ryoo H.D. Shen L. Mann R.S. Aggarwal A.K. Nature. 1999; 397: 714-719Crossref PubMed Scopus (273) Google Scholar, 15Shanmugam K. Featherstone M.A. Saragovi H.U. J. Biol. Chem. 1997; 272: 19081-19087Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Thus, interactions with heterologous proteins may provide the functional specificity requisite for Hox function in vivo. Despite the potential importance of these interactions, very little is known about the range of Hox protein partners or the role of these partners in development. Ultrabithorax (Ubx) is a Hox protein that orchestrates development of the posterior thorax of Drosophila melanogaster. Ubx specifies parasegments 5 and 6 and contributes to the differentiation of more posterior segments (6Bondos S.E. Tan X.-X. Crit. Rev. Eukaryotic Gene Expression. 2001; 11: 145-171Crossref PubMed Google Scholar, 16Lewis E.B. Nature. 1978; 276: 565-570Crossref PubMed Scopus (2601) Google Scholar, 17Beachy P.A. Helfand S.L. Hogness D.S. Nature. 1985; 313: 545-551Crossref PubMed Scopus (219) Google Scholar, 18Christen B. Bienz M. Mech. Dev. 1992; 39: 73-80Crossref PubMed Scopus (11) Google Scholar, 19Graba Y. Aragnol D. Laurenti P. Garzino V. Charmot D. Berenger H. Pradel J. EMBO J. 1992; 11: 3375-3384Crossref PubMed Scopus (60) Google Scholar, 20Castelli-Gair J. Greig S. Micklem G. Akam M. Development. 1994; 120: 1983-1995PubMed Google Scholar). Within these regions, Ubx influences midgut, central nervous system, peripheral nervous system, leg, and haltere development (6Bondos S.E. Tan X.-X. Crit. Rev. Eukaryotic Gene Expression. 2001; 11: 145-171Crossref PubMed Google Scholar, 18Christen B. Bienz M. Mech. Dev. 1992; 39: 73-80Crossref PubMed Scopus (11) Google Scholar, 19Graba Y. Aragnol D. Laurenti P. Garzino V. Charmot D. Berenger H. Pradel J. EMBO J. 1992; 11: 3375-3384Crossref PubMed Scopus (60) Google Scholar, 20Castelli-Gair J. Greig S. Micklem G. Akam M. Development. 1994; 120: 1983-1995PubMed Google Scholar). Several functional domains have been identified in Ubx (Fig. 1). Our previous work has shown that a transcription activation domain resides between residues 159 and 242, and transcription activation levels are further enhanced by inclusion of amino acids 68–158 (21Tan X.-X. Bondos S. Li L. Matthews K.S. Biochemistry. 2002; 41: 2774-2785Crossref PubMed Scopus (21) Google Scholar). Immediately following the activation domain is the YPWM motif and the homeodomain. The C terminus of the protein contains a glutamine/alaninerich region that contributes to transcription repression (22Ronshaugen M. McGinnis N. McGinnis W. Nature. 2002; 415: 914-917Crossref PubMed Scopus (298) Google Scholar, 23Galant R. Carroll S.B. Nature. 2002; 415: 910-913Crossref PubMed Scopus (262) Google Scholar). Although aspects of the transcription regulatory domains have been identified, the mechanisms, including protein interactions, that mediate these functions remain unknown. To identify potential protein partners that may modulate the function of Ubx, we have performed a yeast two-hybrid screen. A partner prominently identified in our two-hybrid cDNA library screen was Disconnected Interacting Protein 1 (DIP1), which contains a putative nuclear localization signal and two double-stranded RNA-binding domains (dsRBDs). Global overexpression of DIP1 causes embryonic lethality (24De Sousa D. Mukhopadhyay M. Pelka P. Zhao X. Dey B.K. Robert V. Pélisson A. Bucheton A. Campos A.R. J. Biol. Chem. 2003; 278: 38040-38050Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). However, specific expression of DIP1 in the eye-antennal imaginal disc generates duplications of antenna and mouth structures and loss of the arista, reflecting aberrant expression of homothorax and spalt major and duplication of the distalless expression domain (24De Sousa D. Mukhopadhyay M. Pelka P. Zhao X. Dey B.K. Robert V. Pélisson A. Bucheton A. Campos A.R. J. Biol. Chem. 2003; 278: 38040-38050Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). DIP1 also interacts with other transcription regulators, including Disco and Su(var)3–9 (24De Sousa D. Mukhopadhyay M. Pelka P. Zhao X. Dey B.K. Robert V. Pélisson A. Bucheton A. Campos A.R. J. Biol. Chem. 2003; 278: 38040-38050Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 25Krauss V. O'Grady M. Hoffmann J. Schotta G. Fischer A. Kuhfittig S. Dorn R. Grigliatti T.A. Reuter G. 17th European Drosophila Research Conference in Edinburgh (UK). 2001; (F8, September 1–5, 2001, Edinburgh, UK)Google Scholar). Thus DIP1, like Ubx, can alter transcription regulation to impact developmental programs, although the molecular mechanism is unknown. Herein, we detail a physical interaction between Ubx and DIP1 using modified yeast two-hybrid methods, phage display, immunoprecipitation, GST pull-down assays, and gel retardation supershifts. In addition, a genetic interaction between ubx and DIP1 is established. Formation of a DIP1 ·Ubx ·DNA ternary complex and the demonstration that DIP1 represses transcription activation by Ubx in a modified yeast one-hybrid assay suggest a physiological role for the interaction. Intriguingly, DIP1 is shown to bind dsRNA with extremely high affinity. The strong interaction of a Hox protein with an RNA-binding protein implies that influences on Hox activity may be wider than previously appreciated. The “interaction trap” method developed by the Brent Laboratory for yeast two-hybrid screens (26Golemis E.A. Serebriiskii I. Finley Jr., R.L. Kolonin M.G. Gyuris J. Brent R. Current Protocols in Molecular Biology. John Wiley & Sons, 1999: 20.1.1-20.1.40Google Scholar) has been utilized in these experiments. Matchmaker LexA two-hybrid system (Clontech) was used, and a 0–12 h D. melanogaster embryonic cDNA library (27Finley Jr., R.L. Thomas B.J. Zipursky S.L. Brent R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3011-3015Crossref PubMed Scopus (82) Google Scholar) was a gift from Roger Brent (The Molecular Sciences Institute, Berkeley, CA). The plasmid pLexA-UbxΔN216 (21Tan X.-X. Bondos S. Li L. Matthews K.S. Biochemistry. 2002; 41: 2774-2785Crossref PubMed Scopus (21) Google Scholar) contains coding sequences for amino acids 216–389 of the Ubx Ib isoform fused to the LexA DNA binding domain, a construct initially used to eliminate transcriptional activation by Ubx Ib alone. Using standard methods (28Gietz R.D. Schiestl R.H. Yeast. 1991; 7: 253-263Crossref PubMed Scopus (368) Google Scholar, 29Chen D.-C. Yang B.-C. Kuo T.-T. Curr. Genet. 1992; 21: 83-84Crossref PubMed Scopus (586) Google Scholar), the yeast strain EGY48 was stably co-transformed with the plasmid pLexA-UbxΔN216 and the reporter plasmid p8op-lacZ prior to the introduction of the D. melanogaster embryonic cDNA library fused to the B42 activation domain in the pJG42AD vector. Transformants were plated on SD minimal medium lacking uracil, histidine, and tryptophan and containing galactose as the carbon source. Positive colonies were selected by blue color, and DNA was isolated and sequenced. Full-length constructs of Ubx Ib and DIP1-c isoforms were also utilized in this assay. The Ubx Ib ·DIP1-c interaction was reassayed in the yeast two-hybrid system using Ubx proline mutant 1 (21Tan X.-X. Bondos S. Li L. Matthews K.S. Biochemistry. 2002; 41: 2774-2785Crossref PubMed Scopus (21) Google Scholar), a full-length Ubx Ib construct incapable of transcription activation. Finally, the yeast two-hybrid screen was repeated using pLexA-DIP1 and pJG42AD-Ubx. In this reverse yeast two-hybrid assay, yeast were plated on medium containing glucose to inhibit expression of the toxic Ubx-B42 chimera. Once the colonies were well formed, they were replica-plated onto galactose plates to induce protein expression. The overlapping cDNA and expressed sequence tags from the Berkeley FlyBase were aligned and combined using DNA Strider 1.2. Homology searches of DIP1 were completed using the Prosite Data base (30Hofmann K. Bucher P. Falquet L. Bairoch A. Nucleic Acids Res. 1999; 27: 215-219Crossref PubMed Scopus (1005) Google Scholar) and the Blast program (31Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59926) Google Scholar). The T7Select Phage Display System (Novagen) was used according to the manual. DIP1-c, the longest isoform, was cloned into T7Select 1–1b vector between the EcoRI and XhoI restriction sites and was fused to the region encoding the C terminus of the 10B capsid protein gene of T7 phage to display, on average, ≤1 DIP1-c protein on each product phage as a fusion protein. Phage were amplified, and a specific amount of phage lysate containing either 500 or 1,000 phages was applied to the surface of a Petri dish upon which cell extracts containing biotinylated Ubx Ib had been immobilized (see below). Controls utilized cell extracts with vector lacking the Ubx Ib coding region. The dish was washed five times with 100 mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.1% Tween 20. Phage that had been captured by Ubx Ib were eluted with 1% SDS. Two 20-min elutions at room temperature were collected and were diluted 20-fold with LB medium. The phage lysate was titered with Escherichia coli BLT5615 both before and after biopanning. These data were used to calculate the percentage of phage retained. Ubx Ib, cloned into pET3c between the NdeI and BamHI restriction sites, was a gift from Philip A. Beachy (Johns Hopkins University). Ubx Ib was expressed in E. coli BL21(DE3)pLysS and purified as described previously (32Bondos S.E. Bicknell A. Anal. Biochem. 2003; 316: 223-231Crossref PubMed Scopus (190) Google Scholar). To produce biotinylated Ubx Ib for phage display assays, Ubx Ib was cloned into the pDW363 vector (33Tsao K.-L. DeBarbieri B. Michel H. Waugh D.S. Gene (Amst.). 1996; 169: 59-64Crossref PubMed Scopus (80) Google Scholar) between the EcoRI and BamHI restriction sites. Biotinylated Ubx Ib was expressed in E. coli AR120. Cell growth and protein induction were performed as described (32Bondos S.E. Bicknell A. Anal. Biochem. 2003; 316: 223-231Crossref PubMed Scopus (190) Google Scholar). Cell pellets from a 200-ml growth were resuspended in 2 ml of 10% sucrose, 50 mm Tris-HCl, pH 7.4, with 0.5 mm PMSF and 0.8 mg/ml lysozyme and frozen at -80 °C. The frozen cell pellet was thawed on ice and diluted with an equal volume of 10% sucrose, 50 mm Tris-HCl, pH 7.4, 0.8 m NaCl, 20 mm EDTA, 4 mm DTT, and 10 μl of 100 mm PMSF. The cells were incubated on ice for 45 min. Subsequently, 4 μl of 10 mg/ml DNase I was added before a further 30-min incubation at room temperature. The slurry was centrifuged for 30 min at 11,000 × g, and the supernatant was applied to avidin-coated Petri dishes to immobilize biotinylated Ubx Ib. The plates were washed with 100 mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.1% Tween 20, to remove all bacterial proteins prior to the addition of phage. DIP1-c was cloned between the NdeI and XhoI restriction sites in the pET28a vector (Novagen) to express an N-terminally His6-tagged DIP1-c in E. coli BL21(DE3)pLysS. Overnight Luria broth culture (8 ml) was used to inoculate each of 12 LB cultures (1 liter) and grown at 37 °C. At midlog phase, DIP1-c expression was induced with 1 mm isopropyl-β-d-thiogalactopyranoside, and the cells were harvested 2 h later. Cells collected from 2-liter growths and stored at -20 °C were thawed on ice and lysed in lysis buffer (50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 1 mm imidazole, 5% glucose, 10 mm DTT). After lysis, DNase I and RNase A (each 20 mg/ml) were added. The lysate was centrifuged at 13,000 × g for 15 min, and the supernatant was filtered before loading onto a 5-ml HiTRAP heparin column (Amersham Biosciences) at 4 °C on an Akta fast protein liquid chromatography apparatus (Amersham Biosciences). The heparin column was pre-equilibrated with Buffer A (50 mm NaH2PO4, pH 8.0, 100 mm NaCl, 5% glucose, 10 mm DTT, passed through 0.2-μm filter, and degassed) and washed with Buffer A after loading the protein. DIP1-c was eluted with an 8-column volume gradient of Buffer A to Buffer A with 1 m NaCl, and fractions were analyzed by SDS-PAGE. DIP1-c fractions were pooled and loaded onto a ∼2.5-ml Ni2+-nitrilotriacetic acid-agarose (Qiagen) column pre-equilibrated with lysis buffer at 4 °C. The column was washed with lysis buffer with 20 mm imidazole, and DIP1-c was eluted in lysis buffer with 100 mm imidazole. Fractions were analyzed by SDS-PAGE. DIP1-c fractions were dialyzed twice at 4 °C against 1-liter exchanges (1 h each) of 20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 2.5 mm CaCl2, 5% glucose, 10 mm DTT. Biotinylated thrombin (Novagen) was added at a 1:100 dilution, mixed well by inverting the tube, and allowed to cleave His6-DIP1-c for 16 h at 4 °C. The protein mixture was mixed with 100 μl of avidin (Novagen) and 500–1000 μl of Ni2+-nitrilotriacetic acid-agarose resin slurries and incubated at 4 °C for 30 min. Centrifugation at 2,200 × g for about 10 s removed the resins along with biotinylated thrombin and His6 tag released from DIP1-c. The resulting DIP1-c was dialyzed at 4 °C against two 1-liter exchanges (1 h each) of 50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 5% glucose, 10 mm DTT and stored at 4 °C for up to 1 week because freezing abrogates protein function. Every other day, solid DTT was introduced to about 100 μm to prevent oxidation effects on protein function. Equimolar aliquots of purified Ubx Ib, purified DIP1-c, 2H.10“7” anti-Ubx antibody, and 10H.7 anti-Ubx antibody were combined and incubated with gentle rocking at 4 °C overnight. Hybridoma cells lines producing these antibodies were a gift from A. Javier Lopez (The John Hopkins School of Medicine) (34Lopez A.J. Hogness D.S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9924-9928Crossref PubMed Scopus (43) Google Scholar). Control incubations included either Ubx Ib or DIP1-c alone with the anti-Ubx antibodies. Each mixture was centrifuged at 16,000 × g. The supernatant was removed, and the pellet was washed with 50 μl of phosphate-buffered saline buffer with Tween 20 and recentrifuged for 5 min to dilute any remaining supernatant. The supernatant, wash, and pellet fractions were analyzed by SDS-PAGE and Western blotting. The coding region of full-length Ubx Ib was subcloned into the pGEX-6P-2 vector (Amersham Biosciences, Inc.) via the EcoRI and XhoI restriction sites to produce pGEX-Ubx. The vector was transformed into E. coli BL21(DE3) cells. Cells were grown in 1 liter of LB at 37 °C to an absorbance at 600 nm of 0.3–0.4. Expression of GST-Ubx was induced with the addition of 1 mm isopropyl-β-d-thiogalactopyranoside followed by growth for an additional 2 h. Cells (200 ml) were harvested at 2,500 × g for 5 min, and the cell pellets were resuspended in 2 ml of 10% (w/v) sucrose, 50 mm Tris-HCl, pH 7.4, 0.8 mg/ml lysozyme, with 10 μl of 100 mm PMSF. Cell pellets were stored at -20 °C. Cell pellets thawed on ice were diluted with an equal volume of 10% (w/v) sucrose, 50 mm Tris-HCl, pH 7.4, 0.8 m NaCl, 20 mm EDTA, 4 mm DTT, with 10 μlof100 mm PMSF. After 45 min, 4 μl of 10 mg/ml DNase I were added, and the lysate was incubated at room temperature for 30 min. The slurry was centrifuged at 3,000 × g for 30 min, and the supernatant was used immediately. DIP1-c was either added as 200 μg of purified protein (full-length only, with or without His6 tag) or as an in vitro transcription/translation reaction (full-length and DIP1-c deletions, TNT T7 PCR Quick Master Mix; Promega). Template DNA for the TNT reaction was amplified by PCR in which the 5′ primer contained the sequence 5′-GGATCCTAATACGACTCACTATAGGAACAGCC(C/A)CCATGG-3′ required to initiate transcription and translation. The resulting products were cloned into the pGEM-T vector (Promega), and 2 μg of purified DNA digested with BamHI was used in two reactions for each DIP1-c deletion. The TNT products were tested for expression levels, and then divided between GST pull-down experiments with cell lysates expressing GST-Ubx or lysates from cells without the pGEX-Ubx vector as a negative control. The GST pull-down protocol was followed as described by Amersham Biosciences using Glutathione Sepharose™ 4b beads. The Drosophila line containing the UAS-DIP1-c insertion was created by Alain Pélisson (CNRS, France) and obtained from Ana Campos (McMaster University, Hamilton, Canada). The Df(1) LB6/Dp(1;Y) line was provided by Bloomington Stock Center (stock number 5999). The P{GawB}BxMS1096 Gal4 flies were acquired from Sean Carroll (University of Wisconsin, Madison, WI), and flies containing the Ubxbx-34e gypsy insertion were received from Bloomington Stock Center (stock number 457). The S880 wild-type fly line was obtained from Michael Stern (Rice University, Houston, Texas). DIP1-c was ectopically expressed in the wing and haltere using the Gal4-UAS system (35Brand A. Perrimon N. Development. 1993; 118: 401-415Crossref PubMed Google Scholar) by crossing MS1096-Gal4 virgins to UAS-DIP1-c males, producing progeny heterozygous for both elements. Progeny exhibited a fully penetrant shriveled wing phenotype, which was lethal in most males, probably due to strong expression of DIP1-c in males, since MS1096-Gal4 is on the X chromosome. To evaluate a genetic interaction, heterozygous female virgins from the previous cross were mated to males homozygous for Ubxbx-34e, although the same results are obtained when heterozygous males and Ubxbx-34e virgins are used. All progeny therefore harbored the Ubxbx-34e insertion, and progeny that also contained the Gal4 and UAS elements were identified by the shriveled wing phenotype generated by DIP1-c ectopic expression (see “Results”). Genetic interactions were also evaluated in crosses between Ubxbx-34e homozygote virgins and Dp(1;Y) males. Pictures of halteres remaining on the fly and dissected wings and halteres were acquired with a Leica MZF1111 microscope fitted with a Zeiss AxioCam MRC digital camera and using Axiovision software. Flies were submerged in ethanol to dissect wings and halteres, which were mounted in 80% glycerol. Images of dissected halteres were also acquired using a Zeiss AxioPlan 2 microscope and Metamorph software. RNA purification and Northern blotting of the S880 line of D. melanogaster followed previously described procedures (36Soehnge H. Huang X. Becker M. Whitley P. Conover D. Stern M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13262-13267Crossref PubMed Scopus (34) Google Scholar) using the entire DIP1 gene as a probe. The Ambion RETROscript kit was used for reverse transcriptase PCR, and random decamers were used for priming first strand synthesis. Primers directed at the full-length DIP1-c sequence were used for all subsequent rounds of PCR. Otherwise, protocols in the Ambion manual were followed. Poly(I)-poly(C) dsRNA and poly(C) single-stranded RNA (Amersham Biosciences) were hydrolyzed according to Ref. 37Krovat B.C. Jantsch M.F. J. Biol. Chem. 1996; 271: 28112-28119Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar and end-labeled with [γ-32P]ATP (MP Biomedicals, Inc.). The Northwestern blotting protocol (38Ho C.K. Shuman S. Virology. 1996; 217: 272-284Crossref PubMed Scopus (41) Google Scholar) was adapted as follows. His6-DIP1-c and BSA were applied to a nitrocellulose membrane (0.45-μm pore size; Schleicher & Schuell) in separate rows containing 0.43, 0.87, 1.74, 3.04, 4.35, 8.69, 13.0, and 17.4 μg of protein using dot blot microfiltration. Membranes were then incubated in 10 mm Tris-HCl, pH 8.0, 25 mm KCl, 10 mm NaCl, 10% glycerol, 0.5 mm DTT, 0.1 mm EDTA, 0.04% BSA for 1 h at room temperature with constant agitation, followed by a 2-h incubation in a rotating hybridization chamber with 1 × 105 cpm/ml 32P-labeled poly(I)-poly(C) at 2.24 × 10-10m or poly(C) at 4.95 × 10-9m. After three washes in buffer (10 min of incubation/wash), membranes were air-dried and exposed to a Fuji phosphorimaging plate for 16 h. Data were analyzed using MacBAS version 2.0 software. Several RNAs, used for gel retardation experiments, were produced by in vitro transcription. Human Immunodeficiency Virus (HIV) TAR RNA—The DNA template contained the sequence of the TAR RNA (5′-GGCAGAUCUGAGCCUGGGAGCUCUCUGCC-3′) behind the T7 RNA polymerase (RNAP) promoter (5′-TAATACGACTCACTATAG-3′). This template (1 μm), resuspended in water, was annealed with the T7 primer oligonucleotide (1.2 μm). Adenovirus VA1 RNA—The template was amplified by PCR from pVA1, a gift from Goran Akusjarvi (Uppsala University, Sweden). The primers, 5′-ATTAATACGACTCACTATAGGGGCACTCTTCCGTGGTCTGGTG-3′ and 5′-AAAAGGAGCGCTCCCCCGTTGTC-3′, were used to generate a 182-base pair product that contains the VA1 sequence under the T7 RNAP control. The PCR product was purified using the Qiagen PCR Clean-up kit, and the DNA template was resuspended in water. In Vitro Transcription—Each transcription reaction consisted of 30 mm Tris-HCl, pH 8.1, 2 mm spermidine, 0.01% Triton X-100, 25 mm MgCl2, each NTP at 4 mm, 10 mm DTT, 1 μm DNA template (HIV TAR or adenovirus VA1), and 50 μg/ml T7 RNAP (a gift from Yousif Shamoo, Rice University). The reactions were incubated at 37 °C for 3 h. To stop the reactions, an equal amount of 2× formamide solution (90% (v/v) formamide, 1× TBE (39Sambrook J. Fritsch E.F. Maniatis F. Molecular Cloning: A Laboratory Manual, 2nd Ed. 1989; : A.2.5-A.2.6, Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar), 25 mm EDTA, 0.02% bromphenol blue, 0.01% xylene cyanol) was added, and the samples were boiled for 1 min. The reactions we