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Nucleic Acids Res. 2004; 32(18): 5636–5648.
Published online Oct 19, 2004. doi:  10.1093/nar/gkh895
PMCID: PMC524303

Characterization of dRFX2, a novel RFX family protein in Drosophila

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 (79) 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 (1214).

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) (1517), E2F-recognition sites (18) and CFDD [common regulatory factor for DNA replication and DREF (DRE-binding factor) genes] 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 (2528). In addition, transcription of DNA polymerase α 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 α 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 (3436). 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 (3842), 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 (4345). 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 BigDye™ Terminator v3.0 Cycle sequencing Standard kit (Applied Biosystems) using an ABI PRISM™ 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 (λACT-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 β-mercaptoethanol, pH 7.0) containing 0.03% 5-bromo-4-chloro-3-indolyl-β-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 λEMBL3 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 [α-32P]dCTP using a random priming method (55). Plaque hybridizations were carried out at 42°C in a solution containing 20% formamide, 3× SSC, 5× 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 β-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 β-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-β-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 β-galactosidase activity was defined as absorbance units per hour per milligram of protein. To correct for endogenous β-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.

β-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-β-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 × 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 [FlyBase (http://flybase.net/) ID, FBrf0115338] 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 × 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 (Figure1,1, 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 ...

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 Figure2B,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 (Figure2B,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 (Figure2A2A 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 ...

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 (Figure2A)2A) and the resultant mutant oligonucleotides were used as competitors in the band mobility shift analysis using GST–dRFX2202–480 fusion protein (Figure (Figure2C).2C). The mutant oligonucleotides mut1 and mut2 did not compete at all (Figure (Figure2C,2C, lanes e–j), while the other mutant oligonucleotides competed for the binding as effectively as the wild-type −124/−98 oligonucleotide (Figure (Figure2C,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 (Figure22C).

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 (Figure2D).2D). EP oligonucleotide competed effectively to binding reaction between EP probe and either GST–dRFX2202–480 or GST–dRFX fusion protein (Figure (Figure2D,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 (Figure2D,2D, lanes a and c). However, when GST–dRFX fusion protein was applied, the shifted band did not disappear (Figure (Figure2D,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 (Figure3A).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 β-galactosidase assays at various developmental stages (Figure (Figure33B).

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 ...

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 (Figure3B,3B, panels a–c). Much more extensive reduction of the lacZ expression was observed in larvae carrying mutations in the dRFX2 site (Figure (Figure3B,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 (Figure3B,3B, panel c). In contrast, only a marginal effect on the lacZ expression was observed with a mutation outside the dRFX2 site (mut3) (Figure (Figure3B,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 (Figure3B).3B). Since the extent of the reduction was most prominent in larvae, β-galactosidase activity was demonstrated in dissected third instar larval tissues (Figure (Figure3C).3C). In transgenic third instar larvae carrying a wild-type construct, high lacZ-staining signal was observed in the salivary glands (Figure (Figure3C,3C, panel a). The larvae having mutations in the dRFX2 site (mut1 and mut2) exhibited extensively reduced staining signals (Figure (Figure3C,3C, panels b and c). In contrast, no reduction of staining was observed with lines carrying a mutation outside the dRFX2 site (mut3, Figure Figure3C,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 (Figure2).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 (Table1),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 (Table1).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 (Table11 and Figure Figure4A,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 (Figure4B).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-dRFX2 ...

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 (Figure4A,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 (Figure4A,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 (Figure44B).

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 (Figure5A)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 (Figure5B).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/+, +; ( ...

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 β-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 β-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 β-galactosidase antibody.

When dRFX2202–480 was expressed in eye imaginal discs, photoreceptors of such flies [early R8 (Figure (Figure6A),6A), R3/R4/R1/R6 (Figure (Figure6B)6B) and R3/R4/R7 (Figure (Figure6C)]6C)] 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 ...

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 (Figure7A,7A, panel a). In contrast, eye discs of flies expressing dRFX2202–480 showed a significant increase of apoptotic cells posterior to the MF (Figure (Figure7A,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 (Figure7B).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-dRFX2 ...

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 α and δ, 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 [(37) and in this study]. 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.

[Supplementary Material]

ACKNOWLEDGEMENTS

We thank Dr Y. Hiromi for providing enhancer trap lines, Dr M. Inagaki for the anti-HA antibody, Dr S. Elledge for the λACT-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.

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