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J. Biol. Chem., Vol. 280, Issue 37, 32090-32100, September 16, 2005

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Different Roles of N-terminal and C-terminal Halves of HIRA in Transcription Regulation of Cell Cycle-related Genes That Contribute to Control of Vertebrate Cell Growth^*

Ahyar Ahmad, Hidehiko Kikuchi, Yasunari Takami¶||, and Tatsuo Nakayama¶||1

From the Department of Life Science, Frontier Science Research Center, ^¶Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Miyazaki Medical College, University of Miyazaki, 5200, Kihara, Kiyotake, Miyazaki 889-1692, Japan, ^||CREST, Japan Science and Technology, Kazusa, Kisarazu, Chiba 292-0818, Japan, and the Department of Chemistry, Hasanuddin University, Makassar 90245, Indonesia

Received for publication, February 7, 2005 , and in revised form, July 12, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
We reported previously that chicken HIRA, a homolog of Saccharomyces cerevisiae transcriptional co-repressors Hir1p and Hir2p, possesses seven WD dipeptide motifs and an LXXLL motif in its N-terminal and C-terminal halves, respectively, required for transcription regulations. Here, by using the gene targeting technique, we generated the homozygous HIRA-deficient DT40 mutant HIRA. The HIRA deficiency caused slightly delayed cell growth and affected the opposite transcriptions of cell cycle-related genes, i.e. repressions for P18, CDC25B, and BCL-2, activations for P19 and cyclin A, and histones H2A, H2B, H3, and H4. These altered expressions were completely revived by the artificial stable expression of hemagglutinin-tagged HIRA in HIRA. The ability to rescue the delayed growth rate was preferentially aided by the N-terminal half instead of the C-terminal half. We cloned the chicken P18 genomic DNA, and we established that its promoter was located surrounding the sequence GCGGGCGC at positions –1157 to –1150. Chromatin immunoprecipitation assay revealed that the N-terminal half interacted directly or indirectly with the putative promoter region of the p18 gene, resulting in up-regulation of the gene. These results indicated that the N-terminal half of HIRA should contribute positively to the growth rate via up-regulation of a set of cell cycle-related genes, whereas the C-terminal half down-regulated another set of them without exhibiting any effect on the cell growth.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Two transcriptional co-repressors, Hir1p and Hir2p, that appear to act on the chromatin structure in Saccharomyces cerevisiae repress the transcription of one copy each of histone H2A and H2B genes located at the HTA1–HTB1 locus throughout the cell cycle and transiently recruit a SWI/SNF chromatin remodeling complex, thereby leading to transcription activation at this locus at the G₁/S transition (1–4). A deficiency of the hir genes or cac1, -2, and -3 genes encoding S. cerevisiae homologs of p150, p60, and p48 subunits of CAF-1 (chromatin assembly factor-1) (5–11) results in a synergistic defect in silencing at both telomeres and silent mating type loci (3, 12). For such silencing, CAF-1 mutants devoid of PCNA² binding ability require Hir/Asf-1 (anti-silencing function-1) proteins (13). Moreover, Hir and CAF-1 proteins have been reported to be involved in building functional kinetochores in S. cerevisiae (14). The Schizosaccharomyces pombe HIRA-like protein Hip1 is required for the periodic expression of histone genes and contributes to the function of complex centromeres (15).

The mammalian HIRA family (HIR is an acronym for histone regulator) has been named for its homology to Hir1p and Hir2p and was cloned from the DiGeorge syndrome (DGS) critical region (16–19). This DGS candidate gene is required for cardiac outflow tract septation (19) and is expressed in embryonic structures affected in human CATCH22 patients (20). Targeted mutagenesis of the HIRA gene has been reported to result in gastrulation defects and patterning abnormalities of mesoendodermal derivatives prior to early embryonic lethality (21). HIRA directly interacts with the transcription factor Pax3 haplo-insufficiency that results in the mouse splotch and human Waardenburg syndrome (WSI and WSII) phenotypes (22). These results, together with others, suggested that altered stoichiometry of HIRA-containing complexes should be important for the development of structures affected in WS and DGS.

The relationship in yeast indicated that HIRA and CAF-1 could play overlapping roles in chromatin dynamics and was consistent with other findings that mammalian HIRA exhibited binding ability as to histones (23) and ASF-1 (24), and ASF-1 then interacted directly with CAF-1p60 (25, 26). The HIRA protein family with seven WD repeat domains was predicted to form a -propeller structure, which may be involved in protein-protein interactions, in its N-terminal half most similar to Hir1p and CAF-1p60 (2, 3, 19, 21). The C-terminal half, similar to Hir2p, is responsible for its binding ability as to homeodomains, HIRA-interacting protein 3 (HIRIP3), and core histones (22, 23). Hence, as with CAF-1 and ASF-1, HIRA is known to act as a histone deposition factor (chaperone).

In recent years, the participatory roles of the mammalian HIRA family in chromatin assembly, cell cycle progression, and transcription regulation of histone genes have gradually been elucidated. The ectopic expression of HIRA, a novel cyclin-cdk2 substrate, in cells blocks S phase progression (27, 28). The ectopic expression of human HIRA represses transcription of histones, and in turn this triggers a concerted block of DNA synthesis. Coupling of DNA synthesis and histone synthesis in S phase is independent of cyclin/cdk2 activity and presumably contributes to the prompt and orderly assembly of newly replicated DNA into chromatin (29). The Xenopus HIRA facilitates chromatin assembly independent of DNA synthesis in vitro, indicating that HIRA functions as a histone chaperone for DNA replication-uncoupled nucleosome assembly (30). This notion is further supported by a finding that CAF-1 and HIRA are contained in the histone H3.1 (replication-coupled) and H3.3 (replication-uncoupled) complexes, respectively (31). On the other hand, we cloned cDNA encoding chicken HIRA, and we demonstrated that WD dipeptide motifs in the N-terminal half of HIRA and an LXXLL motif in the C-terminal half are necessary for its in vitro and in vivo interactions with CAF-1p48 and histone deacetylase-2 (HDAC-2), respectively. Furthermore, our results revealed that the WD dipeptide motifs and the LXXLL motif are individually necessary for global transcription regulation in vivo, indicating that the two halves should be differentially involved in numerous DNA-utilizing processes, probably through distinct participations in transcription regulations (32, 33).

In this study we generated the homozygous HIRA-deficient DT40 mutant HIRA. The HIRA deficiency caused not only the delayed cell growth but also the opposing influences on transcriptions of cell cycle-related genes, i.e. repressions for P18, CDC25B, and BCL-2 and activations for P19, cyclin A, and core histones. These altered transcription patterns and delayed growth rates were completely revived by the expression of HA-tagged HIRA protein in HIRA. Most interestingly, the compensatory ability for delayed growth rate was preferentially based on the N-terminal half of HIRA but not on the C-terminal half. The putative promoter of the chicken P18 gene was located surrounding the sequence GCGGGCGC at positions –1157 to –1150. The N-terminal half of HIRA then interacted with this putative promoter region and thereby up-regulated the P18 gene. Based on these results, we propose possible involvements of the N-terminal and C-terminal halves of HIRA in both cell growth and transcription regulations of cell cycle-related genes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Cloning and Mapping of Chicken Genomic HIRA and P18 Genes—By using chicken HIRA cDNA (33) and 700-bp P18 cDNA fragments amplified by reverse transcription (RT)-PCR as probes, genomic DNA clones specific for HIRA and P18 were isolated on the screening of a DT40 genomic DNA library in a FIXII as described (34, 35). The organizations of the genomic HIRA and P18 genes obtained were determined by the PCR and partial sequencing protocol (Amersham Biosciences).

Cell Cultures—DT40 cells and all subclones were cultured in Dulbecco's modified medium as described (34, 35). At the indicated times, cells were counted to determine the growth rate.

Gene Constructs, Transfection, Mutant Isolation, and Southern Blotting—We constructed two targeting vectors pHIRA/neo and pHIRA/hisD for the disruption of the HIRA gene as follows. First, we cloned the 4.6-kb NotI/EcoRI fragment containing exons 1 and 2 from the HIRA genomic clone into pBluescript II (Stratagene) to yield pB(II)HIRA-4.6kb. The 7-kb region of the genomic HIRA gene containing exons 7–12 was amplified by PCR, using a sense primer from exon 7 (5'-GAAATTCTTGCCACTTTGAAAGGGCATTCT-3'), an antisense primer from exon 12 (5'-CTCTAAGCTCTCTCCATTGACCATGCTTGC-3'), and the genomic clone as a template. After blunt-ending with a blunt-ending kit (Takara), this fragment was cloned into the HincII-digested pBluescript II to yield pB(II)HIRA-7kb. From the pB(II)HIRA-7kb plasmid, we excised the 2.3-kb EcoRV fragment of the genomic HIRA gene containing exon 10, and we then inserted it into EcoRV-digested pB(II)HIRA-4.6kb to yield pB(II)HIRA-6.9kb plasmid. Finally, we inserted a blunt-ended cassette carrying the neo or hisD gene driven by the chicken -actin promoter (35) into the EcoRI/T4 polymerase site of pB(II)HIRA-6.9kb plasmid to yield pHIRA/neo or pHIRA/hisD.

Transfection was carried out essentially as described (36, 37). To obtain HIRA cells, transfectants with the pHIRA/neo construct were first selected in medium containing 2 mg of neomycin (neo; G-418) per ml. We transfected the pHIRA/hisD construct into clones, in which one of the two HIRA alleles had already been disrupted, and we selected stable transfectants in medium containing 0.8 mg of histidinol and 2 mg of neo per ml, respectively.

Genomic DNA from DT40 and drug-resistant clones was digested with EcoRI, separated in a 0.8% agarose gel, transferred to a Hybond N+ membrane, and then hybridized with ³²P-labeled probe HIRA-5', HIRA-3', neo, or hisD as described (34, 35, 38).

Semi-quantitative RT-PCR—Total RNA was isolated from exponentially growing DT40 and HIRA cells (1 x 10⁷) with or without appropriate HIRA derivates, using TRIzol Reagent kit (Invitrogen). Reverse transcription was performed with a first strand cDNA synthesis kit (ReverTra Ace- Toyobo) at 42 °C for 20 min, followed by heating at 99 °C for 5 min. PCRs were carried out at 96 °C for 20 s, 60 °C for 30 s, and 72 °C for 30 s for the indicated cycles for appropriate genes, using appropriate sense primers and antisense primers. Chicken GAPDH gene was used as the internal control. The nucleotide sequences of all amplified RT-PCR products were confirmed as described (39).

Expression of Exogenous HA-tagged HIRA in HIRA-deficient Clones—HIRA cells (cl2-14) were stably transfected with a linearized p-HAHIRA, its derivates, or vector alone p-HA(–/–), which are expressed under the control of the -actin promoter as described (35), and then stable transfectants were selected in medium containing 0.4 mg/ml puromycin (Sigma). Of the clones obtained, some clones (designated by appropriate names for HA-HIRA and its derivates) that overexpressed the HA-tagged HIRA and its derivates in the absence of puromycin were further selected. The other vectors, such as a linearized ptet-HA-HIRA, its derivates, and ptet-TA-bleo, which are expressed under the control of the tetracycline (tet) operator and cytomegalovirus minimal promoter (35, 40), were stably co-transfected into HIRA cells (cl2-14), and then the transfected clones were selected in medium containing 0.3 mg/ml Zeocin (Stratagene). Of the clones obtained, some clones (designated by appropriate names for HA-HIRA and its derivates) that over-expressed the HA-tagged HIRA and its derivates in the absence or presence of tet were further selected.

The expression of recombinant HIRA and its derivates in HIRA cells was verified by Western blotting using anti-HA antiserum as a primary antibody. The expression was confirmed by RT-PCR using primer pairs HIRA-5' (obtained using a sense primer 5'-GGAAAGCCCATATTTTCAGTGGACATTCAC-3' from exon 2 and an antisense primer 5'-CTAAGTGATTGTCCATCTGACAAAGCATC-3' from exon 4), using a Superscript One-step RT-PCR kit (Takara) under the following conditions: 96 °C for 1 min, followed by the treatment of 96 °C for 20 s, 60 °C for 30 s, and 72 °C for 30 s for the indicated cycles.

Luciferase Activity Assay—To identify the putative promoter region(s) of the P18 gene, we first amplified the 2.6-kb upstream SmaI/NcoI fragment by PCR using the P18 genomic clone as a template, and we then cloned the amplified fragment into the XhoI/NcoI sites of the luciferase (Luc) reporter vector pGL3-Basic (Promega) to yield a parental pGL3-Basic p18 (WT) plasmid. By using this parental plasmid, we constructed its derivative plasmids, carrying appropriate 5'-truncated fragments, i.e. pGL3-Basic p18 (ApaI/NcoI), pGL3-Basic p18 (HindIII/NcoI), pGL3-Basic p18 (Bsu36I/NcoI), pGL3-Basic p18 (PstI/NcoI), pGL3-Basic p18 (SmaI/HindIII), and pGL3-Basic p18 (HindIII/Bsu36I) lacking internal HindIII/Bsu36I fragment, by combined digestions with appropriate restriction enzymes. From pGL3-Basic p18 (WT) as a wild type plasmid, we also constructed three point mutation plasmids, pGL3-Basic p18-PM1-(GT-CA), pGL3-Basic p18-PM2-(GCAT), and pGL3-Basic p18-PM3-(GT-CA, GC-AT) by PCR with a QuickChange mutagenesis kit (Stratagene). All resultant constructs were verified by sequencing to ensure their junctions and correct incorporations of point mutations.

View larger version (32K): [in this window] [in a new window]   FIGURE 1. Generation of homozygous HIRA-deficient DT40 mutant. A, genomic HIRA gene organization and schematic diagram of the homologous recombination resulting in disruption of first and second alleles of HIRA. a and b, targeting vectors, pHIRA/neo and pHIRA/hisD constructs. c, HIRA locus in the genome of DT40 cells. Locations of exons are indicated by closed boxes with appropriate designations. d and e, HIRA locus in DT40 subclones after targeted integration of the pHIRA/neo and pHIRA/hisD constructs. Locations of probes HIRA-5', HIRA-3', neo, and hisD are indicated by open boxes. Only relevant restriction sites are indicated. Possible relevant fragments obtained on EcoRI digestion are shown with their lengths in kb. B, Southern blotting of homologous recombination events in HIRA. Genomic DNAs were prepared from DT40 (+/+), 1/2HIRA (cl2, cl3, and cl2-17; +/–), and HIRA (cl2-14 and cl2-16; –/–) cells. The EcoRI fragments were analyzed with probe HIRA-5', HIRA-3', neo, or hisD.

Chromatin Immunoprecipitation (Chip) Assay—Chip assay was performed with a modification as described (41). DT40 and HIRA cells containing HA-tagged HIRA or its derivates were grown until 1 x 10⁸ in two 15-cm dishes and then cross-linked by addition of formaldehyde (to a final concentration of 1%) at room temperature for 10 min. After quenching of the reaction by addition of Gly to 0.125 M at room temperature for 5 min, cells were washed with 1x phosphate-buffered saline containing 0.1% AMBSF (Merck) and 0.5% protease inhibitors mixture (Sigma), collected by centrifugation and rocked in swelling buffer containing 25 mM HEPES (pH 7.5), 1 mM EDTA, 0.2% Triton X-100, 0.1% AMBSF (Merck) and 0.5% protease inhibitors mixture (Sigma) at 4 °C for 5 min. After centrifugation at 2,000 rpm for 5 min, the resulting nuclei pellet was washed with 1x phosphate-buffered saline containing 0.1% AMBSF (Merck) and 0.5% protease inhibitors mixture (Sigma), collected by centrifugation at 2,000 rpm for 10 min, resuspended in 0.4 ml of sonication buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 0.2% SDS, 5 mM EDTA, 1% Triton X-100, 0.1% AMBSF (Merck), and 0.5% protease inhibitors mixture (Sigma)), and sonicated using Bioruptor (Cosmo Bio), a device for cell disruption, on cold water (6–8 °C) so that the average length of chromatin DNA fragments was 250–1000 bp. After centrifugation at 15,000 rpm for 15 min at 4 °C, the supernatant containing the DNA-protein complexes was collected, and then 350 µl of buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% AMBSF (Merck) and 0.5% protease inhibitors mixture (Sigma)) was added, and then aliquots (40 µl) of the resultant solution were used as input. Immunoprecipitations were carried out, using 50 µl of a 50% slurry of anti-HA-conjugated agarose beads (Santa Cruz Biotechnology) and/or anti-FLAG-conjugated agarose beads (Sigma) as a negative control for 2 h at 4°C, and finally the immunoprecipitate was pelleted at 12,000 rpm for 20 s. The immuno-precipitated material was washed 11 times with a washing buffer and eluted with an elution buffer (0.1% NaHCO₃ and 1% SDS). The cross-link was reversed by addition of NaCl (to a final concentration of 200 mM), incubation at 65 °C for 4–6 h, and treatment with 2 µl of 20 mg/ml proteinase K (Takara) at 55 °C for 1 h. The resultant samples were then extracted with phenol/chloroform followed by ethanol precipitation. Pellets were resuspended in 50 µl of TE buffer containing 100 µg/ml RNase and assayed by semi-quantitative PCR with Ex TaqHS DNA polymerase (Takara) using the following primers: p18UR1, 5'-TGTGAGCAGGGTGGGTTGT-3' and 5'-CATATTAAAGCCTTTGGCCGG-3'; p18UR2, 5'-GATAAGCGCCCGAGCGTAC-3' and 5'-AGCGAGGAGCCGAGAATAACTT-3'; p18UR3, 5'-AGGTTGCTTCCGCGTCC-3' and 5'-TCGGCAACCCCGAGATC-3'; p18UR4, 5'-GTTTCCTTCCCCCGGAGAG-3' and 5'-GTTGTACCACCGCCGTTCA-3'; and -actin, 5'-GCACTGTGGAATCAAAAAGGG-3' and 5'-CCCATCGCTGCACAAAATAAT-3'. PCR products were electrophoresed on 1.5% agarose gels. Each experiment was performed at least three times.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Generation of Homozygous HIRA-deficient DT40 Mutants—To clarify the molecular mechanism underlying the participation of HIRA on the cell growth and transcription regulations in vivo, we generated the homozygous HIRA-deficient DT40 mutant. By using the chicken HIRA cDNA (33) as a probe, we first screened a DT40 genomic library to isolate 11 different genomic DNAs specific for the chicken HIRA. Detailed analyses, including the PCR method followed by sequencing, revealed that the partial genomic DNA of about 20 kb in length comprises 12 exons, as schematically depicted with restriction map in Fig. 1A-c. Next, we transfected DT40 cells with the pHIRA/neo construct (Fig. 1A-a). After integration of this targeting vector into the HIRA locus, in two of the stable transfectants selected with neo, probe HIRA-5', originating from the upstream of exon 1, hybridized to the 12.5-kb EcoRI fragment, in addition to the endogenous 7-kb EcoRI fragment (Fig. 1, A-d and B). This result was confirmed by the finding that probe neo only hybridized to the 12.5-kb EcoRI fragment. One of the neoresistant clones (+/–, cl2) was chosen for second round of transfection with the pHIRA/hisD construct (Fig. 1A-b). As expected, after integration of the targeting vector into the remaining HIRA allele, in two of the analyzed clones (–/–, cl2-14 and cl2-16), probe HIRA-5' newly hybridized to the 9-kb EcoRI fragment, in addition to the 12.5-kb EcoRI fragment. This result was confirmed by finding that probe hisD only hybridized to the 9-kb EcoRI fragment, and probe HIRA-3' newly hybridized to the 4.5-kb EcoRI fragment, in addition to the 12.5-kb EcoRI fragment (Fig. 1, A-e and B). To determine whether or not the HIRA gene was really disrupted in the corresponding DT40 mutants, we measured the steady-state mRNA level of HIRA by RT-PCR by using appropriate primers. The mRNA level in the heterozygous mutant, 1/2HIRA, was about 50% that in DT40 cells (data not shown), and in the homozygous mutant, HIRA, no band was detected (see below).

Effect of the HIRA Deficiency on Expressions of Core Histone and Cell Cycle-related and Apoptosis-related Genes—Because HIRA has been reported to repress transcriptions of core histone genes (29), we examined whether or not the HIRA deficiency affects the expressions of these genes in the DT40 cell line by RT-PCR by using appropriate primers (39). As shown in Fig. 2A, the gene expressions of core histones H2A, H2B, H3, and H4 were obviously increased in HIRA cells (cl2-14). The elevated expressions of all core histone genes were mostly suppressed in the presence of full-length HA-tagged HIRA expressed stably in HIRA cells (cl2-14-p-HAHIRAF-6), although the mutant cells transfected with vector p-HA-2 as a control showed no influence on all these genes (cl2-14-p-HA-2), indicating that HIRA surely exhibits the ability to repress the transcriptions of core histone genes.

View larger version (35K): [in this window] [in a new window]   FIGURE 2. Influence of HIRA deficiency on gene expressions of core histones and cell cycle-related and apoptosis-related factors. Total RNAs were extracted from DT40 (+/+), HIRA (cl2-14; –/–), HIRA carrying p-HA-2 (cl2-14-p-HA-2; –/–), and HIRA carrying p-HAHIRAF-6 (cl2-14-p-HAHIRAF-6; –/–), and the mRNA levels were then determined by RT-PCR or semi-quantitative RT-PCR using the appropriate primers. A, RT-PCR for core histones H2A, H2B, H3, and H4 and GAPDH. The 28 S and 18 S rRNA bands stained by ethidium bromide show almost equal loading of total RNAs used for RT-PCR. B, RT-PCR for cell cycle-related and apoptosis-related factors such as cyclins B2, B3, C, D1, D2, D3, E, and G1, c-JUN, HST2, E2F-1, E2F-4, PCNA, P130, P107, RB, bcl-xl, cdcs 2 and 20, and cdks 2, 5–7, and ASP19, and P27, and GAPDH. C, semi-quantitative RT-PCR for cell cycle-related and apoptosis-related factors such as P18, BCL-2, CDC25B, cyclin A, and P19, together with GAPDH.

View larger version (52K): [in this window] [in a new window]   FIGURE 3. Influences of artificially expressed full-length, N-terminal, and C-terminal halves of HIRA on gene expressions altered in HIRA-deficient mutant and on growth rate. A and B, three different HIRA(–/–) cell lines, cl2-14-ptet-HAHIRAN-1, cl2-14-ptet-HAHIRAC-13, and cl2-14-ptet-HAHIRAF-3, respectively, which expressed HA-tagged N-terminal and C-terminal halves and the full length of HIRA, together with DT40 and HIRA mutant cell lines (cl2-14 and cl2-16; –/–), were grown in the absence (–) or presence (+) of tet for 48 h. Total proteins and RNAs were analyzed by Western blotting using anti-HA antiserum and by semi-quantitative RT-PCR using appropriate primers. Yeast RNA was used as control. A, Western blotting for the HA-tagged HIRA and its derivates. B, semi-quantitative RT-PCR together with GAPDH. a, HIRA as a control. b, P18, BCL-2, and CDC25B, respectively, which were down-regulated in HIRA cells. c, cyclin A and P19, respectively, which were up-regulated in HIRA cells. d, core histones H2A, H2B, H3, and H4, respectively, which were up-regulated in HIRA cells. C, three different HIRA(–/–) cell lines, cl2-14-p-HAHIRAN-3, cl2-14-p-HAHIRAC-18, and cl2-14-p-HAHIRAF-6, respectively, which expressed HA-tagged N-terminal and C-terminal halves and the full length of HIRA, together with DT40, HIRA (cl2-14; –/–), and HIRA carrying vector p-HA-2 (cl2-14-p-HA-2; –/–), were grown in the absence of puromycin for 48 h. a, total proteins and RNAs were analyzed by Western blotting using anti-HA antiserum and by RT-PCR using appropriate primers. Upper panel, Western blotting for the HA-tagged HIRA and its derivates. Lower panel, RT-PCR for HIRA and GAPDH as a control. b, DT40 and five subclones were grown, and cell numbers were determined at the indicated times. Numbers of the cells are plotted on a log phase. Values are the averages for three independent experiments. Symbols for the cell lines are shown to the right.

View larger version (29K): [in this window] [in a new window]   FIGURE 4. Identification of putative 5' upstream promoter region of the P18 genomic gene. A, organization of chicken p18 genomic DNA. The p18 genomic DNA comprises two exons and is about 4.7 kb in length, and the locations of exons 1 and 2 are schematically indicated with initiation and stop codons, as well as about the 2.56-kb upstream region. B, identification of the putative p18 promoter region. Schematic diagrams of the transfected chimeric constructs carrying the Luc gene are shown to the left. All deletion mutants were generated from their parental plasmid exhibiting about a 2.56-kb upstream region, as described under "Experimental Procedures," and are depicted schematically below the parental plasmid with the appropriate restriction sites. The lengths of the upstream regions from the translation initiation site are indicated by negative numbers on the left. Effects of deletions on the p18 promoter activities are summarized on the right. The activities of the deletion mutants were assayed as relative Luc activities in HeLa cells. Values that were calibrated for transfection efficiency by the Renilla Luc activity represent the average data from 3 to 9 independent transfections. Two constructs (lanes 1 and 9) were the pGL3-Basic vector as a positive control, carrying an SV40 promoter and the promoter-less pGL3-Basic vector as a negative control. Luc activity for the pGL3-Basic vector was used as 100%. C, nucleotide sequences of putative 5' upstream promoter region from positions –1240 to –971 of the p18 gene. The GGGTGG and GCGGGCGC sequences are boxed.

View larger version (24K): [in this window] [in a new window]   FIGURE 5. Necessity of N-terminal half of HIRA for transcription activation on minimal promoter element of the p18 gene. A, schematic diagrams of the wild type promoter region of the p18 gene with mutant promoters. Nucleotide sequences of wild type promoter (p18 WT promoter) containing GGGTGG and GCGGGCGC sequences are shown. Mutated sites within the wild type promoter sequence are underlined, and then the appropriate mutant promoters are indicated as p18 PM1, PM2, and PM3 promoters, respectively. B, relative promoter activities of three mutant promoters PM1, PM2, and PM3. C, necessity of the HIRA N-terminal half for transcription activation and minimal wild type promoter element of the p18 gene. The Luc activity obtained without HIRA (control) was used as 100%. D–F, reporter plasmids pGL3 p18 PM1, PM2, and PM3, respectively, carrying p18 PM1, PM2, and PM3 promoters, were used instead of the reporter plasmid pGL3 p18 WT in C. Expression of HA-tagged HIRA and its derivates co-transfected with p18 wild type and various mutant plasmids was verified by Western blotting using anti-HA antiserum (data not shown). Each Luc activity in cells expressing only endogenous HIRA is shown as in B.

Next, we examined the influence of the N-terminal and C-terminal halves on transcription regulations of differentially expressed cell cycle-related and apoptosis-related genes. The steady-state mRNA levels of P18, BCL-2, and CDC25B in HIRA cells carrying ptet-HAHIRAN-1 were maintained but decreased to the levels of the control null mutant when treated with the drug (Fig. 3B-b, 1st to 4th and 9th to 12th lanes). Similarly, the transcript levels of cyclin A and P19, as well as those of histones H2A, H2B, H3, and H4, in HIRA cells transfected with ptet-HAHIRAN-1, remained the same as found in the control null mutant and did not change even in the presence of the drug (Fig. 3, B-c and B-d, 1st to 4th and 9th to 12th lanes).

On the other hand, when compared with DT40 cells, HIRA cells with ptet-HAHIRAC-13 showed diminished mRNA levels of P18, BCL-2, and CDC25B and maintained almost similar levels to those in the control null mutant, without any change even in the presence of the drug (Fig. 3B-b, 1st, 2nd, 5th, 6th, 9th, 10th, 13th, and 14th lanes). Although HIRA cells carrying ptet-HAHIRAC-13 exhibited no alterations in expressions of cyclin A and P19, like those of histones H2A, H2B, H3, and H4, in the absence of tet, the tet treatment increased their expression at the levels of the control null mutant (Fig. 3, B-c and B-d, 1st, 2nd, 5th, 6th, 9th, 10th, 13th, and 14th lanes). Therefore, the N-terminal and C-terminal halves deliver individually different influences on transcription regulations, i.e. the former up-regulates the expressions of P18, BCL-2, and CDC25B genes, and the latter down-regulates those of cyclin A, P19, and histones H2A, H2B, H3, and H4 genes.

The N-terminal Half of HIRA Is Involved in Control of the Growth Rate—The growth rate of HIRA cells (cl2-14) decreased slightly, the doubling times being about 15 h (Fig. 3C-b) compared with that of the GCN5-deficient mutant (17 h) (39). To confirm this result and, if so, to assess which half of the N and C terminus of HIRA participates in controlling the growth rate, we transfected p-HA-HIRA(F), p-HAHIRA(N), and p-HA-HIRA(C), as well as vector p-HA-2(–/–) as a control, into HIRA cells (cl2-14) to yield three different HIRA cell lines for stable expressions of full-length and N-terminal and C-terminal halves of HIRA, respectively. RT-PCR using HIRA-5' primers and/or Western blotting using anti-HA antibody showed that three resultant cell lines, cl2-14-p-HAHIRAF-6, cl2-14-p-HAHIRAN-3, and cl2-14-p-HAHIRAC-18, respectively, stably expressed the full-length N-terminal half and C-terminal half of HIRA (Fig. 3C-a), and these results agreed nearly with those in Fig. 3, A and B-a.

View larger version (37K): [in this window] [in a new window]   FIGURE 6. Interaction of N-terminal half of HIRA with promoter region of the p18 gene. Binding ability of HIRA and its N-terminal and C-terminal halves with the p18 promoter region was studied by Chip assays. A, stable expression of HA-tagged full length (lane 3), N-terminal (lane 1), and C-terminal (lane 2) halves of HIRA for Chip assays, together with that for DT40 alone as a control (lane 4), was analyzed by Western blotting using anti-HA antiserum as in Fig. 3C. B, schematic mapping of the chicken p18 5'-flanking region. Four upstream regions UR1, UR2, UR3 and UR4, together with promoter region, are shown with corresponding nucleotide numbers. C, interaction of the N-terminal half of HIRA with upstream regions UR2 and UR3 of the p18 gene. Chip assays were performed using anti-HA antibody or anti-FLAG antibody as a control. PCR primers for four upstream regions UR1, UR2, UR3, and UR4, as in B, or -actin promoter were used for amplification to detect DNA fragments involved in corresponding immunoprecipitates. Input lanes represent 8% of the supernatant carrying the DNA-protein complex used in Chip assays.

The N-terminal half participates in control of the growth rate, probably through alterations in the expressions of the cell cycle-related genes, including P18 and CDC25B. On the other hand, the C-terminal half, which should be involved in the transcriptions of P19, cyclin A, and core histones H2A, H2B, H3, and H4, does not contribute to the growth rate at all. The amplitude of delayed growth rate due to the HIRA deficiency was relatively less than that linked to the GCN5 deficiency accompanied by alterations in transcription of numerous cell cycle-related genes (39), indicating that HIRA acts in the fine controlling of cell growth. The apoptotic cell death was not seriously affected even though the BCL-2 gene, an apoptosis-related gene, was repressed as evident in HIRA-deficient cells. This can be explained as DT40 cells contain BCL-XL, another apoptosis-related gene, that participates mainly in programming the apoptosis pathway, and its transcription is then preferentially controlled by GCN5 (39).

View larger version (19K): [in this window] [in a new window]   FIGURE 7. A model for different participations of N-terminal and C-terminal halves of HIRA in control of vertebrate cell growth.

The Luc activity of the parental pGL3-Basic p18 (WT), carrying the 2.6-kb SmaI/NcoI fragment, was comparable (53%) with that of the pGL3-Basic vector driven by the SV40 promoter (Fig. 4B, lanes 1 and 2), indicating that the 2.6-kb upstream fragment of the P18 gene contained a potential promoter element(s). The Luc activities (63 and 60%) of two mutants pGL3-Basic p18 (ApaI/NcoI) and pGL3-Basic p18 (HindIII/NcoI), lacking fragments from positions –2557 to –1756 and positions –2557 to –1241, were virtually similar to that of the parental one (Fig. 4B, lanes 3 and 4). As expected, the mutant pGL3-Basic p18 (SmaI/HindIII), bearing the fragment from positions –2557 to –1241, exhibited no Luc activity (Fig. 4B, lane 7), indicating that the region between positions –2557 and –1241 contained no promoter sequences. On the other hand, the mutant pGL3-Basic p18 (Bsu36I/NcoI), carrying the fragment from positions –970 to –1, as well as another mutant pGL3-Basic p18 (PstI/NcoI), carrying the fragment from positions –284 to –1, showed no activity (Fig. 4B, lanes 5 and 6), indicating that the putative promoter sequence of the P18 gene should be located within the region between positions –1240 and –971. This result was confirmed by the finding that the mutant pGL3-Basic p18 (HindIII/Bsu36I), carrying two fragments from positions –2557 to –1241 and positions –970 to –1 but lacking the fragment from positions –1240 to –971, lost the Luc activity completely (Fig. 4B, lane 8). As shown in Fig. 4C, two sequences, GGGTGG and GCGGGCGC, were located at positions –1203 to –1198 and –1157 to –1150 in this putative promoter region.

Promoter Element of the P18 Gene and Transcription Activation Ability of N-terminal Half of HIRA—Not only to identify the minimal promoter element of the P18 gene but also to determine whether or not the N-terminal half of HIRA possesses transcription activation ability for this element, we generated three mutant plasmids pGL3-Basic p18-PM1-(GT-CA), pGL3-Basic p18-PM2-(GC-AT), and pGL3-Basic p18-PM3-(GT-CA, GC-AT), respectively, possessing putative mutant promoters PM1, PM2, and PM3, with point mutations at appropriate positions depicted in Fig. 5A. Relative promoter activities of these three PM1, PM2, and PM3 promoters in cells expressing only endogenous HIRA were 45, 58, and 79%, respectively, that of wild type promoter (Fig. 5B).

We then studied the effects of the full-length N-terminal half (amino acids 1–443) and C-terminal half (amino acids 444–1019) of HIRA on the activity of the wild type and putative mutant promoters. As shown in Fig. 5C, for the wild type promoter the full length activated the promoter activity to 400% that of the control (Fig. 5C, lanes 1 and 4). The N-terminal half increased the promoter activity to 400% (Fig. 5C, lane 2), which was comparable with that of the full-length, although the C-terminal half exhibited no effect (lane 3). The effect of the N-terminal half on the activity of the PM1 promoter, carrying mutations within the sequence GGGTGG, was comparable with that of the full-length promoter (Fig. 5D, lanes 2 and 4), and then the enhancement was almost similar to that for the wild type promoter, whereas the C-terminal half exhibited no effect (lane 3). On the other hand, for the PM2 promoter, carrying mutations within the sequence GCGGGCGC, neither the N-terminal half, the C-terminal half, nor full length of HIRA exhibited any effects on the promoter activity (Fig. 5E, lanes 2–4). As expected, no effects of the N-terminal half, the C-terminal half, and full length were also observed for the activity of the PM3 promoter, carrying mutations within both the sequences GGGTGG and GCGGGCGC (Fig. 5F, lanes 2–4). These results indicate not only that the promoter element of the P18 gene should be located surrounding the sequence GCGGGCGC at positions –1157 to –1150, but also that the N-terminal half, like the full length, instead of C-terminal half, possessed its ability to activate the P18 gene promoter, when the nucleotide sequence is wild type form but not mutant one, whereas the C-terminal half exhibits no influence at all. However, these results did not reflect each promoter activity in cells expressing only endogenous HIRA. A difference in the affinities for endogenous HIRA and exogenous HIRA to these elements cannot be ruled out.

The N-terminal Half of HIRA Interacts with Putative Promoter Region of the P18 Gene—Next, we examined the interaction of HIRA (and its N-terminal and C-terminal halves) with the putative promoter region of the P18 gene. We chose four upstream regions, UR1, UR2 that mostly overlapped the putative promoter, UR3, and UR4, which were located at positions –429 to –379, –1269 to –1113, –1563 to –1390, and –2126 to –1961, respectively, because of their advantages over other regions to be amplified by PCR (Fig. 6B). We then constructed three different types of HIRA cell lines, cl2-14-p-HA-HIRA(full), cl2-14-p-HA-HIRA-(1–443), and cl2-14-p-HA-HIRA-(444–1019), respectively, which expressed stably HA-tagged full-length, N-terminal half, and C-terminal half of HIRA under the control of the -actin promoter.

To determine whether or not HIRA and its derivates interact with these upstream regions of the P18 gene, the Chip assay was performed on these three HIRA cell lines with the control cl2-14-p-HA-2(–) clone. In three cases of the chromatin complex after sonication, the complex with anti-HA beads, the eluate from the complex with anti-HA beads, but not in the case of complex with the anti-HA beads after elution, Western blotting using anti-HA antibody showed that N-terminal half, the C-terminal half, and the full length were well expressed in the corresponding clones (Fig. 6A, columns 1–3). The Chip assay performed on cl2-14-p-HA-HIRA(full), using anti-HA antibody and semi-quantitative PCR, showed positive bands for the upstream regions UR2 and UR3 that was close to UR2 but not for UR1 and UR4 (Fig. 6C). Similarly, the Chip assay carried out on cl2-14-p-HA-HIRA-(1–443) generated positive bands for upstream regions UR2 and UR3 but not for UR1 and UR4, although the assay performed on cl2-14-p-HA-HIRA-(444–1019), like on control clone cl2-14-p-HA-2(–), showed no bands for all upstream regions UR1, UR2, UR3, and UR4. The control Chip assay, using anti-FLAG antibody instead of anti-HA antibody, also showed no bands for all four upstream regions. These results suggest not only that the full-length of HIRA interacts directly or indirectly with the upstream region UR2 at positions –1269 to –1113, which mostly overlapped the putative promoter region at positions –1240 to –971, but also that this interaction is preferentially attributable to the N-terminal half. The reason for the positive signal for the upstream region UR3 might be that UR3 could also have cross-linked with HIRA or the N-terminal half (probably the presumable complex containing it) and then was easily amplified by PCR, because it is closely located to UR2, and the average length of the chromatin DNA complex was 250–1000 bp. Specifically, the participation of the N-terminal half of HIRA (and also the full-length) in the up-regulation of the P18 gene is based on its binding ability to the putative promoter region at positions –1240 to –971, probably to the promoter element at positions –1157 to –1150.

In summary, based on these results and others reported previously (31, 32), we propose a model for the possible role of HIRA linked to a fine control of the growth of vertebrate cells (Fig. 7). HIRA is doubly and oppositely involved in the transcription regulations of cell cycle-related and apoptosis-related genes, as well as core histone genes, and these different participations are then divided into two ways because of the N-terminal and C-terminal halves, respectively.. The N-terminal half binds to the putative promoter region (probably the minimal promoter element) of the P18 gene, as well as promoters of CDC25B and BCL-2 genes, directly or indirectly through formation of complex(es) with or without CAF-1p48 and/or other proteins, resulting in up-regulations of these cell cycle-related and apoptosis-related genes. On the other hand, the C-terminal half possibly binds to the putative promoter regions of the P19 and cyclin A genes, like those of histones H2A, H2B, H3, and H4 genes, directly or indirectly through formation of complex(es) with or without HDAC-2 (and/or HDAC-1) and/or other proteins, resulting in down-regulations of genes encoding these cell cycle-related factors and core histones. Thus, HIRA contributes to a minute control of the growth (and also cell cycle) of vertebrate cells, preferentially through transcription regulations of a set of the cell cycle-related genes, with the fine-tuning of distinct participations of its N-terminal and C-terminal halves. On the other hand, it is unlikely that the BCL-2 gene, activated by HIRA, acts as a prime candidate for switching apoptotic DT40 cell death. Rather, the BCL-XL gene of DT40 cells, the transcription of which is preferentially controlled by GCN5, is likely to be a predominant factor linked to an apoptosis process. The influence on the down-regulation of all core histone genes by HIRA remains to be resolved. To clarify exactly how the HIRA participates in the fine control of both the cell cycle progression and the growth of vertebrate cells, we are now cloning the genomic CDC25B DNA and studying the interaction of the N-terminal half of HIRA with its promoter region, because it predominantly facilitates the cell cycle progression through dephosphorylation of cdks.

	FOOTNOTES

 
^* This work was supported in part by The 21st Century COE Program (Life Science), a grant-in-aid for scientific research from Ministry of Education, Culture, Sports, Science, and Technology, and grants from the Japan Society for the Promotion of Science and CREST from Japan Science and Technology of Japan. 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.

¹ To whom correspondence should be addressed: Dept. of Life Science, Frontier Science Research Center, Miyazaki Medical College, University of Miyazaki, Miyazaki 889-1692, Japan. Tel.: 81-985-3127; Fax: 81-985-6503; E-mail: tnakayam{at}med.miyazaki-u.ac.jp.

² The abbreviations used are: PCNA, proliferating cell nuclear antigen; DGS, DiGeorge syndrome; Chip, chromatin immunoprecipitation; GAPDH, glyceraldehydephosphate dehydrogenase; Luc, luciferase; neo, neomycin; RT, reverse transcription; WS, Waardenburg syndrome; HA, hemagglutinin; WT, wild type; AMBSF, 4-(2-aminoethyl)-benzolsulfonylfluorid-hydrochloride.

	ACKNOWLEDGMENTS

 
We thank H. K. Barman for editorial reading of the manuscript.

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