Transcriptional regulation of the Drosophila caudal homeobox gene by D
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《核酸研究医学期刊》
Department of Molecular Biology, College of Natural Science and 1 Research Institute of Genetic Engineering, Pusan National University, Busan 609-735, Korea, 2 Division of Biotechnology, Faculty of Textile Sciences, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan and 3 Faculty of Science, Japan Women's University, 2-8-1 Mejirodai, Bunkyouku, Tokyo 112-8679, Japan
* To whom correspondence should be addressed. Tel: +82 51 510 2278; Fax: +82 51 513 9258; Email: mayoo@pusan.ac.kr
ABSTRACT
The caudal-related homeobox transcription factors are required for the normal development and differentiation of intestinal cells. Recent reports indicate that misregulation of homeotic gene expression is associated with gastrointestinal cancer in mammals. However, the molecular mechanisms that regulate expression of the caudal-related homeobox genes are poorly understood. In this study, we have identified a DNA replication-related element (DRE) in the 5' flanking region of the Drosophila caudal gene. Gel-mobility shift analysis reveals that three of the four DRE-related sequences in the caudal 5'-flanking region are recognized by the DRE-binding factor (DREF). Deletion and site-directed mutagenesis of these DRE sites results in a considerable reduction in caudal gene promoter activity. Analyses with transgenic flies carrying a caudal–lacZ fusion gene bearing wild-type or mutant DRE sites indicate that the DRE sites are required for caudal expression in vivo. These findings indicate that DRE/DREF is a key regulator of Drosophila caudal homeobox gene expression and suggest that DREs and DREF contribute to intestinal development by regulating caudal gene expression.
INTRODUCTION
Homeobox genes encode DNA-binding proteins that play crucial roles during development in defining the body plan and determining cell fate (1–3). For example, the Drosophila caudal gene participates in defining the anteroposterior axis during early embryogenesis, and its expression is maintained in several organs including gut, gonads and compartments of genital discs (4,5). Evidence of the primary role played by the maternal and zygotic expression of the caudal gene in gut organogenesis and differentiation has recently been provided, since the caudal gene transactivates several genes involved in intestinal development, such as folded gastrulation, fork head and wingless (6). A recent report suggests that the Drosophila caudal is involved in the innate immune system and is responsible for the constitutive, tissue-specific local expression of antimicrobial peptides (7). Several vertebrates have been characterized, including Xcad-1, Xcad-2 and Xcad-3 in Xenopus; CdxA, CdxB and CdxC in chicken; Cdx-1, Cdx-2 and Cdx-4 in mouse; and CDX1 and CDX2 in human (8). CDX1 and CDX2 are known to play essential roles in regulating cell proliferation and differentiation in the intestine (8,9), and the expression of these genes is altered in gastrointestinal cancers (10–12). Although the human CDX2 gene is a known target of PTEN/phosphatidylinositol 3-kinase signaling via an NF-kappa B (NF-B)-dependent pathway (13), little is known about other mechanisms that are likely to regulate caudal-related homeobox genes. Our present interest in characterizing additional caudal regulatory mechanisms stems from our observation that four putative DRE-related sequences are present in the 5'-flanking region of the caudal gene.
DRE is known to be important for the expression of DNA replication-related genes (DNA polymerase subunits and PCNA) as well as various cell cycle- and cell proliferation-related genes in Drosophila, including dE2F, cyclin A and D-raf (14–18). The transcription factor, DREF, binds specifically to the DRE sequence as an 80 kDa homodimer (19) and a human homolog of Drosophila DRE-binding factor (DREF) has been identified previously (20). The DRE/DREF regulatory system has also been implicated as a target of differentiation signals. For example, the zerknüllt (zen) gene encodes a homeodomain-containing protein that is expressed in the dorsal region of the early embryo at the cellular blastoderm stage and is involved in differentiation of the amnioserosa and the optic lobe (21,22). Zen expression represses DNA replication-related genes by reducing DREF activity in cultured cells (23,24). Hence, the DRE/DREF regulatory system may co-regulate and thereby coordinate growth- and differentiation-signaling pathways.
In the present study, we have investigated whether the caudal gene is regulated by the DRE/DREF regulatory system. Our results indicate that DREF binds to three DRE sites in the caudal gene promoter and that the DRE sites are required for efficient promoter activity.
MATERIALS AND METHODS
Oligonucleotides
The following double-stranded oligonucleotides containing a 6 bp linker sequence recognized by BglII and BamHI were synthesized chemically. Sequences containing four potential DRE sites or their base substitutional mutants in the caudal promoter region were defined as follows: cad-DRE1 wild-type (wt), 5'-gatccCGAAATATTATCGATATCTCGGTCa-3' and 3'-gGCTTTATAATAGCTATAGAGCCAGtctag-5'; cad-DRE1 mutant (mut), 5'-gatccCGAAATATTTAGGATATCTCGGTCa-3' and 3'-gGCTTTATAAATCCTATAGAGCCAGtctag-5'; cad-DRE2 wt, 5'-gatccACACGGTATATGGATATGGAGAATa-3' and 3'-gTGTGCCATATACCTATACCTCTTAtctag-5'; cad-DRE2 mut, 5'-gatccACACGGTATTACGATATGGAGAATa-3' and 3'-gTGTGCCATAATGCTATACCTCTTAtctag-5'; cad-DRE3 wt, 5'-gatccGAAATAATAATCGATATTTCAATTa-3' and 3'-gCTTTATTATTAGCTATAAAG-TTAAtctag-5'; cad-DRE3 mut, 5'-gatccGAAATAATATAGGATATTTCAATTa-3' and 3'-gCTTTATTATATCCTATAAAGTTAAtctag-5'; cad-DRE4 wt, 5'-gatccCTGTGTGGTATCGAAATTCTTGCGa-3' and 3'-gGACACACCATAGCTTTAAGAACGCtctag-5'; cad-DRE4 mut, 5'-gatccCTGTGTGGTTAGGAAATTCTTGCGa-3' and 3'-gGACACACCAATCCTTTAAGAACGCtctag-5'.
Double-stranded oligonucleotides for site-directed mutagenesis were as follows: cad-DRE1-SM, 5'-GCGTTTCGAAATATTTAGGATATCTCGGTCG-3' and 3'-CGCAAAGCTT-TATAAATCCTATAG-AGCCAGC-5'; cad-DRE3-SM, 5'-GTCGTTATGAAATAATATAGGATATTTCAATTCCACCC-3' and 3'-CAGCAATACTTTATTATATCCTATAAAGTTAAGGTGGG-5'; cad-DRE4-SM, 5'-GGAGT-AGCTGTGTGGTTAGGAAATTCTTGCG-3' and 3'-CCTCATCGACACACCAATCCTTTAAGAACGC-5'. Mutated bases are underlined and lower case letters indicate the linker sequences recognizable by BglII and BamHI. Specific primers for RT–PCR of DREF and rp49 were described previously (25).
Plasmid construction
pcad-Luc contains PCR-amplified 3468 bp fragments spanning from 3303 bp upstream and 165 bp downstream (–3303 to +165) with respect to the maternal transcription initiation site of Drosophila caudal gene. The amplified products were inserted into the KpnI–XhoI sites of pGL2-basic promoter plasmid (Promega). The deletion constructs were generated by digestion of pcad-Luc with PvuII–SmaI, BclI–SmaI, SwaI–SmaI, NruI–SmaI and AatII–SmaI. The resulting constructs were designated pcad(–1795/+165)-Luc, pcad(–1032/+165)-Luc, pcad(–627/+165)-Luc and pcad (–125/+165)-Luc, respectively. To construct the pcad-lacZ and pcadDmut7-lacZ for transgenic flies, the caudal promoter region (–2648 to +165) with or without base-substituted mutations in three of the four DRE sites was inserted into the KpnI–EcoRI sites of the plasmid pCaSpeR-AUG-?gal.
Site-directed mutagenesis
To obtain mutant reporter plasmids carrying base-substitution mutations in the DRE sites in the 5'-flanking region of the Drosophila caudal gene, mutagenesis reactions were carried out on double-stranded DNA of pcad-Luc using a QuickChangeTM Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions. The mutations as well as the fidelity of the rest of the DNA were confirmed by sequencing.
Cell culture, DNA transfection and luciferase assay
Drosophila Kc and S2 cells were grown at 25°C in M3 (BF) medium (Sigma) (26) supplemented with 2% fetal bovine serum (FBS) and 0.5% penicillin/streptomycin (Gibco BRL), and 10% FBS and 1% penicillin/streptomycin, respectively. Transfection of various DNA mixtures into Kc cells was performed using dimethyldioctadecyl ammonium bromide (DDAB) (27) and the cells were harvested 48 h thereafter. The luciferase assay was carried out by means of a Luciferase Assay System (Promega), as described previously (28). Luciferase activity was normalized with ?-galactosidase activity to correct for transfection efficiency.
Preparation of nuclear extracts
Preparation of nuclear extracts from Drosophila Kc cells was performed as described previously (14) with modifications. Briefly, cells were rinsed once using ice-cold phosphate-buffered saline (PBS). Cells were collected by centrifugation at 4000 r.p.m. for 5 min, resuspended in buffer A (10 mM HEPES, 1.5 mM MgCl2, 10 mM NaCl and 0.25% NP-40, pH 7.5) and incubated on ice for 5 min, followed by centrifugation at 4000 r.p.m. for 5 min. The supernatant (cytosolic extracts) was removed and the nuclei were extracted with buffer C (20 mM HEPES, 25% glycerol, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA and 0.25% NP-40, pH 7.5). The nuclei were vortexed vigorously several times for 20 min, followed by centrifugation at 14 000 r.p.m. for 5 min. The supernatant (nuclear extract) was transferred into fresh tubes and diluted 1:2 with buffer D (20 mM HEPES, 50 mM KCl, 0.2 mM EDTA and 20% glycerol, pH 7.5) and frozen at –80°C until use.
Expression and purification of recombinant protein
GST–DREF fusion proteins were expressed in Eschericha coli as described earlier (19) and partially purified using glutathione sepharose 4B matrix (Amersham Pharmacia). The E.coli cells were collected and resuspended in PBS containing 0.5 mM PMSF, 1 μg/ml of aprotinin, 1 μg/ml of leupeptin and 1 μg/ml of pepstatin A, sonicated and then centrifuged at 15 000 r.p.m. for 15 min. The glutathione sepharose 4B slurry was added into supernatant and incubated at room temperature for 30 min. The sepharose matrix was pelleted by centrifugation and washed three times with PBS. The fusion protein was eluted with elution buffer (50 mM Tris–HCl and 10 mM reduced glutathione, pH 8.0) and stored at –80°C.
Electrophoretic gel-mobility shift assay (EMSA)
EMSA was performed as described previously (29). Kc cell nuclear extracts or GST–DREF fusion proteins were incubated in 10–20 μl of reaction mixture containing 10 mM HEPES (pH 7.6), 50 mM KCl, 1 mM EDTA, 5% glycerol and 0.5 μg of poly(dI–dC) for 10–20 min at room temperature. Unlabeled competitor oligonucleotides were also added at this step. Each of the 32P-labeled oligonucleotides (1 x 105 c.p.m.) was added and the mixture was incubated further for 20 min at room temperature. In the case of gel-shift assay performed with anti-DREF monoclonal antibody No. 4 (mAb 4) (19), Kc cell nuclear extract was incubated in a reaction mixture described above with mAb 4 for 20 min before the addition of radiolabeled oligonucleotides. The retarded bands were resolved electrophoretically on a 4–6% non-denaturing Tris–borate–EDTA polyacrylamide gel. The gels were dried and autoradiographed with X-ray film or analyzed with a BAS 2000 imaging analyzer.
Establishment of transgenic flies and fly stocks
Fly stocks were maintained at 25°C on standard food. To establish transgenic flies carrying pcad-lacZ or pcadDmut7-lacZ, P element-mediated germ line transformation was carried out as described previously (30). Five independent lines were obtained with pcad-lacZ and four independent lines with pcadDmut7-lacZ constructs. The line carrying the same fusion genes showed similar lacZ expression patterns. For ectopic expression of DREF using GAL4-UAS system, Hsp70-GAL4 (hs-GAL4) provided by the Bloomington Stock Center and the transgenic flies carrying UAS-DREF on the second chromosome described previously (31) were used.
Ectopic expression of DREF by heat shock induction
Females carrying homozygous hs-GAL4 in the third chromosome were crossed with males carrying homozygous UAS-DREF in the second chromosome. The progeny third instar larvae were heat-shocked at 37°C for 45 min and then returned to 25°C.
RT–PCR
Total RNA from larvae was isolated with Trizol Reagent (Molecular Research Center, Inc.) according to the protocol furnished by the manufacturer and cDNAs were synthesized with M-MLV-RT (reverse transcriptase) (Promega). The RT–PCR products were analyzed on agarose gels stained with ethidium bromide.
X-gal staining
The tissues were dissected and fixed for 15 min in PBS (130 mM NaCl, 7 mM Na2HPO4·2H2O and 3 mM NaH2 PO4·2H2O, pH 7.5) containing 1% glutaraldehyde, washed in PBS, and immersed in 0.2% X-gal (5-bromo-4-chloro-3-indolyl-?-D-galactopyranocyte) in staining buffer containing 6.1 mM K4Fe(CN)6, 6.1 mM K3Fe(CN)6, 1 mM MgCl2, 150 mM NaCl, 10 mM Na2HPO4 and 10 mM NaH2PO4. Incubation was in the dark at 37°C.
Quantitative measurement of ?-galactosidase activity in extracts
Quantitative measurements of ?-galactosidase activity in extracts prepared from Drosophila bodies were carried out as described previously (32). The ?-galactosidase activity was defined as absorbance units per milligram of protein per hour.
RESULTS
Location of four potential DRE-related sequences in the Drosophila caudal gene promoter
Four putative DRE-related sequences similar to the consensus DRE sequences, TATCGATA, were found in the 5'-flanking region of the caudal gene by a public database search (GenBank accession no. AE003668 ) (Figure 1). They were designated cad-DRE1, cad-DRE2, cad-DRE3 and cad-DRE4. To examine whether DREF can recognize the putative binding sites found in the caudal promoter region, gel-mobility shift assays were carried out by using four oligonucleotides containing these DRE sites and a recombinant fusion protein containing the DNA-binding region of DREF (GST–DREF1-125) (17). The oligonucleotide TBP-DRE1 which contains DRE sequences of the Drosophila TBP gene (29), was used as a positive control. Binding complexes were detected when the assays were performed with labeled cad-DRE1 wt, cad-DRE3 wt and cad-DRE4 wt oligonucleotides as probes but not with labeled cad-DRE2 wt (Figure 2). Assays were performed using Kc cell nuclear extracts. As expected, protein–DNA complexes were observed when the assays were performed with labeled cad-DRE1 wt, cad-DRE3 wt and cad-DRE4 wt oligonucleotides as probes but not with labeled cad-DRE2 wt (Figure 3A). Complex formation was diminished by the addition of excess unlabeled cad-DRE1 wt, cad-DRE3 wt and cad-DRE4 wt. However, mutated oligonucleotides carrying base substitutions in the DRE sequences fail to compete for binding. Binding affinity of cad-DRE4 was weak. By the addition of the anti-DREF monoclonal antibody No. 4 (mAb 4) to the binding reaction, the complexes formed with cad-DRE1 and cad-DRE3 were supershifted (Figure 3B and C). It was previously reported that mAb 4 supershifts protein–DNA complexes formed with DRE sites from other Drosophila gene promoter (15,19). These results indicate that DREF binds to three of the four DRE-related sites in the promoter region of the Drosophila caudal gene in a sequence-specific manner.
Figure 1. The nucleotide sequences and relative positions of DRE-related sequences in the 5'-flanking region of Drosophila caudal gene promoter. The four potential DRE sites in the Drosophila caudal promoter, termed cad-DRE1, cad-DRE2, cad-DRE3 and cad-DRE4, are shown. The maternal transcription initiation site is indicated by the arrowhead and designated +1. Lower case letters indicate mutated bases in the DRE sites with numbering relative to the maternal transcription initiation site. The zygotic transcription initiation site is indicated by +1(Z, –159).
Figure 2. Complex formation between putative DRE sites in the caudal promoter and GST–DREF1-125. Radiolabeled double-stranded cad-DRE oligonucleotides were incubated with GST–DREF1-125 fusion proteins in the presence of increasing amounts of competitor oligonucleotides. Lanes 1, 6, 11 and 16, GST only. Lanes 2, 7, 12 and 17, minus competitor. Lanes 3, 8, 13 and 18, unlabeled competitor oligonucleotides with the wt cad-DRE sequences. Lanes 4, 9, 14 and 19, unlabeled competitor oligonucleotides with the mutant having three base changes in cad-DRE sequences (mut). Lanes 5, 10, 15 and 20, unlabeled competitor oligonucleotides with the wt TBP-DRE1 sequences. TBP-DRE1, oligonucleotide containing DRE site 1 of the Drosophila TBP gene promoter.
Figure 3. Complex formation between putative DRE sites in the caudal promoter and Kc cell nuclear extracts factors. (A) Radiolabeled double-stranded cad-DRE oligonucleotides were incubated with Kc cell nuclear extracts in the absence or presence of unlabeled TBP-DRE1 wt competitor oligonucleotides. Radiolabeled cad-DRE1 (B) or cad-DRE3 (C) oligonucleotides were incubated with Kc cell nuclear extracts in the absence or presence (lanes 16 and 21) of anti-DREF monoclonal antibody No. 4 (mAb 4). Lanes 12 and 17, no extract added. Lanes 13 and 18, binding without competitor. Lanes 14 and 19, unlabeled competitor oligonucleotides with the wt cad-DRE sequences. Lanes 15 and 20, unlabeled competitor oligonucleotides with mutant having three base changes in cad-DRE sequences (mut).
Role of the DRE-related sequences on caudal promoter activity
Because DREF binds to three potential DRE-related sequences in the 5'-flanking region of the caudal gene, we analyzed the significance of the DRE sites for caudal expression. A series of promoter deletion mutants was generated in a reporter plasmid containing the caudal promoter region (–3303/+165) upstream of a luciferase reporter gene. The resulting constructs were designated pcad(–1795/+165)-Luc, pcad(–1032/+165)-Luc, pcad(–627/+165)-Luc and pcad(–125/+165)-Luc, respectively (Figure 4). In transient transfection assays performed in Drosophila S2 cells, little difference in luciferase activity was observed between constructs extending to –1795 or to –3303, indicating that the –3303 to –1795 region has no detectable influence on promoter activity in this setting. Both of these constructs contain all the four DRE sites.
Figure 4. Analysis of caudal promoter deletion constructs. (A) Schematic representation of caudal promoter deletion constructs containing progressively shorter sequences at the 5'-end generated by restriction enzyme digestion. The putative DRE sites and transcription start sites are shown. (B) Transfections were performed with Drosophila S2 cells and promoter activities measured as luciferase activities normalized to ?-galactosidase activities 48 h after transfection. The mean activities ± SE from three independent transfections are shown.
However, further deletion extending to –1032, which removes DRE3 and DRE4, greatly decreased luciferase activity, suggesting that the promoter region containing the DRE3 and DRE4 sites are necessary for positive regulation of caudal transcription. Additional removal of the DRE2 site in the –627 construct did not diminish luciferase activity beyond the reduction observed with the –1032 construct. Further reduction of reporter activity was detected in the –125 construct in which the zygotic transcription start site is compromised.
To further examine the function of the DRE sites in the caudal promoter, mutant reporter plasmids bearing the same mutations in DRE1, DRE3 and DRE4 as those used in the gel-mobility shift assay were constructed and transient transfection assays were performed in Drosophila S2 cells. The DRE2 site was excluded from the analysis because complex formation with DREF was not detected with this site (Figures 2 and 3). Single point mutation in the DRE1, DRE3 or DRE4 sites all reduced luciferase activity to some extent (Figure 5). This effect was enhanced by mutations of either two or all the three DREs. Thus, DRE1, DRE3 and DRE4 are all likely to contribute to caudal gene activation.
Figure 5. Effects of DRE-related sequences on caudal gene promoter activity. (A) Schematic features of the promoter–luciferase fusion plasmids are illustrated. DRE-related sequences are indicated by open box and mutated DREs are marked by crossed box. (B) Transfections were performed with Drosophila S2 cells and promoter activities measured as luciferase activities normalized to ?-galactosidase activities 48 h after transfection. The mean activities ± SE from three independent transfections are shown.
In vivo roles of DRE sites in caudal gene expression
To investigate the role of the DRE sites in caudal gene expression in vivo, we established transgenic flies carrying a cad–lacZ fusion gene (caudal promoter region, –2648 to +165, fused to lacZ), or cadDmut7–lacZ (a cad–lacZ fusion gene derivative with base-substitution mutations in all three DRE sites). Drosophila caudal is known to be expressed in the gut, gonads and compartments of the genital discs of third instar larvae (5) and in the anal plates and hindgut of adult flies (33). In addition, endogenous caudal mRNA is expressed in the salivary gland and ejaculatory duct (7). Therefore, we compared expression patterns of lacZ in the gut, salivary glands, genital discs and the ejaculatory ducts of third instar larvae and adult flies carrying cad–lacZ or cadDmut7–lacZ by X-gal staining. Relative to the cad–lacZ control, lacZ staining in cadDmut7–lacZ flies was markedly reduced in the larval hindgut, salivary gland, adult hindgut, ovary and ejaculatory duct (Figure 6A). Quantitative ?-galactosidase activities of the transgenic larvae and adults bearing cad–lacZ or cadDmut7–lacZ fusion gene were examined. The mutation in the DREs reduced ?-galactosidase expression to 75% in third instar larvae and up to 46–53% in adults (Figure 6B). This result indicates that the DRE sites are required for tissue-specific expression of the caudal gene in vivo.
Figure 6. Effects of DRE-related sequences on caudal gene expression in vivo. (A) ?-Galactosidase expression in the transgenic flies carrying cad–lacZ or cadDmut7–lacZ was detected by X-gal staining. The third instar larvae or adults were dissected and stained with 0.2% X-gal solution in the dark at 25°C overnight. Reduced expression of the cadDmut7–lacZ is apparent in the larval hindgut, salivary gland, adult hindgut, ovary and ejaculatory duct compared with cad–lacZ. (B) Quantitative ?-galactosidase activities of the transgenic flies bearing cad–lacZ or cadDmut7–lacZ fusion gene. Crude extracts were prepared from third instar larvae or 5-day-old adult transgenic flies as described under Materials and Methods. The ?-galactosidase activities are expressed as absorbance units at 574 nm/h/mg of protein. Average values obtained from three independent experiments with ± SE values are shown.
DREF overexpression stimulates caudal gene transcription
To investigate a role for DREF in caudal gene expression, ectopic in vivo expression of DREF was performed with the GAL4-UAS system (34,35). Transgenic flies carrying UAS-DREF were crossed with transgenic flies carrying GAL4 cDNA placed under the control of the hsp70 gene promoter (hs-GAL4). Ectopic expression of DREF in the transgenic larvae after heat shock was confirmed by RT–PCR (Figure 7). The DREF mRNA in larvae carrying single copies of hs-GAL4 and UAS-DREF was detected at 1 h after heat shock and its level increased with time, reaching a maximum level at 3 h. We then examined caudal expression in larvae carrying hs-GAL4 and UAS-DREF by RT–PCR. The expression level of caudal was increased at 3 h after heat shock (Figure 7). Quantitative analysis of ?-galactosidase activity in total crude extracts of the larvae of a UAS-DREF/+;cad–lacZ/hs-GAL4 line was carried out. The level of ?-galactosidase activity in heat-shocked UAS-DREF/+;cad–lacZ/hs-GAL4 larvae was moderately but reproducibly higher than that in heat-shocked +/+;cad–lacZ/hs-GAL4 larvae (Figure 8A). The overexpression of DREF in the heat-shocked larvae UAS-DREF/+;cad–lacZ/hs-GAL4 was confirmed by RT–PCR (Figure 8B). These results indicate that DREF overexpression stimulates Drosophila caudal gene expression in vivo.
Figure 7. Expression of DREF and caudal in UAS-DREF/+;hs-GAL4/+ third instar larvae. Total RNA was prepared from the third instar larvae carrying hs-GAL4 and UAS-DREF after heat shock at 37°C for 45 min and incubated at 25°C for various time periods. RT–PCR was performed to determine DREF and caudal mRNA levels. The line carrying hs-GAL4/+ was used as a control and processed in the same way as that carrying UAS-DREF/+;hs-GAL4/+.
Figure 8. DREF overexpression stimulates the caudal gene promoter activity in vivo. (A) The ?-galactosidase activities of third instar larvae from +/+;cad–lacZ/hs-GAL4 or UAS-DREF/+;cad–lacZ/hs-GAL4 lines. Crude extracts were prepared after 45 min of heat shock at 37°C and incubated at 25°C for an additional 8 h. Average values obtained from three independent experiments with ± SE values are shown. (B) DREF mRNA overexpression by heat shock induction. Total RNA was prepared from the third instar larvae carrying +/+;cad–lacZ/hs-GAL4 (a) or UAS-DREF/+;cad–lacZ/hs-GAL4 (b) following 45 min of heat shock at 37°C and incubation at 25°C for an additional 3 h, after which DREF mRNA levels were measured by RT–PCR.
DISCUSSION
The Drosophila homeobox gene caudal is involved in the determination of body plan (1–3), gut development (6) and analia structure formation in adult flies (33). Caudal is known to be expressed in the gut, gonads and compartments of the genital discs of third instar larvae (5), in the anal plates and hindgut of adult flies (33), as well as in the salivary gland and ejaculatory duct (7). However, the molecular mechanisms of Drosophila caudal gene regulation are not well defined. In this study, we found that four DRE-related sites are located in the 5'-flanking regions of Drosophila caudal gene and showed that three out of the four sites are recognized by DREF. Furthermore, we have demonstrated the requirement of DRE sites for in vivo expression of the caudal gene. Thus the Drosophila caudal gene is positively regulated by DRE/DREF system at the transcriptional level.
It is well known that the DRE/DREF system activates transcription of a wide variety of DNA replication- and cell-cycle-related genes (16–19). In addition, Hart et al. (36,37) proposed a novel function of DREF as an antagonist of the chromosomal BEAF (boundary element-associated factor), which is involved in the boundary activity of the scs' element of the Drosophila 87A7 hsp70 locus. We previously reported that the caudal gene transactivates E2F gene expression in Drosophila (38) and here showed that the caudal homeobox gene expression is governed by DREF. Therefore, the present study supports the notion that the DRE/DREF system may be a master regulatory mechanism for the coordinated expression of cell proliferation-related genes.
Our results from analyses with transgenic flies carrying cad–lacZ fusion genes with or without mutation in the DRE sites show that the lacZ staining signals of cadDmut7–lacZ were decreased in several tissues including hindgut (Figure 6A). In particular, decreased ?-galactosidase expression of cadDmut7–lacZ in the hindgut suggests that DREF might be involved in hindgut development by regulating the caudal gene. In mammals, CDX1 and CDX2, mammalian homologs of the Drosophila caudal gene, are also expressed in the intestine and colon where they appear to be involved in the regulation of cell proliferation and differentiation (8,9,39). The importance of CDX1 and CDX2 in cell proliferation and differentiation is also supported by data from the study of several cancer cell lines (40,41). A human homolog (hDREF/KIAA0785) of Drosophila DREF has been identified. In addition, it has been reported that the histone 1 gene is transactivated by hDREF, suggesting that hDREF may play a role in regulating human genes related to cell proliferation (20). Therefore, we examined the possibility that hDREF binding sites might be located in the human CDX1 promoter and found candidate hDREF binding sites in the 5'-flanking region of human CDX1 gene. Recently, we reported that the DNA binding activity of DREF is regulated by a redox mechanism (28) and that DREF is a key regulator of Drosophila catalase gene expression (25), suggesting that expression of DREF target gene expression may be regulated by the intracellular redox state through modulation of DREF binding activity. The down-regulation of CDX1 and CDX2 is also known to be associated with colon cancers (42–45). Therefore, the presence of candidate hDREF binding sites in the 5'-flanking region of CDX1 raises interesting questions concerning the transcriptional regulation of CDX gene by the human DRE/DREF system. In particular, alteration of CDX expression in colon cancer may be associated with aberrant redox regulation of hDREF function.
Caudal has recently been shown to function as an innate immune response transcriptional modulator that controls the constitutive local expression of antimicrobial peptides cecropin and drosomycin in a tissue-specific manner (7). For example, caudal is absolutely necessary for constitutive expression of Cecropin and Drosomycin in ejaculatory duct or salivary gland, respectively (7). Our results demonstrate a marked reduction of caudal reporter expression in salivary gland and ejaculatory duct of flies carrying cadDmut7–lacZ (Figure 6A). Therefore, we examined the 5'-flanking region of the Cecropin gene, and detected DRE-related sequences. These findings suggest that in addition to controlling caudal gene expression and its possible role in human CDX gene regulation, the DRE/DREF system may function in the innate immune response of Drosophila.
ACKNOWLEDGEMENTS
We thank Dr P. Sherwood for critical reading of the manuscript and Bloomington Stock Center for the hs-GAL4 strain. This work was supported by a grant (JR 080) from the Korea Science and Engineering Foundation to M.-A.Y. and grants-in-aid from the Ministry of Education, Science, Sports and Culture, Japan. Y.-J.C. was supported by the Brain Korea 21 project in 2003.
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* To whom correspondence should be addressed. Tel: +82 51 510 2278; Fax: +82 51 513 9258; Email: mayoo@pusan.ac.kr
ABSTRACT
The caudal-related homeobox transcription factors are required for the normal development and differentiation of intestinal cells. Recent reports indicate that misregulation of homeotic gene expression is associated with gastrointestinal cancer in mammals. However, the molecular mechanisms that regulate expression of the caudal-related homeobox genes are poorly understood. In this study, we have identified a DNA replication-related element (DRE) in the 5' flanking region of the Drosophila caudal gene. Gel-mobility shift analysis reveals that three of the four DRE-related sequences in the caudal 5'-flanking region are recognized by the DRE-binding factor (DREF). Deletion and site-directed mutagenesis of these DRE sites results in a considerable reduction in caudal gene promoter activity. Analyses with transgenic flies carrying a caudal–lacZ fusion gene bearing wild-type or mutant DRE sites indicate that the DRE sites are required for caudal expression in vivo. These findings indicate that DRE/DREF is a key regulator of Drosophila caudal homeobox gene expression and suggest that DREs and DREF contribute to intestinal development by regulating caudal gene expression.
INTRODUCTION
Homeobox genes encode DNA-binding proteins that play crucial roles during development in defining the body plan and determining cell fate (1–3). For example, the Drosophila caudal gene participates in defining the anteroposterior axis during early embryogenesis, and its expression is maintained in several organs including gut, gonads and compartments of genital discs (4,5). Evidence of the primary role played by the maternal and zygotic expression of the caudal gene in gut organogenesis and differentiation has recently been provided, since the caudal gene transactivates several genes involved in intestinal development, such as folded gastrulation, fork head and wingless (6). A recent report suggests that the Drosophila caudal is involved in the innate immune system and is responsible for the constitutive, tissue-specific local expression of antimicrobial peptides (7). Several vertebrates have been characterized, including Xcad-1, Xcad-2 and Xcad-3 in Xenopus; CdxA, CdxB and CdxC in chicken; Cdx-1, Cdx-2 and Cdx-4 in mouse; and CDX1 and CDX2 in human (8). CDX1 and CDX2 are known to play essential roles in regulating cell proliferation and differentiation in the intestine (8,9), and the expression of these genes is altered in gastrointestinal cancers (10–12). Although the human CDX2 gene is a known target of PTEN/phosphatidylinositol 3-kinase signaling via an NF-kappa B (NF-B)-dependent pathway (13), little is known about other mechanisms that are likely to regulate caudal-related homeobox genes. Our present interest in characterizing additional caudal regulatory mechanisms stems from our observation that four putative DRE-related sequences are present in the 5'-flanking region of the caudal gene.
DRE is known to be important for the expression of DNA replication-related genes (DNA polymerase subunits and PCNA) as well as various cell cycle- and cell proliferation-related genes in Drosophila, including dE2F, cyclin A and D-raf (14–18). The transcription factor, DREF, binds specifically to the DRE sequence as an 80 kDa homodimer (19) and a human homolog of Drosophila DRE-binding factor (DREF) has been identified previously (20). The DRE/DREF regulatory system has also been implicated as a target of differentiation signals. For example, the zerknüllt (zen) gene encodes a homeodomain-containing protein that is expressed in the dorsal region of the early embryo at the cellular blastoderm stage and is involved in differentiation of the amnioserosa and the optic lobe (21,22). Zen expression represses DNA replication-related genes by reducing DREF activity in cultured cells (23,24). Hence, the DRE/DREF regulatory system may co-regulate and thereby coordinate growth- and differentiation-signaling pathways.
In the present study, we have investigated whether the caudal gene is regulated by the DRE/DREF regulatory system. Our results indicate that DREF binds to three DRE sites in the caudal gene promoter and that the DRE sites are required for efficient promoter activity.
MATERIALS AND METHODS
Oligonucleotides
The following double-stranded oligonucleotides containing a 6 bp linker sequence recognized by BglII and BamHI were synthesized chemically. Sequences containing four potential DRE sites or their base substitutional mutants in the caudal promoter region were defined as follows: cad-DRE1 wild-type (wt), 5'-gatccCGAAATATTATCGATATCTCGGTCa-3' and 3'-gGCTTTATAATAGCTATAGAGCCAGtctag-5'; cad-DRE1 mutant (mut), 5'-gatccCGAAATATTTAGGATATCTCGGTCa-3' and 3'-gGCTTTATAAATCCTATAGAGCCAGtctag-5'; cad-DRE2 wt, 5'-gatccACACGGTATATGGATATGGAGAATa-3' and 3'-gTGTGCCATATACCTATACCTCTTAtctag-5'; cad-DRE2 mut, 5'-gatccACACGGTATTACGATATGGAGAATa-3' and 3'-gTGTGCCATAATGCTATACCTCTTAtctag-5'; cad-DRE3 wt, 5'-gatccGAAATAATAATCGATATTTCAATTa-3' and 3'-gCTTTATTATTAGCTATAAAG-TTAAtctag-5'; cad-DRE3 mut, 5'-gatccGAAATAATATAGGATATTTCAATTa-3' and 3'-gCTTTATTATATCCTATAAAGTTAAtctag-5'; cad-DRE4 wt, 5'-gatccCTGTGTGGTATCGAAATTCTTGCGa-3' and 3'-gGACACACCATAGCTTTAAGAACGCtctag-5'; cad-DRE4 mut, 5'-gatccCTGTGTGGTTAGGAAATTCTTGCGa-3' and 3'-gGACACACCAATCCTTTAAGAACGCtctag-5'.
Double-stranded oligonucleotides for site-directed mutagenesis were as follows: cad-DRE1-SM, 5'-GCGTTTCGAAATATTTAGGATATCTCGGTCG-3' and 3'-CGCAAAGCTT-TATAAATCCTATAG-AGCCAGC-5'; cad-DRE3-SM, 5'-GTCGTTATGAAATAATATAGGATATTTCAATTCCACCC-3' and 3'-CAGCAATACTTTATTATATCCTATAAAGTTAAGGTGGG-5'; cad-DRE4-SM, 5'-GGAGT-AGCTGTGTGGTTAGGAAATTCTTGCG-3' and 3'-CCTCATCGACACACCAATCCTTTAAGAACGC-5'. Mutated bases are underlined and lower case letters indicate the linker sequences recognizable by BglII and BamHI. Specific primers for RT–PCR of DREF and rp49 were described previously (25).
Plasmid construction
pcad-Luc contains PCR-amplified 3468 bp fragments spanning from 3303 bp upstream and 165 bp downstream (–3303 to +165) with respect to the maternal transcription initiation site of Drosophila caudal gene. The amplified products were inserted into the KpnI–XhoI sites of pGL2-basic promoter plasmid (Promega). The deletion constructs were generated by digestion of pcad-Luc with PvuII–SmaI, BclI–SmaI, SwaI–SmaI, NruI–SmaI and AatII–SmaI. The resulting constructs were designated pcad(–1795/+165)-Luc, pcad(–1032/+165)-Luc, pcad(–627/+165)-Luc and pcad (–125/+165)-Luc, respectively. To construct the pcad-lacZ and pcadDmut7-lacZ for transgenic flies, the caudal promoter region (–2648 to +165) with or without base-substituted mutations in three of the four DRE sites was inserted into the KpnI–EcoRI sites of the plasmid pCaSpeR-AUG-?gal.
Site-directed mutagenesis
To obtain mutant reporter plasmids carrying base-substitution mutations in the DRE sites in the 5'-flanking region of the Drosophila caudal gene, mutagenesis reactions were carried out on double-stranded DNA of pcad-Luc using a QuickChangeTM Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions. The mutations as well as the fidelity of the rest of the DNA were confirmed by sequencing.
Cell culture, DNA transfection and luciferase assay
Drosophila Kc and S2 cells were grown at 25°C in M3 (BF) medium (Sigma) (26) supplemented with 2% fetal bovine serum (FBS) and 0.5% penicillin/streptomycin (Gibco BRL), and 10% FBS and 1% penicillin/streptomycin, respectively. Transfection of various DNA mixtures into Kc cells was performed using dimethyldioctadecyl ammonium bromide (DDAB) (27) and the cells were harvested 48 h thereafter. The luciferase assay was carried out by means of a Luciferase Assay System (Promega), as described previously (28). Luciferase activity was normalized with ?-galactosidase activity to correct for transfection efficiency.
Preparation of nuclear extracts
Preparation of nuclear extracts from Drosophila Kc cells was performed as described previously (14) with modifications. Briefly, cells were rinsed once using ice-cold phosphate-buffered saline (PBS). Cells were collected by centrifugation at 4000 r.p.m. for 5 min, resuspended in buffer A (10 mM HEPES, 1.5 mM MgCl2, 10 mM NaCl and 0.25% NP-40, pH 7.5) and incubated on ice for 5 min, followed by centrifugation at 4000 r.p.m. for 5 min. The supernatant (cytosolic extracts) was removed and the nuclei were extracted with buffer C (20 mM HEPES, 25% glycerol, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA and 0.25% NP-40, pH 7.5). The nuclei were vortexed vigorously several times for 20 min, followed by centrifugation at 14 000 r.p.m. for 5 min. The supernatant (nuclear extract) was transferred into fresh tubes and diluted 1:2 with buffer D (20 mM HEPES, 50 mM KCl, 0.2 mM EDTA and 20% glycerol, pH 7.5) and frozen at –80°C until use.
Expression and purification of recombinant protein
GST–DREF fusion proteins were expressed in Eschericha coli as described earlier (19) and partially purified using glutathione sepharose 4B matrix (Amersham Pharmacia). The E.coli cells were collected and resuspended in PBS containing 0.5 mM PMSF, 1 μg/ml of aprotinin, 1 μg/ml of leupeptin and 1 μg/ml of pepstatin A, sonicated and then centrifuged at 15 000 r.p.m. for 15 min. The glutathione sepharose 4B slurry was added into supernatant and incubated at room temperature for 30 min. The sepharose matrix was pelleted by centrifugation and washed three times with PBS. The fusion protein was eluted with elution buffer (50 mM Tris–HCl and 10 mM reduced glutathione, pH 8.0) and stored at –80°C.
Electrophoretic gel-mobility shift assay (EMSA)
EMSA was performed as described previously (29). Kc cell nuclear extracts or GST–DREF fusion proteins were incubated in 10–20 μl of reaction mixture containing 10 mM HEPES (pH 7.6), 50 mM KCl, 1 mM EDTA, 5% glycerol and 0.5 μg of poly(dI–dC) for 10–20 min at room temperature. Unlabeled competitor oligonucleotides were also added at this step. Each of the 32P-labeled oligonucleotides (1 x 105 c.p.m.) was added and the mixture was incubated further for 20 min at room temperature. In the case of gel-shift assay performed with anti-DREF monoclonal antibody No. 4 (mAb 4) (19), Kc cell nuclear extract was incubated in a reaction mixture described above with mAb 4 for 20 min before the addition of radiolabeled oligonucleotides. The retarded bands were resolved electrophoretically on a 4–6% non-denaturing Tris–borate–EDTA polyacrylamide gel. The gels were dried and autoradiographed with X-ray film or analyzed with a BAS 2000 imaging analyzer.
Establishment of transgenic flies and fly stocks
Fly stocks were maintained at 25°C on standard food. To establish transgenic flies carrying pcad-lacZ or pcadDmut7-lacZ, P element-mediated germ line transformation was carried out as described previously (30). Five independent lines were obtained with pcad-lacZ and four independent lines with pcadDmut7-lacZ constructs. The line carrying the same fusion genes showed similar lacZ expression patterns. For ectopic expression of DREF using GAL4-UAS system, Hsp70-GAL4 (hs-GAL4) provided by the Bloomington Stock Center and the transgenic flies carrying UAS-DREF on the second chromosome described previously (31) were used.
Ectopic expression of DREF by heat shock induction
Females carrying homozygous hs-GAL4 in the third chromosome were crossed with males carrying homozygous UAS-DREF in the second chromosome. The progeny third instar larvae were heat-shocked at 37°C for 45 min and then returned to 25°C.
RT–PCR
Total RNA from larvae was isolated with Trizol Reagent (Molecular Research Center, Inc.) according to the protocol furnished by the manufacturer and cDNAs were synthesized with M-MLV-RT (reverse transcriptase) (Promega). The RT–PCR products were analyzed on agarose gels stained with ethidium bromide.
X-gal staining
The tissues were dissected and fixed for 15 min in PBS (130 mM NaCl, 7 mM Na2HPO4·2H2O and 3 mM NaH2 PO4·2H2O, pH 7.5) containing 1% glutaraldehyde, washed in PBS, and immersed in 0.2% X-gal (5-bromo-4-chloro-3-indolyl-?-D-galactopyranocyte) in staining buffer containing 6.1 mM K4Fe(CN)6, 6.1 mM K3Fe(CN)6, 1 mM MgCl2, 150 mM NaCl, 10 mM Na2HPO4 and 10 mM NaH2PO4. Incubation was in the dark at 37°C.
Quantitative measurement of ?-galactosidase activity in extracts
Quantitative measurements of ?-galactosidase activity in extracts prepared from Drosophila bodies were carried out as described previously (32). The ?-galactosidase activity was defined as absorbance units per milligram of protein per hour.
RESULTS
Location of four potential DRE-related sequences in the Drosophila caudal gene promoter
Four putative DRE-related sequences similar to the consensus DRE sequences, TATCGATA, were found in the 5'-flanking region of the caudal gene by a public database search (GenBank accession no. AE003668 ) (Figure 1). They were designated cad-DRE1, cad-DRE2, cad-DRE3 and cad-DRE4. To examine whether DREF can recognize the putative binding sites found in the caudal promoter region, gel-mobility shift assays were carried out by using four oligonucleotides containing these DRE sites and a recombinant fusion protein containing the DNA-binding region of DREF (GST–DREF1-125) (17). The oligonucleotide TBP-DRE1 which contains DRE sequences of the Drosophila TBP gene (29), was used as a positive control. Binding complexes were detected when the assays were performed with labeled cad-DRE1 wt, cad-DRE3 wt and cad-DRE4 wt oligonucleotides as probes but not with labeled cad-DRE2 wt (Figure 2). Assays were performed using Kc cell nuclear extracts. As expected, protein–DNA complexes were observed when the assays were performed with labeled cad-DRE1 wt, cad-DRE3 wt and cad-DRE4 wt oligonucleotides as probes but not with labeled cad-DRE2 wt (Figure 3A). Complex formation was diminished by the addition of excess unlabeled cad-DRE1 wt, cad-DRE3 wt and cad-DRE4 wt. However, mutated oligonucleotides carrying base substitutions in the DRE sequences fail to compete for binding. Binding affinity of cad-DRE4 was weak. By the addition of the anti-DREF monoclonal antibody No. 4 (mAb 4) to the binding reaction, the complexes formed with cad-DRE1 and cad-DRE3 were supershifted (Figure 3B and C). It was previously reported that mAb 4 supershifts protein–DNA complexes formed with DRE sites from other Drosophila gene promoter (15,19). These results indicate that DREF binds to three of the four DRE-related sites in the promoter region of the Drosophila caudal gene in a sequence-specific manner.
Figure 1. The nucleotide sequences and relative positions of DRE-related sequences in the 5'-flanking region of Drosophila caudal gene promoter. The four potential DRE sites in the Drosophila caudal promoter, termed cad-DRE1, cad-DRE2, cad-DRE3 and cad-DRE4, are shown. The maternal transcription initiation site is indicated by the arrowhead and designated +1. Lower case letters indicate mutated bases in the DRE sites with numbering relative to the maternal transcription initiation site. The zygotic transcription initiation site is indicated by +1(Z, –159).
Figure 2. Complex formation between putative DRE sites in the caudal promoter and GST–DREF1-125. Radiolabeled double-stranded cad-DRE oligonucleotides were incubated with GST–DREF1-125 fusion proteins in the presence of increasing amounts of competitor oligonucleotides. Lanes 1, 6, 11 and 16, GST only. Lanes 2, 7, 12 and 17, minus competitor. Lanes 3, 8, 13 and 18, unlabeled competitor oligonucleotides with the wt cad-DRE sequences. Lanes 4, 9, 14 and 19, unlabeled competitor oligonucleotides with the mutant having three base changes in cad-DRE sequences (mut). Lanes 5, 10, 15 and 20, unlabeled competitor oligonucleotides with the wt TBP-DRE1 sequences. TBP-DRE1, oligonucleotide containing DRE site 1 of the Drosophila TBP gene promoter.
Figure 3. Complex formation between putative DRE sites in the caudal promoter and Kc cell nuclear extracts factors. (A) Radiolabeled double-stranded cad-DRE oligonucleotides were incubated with Kc cell nuclear extracts in the absence or presence of unlabeled TBP-DRE1 wt competitor oligonucleotides. Radiolabeled cad-DRE1 (B) or cad-DRE3 (C) oligonucleotides were incubated with Kc cell nuclear extracts in the absence or presence (lanes 16 and 21) of anti-DREF monoclonal antibody No. 4 (mAb 4). Lanes 12 and 17, no extract added. Lanes 13 and 18, binding without competitor. Lanes 14 and 19, unlabeled competitor oligonucleotides with the wt cad-DRE sequences. Lanes 15 and 20, unlabeled competitor oligonucleotides with mutant having three base changes in cad-DRE sequences (mut).
Role of the DRE-related sequences on caudal promoter activity
Because DREF binds to three potential DRE-related sequences in the 5'-flanking region of the caudal gene, we analyzed the significance of the DRE sites for caudal expression. A series of promoter deletion mutants was generated in a reporter plasmid containing the caudal promoter region (–3303/+165) upstream of a luciferase reporter gene. The resulting constructs were designated pcad(–1795/+165)-Luc, pcad(–1032/+165)-Luc, pcad(–627/+165)-Luc and pcad(–125/+165)-Luc, respectively (Figure 4). In transient transfection assays performed in Drosophila S2 cells, little difference in luciferase activity was observed between constructs extending to –1795 or to –3303, indicating that the –3303 to –1795 region has no detectable influence on promoter activity in this setting. Both of these constructs contain all the four DRE sites.
Figure 4. Analysis of caudal promoter deletion constructs. (A) Schematic representation of caudal promoter deletion constructs containing progressively shorter sequences at the 5'-end generated by restriction enzyme digestion. The putative DRE sites and transcription start sites are shown. (B) Transfections were performed with Drosophila S2 cells and promoter activities measured as luciferase activities normalized to ?-galactosidase activities 48 h after transfection. The mean activities ± SE from three independent transfections are shown.
However, further deletion extending to –1032, which removes DRE3 and DRE4, greatly decreased luciferase activity, suggesting that the promoter region containing the DRE3 and DRE4 sites are necessary for positive regulation of caudal transcription. Additional removal of the DRE2 site in the –627 construct did not diminish luciferase activity beyond the reduction observed with the –1032 construct. Further reduction of reporter activity was detected in the –125 construct in which the zygotic transcription start site is compromised.
To further examine the function of the DRE sites in the caudal promoter, mutant reporter plasmids bearing the same mutations in DRE1, DRE3 and DRE4 as those used in the gel-mobility shift assay were constructed and transient transfection assays were performed in Drosophila S2 cells. The DRE2 site was excluded from the analysis because complex formation with DREF was not detected with this site (Figures 2 and 3). Single point mutation in the DRE1, DRE3 or DRE4 sites all reduced luciferase activity to some extent (Figure 5). This effect was enhanced by mutations of either two or all the three DREs. Thus, DRE1, DRE3 and DRE4 are all likely to contribute to caudal gene activation.
Figure 5. Effects of DRE-related sequences on caudal gene promoter activity. (A) Schematic features of the promoter–luciferase fusion plasmids are illustrated. DRE-related sequences are indicated by open box and mutated DREs are marked by crossed box. (B) Transfections were performed with Drosophila S2 cells and promoter activities measured as luciferase activities normalized to ?-galactosidase activities 48 h after transfection. The mean activities ± SE from three independent transfections are shown.
In vivo roles of DRE sites in caudal gene expression
To investigate the role of the DRE sites in caudal gene expression in vivo, we established transgenic flies carrying a cad–lacZ fusion gene (caudal promoter region, –2648 to +165, fused to lacZ), or cadDmut7–lacZ (a cad–lacZ fusion gene derivative with base-substitution mutations in all three DRE sites). Drosophila caudal is known to be expressed in the gut, gonads and compartments of the genital discs of third instar larvae (5) and in the anal plates and hindgut of adult flies (33). In addition, endogenous caudal mRNA is expressed in the salivary gland and ejaculatory duct (7). Therefore, we compared expression patterns of lacZ in the gut, salivary glands, genital discs and the ejaculatory ducts of third instar larvae and adult flies carrying cad–lacZ or cadDmut7–lacZ by X-gal staining. Relative to the cad–lacZ control, lacZ staining in cadDmut7–lacZ flies was markedly reduced in the larval hindgut, salivary gland, adult hindgut, ovary and ejaculatory duct (Figure 6A). Quantitative ?-galactosidase activities of the transgenic larvae and adults bearing cad–lacZ or cadDmut7–lacZ fusion gene were examined. The mutation in the DREs reduced ?-galactosidase expression to 75% in third instar larvae and up to 46–53% in adults (Figure 6B). This result indicates that the DRE sites are required for tissue-specific expression of the caudal gene in vivo.
Figure 6. Effects of DRE-related sequences on caudal gene expression in vivo. (A) ?-Galactosidase expression in the transgenic flies carrying cad–lacZ or cadDmut7–lacZ was detected by X-gal staining. The third instar larvae or adults were dissected and stained with 0.2% X-gal solution in the dark at 25°C overnight. Reduced expression of the cadDmut7–lacZ is apparent in the larval hindgut, salivary gland, adult hindgut, ovary and ejaculatory duct compared with cad–lacZ. (B) Quantitative ?-galactosidase activities of the transgenic flies bearing cad–lacZ or cadDmut7–lacZ fusion gene. Crude extracts were prepared from third instar larvae or 5-day-old adult transgenic flies as described under Materials and Methods. The ?-galactosidase activities are expressed as absorbance units at 574 nm/h/mg of protein. Average values obtained from three independent experiments with ± SE values are shown.
DREF overexpression stimulates caudal gene transcription
To investigate a role for DREF in caudal gene expression, ectopic in vivo expression of DREF was performed with the GAL4-UAS system (34,35). Transgenic flies carrying UAS-DREF were crossed with transgenic flies carrying GAL4 cDNA placed under the control of the hsp70 gene promoter (hs-GAL4). Ectopic expression of DREF in the transgenic larvae after heat shock was confirmed by RT–PCR (Figure 7). The DREF mRNA in larvae carrying single copies of hs-GAL4 and UAS-DREF was detected at 1 h after heat shock and its level increased with time, reaching a maximum level at 3 h. We then examined caudal expression in larvae carrying hs-GAL4 and UAS-DREF by RT–PCR. The expression level of caudal was increased at 3 h after heat shock (Figure 7). Quantitative analysis of ?-galactosidase activity in total crude extracts of the larvae of a UAS-DREF/+;cad–lacZ/hs-GAL4 line was carried out. The level of ?-galactosidase activity in heat-shocked UAS-DREF/+;cad–lacZ/hs-GAL4 larvae was moderately but reproducibly higher than that in heat-shocked +/+;cad–lacZ/hs-GAL4 larvae (Figure 8A). The overexpression of DREF in the heat-shocked larvae UAS-DREF/+;cad–lacZ/hs-GAL4 was confirmed by RT–PCR (Figure 8B). These results indicate that DREF overexpression stimulates Drosophila caudal gene expression in vivo.
Figure 7. Expression of DREF and caudal in UAS-DREF/+;hs-GAL4/+ third instar larvae. Total RNA was prepared from the third instar larvae carrying hs-GAL4 and UAS-DREF after heat shock at 37°C for 45 min and incubated at 25°C for various time periods. RT–PCR was performed to determine DREF and caudal mRNA levels. The line carrying hs-GAL4/+ was used as a control and processed in the same way as that carrying UAS-DREF/+;hs-GAL4/+.
Figure 8. DREF overexpression stimulates the caudal gene promoter activity in vivo. (A) The ?-galactosidase activities of third instar larvae from +/+;cad–lacZ/hs-GAL4 or UAS-DREF/+;cad–lacZ/hs-GAL4 lines. Crude extracts were prepared after 45 min of heat shock at 37°C and incubated at 25°C for an additional 8 h. Average values obtained from three independent experiments with ± SE values are shown. (B) DREF mRNA overexpression by heat shock induction. Total RNA was prepared from the third instar larvae carrying +/+;cad–lacZ/hs-GAL4 (a) or UAS-DREF/+;cad–lacZ/hs-GAL4 (b) following 45 min of heat shock at 37°C and incubation at 25°C for an additional 3 h, after which DREF mRNA levels were measured by RT–PCR.
DISCUSSION
The Drosophila homeobox gene caudal is involved in the determination of body plan (1–3), gut development (6) and analia structure formation in adult flies (33). Caudal is known to be expressed in the gut, gonads and compartments of the genital discs of third instar larvae (5), in the anal plates and hindgut of adult flies (33), as well as in the salivary gland and ejaculatory duct (7). However, the molecular mechanisms of Drosophila caudal gene regulation are not well defined. In this study, we found that four DRE-related sites are located in the 5'-flanking regions of Drosophila caudal gene and showed that three out of the four sites are recognized by DREF. Furthermore, we have demonstrated the requirement of DRE sites for in vivo expression of the caudal gene. Thus the Drosophila caudal gene is positively regulated by DRE/DREF system at the transcriptional level.
It is well known that the DRE/DREF system activates transcription of a wide variety of DNA replication- and cell-cycle-related genes (16–19). In addition, Hart et al. (36,37) proposed a novel function of DREF as an antagonist of the chromosomal BEAF (boundary element-associated factor), which is involved in the boundary activity of the scs' element of the Drosophila 87A7 hsp70 locus. We previously reported that the caudal gene transactivates E2F gene expression in Drosophila (38) and here showed that the caudal homeobox gene expression is governed by DREF. Therefore, the present study supports the notion that the DRE/DREF system may be a master regulatory mechanism for the coordinated expression of cell proliferation-related genes.
Our results from analyses with transgenic flies carrying cad–lacZ fusion genes with or without mutation in the DRE sites show that the lacZ staining signals of cadDmut7–lacZ were decreased in several tissues including hindgut (Figure 6A). In particular, decreased ?-galactosidase expression of cadDmut7–lacZ in the hindgut suggests that DREF might be involved in hindgut development by regulating the caudal gene. In mammals, CDX1 and CDX2, mammalian homologs of the Drosophila caudal gene, are also expressed in the intestine and colon where they appear to be involved in the regulation of cell proliferation and differentiation (8,9,39). The importance of CDX1 and CDX2 in cell proliferation and differentiation is also supported by data from the study of several cancer cell lines (40,41). A human homolog (hDREF/KIAA0785) of Drosophila DREF has been identified. In addition, it has been reported that the histone 1 gene is transactivated by hDREF, suggesting that hDREF may play a role in regulating human genes related to cell proliferation (20). Therefore, we examined the possibility that hDREF binding sites might be located in the human CDX1 promoter and found candidate hDREF binding sites in the 5'-flanking region of human CDX1 gene. Recently, we reported that the DNA binding activity of DREF is regulated by a redox mechanism (28) and that DREF is a key regulator of Drosophila catalase gene expression (25), suggesting that expression of DREF target gene expression may be regulated by the intracellular redox state through modulation of DREF binding activity. The down-regulation of CDX1 and CDX2 is also known to be associated with colon cancers (42–45). Therefore, the presence of candidate hDREF binding sites in the 5'-flanking region of CDX1 raises interesting questions concerning the transcriptional regulation of CDX gene by the human DRE/DREF system. In particular, alteration of CDX expression in colon cancer may be associated with aberrant redox regulation of hDREF function.
Caudal has recently been shown to function as an innate immune response transcriptional modulator that controls the constitutive local expression of antimicrobial peptides cecropin and drosomycin in a tissue-specific manner (7). For example, caudal is absolutely necessary for constitutive expression of Cecropin and Drosomycin in ejaculatory duct or salivary gland, respectively (7). Our results demonstrate a marked reduction of caudal reporter expression in salivary gland and ejaculatory duct of flies carrying cadDmut7–lacZ (Figure 6A). Therefore, we examined the 5'-flanking region of the Cecropin gene, and detected DRE-related sequences. These findings suggest that in addition to controlling caudal gene expression and its possible role in human CDX gene regulation, the DRE/DREF system may function in the innate immune response of Drosophila.
ACKNOWLEDGEMENTS
We thank Dr P. Sherwood for critical reading of the manuscript and Bloomington Stock Center for the hs-GAL4 strain. This work was supported by a grant (JR 080) from the Korea Science and Engineering Foundation to M.-A.Y. and grants-in-aid from the Ministry of Education, Science, Sports and Culture, Japan. Y.-J.C. was supported by the Brain Korea 21 project in 2003.
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