From the
Division of Biochemistry, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464-8681 and
Department of Pathology, Nagoya Graduate School of Medicine, Showa-ku, Nagoya 466-8550, Japan
Received for publication, March 26, 2003
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ABSTRACT |
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INTRODUCTION |
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Recently, Hart et al. (11, 12) proposed a novel function of DREF as an antagonist of the boundary element-associated factor (BEAF), which is involved in the boundary activity of the scs' region of the Drosophila 87A7 hsp70 gene (5, 11, 12). Staining of polytene chromosomes with anti-DREF and anti-BEAF antibodies revealed about 50% of signals for the two proteins to overlap. Furthermore, using a chromatin precipitation method, they demonstrated that DREF could bind to the same sequences as BEAF. From these results, they hypothesized that competition of binding between DREF and BEAF is important for the regulation of activity at the chromatin boundary.
More recently, we have established transgenic flies, in which ectopic expression of DREF was targeted to the eye imaginal discs (13). Adult flies expressing DREF exhibited a severe rough eye phenotype. We found that this was significantly suppressed by a half-dose reduction of any of the trithorax-group genes, brahma (14), moira (15), and osa (16), involved in determining chromatin structure or chromatin remodeling, whereas reduction of Distal-less, a transcription factor involved in proximal/distal pattern formation (17), enhanced the DREF-induced rough eye phenotype (18). These results suggest a possibility that DREF activity is regulated by protein complexes that play a role in determining chromatin structure (for example, establishment, maintenance, or cancellation of the chromatin boundary) such as those involving BEAF (5, 11, 12) and Polycomb/trithorax group proteins (19).
Despite its obvious potential importance, identification of a mammalian DRE/DREF system has not yet been achieved. To obtain clues for cDNA cloning of mammalian DREF, we isolated a gene for DREF from Drosophila virilis (13) and determined highly conserved regions in DREFs of this species and Drosophila melanogaster (Dm) (8). Comparison of deduced amino acid sequences for the two species allowed us to identify three highly conserved regions, CR1, CR2, and CR3 (13). A BLAST search with the amino acid sequence of DmCR1, a domain required for DNA binding and homodimer formation (8), hit a candidate human DREF protein registered to data bases as KIAA0785 (20). Comparison of amino acid sequences of KIAA0785 protein and the two Drosophila DREFs revealed obvious conservation of CR1, CR2, and CR3. In the present study, we characterized KIAA0785 protein as a human homologue of DREF (hDREF) and demonstrated sequence-specific DNA binding activity and fluctuation of expression during the cell cycle. Furthermore, we found that hDREF/KIAA0785 binds to the promoter region of the histone H1 gene and stimulates its promoter activity.
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EXPERIMENTAL PROCEDURES |
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OligonucleotidesDouble-stranded oligonucleotides containing the hDREF/KIAA0785 binding sequence (H1) in the promoter region of human histone H1 gene (referred to as FNC16/H1.5) (position at 401 to 371 with respect to translational start codon) (21, 22) and its base substitution sequences (H1m), shown below, were synthesized: H1, 5'-GATCTAGTAGAAATTTATCGCGACACGCTACTAACG and 5'-GATCCGTTAGTAGCGTGTCGCGATAAATTTCTACTA; H1m, 5'-GATCTAGTAGAAATTTATatatACACGCTACTAACG and 5'-GATCCGTTAGTAGCGTGTatatATAAATTTCTACTA. In addition, the following double-stranded oligonucleotides containing the hDREF/KIAA0785 binding sequence and base-substituted derivatives were synthesized: hDRE, 5'-GATCCACATGTCGCGACAGGTACCAGCTGA and 5'-GATCTCAGCTGGTACCTGTCGCGACATGTG; mut1, 5'-GATCCACAgtTCGCGACAGGTACCAGCTGA and 5'-GATCTCAGCTGGTACCTGTCGCGAacTGTG; mut2, 5'-GATCCACATGgaGCGACAGGTACCAGCTGA- and 5'-GATCTCAGCTGGTACCTGTCGCtcCATGTG; mut3, 5'-GATCCACATGTCtaGACAGGTACCAGCTGA and 5'-GATCTCAGCTGGTACCTGTCtaGACATGTG; mut4, 5'-GATCCACATGTCGCtcCAGGTACCAGCTGA and 5'-GATCTCAGCTGGTACCTGgaGCGACATGTG; mut5, 5'-GATCCACATGTCGCGAacGGTACCAGCTGA and 5'-GATCTCAGCTGGTACCgtTCGCGACATGTG; mut6, 5'-GATCCACATGTCGCGAtAGGTACCAGCTGA and 5'-GATCTCAGCTGGTACCTaTCGCGACATGTG; mut7, 5'-GATCCACATGTCGCGcacGGTACCAGCTGA and 5'-GATCTCAGCTGGTACCgtgCGCGACATGTG; mut8, 5'-GATCCACAgtgatCGACAGGTACCAGCTGA and 5'-GATCTCAGCTGGTACCTGTCGatcacTGTG; mut9, 5'-GATCCACATGTacgtACAGGTACCAGCTGA and 5'-GATCTCAGCTGGTACCTGTacgtACATGTG; mut10, 5'-GATCCACATtgCGCGcaAGGTACCAGCTGA and 5'-GATCTCAGCTGGTACCTtgCGCGcaATGTG. Regions corresponding to the 10-bp binding core sequence for hDREF/KIAA0785 are shown in bold letters, with nucleotides substituted for the wild-type sequence in lowercase with underlining.
PCR primers (primer F, 5'-GCTGCAGTTGCACTGAATTCGCCTC; primer R, 5'-CAGGTCAGTTCAGCGGATCCTGTCG) and an oligonucleotide carrying defined ends and a 26-nucleotide region of degeneracy (R76, 5'-CAGGTCAGTTCAGCGGATCCTGTCG-(N)26-GAGGCGAATTCAGTGCAACTGCAGC) were synthesized and used for the CASTing experiments.
A full-length cDNA for hDREF/KIAA0785 was generated by PCR using primers hDREF5', 5'-GGAgctagcATGGAGAATAAAAGCCTGGAG, and hDREF3', 5'-GCTctcgagCTACAGGAAGCTGCTGTCCCT. Recognition sites for NheI in hDREF5' and XhoI in hDREF3' are shown by lowercase letters with underlining.
The promoter region (537 to 1) of the human histone H1 gene was generated by PCR using primers H1-p-5', 5'-AATgctagcGTCCTGTGCCTGTGTTAC, and H1-p-3', 5'-TATctcgagGGTGGCAAGAAACTGCTAG. Recognition sites for NheI in H1-p-5' and XhoI in H1-p-3' are shown by lowercase letters with underlining. A full-length cDNA for the human histone H1 gene (22) was generated by RT-PCR using primers 5'-H1, 5'-ATGTCGGAAACCGCTCCTG, and 3'-H1, 5'-CTACTTCTTTTTGGCAGCC.
Plasmid ConstructionKIAA0785 cDNA/pBluescript ks() was obtained from Kazusa DNA Research Institute (20) and used as a PCR template to generate a full-length cDNA for hDREF/KIAA0785. The obtained cDNA was blunt-ended with a DNA blunting kit (Takara) and inserted into the SmaI site of pGEX-2T (Amersham Biosciences) to create pGST-hDREF/KIAA0785. A plasmid expressing hDREF/KIAA0785 was constructed by amplifying the full-length cDNA of hDREF/KIAA0785 by the PCR method using KIAA0785 cDNA/pBluescript ks() as a template, cutting with NheI and XhoI and inserting into the NheI-XhoI site of pcDNA3-HA. To construct the expression plasmid CR1hDREF/KIAA0785/pcDNA3-HA, cDNA encoding the CR1 region (1140 amino acid residue) of hDREF/KIAA0785 was amplified by PCR and inserted into the blunt-ended XhoI site of pcDNA3-HA.
The H1-p/pGL3 reporter plasmid was constructed as follows. The promoter region (537 to 1) of the human histone H1 gene (21) was amplified by PCR using genomic DNA from HeLa cells as template, cut with NheI and XhoI, and inserted into the NheI-XhoI site of pGL3 promoter vector (Promega), which contains the SV40 promoter placed upstream of the firefly luciferase gene. Then, the SV40 promoter was removed by cutting with HindIII and BglII. The obtained DNA fragment was blunt-ended with a DNA blunting kit (Takara) and self-ligated. All plasmids were propagated in Escherichia coli XL-1 Blue, isolated by standard procedures (23) and further purified using a Qiagen Plasmid Midi kit (Qiagen Inc.).
Expression of GST Fusion ProteinsExpression and purification of GST-hDREF/KIAA0785 fusion proteins in E. coli XL-1 Blue were carried out as described elsewhere (8). Lysates of cells were prepared by sonication in Buffer D containing 0.6 M KCl, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml each of pepstatin, leupeptin, and aprotinin, and cleared by centrifugation at 12,000 x g for 20 min at 4 °C before application to a glutathione-Sepharose (Amersham Biosciences) column to purify the GST-hDREF/KIAA0785 fusion protein. Control GST protein was expressed and purified in the same way.
AntibodyThe purified GST-hDREF/KIAA0785 fusion protein was used to elicit polyclonal antibody production in a rabbit. Polyclonal antibodies reacting with GST-hDREF/KIAA0785 were purified by sequential passage through affinity resin covalently conjugated with GST or GST-hDREF/KIAA0785.
ImmunoblottingPolypeptides in E. coli cell extracts or total cell extracts were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes in a solution containing 50 mM borate-NaOH buffer (pH 9.0) and 20% methanol at 4 °C for 16 h. Membranes were blocked with TBS (50 mM Tris-HCl (pH 8.3) and 150 mM NaCl) containing 10% skim milk for 1 h at room temperature and then incubated with the rabbit anti-hDREF/KIAA0785 polyclonal antibody for 1 h at room temperature. After extensive washing with TBS, membranes were incubated with an alkaline phosphatase-conjugated goat anti-rabbit IgG (used at a dilution of 1:2,000) (Promega) for 1 h at room temperature. After further extensive washing with TBS, color was developed in a solution containing 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl2, 0.34 mg/ml nitro blue tetrazolium salt, and 0.175 mg/ml 5-bromo-4-chloro-3-indoryl phosphate toluidinium salt.
Cell Cycle Analysis with Flow CytometryCells were harvested by trypsinization, and the nuclei were stained with propidium iodide using a CycleTest kit (BD Biosciences) according to the manufacturer's instructions. Analysis was with a FACScan (BD Biosciences), and the percentages in each phase were calculated with the SOBR model in the CELFIT program.
Analysis of the Cellular Localization of hDREF/KIAA0785Triton X-100-extracted nuclei were prepared as follows. HeLa cells cultured in 100-mm plates were washed three times with ice-cold phosphate-buffered saline (PBS) and lysed for 10 min on ice with 1 ml of ice-cold CSK buffer (10 mM Pipes (pH 6.8), 150 mM NaCl, 300 mM sucrose, 1 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol) (24) containing proteinase inhibitors and 0.5% Triton X-100. After low speed centrifugation (3,000 rpm, 3 min at 4 °C), the nuclei were once more extracted with 1 ml of ice-cold CSK buffer containing 0.5% Triton X-100.
DNase I digestion of Triton X-100-treated nuclei was performed as follows. Triton X-100-extracted nucleus suspension was prepared as described above. The samples (100 µl) were supplemented with 1 mM ATP and 1,000 units/ml DNase I, followed by incubation for 1 h at room temperature. To examine the extent of hDREF/KIAA0785 solubilization by salt, Triton X-100-treated nuclei (100 µl) were suspended in CSK containing NaCl at concentrations of 0.15, 0.35, or 0.55 M, followed by incubation for 1 h on ice. The pellet fractions and supernatants were then separated by low speed centrifugation, the former being resuspended in 100 µl of the buffer, and the latter was clarified by centrifugation at 100,000 rpm for 15 min using a Beckman TLA100.1 rotor. The samples were then added to the same volumes of 2x SDS sample buffer (1x solution (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 5% -mercaptoethanol, 10% glycerol, 0.01% bromphenol blue)), boiled, and analyzed by immunoblotting.
Immunofluorescence AnalysisCells grown on 13-mm coverglasses were fixed for 20 min in 100% methanol at 20 °C and then permeabilized with 0.2% Triton X-100 for 2 min. All staining procedures were carried out at room temperature. The samples were incubated with either anti-hDREF/KIAA0785 IgG or normal rabbit IgG at a 1:400 dilution in PBS with 10% normal goat serum for 1 h. After washing three times with PBS, the samples were reacted with Alexa 488-conjugated goat anti-rabbit IgG antibody (Molecular Probes) at a 1:400 dilution in PBS with 10% normal goat serum for 1 h. After washing a further three times with PBS, the samples were mounted and analyzed by conventional microscopy.
Selection of hDREF/KIAA0785 Binding Sites with CASTing ExperimentsA 32P-labeled double-stranded degenerate oligonucleotide was prepared by incubating 50 µl of reaction mixture containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 50 µM each of dATP, dGTP, and dTTP, 20 µM [-32P]dCTP (740 kBq), 100 µM each of primers F and R76 oligonucleotide, and 2 units of the Klenow fragment of DNA polymerase I at 37 °C for 1 h. Binding reactions were performed by adding GST-hDREF/KIAA0785 (0.4 µg) to Buffer D (20 mM Hepes (pH 7.9), 120 mM KCl, 1 mM DTT, 0.1% Tween 80, and 12% glycerol) containing 400 ng of poly(dI-dC), 400 ng of sonicated salmon sperm DNA, 50 µg of bovine serum albumin, and 32P-labeled double-stranded degenerate oligonucleotide (1.8 ng) and incubating at 4 °C for 30 min. Then, glutathione-Sepharose beads (10 µl) were added with incubation at 4 °C for an additional 1 h. The DNA-protein complexes were then precipitated by centrifugation, and the pellets were washed six times with Buffer D. After elution with 100 µl of Buffer D containing 5 mM reduced glutathione, the oligonucleotides were recovered by phenol extraction and ethanol precipitation and amplified by PCR in 20 µl of a mixture containing 300 ng of primers F and R, 20 µM [
-32P]dCTP (740 kBq), 50 µM each of dATP, dGTP, and dTTP, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, and 0.5 unit of Taq polymerase (Roche Molecular Biochemicals). DNAs were amplified by 20 cycles of 1 min at 94 °C, 30 s at 60 °C, and 1 min at 72 °C, purified by passage through a Sephacryl-200 spin column, precipitated with ethanol and dissolved in 50 µl of a buffer containing 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. The amount of amplified oligonucleotide was quantified, and a portion corresponding to 2 ng was used in subsequent CASTing cycles. After five cycles of CASTing, the radiolabeled oligonucleotides (1.5 x 105 cpm) were used as probes in an electrophoretic mobility shift assay (EMSA). Oligonucleotides binding to GST-hDREF/KIAA0785 were excised from gels, eluted overnight in 0.2 ml of a solution containing 0.2 M NaCl, 20 mM EDTA, and 0.1% SDS at 37 °C, extracted once with phenol, and then precipitated with ethanol.
DNAs were amplified by PCR as described above, digested with EcoRI and BamHI, and then subcloned into EcoRI and BamHI sites of the pBluescript vector. Nucleotide sequences of 50 independent clones were determined.
Preparation of HeLa Cell Nuclear ExtractA nuclear extract was prepared by a modified Dignam method (25). Cultured HeLa cells in suspension were collected by centrifugation and washed with PBS. Two packed volumes of Buffer A containing 10 mM Hepes (pH 7.9), 1.5 mM MgCl2,10mM KCl, 0.5 mM DTT, and proteinase inhibitor mixture (1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 2 µg/ml pepstatin, 0.75 µg/ml aprotinin) were added to swell the cells on ice for 10 min. A Dounce homogenizer (size B) was then used to rupture the cell membranes, and nuclei were collected at 1,600 x g at 4 °C and resuspended in high salt Buffer B containing 20 mM Hepes (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and the proteinase inhibitor mixture to extract nuclear proteins by rocking at 4 °C for 1 h before centrifugation at 100,000 x g for 5 min. The supernatant was dialyzed in Buffer C (20 mM Hepes (pH 7.9), 50 mM NaCl, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 20% glycerol), clarified by centrifugation, and stored at 80 °C in small aliquots. The protein concentration of the extract was 4.8 mg/ml.
Electrophoretic Mobility Shift AssayEMSA were performed as described earlier (1), with minor modifications. A reaction mixture containing 20 mM Hepes (pH 7.6), 150 mM KCl, 0.1 mM EDTA, 0.5 mM DTT, 10% glycerol, and 1 µg of salmon sperm DNA was used with purified GST-hDREF/KIAA0785. Another reaction mixture containing 15 mM Hepes (pH 7.6), 120 mM KCl, 0.1 mM EDTA, 1 mM DTT, 2% glycerol, and 1 µg of poly(dI-dC) DNA was employed for EMSA with the HeLa cell nuclear extracts. 32P-Labeled probes (10,000 cpm) were incubated in 15 µl of reaction mixture. When necessary, unlabeled DNA fragments were added as competitors at this step. Then, GST-hDREF/KIAA0785 fusion proteins (20 ng) or aliquots of the HeLa cell nuclear extract (5 µg protein) were added, and the reaction mixture was incubated for 15 min on ice. In experiments with antibodies, the HeLa cell nuclear extract was preincubated with the antibody for 1 h 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.
DNA Transfection into Cells and Luciferase AssaysHeLa cells were plated at about 6 x 104 cells per well in 24-well culture plates for 16 h. 100 ng of firefly luciferase reporter plasmid and 10 ng of pRL-TK plasmid (Promega), carrying sea pansy luciferase under the control of the herpes simplex virus tk gene promoter as an internal control, were co-transfected into cells using LipofectAMINE Plus reagent (Invitrogen) (26). In the co-transfection experiment with the expression plasmid, reporter plasmids were co-transfected with hDREF/KIAA0785/pcDNA3-HA or CR1hDREF/KIAA0785/pcDNA3-HA with pRL-TK as an internal control. The total amount of expression plasmid was adjusted to 410 ng by addition of pcDNA3-HA. After DNA transfection, cells were cultured for 48 h, and luciferase activity was measured with a dual-luciferase reporter assay system (Promega). Firefly luciferase activity was normalized to sea pansy luciferase activity. Transfections were performed several times with at least three independent plasmid preparations.
RT-PCR AnalysisTotal RNA was isolated using RNeasy Mini Kit (Qiagen Inc.), and 250 ng of total RNA was subjected to RT-PCR analyses using a high fidelity RNA PCR kit (Takara). The cDNAs were generated using random 9-mers as primers. The PCR condition was 4550 cycles at 94 °C for 1 min, 53 °C for 1 min, and 72 °C for 1 min using 5'-H1 and 3'-H1 as primers. All the PCR were performed within the range of linear amplification. The PCR products were electrophoretically separated in a 2% agarose gel, stained with ethidium bromide, and quantified using ImageMaster VDS-CL (Amersham Biosciences).
siRNA TransfectionsiRNAs (short interfering double-stranded RNAs) against hDREF/KIAA0785 were chemically synthesized by Dharmacon Research, Inc. The sequences for hDREFsiRNA1 and hDREFsiRNA2 correspond to regions +221 to +241 and +132 to +152 with respect to the translational start codon, respectively. Scramble siRNA (Dharmacon Research, Inc.) was used as a negative control. siRNAs were transfected into 30% confluent HeLa cells using Oligofectamine (Invitrogen) (27), and cells were incubated for 72 h at 37 °C.
5-Bromo-2'-deoxyuridine (BrdUrd) LabelingHeLa cells were incubated in the presence of 20 µg/ml BrdUrd (Roche Molecular Biochemicals) for 1 h. The samples were fixed in methanol for 1 h at 20 °C and further fixed in 80% ethanol-50 mM glycine buffer (pH 2.0) at 20 °C for 2 h. Incorporated BrdUrd was visualized using an anti-BrdUrd antibody and an alkaline phosphatase detection kit (Roche Molecular Biochemicals). The time of color development for alkaline phosphatase was precisely regulated to be identical for all samples.
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RESULTS |
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Interestingly, significant similarities with other proteins were also found by the BLAST search using the DmCR1 amino acid sequence as a query. As shown in Fig. 1B, KIAA0637 (AB014537 [GenBank] ) polypeptide (1171 amino acids) contains four CR1-like sequences (29) and the N-terminal DNA binding region of the Drosophila BEAF32B (283 amino acids) (11). Alignment of amino acid sequences of the polypeptides revealed complete conservation of cysteine and histidine residues (shown by asterisks) and high conservation of surrounding amino acid residues, suggesting DNA binding activity in the N-terminal region containing C2H2-type zinc finger structure.
It should be noted that Esposito et al. (30) isolated a cDNA and the gene encoding KIAA0785 protein and called it Tramp. In the meantime we obtained KIAA0785 cDNA from the Kazusa DNA Research Institute and started functional analysis. They reported the Tramp gene to be localized on the X and Y chromosomes, and the amino acid sequence of the Tramp protein shows similarity with Drosophila DREF, although they did not demonstrate any biological activity. hDREF/KIAA0785 Protein Is Localized in the Nuclei of HeLa CellsHart et al. and ourselves (8, 12) have demonstrated that DmDREF is a ubiquitous nuclear protein. We raised a rabbit polyclonal antibody against bacterially produced GST-fused-hDREF/KIAA0785 protein. IgGs specifically recognizing hDREF/KIAA0785 protein were purified by serial passage of rabbit serum through affinity columns coupled with GST and GST-hDREF/KIAA0785 proteins. Immunoblot analysis revealed the antibody to react specifically with an 80-kDa polypeptide in a HeLa cell extract (Fig. 2A, lane 5). Preincubation of the antibody with GST-hDREF/KIAA0785 resulted in no detection (data not shown).
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To investigate the subcellular localization of hDREF/ KIAA0785 protein, we immunostained HeLa cells. Signals for hDREF/KIAA0785 proteins were detected in nuclei (Fig. 2B, panel a). To clarify whether hDREF/KIAA0785 is associated with a specific nuclear structure, HeLa cells treated with 0.2% Triton X-100 were immunostained. Significant amounts of fluorescence were then found in granular structures (Fig. 2B, panel b), and the signals were resistant to treatment with 50 µg/ml pancreatic DNase I (data not shown), suggesting that at least a part of hDREF/KIAA0785 may be tightly bound to nuclear structures.
Subcellular localization of hDREF/KIAA0785 was also investigated with biochemical cell fractionation of HeLa cells into cytosolic and nuclear fractions by treatment with hypotonic buffer and low speed centrifugation. As shown in Fig. 2C, hDREF/KIAA0785 protein was detected in both cytosolic (lane 2) and nuclear (lane 3) fractions but was mainly localized in the nuclear fraction (lane 3). Subnuclear localization of hDREF/KIAA0785 protein was further examined with use of detergent, salt, and nuclease. HeLa cells were first treated with CSK buffer containing 0.5% Triton X-100 and 0.15 M NaCl, which extracts not only cytoplasmic but also nuclear proteins not tightly bound to nuclear structures, and then the residual nuclei were treated with DNase I and/or various concentrations of NaCl. DNase treatment did not liberate hDREF/KIAA0785 from nuclei (Fig. 2C, lanes 47), although DNase treatment under this condition liberated about two-thirds of core histones and DNA from the extracted nuclei (31). NaCl treatment at 0.15 M did not release hDREF/KIAA0785, but two-thirds and almost all of hDREF/KIAA0785 were released from nuclei in the presence of 0.35 and 0.55 M NaCl, respectively (Fig. 2C, lanes 10 and 11). These results indicate that hDREF/KIAA0785 protein is associated with granular structures in a detergent- and nuclease-resistant manner.
Determination of Consensus Sequences for hDREF/KIAA0785 BindingAs described above, hDREF/KIAA0785 protein is localized in nuclei, like DmDREF (8). Therefore, we addressed the question of whether hDREF/KIAA0785 protein can directly bind to DNA and if so to which nucleotide sequence. For this purpose, we performed a series of CASTing experiments with a double-stranded oligonucleotide with defined ends for which PCR primers were available and a degenerate central core region of 26 nucleotides. Purified GST-hDREF/KIAA0785 fusion protein was incubated with degenerate double-stranded oligonucleotides and affinity-purified with glutathione-Sepharose beads. Unbound DNA was removed by washing, and bound DNA was amplified by PCR. Several cycles of affinity purification and PCR amplification were performed. Obtained DNA segments were cloned into pBluescript, and 50 independent isolates were sequenced. The relevant features of the determined sequences of 10 bp are shown in Fig. 3A. The consensus nucleotide sequence for hDREF/KIAA0785 protein is a palindromic 5'-TGTCG(C/) TGA(C/T)A. Notably, the half-site of this sequence (5'-(C/T)GA(C/T)A) matches five of eight nucleotides of the DmDREF binding sequence (5'-TATCGATA) (8) and the main recognition motif of BEAF (5'-CGATA) (11) (Fig. 3B).
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Next we tried to detect hDREF/KIAA0785 binding activity in HeLa cell nuclear extracts by EMSA. Oligonucleotides (hDRE) containing the 10-bp palindromic sequence (5'-TGTCGCGACA) preferentially recognized by GST-hDREF/KIAA0785 protein was synthesized and used for EMSA as a probe (Fig. 4A). As shown in Fig. 4B, two protein-DNA complexes were formed with HeLa cell nuclear extracts (lanes 1 and 7). Therefore, we examined whether these shifted bands are complexes containing hDREF/KIAA0785 protein by EMSA experiments with anti-hDREF/KIAA0785 antibody. Preincubation of HeLa cell nuclear extracts with the rabbit anti-hDREF/KIAA0785 antibody eliminated the signals in a dose-dependent manner (Fig. 4B, lanes 812), whereas addition of normal rabbit IgG had no effect (Fig. 4B, lanes 26). The result indicates that these two DNA-protein complexes formed between hDRE oligonucleotides and HeLa cell nuclear extract contain hDREF/KIAA0785 protein, although it is not clear whether there are qualitative differences and what are the components of these two kinds of DNA-protein complexes.
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Given the preference for the 10-bp nucleotide sequence selected by CASTing using GST-hDREF/KIAA0785, we further examined the nucleotide sequence sufficient for binding using HeLa cell nuclear extract. A series of oligonucleotides with base substitutions inside the hDREF/KIAA0785 binding site (mut1mut10) were synthesized and used for EMSA as competitors (Fig. 4A). As shown in Fig. 4C, the two shifted bands were diminished by adding an excess amount of unlabeled oligonucleotides of wild-type (hDRE) (lanes 2 and 3) and mut6 with a single base substitution at the ninth C to T of the hDREF/KIAA0785 binding sequence (lanes 14 and 15). The mut2, mut3, and mut4 with two base substitution mutations in the center region of the hDREF/KIAA0785 binding site did not compete for the binding at all (lanes 611), and excess amounts of mut7 (lanes 16 and 17) and mut10 (lanes 22 and 23), with base substitutions inside the binding site, slightly competed, whereas the other mutations in the end of the binding site (mut1, mut5, mut8, and mut9) retained competition ability (lanes 4, 5, 12, 13, 16, 17, 20, and 21). These results indicate that the 6-bp sequence 5'-TCG(C/T)GA in the center of the hDREF/KIAA0785 binding site plays an important role in hDREF/KIAA0785 binding.
Search for Genes Carrying hDREF/KIAA0785 Binding SitesTo identify genes that might be under the control of hDREF/KIAA0785 protein, we searched the GenBankTM and EMBL data bases using BLAST and the TargetFinder network service (Telethon Institute of Genetics and Medicine). The simple BLAST program revealed that there exist more than 500 TGTCG(C/T)GA(T/C)A-like sequences (counting sequences with more than 7 bp of the 10-bp consensus sequence) in the human genome, and results for selected examples involving promoter regions are listed in Table I. Interestingly, we found hDREF/KIAA0785 binding sequences in a variety of genes related to cell proliferation, as reported for DmDREF (32). We categorized these genes by its function as follows: DNA replication (DNA synthesis), DNA repair, cell cycle regulation, transcription, regulation of chromatin structure, and protein synthesis. It should be noted that we also found DRE in the promoter regions of Drosophila genes homologous to those for human topoisomerase II (33), DNA polymerase
(34), c-Myb (35), rRNA (36), TGF-
(37), and K-ras (38). The findings suggest that hDREF/KIAA0785 might be a functional homologue of DmDREF.
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The Promoter Region of the Histone H1 Gene Specifically Forms a Complex with hDREF/KIAA0785 ProteinBecause we demonstrated that DmDREF regulates the expression of Drosophila genes related to DNA replication and cell proliferation (1, 7, 3941), we examined whether hDREF/KIAA0785 regulates human DNA replication-related genes. We focused on the histone H1 gene (referred to as FNC16/H1.5), because a single 10-bp sequence completely matching the hDREF/KIAA0785 binding sequence was found in its promoter region, which has been cloned and for which several transcriptional regulatory elements have been characterized (21, 22).
The hDREF/KIAA0785 binding sequence is positioned at 390 to 381 with respect to the translation start codon. We first examined whether the hDREF/KIAA0785 protein binds. Oligonucleotides containing the putative binding sequence in the histone H1 promoter (H1) or with base substitutions inside the hDREF/KIAA0785 binding site (H1m) were chemically synthesized and used for EMSA (Fig. 5A). As shown in Fig. 5B, four protein-DNA complexes were formed with the HeLa cell nuclear extract and the radiolabeled H1 as a probe (lane 1). Signals for the slowest and the second slowest migrating bands were diminished by adding excess amount of unlabeled H1 oligonucleotide (Fig. 5B, lanes 2 and 3) or oligonucleotides containing the consensus hDREF/KIAA0785 binding sequence (hDRE and mut6) (Fig. 5B, lanes 69) but not by adding oligonucleotide H1m (Fig. 5B, lanes 4 and 5). Furthermore, the signals of the two retarded bands were diminished by preincubation with the rabbit anti-hDREF/KIAA0785 antibody in a dose-dependent manner (Fig. 5C, lanes 812), whereas addition of normal rabbit IgG had no effect (Fig. 5C, lanes 26).
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hDREF/KIAA0785 Protein Stimulates Histone H1 Gene Promoter ActivityTo address the function of hDREF/KIAA0785 with regard to transcription of the histone H1 gene, we examined its effects on promoter activity. A DNA region containing the histone H1 promoter (537 to 1) was amplified by PCR using genomic DNA from HeLa cells and cloned into a plasmid carrying the firefly luciferase reporter gene (H1-p/pGL3). Transient co-expression experiments with H1-p/pGL3 reporter plasmid and an hDREF/KIAA0785-bearing plasmid revealed that expression of the latter stimulated the H1 promoter activity in a dose-dependent manner (Fig. 6A). In contrast, expression of a deletion mutant for hDREF/KIAA0785 encoding N-terminal amino acid residues corresponding to the CR1 region did not result in activation (Fig. 6B). The results indicate that hDREF/KIAA0785 protein positively regulates the histone H1 gene promoter.
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Expression of hDREF/KIAA0785 Protein Is Induced in G1-S Phase of the Cell CycleBecause transcription of the histone H1 gene is transiently induced during G1-S phase (42, 43), we examined fluctuation of hDREF/KIAA0785 protein during the cell cycle by immuno-Western blotting. We used primary cultures of normal human lung fibroblasts (HEL cells) for this analysis, because they can be readily induced to enter a quiescent state by serum deprivation, re-entering the cell cycle on addition of 10% fetal calf serum. We checked cell cycle progression after release from serum starvation by staining cells with propidium iodine and applying to flow cytometry (Fig. 7A) and by staining cells with anti-PCNA antibody, which is known as aG1-S phase marker (Fig. 7C) (44). Both analyses revealed that more than 85% of HEL cells arrested by 72 h of serum starvation synchronously re-enter the cell cycle after adding serum to the culture. As shown in Fig. 7, the total amount of hDREF/KIAA0785 protein in quiescent HEL cells after 48 h of serum starvation was significantly reduced in comparison with that of asynchronous cultures of HEL cells (Fig. 7B, a). Levels began to increase in cells at 14 h after serum stimulation, reaching a maximum in S/G2-cell enriched conditions after 1618 h, and then gradually decreasing (Fig. 7B, a). Immunostaining of HEL cells also showed that expression of hDREF/KIAA0785 protein is regulated in line with the proliferating stage. Decrease in the amount of nuclear fluorescence with the anti-hDREF/KIAA0785 antibody was observed in quiescent cells in serum-deprived culture. Release from serum starvation induced the reappearance of strong fluorescence within 14 h (data not shown). As shown in panel c of Fig. 7C, more than 70% of cells were then stained with hDREF/KIAA0785 antibody, and this expression pattern was similar to those of PCNA (Fig. 7C, panel d). The period of induction of hDREF/KIAA0785 expression after serum stimulation seems to correspond well with the G1-S phase in the cell cycle (Fig. 7, A and B, a). The results thus indicate that expression of hDREF/KIAA0785 is induced during the G1-S transition.
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The expression pattern of the histone H1 gene after serum stimulation was also examined by RT-PCR analysis. As reported previously (59), the signal for histone H1 mRNA was not detected in serum-starved cells. Expression of histone H1 mRNA was induced at 14 h after serum release, and the expression level reached a maximum at 1820 h (Fig. 7B, b). Fluctuation of histone H1 mRNA was quite similar to that of hDREF/KIAA0785 expression.
Reduction of hDREF/KIAA0785 Expression Caused Repression of DNA Synthesis and the Histone H1 Gene Expression The above results suggest a possibility that hDREF/KIAA0785 protein might play a role in G1-S progression. To address this question, we performed RNA interference targeting endogenous hDREF/KIAA0785 in HeLa cells. siRNAs against hDREF/KIAA0785 (hDREFsiRNA1 and hDREFsiRNA2) were transfected, and the expression amounts of hDREF/KIAA0785 protein were determined. Immunoblot analysis revealed that amounts of hDREF/KIAA0785 protein were reduced to 12 and 70% by transfection of hDREFsiRNA1 and hDREFsiRNA2, respectively, relative to Scramble siRNA transfection (Fig. 8A, a). Immunofluorescence staining also demonstrated that both hDREFsiRNA1 and hDREFsiRNA2 specifically reduced signals for hDREF/KIAA0785 protein in nuclei (Fig. 8A, panels be). Under this condition, expression levels of histone H1 mRNA were measured by quantitative RT-PCR analysis. Introduction of hDREFsiRNA1 and hDREFsiRNA2 decreased amounts of histone H1 mRNA by 74 and 32% compared with Scramble siRNA transfection, respectively (Fig. 8B). Evidence strongly suggests that transcription of the histone H1 gene might be under the control of hDREF/KIAA0785.
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Next we examined the effect of hDREF/KIAA0785 reduction on G1-S progression using the BrdUrd labeling method. hDREFsiRNAs-transfected cells failed to stain with BrdUrd (Fig. 8C, panels c and d), whereas about 30% of control cells incorporated BrdUrd (Fig. 8C, panels a and b). Noticeably, we observed that not a few cells transfected with hDREFsiRNAs exhibited flattened and enlarged morphology (Fig. 8, A (panels d and e) and C (panels c and d)) and began to die after 5 days after transfection (date not shown). Thus, we concluded that hDREF/KIAA0785 might play an important role in G1-S progression.
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DISCUSSION |
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We further detected hDREF/KIAA0785 DNA binding activity using HeLa cell nuclear extracts and determined the required nucleotide sequences by EMSA with oligonucleotides carrying a series of base substitution mutations as competitors. The results indicate that the binding sequence with the highest affinity to hDREF/KIAA0785 protein is 5'-TGTCG(C/T)GA (C/T)A, and particularly the 6-bp sequence in the center (5'-TCG(C/T)GA) might be important for hDREF/KIAA0785 binding.
It is noteworthy that the consensus binding sequence for hDREF/KIAA0785 contains a CGCG sequence in its center, this being frequent in the promoter regions of ubiquitous-expressing house-keeping genes in the mammalian genome (45). Generally, non-methylated CpG is mainly found in CpG islands of promoter regions (45). Methylation prevents methylation-sensitive transcription factors such as E2F from binding and strongly suppresses gene expression (46, 47). In contrast, non-sensitive transcription factors such as Sp1 can bind to methylated CpG islands and activate transcription (48, 49). We obtained results indicating that hDREF/KIAA0785 can bind to TGTCGCGACA and its methylated form, TGTmCGmCGACA, with almost the same affinity2 so that it is thus likely that hDREF/KIAA0785 can regulate a wide variety of genes containing both hypo- and hypermethylated CpG islands.
Our search for the hDREF/KIAA0785 binding sequence in the human genome revealed its presence in the promoter regions of many kinds of genes involved in DNA replication, DNA repair, cell cycle regulation, and transcription. We have already reported this to be the case for DmDREF (32), and some of the genes have been demonstrated to be actually under the control of the DRE/DREF system (1, 7, 3941). The gene catalogue obtained by data base searching suggests that hDREF/ KIAA0785 may be a functional homologue of DmDREF and may similarly take part in the transcriptional regulation of genes related to cell proliferation.
We here focused on the human histone H1 gene (FNC16/H1.5), because it carries a nucleotide sequence completely matching the 10-bp sequence exhibiting the highest affinity to the hDREF/KIAA0785 and is well known as a gene whose expression is stringently coupled with DNA replication. Several groups have extensively analyzed the 180-bp region from the cap site of the histone H1 promoter and found two elements that might be important in the S phase-specific H1 transcription; an AC box, 5'-AAACACA-3', and a CCAAT box, 5'-ACCAATCACA-3' (5053), exist at 176 to 167 and 112 to 103, respectively (21). The putative hDREF/KIAA0785 binding site is located at 390 to 381 in a more distal region that has not yet been characterized by anyone. Therefore, we cloned the promoter region (537 to 1) of the histone H1 gene (FNC16/H1.5) and examined whether it is regulated by hDREF/KIAA0785. We can conclude that hDREF/KIAA0785 specifically binds to the human histone H1 gene promoter and stimulates its activity for the following two reasons. 1) EMSA experiments with anti-hDREF/KIAA0785 antibody or competitor oligonucleotides demonstrated that hDREF/KIAA0785 protein specifically binds to the nucleotide sequence at 401 to 371. 2) Transient co-transfection assays revealed that expression of full-length hDREF/KIAA0785 protein stimulates the histone H1 gene promoter activity in dose-dependent manner, whereas expression of a truncated form of hDREF/KIAA0785 does not.
Promoters of most DNA replication-dependent human H4, H3, H2A, H2B, and H1 histone genes do not contain a typical E2F binding site, although the expression of these genes is coordinately controlled in a cell-cycle dependent fashion. Many kinds of elements and trans-acting factors have been identified in vitro using mutation analysis of promoter regions of histone genes and EMSA. For example, the HiNF-D complex (consisting of CDP/Cut, CDC2, and cyclin A, a retinoblastoma-related protein) (5456), HiNF-M/IRF-2 (5658), and HiNF-B/H1TF2 (52, 53) are all suggested to be involved in up-regulation of human histone genes during G1-S transition. Despite much effort to characterize these factors, fluctuation during the cell cycle and roles of each in regulation of cell cycle-specific histone gene expression have not yet been clearly demonstrated, other than for HiNF-M/IRF2 and CDP/Cut protein (55, 56, 58). Western blotting and immunohistochemical analysis here showed that expression of hDREF/KIAA0785 protein is induced by adding serum to serum-starved cultures of normal human fibroblasts, reaching a maximum in S phase. In addition, considering our previous finding that overexpression of DmDREF in the eye imaginal disc induces ectopic DNA synthesis in the post-mitotic cells by up-regulating many DNA replication genes (18), we hypothesize that hDREF/KIAA0785 protein may have an important role in up-regulation of histone gene expression. To directly demonstrate hDREF/KIAA0785 function in the histone H1 gene expression, we tested whether reduction of endogenous hDREF/KIAA0785 protein diminishes the histone H1 gene expression. As expected, introduction of siRNAs against hDREF/KIAA0785 resulted in reduction of histone H1 mRNA. Therefore, we concluded that hDREF/KIAA0785 might regulate the histone H1 gene expression. Interestingly, we found that the histone H4 genes, organized in the cluster containing the histone H1 gene (FNC16/H1.5), also have hDREF/KIAA0785 putative binding sites.
Knock-down of hDREF/KIAA0785 protein resulted in inhibition of G1-S progression. We have already reported (13) that expression of a dominant-negative form of DmDREF in cells of eye imaginal discs inhibited G1-S progression in the second mitotic wave. Similar phenotype induced by knock-down of hDREF/KIAA0785 and DmDREF protein also provide a piece of evidence that hDREF/KIAA0785 might be the DmDREF homologue.
In summary, we here characterized hDREF/KIAA0785 protein as a human homologue of DmDREF and demonstrated that it might be a positive transcriptional regulatory factor. We also generated evidence suggesting that the human histone H1 gene is one of the targets of hDREF/KIAA0785. Although the data in the present study potentially suggests that hDREF/KIAA0785 might be the homologue of DmDREF, we cannot rule out a possibility that hDREF/KIAA0785 is not the "true" homologue. To answer the issue, we should provide evidence that hDREF/KIAA0785 possesses similar activity as DmDREF. We are now trying to establish that transgenic flies expressing chimera hDREF/KIAA0785 protein swapped its N-terminal DNA binding domain with that of DmDREF. Furthermore, studies on other genes with putative hDREF/KIAA0785 protein binding sequence should increase our understanding of the biological function of hDREF/KIAA0785.
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Tel.: 81-52-762-6111 (ext. 7220); Fax: 81-52-763-5233; E-mail: fsegawa{at}aichi-cc.jp.
1 The abbreviations used are: PCNA, proliferating cell nuclear antigen; BEAF, boundary element-associated factor; Dm, Drosophila melanogaster; CR, conserved region; HEL, human embryonic lung fibroblast; RT, reverse transcription; HA, hemagglutinin; GST, glutathione S-transferase; PBS, phosphate-buffered saline; Pipes, 1,4-piperazinediethanesulfonic acid; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay.
2 F. Hirose and N. Ohshima, manuscript in preparation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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