GATA Factor Translation Is the Final Downstream Step in the Amino Acid/Target-of-Rapamycin-mediated Vitellogenin Gene Expression in the Anautogenous Mosquito Aedes aegypti
2006; Elsevier BV; Volume: 281; Issue: 16 Linguagem: Inglês
10.1074/jbc.m601517200
ISSN1083-351X
AutoresJong-Hwa Park, Geoffrey M. Attardo, Immo A. Hansen, Alexander S. Raikhel,
Tópico(s)Insect Resistance and Genetics
ResumoIngestion of blood is required for vector mosquitoes to initiate reproductive cycles determining their role as vectors of devastating human diseases. Nutritional signaling plays a pivotal role in regulating mosquito reproduction. Transcription of yolk protein precursor genes is repressed until mosquitoes take blood. Previously, we have shown that to signal the presence of blood in the gut, mosquitoes utilize the target-of-rapamycin (TOR) pathway. The TOR signaling pathway transduces the amino acid signal activating the major yolk protein precursor gene, vitellogenin (Vg). Here we report the identification of a GATA factor (AaGATAa) that is synthesized after a blood meal and acts as a transcriptional activator of Vg. We showed that AaGATAa bound specifically to GATA-binding sites present in the proximal promoter region of the Vg gene and positively regulated Vg expression in transfection assays. RNA interference-mediated knock down of AaGATAa transcript resulted in a significant inhibition of Vg expression in both fat-body tissue culture and blood-fed mosquitoes. AaGATAa mRNA accumulated in the fat body prior to blood feeding. However, translation of GATA was activated by blood feeding because the GATA protein increased dramatically in the fat body of blood-fed mosquitoes. This increase was also reproduced in the fat-body culture stimulated with amino acids. GATA translation was inhibited by rapamycin and cycloheximide as well as by RNA interference-mediated knock down of S6 kinase. These experiments have revealed that the TOR signaling pathway induced by nutritional signaling regulates the translation of a GATA factor, which is the specific transcriptional activator of the Vg gene. Ingestion of blood is required for vector mosquitoes to initiate reproductive cycles determining their role as vectors of devastating human diseases. Nutritional signaling plays a pivotal role in regulating mosquito reproduction. Transcription of yolk protein precursor genes is repressed until mosquitoes take blood. Previously, we have shown that to signal the presence of blood in the gut, mosquitoes utilize the target-of-rapamycin (TOR) pathway. The TOR signaling pathway transduces the amino acid signal activating the major yolk protein precursor gene, vitellogenin (Vg). Here we report the identification of a GATA factor (AaGATAa) that is synthesized after a blood meal and acts as a transcriptional activator of Vg. We showed that AaGATAa bound specifically to GATA-binding sites present in the proximal promoter region of the Vg gene and positively regulated Vg expression in transfection assays. RNA interference-mediated knock down of AaGATAa transcript resulted in a significant inhibition of Vg expression in both fat-body tissue culture and blood-fed mosquitoes. AaGATAa mRNA accumulated in the fat body prior to blood feeding. However, translation of GATA was activated by blood feeding because the GATA protein increased dramatically in the fat body of blood-fed mosquitoes. This increase was also reproduced in the fat-body culture stimulated with amino acids. GATA translation was inhibited by rapamycin and cycloheximide as well as by RNA interference-mediated knock down of S6 kinase. These experiments have revealed that the TOR signaling pathway induced by nutritional signaling regulates the translation of a GATA factor, which is the specific transcriptional activator of the Vg gene. Anautogenous mosquitoes are effective disease vectors because adult females require vertebrate blood for their reproductive cycles. The need for blood brings mosquitoes into repeated contact with multiple host organisms, making mosquitoes an effective vehicle through which pathogens can be spread from host to host. Initiation of vitellogenesis by blood feeding is a key event in the reproductive cycle of anautogenous mosquitoes. This process involves the synthesis and secretion of yolk protein precursors (YPPs) 5The abbreviations used are: YPP, yolk protein precursor; 20E, 20-hydroxyecdysone; AA, amino acid; DAPi, 4′,6-diamidino-2-phenylindole; EcR, ecdysone receptor; PBM, postblood meal; RT, reverse transcription; TOR, target-of-rapamycin; USP, ultraspiracle; Vg, vitellogenin; EMSAs, electrophoretic mobility shift assays; RNAi, RNA interference; S6K, S6 kinase; AaS6K, A. aegypti S6K; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling; PBS, phosphate-buffered saline; dsRNA, double-stranded RNA; GST, glutathione S-transferase. 5The abbreviations used are: YPP, yolk protein precursor; 20E, 20-hydroxyecdysone; AA, amino acid; DAPi, 4′,6-diamidino-2-phenylindole; EcR, ecdysone receptor; PBM, postblood meal; RT, reverse transcription; TOR, target-of-rapamycin; USP, ultraspiracle; Vg, vitellogenin; EMSAs, electrophoretic mobility shift assays; RNAi, RNA interference; S6K, S6 kinase; AaS6K, A. aegypti S6K; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling; PBS, phosphate-buffered saline; dsRNA, double-stranded RNA; GST, glutathione S-transferase. on a massive scale by the fat body, a tissue analogous to vertebrate liver and white fat tissue. The YPPs accumulate in developing oocytes (1Raikhel A.S. Dhadialla T.S. Annu. Rev. Entomol. 1992; 37: 217-251Crossref PubMed Scopus (561) Google Scholar, 2Raikhel A.S. Kokoza V.A. Zhu J. Martin D. Wang S.F. Li C. Sun G. Ahmed A. Dittmer N. Attardo G. Insect Biochem. Mol. Biol. 2002; 32: 1275-1286Crossref PubMed Scopus (172) Google Scholar). The main YPP genes activated during vitellogenesis are vitellogenin (Vg), vitellogenic carboxypeptidase (VCP), vitellogenic cathepsin B (VCB), and lipophorin (Lp). The Vg gene is the most highly expressed and best characterized YPP; it is controlled by the combined inputs of the steroid hormone 20-hydroxyecdysone (20E) cascade and nutritional signaling. The 2.1-kb upstream regulatory region of the Vg gene contains three regulatory units with binding sites for the ecdysteroid receptor complex (EcR·USP), the products of the 20E-stimulated early genes E74 and E75, GATA-type transcription factors, and several factors determining specificity of fat-body expression (3Kokoza V.A. Martin D. Mienaltowski M.J. Ahmed A. Morton C.M. Raikhel A.S. Gene (Amst.). 2001; 274: 47-65Crossref PubMed Scopus (122) Google Scholar). These regions are responsible for tissue- and stage-specific expression and hormonal enhancement of the Vg gene expression. The hormonal cascade triggered by blood feeding leads to the elevation of the endogenous steroid hormone 20E, which acts upon the Vg gene through the nuclear hormone receptor heterodimer EcR (ecdysone receptor)/USP (ultraspiracle, an insect homologue of the mammalian retinoid X receptor). The binding of EcR·USP ecdysone at an EcR·USP ecdysone-response element directly activates Vg gene transcription (4Martin D. Wang S.F. Raikhel A.S. Mol. Cell. Endocrinol. 2001; 28: 75-86Crossref Scopus (117) Google Scholar, 5Wang S.-F. Zhu J. Martin D. Raikhel A.S. Raikhel A.S. Sappington T.W. Progress in Vitellogenesis. 12, Part B. Science Publishers, Inc., Enfield, UK2005: 69-93Google Scholar). 20E stimulation also works through a hierarchy of 20E-activated intermediate genes (E74 and Broad) called early genes that code for transcription factors that bind in another unit of the regulatory region (6Sun G. Zhu J. Raikhel A.S. Mol. Cell. Endocrinol. 2004; 218: 95-105Crossref PubMed Scopus (23) Google Scholar, 7Chen L. Zhu J. Sun G. Raikhel A.S. J. Mol. Endocrinol. 2004; 33: 743-761Crossref PubMed Scopus (58) Google Scholar). In addition to hormone-specific gene regulation, transcriptional activation of the Vg gene is regulated by GATA factors. The Vg regulatory region contains 12 putative GATA-binding sites, which lead to the hypothesis that GATA factors are important for tissue specificity as well as for high levels of Vg expression (3Kokoza V.A. Martin D. Mienaltowski M.J. Ahmed A. Morton C.M. Raikhel A.S. Gene (Amst.). 2001; 274: 47-65Crossref PubMed Scopus (122) Google Scholar). GATA factors are a ubiquitous family of transcription factors, conserved from yeast to vertebrates, which regulate a variety of developmental processes (8Lowry J.A. Atchley W.R. J. Mol. Evol. 2000; 50: 103-105Crossref PubMed Scopus (209) Google Scholar, 9Patient R.K. McGhee J.D. Curr. Opin. Genet. Dev. 2002; 12: 416-422Crossref PubMed Scopus (448) Google Scholar). GATA factors have been shown to play critical roles in development, differentiation, and control of cell proliferation. GATA factors typically consist of either one or two conserved zinc finger DNA binding domains with characteristic CX2CX17CX2C motifs, followed by a basic region, whereas the rest of the protein is typically not conserved. The nonconserved regions share little or no sequence similarity, and their functions are poorly defined (8Lowry J.A. Atchley W.R. J. Mol. Evol. 2000; 50: 103-105Crossref PubMed Scopus (209) Google Scholar). These factors recognize and bind with high affinity to the DNA consensus motif (A/T) GATA(A/G) (10Orkin S.H. Blood. 1992; 80: 575-581Crossref PubMed Google Scholar). In previous work, we cloned a two-zinc finger GATA factor (AaGATAr) that has the ability to repress transcription and specifically inhibit 20E-mediated activation of the Vg gene (11Martin D. Piulachs M.D. Raikhel A.S. Mol. Cell. Biol. 2001; 21: 164-174Crossref PubMed Scopus (38) Google Scholar). It has been hypothesized that another GATA factor may be involved in positive regulation of Vg gene expression. Nutrients have long been suspected of playing a significant role in the regulation of egg development in mosquitoes. The total amino acid (AA) concentration in the mosquito hemolymph is significantly increased within 8 h after a blood meal (12Uchida K. Ohmori D. Yamakura F. Suzuki K. J. Med. Entomol. 1990; 27: 302-308Crossref PubMed Scopus (24) Google Scholar). A number of AAs are essential for oogenesis, and a steady infusion of a balanced AA mixture into the hemolymph can stimulate egg development in a variety of mosquito species (13Uchida K. Oda T. Matsuoka H. Moribayashi A. Ohmori D. Eshita Y. Fukunaga A. J. Med. Entomol. 2001; 38: 572-575Crossref PubMed Scopus (26) Google Scholar). We have shown that AAs induce transcription of the Vg gene in the fat body. Furthermore, we have discovered that a conserved pathway (target-of-rapamycin or TOR pathway) is utilized by mosquitoes to signal the presence of blood in their gut. The TOR signaling pathway transduces the AA signal to regulate Vg gene expression and activate egg development after blood feeding. We have demonstrated that RNA interference-mediated knock down of TOR resulted in blockage of these blood meal-activated events (14Hansen I.A. Attardo G.M. Park J.H. Peng Q. Raikhel A.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10626-10631Crossref PubMed Scopus (176) Google Scholar). Recently, we have established that the downstream step that is controlled by TOR is the phosphorylation of S6 kinase (15Hansen I.A. Attardo G.M. Roy S.G. Raikhel A.S. J. Biol. Chem. 2005; 280: 20565-20672Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). This finding strongly suggests that underlying the AA/TOR nutritional signaling and mediated by the phosphorylation of S6 kinase is a specific translational control adapted to the needs of anautogenous mosquitoes in initiating the blood-dependent reproductive cycle. In this work, we cloned the AaGATAa factor and demonstrated that it is an activator of Vg gene translation. Furthermore, we show that translation of the AaGATAa factor is the specific downstream step in the AA/TOR pathway that is mediated by S6 kinase. Animals and Fat-body Culture—The Aedes aegypti mosquito strain UGAL/Rockefeller was reared according to the method described by Hays and Raikhel (16Hays A.R. Raikhel A.S. Roux's Arch. Dev. Biol. 1990; 199: 114-121Crossref Scopus (84) Google Scholar). Vitellogenesis was initiated by allowing mosquitoes to feed on an anesthetized white rat 4-6 days after eclosion. The abdominal walls with adhering fat bodies (hereinafter referred to as the fat body) were incubated in an organ culture system as described previously (17Raikhel A.S. Deitsch K.W. Sappington T.W. Crampton J.M. Beard C.B. Louis C.C. The Molecular Biology of Insect Disease Vectors: A Method Manual. Chapman and Hall Ltd., London1997: 507-522Crossref Google Scholar). Cloning and Sequencing of AaGATAa—Two degenerate oligonucleotides were designed from highly conserved AA sequences of the GATA C-terminal zinc finger region. The sequences of the oligonucleotides were as follows: sense oligonucleotide, 5′-GAT ATG TCA TGY DCS AAC TG; and antisense oligonucleotide, 5′-AAT GGT GTC CTT YYT CAT CG, where Y is C or T, and D is A, T, or G. A 140-bp PCR product was obtained by degenerate RT-PCR using the above two oligonucleotides and cDNA made from previtellogenic fat-body total RNA isolated from the previtellogenic fat body. The 5′- and 3′-ends of the cDNAs were amplified by means of rapid amplification of cDNA ends PCR using a Generacer Core kit (Invitrogen). The full-length cDNA was amplified using RT-PCR with total RNA prepared from previtellogenic fat bodies and was cloned into the pCR4-TOPO vector (Invitrogen). DNA sequences were analyzed by Chromas (version 2.22) and BLAST (National Center for Biotechnology Information, National Institutes of Health). RNA Extraction, Reverse Transcription, and Real Time PCR—Total RNA from mosquitoes or dissected mosquito tissues (fat body, ovary, gut) was extracted using the Trizol method (Invitrogen) after homogenization with a motor-driven pellet pestle mixer. Aliquots of 2 μg of total RNA treated with amplification-grade RNase-free DNase I (Invitrogen) were used in cDNA synthesis reactions using an Omniscript reverse transcriptase kit (Qiagen, Valencia, CA). Reverse transcription was carried out according to the manufacturer's protocol in 40-μl reaction mixtures containing random primer or oligo(dT) primer at 37 °C for 1 h. PCR products were obtained from the PCR using a HotStarTaq Master Mix kit (Qiagen), and 2 μl of cDNA was subjected to PCR by using specific primers. PCR products were separated on a 1% agarose gel. Real time PCR was performed using the iCycler iQ system (Bio-Rad), and reactions were performed in 96-well plates with a QuantiTect SYBR PCR kit (Qiagen). Quantitative measurements were performed in triplicate and normalized to the internal control of β-actin mRNA for each sample. Primers were as follows: actin forward, 5′-GAC TAC CTG ATG AAG ATC CTG AC; actin reverse, 5′-GGC ACA GCT TCT CCT TAA TGT CAC; Vg forward, 5′-GCA GGA ATG TGT CAA GCG TGA AG; Vg reverse, 5′-ACG AGG ACG AAG AAT CGG AAG AG; AaGATAa forward, 5′-GAC GGC CTT CCA CAA GTG TAC; and AaGATAa reverse, 5′-GGT GCG AGT TGT CAA GGA ATT G. All reactions were run with 2 μl of cDNA and 0.5 μm primers per reaction. Standard curves used to quantify relative concentrations were made from 10-fold serial dilutions of cDNA pools containing high concentrations of the gene of interest or were from a plasmid standard. Real time data were collected and analyzed using iCycler iQ real time detection system software version 3.0. In Vitro Transcription and Translation—PCR product containing the entire AaGATAa cDNA was cloned into pcDNA3.1/Zeo(+) plasmid. The TnT® system (Promega, San Luis Obispo, CA) was used for in vitro transcription and translation of AaGATAa in rabbit reticulocyte lysate, utilizing the T7 promoter. To monitor the in vitro reaction, the synthesized proteins were labeled with [35S]methionine (1,200 Ci/mmol), and the radiolabeled products were visualized by means of electrophoresis and autoradiography. Electrophoretic Mobility Shift Assay—Electrophoretic mobility shift assays (EMSAs) were carried out as described previously (22Abel T. Michelson A.M. Maniatis T. Development (Camb.). 1993; 119: 623-633Crossref PubMed Google Scholar), with some modification, in a 10-μl volume containing 2 μl of TnT-expressed AaGATAa, 5× gel shift-binding buffer (Promega), and the indicated amount of competitor DNA oligonucleotides obtained from the regulatory region of the Vg gene. After 10 min of incubation at room temperature, 32P-labeled DNA probe was added, and the incubation was continued for another 10 min at room temperature. Free and protein-bound DNA was separated on a 5% nondenaturing polyacrylamide gel, and the gel was then dried and autoradiographed. DNA probes for EMSA were made by annealing together complementary oligonucleotide, and they were end-labeled by T4 polynucleotide kinase using [γ-32P]ATP (PerkinElmer Life Sciences). Site-directed Mutagenesis—GATA sequences present in the Vg gene regulatory region of the p0.6Vg-Luc vector were changed to AATA using a QuickChange site-direct mutagenesis kit (Stratagene, La Jolla, CA). For site-directed mutagenesis, complementary oligonucleotides carrying the designed nucleotide changes were used to amplify the entire p0.6Vg-Luc template. The amplified DNA mixture was treated with DpnI to digest the parental, methylated DNA; the DpnI-digested DNA was used to transform Escherichia coli XL-1 Blue, and mutated GATA-binding sites were checked by sequencing. In Vitro Transfection Assay Using Drosophila Cells—The AaGATAa expression plasmid, pAc5.1-AaGATAa, was created by cloning the full-length AaGATAa cDNA into pAc5.1/V5/His(A) (Invitrogen) under the control of the actin 5C promoter. The 0.6- or 2.1-kb regulatory region of the Vg gene was inserted into pGL3/firefly luciferase vector (Promega) to form the reporter construct p0.6Vg-Luc or p2.1Vg-Luc, respectively (4Martin D. Wang S.F. Raikhel A.S. Mol. Cell. Endocrinol. 2001; 28: 75-86Crossref Scopus (117) Google Scholar). Transfections were carried out using Drosophila Kc cells (L57-3-11), as described by Wang et al. (18Wang S.F. Miura K. Miksicek R.J. Segraves W.A. Raikhel A.S. J. Biol. Chem. 1998; 273: 27531-27540Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), with minor modification. The Drosophila L57-3-11 cell line was maintained at 27 °C in Drosophila-SFM (Invitrogen) containing 5% fetal bovine serum (19Hu X. Cherbas L. Cherbas P. Mol. Endocrinol. 2003; 17: 716-731Crossref PubMed Scopus (139) Google Scholar). Transfection was conducted with Cellfectin reagent (Invitrogen) with a DNA-to-lipid ratio of 1:4 (w/w). Luciferase enzyme activity was assayed with a dual-luciferase reporter assay system (Promega) and detected with Lumimark (Bio-Rad). β-Galactosidase enzyme activity was analyzed using a β-galactosidase enzyme assay system (Promega). RNA Interference—For production of AaGATAa-specific, double-stranded RNA (dsRNA), an AaGATAa-specific cDNA region (∼130 bp) was cloned into the pLitmus 28i vector, and AaGATAa dsRNA was produced by means of in vitro transcription with T7 RNA polymerase using the Hiscribe RNAi transcription kit (New England Biolabs). By using the same technique, a 500-bp cDNA fragment cloned in pLitmus 28i was used for the production of dsRNA against AaS6K. 0.8-kb Mal dsRNA was produced from the pLitmus 28i-Mal (containing a nonfunctional portion of the MalE gene) and used as a control in the RNA interference experiment. Approximately 1 μg of dsRNA in 0.3 μl of distilled H2O was injected into the thorax of CO2-anesthetized female mosquitoes 1 or 2 days after emergence. The dsRNA-injected mosquitoes were allowed to recover for 3 days and then were dissected for fat-body culture or fed blood to induce vitellogenesis. GATA Antibody—Antibody against a region present in both the AaGATAa and AaGATAr proteins was made using a bacterially expressed GST fusion protein. The GST fusion construct was created in the vector pGex-4T1 (GE Healthcare BioSciences Corp., Piscataway, NJ) by cloning in the region of the AaGATAa gene coding for AAs 601-861. This region is also shared by the AaGATAr protein; however, generation of an antibody specific to AaGATAa was impossible because of the short length of the sequence unique to AaGATAa. Transformed cells with GST fusion construct were tested for expression of the fusion protein by SDS-PAGE comparison of isopropyl 1-thio-β-d-galactopyranoside-induced and uninduced cell extracts. The expression of GST-fused AaGATA was tested by Western blot analysis using GST antibody and was purified by glutathione-agarose beads. The purified fraction was separated by means of SDS-PAGE, and the band containing the predicted protein was excised and sent to Cocalico Biologicals, Inc., for antibody production. Antibody was purified from antisera produced in five guinea pigs by antigen affinity column purification using a GST orientation kit (Pierce). Protein Extraction and Western Blot Analysis—Total protein extracts were prepared using an extraction method described elsewhere (20Carney G.E. Bender M. Genetics. 2000; 154: 1203-1211Crossref PubMed Google Scholar). Fifty fat bodies were ground in a cracking buffer (0.125 m Tris-HCl, pH 6.8, 5% β-mercaptoethanol, 2% SDS, 4 m urea). After 2 min of heating, the soluble protein fraction was separated by centrifugation. Preparation of nuclear extracts from fat bodies was carried out in accordance with the method described previously (21Miura K. Wang S.F. Raikhel A.S. Mol. Cell. Endocrinol. 1999; 156: 111-120Crossref PubMed Scopus (32) Google Scholar, 22Abel T. Michelson A.M. Maniatis T. Development (Camb.). 1993; 119: 623-633Crossref PubMed Google Scholar), with minor modification. Fifty fat bodies frozen in liquid nitrogen were homogenized in 1 ml of 10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, and 1 mm dithiothreitol. The homogenate was kept on ice for 15 min before being lysed by the addition of 0.6% (v/v) Nonidet P-40, and nuclei were pelleted by centrifugation at 13,000 rpm for 5 min. The nuclear pellet was resuspended in 50 μl of 20 mm HEPES, pH 7.9, 0.4 m NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, and 10% (v/v) glycerol. Nuclei were lysed by shaking for 30 min at 4 °C. The lysate was centrifuged at 13,000 rpm for 15 min to remove cell debris, and a protease inhibitor mixture (Roche Applied Science) was added to all buffers in accordance with the manufacturer's instructions. Total or nuclear protein extracts were separated on a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked in 5% nonfat dry milk in PBST (137 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.8 mm KH2PO4, pH 7.4, 0.05% Tween 20) and then incubated with GATA antibody (1:200 dilution). After the incubation of peroxidase-conjugated goat anti-guinea pig antibody (1:1000 dilution), a signal was detected using West Pico chemiluminescent substrate (Pierce). Immunohistochemistry—Dissected fat bodies were fixed overnight at 4 °C with 4% paraformaldehyde in PBS. After multiple washes with PAT solution (PBS, 1% albumin, 0.5% Triton X-100), the samples were submitted to a graded ethanol series (20, 40, 60, 80, 100, 80, 60, 40, and 20%, PBS for 15 min each). After multiple washes with PAT, the samples were treated with 3% goat serum in PAT solution for 2 hat room temperature and then washed three times with PAT. Subsequent incubation with GATA antibody (diluted 1:50) lasted 2 days at 4 °C. After multiple washes with PAT, the fat bodies were incubated overnight at 4 °C with Alexa Fluor 488 goat anti-guinea pig IgG (diluted 1:100; Invitrogen). Following thorough washing with PAT, the fat bodies were treated with DAPi (4′,6-diamidino-2-phenylindole) solution (10 μg/ml) for 1 h. After washing with PAT, the fat bodies were mounted with Gel/Mount (EMScience, Gibbstown, NJ) for microscopic analysis. Samples were examined under a Leica TCS SP2 confocal microscope (Leica Microsystems) at the Center for Plant Cell Biology/Institute for Integrative Genome Biology Core Facility. Chromosomal DNA Fragmentation Assay—Apoptosis-induced nuclear DNA fragmentation was assayed via terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) using the ApoAlert DNA fragmentation assay kit (Clontech), following the manufacturer's protocol. AaGATAa and Its Expression in the Mosquito Fat Body—A GATA factor (AaGATAa) containing a single zinc finger motif was cloned by means of degenerate and rapid amplification of cDNA ends PCR, as described under “Experimental Procedures.” The sequence data have been published at the DDBJ/EMBL/GenBank™ data base under accession number AY439009. The AaGATAa mRNA encodes a protein with 1,003 AAs and a relative molecular mass of 120 kDa. AaGATAa is an isoform of the two-zinc finger AaGATAr transcription factor and shares the majority of its sequence with it, with the exception of a short stretch of AAs that replace the N-terminal zinc finger normally found in AaGATAr. AaGATAa shares a high homology with DmGATAb (serpent) from Drosophila melanogaster (22Abel T. Michelson A.M. Maniatis T. Development (Camb.). 1993; 119: 623-633Crossref PubMed Google Scholar). In a semi-quantitative RT-PCR experiment using primers against the AaGATAa-specific region, AaGATAa mRNA was found in previtellogenic and vitellogenic mosquito fat bodies and ovaries; the levels expressed in the fat bodies were higher than those in the ovaries. After the blood meal, the mRNA levels decreased (data not shown). Real time PCR was used to obtain a detailed expression profile of AaGATAa in the fat body of the adult female mosquito. Total RNA was obtained from mosquito fat bodies dissected at 24-h intervals during the previtellogenic state and at either 6- or 12-h intervals during the vitellogenic state. Equal amounts of total RNA obtained from groups of 10 fat bodies were used to synthesize cDNA. AaGATAa mRNA levels were determined using primers designed from the AaGATAa-specific region. During previtellogenesis, the AaGATAa mRNA level gradually increased in the fat body (Fig. 1). In contrast, it decreased PBM, declining significantly between 3 and 24 h PBM, which is the time of active expression of the Vg gene. This AaGATAa expression pattern is different from that of AaGATAr, which functions as a repressor of Vg expression (23Attardo G.M. Higgs S. Klingler K.A. Vanlandingham D.L. Raikhel A.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13374-13379Crossref PubMed Scopus (57) Google Scholar). AaGATAr mRNA is expressed constitutively in previtellogenesis and increases at the end of vitellogenesis. AaGATAa Binds Specific GATA Sites in the Vg Gene Regulatory Region—The AaGATAa cDNA sequence was translated by a coupled in vitro transcription and translation system and labeled by [35S]methionine. A band of ∼120 kDa was detected by means of SDS-PAGE using 10% acrylamide gel followed by exposure to x-ray film (data not shown). This size is similar to the predicted AaGATAa molecular weight. The TnT-expressed AaGATAa was used in EMSA to identify the binding specificity of AaGATAa to the GATA-binding sites present in the Vg regulatory region. Nine GATA-like binding sites are located within the 2-kb upstream region of the Vg gene (Fig. 2A). Nine GATA oligonucleotides were designed from 12 putative GATA-binding sites within the regulatory region of the Vg gene (Fig. 2B). The box-A sequence from the Drosophila mulleri alcohol dehydrogenase gene was labeled with 32P and used as a probe. This sequence had been shown previously to bind a truncated version of the Drosophila GATAb protein (24Waltzer L. Bataille L. Peyrefittee S. Haenlin M. EMBO J. 2002; 21: 5477-5486Crossref PubMed Scopus (79) Google Scholar). The TnT-expressed AaGATAa formed a binding complex with the box-A probe (Fig. 2C, lane 2), and the specificity of this complex was confirmed by its competition with a 50-fold molar excess of the box-A DNA (Fig. 2C, 3rd lane). To demonstrate the different relative affinities of AaGATAa binding to different GATA-binding sites within the regulatory region of the Vg gene, double-stranded oligonucleotides containing GATA-binding sites presented in the regulatory region of the Vg gene were added as competitors in the EMSA. The binding complex formed by AaGATAa and the box-A probe was competed away with the unlabeled GATA 1-3 oligonucleotides (Fig. 2C, 4th to 12th lanes). To confirm the binding specificity of AaGATAa on the Vg gene regulatory region, TnT-expressed AaGATAa was tested by means of EMSA using the box-A sequence and GATA oligonucleotides as probes for direct binding (Fig. 2D). Binding affinities between AaGATAa and GATA oligonucleotide probes were highest in lanes in which GATA oligonucleotides 1 and 2 were used as probes. This result matched those obtained from the competitive EMSA (Fig. 2C). The GATA 1 and 2 oligonucleotides contained GATA-binding sites located at -115 to -118 and -161 to -164, respectively. The GATA 3 oligonucleotide was shown to contain two GATA-binding sites (-412 to -415 and -429 to -432). These results indicate that AaGATAa specifically binds to GATA-binding sites located in the proximal region (-115 to -440) of the Vg gene regulatory region (Fig. 2A). AaGATAa Activates the Vg Regulatory Region in Cell Transfection Assays—Cell transfection assays utilizing Drosophila cells were used to investigate the function of AaGATAa in Vg gene expression. The Drosophila L57-3-11 cells used in these transfection experiments are EcR-deficient and are derived from the Kc 167 cell line via parahomologous targeting (19Hu X. Cherbas L. Cherbas P. Mol. Endocrinol. 2003; 17: 716-731Crossref PubMed Scopus (139) Google Scholar). We utilized luciferase reporter constructs driven by the 0.6- or 2.1-kb regulatory region of the Vg gene (4Martin D. Wang S.F. Raikhel A.S. Mol. Cell. Endocrinol. 2001; 28: 75-86Crossref Scopus (117) Google Scholar). Co-transfection of the p0.6Vg-Luc reporter with the AaGATAa expression plasmid (pAc5.1-AaGATAa) resulted in a 40-fold enhancement of the basal luciferase activity (Fig. 3A). When the p2.1Vg-Luc reporter was co-transfected with pAc5.1-AaGATAa, the resulting luciferase activity was
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