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Originally published In Press as doi:10.1074/jbc.M607180200 on January 5, 2007

J. Biol. Chem., Vol. 282, Issue 10, 7563-7575, March 9, 2007
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Human DNA Replication-related Element Binding Factor (hDREF) Self-association via hATC Domain Is Necessary for Its Nuclear Accumulation and DNA Binding*

Daisuke Yamashita1, Hirofumi Komori, Yoshiki Higuchi, Tomohiro Yamaguchi, Takashi Osumi, and Fumiko Hirose2

From the Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori, Hyogo 678-1297, Japan

Received for publication, July 28, 2006 , and in revised form, January 2, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously demonstrated that hDREF, a human homologue of Drosophila DNA replication-related element binding factor (dDREF), is a DNA-binding protein predominantly distributed with granular structures in the nucleus. Here, glutathione S-transferase pulldown and chemical cross-linking assays showed that the carboxyl-terminal hATC domain of hDREF, highly conserved among hAT transposase family members, possesses self-association activity. Immunoprecipitation analyses demonstrated that hDREF self-associates in vivo, dependent on hATC domain. Moreover, analyses using a series of hDREF mutants carrying amino acid substitutions in the hATC domain revealed that conserved hydrophobic amino acids are essential for self-association. Immunofluorescence studies further showed that all hDREF mutants lacking self-association activity failed to accumulate in the nucleus. Self-association-defective hDREF mutants also lost association with endogenous importin beta1. Moreover, electrophoretic gel-mobility shift assays revealed that the mutations completely abolished the DNA binding activity of hDREF. These results suggest that self-association of hDREF via the hATC domain is necessary for its nuclear accumulation and DNA binding. We also found that ZBED4/KIAA0637, another member of the human hAT family, also self-associates, again dependent on the hATC domain, with deletion resulting in loss of efficient nuclear accumulation. Thus, hATC domains of human hAT family members appear to have conserved functions in self-association that are required for nuclear accumulation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Drosophila DNA replication-related element binding factor (dDREF)3 is a transcriptional factor required for expression of genes related to DNA replication such as proliferating cell nuclear antigen and DNA polymerase {alpha} and dE2F (1-4). In vivo and in vitro studies from our and other groups have shown that dDREF positively regulates processes involved not only in DNA replication but also cell growth and differentiation (5, 6). It has been reported that dDREF is a component of a transcription initiation complex containing Drosophila TATA-binding protein-related factor 2 (dTRF2), so that it might direct dTRF2 complexes to dDRE-containing genes (7). In addition, dDREF might function as an antagonist of the boundary element-associated factor, which is involved in establishing insulators of several hundred regions of the Drosophila genome (8, 9) and plays a role in the determination of chromatin structures by interacting with Drosophila Mi-2 (10). The available data suggest that dDREF makes important contributions to the regulation of higher order chromatin structures.

We have identified a human homologue of dDREF (hDREF; also called as ZBED1, ALTE, TRAMP, and KIAA0785) by a computer search for amino acid sequences homologous to three highly conserved regions (CR1, CR2, and CR3) found in Drosophila melanogaster and Drosophila virilis DREFs (11, 12). In a previous study (12) we determined a consensus nucleotide sequence for hDREF binding (hDRE, 5'-TGTCG(C/T)GA(C/T)A-3') and showed its presence in promoter regions of human genes involved in cell proliferation and cell cycle regulation, similar to dDRE sequences in Drosophila. Indeed, we could demonstrate that hDREF is a potent activator of the histone H1 gene, whose expression is stringently coupled with DNA replication. Importantly, RNA interference against hDREF resulted in inhibition of S-phase entry. These results suggest that hDREF regulates the expression of human genes related to cell proliferation, similar to dDREF.

As evidenced by the similarity in amino acid sequences, hDREF is a member of the hAT transposase family (13), like fruit fly hobo, maize Activator, and snapdragon Tam3, defined by sharing close structural and functional characteristics with McClintock's Activator (14). hAT transposons are known to exist in genomes of fungi, nematodes, and fish as well as man. CR1 and CR3 of hDREF correspond to two domains conserved among the family, the amino-terminal BED zinc finger and carboxyl-terminal hATC domains, respectively. The BED (boundary element-associated factor and DREF) zinc finger domain (conserved domain data base (CDD) accession number pfam02892) is thought to be a DNA binding domain of chromatin boundary element-binding proteins and transposases (15). Indeed, our and other groups demonstrated earlier that BED zinc finger domains of dDREF and boundary element-associated factor possess DNA binding activity (16, 17). The hATC domain (CDD accession number pfam05699) has been designated as a dimerization domain as assessed by in vitro cross-linking experiments and yeast two-hybrid assays using the family members Activator and housefly Hermes (18, 19). However, the functions of hATC domains of the human hAT family members have hitherto not been elucidated.

Despite the importance of hDREF for cell proliferation, the mechanisms underlying its regulation are poorly understood. In this study we demonstrated that hDREF self-associates in vivo and that conserved hydrophobic amino acids in the hATC domain are essential for this purpose. Interestingly, hDREF mutants that fail to self-associate neither accumulate in the nucleus nor bind to hDRE sequences.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—The mammalian expression plasmid pcDNA3-HA-hDREF (amino acids (aa) 1-694) was described previously (12). The pcDNA3-HA vector contains a sequence encoding the hemagglutinin (HA) peptide, YPYDVPDYA, and both T7 and cytomegalovirus promoters. For constructing carboxyl-terminal-truncated mutants of pcDNA3-HA-hDREF, cDNAs encoding the truncated polypeptides were amplified by PCR with appropriate phosphorylated oligonucleotides, digested with NheI, and inserted between the NheI and blunt-ended XbaI sites of pcDNA3-HA vector. Base substitutions were made by site-specific mutagenesis employing the overlap extension method (20) with a combination of the appropriate oligonucleotides and confirmation by DNA sequencing. Amplified DNA fragments carrying base substitutions in full-length hDREF cDNA were then digested with NheI and ApaI and then inserted between the NheI and ApaI sites of the pcDNA3-HA vector. To construct pcDNA3-HA-hDREF ({Delta}hATC) (the deleted cDNA region encoding aa 571-651), a cDNA fragment encoding aa 1-570 was amplified by PCR with phosphorylated oligonucleotides containing EcoRV site at the 5'-end, digested with NheI, and inserted between the NheI and blunt-ended XhoI sites of the pcDNA3-HA vector. A cDNA fragment encoding aa 652-694 was amplified by PCR, digested with EcoRV and ApaI, and inserted between the EcoRV and ApaI sites of pcDNA3-HA-hDREF (aa 1-570). For constructing pEYFP-hDREF (aa 1-694), a cDNA fragment encoding aa 1-694 obtained by digesting pcDNA3-HA-hDREF (aa 1-694) with XhoI and ApaI was inserted between the SalI and ApaI sites of the pEYFP-C1 vector (Clontech), which contains the cytomegalovirus promoter and an enhanced yellow fluorescent protein (YFP) coding sequence. For construction of pEYFP-hDREF (aa 1-624), a cDNA fragment encoding aa 1-624 obtained by digesting pcDNA3-HA-hDREF (aa 1-694) with XhoI and BamHI was inserted between the SalI and BamHI sites of the pEYFP-C1 vector. For constructing pEYFP-hDREF (aa 520-551), a cDNA fragment encoding aa 520-551 obtained by digesting pcDNA3-HA-hDREF (aa 1-694) with BglII was blunt-ended with the Klenow fragment and then inserted into the SmaI site of the pEYFP-C1 vector. pEYFP-hDREF (aa 106-624) was constructed by digesting pEYFP-hDREF (aa 1-624) with SmaI and XhoI, filling in the sticky end with the Klenow fragment, and self-ligation. pEYFP-hDREF (aa 567-651) was constructed by amplifying a cDNA encoding aa 1-651 by PCR with pcDNA3-HA-hDREF (aa 1-694) as a template, digesting the PCR product with SphI, creating a blunt end by a filling-in reaction with the Klenow fragment, and then insertion into the blunt-ended EcoRI site of the pEYFP-C1 vector. For constructing pcDNA3-HA-hDREF (aa 567-651), a cDNA fragment encoding aa 567-651 obtained by digesting pEYFP-hDREF (aa 567-651) with XhoI and ApaI was inserted between the XhoI and ApaI sites of the pcDNA3-HA vector. To construct pEGFP-GST-hDREF (aa 520-551), a cDNA fragment encoding aa 520-551, obtained by digesting pEYFP-hDREF (aa 520-551) with EcoRI and BamHI, was inserted between the EcoRI and BamHI sites of the pEGFP-GST vector (a gift from Drs. Okada and Yagisawa) (21), which contains the cytomegalovirus promoter and coding sequences for an enhanced green fluorescent protein (GFP) and glutathione S-transferase (GST). For constructing pGEX-4T-2-hDREF (aa 567-651) and pMAL-c2-hDREF (aa 567-651), a cDNA fragment encoding aa 567-651 obtained by digesting pEYFP-hDREF (aa 567-651) with BglII and SalI was inserted between the BamHI and SalI sites of the pGEX-4T-2 vector (GE Healthcare), which contains a GST coding sequence, and pMAL-c2 vector (New England Biolabs), which contains an maltose-binding protein (MBP) coding sequence, respectively.

ZBED4/KIAA0637 cDNA in pBluescript KS(-) (22) was obtained from Kazusa DNA Research Institute. pcDNA3-HA-ZBED4/KIAA0637 (aa 1-1171) was constructed by amplifying full-length ZBED4/KIAA0637 cDNA by PCR in pBluescript KS(-) as a template, digesting the product with NheI and XbaI, and insertion between the NheI and XbaI sites of the pcDNA3-HA vector. To construct pcDNA3-HA-ZBED4/KIAA0637 ({Delta}hATC) (the deleted cDNA region encoding aa 1086-1171), a cDNA fragment encoding aa 1-1085 was amplified by PCR, digested with NheI and ApaI, and inserted between the NheI and ApaI sites of the pcDNA3-HA vector. For constructing pEYFP-ZBED4/KIAA0637 (aa 1-1171 or {Delta}hATC), cDNA fragments encoding aa 1-1171 or 1-1085, obtained by digesting pcDNA3-HA-ZBED4/KIAA0637 (aa 1-1171 or {Delta}hATC) with NheI, filling in the sticky end with Klenow fragment, and digesting with ApaI, were inserted between the ApaI and blunt-ended EcoRI sites of the pEYFP-C1 vector.

To construct phGFP105-RanQ69L, a cDNA fragment encoding human RanQ69L mutant obtained by digesting pQE-80L/RanQ69L (a gift from Dr. Imamoto) (23) with BamHI and EcoRI was inserted between the BglII and EcoRI sites of the phGFP105-C1 vector expressing a GFP mutant protein with an increased brightness (24). All PCR reactions described above were performed with KOD-plus DNA polymerase (Toyobo). All plasmids were purified using a Qiagen plasmid kit (Qiagen).

Oligonucleotides—The following oligonucleotides were used for constructing the carboxyl-terminal-truncated mutants of pcDNA3-HA-hDREF, 5'-tacggtgggaggtctatata-3' as a common forward primer, and 1-651 (5'-gcgttctcatacagaaacacc-3'), 1-624, (5'-gatccgaagagacgctcaggg-3'), 1-551 (5'-atctcggccagcatgttgttg-3'), and 1-523 (5'-accgggaagatcttgtcctcag-3') as reverse primers for the mutants indicated.

For creating base-substituted mutants of pcDNA3-HA-hDREF, 5'-tacggtgggaggtctatata-3' and 5'-agtcgaggctgatcagcgagc-3' were used as the common forward and reverse primers, respectively. The following oligonucleotides were used as site-specific forward and reverse primers: K530A/K531A/R534A, 5'-gcctcccgtcGCGGCGctcatgGCGacatccacg-3' and 5'-cgtggatgtCGCcatgagCGCCGCgacgggaggc-3'; W590A/W591A, 5'-gaccccctcaagGCGGCGtcagaccgcctg-3' and 5'-caggcggtctgaCGCCGCcttgagggggtc-3'; P599A, 5'-gccctcttcGCCctgctgccca-3' and 5'-tgggcagcagGGCgaagagggc-3'; L600A/L601A, 5'-cctcttccccGCGGCGcccaaggtgc-3' and 5'-gcaccttgggCGCCGCggggaagagg-3'; V604A/L605A, 5'-gctgcccaagGCGGCGcagaagtac-3' and 5'-gtacttctgCGCCGCcttgggcagc-3'; LLVL/AAAA, 5'-cctcttccccGCGGCGcccaaggcgg-3' and 5'-ccgccttgggCGCCGCggggaagagg-3'; E619A/R620A, 5'-gtcgcccctGCGGCTctcttcggatc-3' and 5'-gatccgaagagAGCCGCaggggcgac-3'; F622A, 5'-tgagcgtctcGCCggatccgcc-3' and 5'-ggcggatccGGCgagacgctca-3'; R633A, 5'-cagcgccaagGCGaaccggctg-3' and 5'-cagccggttCGCcttggcgctg-3'. As a template, pcDNA3-HA-hDREF (V604A/L605A) was employed for constructing pcDNA3-HA-hDREF (LLVL/AAAA), whereas pcDNA3-HA-hDREF (wild type) was used for constructing other pcDNA3-HA-hDREF mutants. Underlining and capital letters denote the substituted nucleotides and the codons encoding the substituted amino acids, respectively. For creating the hATC domain-deleted mutant of pcDNA3-HA-hDREF, the following oligonucleotides containing EcoRV sites were used: 1-570, 5'-agatatcctccaccacctgggcatg-3'; 651-694, 5'-tatagatatccggagtggggcagaggcg-3'.

For creating a full-length construct of pcDNA3-HA-ZBED4/KIAA0637, 5'-tgggctagcatggagaataacttgaaaact-3' and 5'-tcgtctagatcaatactgaaagtatattaa-3' were used as forward and reverse primers. For constructing the hATC domain-deleted mutant of pcDNA3-HA-ZBED4/KIAA0637, 5'-atagggccctcacaccatggcttcaggcag-3', containing an ApaI site, was used.

Cell Culture, Synchronization, and DNA Transfection—HeLa cells were cultured in Ham's F-12 medium containing 10% fetal bovine serum at 37 °C under 5% CO2. To obtain populations of cells in mitosis, synchronization was performed with a double thymidine block. HeLa cells were blocked for 20 h in medium containing 2.5 mM thymidine (Wako), released for 9 h by washing out the medium, and then blocked again in medium containing 2.5 mM thymidine for 16 h to arrest all the cells at the beginning of S phase. The cells released from the second block then progressed through G2 phase and mitosis synchronously.

Transfection was carried out by the calcium phosphate method as described previously (25). The DNA/calcium phosphate precipitates were removed 4 h after the addition of precipitates, and the cells were cultured in fresh medium for 20 h. Precipitates containing 3, 6, 12, and 24 µg of expression plasmids in total were added to HeLa cells in 12- and 6-well plates and 6- and 10-cm dishes, respectively. The lack of expression plasmids was compensated with an empty vector, pBluescript KS(-) (Stratagene).

Antibodies—The following antibodies were used: rabbit anti-hDREF polyclonal antibody (12); rabbit anti-GFP polyclonal antibody (632459) from Clontech; rat anti-HA monoclonal antibody (3F10) from Roche Applied Science; rabbit anti-phospho-histone H3 (Ser-10) polyclonal antibody (mitosis marker, 06-570) from Upstate%20Biotechnology">Upstate Biotechnology, Inc.; goat anti-karyopherin (importin) beta1 polyclonal antibody (C-19) from Santa Cruz Biotechnology, Inc.; rabbit anti-MBP antiserum (E8030) from New England Biolabs; normal goat IgG from Sigma; anti-rabbit IgG species-specific antibody linked to horseradish peroxidase (HRP) (NA934) from GE Healthcare; anti-rat IgG species-specific antibody linked to HRP (712-035-153) from Jackson ImmunoResearch; anti-goat IgG species-specific antibody linked to HRP (sc-2020) from Santa Cruz; anti-rat and anti-rabbit species-specific antibodies linked to Alexa fluor 594 (A-11007) and 488 (A-11034) dyes, respectively, from Molecular Probes. Expression of GST-ZBED4/KIAA0637 fusion protein, production of antibody against GST-ZBED4/KIAA0637, and affinity purification of anti-ZBED4/KIAA0637 IgG were performed as described previously (12).

Immunoprecipitation and Western Blotting—Cells were washed with phosphate-buffered saline (PBS) and lysed on ice in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Nonidet P-40, 1 mM dithiothreitol, 0.1 mM EDTA, 50 µg/ml amidino-phenylmethanesulfonyl fluoride, and a protease inhibitor mixture (P8340, Sigma)) for 30 min. The lysates were passed through 29-gauge needles 10 times and then cleared by centrifugation, and the supernatants were incubated with protein G-Sepharose 4 Fast Flow (GE Healthcare) for 1 h at 4°C. The samples were centrifuged to remove nonspecific proteins binding to the beads, and the supernatants were incubated with an appropriate antibody for 1 h at 4°C and then with protein G-Sepharose 4 Fast Flow overnight at 4 °C. The immunoprecipitates were washed with lysis buffer without protease inhibitors, and the immunoprecipitated proteins were eluted from the beads by boiling for 5 min in SDS-PAGE sample buffer. Proteins were separated by SDS-PAGE under standard conditions, and the resolved proteins were transferred to Immobilon-NC membranes (Millipore) using a semi-dry apparatus. The membranes were then probed with the indicated primary antibodies in PBS containing 5% skimmed milk and 0.05% Tween 20 and finally with appropriate secondary antibodies linked to horseradish peroxidase. Bands were detected using an ECL kit (GE Healthcare).

Chemical Cross-linking Assay—HeLa cells transfected with pcDNA3-HA-hDREF (aa 567-651) were harvested in PBS supplemented with the protease inhibitor mixture. Collected cells were subjected to chemical cross-linking by the addition of disuccinimidyl suberate (Pierce) dissolved in dimethyl sulfoxide (Me2SO) (5 mg/ml) at final concentrations of 0.17 mg/ml. Cross-linking reactions were performed at 25 °C for 30 min and terminated by the addition of SDS-PAGE sample buffer followed by boiling for 5 min. Proteins were separated by SDS-PAGE, and HA-hDREF (aa 567-651) was detected by Western blotting as described above.

GST Pulldown Assay—Recombinant GST- and MBP-fused proteins were purified using glutathione-Sepharose 4B (GE Healthcare) and amylose resin (New England Biolabs) according to the manufacturer's instructions. Approximately 100 ng of purified recombinant MBP or MBP-hDREF (aa 567-651) proteins was added to 16 µl of the glutathione-Sepharose 4B beads bound to 1 µg of recombinant GST or GST-hDREF (aa 567-651) proteins and incubated in buffer A (20 mM Tris-HCl (pH 7.4), 200 mM NaCl and 1 mM EDTA) supplemented 0.5% (v/v) Triton X-100, 1 mM dithiothreitol, the protease inhibitor mixture, and 1 mM phenylmethanesulfonyl fluoride for 6 h at 4 °C. The beads were washed 5 times with buffer A supplemented 0.5% (v/v) Triton X-100. Bound proteins to the beads were eluted by boiling for 5 min in SDS-PAGE sample buffer. Half of each sample was analyzed by Western blotting with anti-MBP antiserum.

Blue Native (BN)-PAGE and Two-dimensional BN-PAGE/SDS-PAGE—Samples for two-dimensional BN-PAGE/SDS-PAGE were prepared as follows. Subconfluent HeLa cells cultured in 6-cm dishes were collected in a Tris-buffered salinebased buffer (20 mM Tris-HCl (pH 7.0), 150 mM NaCl, 1 mM dithiothreitol, 0.1 mM EDTA, 10% glycerol, 50 µg/ml amidino-phenylmethanesulfonyl fluoride, and the protease inhibitor mixture) and sonicated 10 times for 20 s each with 1-min intervals using a Bioruptor (CosmoBio). The lysates were centrifuged at 12,500 x g at 4 °C for 10 min, and protein amounts in the supernatants were measured with a Bio-Rad protein assay kit (Bio-Rad) using bovine serum albumin as a standard. Supernatant samples containing 80 µg of proteins were subjected to BN-PAGE. BN-gels were prepared as described (26), and acrylamide gels with 5-13% gradients were used as native separating gels. A high molecular weight calibration kit for electrophoresis (GE Healthcare) in solution (20 mM Tris-HCl (pH 7.0) and 150 mM NaCl) was used for molecular mass markers (thyroglobulin, 669 kDa; ferritin, 440 kDa; catalase, 232 kDa; lactate dehydrogenase, 140 kDa; bovine serum albumin, 67 kDa). Electrophoresis was performed at 50 V and 4 °C while the samples moved in the stacking gel and then at 250 V and 4 °C while the samples migrated in the separating gel.

For further separation in second-dimension SDS-PAGE, the lanes from first-dimension BN-PAGE were cut out, dried in a DNA SpeedVac (Savant) for 30 min, and placed onto a second-dimension SDS-polyacrylamide gel of the same thickness and overlaid with 1% H14-agarose (TaKaRa) containing 1-fold SDS-PAGE sample buffer. SDS-PAGE was performed at 1 mA during stacking and at 10 mA during separation. Western blotting was performed as described above.

Immunofluorescence and Fluorescence Microscopy—For observing YFP- and GFP-fused proteins, cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 20 min. Cells were again washed with PBS and finally mounted with 90% glycerol in PBS containing 25 mg/ml 1,4-diazabicyclo-[2.2.2]octane.

For immunofluorescence, cells were washed with PBS, fixed with 2% paraformaldehyde in PBS for 20 min, permeabilized in PBS containing 0.2% Triton X-100 for 20 min, and then treated with 2% bovine serum albumin, PBS blocking buffer before incubation with primary antibodies in the same buffer for 1 h at 37 °C. Then they were washed with PBS and incubated with secondary antibody in 2% bovine serum albumin, PBS for 1 h at 37 °C. DNA was stained with Hoechst dye or propidium iodide when necessary, and samples were finally mounted with 90% glycerol in PBS containing 25 mg/ml 1,4-diazabicyclo-[2.2.2]octane. With an IX-70 microscope (Olympus) or with a LSM510 confocal microscope (Carl Zeiss), fluorescence images from more than 100 cells expressing HA-tagged YFP- or GFP-fused proteins with moderate expression levels were examined for each experiment, and representative examples are illustrated in the figures as indicated in the legends to Figs. 3, 4, 5, and 7.

In Vitro Transcription/Translation and the Electrophoretic Gel Mobility Shift Assay (EMSA)—In vitro transcription/translation of HA-hDREF was performed using the TNT T7 Quick Coupled Transcription/Translation system (Promega) according to the manufacturer's protocol. EMSA was performed as described previously (12) using in vitro synthesized HA-hDREF proteins.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The hATC Domain-mediated Self-association of hDREF in Vivo—Inspection of the hDREF amino acid sequence by BLAST search revealed correspondence of the region spanning aa 571-651 near the carboxyl terminus with a hATC domain (Fig. 1, A and B). Because hATC domains of Activator and Hermes have been shown to be involved in dimerization or multimerization (18, 19), we first examined whether the hATC domain of hDREF could bind to itself in vitro and in vivo.To examine in vitro self-association of the hATC domain, we performed a GST pulldown assay (Fig. 1C). Immunoblotting with anti-MBP antiserum revealed that purified MBP-hDREF (aa 567-651), but not MBP, was pulled down by GST-hDREF (aa 567-651) (lanes 3 and 6), indicating that the hATC domain could bind to itself in vitro. To examine in vivo self-association of the hATC domain, we performed an in vivo cross-linking experiment using HeLa cells expressing HA-tagged the region containing the hATC domain (Fig. 1D). A signal of HA-hATC in a whole cell extract without cross-linking was only detected at the position corresponding to a monomer (lane 1). In a whole cell extract with cross-linking, we observed two bands at the positions corresponding to a monomer and a dimer of HA-hATC (lane 2). Thus, we concluded that the hATC domain of hDREF could self-associate by itself in vitro and in vivo. We next examined whether full-length hDREF self-associates in vivo. Whole cell extracts from HeLa cells simultaneously overexpressing HA-hDREF and YFP-fused hDREF (YFP-hDREF) were subjected to immunoprecipitation with anti-HA antibodies, and the immunoprecipitates were resolved in SDS-PAGE followed by Western blotting using anti-GFP and anti-HA antibodies (Fig. 1E). Immunoblotting with anti-HA antibodies revealed that the amounts of immunoprecipitated HA-hDREF mutants were comparable with that of the wild type (lower panels in lanes 5-8 and 15-20). Immunoblotting with anti-GFP antibodies revealed that the wild-type YFP-hDREF was immunoprecipitated together with the wild-type HA-hDREF (lane 5), indicating that full-length hDREF self-associates in vivo.

We next investigated whether the hATC domain is necessary for the self-association. A whole cell extract from HeLa cells simultaneously overexpressing wild-type YFP-hDREF and HA-hDREF ({Delta}hATC), the latter being a mutant lacking the hATC domain (Fig. 1B), was subjected to immunoprecipitation with anti-HA antibodies. Immunoblotting with anti-GFP antibodies revealed that the wild-type YFP-hDREF did not co-immunoprecipitate with the HA-hDREF ({Delta}hATC) (Fig. 1E, lane 6), indicating that hATC domain is required for the self-association.

Several amino acid residues are highly conserved among the hATC domains of proteins belonging to the hAT transposase family (hobo, Activator, Tam3, Hermes, ZBED4/KIAA0637, and hDREF) (Fig. 1A). To examine whether these highly conserved amino acids are involved in hDREF self-association, we constructed a series of base-substitution mutants of pcDNA3-HA-hDREF, including amino acid substitutions with alanine for residues Trp-590 and Trp-591 (W590A/W591A), Pro-599 (P599A), Leu-600 and Leu-601 (L600A/L601A), Val-604 and Leu-605 (V604A/L605A), Leu-600, Leu-601, Val-604, and Leu-605 (LLVL/AAAA), Glu-619 and Arg-620 (E619A/R620A), Phe-622 (F622A), and Arg-633 (R633A) (Fig. 1B). Immunoprecipitation was performed with anti-HA antibodies against whole cell extracts from HeLa cells simultaneously overexpressing one of these HA-hDREF mutants and wild-type YFP-hDREF. Immunoblotting with anti-GFP antibodies revealed that the wild-type YFP-hDREF did not co-immunoprecipitate with the W590A/W591A, LLVL/AAAA, or L600A/L601A HA-hDREF mutants (Fig. 1E, lanes 7, 8, and 16). The V604A/L605A HA-hDREF mutant weakly associated with the wild-type YFP-hDREF (lane 17), whereas other HA-hDREF mutants associated with the wild-type YFP-hDREF at comparable levels with that of the wild-type HA-hDREF (lanes 15 and 18-20). These results indicate that the conserved hydrophobic amino acids in hATC domain (Trp-590, Trp-591, and Leu-601) are critical for hDREF self-association.


Figure 1
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  		FIGURE 1.
The hATC domain is required for in vivo self-association and in vitro multimerization of hDREF. A, alignment of hATC domains was accomplished using Swiss-Prot accession numbers: hobo, P12258; Activator, P08770; Tam3, Q38743; Hermes, Q25438; ZBED4/KIAA0637, O75132; hDREF, O96006. Conserved amino acids characterized in this study are shown with large letters. Amino acids located in the interaction interface of Hermes are shown with underlining. The consensus amino acid sequence of the hATC domain (consensus) is also shown. B, schematic representation of the hDREF domain structure (BED and hATC). The hDREF mutants are also listed. C, GST pulldown assay. Glutathione-Sepharose beads bound to GST (GST) or GST-hDREF (aa 567-651) (GST-hATC) was incubated with MBP (MBP) or MBP-hDREF (aa 567-651) (MBP-hATC) as indicated. Proteins bound to the beads were recovered, resolved by SDS-PAGE, and then analyzed by immunoblotting with anti-MBP antiserum (IB, {alpha}MBP, upper panel) or Coomassie Brilliant Blue staining (CBB, lower panel). Input indicates 20% input of the purified MBP and MBP-hDREF (aa 567-651) used for GST pull down. D, HeLa cells in 6-cm dish were transfected with 12 µg of pcDNA3-HA-hDREF (aa 567-651). One-third of the collected cells was treated with Me2SO (-) or with a cross-linker, disuccinimidyl suberate (DSS), at a final concentration of 0.17 mg/ml (+), respectively. The protein samples from the cross-linked cells were analyzed by immunoblotting with anti-HA antibodies. A schematic representation of HA-hDREF (aa 567-651) is also shown. E, HeLa cells in 6-cm dishes were co-transfected with 6 µg each of wild type, hATC domain-lacking ({Delta}hATC), or amino acid substitution mutants of pcDNA3-HA-hDREF (HA-hDREF) and pEYFP-hDREF (YFP-hDREF) as indicated, and immunoprecipitation using anti-HA antibodies was performed (IP, {alpha}HA). The 5% input and immunoprecipitates were analyzed by immunoblotting with anti-GFP antibodies ({alpha}GFP, upper panels). The same membrane was treated with a stripping buffer (62.5 mM Tris-HCl (pH 6.9), 2% SDS, and 100 mM beta-mercaptoethanol) and then examined by immunoblotting with anti-HA antibodies ({alpha}HA, lower panels). F, HA-hDREF was synthesized in vitro in the presence of [35S]methionine/cysteine. Radiolabeled hDREF proteins (wild type, W590A/W591A, or LLVL/AAAA mutants) in 25 mM Hepes (pH 7.9), 15% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1% Triton X-100, and 100 mM NaCl were resolved by BN-PAGE and detected by autoradiography. Mock denotes the in vitro transcription/translation reaction without template. The open and filled arrowheads indicate bands for the wild type and two substitution mutants of HA-hDREF, respectively.

 


Figure 2
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  		FIGURE 2.
hDREF exists as higher molecular weight complexes in vivo. A, a whole cell extract (80 µg) of asynchronous HeLa cells was subjected to two-dimensional BN-PAGE/SDS-PAGE and analyzed by immunoblotting using anti-hDREF antibodies. The open arrowhead indicates a single spot at 220 kDa. B, indirect immunofluorescence experiments were carried out with HeLa cells at 14 h (Interphase) or 9 h (mitosis) after release from the second thymidine block. Endogenous hDREF was stained with the anti-hDREF primary and Alexa fluor 488-conjugated secondary antibodies (hDREF), and DNA was stained with PI (PI). The individual stages within mitosis were selected according to the chromosomal morphology. Typical confocal microscopic images are shown. Bar in hDREF panels, 10 µm. C, whole cell extracts (25 µg) of asynchronous HeLa cells (Asynchronous) and HeLa cells at 9 h after release from the second thymidine block (Synchronous) were mixed with SDS-PAGE sample buffer and then boiled at 100 °C for 5 min. These protein samples were analyzed by immunoblotting (IB) using anti-hDREF (top panel) and anti-phosphorylated histone H3 (Ser-10) antibodies (middle panel), indicative of mitosis. Amounts of proteins loaded were confirmed by Ponceau staining of the membrane (bottom panel). D, a whole cell extract (80 µg) of HeLa cells at 9h after release from the second thymidine block was subjected to two-dimensional BN-PAGE/SDS-PAGE and analyzed by immunoblotting with the anti-hDREF antibodies. Open and filled arrowheads indicate spots at 220 and 170 kDa, respectively.

 
hDREF Multimerization in Vitro—Next, we examined whether full-length hDREF can form multimers. In vitro synthesized, 35S-labeled hDREF was analyzed using BN-PAGE (Fig. 1F) (26, 27), allowing rough separation of native protein complexes on the basis of size (28). We observed a signal for wild-type hDREF at a position corresponding to 200 kDa (lane 2), likely representing a dimer because hDREF is a 80 kDa polypeptide (12). Signals at the positions of 500 and 670 kDa and additional higher molecular weight bands were also observed (lane 2). This result suggests that full-length hDREF can form multimers in vitro. In contrast, BN-PAGE analysis with in vitro synthesized W590A/W591A and LLVL/AAAA mutants, which failed to self-associate in vivo, exhibited only one major band at the position of 120 kDa (lanes 3 and 4), likely representing the monomer of hDREF. These data suggest that the conserved hydrophobic amino acids in the hATC domain are responsible for full-length hDREF multimerization in vitro, consistent with the in vivo self-association data (Fig. 1E).

Endogenous hDREF Exists As Higher Molecular Weight Complexes in Vivo—To gain insight into hDREF-containing protein complexes in vivo under native conditions, we performed two-dimensional BN-PAGE/SDS-PAGE with a whole extract from asynchronous HeLa cells (Fig. 2A). Immunoblotting with anti-hDREF antibodies revealed a broad band in the region larger than 440 kDa and a single spot at the position of 220 kDa, suggesting that endogenous hDREF in fact exists as higher molecular weight complexes in asynchronous cells.

Immunofluorescence analysis using anti-hDREF antibodies revealed nuclear accumulation of endogenous hDREF in interphase HeLa cells, whereas endogenous hDREF was absent from condensed chromosomes during mitosis (Fig. 2B; also see Ref. 12). Accordingly, we focused on whether protein complexes containing hDREF vary in size and composition during the cell cycle. Immunoblotting using anti-phosphorylated histone H3 (Ser-10) antibodies revealed the degree of phosphorylation of Ser-10 of histone H3, a hallmark of mitosis, to be much higher in cells at 9 h after release from the second thymidine block than in asynchronous cells (Fig. 2C, middle panel), indicating a large population of synchronized cells to be undergoing mitosis. Immunoblotting with anti-hDREF antibodies showed that almost the same amounts of hDREF were expressed between the asynchronous and synchronous HeLa cells (top panel). When two-dimensional BN-PAGE/SDS-PAGE using a whole extract from synchronous HeLa cells was performed (Fig. 2D), a single spot at a position of 220 kDa was comparable with that in asynchronous cells (Fig. 2A), whereas the broad band signals in regions over 440 kDa were significantly diminished, and a new spot appeared at the position of 170 kDa (Fig. 2D). These results indicate that the compositions of higher molecular weight complexes containing hDREF dynamically change in a cell cycle-dependent manner, although we do not know at present the identities of the two spots at positions 170 and 220 kDa.


Figure 3
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  		FIGURE 3.
The hATC domain is required for nuclear accumulation of hDREF. A, wild type, hATC domain-lacking ({Delta}hATC), or substitution mutants of pcDNA3-HA-hDREF (400 ng) were transfected into HeLa cells in 12-well plates, and expressed HA-tagged proteins were visualized by immunofluorescence using anti-HA primary and Alexa fluor 594-conjugated secondary antibodies. The immunofluorescence images shown in panels a-j are representative of 80, 70, 95, 85, 80, 80, 90, 90, 80, and 90% of the cells observed under the IX-70 microscope, respectively (magnification, 600x). B, HeLa cells in 10-cmdishes were transfected with 24 µg of wild type or W590A/W591A mutants of pcDNA3-HA-hDREF (HA-hDREF). One-third of each collected lysate was immunoprecipitated (IP) using normal goat IgG or anti-importin beta1 antibodies, and the 5% input and immunoprecipitates were analyzed by immunoblotting (IB) with anti-HA antibodies (upper panel). The same membrane was stripped as for Fig. 1E and then examined by immunoblotting with anti-import in beta1 antibodies (lower panel). C, HeLa cells in 12-well plates were co-transfected with 2.5 µg each of phGFP105-C1 (GFP) or phGFP105-RanQ69L (GFP-RanQ69L) and 0.5 µg of wild type of pcDNA3-HA-hDREF. HA-hDREF was visualized by immunofluorescence using anti-HA primary and Alexa fluor 594-conjugated secondary antibodies. The immunofluorescence images of HA-hDREF shown in panels a and d are representative of 80 and 65% of the cells observed under the IX-70 microscope, respectively (magnification, 600x).

 
The hATC Domain Is Necessary for hDREF Nuclear Accumulation—We previously reported that hDREF is predominantly accumulated in the nucleus and associated with granular structures (12). We, hence, investigated whether hDREF self-association is involved in the nuclear accumulation and the granular pattern formation. Wild-type HA-hDREF and its mutants listed in Fig. 1B were expressed in HeLa cells, and subcellular distributions of the proteins were examined by immunofluorescence microscopy with anti-HA antibodies (Fig. 3A). Wild-type HA-hDREF was mainly distributed in the nucleus with a granular pattern (panel a). It should be noted that the subcellular distribution of full-length hDREF was assessed with cells expressing hDREF at moderate expression levels, because wild-type hDREF tends to form large granules in the cytoplasm when expressed at higher levels (data not shown). The deletion mutant lacking the hATC domain ({Delta}hATC) and substitution mutants W590A/W591A, L600A/L601A, and LLVL/AAAA exhibited diffuse cytoplasmic distribution (panels b, c, e, and g). In contrast, the V604A/L605A mutant was detected both in the nucleus with a granular pattern and in the cytoplasm with diffuse distribution (panel f). Other mutants were accumulated in the nucleus with a granular pattern similar to the wild type (panels d, h, i, and j). Hence, both the efficiencies of nuclear accumulation and granular pattern formation proved to be closely correlated with the hDREF self-association level (Fig. 1E).

The above-described results raise the question of whether hDREF self-association is required for efficient nuclear import. To answer this, we examined the association between hDREF and importin beta1, a major component of the nuclear import complex (for a review, see Ref. 29). Whole cell extracts from HeLa cells overexpressing wild-type HA-hDREF or its W590A/W591A mutant were subjected to immunoprecipitation with anti-importin beta1 antibodies. Immunoblotting with anti-HA antibodies revealed the wild-type HA-hDREF to immunoprecipitate together with endogenous importin beta1 (Fig. 3B, lane 3), whereas negligible amounts of W590A/W591A mutant associated with endogenous importin beta1 (lane 6). To confirm that hDREF nuclear import is indeed dependent on importin beta, we investigated the effect of a dominant-negative mutant of Ran deficient in GTPase activity (RanQ69L) on the nuclear accumulation of HA-hDREF because Ran-GTP is a form that normally prevents importin beta action within the cell (30-32). Immunofluorescence analysis showed that the nuclear accumulation of HA-hDREF was perturbed by co-expression of GFP-tagged RanQ69L mutant (Fig. 3C, panel d). These results suggest that hATC domain-mediated hDREF self-association is necessary for efficient nuclear import mediated by importin beta1.


Figure 4
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  		FIGURE 4.
Analysis of the subcellular distribution of truncated hDREF mutants. A, HeLa cells in 12-well plates were transfected with 400 ng of wild type (1-694, panel a) or carboxyl-terminally truncated mutants (panels b-e) of pcDNA3-HA-hDREF. Expressed HA-tagged proteins were visualized by immunofluorescence using anti-HA primary and Alexa fluor 594-conjugated secondary antibodies. The immunofluorescence images shown in panels a-e are representative of 80, 80, 90, 80, and 80% of the cells observed under the IX-70 microscope, respectively (magnification, 600x). A schematic representation of the HA-hDREF constructs used is also given. B, wild type (1-694, panel a) or truncated mutants (panels b and c) of pEYFP-hDREF (400 ng) were transfected into HeLa cells in 12-well plates. The fluorescence images of expressed YFP-fused proteins shown in panels a-c are representative of 90, 90, and 90% of the cells observed under the IX-70 microscope, respectively (magnification, 600x). A schematic representation of the YFP-hDREF constructs used is also given. C, wild type (panel b) of pEYFP-hDREF (aa 567-651) or pEYFP-C1 (panel a) (6 µg) were transfected into HeLa cells in 6-well plates, and the subcellular distributions of the expressed proteins are analyzed in living cells. The fluorescence images of expressed YFP-fused proteins shown in panels a and b are representative of 100 and 70% of the cells observed under the IX-70 microscope, respectively (magnification, 200x). A schematic representation of YFP-hDREF (aa 567-651) is also given. D, HeLa cells in 6-cm dishes were co-transfected with 6 µg each of wild type or carboxyl-terminal-truncated mutants of pcDNA3-HA-hDREF (HA-hDREF) and wild-type pEYFP-hDREF (YFP-hDREF) as indicated. Immunoprecipitation and immunoblotting (IB) were performed as for Fig. 1E.

 
Analysis of the Subcellular Distribution of Truncated hDREF Mutants—We further investigated the role of hDREF carboxylterminal region containing the hATC domain in nuclear accumulation and granular pattern formation using a series of the truncation mutants. Wild-type HA- or YFP-hDREF or their truncation mutants were expressed in HeLa cells, and the subcellular distributions were examined (Fig. 4, A-C). Full-length HA- or YFP-hDREF (aa 1-694) and a carboxyl-terminal-truncated HA-hDREF (aa 1-651) were mainly accumulated in the nucleus with granular patterns (Fig. 4, A, panels a and b, and B, panel a). HA- or YFP-hDREF (aa 1-624), which was further truncated in the carboxyl-terminal region (aa 625-651) of the hATC domain, showed diffuse nuclear accumulation (Fig. 4, A, panel c, and B, panel b), suggesting that the region spanning aa 625-651 is required for granular pattern formation. HA-hDREF (aa 1-551) completely deleted for the hATC domain exhibited diffuse distribution both in the nucleus and the cytoplasm (Fig. 4A, panel d), whereas HA-hDREF (aa 1-523) was diffusely distributed predominantly in the cytoplasm (panel e), suggesting that the region spanning aa 524-551 is necessary for nuclear localization of hDREF. YFP-hDREF (aa 106-624), lacking the BED zinc finger domain that proved to be a DNA binding domain of hDREF (data not shown), was localized in the nucleus (Fig. 4B, panel c), suggesting that hDREF DNA-binding activity itself is not necessary for nuclear accumulation.

To examine whether the hATC domain is sufficient for nuclear accumulation, we analyzed the in vivo distribution of YFP attached with the region spanning aa 567-651 of hDREF (YFP-hDREF (aa 567-651)) containing the hATC domain (aa 571-651) (Fig. 4C). The wild-type YFP-hDREF (aa 567-651) exhibited a large cytoplasmic granule-like pattern around the nucleus (panel b), whereas YFP protein itself was evenly distributed in the nucleus and the cytoplasm (panel a). Thus, the hATC domain itself is necessary but not sufficient for nuclear accumulation.

The distribution analysis using these truncation mutants suggests that the amino-terminal region (aa 571-624) and the carboxyl-terminal region (aa 625-651) of the hATC domain are necessary for nuclear accumulation and granular pattern formation, respectively. We, hence, examined the relationship between cellular distribution and in vivo self-association using these mutants. Whole cell extracts from HeLa cells simultaneously overexpressing one of the carboxyl-terminal-truncated HA-hDREF mutants used for Fig. 4A and YFP-hDREF were subjected to immunoprecipitation with anti-HA antibodies (Fig. 4D). Immunoblotting with anti-GFP antibodies revealed that the wild-type YFP-hDREF immunoprecipitated together with the HA-hDREF (aa 1-651) and HA-hDREF (aa 1-624), the latter exhibiting weaker association than the former (lanes 6 and 7), whereas HA-hDREF (aa 1-551) did not (lane 8). Taken together, these results indicate that the amino-terminal region (aa 571-624) of hATC domain is necessary for self-association and nuclear accumulation and that the carboxyl-terminal region (aa 625-651) is necessary for efficient self-association and granular pattern formation.


Figure 5
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  		FIGURE 5.
Identification of a NLS in hDREF. A, a 400-ng aliquot of K530A/K531A/R534A mutant of pcDNA3-HA-hDREF (HA-hDREF, aa 1-694) was transfected into HeLa cells in 12-well plates. The expressed mutant protein was visualized by immunofluorescence using anti-HA primary and Alexa fluor 594-conjugated secondary antibodies ({alpha}HA), and DNA was stained with Hoechst dye (Hoechst). A representative immunofluorescence image of 80% of cells studied under the IX-70 microscope is shown (magnification, 600x). Alignment of amino acid sequences of the region spanning aa 520-551 of the wild type and the K530A/K531A/R534A mutant is also shown. B, HeLa cells in 12-well plates were transfected with 400 ng of pEGFP-GST (GFP-GST, panel a), wild-type pEGFP-GST-hDREF (GFP-GST-hDREF, aa 520-551), or its K530A/K531A/R534A mutant (panels b and c). The fluorescence images of expressed GFP-fused proteins shown in panels a-c are representative of 80, 90, and 80% of the cells observed under the IX-70 microscope, respectively (magnification, 600x). C, HeLa cells in 6-cm dishes were co-transfected with 6 µg each of the wild type or the K530A/K531A/R534A mutant of pcDNA3-HA-hDREF (HA-hDREF) and wild-type pEYFP-hDREF (YFP-hDREF) as indicated. Immunoprecipitation (IP) and immunoblotting (IB) were performed as for Fig. 1E.

 
The Nuclear Localization Signal of hDREF—Based on the indication that the region spanning aa 524-551 is required for nuclear localization of hDREF (Fig. 4A, panels d and e), we investigated whether it acts as a nuclear localization signal (NLS). By inspecting the amino acid sequence, we found a cluster of basic amino acids at positions Lys-530, Lys-531, and Arg-534 (Fig. 5A), resembling the classical NLS, which is a cluster of basic amino acid residues in the form, K(K/R)X(K/R) (33-36). To examine whether this cluster functions as an NLS, we replaced these basic residues with alanine in HA-hDREF (K530A/K531A/R534A) and expressed the protein in HeLa cells. Immunofluorescence analysis revealed cytoplasmic distribution with a granular pattern (Fig. 5A), indicating the clustered basic amino acids are necessary for the nuclear targeting of full-length hDREF. We next examined whether this region is sufficient to direct a non-nuclear protein to the nucleus. HeLa cells were transfected with a plasmid encoding GFP-GST heterologous protein fused to hDREF (aa 520-551) and its mutated version, and the subcellular distribution of these fusion proteins was assessed (Fig. 5B). GFP-GST itself showed a cytoplasmic distribution (panel a; also see Ref. 21). However, GFP-GST-hDREF (aa 520-551, wild type) was localized in the nucleus (panel b), whereas the K530A/K531A/R534A mutant was not (panel c). Taken together, these results indicate that the cluster of basic amino acid residues (Lys-530, Lys-531, and Arg-534) within aa 520-551 is a bona fide classical NLS of hDREF.

Next, we determined whether the NLS is required for hDREF self-association by immunoprecipitation with anti-HA antibodies using whole cell extracts from HeLa cells simultaneously overexpressing the K530A/K531A/R534A mutants of HA-hDREF and wild-type YFP-hDREF. Immunoblotting with anti-GFP antibodies revealed that the K530A/K531A/R534A HA-hDREF mutant associated with the wild-type YFP-hDREF (Fig. 5C, lane 4), indicating that the NLS does not contribute to hDREF self-association.

The hATC Domain Is Necessary for DNA Binding of hDREF in Vitro—The BED zinc finger domain is thought to be involved in DNA binding of chromatin boundary element-binding proteins and transposases (for a review, see Ref. 15). hDREF contains a BED zinc finger domain at the amino terminus (aa 23-72; Fig. 1B). EMSA revealed that GST-hDREF (aa 1-140) binds to the hDRE of the histone H1 gene (data not shown), indicating that this region is sufficient for specific DNA binding in vitro. Because our previous study showed that dDREF probably binds to dDRE as a homodimer (1), we hypothesized that hATC domain-mediated multimerization is required for DNA binding of full-length hDREF. We performed EMSA using 32P-labeled oligonucleotide containing hDRE of the histone H1 gene and in vitro synthesized hDREF (Fig. 6). Three shifted bands were detected when wild-type hDREF protein was mixed with the H1 hDRE probe (lane 2, filled and open arrowheads), whereas the hATC domain-deleted ({Delta}hATC), W590A/W591A, or LLVL/AAAA mutants completely lost the DNA binding activity (lanes 6-8). The HA-hDREF (aa 1-651) and HA-hDREF (aa 1-624) bound to the hDRE (lanes 3 and 4), whereas HA-hDREF (aa1-551) did not (lane 5). It should be noted that two slowly migrated bands (open arrowheads) were detected only when wild-type hDREF was used. These results indicate that hATC domain is necessary for DNA binding of hDREF in vitro and suggest that the region spanning aa 652-694 is involved in formation of high order complexes consisting hDREF and the binding sequences.

The hATC Domain of ZBED4/KIAA0637 Is Also Necessary for Self-association and Efficient Nuclear Accumulation—An important question raised by the above results is whether the hATC domain is generally required for nuclear accumulation of human hAT family proteins. We, therefore, studied another hAT family protein, ZBED4/KIAA0637, structurally belonging to this transposase family (22, 37) and containing four BED zinc finger domains (aa 118-166, 288-336, 459-506, and 561-609) and a hATC domain (aa 1086-1164), based on BLAST search (Fig. 7C). Immunofluorescence analysis using anti-ZBED4/KIAA0637 antibodies in HeLa cells showed localization in the nucleus in interphase cells (Fig. 7A) and absence from chromosomes during mitosis (data not shown). Immunoprecipitation with anti-HA antibodies performed against whole cell extracts from HeLa cells co-expressing HA-ZBED4/KIAA0637 and YFP-ZBED4/KIAA0637 (Fig. 7B) and immunoblotting with anti-GFP antibodies showed the wild-type YFP-ZBED4/KIAA0637 to immunoprecipitate together with the wild-type HA-ZBED4/KIAA0637 (lane 3), whereas the YFP-ZBED4/KIAA0637 ({Delta}hATC), a mutant lacking the hATC domain, did not (lane 4), indicating that ZBED4/KIAA0637 also self-associates, and the association depends on the hATC domain. Fluorescence microscopic analysis (Fig. 7C) further revealed that both wild-type HA- and YFP-ZBED4/KIAA0637 were accumulated in the nucleus (panels a and c). In contrast, hATC domain-deleted mutants ({Delta}hATC) of HA- and YFP-ZBED4/KIAA0637 were evenly distributed both in the nucleus and cytoplasm in 30 and 50% of cells examined, respectively (panels b and d), but accumulated in the nucleus in the remaining cells (data not shown), indicating that the hATC domain is required for efficient nuclear accumulation of ZBED4/KIAA0637. These results suggest that the role of the hATC domain in nuclear accumulation is conserved between hDREF and ZBED4/KIAA0637.


Figure 6
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  		FIGURE 6.
The hATC domain is necessary for DNA binding of hDREF. EMSA was performed using 32P-labeled hDRE of histone H1 gene as a probe and in vitro synthesized HA-hDREF. The filled, opened arrowheads and asterisks indicate a main band, two slowly migrated bands, and a nonspecific band, respectively. Wild-type (wild type (1-694)), carboxyl-terminal-truncated mutants (1-651, 1-624, or 1-551), hATC domain-lacking ({Delta}hATC), or substitution mutants (W590A/W591A or LLVL/AAAA) of pcDNA3-HA-hDREF or pcDNA3-HA (HA) was used.

 
Given that both hDREF and ZBED4/KIAA0637 self-associate via their hATC domains (Figs. 1E and 7B), we examined whether they can heterotypically interact. Immunoprecipitation was performed against a whole cell extract from HeLa cells co-expressing HA-hDREF and YFP-ZBED4/KIAA0637. The wild-type YFP-ZBED4/KIAA0637 was not immunoprecipitated together with the wild-type HA-hDREF (Fig. 7D, lane 4), indicating no direct association.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this work we studied the functions of hATC domain by detailed mutagenesis analyses and found that highly conserved hydrophobic amino acids (Trp-590, Trp-591, and Leu-601) among hATC domains of hAT family members are essential for hDREF self-association in vivo. Importantly, the results using the substitution and truncated mutants provide evidence that the hATC domain (aa 571-651) consists of two functionally distinct regions; (i) the amino-terminal region (aa 571-624) is essential for hDREF self-association, nuclear accumulation, and DNA binding, (ii) the carboxyl-terminal region (aa 625-651) is involved in the efficient self-association (or stabilization of self-association) and granular pattern formation. Moreover, we identified the region spanning aa 652-694 as a region to form high order complexes of hDREF with the binding sequences and a classical form of NLS within the region spanning aa 520-551.

Among the hydrophobic amino acids (Trp-590, Trp-591, and Leu-601) essential for hDREF self-association, Leu-601 corresponds to Leu-554 of Hermes, which is critical for the homotypic interaction of this protein as assessed by yeast two-hybrid assay (19). However, amino acid substitutions with alanine for Trp-690 and Trp-691 of Activator, which correspond to Trp-590 and Trp-591 of hDREF, did not affect dimer formation (18). Even though conserved, the roles of these amino acid residues might differ in different proteins. It is also be possible that the different experimental conditions employed in these studies (immunoprecipitation in vivo versus cross-linking in vitro, full-length protein versus partial polypeptides, etc.) affected the results. An interesting pointer can be gleaned from the recently determined Hermes crystal structure (38); the hydrophobic amino acids essential for hDREF self-association (Trp-590, Trp-591, and Leu-601) correspond to the residues (Trp-543, Trp-544, and Leu-554) located in the hydrophobic core close to the interaction interface of Hermes (Fig. 8). This raises the possibility that impairment of hDREF self-association by the above mutations is due to a structural change in the interaction interface caused by defective formation of the hydrophobic core. Moreover, the fact that hDREF and ZBED4/KIAA0637 do not interact with each other (Fig. 7D), suggests that each protein specifically self-associates via its hATC domain. Interestingly, the amino acids located in the interface of Hermes (Glu-530, Asp-537, Leu-546, Lys-549, and Lys-550) (38) are not conserved among the hAT family (Figs. 1A and 8). More details of mechanism of self-association of hDREF should be provided by identification of the amino acids constituting the interaction interface by x-ray crystallography.


Figure 7
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  		FIGURE 7.
Analysis of the ZBED4/KIAA0637 protein. A, indirect immunofluorescence experiments were carried out with HeLa cells. Endogenous ZBED4/KIAA0637 was stained with the anti-ZBED4/KIAA0637 primary and Alexa fluor 488-conjugated secondary antibodies (ZBED4) and DNA with PI (PI). Typical confocal microscopic images are shown. Bar in the ZBED4 panel, 10 µm. B, HeLa cells in 10-cm dishes were co-transfected with 16 µg of pcDNA3-HA-ZBED4/KIAA0637 (HA-ZBED4) and 8 µg of pEYFP-ZBED4/KIAA0637 (YFP-ZBED4) as indicated. At 48 h after transfection, immunoprecipitation (IP) and immunoblotting (IB) were performed as for Fig. 1E. C, wild type or the hATC domain-lacking mutant ({Delta}hATC) of pcDNA3-HA-ZBED4/KIAA0637 (HA-ZBED4) or pEYFP-ZBED4/KIAA0637 (YFP-ZBED4) (400 ng) was transfected into HeLa cells in 12-well plates. Expressed HA-ZBED4/KIAA0637 proteins were visualized by immunofluorescence using anti-HA primary and Alexa fluor 594-conjugated secondary antibodies (panels a and b). The fluorescence images shown in panels a-d are representative of 100, 30, 100, and 50% of the cells observed under the IX-70 microscope, respectively (magnification, 600x). A schematic representation of ZBED4/KIAA0637 domain structure is also shown. D, HeLa cells in 6-cm dishes were co-transfected with 3 µg of pcDNA3-HA-hDREF (HA-hDREF) and 9 µg of pEYFP-ZBED4/KIAA0637 (YFP-ZBED4) as indicated. Immunoprecipitation and immunoblotting were performed as for Fig. 1E.

 


Figure 8
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  		FIGURE 8.
The conserved hydrophobic amino acids are located in the hydrophobic core close to the interaction interface of Hermes. The hATC domain (aa 523-604) is shown in blue. The amino acids involved in the hydrophobic core (W543, W544, and L554) and in the direct interaction (E530, D537, L546, K549, and K550), and the conserved amino acid, P552, are shown in orange, gray, and red, respectively.

 
Several subnuclear structures microscopically observed in granular patterns, such as Cajal, promyelocytic leukemia, and polycomb bodies, have been well described (39). Although they consist of complex arrays of proteins, a common feature of individual core proteins is self-association; e.g. Coilin for Cajal bodies (40), promyelocytic leukemia (PML) for PML bodies (41), and RING1 and BMI1 for polycomb bodies (42). Although we cannot determine at present whether hDREF associates with any of the above structures or forms granular structures as a core protein, the close correlation between levels of self-association and the efficiency of granular pattern formation (Figs. 1E, 3A, 4A, and 4D) strongly indicates that hDREF possesses a common feature of core proteins. Interestingly, the NLS-deficient hDREF mutant that retains the hATC domain intact (Fig. 5A) and even the hATC domain by itself (Fig. 4C) exhibited granular patterns in the cytoplasm, implying an importance of hATC domain-mediated self-association for this distribution pattern, although of course the protein compositions of the nuclear and cytoplasmic granules might differ. We speculate that nuclear granular structures containing hDREF would be formed by self-association of hDREF into a core and further association with other proteins. Because endogenous hDREF exists as high molecular weight complexes (Fig. 2), elucidation of the protein composition of hDREF-containing granules should help clarify the functions of such structures as well as hDREF itself.

The correlation between self-association and nuclear accumulation of hDREF and the linkage with importin beta1 strongly suggest that self-association via hATC domain is required for nuclear accumulation of hDREF and that hDREF self-associates before nuclear import (Figs. 1E and 3). The mechanisms underlying formation of cargo/importin {alpha}-importin beta complexes by proteins containing a classical NLS and translocation into the nucleus are well known (29, 43). Our NLS-deficient hDREF construct, which failed to localize into the nucleus (Fig. 5A), however, bound to endogenous importin beta1 at a comparable level with the wild-type HA-hDREF,4 suggesting that only importin {alpha} to hDREF NLS has an essential role in hDREF nuclear import. The importin {alpha} family of receptors of classical NLS comprises many members (43). It is an intriguing question which form(s) might interact with hDREF NLS and mediate nuclear import of the protein in conjunction with importin beta1. Interestingly, the hATC domain of ZBED4/KIAA0637 is also required for efficient nuclear accumulation (Fig. 7C), indicating conservation of this role.

It is generally believed that all the human hAT transposases are currently inactive (37, 44), and indeed, Asp-248 in the DDE motif essential for transposase activity of Hermes (38, 45) is not conserved in hDREF. It has been speculated that the human hAT family including hDREF plays a role in gene expression and genome stability (14). Because EMSA showed that hDREF binds to the hDREs as some high order complexes in vitro (Fig. 6), a more detailed study for hDREF binding manner to hDREs in vitro and in vivo should be addressed in future. In conclusion, the present study identified the highly conserved hATC domain as the region for self-association, nuclear accumulation, DNA binding, and granular pattern formation, indicating that future detailed functional analysis of the human hAT family members is warranted.


   FOOTNOTES
 
* This work was supported by grants-in-aid for Scientific Research from the Japan Society for the Promotion of Science and the 21st Century Centers of Excellence Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by a Research Fellowship of Japan Society for the Promotion of Science for Young Scientists. Back

2 To whom correspondence should be addressed. Tel.: 81-791-58-0194; Fax: 81-791-58-0193; E-mail: fhirose{at}sci.u-hyogo.ac.jp.

3 The abbreviations used are: dDREF, Drosophila DNA replication-related element binding factor; hDREF, human DREF; CR, conserved region; hAT, hobo, Activator, and Tam3; CDD, conserved domain database; aa, amino acids; HA, hemagglutinin; YFP, yellow fluorescent protein; GFP, green fluorescent protein; GST, glutathione S-transferase; MBP, maltose-binding protein; PBS, phosphate-buffered saline; BN, blue native; EMSA, electrophoretic gel-mobility shift assay; NLS, nuclear localization signal. Back

4 D. Yamashita and F. Hirose, unpublished data. Back


   ACKNOWLEDGMENTS
 
We are grateful to Drs. Masashi Okada (Emory University School of Medicine), Hitoshi Yagisawa, Yasuhiro Kashino, and Yohei Ikeda (University of Hyogo) for valuable discussion, providing reagents, and supporting techniques, Dr. Osamu Ohara (Kazusa DNA Research Institute) for ZBED4/KIAA0637 cDNA, Dr. Naoko Imamoto (Riken) for RanQ69L cDNA, and Dr. Malcolm Moore for critical reading of the manuscript.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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