Characterization of dRFX2, a novel RFX family protein in Drosophila
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《核酸研究医学期刊》
Department of Applied Biology, Faculty of Textile Science and 1 Venture Laboratory, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan and 2 Division of Biochemistry, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, 464-8681, Japan
* To whom correspondence should be addressed. Tel: +81 75 724 7781; Fax: +81 75 724 7769; Email: myamaguc@ipc.kit.ac.jp
Present addresses: Masaki Kato, Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, 725 North Wolfe Street PCTB 714 Baltimore, MD 21205-2185, USA
Hideki Yoshida, Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan
ABSTRACT
A transcriptional regulatory element was identified in the region between URE (upstream regulatory element) and DRE (DNA replication-related element) in the Drosophila PCNA gene promoter. This element plays an important role in promoter activity in living flies. A yeast one-hybrid screening using this element as a bait allowed isolation of a cDNA encoding a protein which binds to the element in vitro. Nucleotide sequence analyses revealed that the cDNA encodes a novel protein containing a characteristic DNA-binding domain conserved among the regulatory factor X (RFX) family proteins. We termed this protein Drosophila RFX2 (dRFX2) and this element dRFX2 site. To investigate the function of dRFX2 in vivo, we took the strategy of analyzing the dominant negative effects against the endogenous dRFX2. Transgenic flies were established in which expression of HA-dRFX202–480 carrying the amino acid sequences from 202 to 480 containing the RFX domain (DNA-binding domain) of dRFX2 was targeted to the cells in the eye imaginal discs. In the eye imaginal disc expressing the HA-dRFX202–480, the G1–S transition and/or the progression of S phase were/was interrupted, and the ectopic apoptosis was induced, though photoreceptor cells differentiated normally. These results indicate that dRFX2 plays a role in G1–S transition and/or in progression of S phase.
INTRODUCTION
The proliferating cell nuclear antigen (PCNA) is required for replication of simian virus 40 (1) as well as cellular DNA (2,3). It functions as a sliding clamp at DNA replication forks (4) and is also involved in DNA repair (5,6) and cell cycle regulation (7–9) by interacting with various enzymes and regulatory proteins (10,11). Interaction of PCNA with the chromatin assembly factor 1 (CAF-1) suggests another role in marking of DNA for chromatin assembly during the S phase of the cell cycle (12–14).
Previous studies on the Drosophila PCNA and other DNA replication-related genes have revealed that there are at least three common regulatory elements in the promoters of Drosophila DNA replication-related genes, DRE (DNA replication-related element) (15–17), E2F-recognition sites (18) and CFDD recognition sites (19). DRE appears to be an important regulatory element not only for DNA replication-related genes but also for various other cell cycle- (20) and proliferation-related genes (21,22). At least two different protein factors, DREF (15,23) and boundary element-associated factor of 32 kDa (BEAF32) (24) can bind to DRE.
E2F sites are essential for PCNA gene promoter activity throughout development (18). However, they themselves do not appear to be sufficient for PCNA gene promoter activity during embryonic and larval stages, since this was completely abolished by deletion of the upstream region containing the DRE sequence (18). The Drosophila E2F family proteins (dE2F1 and dE2F2) form complexes with their heterodimer partner dDP and bind to E2F sites (25–28). In addition, transcription of DNA polymerase and PCNA is completely lost in dE2F1 or dDP mutant embryos after division cycle 16, indicating that the two proteins are essential for transcription of these DNA replication-related genes (29,30). In contrast, a dE2F2 mutant fly demonstrates upregulation of E2F target genes (31,32). Therefore, dE2F1 and dE2F2 appear to have opposing functions during Drosophila development (32,33).
The PCNA gene promoter has three CFDD sites (site 1, 2 and 3), one of these (site 1) could be demonstrated to play an important role in promoter activity in both cultured cells and living flies. CFDD sites are present in promoters of the DNA polymerase and DREF genes, although specific CFDD binding factors have yet to be identified (19). The Drosophila PCNA gene promoter contains an additional upstream regulatory element (URE) to which a transcription factor Grainyhead (GRH/NTF-1) binds (34–36). URE in addition to the E2F sites, CFDD sites and DRE appear to be essential for activation of the PCNA gene promoter in larvae (37).
In the present study, we have identified a novel regulatory element in the region between URE and DRE in the PCNA gene promoter. A yeast one-hybrid screening using this element as bait allowed isolation of a cDNA encoding a protein which can bind to the element in vitro and which contains a characteristic DNA-binding domain conserved among the regulatory factor X (RFX) family proteins (38–42), known to be present in yeast, fungi, nematode, mouse, human and Drosophila. Therefore, we termed this protein dRFX2 and this element the dRFX2 site. The RFX-binding site has been suggested to be involved in regulation of the human PCNA gene (43–45). The function of some of RFX family proteins might be conserved during evolution. Over-expression of the DNA-binding domain of dRFX2 in imaginal discs inhibited DNA synthesis but exerted no effects on photoreceptor cell differentiation. These results indicate that dRFX2 plays a role in the G1–S transition and/or in S-phase progression.
MATERIALS AND METHODS
Oligonucleotides
The sequences of double-stranded oligonucleotides containing URE (UREL), DRE (DRE-P), CFDD-binding site 1 (CFDD-1) or E2F-recognition sites (E2F-P) in the PCNA gene were described earlier (16,18,19,36).
The sequences of double-stranded oligonucleotides containing the dRFX2 site (–124/–98) or base-substituted derivatives in the PCNA gene promoter were defined as follows.
–124WT gatccGTTGGCAGGCCGCTCGCTGCCTGCTATagCAACCGTCCGGCGAGCGACGGACGATAt
–124MUT1 gatcctggttCAGGCCGCTCGCTGCCTGCTATagaccaaGTCCGGCGAGCGACGGACGATAt
–124MUT2 gatccGTTGGacttCCGCTCGCTGCCTGCTATagCAACCtaggGGCGAGCGACGGACGATAt
–124MUT3 gatccGTTGGCAGGaataTCGCTGCCTGCTATagCAACCGTCCttatAGCGACGGACGATAt
–124MUT4 gatccGTTGGCAGGCCGCgataTGCCTGCTATagCAACCGTCCGGCGctatACGGACGATAt
–124MUT5 gatccGTTGGCAGGCCGCTCGCgtaagGCTATagCAACCGTCCGGCGAGCGcattcCGATAt
–124MUT6 gatccGTTGGCAGGCCGCTCGCTGCCTtagcgagCAACCGTCCGGCGAGCGACGGAatcgct
Nucleotides substituted for the wild-type sequence are shown by small letters with underlining. To obtain fragments containing base-substitutions in the PCNA gene promoter, the following primers were synthesized and used for PCR.
124MUT1P CTATCGATAGCAGGCAGCGAGCGGCCTGaaccaTGGTTTG
124MUT2P CTATCGATAGCAGGCAGCGAGCGGaagtCCAACTGGTTTG
124MUT3P CTATCGATAGCAGGCAGCGAtattCCTGCCAACTGGTTTG
Plasmid construction
A fragment from –168 to –91 having base-substituted mutations was generated by the PCR method using p5'–168DPCNACAT (46) as a template with primers –124MUT1P and SalI (–168) (16). The plasmid p5'–168DPCNAlacZW8HS contains a PCNA gene fragment spanning from –168 to +137 fused with the lacZ gene in a P-element vector (46). To create mutated derivatives in P-element vector backbones, fragments having various mutations in the region from –124 to –112 of the PCNA gene promoter were isolated from CAT plasmids by digestion with SalI (–168) and SacII (+24), and inserted between the XhoI (–607) and SacII (+24) sites of p5'–607DPCNAlacZW8HS (47).
pACT-dRFX2202–589, which was isolated by one-hybrid screening, was digested with XhoI and the isolated dRFX2 cDNA fragment was inserted into the SalI site of pGEX-4T-1 (Amersham Pharmacia Biotech) to create pGST-dRFX2202–480, or the XhoI site of pUAST-HA to create pUAS-HA-dRFX2202–480. Full length of dRFX2 cDNA was isolated by 5'-RACE (Invitrogen) using total RNAs from larvae and the primer carrying the sequence, 5'-ACCGTAATATTCTAGAGG. The isolated full-length dRFX2 cDNA was then inserted into the XhoI site of pUAST-HA to create pUAS-HA-dRFX2FL.
The GAL4-coding sequence was isolated using PCR with the primers 5'-ATCGAATTCGGTACCAGATGAAGCTACTGTCTTCTATCGA and 5'-ATAAGATCTGCGGCCGCTTACTCTTTTTTTGGGTTTGGTG, and pGaTB plasmid (48) as a template. Products were gel-purified, cut with EcoRI and BglII, and cloned into EcoRI–BglII sites of the pGMR vector (49).
All plasmids were propagated in Escherichia coli (E.coli) XL-1 Blue, isolated by standard procedures (50) and further purified using a Qiagen Plasmid Midi Kit (Qiagen). The DNA sequencing was carried out with a BigDyeTM Terminator v3.0 Cycle sequencing Standard kit (Applied Biosystems) using an ABI PRISMTM 310 NT Genetic Analyzer (Applied Biosystems). When necessary, chemically synthesized oligonucleotides (17mer) were used as sequencing primers.
Expression of GST fusion proteins
Expression of GST–dRFX2202–480 fusion protein in E.coli XL-1 Blue was carried out as described elsewhere (51). Lysates of cells were prepared by sonication in buffer D containing 0.6 M KCl, 1 mM PMSF, and 1 μg/ml each of pepstatin, leupeptin and aprotinin. Lysates were cleared by centrifugation at 12 000 g for 20 min at 4°C and applied to glutathione–Sepharose (Amersham Biotech) to purify the GST–dRFX2202–480 fusion protein as described elsewhere (51). GST protein and GST–dRFX were expressed and purified in the same way.
One-hybrid screening
The MATCHMAKER one-hybrid system from Clontech was applied to isolate a cDNA encoding the protein responsible for dRFX2 site-binding activity. The MATCHMAKER one-hybrid system protocol was used to prepare the target-reporter constructs, to integrate these constructs into Saccharomyces cerevisiae strain YM4271 (his– ura– leu–), and to screen an activation domain (AD) fusion library (ACT-cDNA library) from Drosophila third instar larvae (kindly supplied by Dr Elledge). Four tandem copies of the double-stranded oligonucleotide, –124WT (4-dRFX2 sites) were placed upstream of the marker genes of both the pHISi-1 and pLacZi plasmids (Clontech). The two target-reporter constructs were transformed into S.cerevisiae strain YM4271 in a consecutive manner to produce a dual reporter strain. This was transformed with the pACT-cDNA library, and his+ ura+ leu+ transformants grown in synthetic dropout (SD) medium containing 30 mM 3-aminotriazole (3-AT) were selected. Each colony was streaked on SD agar medium without histidine but containing 30 mM 3-AT and incubated for 3 days at 30°C. A dry NEF-978X filter (NEN Research Products) was placed over the surface of each agar plate containing transformants. The filters were lifted off the agar plate and dipped in liquid nitrogen. The frozen colonies were then thawed at room temperature and the filters were overlaid onto Whatman number 5 filters that had been soaked in Z buffer (60 mM Na2HPO4·7H2O, 40 mM NaH2PO4·H2O, 10 mM KCl, 1 mM MgSO4·7H2O, 50 mM ?-mercaptoethanol, pH 7.0) containing 0.03% 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside (X-Gal) at 30°C for 5–8 h. Positive blue colonies were selected. To confirm sequence-specific interactions, plasmid DNA for each candidate was recovered in E.coli DH5 (Competent high, TOYOBO) followed by retransformation into yeast strains containing the 4-dRFX2 sites reporter.
Southern and northern blot analysis
DNA was extracted from Drosophila bodies at various developmental stages and digested with BamHI, then separated by agarose gel electrophoresis and blotted onto a GeneScreen Plus membrane (New England Nuclear) using a VacuGene blotting apparatus (LKB). The membrane was sequentially hybridized with a 32P-labeled probe containing the dRFX2 cDNA or the PCNA cDNA. The blot was then subjected to hybridization analysis, under the conditions described earlier (52).
Total cellular RNA was isolated from Drosophila bodies at various developmental stages using TRIZOL (Invitrogen), then separated by agarose gel electrophoresis and blotted onto a GeneScreen Plus membrane. The membrane was sequentially hybridized with a 32P-labeled probe, which recognizes mRNA for each of dRFX2, dRFX (42), PCNA (53) or RP-49 (54). The hybridization and washing conditions were as described previously (52).
Isolation of the Drosophila gene for dRFX2
A genomic library that was constructed by inserting the partially Sau3AI-digested DNA from Drosophila melanogaster Oregon-R into the BamHI site of EMBL3 was used (52). Clones containing the dRFX2 gene sequence were isolated by screening the library. The XhoI fragment from the dRFX2 cDNA clone, which was isolated by one-hybrid screening, was used as a probe, labeled with dCTP using a random priming method (55). Plaque hybridizations were carried out at 42°C in a solution containing 20% formamide, 3x SSC, 5x Denhardt's solution, 10% dextran sulfate, 1% SDS, 1 mM sodium pyrophosphate, 100 μg/ml heat-denatured salmon sperm DNA and the probe, as described previously (52).
Band mobility shift assays
Band mobility shift analysis was performed as described earlier (15) with minor modifications. An aliquot of 2 μl of 32P-labeled probes (10 000 c.p.m.) was mixed with 10 μl of binding buffer containing 25 mM HEPES (pH 7.6), 150 mM KCl, 0.1% Triton X-100, 1 mM EDTA, 1 mM DTT and 10% glycerol, then 1 μl of 0.5 mg/ml poly (dI–dC) was added, and the probe mixture was incubated on ice for 5 min. When necessary, unlabeled oligonucleotides were added as competitors at this step. Purified GST–dRFX2 fusion protein was diluted with buffer containing 25 mM HEPES (pH 7.6), 150 mM KCl, 0.1% Triton X-100, 1 mM EDTA, 1 mM DTT, 10% glycerol. Two μl of each fusion protein was added to the probe mixture, then incubated for 15 min on ice. DNA–protein complexes were electrophoretically resolved on 4% polyacrylamide gels in 100 mM Tris-borate (pH 8.3), 2 mM EDTA containing 2.5% glycerol at 25°C. The gels were dried and then autoradiographed.
Fly stocks and establishment of transgenic flies
Fly stocks were maintained at 25°C on standard food. Canton S flies were used as the wild-type strain. Enhancer trap lines carrying the lacZ markers D120 (inserted in scabrous) (56), AE127 (inserted in seven-up) (57) and P82 (58) were obtained from Dr Y. Hiromi. These lines express the ?-galactosidase marker in photoreceptor cells (R) of early R8, R3/R4/R1/R6 and R3/R4/R7, respectively. The GAL4 driver strains were obtained from the Bloomington Stock Center or the Kyoto Institute of Technology Stock Center. Transgenic flies were established with P-element-mediated germ line transformation as described earlier (59), and F1 transformants were selected on the basis of white eye color rescue (60). Transgenic flies for pGMR-GAL4 were used as described previously (61). Established transgenic fly strains and their chromosomal linkages are listed in Supplementary Materials 2 and 3.
Analysis of PCNA-lacZ expression patterns
Quantitative measurement of ?-galactosidase activity in extracts was carried out as described previously (62). Male transgenic flies were crossed with wild-type females, and groups of 50–100 individual dechorionated embryos, larvae, pupae and adult flies were homogenized in 500 μl of ice-cold assay buffer (50 mM potassium phosphate, pH 7.5/1 mM MgCl2). Homogenates were centrifuged at 10 000 g at 4°C for 5 min. For each assay, 50–200 μl of supernatant was added to give 1 ml of assay buffer containing 1 mM chlorophenol red-?-D-galactopyranoside substrate (CPRG; Roche). Reaction incubations were at 37°C in the dark. Substrate conversion was measured at 574 nm using a spectrophotometer at 0.25, 0.5, 0.75, 1 and 1.5 h after addition of the extract, and the rate of color development was linear. The ?-galactosidase activity was defined as absorbance units per hour per milligram of protein. To correct for endogenous ?-galactosidase activity, extracts from the wild-type strain were included in each experiment and the observed background reading was subtracted from readings obtained with each transformant line. Deviation among independent strains was <30% (18). The protein concentrations of the extracts were determined by Bio-Rad protein assay.
?-Galactosidase activity of larval tissues was visualized as described elsewhere (16). After dissection, tissues were incubated in fixative (12 mM sodium cacodylate buffer, pH 7.3/26% glutaraldehyde) for 15 min at room temperature. Treated tissues were then incubated with a staining solution containing 0.2% X-Gal in the dark at 37°C for 5–16 h. For photography, tissues were immersed in glycerol, mounted on slides and photographed with an Olympus microscope (BX-50) using Tri X pan 400 films (Kodak).
Scanning electron microscopy
Adult flies were anesthetized, mounted and observed under a Hitachi S-3000 scanning electron microscope.
5-Bromo-2'-deoxyuridine (BrdU) labeling
Detection of cells in S phase was performed using a BrdU-labeling method as described previously with minor modifications (63). Third instar larvae were dissected in Drosophila Ringer's solution and the imaginal discs were suspended in Grace's insect medium, then incubated in the presence of 75 μg/ml BrdU (Roche) for 40 min at 25°C. The samples were fixed in Carnoy's fixative (ethanol/acetic acid/chloroform, 6:1:3) for 20 min at 25°C, and further fixed in 80% ethanol/50 mM glycine buffer (pH 2.0) at –20°C overnight. Incorporated BrdU was visualized using an anti-BrdU antibody and an alkaline phosphatase detection kit (Roche). The time of color development was identical for all samples.
Apoptosis assay
Third instar larvae were dissected in Drosophila Ringer's solution and the imaginal discs were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at 25°C. After being washed with PBS, the samples were permeabilized by incubation in a solution containing 0.1% sodium citrate and 0.1% Triton X-100 on ice for 30 min. After extensive washing, the TUNEL reaction was carried out using an in situ Cell Death Detection Kit, POD (Roche) according to the manufacturer's recommendations.
Immunohistology
The third instar larvae were dissected and eye imaginal discs were immuno-stained by the method described previously with minor modifications (64). Samples were incubated with mouse anti-?-galactosidase monoclonal antibody (Promega) at a 1:500 dilution or culture supernatant of hybridoma cells producing mouse anti-HA monoclonal antibody (supplied by Dr M. Inagaki) at a 1:100 dilution. The second antibody, an alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G (IgG) (Promega), was used at a 1:500 dilution. In the case of immuno-staining photoreceptor cells, the F1 third instar larvae from the cross of GMR-GAL4; +, or GMR-GAL4; UAS-HA-dRFX2202–480/CyO with each enhancer trap line were dissected.
RESULTS
Identification of binding factor to the region between URE and DRE in the PCNA gene promoter
In previous studies, we found the consensus binding sequence for Zeste protein in the region between URE and DRE (46). To isolate a cDNA encoding a binding factor, we carried out a yeast one-hybrid screen of a Drosophila third instar larvae cDNA library using four tandem copies of the sequence spanning from –124 to –98 as the target binding sequence. On screening 3.1 x 106 independent clones, 52 independent positive clones were identified. Nucleotide sequences of these clones were determined. One clone did not show any match to any sequence in the Drosophila genome but was found to have 57% identity with DNA-binding domain of Drosophila transcription factor, dRFX (Supplementary Material 1) (42). We therefore designated this novel factor as dRFX2. The rest of clones were cDNAs for the ribosomal protein, RpL22 and the cDNA for Zeste protein was not found.
By conducting a 5'-RACE (Invitrogen) using total RNAs from larvae and a primer carrying the sequence, 5'-ACCGTAATATTCTAGAGG, full-length dRFX2 cDNA was isolated. In addition, using the dRFX2 cDNA as a probe, we screened a Drosophila genomic library (52). On screening 4.6 x 105 clones, one containing a sequence of the dRFX2 gene was isolated and its nucleotide sequence was determined (Supplementary Material 1, DDBJ accession no.: AB190179 ). Comparison of nucleotide sequences of the dRFX2 cDNA (2526 bp) and the dRFX2 gene suggested that the latter is intronless. The deduced dRFX2 protein consists of 842 amino acids. Multiple alignment with other RFX family proteins from yeast, nematode, Drosophila and human showed the amino acid sequence from 388 to 463 of dRFX2 to share 33% identity and 57% similarity with hRFX1, the prototype of RFX family proteins, and 39.5% and 61.8% with Sak1, an RFX protein in Schizosaccharomyces pombe (Supplementary Material 1). This domain is highly conserved in the RFX family proteins, and demonstrated to be a DNA-binding domain (DBD). Arginine at position 406 (R406), K432, K435, R444, R445, R449, R453, Y454 and Y456 are highly conserved among RFX family proteins, and in a crystallographic structure of the mammalian RFX1 DBD, make direct or water-mediated DNA contacts (65).
We examined the dRFX2 expression pattern during Drosophila development by northern blot hybridization. Using dRFX2 cDNA as a probe, a major transcript of 3.4 kb was detected in early embryos and larvae (Figure 1, second panel). Apparently, no 3.4 kb transcript was detected in pupae, adults and cultured Kc cells. Although the 3.4 kb transcript is barely detectable in third instar larvae, a minor transcript of 6.1 kb was detected in both second and third instar larvae. The 6.1 kb band may represent an alternative form of dRFX2 mRNA such as a transcript derived from an alternative promoter, since the dRFX2 cDNA clone was identified in the one-hybrid screen using a third instar cDNA library as described above. The cDNAs for dRFX and PCNA were used as reference probes. The dRFX mRNA of 4.4 kb was expressed throughout all developmental stages as reported previously, although its level was fluctuated (42). PCNA mRNA of 1.1 kb was found to be expressed in line with cell proliferation during development as described previously (53). The RP-49 cDNA was used as an internal control to monitor the integrity of mRNA (54). These results indicate that dRFX2 is expressed in embryos and larvae with highest expression in second instar larvae.
Figure 1. Northern blot hybridization analysis: 50 μg of total RNA was loaded in each lane; dRFX2 cDNA was used as a probe (second). As reference probes, cDNAs for dRFX (top) and PCNA (third) were employed. The RP-49 cDNA (bottom) was used as an internal control.
Nucleotide sequences required for binding to dRFX2
Multiple alignment of dRFX2 protein with RFX family proteins revealed high conservation in the region corresponding to the DNA-binding domain, suggesting that dRFX2 carrying this region could bind to DNA. dRFX2202–480, the amino acids from 202 to 480, was therefore expressed as a fusion protein with GST (GST–dRFX2202–480) and band mobility shift assays were carried out. As shown in Figure 2B, a DNA–protein complex was detected with the –124/–98 oligonucleotide containing the region between –124 and –98 of the PCNA gene. To examine specific binding of dRFX2, oligonucleotides containing –149/–118 (36), –108/–85 (15) and –87/–62 (19) were employed as competitors (Figure 2B, lanes e–j and l–n), spanning the binding site between –130 and –119 (URE), –100 and –93 (DRE), and –84 and –77 (CFDD site 1) of the PCNA gene promoter, respectively. None of these competitors competed for the binding (Figure 2A and B). Therefore, the region between –124 and –98 is specifically required for dRFX2-binding.
Figure 2. DNA-binding activity of GST–dRFX2. (A) Organization of Drosophila PCNA gene promoter elements and nucleotide sequences in and around the dRFX2 site. Nucleotide positions of each promoter element and their sequences are shown. The substituted bases are boldface. Results of band mobility shift assays are shown on the right. Gray box, dRFX2 site; open box, URE; stippled box, DRE. (B and C), Radiolabeled double-stranded oligonucleotides carrying the wild-type –124/–98 site were incubated with 0.2 μg of the purified GST–dRFX2 fusion protein in the absence (0), or the presence of a competitor (indicated at the top of each lane, at 100, 200 or 400 ng). In (B), competitors were –124/–98 oligonucleotides containing dRFX2-binding site, –148/–118 oligonucleotides containing URE (36), –108/–85 oligonucleotides containing DRE (15), and –7/–62 oligonucleotides containing the CFDD site1 (19). In (C), –124/–98 oligonucleotides carrying base-substitution mutations were used. Nucleotide sequences of each are shown in panel A. (D) Binding specificity of GST–dRFX and GST–dRFX2 fusion proteins on complex formation with the dRFX2 site or EP sequence. Radiolabeled –124/–98 oligonucleotides were incubated with 0.2 μg each of purified fusion protein (see bottom of each lane) in the presence or absence (0) of the indicated amounts of competitor oligonucleotides containing EP sequence or –124/–98 sequence (see top of each lane).
To determine in detail the nucleotide sequence required for binding to dRFX2, various base-substituted mutations were introduced into the region between –124 and –98 (Figure 2A) and the resultant mutant oligonucleotides were used as competitors in the band mobility shift analysis using GST–dRFX2202–480 fusion protein (Figure 2C). The mutant oligonucleotides mut1 and mut2 did not compete at all (Figure 2C, lanes e–j), while the other mutant oligonucleotides competed for the binding as effectively as the wild-type –124/–98 oligonucleotide (Figure 2C, lanes b–d, l–q and s–x). These results indicate that the sequence 5'-GTTGGCAGG between –124 and –116 plays an important role in the dRFX2-binding (Figure 2C).
We were also interested in whether dRFX can bind to the region between –124 and –116. When band mobility shift assays were performed using the –124/–98 oligonucleotide and GST–dRFX fusion protein, no band shift was detected (data not shown). Band mobility shift analysis was also conducted with the EP probe (66) carrying the binding consensus sequence for the mammalian RFX family proteins and GST–dRFX2202–480 or GST–dRFX fusion protein, and EP oligonucleotide or –124/–98 oligonucleotide was added in the binding reaction as a competitor (Figure 2D). EP oligonucleotide competed effectively to binding reaction between EP probe and either GST–dRFX2202–480 or GST–dRFX fusion protein (Figure 2D, lanes b and e), suggesting that both dRFX and dRFX2 have strong affinity to EP oligonucleotides. When we used GST–dRFX2202–480 fusion protein, the –124/–98 oligonucleotide competed effectively (Figure 2D, lanes a and c). However, when GST–dRFX fusion protein was applied, the shifted band did not disappear (Figure 2D, lanes d and f). These results suggest that the region between –124 and –116 is bound exclusively with dRFX2, and we thus termed this the dRFX2 site.
Role of the dRFX2 site in the dPCNA gene promoter in living flies
Previously, we established transgenic flies carrying PCNA [–168 to +137) and lacZ fusion genes (46). To examine the role of the dRFX2 site in the PCNA promoter activity during Drosophila development, we generated PCNA–lacZ fusion genes carrying base-substituted mutations in and around the dRFX2 site (Figure 3A). These fusion genes were then introduced into flies by germ-line transformation. Established transgenic fly lines and their chromosomal linkages are listed in Supplementary Material 2. Activities of the modified promoters were then monitored by quantitative ?-galactosidase assays at various developmental stages (Figure 3B).
Figure 3. Role of dRFX2 site in PCNA gene promoter activity. (A) Nucleotide sequences of the region between –130 and –98 of the wild-type PCNA gene promoter and its base-substituted mutants are shown. The substituted bases are boldface. Promoter activity in transgenic flies is also indicated. Gray box, dRFX2 site; open box, URE. (B) Male transgenic flies were crossed with female wild-type flies, and extracts were prepared from Drosophila bodies at various stages of development. The ?-galactosidase activities in the extracts are expressed as absorbance units/h/mg of protein. (C) Salivary glands were dissected from third instar lavae of the progeny from crosses described in (B). Salivary glands from flies carrying: (a) p5'-168DPCNAlacZW8HS (–168); (b) p5'-168mut1DPCNAlacZW8HS (mut1); (c) p5'-168mut2DPCNAlacZW8HS (mut2); (d) p5'-168mut3DPCNAlacZW8HS (mut3). Staining signals in the ducts appear to be non-specific.
In flies carrying the PCNA gene promoter region up to position –168, base-substitution mutations in the dRFX2 site (mut1 and mut2) reduced the lacZ expression to 50% in embryos (Figure 3B, panels a–c). Much more extensive reduction of the lacZ expression was observed in larvae carrying mutations in the dRFX2 site (Figure 3B, panels a–c). Especially in second and third instar larvae carrying base-substitution mutations in the region between –116 to –120, the lacZ expression was barely detectable (Figure 3B, panel c). In contrast, only a marginal effect on the lacZ expression was observed with a mutation outside the dRFX2 site (mut3) (Figure 3B, panel d). In pupae and adults, there were no remarkable differences between flies carrying the wild-type promoter and those carrying base-substituted derivatives (Figure 3B). Since the extent of the reduction was most prominent in larvae, ?-galactosidase activity was demonstrated in dissected third instar larval tissues (Figure 3C). In transgenic third instar larvae carrying a wild-type construct, high lacZ-staining signal was observed in the salivary glands (Figure 3C, panel a). The larvae having mutations in the dRFX2 site (mut1 and mut2) exhibited extensively reduced staining signals (Figure 3C, panels b and c). In contrast, no reduction of staining was observed with lines carrying a mutation outside the dRFX2 site (mut3, Figure 3C, panel d). We carried out the same examination on probable neuroblasts in the central nervous system (CNS) of third instar larvae and obtained similar results (data not shown). Thus, an important role of the dRFX2 site for the dPCNA gene promoter activity, especially at larval stages, was demonstrated in living flies.
Expression of the DNA-binding domain (amino acid residues 202–480) of dRFX2 in transgenic flies
In our previous studies, transgenic flies expressing DBD of the transcription factor DREF were successfully used to examine function of endogenous DREF by inhibiting its activity in a dominant negative manner (67). To investigate the function of dRFX2 in living flies, we took a similar approach. Band mobility shift assay using the dRFX2 site revealed that the region from amino acid 202 to 480 of dRFX2 contains the DNA-binding activity (Figure 2). Therefore, the region containing this putative DNA-binding domain of dRFX2 with HA-tag (HA-dRFX2202–480) was subcloned into the pUAST vector, and the plasmid was named pUAS-HA-dRFX2202–480. We established 11 independent transgenic fly lines carrying UAS-HA-dRFX2202–480 (Supplementary Material 3). These transgenic flies were then crossed with transgenic flies carrying GAL4 cDNA placed under the control of promoters that function in specific tissues or at specific developmental stages (Table 1), and in their progenies the effects of expression of dRFX2202–480 on the development of Drosophila were examined (48).
Table 1. Summary of effects in expression of HA-dRFX2202–480 with each GAL4 driver line
Over-expression of dRFX2202–480 with the Cg-GAL4 driver (Cg-GAL4>HA-dRFX2202–480) caused the generation of melanotic masses in larvae, and flies died in the larval stage. The Cg-GAL4 driver strain expresses GAL4 in blood cells. With dll-GAL4>HA-dRFX2202–480 and en-GAL4>HA-dRFX2202–480, death occurred in the embryonic stage (Table 1). While no detectable phenotype was observed with twi-GAL4> HA-dRFX2202–480, 71B-GAL4>HA-dRFX2202–480 and act88F-GAL4>HA-dRFX2202–480. The GMR-GAL4>HA-dRFX2202–480 flies exhibited severely abnormal eye morphology with a rough appearance (Table 1 and Figure 4A, panels c and d). The ommatidia lacked their regular hexagonal shape and appeared to be fused. Either additional or missing bristles were apparent. Over-expression of HA-dRFX2202–480 in the eye imaginal disc was confirmed by immuno-staining with an anti-HA antibody (Figure 4B). When the copy number of UAS-HA-dRFX2202–480 was increased to two, embryonic lethality resulted.
Figure 4. Expression of HA-dRFX2202–480 in eye imaginal disc induces a rough eye phenotype. (A) Scanning electron micrographs of adult compound eyes. (a) and (b), GMR-GAL4/+, +; (c) and (d), GMR-GAL4/+, UAS-HA-dRFX2202–480/+; (e) and (f), GMR-GAL4/+, UAS-HA-dRFX2202–480/UAS-HA-dRFX2FL; (g) and (h), GMR-GAL4/+, UAS-HA-dRFX2FL/+. Magnification: (a, c, e and g), 200x; (b, d, f and h), 1000x. The flies developed at 28°C. (B) Immuno-staining of eye imaginal discs with anti-HA antibody. (a), GMR-GAL4/+, +/+; (b), GMR-GAL4/+, UAS-HA-dRFX2202–480/+. The anti-HA signal was detected in the region posterior to the MF. MF, morphogenetic furrow.
We have also established 11 independent transgenic fly lines carrying UAS-HA-dRFX2FL (Supplementary Material 3). Over-expression of dRFX2 protein with GMR-GAL4 driver induced apparently weak rough eye phenotype which is indistinguishable from that induced by expression of GAL4 alone (Figure 4A, panels a, b, g and h). However, when the GMR-GAL4; UAS-HA-dRFX2202–480/CyO was crossed with UAS-dRFX2FL, the rough eye phenotype induced by dRFX2202–480 was effectively suppressed (Figure 4A, panels e and f), indicating that the effect of expression of the dRFX2-DBD on eye morphology is truly the dominant negative effect against the endogenous dRFX2. In the following studies, we focused on analyses of the rough eye phenotype induced by dRFX2202–480, since cell proliferation and differentiation during Drosophila eye development have been well characterized (68).
Ectopic expression of dRFX2202–480 in eye imaginal discs can inhibit cell cycle progression
In the third instar larvae, the morphogenetic furrow (MF) appears at the posterior end of the eye imaginal discs, and slowly moves in the anterior direction. Cells in front of the MF proliferate asynchronously, while those on the MF are arrested synchronously at G0/G1 phase. Cells behind the MF either leave the cell cycle and differentiate into photoreceptors of the adult ommatidium, or undergo one more cell division. This cell cycle is a final synchronous round, and produces an S-phase band (the second mitotic wave), then these cells form a reservoir of cells for subsequent differentiation events. Therefore, no mitotic cells behind the second mitotic wave are seen (68).
In GMR-GAL4, the promoter carrying transcription factor Glass-binding sites was used for the expression of GAL4 in the region within and posterior to the MF (49). The flies with GMR-GAL4>HA-dRFX2202–480 expressed HA-dRFX2202–480 in the region within and posterior to the furrow that was visualized by immuno-staining with an anti-HA antibody (Figure 4B).
To examine the effect of dRFX2202–480 expression on DNA synthesis in the eye imaginal disc cells, we visualized the cells in S phase with BrdU incorporation. In eye discs of flies expressing GAL4 alone, BrdU incorporation was observed anterior to the MF and in a stripe just posterior to the MF, indistinguishable from the pattern of BrdU incorporation in wild-type fly discs reported previously (Figure 5A) (61). In HA-dRFX2202–480-expressing discs, extensive reduction of BrdU signals in the synchronized S-phase zone behind the MF was evident (Figure 5B). The results indicate that ectopic expression of HA-dRFX2202–480 can inhibit entry into the S phase or progress through the S phase in the eye imaginal disc, suggesting that endogenous dRFX2 plays a role in the G1–S transition or S-phase progression. In addition, slight reduction of BrdU signals was also observed in the region anterior to the MF where the GMR promoter was inactive, suggesting some non-cell autonomous regulations in eye imaginal discs. Further analyses are necessary to clarify this point.
Figure 5. Expression of HA-dRFX2202–480 reduces the number of S-phase cells in eye imaginal discs. After incorporation of BrdU for 40 min, the eye imaginal discs were immuno-stained with anti-BrdU antibody. (A) GMR-GAL4/+, +; (B) GMR-GAL4/+, UAS-HA-dRFX2202–480/+. MF, morphogenetic furrow.
Ectopic expression of dRFX2202–480 in eye imaginal discs does not interfere with photoreceptor cell differentiation
Photoreceptor cells have been found to be generated sequentially: R8 is generated first, with movement posterior from the MF, then cells are added pairwise (R2 and R5, R3 and R4, and R1 and R6), and R7 is the last photoreceptor to be added to the precluster (68). Several enhancer trap lines expressing a nucleus-localized form of ?-galactosidase depend on the specific enhancer-promoter located nearby the P-element. They were used here to identify each photoreceptor cell. We used three enhancer trap lines, D120 (inserted in scabrous) (56), AE127 (inserted in seven-up) (57) and P82 (58), specifically expressing the ?-galactosidase marker in photoreceptor cells of early R8, R3/R4/R1/R6 and R3/R4/R7, respectively. The imaginal discs of F1 larvae from mating of each enhancer trap line and transgenic fly expressing dRFX2202–480 in developing eye were immunohistologically stained with an anti ?-galactosidase antibody.
When dRFX2202–480 was expressed in eye imaginal discs, photoreceptors of such flies showed essentially the same staining pattern as those of control flies (data not shown). From these results, we conclude that ectopic expression of dRFX2202–480 does not interfere with differentiating photoreceptor cells in eye discs.
Figure 6. Effects of HA-dRFX2202–480 on photoreceptor cell development. (a) Males of each photoreceptor cell-specific enhancer trap line were crossed with females of GMR-GAL4 or GMR-GAL4; UAS-HA-dRFX2202–480/CyO, ActGFP. The enhancer trap lines were D120 (A), AE127 (B) and P82 (C), and expressed ?-galactosidase in R8, R3/4/1/6 and R3/4/7, respectively. Eye imaginal discs of these GFP-negative progenies were stained with anti-?-GAL antibody (Promega). R8, R3/4/1/6 and R3/4/7 were stained as well as the wild type (data not shown). (b) High-magnification appearance.
Ectopic expression of dRFX2202–480 in eye imaginal discs can induce apoptosis
Ectopic expression of dRFX2202–480 in eye imaginal discs induced an abnormal S phase. Because it has been reported that disorder of cell cycle progression and disturbance of differentiation processes cause apoptosis (69), we investigated this process in eye imaginal discs expressing dRFX2202–480 by the TUNEL method. In eye discs of flies expressing GAL4 alone, there was very limited cell death (Figure 7A, panel a). In contrast, eye discs of flies expressing dRFX2202–480 showed a significant increase of apoptotic cells posterior to the MF (Figure 7A, panel b). Moreover, when adult flies expressing dRFX2202–480 were crossed with those expressing inhibitors of apoptosis in Drosophila, p35 (70), DIAP1 and DIAP2 (71), the rough eye phenotype was suppressed in their progeny (Figure 7B). These results indicate that ectopic expression of dRFX2202–480 in eye imaginal discs can induce apoptosis.
Figure 7. Expression of HA-dRFX2202–480 induces ectopic apoptosis in eye imaginal discs. (A) Detection of apoptotic cells in eye imaginal discs with TUNEL assays. (a), GMR-GAL4/+, +; (b), GMR-GAL4/+, UAS-HA-dRFX2202–480/+. Expression of HA-dRFX2 increased the signals (see bracket). MF, morphogenetic furrow. (B) Apoptosis inhibitors, p35 (70), DIAP1 and DIAP2 (71) suppress the HA-dRFX2202–480-induced rough eye phenotype. (a), GMR-GAL4/+, UAS-HA-dRFX2202–480/UAS-LacZ; (b), GMR-GAL4/+, UAS-HA-dRFX2202–-480/+, UAS-p35/+; (c), GMR-GAL4/+, UAS-HA-dRFX2202–480/+, GMR-DIAP1/+; (d), GMR-GAL4/+, UAS-HA-dRFX2202–480/+, GMR-DIAP2/+.
DISCUSSION
The Drosophila PCNA gene promoter contains several regulatory elements such as E2F sites (18), DRE (15), CFDD sites (19) and URE (36). In the present study, we found a novel regulatory element, dRFX2 site in the region between DRE and URE. The nucleotide sequence required for dRFX2-binding was determined to be 5'-GTTGGCAGG that matches eight out of nine bases of the sequence 5'-GTNRCCNNRG that spans half of the binding consensus sequence for human RFX family proteins, 5'-GTNRCC/N-N0–3-RGYAAC (72). E2F sites, DRE (15) and CFDD sites (19) are found in common in various DNA replication-related genes in Drosophila, while URE appears to be unique to the PCNA gene (36,73). Although we have searched for dRFX2-binding sequences in the Drosophila genome database, no perfect match was apparent within the region between –250 and +50 of DNA replication-related genes such as orc1-6, mcm2-7, dup/Cdt1, CG5790/Cdc7, DNA polymerase and , and gnf1/RFC140. PCNA is well known to act as a sliding clamp in DNA replication, but can also interact with many other protein factors involved in various other biological functions (74). The endogenous dRFX2, therefore, might take a part in regulation of PCNA gene expression related to not only DNA replication but also other biological functions.
We have found that the nucleotide sequence of the dRFX2 site shares 6 out of 9 bases (the region between –124 and –119 of the PCNA gene) with the sequence of the GRH-binding site (URE) (36,37). In this study, we carried out yeast one-hybrid screening using the sequence spanning from –124 to –98 as the target site and isolated only a dRFX2 cDNA and not a GRH cDNA. A previous study revealed that the region between –130 and –123 is more important for GRH-binding than that between –122 and –118 (36). On the other hand, in this study, in the band mobility shift assays with GST-dRFX2 and the oligonucleotide –124/–98 as a probe, the oligonucleotide mut1 containing the mutation in the region between –124 and –120 did not compete for binding at all. Therefore, the shared nucleotide sequences appear more important for dRFX2- than GRH-binding.
The full-length dRFX2 cDNA was isolated by conducting 5'-RACE and screening of the Drosophila genomic DNA library allowed us to determine genomic clones spanning the dRFX2 gene and their nucleotide sequences. Although the sequence could not be found in the Drosophila database, the FlyBase, Southern blot hybridization analysis revealed that the dRFX2 is the gene of Drosophila melanogaster. By Southern blot analysis of the genomic DNA from Drosophila adults cut with BamHI using the dRFX2 cDNA as a probe, a single band of 7.7 kb was detected (data not shown), suggesting the occurrence of a single copy of the dRFX2 gene per genome. It should be noted that the signal was much weaker than for genes located in euchromatin regions such as the dPCNA gene. Genomic DNA in euchromatin region is highly amplified in Drosophila adult tissues. Moreover, nucleotide sequences in and around the dRFX2 gene contain many repetitive sequences, which are characteristic of the heterochromatin region. The fact that they were found to be five times more frequent in the dRFX2 gene region than in the dRFX gene region suggests that the former is located in heterochromatin.
Northern blot hybridization analysis revealed the highest expression of dRFX2 in larval stages, correlating with findings for function. Our previous studies indicated that the region containing the transcription initiation site and the E2F sites is sufficient for maternal expression in the ovary. In addition, DRE is required for promoter activity in embryos. In larval stages, the dRFX2 site in addition to the E2F sites, CFDD sites, DRE and URE appear to be essential for activation of the PCNA gene promoter . Therefore, as development advances, the promoter may require more and more elements to exhibit high activity.
In Drosophila, the dRFX gene has already been established as an RFX family gene. The dRFX gene is expressed in type I sensory neuron lineage of the peripheral nervous system throughout development (75), and dRFX mutants exhibit defects in the morphogenesis of the sensory cilium (76). dRFX is homologous to DAF-19 (RFX of Caenorhabditis elegans) and to human RFX1 to 3 (hRFX1-3) (76). The hRFX1 protein was the first representative of the RFX family proteins to be isolated and has been used as the prototype. In addition to the RFX domain, hRFX1 has other conserved domains containing dimerization domain, shared with dRFX. dRFX2 has the RFX domain but lacks these motifs. Therefore, it may be not be likely that dRFX2 forms heterodimer with dRFX. The amino acid sequence of dRFX2 has highest homology with Sak1 and Crt1 (39.5% identity and 61.8% similarity) among RFX family proteins. Therefore, we conclude that dRFX2 is a new member, belonging to a different group from another Drosophila RFX proteins.
To investigate the function of dRFX2 in living flies, we analyzed dominant negative effects. HA-dRFX2202–480 carrying an amino acid sequence containing the RFX domain (DNA-binding domain) was expressed within and posterior to the MF of the eye imaginal discs. Although the HA-dRFX2202–480 is very likely to be a specific dominant negative form active against endogenous dRFX2, we cannot completely exclude the possibility that this form also exerts some effects against endogenous dRFX, since both dRFX2 and dRFX have affinity to the same nucleotide sequence such as EP probe. In eye imaginal discs expressing HA-dRFX2202–480, the G1–S transition and/or the progression of S phase were/was interrupted, and the ectopic apoptosis was induced, though photoreceptor cells differentiated normally. Since dRFX2202–480 seemed to induce the disturbance of the cell cycle and reduction of the number of cells, cell–cell interactions may have been perturbed so that ectopic apoptosis was induced. Expression of dRFX2202–480 might also induce ectopic apoptosis directly. All components of the adult compound eye, including cone cells, bristles and pigment cells are recruited after photoreceptor cell commitment. In either case, these might not be able to develop normally. Specific antibodies against proteins specifically expressed in each of these cells and enhancer trap lines might be useful to identify these cell types. For genetic screening to identify dRFX2-target genes or interacting proteins in vivo, the transgenic flies established in this study should be useful, and more detailed study of the rough eye phenotype with histological analyses might provide us more information on the roles of endogenous dRFX2.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at NAR Online.
ACKNOWLEDGEMENTS
We thank Dr Y. Hiromi for providing enhancer trap lines, Dr M. Inagaki for the anti-HA antibody, Dr S. Elledge for the ACT-Drosophila cDNA library, and Drs S. Cotterill and M. Moore for comments on the English language used in the manuscript. This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan.
REFERENCES
Tsurimoto,T., Melendy,T. and Stillman,B. ( (1990) ) Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin. Nature, , 346, , 534–539.
Jaskulski,D., DeRiel,K., Mercer,W.E., Calabretta,B. and Baserga,R. ( (1988) ) Inhibition of cellular proliferation by antisense oligodeoxynucleotides to PCNA cyclin. Science, , 240, , 1544–1546.
Liu,Y.C., Marraccino,R.L., Keng,P.C., Bambara,R.A., Lord,E.M., Chou,W.G. and Zain,S.B. ( (1989) ) Requirement for proliferating cell nuclear antigen expression during stages of the Chinese hamster ovary cell cycle. Biochemistry, , 28, , 2967–2974.
Kelman,Z. and O'Donnell,M. ( (1995) ) Structural and functional similarities of prokaryotic and eukaryotic DNA polymerase sliding clamps. Nucleic Acids Res., , 23, , 3613–3620.
Shivji,M.K.K., Kenny,M.K. and Wood,R.D. ( (1992) ) Proliferating cell nuclear antigen is required for DNA excision repair. Cell, , 69, , 367–374.
Smith,M.L., Chen,I.-T., Zhan,Q., Bae,I., Chen,C.-Y., Gilmer,T.M., Kastan,M.B., O'Conner,P.M. and Fornace,A.J.J. ( (1994) ) Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science, , 266, , 1376–1380.
Matsuoka,S., Yamaguchi,M. and Matsukage,A. ( (1994) ) D-type cyclin-binding regions of proliferating cell nuclear antigen. J. Biol. Chem., , 269, , 11030–11036.
Waga,S., Hannon,G.J., Beach,D. and Stillman,B. ( (1994) ) The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature, , 369, , 574–578.
Flores-Rozas,H., Kelman,Z., Dean,F., Pan,Z.-Q., Harper,J.W., Elledge,S.J., O'Donnell,M. and Hurwitz,J. ( (1994) ) Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme. Proc. Natl Acad. Sci. USA, , 91, , 8655–8659.
Kelman,Z. and Hurwitz,J. ( (1998) ) Protein–PCNA interactions: a DNA-scanning mechanism? Trends Biochem. Sci., , 23, , 236–238.
Warbrick,E. ( (1998) ) The puzzle of PCNA's many partners. Bioessays, , 20, , 195–199.
Henderson,D.S., Banga,S.S., Grigliatti,T.A. and Boyd,J.B. ( (1994) ) Mutagen sensitivity and suppression of position-effect variegation result from mutations in mus209, the Drosophila gene encoding PCNA. EMBO J., , 13, , 1450–1459.
Shibahara,K. and Stillman,B. ( (1999) ) Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin. Cell, , 96, , 575–585.
Zhang,Z., Shibahara,K. and Stillman,B. ( (2000) ) PCNA connects DNA replication to epigenetic inheritance in yeast. Nature, , 408, , 221–225.
Hirose,F., Yamaguchi,M., Handa,H., Inomata,Y. and Matsukage,A. ( (1993) ) Novel 8-base pair sequence (Drosophila DNA replication-related element) and specific binding factor involved in the expression of Drosophila genes for DNA polymerase and proliferating cell nuclear antigen. J. Biol. Chem., , 268, , 2092–2099.
Yamaguchi,M., Hayashi,Y., Nishimoto,Y., Hirose,F. and Matsukage,A. ( (1995) ) A nucleotide sequence essential for the function of DRE, a common promoter element for Drosophila DNA replication-related genes. J. Biol. Chem., , 270, , 15808–15814.
Takahashi,Y., Yamaguchi,M., Hirose,F., Cotterill,S., Kobayashi,J., Miyajima,S. and Matsukage,A. ( (1996) ) DNA replication-related elements cooperate to enhance promoter activity of the Drosophila DNA polymerase 73-kDa subunit gene. J. Biol. Chem., , 271, , 14541–14547.
Yamaguchi,M., Hayashi,Y. and Matsukage,A. ( (1995) ) Essential role of E2F recognition sites in regulation of the proliferating cell nuclear antigen gene promoter during Drosophila development. J. Biol. Chem., , 270, , 25159–25165.
Hayashi,Y., Hirose,F., Nishimoto,Y., Shiraki,M., Yamagishi,M., Matsukage,A. and Yamaguchi,M. ( (1997) ) Identification of CFDD (common regulatory factor for DNA replication and DREF genes) and role of its binding site in regulation of the proliferating cell nuclear antigen gene promoter. J. Biol. Chem., , 272, , 22848–22858.
Ohno K., Hirose,F., Sakaguchi,K., Nishida,Y. and Matsukage,A. ( (1996) ) Transcriptional regulation of the Drosophila CycA gene by the DNA replication-related element (DRE) and DRE binding factor (DREF) Nucleic Acids Res., , 24, , 3942–3946.
Ryu,J.-R., Choi,T.-Y., Kwon,E.-J., Lee,W.-H., Nishida,Y., Hayashi,Y., Matsukage,A., Yamaguchi,M. and Yoo,M.-A. ( (1997) ) Transcriptional regulation of the Drosophila-raf proto-oncogene by the DNA replication-related element (DRE)/DRE-binding factor (DREF) system. Nucleic Acids Res., , 24, , 794–799.
Matsukage,A., Hirose,F., Hayashi,Y., Hamada,K. and Yamaguchi,M. ( (1995) ) The DRE sequence TATCGATA, a putative promoter-activating element for Drosophila melanogaster cell-proliferation-related genes. Gene, , 166, , 233–236.
Hirose,F., Yamaguchi,M., Kuroda,K., Omori,A., Hachiya,T., Ikeda,M., Nishimoto,Y. and Matsukage,A. ( (1996) ) Isolation and characterization of cDNA for DREF, a promoter-activating factor for Drosophila DNA replication-related genes. J. Biol. Chem., , 271, , 3930–3937.
Hart,C.M., Cuvier,O. and Laemmli,U.K. ( (1999) ) Identification of a multicopy chromatin boundary element at the borders of silenced chromosomal domains. Chromosoma, , 108, , 375–383.
Ohtani,K. and Nevins,J.R. ( (1994) ) Functional properties of a Drosophila homolog of the E2F1 gene. Mol. Cell. Biol., , 14, , 1603–1612.
Dynlacht,B.D., Brook,A., Dembski,M., Yenush,L. and Dyson,N. ( (1994) ) DNA-binding and trans-activation properties of Drosophila E2F and DP proteins. Proc. Natl Acad. Sci. USA, , 91, , 6359–6363.
Hao,X.F., Alphey,L., Bandara,L.R., Lam,E.W.-F., Glover,D. and La Thangue,N.B. ( (1995) ) Functional conservation of the cell cycle-regulating transcription factor DRTF1/E2F and its pathway of control in Drosophila melanogaster. J. Cell Sci., , 108, , 2945–2954.
Sawado,T., Yamaguchi,M., Nishimoto,Y., Ohno,K., Sakaguchi,K. and Matsukage,A. ( (1998) ) dE2F2, a novel E2F-family transcription factor in Drosophila melanogaster. Biochem. Biophys. Res. Commun., , 251, , 409–415.
Duronio,R.J., O'Farrell,P.H., Xie,J.E., Brook,A. and Dyson,N. ( (1995) ) The transcription factor E2F is required for S phase during Drosophila embryogenesis. Genes Dev., , 9, , 1445–1455.
Royzman,I., Whittaker,A.J. and Orr-Weaver,T.L. ( (1997) ) Mutations in Drosophila DP and E2F distinguish G1-S progression from an associated transcriptional program. Genes Dev., , 11, , 1999–2011.
Cayirlioglu,P., Bonnette,P., Dickson,M.R. and Duronio,R.J. ( (2001) ) Drosophila E2f2 promotes the conversion from genomic DNA replication to gene amplification in ovarian follicle cells. Development, , 28, , 5085–5098.
Frolov,M.V., Huen,D., Stevaux,O., Dimova,D., Balczarek-Strang,K., Elsdon,M. and Dyson,N.J. ( (2001) ) Functional antagonism between E2F family members. Genes Dev., , 15, , 2146–2160.
Stevaux,O., Dimova,D., Frolov,M.V., Taylor-Harding,B., Morris,E. and Dyson,N. ( (2002) ) Distinct mechanisms of E2F regulation by Drosophila RBF1 and RBF2. EMBO J., , 21, , 4927–4937.
Bray,S.J., Burke,B., Brown,N.H. and Hirsh,J. ( (1989) ) Embryonic expression pattern of a family of Drosophila proteins that interact with a central nervous system regulatory element. Genes Dev., , 3, , 1130–1145.
Dynlacht,B.D., Attardi,L.D., Admon,A., Freeman,M. and Tjian,R. ( (1989) ) Functional analysis of NTF-1, a developmentally regulated Drosophila transcription factor that binds neuronal cis elements. Genes Dev., , 3, , 1677–1688.
Hayashi,Y., Yamagishi,M., Nishimoto,Y., Taguchi,O., Matsukage,A. and Yamaguchi,M. ( (1999) ) A binding site for the transcription factor Grainyhead/Nuclear transcription factor-1 contributes to regulation of the Drosophila proliferating cell nuclear antigen gene promoter. J. Biol. Chem., , 274, , 35080–35088.
Yamaguchi,M., Hirose,F. and Matsukage,A. ( (1996) ) Roles of multiple promoter elements of the proliferating cell nuclear antigen gene during Drosophila development. Genes Cells, , 1, , 47–58.
Reith,W., Herrero-Sanchez,C., Kobr,M., Silacci,P., Berte,C., Barras,E., Fey,S. and Mach,B. ( (1990) ) MHC class II regulatory factor RFX has a novel DNA-binding domain and a functionally independent dimerization domain. Genes Dev., , 4, , 1528–1540.
Wu,S.Y. and McLeod,M. ( (1995) ) The sak1+ gene of Schizosaccharomyces pombe encodes an RFX family DNA-binding protein that positively regulates cyclic AMP-dependent protein kinase-mediated exit from the mitotic cell cycle. Mol. Cell. Biol., , 15, , 1479–1488.
Emery,P., Durand,B., Mach,B. and Reith,W. ( (1996) ) RFX proteins, a novel family of DNA binding proteins conserved in the eukaryotic kingdom. Nucleic Acids Res., , 24, , 803–807.
Huang,M., Zhou,Z. and Elledge,S.J. ( (1998) ) The DNA replication and damage checkpoint pathways induce transcription by inhibition of the Crt1 repressor. Cell, , 94, , 595–605.
Durand,B., Vandaele,C., Spencer,D., Pantalacci,S. and Couble,P. ( (2000) ) Cloning and characterization of dRFX, the Drosophila member of the RFX family of transcription factors. Gene, , 246, , 285–293.
Labrie,C., Lee,B.H. and Mathews,M.B. ( (1995) ) Transcription factors RFX1/EF-C and ATF-1 associate with the adenovirus E1A-responsive element of the human proliferating cell nuclear antigen promoter. Nucleic Acids Res., , 23, , 3732–3741.
Lee,B.H., Liu,M. and Mathews,M.B. ( (1998) ) Regulation of the human proliferating cell nuclear antigen promoter by the adenovirus E1A-associated protein p107. J. Virol., , 72, , 1138–1145.
Liu,M., Lee,B.H. and Mathews,M.B. ( (1999) ) Involvement of RFX1 protein in the regulation of the human proliferating cell nuclear antigen promoter. J. Biol. Chem., , 274, , 15433–15439.
Yamaguchi,M., Hirose,F., Nishida,Y. and Matsukage,A. ( (1991) ) Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknullt protein. Mol. Cell. Biol., , 11, , 4909–4917.
Yamaguchi,M., Nishida,Y. and Matsukage,A. ( (1995) ) Role of homeodomain protein binding region in the expression of Drosophila proliferating cell nuclear antigen gene: analysis with transgenic flies. Gene Expr., , 4, , 183–193.
Brand,A.H. and Perrimon,N. ( (1993) ) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development, , 118, , 401–415.
Hay,B.A., Wolff,T. and Rubin,G.M. ( (1994) ) Expression of baculovirus P35 prevents cell death in Drosophila. Development, , 120, , 2121–2129.
Sambrook,J., Fritsch,E.F. and Maniatis,T. ( (1989) ) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Smith,D.B. and Johnson,K.S. ( (1988) ) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene, , 67, , 31–40.
Hirose,F., Yamaguchi,M., Nishida,Y., Masutani,M., Miyazawa,H., Hanaoka,F. and Matsukage,A. ( (1991) ) Structure and expression during development of Drosophila melanogaster gene for DNA polymerase . Nucleic Acids Res., , 19, , 4991–4998.
Yamaguchi,M., Nishida,Y., Moriuchi,T., Hirose,F., Hui,C.-C., Suzuki,Y. and Matsukage,A. ( (1990) ) Drosophila PCNA/cyclin gene: structure and expression during development and specific binding of homeodomain proteins to its 5'-flanking region. Mol. Cell. Biol., , 10, , 872–879.
O'Connell,P.O. and Rosbash,M. ( (1984) ) Sequence, structure, and codon preference of the Drosophila ribosomal protein 49 gene. Nucleic Acids Res., , 12, , 5495–5513.
Feinberg,A.P. and Vogelstein,B. ( (1983) ) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem., , 132, , 6–13.
Mlodzik,M., Baker,N.E. and Rubin,G.M. ( (1990) ) Spacing differentiation in the developing Drosophila eye: a fibrinogen-related lateral inhibitor encoded by scabrous. Genes Dev., , 4, , 1848–1861.
Mlodzik,M., Hiromi,Y., Weber,U., Goodman,C.S. and Rubin,G.M. ( (1990) ) The Drosophila seven-up gene, a member of the steroid receptor gene superfamily, controls photoreceptor cell fates Cell, , 60, , 211–224.
Kramer,S., West,S.R. and Hiromi,Y. ( (1995) ) Cell fate control in the Drosophila retina by the orphan receptor seven-up: its role in the decisions mediated by the ras signaling pathway. Development, , 121, , 1361–1372.
Spradling,A.C. ( (1986) ) P element-mediated transformation. In Roberts,D.B. (ed.), Drosophila: A Practical Approach. IRL Press, Oxford, pp. 175–197.
Robertson,H.M., Preston C.R., Philips,R.W., Johnson-Schlitz,D.M., Benz,W.K. and Engels,W.R. ( (1988) ) A stable genomic source of P element transposase in Drosophila melanogaster. Genetics, , 118, , 461–470.
Yamaguchi,M., Hirose,F., Inoue,Y.H., Shiraki,M., Hayashi,Y., Nishi,Y. and Matsukage,A. ( (1999) ) Ectopic expression of human p53 inhibits entry into S phase and induces apoptosis in the Drosophila eye imaginal disc. Oncogene, , 18, , 6767–6775.
Fridell,Y.-W.C. and Searles,L.L. ( (1992) ) In vivo transcriptional analysis of the TATA-less promoter of the Drosophila melanogaster vermilion gene. Mol. Cell. Biol., , 12, , 4571–4577.
Wilder,E.L. and Perrimon,N. ( (1995) ) Dual functions of wingless in the Drosophila leg imaginal disc. Development, , 121, , 477–488.
Hirose,F., Ohshima,N., Shiraki,M., Inoue,H.Y., Taguchi,O., Nishi,Y., Matsukage A. and Yamaguchi,M. ( (2001) ) Ectopic expression of DREF induces DNA synthesis, apoptosis, and unusual morphogenesis in the Drosophila eye imaginal disc: possible interaction with Polycomb and trithorax group proteins. Mol. Cell. Biol., , 21, , 7231–7242.
Gajiwala,K.S., Chen,H., Cornille,F., Roques,B.P., Reith,W., Mach,B. and Burley,S.K. ( (2000) ) Structure of the winged-helix protein hRFX1 reveals a new mode of DNA binding. Nature, , 403, , 916–921.
Katan-Khaykovich,Y. and Shaul,Y. ( (1998) ) RFX1, a single DNA-binding protein with a split dimerization domain, J. Biol. Chem., , 273, , 24504–24512.
Hirose,F., Yamaguchi,M. and Matsukage,A. ( (1999) ) Targeted expression of the DNA binding domain of DRE-binding factor, a Drosophila transcription factor, attenuates DNA replication of the salivary gland and eye imaginal disc. Mol. Cell. Biol., , 19, , 6020–6028.
Wolff,T. and Ready,D.F. ( (1993) ) The Development of Drosophila melanogaster. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 1277–1326.
Harrington,E.A., Fanidi,A. and Evan,G.I. ( (1994) ) Oncogenes and cell death. Curr. Opin. Genet. Dev., , 4, , 120–129.
Crook,N.E., Clem,R.J. and Miller,L.K. ( (1993) ) An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif. J. Virol., , 67, , 2168–2174.
Hay,B.A., Wassarman,D.A. and Rubin,G.M. ( (1995) ) Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell, , 83, , 1253–1262.
Emery,P., Strubin,M., Hofmann,K., Bucher,P., Mach,B. and Reith,W. ( (1996) ) A consensus motif in the RFX DNA binding domain and binding domain mutants with altered specificity. Mol. Cell. Biol., , 16, , 4486–4494.
Yamaguchi,M., Hayashi,Y., Hirose,F., Nishimoto,Y. and Matsukage,A. ( (1997) ) Distinct roles of E2F recognition sites as positive or negative elements in regulation of the DNA polymerase 180 kDa catalytic subunit gene promoter during Drosophila development. Nucleic Acids Res., , 25, , 3847–3854.
Maga,G. and Hubscher,U. ( (2003) ) Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J. Cell Sci., , 116, , 3051–3060.
Vandaele,C., Coulon-Bublex,M., Couble,P. and Durand,B. ( (2001) ) Drosophila regulatory factor X is an embryonic type I sensory neuron marker also expressed in spermatids and in the brain of Drosophila. Mech.Dev., , 103, , 159–162.
Dubruille,R., Laurencon,A., Vandaele,C., Shishido,E., Coulon-Bublex,M., Swoboda,P., Couble,P., Kernan,M. and Durand,B. ( (2002) ) Drosophila regulatory factor X is necessary for ciliated sensory neuron differentiation. Development, , 129, , 5487–5498.(Kyoko Otsuki, Yuko Hayashi2, Masaki Kato)
* To whom correspondence should be addressed. Tel: +81 75 724 7781; Fax: +81 75 724 7769; Email: myamaguc@ipc.kit.ac.jp
Present addresses: Masaki Kato, Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, 725 North Wolfe Street PCTB 714 Baltimore, MD 21205-2185, USA
Hideki Yoshida, Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan
ABSTRACT
A transcriptional regulatory element was identified in the region between URE (upstream regulatory element) and DRE (DNA replication-related element) in the Drosophila PCNA gene promoter. This element plays an important role in promoter activity in living flies. A yeast one-hybrid screening using this element as a bait allowed isolation of a cDNA encoding a protein which binds to the element in vitro. Nucleotide sequence analyses revealed that the cDNA encodes a novel protein containing a characteristic DNA-binding domain conserved among the regulatory factor X (RFX) family proteins. We termed this protein Drosophila RFX2 (dRFX2) and this element dRFX2 site. To investigate the function of dRFX2 in vivo, we took the strategy of analyzing the dominant negative effects against the endogenous dRFX2. Transgenic flies were established in which expression of HA-dRFX202–480 carrying the amino acid sequences from 202 to 480 containing the RFX domain (DNA-binding domain) of dRFX2 was targeted to the cells in the eye imaginal discs. In the eye imaginal disc expressing the HA-dRFX202–480, the G1–S transition and/or the progression of S phase were/was interrupted, and the ectopic apoptosis was induced, though photoreceptor cells differentiated normally. These results indicate that dRFX2 plays a role in G1–S transition and/or in progression of S phase.
INTRODUCTION
The proliferating cell nuclear antigen (PCNA) is required for replication of simian virus 40 (1) as well as cellular DNA (2,3). It functions as a sliding clamp at DNA replication forks (4) and is also involved in DNA repair (5,6) and cell cycle regulation (7–9) by interacting with various enzymes and regulatory proteins (10,11). Interaction of PCNA with the chromatin assembly factor 1 (CAF-1) suggests another role in marking of DNA for chromatin assembly during the S phase of the cell cycle (12–14).
Previous studies on the Drosophila PCNA and other DNA replication-related genes have revealed that there are at least three common regulatory elements in the promoters of Drosophila DNA replication-related genes, DRE (DNA replication-related element) (15–17), E2F-recognition sites (18) and CFDD recognition sites (19). DRE appears to be an important regulatory element not only for DNA replication-related genes but also for various other cell cycle- (20) and proliferation-related genes (21,22). At least two different protein factors, DREF (15,23) and boundary element-associated factor of 32 kDa (BEAF32) (24) can bind to DRE.
E2F sites are essential for PCNA gene promoter activity throughout development (18). However, they themselves do not appear to be sufficient for PCNA gene promoter activity during embryonic and larval stages, since this was completely abolished by deletion of the upstream region containing the DRE sequence (18). The Drosophila E2F family proteins (dE2F1 and dE2F2) form complexes with their heterodimer partner dDP and bind to E2F sites (25–28). In addition, transcription of DNA polymerase and PCNA is completely lost in dE2F1 or dDP mutant embryos after division cycle 16, indicating that the two proteins are essential for transcription of these DNA replication-related genes (29,30). In contrast, a dE2F2 mutant fly demonstrates upregulation of E2F target genes (31,32). Therefore, dE2F1 and dE2F2 appear to have opposing functions during Drosophila development (32,33).
The PCNA gene promoter has three CFDD sites (site 1, 2 and 3), one of these (site 1) could be demonstrated to play an important role in promoter activity in both cultured cells and living flies. CFDD sites are present in promoters of the DNA polymerase and DREF genes, although specific CFDD binding factors have yet to be identified (19). The Drosophila PCNA gene promoter contains an additional upstream regulatory element (URE) to which a transcription factor Grainyhead (GRH/NTF-1) binds (34–36). URE in addition to the E2F sites, CFDD sites and DRE appear to be essential for activation of the PCNA gene promoter in larvae (37).
In the present study, we have identified a novel regulatory element in the region between URE and DRE in the PCNA gene promoter. A yeast one-hybrid screening using this element as bait allowed isolation of a cDNA encoding a protein which can bind to the element in vitro and which contains a characteristic DNA-binding domain conserved among the regulatory factor X (RFX) family proteins (38–42), known to be present in yeast, fungi, nematode, mouse, human and Drosophila. Therefore, we termed this protein dRFX2 and this element the dRFX2 site. The RFX-binding site has been suggested to be involved in regulation of the human PCNA gene (43–45). The function of some of RFX family proteins might be conserved during evolution. Over-expression of the DNA-binding domain of dRFX2 in imaginal discs inhibited DNA synthesis but exerted no effects on photoreceptor cell differentiation. These results indicate that dRFX2 plays a role in the G1–S transition and/or in S-phase progression.
MATERIALS AND METHODS
Oligonucleotides
The sequences of double-stranded oligonucleotides containing URE (UREL), DRE (DRE-P), CFDD-binding site 1 (CFDD-1) or E2F-recognition sites (E2F-P) in the PCNA gene were described earlier (16,18,19,36).
The sequences of double-stranded oligonucleotides containing the dRFX2 site (–124/–98) or base-substituted derivatives in the PCNA gene promoter were defined as follows.
–124WT gatccGTTGGCAGGCCGCTCGCTGCCTGCTATagCAACCGTCCGGCGAGCGACGGACGATAt
–124MUT1 gatcctggttCAGGCCGCTCGCTGCCTGCTATagaccaaGTCCGGCGAGCGACGGACGATAt
–124MUT2 gatccGTTGGacttCCGCTCGCTGCCTGCTATagCAACCtaggGGCGAGCGACGGACGATAt
–124MUT3 gatccGTTGGCAGGaataTCGCTGCCTGCTATagCAACCGTCCttatAGCGACGGACGATAt
–124MUT4 gatccGTTGGCAGGCCGCgataTGCCTGCTATagCAACCGTCCGGCGctatACGGACGATAt
–124MUT5 gatccGTTGGCAGGCCGCTCGCgtaagGCTATagCAACCGTCCGGCGAGCGcattcCGATAt
–124MUT6 gatccGTTGGCAGGCCGCTCGCTGCCTtagcgagCAACCGTCCGGCGAGCGACGGAatcgct
Nucleotides substituted for the wild-type sequence are shown by small letters with underlining. To obtain fragments containing base-substitutions in the PCNA gene promoter, the following primers were synthesized and used for PCR.
124MUT1P CTATCGATAGCAGGCAGCGAGCGGCCTGaaccaTGGTTTG
124MUT2P CTATCGATAGCAGGCAGCGAGCGGaagtCCAACTGGTTTG
124MUT3P CTATCGATAGCAGGCAGCGAtattCCTGCCAACTGGTTTG
Plasmid construction
A fragment from –168 to –91 having base-substituted mutations was generated by the PCR method using p5'–168DPCNACAT (46) as a template with primers –124MUT1P and SalI (–168) (16). The plasmid p5'–168DPCNAlacZW8HS contains a PCNA gene fragment spanning from –168 to +137 fused with the lacZ gene in a P-element vector (46). To create mutated derivatives in P-element vector backbones, fragments having various mutations in the region from –124 to –112 of the PCNA gene promoter were isolated from CAT plasmids by digestion with SalI (–168) and SacII (+24), and inserted between the XhoI (–607) and SacII (+24) sites of p5'–607DPCNAlacZW8HS (47).
pACT-dRFX2202–589, which was isolated by one-hybrid screening, was digested with XhoI and the isolated dRFX2 cDNA fragment was inserted into the SalI site of pGEX-4T-1 (Amersham Pharmacia Biotech) to create pGST-dRFX2202–480, or the XhoI site of pUAST-HA to create pUAS-HA-dRFX2202–480. Full length of dRFX2 cDNA was isolated by 5'-RACE (Invitrogen) using total RNAs from larvae and the primer carrying the sequence, 5'-ACCGTAATATTCTAGAGG. The isolated full-length dRFX2 cDNA was then inserted into the XhoI site of pUAST-HA to create pUAS-HA-dRFX2FL.
The GAL4-coding sequence was isolated using PCR with the primers 5'-ATCGAATTCGGTACCAGATGAAGCTACTGTCTTCTATCGA and 5'-ATAAGATCTGCGGCCGCTTACTCTTTTTTTGGGTTTGGTG, and pGaTB plasmid (48) as a template. Products were gel-purified, cut with EcoRI and BglII, and cloned into EcoRI–BglII sites of the pGMR vector (49).
All plasmids were propagated in Escherichia coli (E.coli) XL-1 Blue, isolated by standard procedures (50) and further purified using a Qiagen Plasmid Midi Kit (Qiagen). The DNA sequencing was carried out with a BigDyeTM Terminator v3.0 Cycle sequencing Standard kit (Applied Biosystems) using an ABI PRISMTM 310 NT Genetic Analyzer (Applied Biosystems). When necessary, chemically synthesized oligonucleotides (17mer) were used as sequencing primers.
Expression of GST fusion proteins
Expression of GST–dRFX2202–480 fusion protein in E.coli XL-1 Blue was carried out as described elsewhere (51). Lysates of cells were prepared by sonication in buffer D containing 0.6 M KCl, 1 mM PMSF, and 1 μg/ml each of pepstatin, leupeptin and aprotinin. Lysates were cleared by centrifugation at 12 000 g for 20 min at 4°C and applied to glutathione–Sepharose (Amersham Biotech) to purify the GST–dRFX2202–480 fusion protein as described elsewhere (51). GST protein and GST–dRFX were expressed and purified in the same way.
One-hybrid screening
The MATCHMAKER one-hybrid system from Clontech was applied to isolate a cDNA encoding the protein responsible for dRFX2 site-binding activity. The MATCHMAKER one-hybrid system protocol was used to prepare the target-reporter constructs, to integrate these constructs into Saccharomyces cerevisiae strain YM4271 (his– ura– leu–), and to screen an activation domain (AD) fusion library (ACT-cDNA library) from Drosophila third instar larvae (kindly supplied by Dr Elledge). Four tandem copies of the double-stranded oligonucleotide, –124WT (4-dRFX2 sites) were placed upstream of the marker genes of both the pHISi-1 and pLacZi plasmids (Clontech). The two target-reporter constructs were transformed into S.cerevisiae strain YM4271 in a consecutive manner to produce a dual reporter strain. This was transformed with the pACT-cDNA library, and his+ ura+ leu+ transformants grown in synthetic dropout (SD) medium containing 30 mM 3-aminotriazole (3-AT) were selected. Each colony was streaked on SD agar medium without histidine but containing 30 mM 3-AT and incubated for 3 days at 30°C. A dry NEF-978X filter (NEN Research Products) was placed over the surface of each agar plate containing transformants. The filters were lifted off the agar plate and dipped in liquid nitrogen. The frozen colonies were then thawed at room temperature and the filters were overlaid onto Whatman number 5 filters that had been soaked in Z buffer (60 mM Na2HPO4·7H2O, 40 mM NaH2PO4·H2O, 10 mM KCl, 1 mM MgSO4·7H2O, 50 mM ?-mercaptoethanol, pH 7.0) containing 0.03% 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside (X-Gal) at 30°C for 5–8 h. Positive blue colonies were selected. To confirm sequence-specific interactions, plasmid DNA for each candidate was recovered in E.coli DH5 (Competent high, TOYOBO) followed by retransformation into yeast strains containing the 4-dRFX2 sites reporter.
Southern and northern blot analysis
DNA was extracted from Drosophila bodies at various developmental stages and digested with BamHI, then separated by agarose gel electrophoresis and blotted onto a GeneScreen Plus membrane (New England Nuclear) using a VacuGene blotting apparatus (LKB). The membrane was sequentially hybridized with a 32P-labeled probe containing the dRFX2 cDNA or the PCNA cDNA. The blot was then subjected to hybridization analysis, under the conditions described earlier (52).
Total cellular RNA was isolated from Drosophila bodies at various developmental stages using TRIZOL (Invitrogen), then separated by agarose gel electrophoresis and blotted onto a GeneScreen Plus membrane. The membrane was sequentially hybridized with a 32P-labeled probe, which recognizes mRNA for each of dRFX2, dRFX (42), PCNA (53) or RP-49 (54). The hybridization and washing conditions were as described previously (52).
Isolation of the Drosophila gene for dRFX2
A genomic library that was constructed by inserting the partially Sau3AI-digested DNA from Drosophila melanogaster Oregon-R into the BamHI site of EMBL3 was used (52). Clones containing the dRFX2 gene sequence were isolated by screening the library. The XhoI fragment from the dRFX2 cDNA clone, which was isolated by one-hybrid screening, was used as a probe, labeled with dCTP using a random priming method (55). Plaque hybridizations were carried out at 42°C in a solution containing 20% formamide, 3x SSC, 5x Denhardt's solution, 10% dextran sulfate, 1% SDS, 1 mM sodium pyrophosphate, 100 μg/ml heat-denatured salmon sperm DNA and the probe, as described previously (52).
Band mobility shift assays
Band mobility shift analysis was performed as described earlier (15) with minor modifications. An aliquot of 2 μl of 32P-labeled probes (10 000 c.p.m.) was mixed with 10 μl of binding buffer containing 25 mM HEPES (pH 7.6), 150 mM KCl, 0.1% Triton X-100, 1 mM EDTA, 1 mM DTT and 10% glycerol, then 1 μl of 0.5 mg/ml poly (dI–dC) was added, and the probe mixture was incubated on ice for 5 min. When necessary, unlabeled oligonucleotides were added as competitors at this step. Purified GST–dRFX2 fusion protein was diluted with buffer containing 25 mM HEPES (pH 7.6), 150 mM KCl, 0.1% Triton X-100, 1 mM EDTA, 1 mM DTT, 10% glycerol. Two μl of each fusion protein was added to the probe mixture, then incubated for 15 min on ice. DNA–protein complexes were electrophoretically resolved on 4% polyacrylamide gels in 100 mM Tris-borate (pH 8.3), 2 mM EDTA containing 2.5% glycerol at 25°C. The gels were dried and then autoradiographed.
Fly stocks and establishment of transgenic flies
Fly stocks were maintained at 25°C on standard food. Canton S flies were used as the wild-type strain. Enhancer trap lines carrying the lacZ markers D120 (inserted in scabrous) (56), AE127 (inserted in seven-up) (57) and P82 (58) were obtained from Dr Y. Hiromi. These lines express the ?-galactosidase marker in photoreceptor cells (R) of early R8, R3/R4/R1/R6 and R3/R4/R7, respectively. The GAL4 driver strains were obtained from the Bloomington Stock Center or the Kyoto Institute of Technology Stock Center. Transgenic flies were established with P-element-mediated germ line transformation as described earlier (59), and F1 transformants were selected on the basis of white eye color rescue (60). Transgenic flies for pGMR-GAL4 were used as described previously (61). Established transgenic fly strains and their chromosomal linkages are listed in Supplementary Materials 2 and 3.
Analysis of PCNA-lacZ expression patterns
Quantitative measurement of ?-galactosidase activity in extracts was carried out as described previously (62). Male transgenic flies were crossed with wild-type females, and groups of 50–100 individual dechorionated embryos, larvae, pupae and adult flies were homogenized in 500 μl of ice-cold assay buffer (50 mM potassium phosphate, pH 7.5/1 mM MgCl2). Homogenates were centrifuged at 10 000 g at 4°C for 5 min. For each assay, 50–200 μl of supernatant was added to give 1 ml of assay buffer containing 1 mM chlorophenol red-?-D-galactopyranoside substrate (CPRG; Roche). Reaction incubations were at 37°C in the dark. Substrate conversion was measured at 574 nm using a spectrophotometer at 0.25, 0.5, 0.75, 1 and 1.5 h after addition of the extract, and the rate of color development was linear. The ?-galactosidase activity was defined as absorbance units per hour per milligram of protein. To correct for endogenous ?-galactosidase activity, extracts from the wild-type strain were included in each experiment and the observed background reading was subtracted from readings obtained with each transformant line. Deviation among independent strains was <30% (18). The protein concentrations of the extracts were determined by Bio-Rad protein assay.
?-Galactosidase activity of larval tissues was visualized as described elsewhere (16). After dissection, tissues were incubated in fixative (12 mM sodium cacodylate buffer, pH 7.3/26% glutaraldehyde) for 15 min at room temperature. Treated tissues were then incubated with a staining solution containing 0.2% X-Gal in the dark at 37°C for 5–16 h. For photography, tissues were immersed in glycerol, mounted on slides and photographed with an Olympus microscope (BX-50) using Tri X pan 400 films (Kodak).
Scanning electron microscopy
Adult flies were anesthetized, mounted and observed under a Hitachi S-3000 scanning electron microscope.
5-Bromo-2'-deoxyuridine (BrdU) labeling
Detection of cells in S phase was performed using a BrdU-labeling method as described previously with minor modifications (63). Third instar larvae were dissected in Drosophila Ringer's solution and the imaginal discs were suspended in Grace's insect medium, then incubated in the presence of 75 μg/ml BrdU (Roche) for 40 min at 25°C. The samples were fixed in Carnoy's fixative (ethanol/acetic acid/chloroform, 6:1:3) for 20 min at 25°C, and further fixed in 80% ethanol/50 mM glycine buffer (pH 2.0) at –20°C overnight. Incorporated BrdU was visualized using an anti-BrdU antibody and an alkaline phosphatase detection kit (Roche). The time of color development was identical for all samples.
Apoptosis assay
Third instar larvae were dissected in Drosophila Ringer's solution and the imaginal discs were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at 25°C. After being washed with PBS, the samples were permeabilized by incubation in a solution containing 0.1% sodium citrate and 0.1% Triton X-100 on ice for 30 min. After extensive washing, the TUNEL reaction was carried out using an in situ Cell Death Detection Kit, POD (Roche) according to the manufacturer's recommendations.
Immunohistology
The third instar larvae were dissected and eye imaginal discs were immuno-stained by the method described previously with minor modifications (64). Samples were incubated with mouse anti-?-galactosidase monoclonal antibody (Promega) at a 1:500 dilution or culture supernatant of hybridoma cells producing mouse anti-HA monoclonal antibody (supplied by Dr M. Inagaki) at a 1:100 dilution. The second antibody, an alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G (IgG) (Promega), was used at a 1:500 dilution. In the case of immuno-staining photoreceptor cells, the F1 third instar larvae from the cross of GMR-GAL4; +, or GMR-GAL4; UAS-HA-dRFX2202–480/CyO with each enhancer trap line were dissected.
RESULTS
Identification of binding factor to the region between URE and DRE in the PCNA gene promoter
In previous studies, we found the consensus binding sequence for Zeste protein in the region between URE and DRE (46). To isolate a cDNA encoding a binding factor, we carried out a yeast one-hybrid screen of a Drosophila third instar larvae cDNA library using four tandem copies of the sequence spanning from –124 to –98 as the target binding sequence. On screening 3.1 x 106 independent clones, 52 independent positive clones were identified. Nucleotide sequences of these clones were determined. One clone did not show any match to any sequence in the Drosophila genome but was found to have 57% identity with DNA-binding domain of Drosophila transcription factor, dRFX (Supplementary Material 1) (42). We therefore designated this novel factor as dRFX2. The rest of clones were cDNAs for the ribosomal protein, RpL22 and the cDNA for Zeste protein was not found.
By conducting a 5'-RACE (Invitrogen) using total RNAs from larvae and a primer carrying the sequence, 5'-ACCGTAATATTCTAGAGG, full-length dRFX2 cDNA was isolated. In addition, using the dRFX2 cDNA as a probe, we screened a Drosophila genomic library (52). On screening 4.6 x 105 clones, one containing a sequence of the dRFX2 gene was isolated and its nucleotide sequence was determined (Supplementary Material 1, DDBJ accession no.: AB190179 ). Comparison of nucleotide sequences of the dRFX2 cDNA (2526 bp) and the dRFX2 gene suggested that the latter is intronless. The deduced dRFX2 protein consists of 842 amino acids. Multiple alignment with other RFX family proteins from yeast, nematode, Drosophila and human showed the amino acid sequence from 388 to 463 of dRFX2 to share 33% identity and 57% similarity with hRFX1, the prototype of RFX family proteins, and 39.5% and 61.8% with Sak1, an RFX protein in Schizosaccharomyces pombe (Supplementary Material 1). This domain is highly conserved in the RFX family proteins, and demonstrated to be a DNA-binding domain (DBD). Arginine at position 406 (R406), K432, K435, R444, R445, R449, R453, Y454 and Y456 are highly conserved among RFX family proteins, and in a crystallographic structure of the mammalian RFX1 DBD, make direct or water-mediated DNA contacts (65).
We examined the dRFX2 expression pattern during Drosophila development by northern blot hybridization. Using dRFX2 cDNA as a probe, a major transcript of 3.4 kb was detected in early embryos and larvae (Figure 1, second panel). Apparently, no 3.4 kb transcript was detected in pupae, adults and cultured Kc cells. Although the 3.4 kb transcript is barely detectable in third instar larvae, a minor transcript of 6.1 kb was detected in both second and third instar larvae. The 6.1 kb band may represent an alternative form of dRFX2 mRNA such as a transcript derived from an alternative promoter, since the dRFX2 cDNA clone was identified in the one-hybrid screen using a third instar cDNA library as described above. The cDNAs for dRFX and PCNA were used as reference probes. The dRFX mRNA of 4.4 kb was expressed throughout all developmental stages as reported previously, although its level was fluctuated (42). PCNA mRNA of 1.1 kb was found to be expressed in line with cell proliferation during development as described previously (53). The RP-49 cDNA was used as an internal control to monitor the integrity of mRNA (54). These results indicate that dRFX2 is expressed in embryos and larvae with highest expression in second instar larvae.
Figure 1. Northern blot hybridization analysis: 50 μg of total RNA was loaded in each lane; dRFX2 cDNA was used as a probe (second). As reference probes, cDNAs for dRFX (top) and PCNA (third) were employed. The RP-49 cDNA (bottom) was used as an internal control.
Nucleotide sequences required for binding to dRFX2
Multiple alignment of dRFX2 protein with RFX family proteins revealed high conservation in the region corresponding to the DNA-binding domain, suggesting that dRFX2 carrying this region could bind to DNA. dRFX2202–480, the amino acids from 202 to 480, was therefore expressed as a fusion protein with GST (GST–dRFX2202–480) and band mobility shift assays were carried out. As shown in Figure 2B, a DNA–protein complex was detected with the –124/–98 oligonucleotide containing the region between –124 and –98 of the PCNA gene. To examine specific binding of dRFX2, oligonucleotides containing –149/–118 (36), –108/–85 (15) and –87/–62 (19) were employed as competitors (Figure 2B, lanes e–j and l–n), spanning the binding site between –130 and –119 (URE), –100 and –93 (DRE), and –84 and –77 (CFDD site 1) of the PCNA gene promoter, respectively. None of these competitors competed for the binding (Figure 2A and B). Therefore, the region between –124 and –98 is specifically required for dRFX2-binding.
Figure 2. DNA-binding activity of GST–dRFX2. (A) Organization of Drosophila PCNA gene promoter elements and nucleotide sequences in and around the dRFX2 site. Nucleotide positions of each promoter element and their sequences are shown. The substituted bases are boldface. Results of band mobility shift assays are shown on the right. Gray box, dRFX2 site; open box, URE; stippled box, DRE. (B and C), Radiolabeled double-stranded oligonucleotides carrying the wild-type –124/–98 site were incubated with 0.2 μg of the purified GST–dRFX2 fusion protein in the absence (0), or the presence of a competitor (indicated at the top of each lane, at 100, 200 or 400 ng). In (B), competitors were –124/–98 oligonucleotides containing dRFX2-binding site, –148/–118 oligonucleotides containing URE (36), –108/–85 oligonucleotides containing DRE (15), and –7/–62 oligonucleotides containing the CFDD site1 (19). In (C), –124/–98 oligonucleotides carrying base-substitution mutations were used. Nucleotide sequences of each are shown in panel A. (D) Binding specificity of GST–dRFX and GST–dRFX2 fusion proteins on complex formation with the dRFX2 site or EP sequence. Radiolabeled –124/–98 oligonucleotides were incubated with 0.2 μg each of purified fusion protein (see bottom of each lane) in the presence or absence (0) of the indicated amounts of competitor oligonucleotides containing EP sequence or –124/–98 sequence (see top of each lane).
To determine in detail the nucleotide sequence required for binding to dRFX2, various base-substituted mutations were introduced into the region between –124 and –98 (Figure 2A) and the resultant mutant oligonucleotides were used as competitors in the band mobility shift analysis using GST–dRFX2202–480 fusion protein (Figure 2C). The mutant oligonucleotides mut1 and mut2 did not compete at all (Figure 2C, lanes e–j), while the other mutant oligonucleotides competed for the binding as effectively as the wild-type –124/–98 oligonucleotide (Figure 2C, lanes b–d, l–q and s–x). These results indicate that the sequence 5'-GTTGGCAGG between –124 and –116 plays an important role in the dRFX2-binding (Figure 2C).
We were also interested in whether dRFX can bind to the region between –124 and –116. When band mobility shift assays were performed using the –124/–98 oligonucleotide and GST–dRFX fusion protein, no band shift was detected (data not shown). Band mobility shift analysis was also conducted with the EP probe (66) carrying the binding consensus sequence for the mammalian RFX family proteins and GST–dRFX2202–480 or GST–dRFX fusion protein, and EP oligonucleotide or –124/–98 oligonucleotide was added in the binding reaction as a competitor (Figure 2D). EP oligonucleotide competed effectively to binding reaction between EP probe and either GST–dRFX2202–480 or GST–dRFX fusion protein (Figure 2D, lanes b and e), suggesting that both dRFX and dRFX2 have strong affinity to EP oligonucleotides. When we used GST–dRFX2202–480 fusion protein, the –124/–98 oligonucleotide competed effectively (Figure 2D, lanes a and c). However, when GST–dRFX fusion protein was applied, the shifted band did not disappear (Figure 2D, lanes d and f). These results suggest that the region between –124 and –116 is bound exclusively with dRFX2, and we thus termed this the dRFX2 site.
Role of the dRFX2 site in the dPCNA gene promoter in living flies
Previously, we established transgenic flies carrying PCNA [–168 to +137) and lacZ fusion genes (46). To examine the role of the dRFX2 site in the PCNA promoter activity during Drosophila development, we generated PCNA–lacZ fusion genes carrying base-substituted mutations in and around the dRFX2 site (Figure 3A). These fusion genes were then introduced into flies by germ-line transformation. Established transgenic fly lines and their chromosomal linkages are listed in Supplementary Material 2. Activities of the modified promoters were then monitored by quantitative ?-galactosidase assays at various developmental stages (Figure 3B).
Figure 3. Role of dRFX2 site in PCNA gene promoter activity. (A) Nucleotide sequences of the region between –130 and –98 of the wild-type PCNA gene promoter and its base-substituted mutants are shown. The substituted bases are boldface. Promoter activity in transgenic flies is also indicated. Gray box, dRFX2 site; open box, URE. (B) Male transgenic flies were crossed with female wild-type flies, and extracts were prepared from Drosophila bodies at various stages of development. The ?-galactosidase activities in the extracts are expressed as absorbance units/h/mg of protein. (C) Salivary glands were dissected from third instar lavae of the progeny from crosses described in (B). Salivary glands from flies carrying: (a) p5'-168DPCNAlacZW8HS (–168); (b) p5'-168mut1DPCNAlacZW8HS (mut1); (c) p5'-168mut2DPCNAlacZW8HS (mut2); (d) p5'-168mut3DPCNAlacZW8HS (mut3). Staining signals in the ducts appear to be non-specific.
In flies carrying the PCNA gene promoter region up to position –168, base-substitution mutations in the dRFX2 site (mut1 and mut2) reduced the lacZ expression to 50% in embryos (Figure 3B, panels a–c). Much more extensive reduction of the lacZ expression was observed in larvae carrying mutations in the dRFX2 site (Figure 3B, panels a–c). Especially in second and third instar larvae carrying base-substitution mutations in the region between –116 to –120, the lacZ expression was barely detectable (Figure 3B, panel c). In contrast, only a marginal effect on the lacZ expression was observed with a mutation outside the dRFX2 site (mut3) (Figure 3B, panel d). In pupae and adults, there were no remarkable differences between flies carrying the wild-type promoter and those carrying base-substituted derivatives (Figure 3B). Since the extent of the reduction was most prominent in larvae, ?-galactosidase activity was demonstrated in dissected third instar larval tissues (Figure 3C). In transgenic third instar larvae carrying a wild-type construct, high lacZ-staining signal was observed in the salivary glands (Figure 3C, panel a). The larvae having mutations in the dRFX2 site (mut1 and mut2) exhibited extensively reduced staining signals (Figure 3C, panels b and c). In contrast, no reduction of staining was observed with lines carrying a mutation outside the dRFX2 site (mut3, Figure 3C, panel d). We carried out the same examination on probable neuroblasts in the central nervous system (CNS) of third instar larvae and obtained similar results (data not shown). Thus, an important role of the dRFX2 site for the dPCNA gene promoter activity, especially at larval stages, was demonstrated in living flies.
Expression of the DNA-binding domain (amino acid residues 202–480) of dRFX2 in transgenic flies
In our previous studies, transgenic flies expressing DBD of the transcription factor DREF were successfully used to examine function of endogenous DREF by inhibiting its activity in a dominant negative manner (67). To investigate the function of dRFX2 in living flies, we took a similar approach. Band mobility shift assay using the dRFX2 site revealed that the region from amino acid 202 to 480 of dRFX2 contains the DNA-binding activity (Figure 2). Therefore, the region containing this putative DNA-binding domain of dRFX2 with HA-tag (HA-dRFX2202–480) was subcloned into the pUAST vector, and the plasmid was named pUAS-HA-dRFX2202–480. We established 11 independent transgenic fly lines carrying UAS-HA-dRFX2202–480 (Supplementary Material 3). These transgenic flies were then crossed with transgenic flies carrying GAL4 cDNA placed under the control of promoters that function in specific tissues or at specific developmental stages (Table 1), and in their progenies the effects of expression of dRFX2202–480 on the development of Drosophila were examined (48).
Table 1. Summary of effects in expression of HA-dRFX2202–480 with each GAL4 driver line
Over-expression of dRFX2202–480 with the Cg-GAL4 driver (Cg-GAL4>HA-dRFX2202–480) caused the generation of melanotic masses in larvae, and flies died in the larval stage. The Cg-GAL4 driver strain expresses GAL4 in blood cells. With dll-GAL4>HA-dRFX2202–480 and en-GAL4>HA-dRFX2202–480, death occurred in the embryonic stage (Table 1). While no detectable phenotype was observed with twi-GAL4> HA-dRFX2202–480, 71B-GAL4>HA-dRFX2202–480 and act88F-GAL4>HA-dRFX2202–480. The GMR-GAL4>HA-dRFX2202–480 flies exhibited severely abnormal eye morphology with a rough appearance (Table 1 and Figure 4A, panels c and d). The ommatidia lacked their regular hexagonal shape and appeared to be fused. Either additional or missing bristles were apparent. Over-expression of HA-dRFX2202–480 in the eye imaginal disc was confirmed by immuno-staining with an anti-HA antibody (Figure 4B). When the copy number of UAS-HA-dRFX2202–480 was increased to two, embryonic lethality resulted.
Figure 4. Expression of HA-dRFX2202–480 in eye imaginal disc induces a rough eye phenotype. (A) Scanning electron micrographs of adult compound eyes. (a) and (b), GMR-GAL4/+, +; (c) and (d), GMR-GAL4/+, UAS-HA-dRFX2202–480/+; (e) and (f), GMR-GAL4/+, UAS-HA-dRFX2202–480/UAS-HA-dRFX2FL; (g) and (h), GMR-GAL4/+, UAS-HA-dRFX2FL/+. Magnification: (a, c, e and g), 200x; (b, d, f and h), 1000x. The flies developed at 28°C. (B) Immuno-staining of eye imaginal discs with anti-HA antibody. (a), GMR-GAL4/+, +/+; (b), GMR-GAL4/+, UAS-HA-dRFX2202–480/+. The anti-HA signal was detected in the region posterior to the MF. MF, morphogenetic furrow.
We have also established 11 independent transgenic fly lines carrying UAS-HA-dRFX2FL (Supplementary Material 3). Over-expression of dRFX2 protein with GMR-GAL4 driver induced apparently weak rough eye phenotype which is indistinguishable from that induced by expression of GAL4 alone (Figure 4A, panels a, b, g and h). However, when the GMR-GAL4; UAS-HA-dRFX2202–480/CyO was crossed with UAS-dRFX2FL, the rough eye phenotype induced by dRFX2202–480 was effectively suppressed (Figure 4A, panels e and f), indicating that the effect of expression of the dRFX2-DBD on eye morphology is truly the dominant negative effect against the endogenous dRFX2. In the following studies, we focused on analyses of the rough eye phenotype induced by dRFX2202–480, since cell proliferation and differentiation during Drosophila eye development have been well characterized (68).
Ectopic expression of dRFX2202–480 in eye imaginal discs can inhibit cell cycle progression
In the third instar larvae, the morphogenetic furrow (MF) appears at the posterior end of the eye imaginal discs, and slowly moves in the anterior direction. Cells in front of the MF proliferate asynchronously, while those on the MF are arrested synchronously at G0/G1 phase. Cells behind the MF either leave the cell cycle and differentiate into photoreceptors of the adult ommatidium, or undergo one more cell division. This cell cycle is a final synchronous round, and produces an S-phase band (the second mitotic wave), then these cells form a reservoir of cells for subsequent differentiation events. Therefore, no mitotic cells behind the second mitotic wave are seen (68).
In GMR-GAL4, the promoter carrying transcription factor Glass-binding sites was used for the expression of GAL4 in the region within and posterior to the MF (49). The flies with GMR-GAL4>HA-dRFX2202–480 expressed HA-dRFX2202–480 in the region within and posterior to the furrow that was visualized by immuno-staining with an anti-HA antibody (Figure 4B).
To examine the effect of dRFX2202–480 expression on DNA synthesis in the eye imaginal disc cells, we visualized the cells in S phase with BrdU incorporation. In eye discs of flies expressing GAL4 alone, BrdU incorporation was observed anterior to the MF and in a stripe just posterior to the MF, indistinguishable from the pattern of BrdU incorporation in wild-type fly discs reported previously (Figure 5A) (61). In HA-dRFX2202–480-expressing discs, extensive reduction of BrdU signals in the synchronized S-phase zone behind the MF was evident (Figure 5B). The results indicate that ectopic expression of HA-dRFX2202–480 can inhibit entry into the S phase or progress through the S phase in the eye imaginal disc, suggesting that endogenous dRFX2 plays a role in the G1–S transition or S-phase progression. In addition, slight reduction of BrdU signals was also observed in the region anterior to the MF where the GMR promoter was inactive, suggesting some non-cell autonomous regulations in eye imaginal discs. Further analyses are necessary to clarify this point.
Figure 5. Expression of HA-dRFX2202–480 reduces the number of S-phase cells in eye imaginal discs. After incorporation of BrdU for 40 min, the eye imaginal discs were immuno-stained with anti-BrdU antibody. (A) GMR-GAL4/+, +; (B) GMR-GAL4/+, UAS-HA-dRFX2202–480/+. MF, morphogenetic furrow.
Ectopic expression of dRFX2202–480 in eye imaginal discs does not interfere with photoreceptor cell differentiation
Photoreceptor cells have been found to be generated sequentially: R8 is generated first, with movement posterior from the MF, then cells are added pairwise (R2 and R5, R3 and R4, and R1 and R6), and R7 is the last photoreceptor to be added to the precluster (68). Several enhancer trap lines expressing a nucleus-localized form of ?-galactosidase depend on the specific enhancer-promoter located nearby the P-element. They were used here to identify each photoreceptor cell. We used three enhancer trap lines, D120 (inserted in scabrous) (56), AE127 (inserted in seven-up) (57) and P82 (58), specifically expressing the ?-galactosidase marker in photoreceptor cells of early R8, R3/R4/R1/R6 and R3/R4/R7, respectively. The imaginal discs of F1 larvae from mating of each enhancer trap line and transgenic fly expressing dRFX2202–480 in developing eye were immunohistologically stained with an anti ?-galactosidase antibody.
When dRFX2202–480 was expressed in eye imaginal discs, photoreceptors of such flies showed essentially the same staining pattern as those of control flies (data not shown). From these results, we conclude that ectopic expression of dRFX2202–480 does not interfere with differentiating photoreceptor cells in eye discs.
Figure 6. Effects of HA-dRFX2202–480 on photoreceptor cell development. (a) Males of each photoreceptor cell-specific enhancer trap line were crossed with females of GMR-GAL4 or GMR-GAL4; UAS-HA-dRFX2202–480/CyO, ActGFP. The enhancer trap lines were D120 (A), AE127 (B) and P82 (C), and expressed ?-galactosidase in R8, R3/4/1/6 and R3/4/7, respectively. Eye imaginal discs of these GFP-negative progenies were stained with anti-?-GAL antibody (Promega). R8, R3/4/1/6 and R3/4/7 were stained as well as the wild type (data not shown). (b) High-magnification appearance.
Ectopic expression of dRFX2202–480 in eye imaginal discs can induce apoptosis
Ectopic expression of dRFX2202–480 in eye imaginal discs induced an abnormal S phase. Because it has been reported that disorder of cell cycle progression and disturbance of differentiation processes cause apoptosis (69), we investigated this process in eye imaginal discs expressing dRFX2202–480 by the TUNEL method. In eye discs of flies expressing GAL4 alone, there was very limited cell death (Figure 7A, panel a). In contrast, eye discs of flies expressing dRFX2202–480 showed a significant increase of apoptotic cells posterior to the MF (Figure 7A, panel b). Moreover, when adult flies expressing dRFX2202–480 were crossed with those expressing inhibitors of apoptosis in Drosophila, p35 (70), DIAP1 and DIAP2 (71), the rough eye phenotype was suppressed in their progeny (Figure 7B). These results indicate that ectopic expression of dRFX2202–480 in eye imaginal discs can induce apoptosis.
Figure 7. Expression of HA-dRFX2202–480 induces ectopic apoptosis in eye imaginal discs. (A) Detection of apoptotic cells in eye imaginal discs with TUNEL assays. (a), GMR-GAL4/+, +; (b), GMR-GAL4/+, UAS-HA-dRFX2202–480/+. Expression of HA-dRFX2 increased the signals (see bracket). MF, morphogenetic furrow. (B) Apoptosis inhibitors, p35 (70), DIAP1 and DIAP2 (71) suppress the HA-dRFX2202–480-induced rough eye phenotype. (a), GMR-GAL4/+, UAS-HA-dRFX2202–480/UAS-LacZ; (b), GMR-GAL4/+, UAS-HA-dRFX2202–-480/+, UAS-p35/+; (c), GMR-GAL4/+, UAS-HA-dRFX2202–480/+, GMR-DIAP1/+; (d), GMR-GAL4/+, UAS-HA-dRFX2202–480/+, GMR-DIAP2/+.
DISCUSSION
The Drosophila PCNA gene promoter contains several regulatory elements such as E2F sites (18), DRE (15), CFDD sites (19) and URE (36). In the present study, we found a novel regulatory element, dRFX2 site in the region between DRE and URE. The nucleotide sequence required for dRFX2-binding was determined to be 5'-GTTGGCAGG that matches eight out of nine bases of the sequence 5'-GTNRCCNNRG that spans half of the binding consensus sequence for human RFX family proteins, 5'-GTNRCC/N-N0–3-RGYAAC (72). E2F sites, DRE (15) and CFDD sites (19) are found in common in various DNA replication-related genes in Drosophila, while URE appears to be unique to the PCNA gene (36,73). Although we have searched for dRFX2-binding sequences in the Drosophila genome database, no perfect match was apparent within the region between –250 and +50 of DNA replication-related genes such as orc1-6, mcm2-7, dup/Cdt1, CG5790/Cdc7, DNA polymerase and , and gnf1/RFC140. PCNA is well known to act as a sliding clamp in DNA replication, but can also interact with many other protein factors involved in various other biological functions (74). The endogenous dRFX2, therefore, might take a part in regulation of PCNA gene expression related to not only DNA replication but also other biological functions.
We have found that the nucleotide sequence of the dRFX2 site shares 6 out of 9 bases (the region between –124 and –119 of the PCNA gene) with the sequence of the GRH-binding site (URE) (36,37). In this study, we carried out yeast one-hybrid screening using the sequence spanning from –124 to –98 as the target site and isolated only a dRFX2 cDNA and not a GRH cDNA. A previous study revealed that the region between –130 and –123 is more important for GRH-binding than that between –122 and –118 (36). On the other hand, in this study, in the band mobility shift assays with GST-dRFX2 and the oligonucleotide –124/–98 as a probe, the oligonucleotide mut1 containing the mutation in the region between –124 and –120 did not compete for binding at all. Therefore, the shared nucleotide sequences appear more important for dRFX2- than GRH-binding.
The full-length dRFX2 cDNA was isolated by conducting 5'-RACE and screening of the Drosophila genomic DNA library allowed us to determine genomic clones spanning the dRFX2 gene and their nucleotide sequences. Although the sequence could not be found in the Drosophila database, the FlyBase, Southern blot hybridization analysis revealed that the dRFX2 is the gene of Drosophila melanogaster. By Southern blot analysis of the genomic DNA from Drosophila adults cut with BamHI using the dRFX2 cDNA as a probe, a single band of 7.7 kb was detected (data not shown), suggesting the occurrence of a single copy of the dRFX2 gene per genome. It should be noted that the signal was much weaker than for genes located in euchromatin regions such as the dPCNA gene. Genomic DNA in euchromatin region is highly amplified in Drosophila adult tissues. Moreover, nucleotide sequences in and around the dRFX2 gene contain many repetitive sequences, which are characteristic of the heterochromatin region. The fact that they were found to be five times more frequent in the dRFX2 gene region than in the dRFX gene region suggests that the former is located in heterochromatin.
Northern blot hybridization analysis revealed the highest expression of dRFX2 in larval stages, correlating with findings for function. Our previous studies indicated that the region containing the transcription initiation site and the E2F sites is sufficient for maternal expression in the ovary. In addition, DRE is required for promoter activity in embryos. In larval stages, the dRFX2 site in addition to the E2F sites, CFDD sites, DRE and URE appear to be essential for activation of the PCNA gene promoter . Therefore, as development advances, the promoter may require more and more elements to exhibit high activity.
In Drosophila, the dRFX gene has already been established as an RFX family gene. The dRFX gene is expressed in type I sensory neuron lineage of the peripheral nervous system throughout development (75), and dRFX mutants exhibit defects in the morphogenesis of the sensory cilium (76). dRFX is homologous to DAF-19 (RFX of Caenorhabditis elegans) and to human RFX1 to 3 (hRFX1-3) (76). The hRFX1 protein was the first representative of the RFX family proteins to be isolated and has been used as the prototype. In addition to the RFX domain, hRFX1 has other conserved domains containing dimerization domain, shared with dRFX. dRFX2 has the RFX domain but lacks these motifs. Therefore, it may be not be likely that dRFX2 forms heterodimer with dRFX. The amino acid sequence of dRFX2 has highest homology with Sak1 and Crt1 (39.5% identity and 61.8% similarity) among RFX family proteins. Therefore, we conclude that dRFX2 is a new member, belonging to a different group from another Drosophila RFX proteins.
To investigate the function of dRFX2 in living flies, we analyzed dominant negative effects. HA-dRFX2202–480 carrying an amino acid sequence containing the RFX domain (DNA-binding domain) was expressed within and posterior to the MF of the eye imaginal discs. Although the HA-dRFX2202–480 is very likely to be a specific dominant negative form active against endogenous dRFX2, we cannot completely exclude the possibility that this form also exerts some effects against endogenous dRFX, since both dRFX2 and dRFX have affinity to the same nucleotide sequence such as EP probe. In eye imaginal discs expressing HA-dRFX2202–480, the G1–S transition and/or the progression of S phase were/was interrupted, and the ectopic apoptosis was induced, though photoreceptor cells differentiated normally. Since dRFX2202–480 seemed to induce the disturbance of the cell cycle and reduction of the number of cells, cell–cell interactions may have been perturbed so that ectopic apoptosis was induced. Expression of dRFX2202–480 might also induce ectopic apoptosis directly. All components of the adult compound eye, including cone cells, bristles and pigment cells are recruited after photoreceptor cell commitment. In either case, these might not be able to develop normally. Specific antibodies against proteins specifically expressed in each of these cells and enhancer trap lines might be useful to identify these cell types. For genetic screening to identify dRFX2-target genes or interacting proteins in vivo, the transgenic flies established in this study should be useful, and more detailed study of the rough eye phenotype with histological analyses might provide us more information on the roles of endogenous dRFX2.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at NAR Online.
ACKNOWLEDGEMENTS
We thank Dr Y. Hiromi for providing enhancer trap lines, Dr M. Inagaki for the anti-HA antibody, Dr S. Elledge for the ACT-Drosophila cDNA library, and Drs S. Cotterill and M. Moore for comments on the English language used in the manuscript. This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan.
REFERENCES
Tsurimoto,T., Melendy,T. and Stillman,B. ( (1990) ) Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin. Nature, , 346, , 534–539.
Jaskulski,D., DeRiel,K., Mercer,W.E., Calabretta,B. and Baserga,R. ( (1988) ) Inhibition of cellular proliferation by antisense oligodeoxynucleotides to PCNA cyclin. Science, , 240, , 1544–1546.
Liu,Y.C., Marraccino,R.L., Keng,P.C., Bambara,R.A., Lord,E.M., Chou,W.G. and Zain,S.B. ( (1989) ) Requirement for proliferating cell nuclear antigen expression during stages of the Chinese hamster ovary cell cycle. Biochemistry, , 28, , 2967–2974.
Kelman,Z. and O'Donnell,M. ( (1995) ) Structural and functional similarities of prokaryotic and eukaryotic DNA polymerase sliding clamps. Nucleic Acids Res., , 23, , 3613–3620.
Shivji,M.K.K., Kenny,M.K. and Wood,R.D. ( (1992) ) Proliferating cell nuclear antigen is required for DNA excision repair. Cell, , 69, , 367–374.
Smith,M.L., Chen,I.-T., Zhan,Q., Bae,I., Chen,C.-Y., Gilmer,T.M., Kastan,M.B., O'Conner,P.M. and Fornace,A.J.J. ( (1994) ) Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science, , 266, , 1376–1380.
Matsuoka,S., Yamaguchi,M. and Matsukage,A. ( (1994) ) D-type cyclin-binding regions of proliferating cell nuclear antigen. J. Biol. Chem., , 269, , 11030–11036.
Waga,S., Hannon,G.J., Beach,D. and Stillman,B. ( (1994) ) The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature, , 369, , 574–578.
Flores-Rozas,H., Kelman,Z., Dean,F., Pan,Z.-Q., Harper,J.W., Elledge,S.J., O'Donnell,M. and Hurwitz,J. ( (1994) ) Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme. Proc. Natl Acad. Sci. USA, , 91, , 8655–8659.
Kelman,Z. and Hurwitz,J. ( (1998) ) Protein–PCNA interactions: a DNA-scanning mechanism? Trends Biochem. Sci., , 23, , 236–238.
Warbrick,E. ( (1998) ) The puzzle of PCNA's many partners. Bioessays, , 20, , 195–199.
Henderson,D.S., Banga,S.S., Grigliatti,T.A. and Boyd,J.B. ( (1994) ) Mutagen sensitivity and suppression of position-effect variegation result from mutations in mus209, the Drosophila gene encoding PCNA. EMBO J., , 13, , 1450–1459.
Shibahara,K. and Stillman,B. ( (1999) ) Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin. Cell, , 96, , 575–585.
Zhang,Z., Shibahara,K. and Stillman,B. ( (2000) ) PCNA connects DNA replication to epigenetic inheritance in yeast. Nature, , 408, , 221–225.
Hirose,F., Yamaguchi,M., Handa,H., Inomata,Y. and Matsukage,A. ( (1993) ) Novel 8-base pair sequence (Drosophila DNA replication-related element) and specific binding factor involved in the expression of Drosophila genes for DNA polymerase and proliferating cell nuclear antigen. J. Biol. Chem., , 268, , 2092–2099.
Yamaguchi,M., Hayashi,Y., Nishimoto,Y., Hirose,F. and Matsukage,A. ( (1995) ) A nucleotide sequence essential for the function of DRE, a common promoter element for Drosophila DNA replication-related genes. J. Biol. Chem., , 270, , 15808–15814.
Takahashi,Y., Yamaguchi,M., Hirose,F., Cotterill,S., Kobayashi,J., Miyajima,S. and Matsukage,A. ( (1996) ) DNA replication-related elements cooperate to enhance promoter activity of the Drosophila DNA polymerase 73-kDa subunit gene. J. Biol. Chem., , 271, , 14541–14547.
Yamaguchi,M., Hayashi,Y. and Matsukage,A. ( (1995) ) Essential role of E2F recognition sites in regulation of the proliferating cell nuclear antigen gene promoter during Drosophila development. J. Biol. Chem., , 270, , 25159–25165.
Hayashi,Y., Hirose,F., Nishimoto,Y., Shiraki,M., Yamagishi,M., Matsukage,A. and Yamaguchi,M. ( (1997) ) Identification of CFDD (common regulatory factor for DNA replication and DREF genes) and role of its binding site in regulation of the proliferating cell nuclear antigen gene promoter. J. Biol. Chem., , 272, , 22848–22858.
Ohno K., Hirose,F., Sakaguchi,K., Nishida,Y. and Matsukage,A. ( (1996) ) Transcriptional regulation of the Drosophila CycA gene by the DNA replication-related element (DRE) and DRE binding factor (DREF) Nucleic Acids Res., , 24, , 3942–3946.
Ryu,J.-R., Choi,T.-Y., Kwon,E.-J., Lee,W.-H., Nishida,Y., Hayashi,Y., Matsukage,A., Yamaguchi,M. and Yoo,M.-A. ( (1997) ) Transcriptional regulation of the Drosophila-raf proto-oncogene by the DNA replication-related element (DRE)/DRE-binding factor (DREF) system. Nucleic Acids Res., , 24, , 794–799.
Matsukage,A., Hirose,F., Hayashi,Y., Hamada,K. and Yamaguchi,M. ( (1995) ) The DRE sequence TATCGATA, a putative promoter-activating element for Drosophila melanogaster cell-proliferation-related genes. Gene, , 166, , 233–236.
Hirose,F., Yamaguchi,M., Kuroda,K., Omori,A., Hachiya,T., Ikeda,M., Nishimoto,Y. and Matsukage,A. ( (1996) ) Isolation and characterization of cDNA for DREF, a promoter-activating factor for Drosophila DNA replication-related genes. J. Biol. Chem., , 271, , 3930–3937.
Hart,C.M., Cuvier,O. and Laemmli,U.K. ( (1999) ) Identification of a multicopy chromatin boundary element at the borders of silenced chromosomal domains. Chromosoma, , 108, , 375–383.
Ohtani,K. and Nevins,J.R. ( (1994) ) Functional properties of a Drosophila homolog of the E2F1 gene. Mol. Cell. Biol., , 14, , 1603–1612.
Dynlacht,B.D., Brook,A., Dembski,M., Yenush,L. and Dyson,N. ( (1994) ) DNA-binding and trans-activation properties of Drosophila E2F and DP proteins. Proc. Natl Acad. Sci. USA, , 91, , 6359–6363.
Hao,X.F., Alphey,L., Bandara,L.R., Lam,E.W.-F., Glover,D. and La Thangue,N.B. ( (1995) ) Functional conservation of the cell cycle-regulating transcription factor DRTF1/E2F and its pathway of control in Drosophila melanogaster. J. Cell Sci., , 108, , 2945–2954.
Sawado,T., Yamaguchi,M., Nishimoto,Y., Ohno,K., Sakaguchi,K. and Matsukage,A. ( (1998) ) dE2F2, a novel E2F-family transcription factor in Drosophila melanogaster. Biochem. Biophys. Res. Commun., , 251, , 409–415.
Duronio,R.J., O'Farrell,P.H., Xie,J.E., Brook,A. and Dyson,N. ( (1995) ) The transcription factor E2F is required for S phase during Drosophila embryogenesis. Genes Dev., , 9, , 1445–1455.
Royzman,I., Whittaker,A.J. and Orr-Weaver,T.L. ( (1997) ) Mutations in Drosophila DP and E2F distinguish G1-S progression from an associated transcriptional program. Genes Dev., , 11, , 1999–2011.
Cayirlioglu,P., Bonnette,P., Dickson,M.R. and Duronio,R.J. ( (2001) ) Drosophila E2f2 promotes the conversion from genomic DNA replication to gene amplification in ovarian follicle cells. Development, , 28, , 5085–5098.
Frolov,M.V., Huen,D., Stevaux,O., Dimova,D., Balczarek-Strang,K., Elsdon,M. and Dyson,N.J. ( (2001) ) Functional antagonism between E2F family members. Genes Dev., , 15, , 2146–2160.
Stevaux,O., Dimova,D., Frolov,M.V., Taylor-Harding,B., Morris,E. and Dyson,N. ( (2002) ) Distinct mechanisms of E2F regulation by Drosophila RBF1 and RBF2. EMBO J., , 21, , 4927–4937.
Bray,S.J., Burke,B., Brown,N.H. and Hirsh,J. ( (1989) ) Embryonic expression pattern of a family of Drosophila proteins that interact with a central nervous system regulatory element. Genes Dev., , 3, , 1130–1145.
Dynlacht,B.D., Attardi,L.D., Admon,A., Freeman,M. and Tjian,R. ( (1989) ) Functional analysis of NTF-1, a developmentally regulated Drosophila transcription factor that binds neuronal cis elements. Genes Dev., , 3, , 1677–1688.
Hayashi,Y., Yamagishi,M., Nishimoto,Y., Taguchi,O., Matsukage,A. and Yamaguchi,M. ( (1999) ) A binding site for the transcription factor Grainyhead/Nuclear transcription factor-1 contributes to regulation of the Drosophila proliferating cell nuclear antigen gene promoter. J. Biol. Chem., , 274, , 35080–35088.
Yamaguchi,M., Hirose,F. and Matsukage,A. ( (1996) ) Roles of multiple promoter elements of the proliferating cell nuclear antigen gene during Drosophila development. Genes Cells, , 1, , 47–58.
Reith,W., Herrero-Sanchez,C., Kobr,M., Silacci,P., Berte,C., Barras,E., Fey,S. and Mach,B. ( (1990) ) MHC class II regulatory factor RFX has a novel DNA-binding domain and a functionally independent dimerization domain. Genes Dev., , 4, , 1528–1540.
Wu,S.Y. and McLeod,M. ( (1995) ) The sak1+ gene of Schizosaccharomyces pombe encodes an RFX family DNA-binding protein that positively regulates cyclic AMP-dependent protein kinase-mediated exit from the mitotic cell cycle. Mol. Cell. Biol., , 15, , 1479–1488.
Emery,P., Durand,B., Mach,B. and Reith,W. ( (1996) ) RFX proteins, a novel family of DNA binding proteins conserved in the eukaryotic kingdom. Nucleic Acids Res., , 24, , 803–807.
Huang,M., Zhou,Z. and Elledge,S.J. ( (1998) ) The DNA replication and damage checkpoint pathways induce transcription by inhibition of the Crt1 repressor. Cell, , 94, , 595–605.
Durand,B., Vandaele,C., Spencer,D., Pantalacci,S. and Couble,P. ( (2000) ) Cloning and characterization of dRFX, the Drosophila member of the RFX family of transcription factors. Gene, , 246, , 285–293.
Labrie,C., Lee,B.H. and Mathews,M.B. ( (1995) ) Transcription factors RFX1/EF-C and ATF-1 associate with the adenovirus E1A-responsive element of the human proliferating cell nuclear antigen promoter. Nucleic Acids Res., , 23, , 3732–3741.
Lee,B.H., Liu,M. and Mathews,M.B. ( (1998) ) Regulation of the human proliferating cell nuclear antigen promoter by the adenovirus E1A-associated protein p107. J. Virol., , 72, , 1138–1145.
Liu,M., Lee,B.H. and Mathews,M.B. ( (1999) ) Involvement of RFX1 protein in the regulation of the human proliferating cell nuclear antigen promoter. J. Biol. Chem., , 274, , 15433–15439.
Yamaguchi,M., Hirose,F., Nishida,Y. and Matsukage,A. ( (1991) ) Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknullt protein. Mol. Cell. Biol., , 11, , 4909–4917.
Yamaguchi,M., Nishida,Y. and Matsukage,A. ( (1995) ) Role of homeodomain protein binding region in the expression of Drosophila proliferating cell nuclear antigen gene: analysis with transgenic flies. Gene Expr., , 4, , 183–193.
Brand,A.H. and Perrimon,N. ( (1993) ) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development, , 118, , 401–415.
Hay,B.A., Wolff,T. and Rubin,G.M. ( (1994) ) Expression of baculovirus P35 prevents cell death in Drosophila. Development, , 120, , 2121–2129.
Sambrook,J., Fritsch,E.F. and Maniatis,T. ( (1989) ) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Smith,D.B. and Johnson,K.S. ( (1988) ) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene, , 67, , 31–40.
Hirose,F., Yamaguchi,M., Nishida,Y., Masutani,M., Miyazawa,H., Hanaoka,F. and Matsukage,A. ( (1991) ) Structure and expression during development of Drosophila melanogaster gene for DNA polymerase . Nucleic Acids Res., , 19, , 4991–4998.
Yamaguchi,M., Nishida,Y., Moriuchi,T., Hirose,F., Hui,C.-C., Suzuki,Y. and Matsukage,A. ( (1990) ) Drosophila PCNA/cyclin gene: structure and expression during development and specific binding of homeodomain proteins to its 5'-flanking region. Mol. Cell. Biol., , 10, , 872–879.
O'Connell,P.O. and Rosbash,M. ( (1984) ) Sequence, structure, and codon preference of the Drosophila ribosomal protein 49 gene. Nucleic Acids Res., , 12, , 5495–5513.
Feinberg,A.P. and Vogelstein,B. ( (1983) ) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem., , 132, , 6–13.
Mlodzik,M., Baker,N.E. and Rubin,G.M. ( (1990) ) Spacing differentiation in the developing Drosophila eye: a fibrinogen-related lateral inhibitor encoded by scabrous. Genes Dev., , 4, , 1848–1861.
Mlodzik,M., Hiromi,Y., Weber,U., Goodman,C.S. and Rubin,G.M. ( (1990) ) The Drosophila seven-up gene, a member of the steroid receptor gene superfamily, controls photoreceptor cell fates Cell, , 60, , 211–224.
Kramer,S., West,S.R. and Hiromi,Y. ( (1995) ) Cell fate control in the Drosophila retina by the orphan receptor seven-up: its role in the decisions mediated by the ras signaling pathway. Development, , 121, , 1361–1372.
Spradling,A.C. ( (1986) ) P element-mediated transformation. In Roberts,D.B. (ed.), Drosophila: A Practical Approach. IRL Press, Oxford, pp. 175–197.
Robertson,H.M., Preston C.R., Philips,R.W., Johnson-Schlitz,D.M., Benz,W.K. and Engels,W.R. ( (1988) ) A stable genomic source of P element transposase in Drosophila melanogaster. Genetics, , 118, , 461–470.
Yamaguchi,M., Hirose,F., Inoue,Y.H., Shiraki,M., Hayashi,Y., Nishi,Y. and Matsukage,A. ( (1999) ) Ectopic expression of human p53 inhibits entry into S phase and induces apoptosis in the Drosophila eye imaginal disc. Oncogene, , 18, , 6767–6775.
Fridell,Y.-W.C. and Searles,L.L. ( (1992) ) In vivo transcriptional analysis of the TATA-less promoter of the Drosophila melanogaster vermilion gene. Mol. Cell. Biol., , 12, , 4571–4577.
Wilder,E.L. and Perrimon,N. ( (1995) ) Dual functions of wingless in the Drosophila leg imaginal disc. Development, , 121, , 477–488.
Hirose,F., Ohshima,N., Shiraki,M., Inoue,H.Y., Taguchi,O., Nishi,Y., Matsukage A. and Yamaguchi,M. ( (2001) ) Ectopic expression of DREF induces DNA synthesis, apoptosis, and unusual morphogenesis in the Drosophila eye imaginal disc: possible interaction with Polycomb and trithorax group proteins. Mol. Cell. Biol., , 21, , 7231–7242.
Gajiwala,K.S., Chen,H., Cornille,F., Roques,B.P., Reith,W., Mach,B. and Burley,S.K. ( (2000) ) Structure of the winged-helix protein hRFX1 reveals a new mode of DNA binding. Nature, , 403, , 916–921.
Katan-Khaykovich,Y. and Shaul,Y. ( (1998) ) RFX1, a single DNA-binding protein with a split dimerization domain, J. Biol. Chem., , 273, , 24504–24512.
Hirose,F., Yamaguchi,M. and Matsukage,A. ( (1999) ) Targeted expression of the DNA binding domain of DRE-binding factor, a Drosophila transcription factor, attenuates DNA replication of the salivary gland and eye imaginal disc. Mol. Cell. Biol., , 19, , 6020–6028.
Wolff,T. and Ready,D.F. ( (1993) ) The Development of Drosophila melanogaster. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 1277–1326.
Harrington,E.A., Fanidi,A. and Evan,G.I. ( (1994) ) Oncogenes and cell death. Curr. Opin. Genet. Dev., , 4, , 120–129.
Crook,N.E., Clem,R.J. and Miller,L.K. ( (1993) ) An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif. J. Virol., , 67, , 2168–2174.
Hay,B.A., Wassarman,D.A. and Rubin,G.M. ( (1995) ) Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell, , 83, , 1253–1262.
Emery,P., Strubin,M., Hofmann,K., Bucher,P., Mach,B. and Reith,W. ( (1996) ) A consensus motif in the RFX DNA binding domain and binding domain mutants with altered specificity. Mol. Cell. Biol., , 16, , 4486–4494.
Yamaguchi,M., Hayashi,Y., Hirose,F., Nishimoto,Y. and Matsukage,A. ( (1997) ) Distinct roles of E2F recognition sites as positive or negative elements in regulation of the DNA polymerase 180 kDa catalytic subunit gene promoter during Drosophila development. Nucleic Acids Res., , 25, , 3847–3854.
Maga,G. and Hubscher,U. ( (2003) ) Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J. Cell Sci., , 116, , 3051–3060.
Vandaele,C., Coulon-Bublex,M., Couble,P. and Durand,B. ( (2001) ) Drosophila regulatory factor X is an embryonic type I sensory neuron marker also expressed in spermatids and in the brain of Drosophila. Mech.Dev., , 103, , 159–162.
Dubruille,R., Laurencon,A., Vandaele,C., Shishido,E., Coulon-Bublex,M., Swoboda,P., Couble,P., Kernan,M. and Durand,B. ( (2002) ) Drosophila regulatory factor X is necessary for ciliated sensory neuron differentiation. Development, , 129, , 5487–5498.(Kyoko Otsuki, Yuko Hayashi2, Masaki Kato)