Serpent regulates Drosophila immunity genes in the larval fat body through an essential GATA motif
1999; Springer Nature; Volume: 18; Issue: 14 Linguagem: Inglês
10.1093/emboj/18.14.4013
ISSN1460-2075
AutoresUlla-Maja Petersen, Latha Kadalayil, Klaus-Peter Rehorn, Deborah K. Hoshizaki, Rolf Reuter, Ylva Engström,
Tópico(s)Aquaculture disease management and microbiota
ResumoArticle15 July 1999free access Serpent regulates Drosophila immunity genes in the larval fat body through an essential GATA motif Ulla-Maja Petersen Ulla-Maja Petersen Department of Molecular Biology, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden Search for more papers by this author Latha Kadalayil Latha Kadalayil Department of Molecular Biology, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden Search for more papers by this author Klaus-Peter Rehorn Klaus-Peter Rehorn Institut für Genetik, Universität zu Köln, Weyertal 121, D-50931 Köln, Germany Search for more papers by this author Deborah Keiko Hoshizaki Deborah Keiko Hoshizaki Department of Biological Sciences, University of Nevada at Las Vegas, 4505 Maryland Parkway, Box 454004, Las Vegas, NV, 89154-4004 USA Search for more papers by this author Rolf Reuter Rolf Reuter University of Tübingen, Biology, Division of Animal Genetics, Auf der Morgenstelle 28, D-72076 Tübingen, Germany Search for more papers by this author Ylva Engström Corresponding Author Ylva Engström Department of Molecular Biology, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden Search for more papers by this author Ulla-Maja Petersen Ulla-Maja Petersen Department of Molecular Biology, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden Search for more papers by this author Latha Kadalayil Latha Kadalayil Department of Molecular Biology, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden Search for more papers by this author Klaus-Peter Rehorn Klaus-Peter Rehorn Institut für Genetik, Universität zu Köln, Weyertal 121, D-50931 Köln, Germany Search for more papers by this author Deborah Keiko Hoshizaki Deborah Keiko Hoshizaki Department of Biological Sciences, University of Nevada at Las Vegas, 4505 Maryland Parkway, Box 454004, Las Vegas, NV, 89154-4004 USA Search for more papers by this author Rolf Reuter Rolf Reuter University of Tübingen, Biology, Division of Animal Genetics, Auf der Morgenstelle 28, D-72076 Tübingen, Germany Search for more papers by this author Ylva Engström Corresponding Author Ylva Engström Department of Molecular Biology, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden Search for more papers by this author Author Information Ulla-Maja Petersen1, Latha Kadalayil1, Klaus-Peter Rehorn2, Deborah Keiko Hoshizaki3, Rolf Reuter4 and Ylva Engström 1 1Department of Molecular Biology, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden 2Institut für Genetik, Universität zu Köln, Weyertal 121, D-50931 Köln, Germany 3Department of Biological Sciences, University of Nevada at Las Vegas, 4505 Maryland Parkway, Box 454004, Las Vegas, NV, 89154-4004 USA 4University of Tübingen, Biology, Division of Animal Genetics, Auf der Morgenstelle 28, D-72076 Tübingen, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:4013-4022https://doi.org/10.1093/emboj/18.14.4013 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Insects possess a powerful immune system, which in response to infection leads to a vast production of different antimicrobial peptides. The regulatory regions of many immunity genes contain a GATA motif in proximity to a κB motif. Upon infection, Rel proteins enter the nucleus and activate transcription of the immunity genes. High levels of Rel protein-mediated Cecropin A1 expression previously have been shown to require the GATA site along with the κB site. We provide evidence demonstrating that the GATA motif is needed for expression of the Cecropin A1 gene in larval fat body, but is dispensable in adult fat body. A nuclear DNA-binding activity interacts with the Cecropin A1 GATA motif with the same properties as the Drosophila GATA factor Serpent. The GATA-binding activity is recognized by Serpent-specific antibodies, demonstrating their identity. We show that Serpent is nuclear in larval fat body cells and haemocytes both before and after infection. After overexpression, Serpent increases Cecropin A1 transcription in a GATA-dependent manner. We propose that Serpent plays a key role in tissue-specific expression of immunity genes, by priming them for inducible activation by Rel proteins in response to infection. Introduction The invertebrate immune system is non-clonal and in many ways analogous to the innate immune system of vertebrates. The relationship between the immune systems of such diverse phylogenetic groups as insects and mammals has become increasingly evident during the last few years (for a recent review see Medzhitov and Janeway, 1997). In insects, the immune response is induced rapidly upon injury or infection by bacteria, fungi and other pathogens (reviewed in Engström, 1999). Infections of the animal lead to a vast production of antimicrobial peptides such as attacins, cecropins, defensins and diptericin (reviewed in Hultmark, 1993; Boman, 1995; Hoffmann and Reichart, 1997). The genes coding for these peptides are regulated at the level of transcriptional initiation. Several conserved cis-elements have been found in the upstream region of genes that are known to play a role in host defence. The most prominent one is the κB-like element, which has been found in the regulatory regions of all cloned inducible insect immune genes (reviewed in Engström, 1998). In mammals, κB motifs are known to bind transcription factors belonging to the Rel family, such as NF-κB (Leonardo and Baltimore, 1989), and to influence genes of the immune system (Stancovski and Baltimore, 1997). In Drosophila, the κB-like sequence is bound by the Rel proteins dorsal, dorsal-related immunity factor (Dif) and Relish (Steward, 1987; Ip et al., 1993; Dushay et al., 1996). The phenotypes of different mutants affecting the immune response in Drosophila suggest that at least two different signalling pathways are utilized for the induction of antimicrobial peptide genes (Ip et al., 1993; Lemaitre et al., 1995, 1996; Corbo and Levine, 1996; Williams et al., 1997; Wu and Anderson, 1998). It was proposed that one pathway is responsible for the induction of the antibacterial peptides, the other for the induction of the antifungal peptides (Lemaitre et al., 1996). In addition, it was found recently that the antimicrobial response can discriminate between various classes of microorganisms (Lemaitre et al., 1997). A large number of insect immunity genes contain a GATA motif (WGATAR) situated close to the κB motif in their regulatory regions (Kadalayil et al., 1997). Both the κB and the GATA motifs have been shown to be necessary for full Drosophila Cecropin A1 (CecA1) promoter activity in transfection assays (Engström et al., 1993; Petersen et al., 1995; Kadalayil et al., 1997; Roos et al., 1998). By using P-element transformation, we demonstrate in this study that the GATA motif is crucial for the tissue-specific expression of the CecA1 gene in the larval but not in the adult fat body. The cell line malignant blood neoplasm-2 (mbn-2) is of haemocyte origin (Gateff et al., 1980). Treatment of these cells with lipopolysaccharide (LPS) induces an immune response (Samakovlis et al., 1992). In the nuclear fraction of mbn-2 cells, we previously identified a DNA-binding activity that specifically interacts with the Drosophila GATA consensus sequence (Kadalayil et al., 1997). We postulated this GATA-binding activity (GBA) to be a member of the GATA family of transcription factors. GATA transcription factors have been found in many different organisms spanning from yeast to man. In mammals, the GATA motif is found in promoters of erythroid-expressed genes, and the GATA transcription factors are essential for haematopoesis (Orkin, 1995). In Drosophila, three members of this family have been reported: pannier (pnr or dGATAa) regulates the achaete and scute complex (Ramain et al., 1993; Winick et al., 1993), serpent (srp or dGATAb) is necessary for development of many organs, among those the larval fat body and embryonic blood cells (Abel et al., 1993; Rehorn et al., 1996; Sam et al., 1996), and dGATAc, which also is implicated in organogenesis in early embryos (Lin et al., 1995). A partial srp cDNA, named abf, was cloned originally in a screen for factors binding to regulatory regions of the fat body-expressed Drosophila alcohol dehydrogenase (Adh) gene. This cDNA encoded a truncated Srp protein called the A-box-binding factor (ABF). In co-transfection assays, ABF was able to activate an artificial Adh promoter (Abel et al., 1993). Recently, Rehorn et al. (1996) cloned the Drosophila srp locus and isolated a full-length cDNA. On the basis of the biochemical properties of the GBA, we decided to test Srp experimentally as a potential trans-activator of the CecA1 gene. In this study, we show for the first time that the Srp protein is present in tissues with known immunocompetence, such as the larval fat body, where we also show that the GATA motif is essential for CecA1 expression. Larval fat body extracts contain GATA-binding activities. We demonstrate that Srp is identical to or at least a component of the GBA in both mbn-2 cell and larval fat body nuclear extracts. Finally, Srp acts as a positive regulator of the CecA1 gene and is therefore likely to play a key role in the regulation of immunity genes. Results The GATA motif is essential for expression of the CecA1 gene in the larval fat body The importance of the κB-motif for expression of Drosophila immunity genes has been studied both in transgenic animals and in transfection assays (Engström et al., 1993; Kappler et al., 1993; Meister et al., 1994; Petersen et al., 1995; Gross et al., 1996; Roos et al., 1998). The GATA motif has been demonstrated to be necessary for expression of CecA1 reporter constructs in mbn-2 cells (Kadalayil et al., 1997). To investigate the function of the GATA motif for tissue-specific expression in vivo, we generated transgenic flies carrying the pA16 CecA1–lacZ reporter construct, in which the GATA core sequence was altered to CGAG (Figure 1). Transgenic animals carrying the pA16 or the unmodified pA10 reporter construct (Figure 1) were challenged with LPS and the reporter expression was analysed using X-gal as a chromophore. Larvae carrying the pA10 reporter construct mounted strong induction of the reporter gene in the fat body upon LPS injection (Engström et al., 1993; Roos et al., 1998; Figure 2C). Staining of tissues from transgenic larvae carrying the pA16 construct was negative in both unchallenged (Figure 2A) and LPS-treated animals (Figure 2B). In LPS-injected adults, on the other hand, the pA16 construct conferred normal levels of β-gal expression in the fat body (Figure 2E and G). In uninjected adults, only tissues known to possess endogenous β-gal activity stained blue with X-gal (Figure 2D and F). We conclude that the GATA motif is necessary for expression of the CecA1 gene in the larval fat body but not needed for expression in the adult fat body. Figure 1.Schematic representation of the CecA1–lacZ fusion constructs pA10 and pA16 and the expression plasmid pAct-srp. The pA10 and pA16 constructs contain upstream regions of the CecA1 gene and the transcriptional start site (arrow) fused to an SV40 leader, providing a translational start site in-frame with the E.coli lacZ coding sequence (grey box). Numbers refer to position relative to the CAP site. Plasmid pA16 carries mutations in the GATA core sequence (GATA to CGAG). The expression plasmid pAct-srp carries the Srp cDNA under the control of the constitutive Drosophila actin 5C promoter region. Download figure Download PowerPoint Figure 2.The GATA motif is necessary for expression of the CecA1 gene in the third larval stage but not in the adult stage. (A–G) X-gal staining of tissues from transgenic larvae carrying the pA16 (A and B) or the pA10 (C) constructs and whole transgenic adults carrying the pA16 construct(D–G). (F) is an enlargement of (D), and (G) is an enlargement of (E). (B), (C) and (E) were injected with LPS (10 μg/ml) into the body cavity 3–6 h before staining. All tissues were stained overnight. fb, fat body; sgl, salivary gland; mg, midgut. Six independent transgenic strains of pA16 were generated. Five of these strains were homozygous viable and the induction pattern in the larval and adult fat body was analysed in these five strains. None of the strains showed any β-gal activity in the larval fat body, with or without pre-treatment with LPS. Four strains displayed strong induction of the reporter gene in the adult fat body after injection with LPS. One strain did not show any expression in the adult fat body even after immune challenge. This variability presumably is caused by the influence of nearby sequences or chromatin structure (Spradling and Rubin, 1982). One strain was homozygous lethal and was not analysed further. Download figure Download PowerPoint Srp binds to the CecA1 GATA motif We previously demonstrated the presence of a specific GBA in the nucleus of mbn-2 cells (Kadalayil et al., 1997; Figure 3A, lane 1). Our results suggested that the GBA belongs to the family of GATA transcription factors. The embryonic expression pattern of srp and the fact that srp mutants fail to develop fat body and haemocytes led us to investigate Srp as a potential regulator of Drosophila antimicrobial genes. To analyse the DNA-binding properties of Srp, electrophoretic mobility shift assays (EMSAs) were performed with in vitro transcribed and translated srp cDNA. A protein–DNA complex was formed when in vitro-translated Srp was incubated with a 32P-labelled oligonucleotide containing the Drosophila CecA1 GATA site (Figure 3A, lane 2). The Srp-containing complex co-migrated with the GBA (Figure 3A, compare lanes 1 and 2), suggesting that the protein composition is similar or identical in the two complexes. The presence of Srp protein in the in vitro translated sample was confirmed by Western blot analysis developed with a Srp-specific antibody (Figure 4, lane 2). Mock-translated wheat germ extract was also examined for DNA-binding activity and was found to be negative (Figure 3A, lane 6). Figure 3.Srp binds specifically to the CecA1 GATA motif and is a component of the GBA. (A) Electrophoretic mobility shift assay (EMSA) with in vitro translated srp. DNA binding was carried out with nuclear extract (N) (lane 1) or in vitro translated Srp (lanes 2–5) and a 32P-labelled GATA probe (wt). Unlabelled competitors were added to the reactions in lanes 3–5 as indicated (for details, see Materials and methods). As a negative control, the wheat germ extract (WG) was analysed for the presence of GATA-binding activity (lane 6). (B) EMSA with nuclear extract (N) from untreated (lane 1) or LPS-treated (lane 2) mbn-2 cells incubated with a 32P-labelled GATA probe (wt). LPS-treated nuclear extract was pre-incubated with antisera against the DNA-binding domain of Srp (S1) (lane 3), with antisera against the C-terminus of Srp (S2) (lane 4) or with normal rabbit serum (NS) (lane 7). The Srp antibodies S1 and S2 were also incubated with the probe alone (lanes 5–6). The asterisk indicates the supershift of the GBA. The protein–DNA band indicated by an arrowhead is due to unspecific binding. Download figure Download PowerPoint Figure 4.Srp is present in different forms in mbn-2 cells. Western blot analysis with the S2 Srp antiserum. Total cell extracts (T) prepared from mbn-2 cells were analysed for Srp content (lane 1). Several protein bands were identified by the S2 antiserum. The lower band denoted Srp corresponds to a 130 kDa protein and co-migrates with in vitro translated Srp (lane 2). The Srp-specific bands indicated by an arrowhead are due to phosphorylation of the 130 kDa form. The upper band denoted Srp′ indicates the 170 kDa form of Srp. In the mock-translated wheat germ extract (WG), no protein was recognized by the antiserum (lane 3). A parallel Western blot was analysed with another Srp antibody (S1), revealing an identical pattern of bands and thus verifying their identity as Srp (data not shown). Download figure Download PowerPoint The Srp DNA-binding activity was competed effectively by the unlabelled GATA oligonucleotide (Figure 3A, lane 3), but not competed by an oligonucleotide (m1) in which the GATA core sequence had been changed from GATA to CGAG (Figure 3A, lane 4). The conserved GATA motif found in the 5′ region of the Drosophila Cec genes and the diptericin gene is extended on the 3′ side by three bases (A/TGATAAT/GGC/T) (Kadalayil et al., 1997). Competition experiments using an unlabelled oligonucleotide in which GC on the 3′ side has been mutated to TG, keeping the core sequence intact (m2), competed as efficiently as the wild-type (Figure 3A, lane 5). The DNA-binding properties of Srp are the same as those previously determined for the GBA, supporting our theory that Srp protein is a constituent of the GBA (Kadalayil et al., 1997). Srp is a component of the GBA To investigate further the presence of Srp in the GBA, nuclear extracts were pre-treated with two different antisera, S1 [directed against the central domain of Srp including the DNA-binding Zn finger (Sam et al., 1996)] and S2 [directed against the C-terminal 22 amino acids of Srp (Hu, 1995)]. The extracts were then incubated with a 32P-labelled oligonucleotide containing the Drosophila CecA1 GATA motif in order for DNA binding to take place. In the nuclear fraction of untreated and LPS-treated mbn-2 cells, a strong band shift was present, GBA (Figure 3B, lanes 1 and 2). Pre-treatment of the nuclear extracts with the S1 antiserum disrupted the GBA (Figure 3B, lane 3), while the S2 antiserum resulted in a supershift (Figure 3B, lane 4). The two antisera, S1 and S2, were also incubated with oligonucleotide alone to rule out any direct interaction between the antibodies and the oligonucleotide (Figure 3B, lanes 5 and 6). Pre-treatment of the extract with normal serum did not influence the migration of the GBA (Figure 3B, lane 7). We conclude that Srp is, if not identical to, at least a component of the GBA. Expression of srp in an immunoresponsive Drosophila blood cell line Previous studies have shown that srp mRNA is expressed throughout development (Abel et al., 1993; Winick et al., 1993). The srp gene is expressed from the blastodermal stage and is essential for the differentiation of haemocytes (Rehorn et al., 1996). Studies on expression of srp in haemocytes at later stages have not been reported. The mbn-2 cell line is of haemocytic origin (Gateff et al., 1980) and expresses the antimicrobial peptide genes when treated with LPS. Two antisera with different specificities against Srp reacted with the GBA in this cell line (Figure 3B). To investigate the biochemical properties of Srp in the mbn-2 cell line, we performed Western blot analysis on total cell extracts (Figure 4, lane 1) and on in vitro translated Srp (Figure 4, lane 2). The predicted molecular weight of Srp is 102 kDa. Several protein bands were observed in mbn-2 cell extracts using the S2 antiserum (Figure 4, lane 1); incubation with the secondary antibody alone did not generate any protein bands (data not shown). A cluster of bands, denoted Srp (Figure 4, lane 1, arrow and arrowhead), had a slightly slower mobility compared with in vitro translated Srp, which migrates at ∼130 kDa (Figure 4, lane 2). Pre-treatment of the extract with potato acid phosphatase shifted the mobility of these bands to the same as that of the in vitro translated product, suggesting that Srp is phosphorylated (data not shown). The antisera did not cross-react with any protein in the mock-translated wheat germ extract (Figure 4, lane 3); therefore, we conclude that the 130 kDa protein band corresponds to full-length in vitro translated Srp. A band corresponding to a 170 kDa protein, denoted Srp′, was also detected by the S2 antiserum in extracts of mbn-2 cells (Figure 4, lane 1). The same protein bands were visualized using the antibody S1, directed against another domain of Srp, as S2 (data not shown). Therefore, these bands cannot be due to cross-reactivity of the antisera with an unrelated protein, but most likely reveal different forms of Srp. The electrophoresis was carried out under denaturing conditions indicating that the 170 kDa protein band is either a covalently modified form of Srp or the translational product of an alternative mRNA. Lossky and Wensink (1995) reported that alternative srp mRNA forms exist in different tissues. However, Northern blot analysis with total RNA from mbn-2 cells did not reveal any band that would correspond to the 170 kDa form (data not shown). The 170 kDa protein was not detected in fat body extracts from third instar larvae (data not shown). Therefore, we conclude that this is a haemocyte-specific form of Srp. Intracellular localization of Srp The fat body is the main site of antimicrobial peptide synthesis in response to infection (Samakovlis et al., 1990). The expression of srp in the embryonic fat body has been reported from stage 5 (Abel et al., 1993; Rehorn et al., 1996; Sam et al., 1996). To investigate the expression of Srp in the fully developed larval fat body, immunostaining of third instar larvae was performed. Strong Srp staining was seen in the fat body and the staining was more prominent in the nuclei than in the cytoplasm (Figure 5A). In a parallel independent study, it was demonstrated that Srp is expressed in the larval fat body, gonads, gut, lymph glands and in the pericardial cells (Brodu et al., 1999). Srp was localized in the nucleus in all these tissues. Figure 5.Srp is localized to the nucleus. (A and B) Srp immunostaining of third instar larval fat body (A) and of mbn-2 cells (B) using the S2 antiserum. (C) Western blot assay. Cytoplasmic (C) (lanes 1 and 2), nuclear (N) (lanes 3 and 4) and total extracts (T) (lane 5) were prepared from untreated (−) (lanes 1, 3 and 5) and LPS-treated (+) mbn-2 cells (lanes 2 and 4). The lower band denoted Srp corresponds to a 130 kDa protein and co-migrates with in vitro translated srp (see Figure 3). The upper band denoted Srp′ is the 170 kDa form of Srp. Download figure Download PowerPoint Immunostaining of mbn-2 cells demonstrated that Srp is a nuclear protein also in these cells, as was previously found for the GBA. All cells were positive when stained with anti-Srp antiserum, but the intensity of the staining showed some variation (Figure 5B. The Srp staining was more pronounced in the nuclei of some cells (Figure 5B, arrow and arrowhead). To confirm biochemically that Srp is a nuclear protein, Western blot analysis was done on cytoplasmic (Figure 5C, lanes 1 and 2) and nuclear extracts (Figure 5C, lanes 3 and 4) of mbn-2 cells. Srp was present predominantly in the nuclear fractions. Neither the localization nor the intensity of the Srp-specific bands was affected by addition of LPS before harvesting the cells (Figure 5C). We conclude that Srp is a nuclear protein, present in tissues where antimicrobial peptides are being produced. The larval fat body contains nuclear GBA To investigate if the presence of Srp in fat body nuclei correlates with the existence of factors that bind to the GATA sequence, we performed EMSA with nuclear extracts of fat body dissected from third instar larvae. Two different GATA-binding activities (GBA-1 and GBA-2) appeared when fat body extracts were incubated with a 32P-labelled GATA oligonucleotide (Figure 6A, lane 1). Both GBA-1 and GBA-2 were competed effectively by an unlabelled GATA oligonucleotide (Figure 6A, lane 2), but not by the mutated oligonucleotide, m1 (Figure 6A, lane 3). To identify the presence of Srp in GBA-1 and GBA-2, the fat body nuclear extracts were pre-incubated with antisera S1 and S2. Pre-treatment of the fat body nuclear extracts with the S1 antiserum disrupted both GBA-1 and GBA-2 (Figure 6B, lane 3). Pre-treatment of the extracts with the S2 antiserum resulted in a supershift of the GBA-1 (Figure 6B, lane 2, asterisk), while the GBA-2 remained unaffected by this antiserum (Figure 6B, lane 2). We suggest that two forms of Srp exist in larval fat body, and that one form thereof (GBA-2) does not contain the C-terminal 22 amino acids, the epitope to which the S2 antiserum is directed. The mobilities of GBA-1 and GBA from mbn-2 cell nuclear extracts were similar (data not shown). Both complexes reacted with the S1 and S2 antisera, suggesting that GBA-1 and GBA are the same form of Srp. Figure 6.GATA-binding activities in larval fat body extracts. (A) EMSA with nuclear extract from third instar larval fat body (lanes 1–3) and a 32P-labelled GATA probe (wt). Unlabelled competitors were added to the reactions, wt in lane 2 and m1 in lane 3. The protein–DNA band indicated by an open circle is due to unspecific binding. The complex indicated by an arrowhead is an unidentified GATA-binding activity found in some extract preparations. (B) EMSA with nuclear extract from third instar larval fat body (lanes 1–3) and a 32P-labelled GATA probe (wt). Nuclear extract was pre-incubated with antisera against the C-terminus of Srp (S2) (lane 2) or against the DNA-binding domain of Srp (S1) (lane 3). The asterisk indicates the supershift of the GBA-1 (lane 2). To minimize unspecific binding, unlabelled m1 oligonucleotide was added to the binding reactions. Download figure Download PowerPoint Srp activates the CecA1 promoter It was demonstrated previously that the GATA motif is necessary for full Drosophila CecA1 promoter activity in transfection assays of mbn-2 cells (Kadalayil et al., 1997). In order to test if Srp can trans-activate the Drosophila CecA1 promoter, co-transfection assays were performed. The srp cDNA was inserted into the expression vector pAct5C and co-transfected with the pA10 or pA16 reporter constructs in the mbn-2 cell line (Figure 1). Increasing amounts of the expression plasmid pAct–srp were transfected with a constant amount of reporter plasmid pA10, and led to significantly increased levels of β-gal activity in the cell extracts (Figure 7A). Co-transfections of 2 μg of expression plasmid pAct–Srp and the reporter plasmid pA16 (Figure 7A), carrying a mutated GATA motif, yielded 35% β-gal activity as compared with pA10 (Figure 7A). Addition of LPS to the media before harve sting the cells did not significantly increase the β-gal activity (Figure 7A, grey bars) in comparison with the unchallenged cells (Figure 7A, white bars). The results from the co-transfection experiments were confirmed by utilizing the GAL4/UAS system to overexpress Srp in vivo (Figure 7B). The level of β-gal activity in extracts from transgenic larvae carrying pA10, UAS-Srp and hs-GAL4 showed a 2- to 3-fold enhancement when the larvae were subjected to heat shock as compared with untreated animals. Enhanced β-gal activity was also found in larvae carrying pA10, UAS-Srp and the GAL4 enhancer trap line c729, which constitutively expresses GAL4 in the fat body. The activity was 2- to 3-fold higher as compared with larvae carrying pA10 alone (Figure 7B. We conclude that Srp acts as a positive regulator of the Drosophila CecA1 promoter and therefore is likely to be a key regulator of antimicrobial peptide gene expression. Figure 7.Srp activates Drosophila CecA1 expression. (A) β-gal expression in co-transfection assays. mbn-2 cells were co-transfected with 1 μg of reporter plasmid pA10 and increasing amounts of expression plasmid pAct-srp as indicated, or with 1 μg of reporter plasmid pA16 and 2 μg of pAct-srp. The cells were incubated with (grey bars) or without (white bars) LPS (10 μg/ml) 4 h prior to harvest. The results shown are the average of at least four independent experiments with standard deviation indicated as T-bars. (B) β-gal activity in extracts from transgenic larvae carrying the CecA1–lacZ reporter construct pA10, and UAS-Srp driven by the heat shock-inducible hs-Gal489-2-1, or the enhancer trap GAL4 line c729, which constitutively express GAL4 in the fat body. Heat shock (+HS) was carried out at 37°C for 1 h and larvae were allowed to recover for 3 h. Download figure Download PowerPoint Discussion Several conclusions can be drawn based on the results presented in this study. (i) The GATA motif is essential for expression of the CecA1 gene in larval fat body but dispensable for the infection-dependent expression in adult fat body. (ii) The GATA transcription factor Srp is expressed and localized in the nucleus of immunocompetent tissues. (iii) Srp shares DNA-binding properties with the GBA found in mbn-2 cell and larval fat body nuclear fractions. (iv) Two different antibodies directed towards different parts of Srp reacted with the GBA by destroying the complex or causing it to shift in a DNA-binding assay. (v) Overexpression of Srp in larval fat body and in mbn-2 cells led to activation of the CecA1 promoter in the absence of infection. These results led us to conclude that Srp is the main component of the GBA. The in vivo importance of the GATA motif The GATA sequence is present in proximity to a κB site in all the known regulatory regions of antimicrobial genes in Drosophila melanogaster and in several other insects as well. In an earlier study, we have shown by transfection assays that the GATA sequence is needed for proper CecA1 promoter activity in the mbn-2 cell line (Kadalayil et al.,
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