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J. Biol. Chem., Vol. 277, Issue 31, 27668-27681, August 2, 2002

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A 63-Base Pair DNA Segment Containing an Sp1 Site but Not a Canonical E2F Site Can Confer Growth-dependent and E2F-mediated Transcriptional Stimulation of the Human ASK Gene Encoding the Regulatory Subunit for Human Cdc7-related Kinase^*

Masayuki Yamada, Noriko Sato^§, Chika Taniyama^§¶, Kiyoshi Ohtani, Ken-ichi Arai^§¶, and Hisao Masai^§**

From the  Department of Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, the ^§ Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, ^¶ CREST, Japan Science and Technology, Tokyo 108-8639, and the  Human Gene Sciences Center, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan

Received for publication, March 25, 2002, and in revised form, May 8, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cdc7-Dbf4 kinase complexes, conserved widely in eukaryotes, play essential roles in initiation and progression of the S phase. Cdc7 kinase activity fluctuates during cell cycle, and this is mainly the result of oscillation of expression of the Dbf4 subunit. Therefore, it is crucial to understand the mechanisms of regulation of Dbf4 expression. We have isolated and characterized the promoter region of the human ASK gene encoding Dbf4-related regulatory subunit for human Cdc7 kinase. We have identified a 63-base pair ASK promoter segment, which is sufficient for mediating growth stimulation. This minimal promoter segment (MP), containing an Sp1 site but no canonical E2F site, can be activated by ectopic E2F expression as well. Within the 63-base pair region, the Sp1 site as well as other elements are essential for stimulation by growth signals and by E2F, whereas an AT-rich sequence proximal to the coding region may serve as an element required for suppression in quiescence. Gel shift assays in the presence of an antibody demonstrate the presence of E2F1 in the protein-DNA complexes generated on the MP segment. However, the complex formation on MP was not competed by a DHFR promoter fragment, known to bind to E2F, nor by a consensus E2F binding oligonucleotide. Gel shift assays with point mutant MP fragments indicate that a non-canonical E2F site in the middle of this segment is critical for generation of the E2F complex. Our results suggest that E2F regulates the ASK promoter through an atypical mode of recognition of the target site.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

One of the key problems in cell proliferation is to achieve faithful and coordinated replication of their genomes. The replication of chromosomal DNA of eukaryotic cells is initiated from multiple replication origins. Thus, precise regulation of firing individual origins during S phase is critical for regulated growth of eukaryotic cells. Mechanisms of the initiation of DNA replication in eukaryotic cells are best understood in the budding yeast Saccharomyces cerevisiae (1-4). Initiation of DNA replication is controlled by regulated assembly of a pre-replicative complex (pre-RC)¹ at each replication origin. The pre-RC includes at least two protein complexes, the minichromosome maintenance (MCM) complex and the origin recognition complex (ORC), together with Cdc6 proteins (4). The six-subunit ORC binds specifically to S. cerevisiae replication origins throughout the cell cycle. ORC recruits Cdc6, which in turn promotes loading of the MCM complex onto chromatin. Association of these proteins with chromatin during G₁ renders it competent to respond to the second set of factors, the activation of which in late G₁ is thought to trigger initiation. This second set of proteins includes a family of cyclin-dependent kinase (Cdk) and the Cdc7 kinase, the activities of which depend on G₁/S-specific cyclin regulatory subunits (Clb5 and Clb6) and on the regulatory subunit called Dbf4, respectively (5-7).

It is well established that Cdks play critical roles in cell cycle progression. G₁ Cdk activity is essential for progression from G₀ or G₁ to S phase. In mammalian cells, inhibition of either cyclin D- or cyclin E-dependent kinase activity prevents S phase induction (8, 9). At least part of the activities of these kinases is involved in the phosphorylation of retinoblastoma (Rb) protein, a tumor suppressor. The major role of Rb and Rb family members (p107 and p130) in cell growth control is the regulation of E2F transcription factors. It is now well known that E2F is essential for coordinating transcription during the mammalian cell cycle (10-12). A number of genes have been found to be regulated by E2F. These include DNA polymerase  (13, 14), thymidine kinase (15), dihydrofolate reductase (DHFR) (16), cyclin E (17-19), E2F1 (20, 21), E2F2 (22), HsOrc1 (23), HsCdc6 (24-26), and MCM5/6 (27). The striking property of E2F proteins is that they can drive quiescent cells into S phase (28-32). Because mutational analyses have shown that the ability of E2F proteins to drive cells into S phase requires their DNA binding and transactivation domains, and there is strong correlation between the ability of E2F to activate transcription of target genes and that to promote DNA synthesis (28, 29), it has been assumed that the activation of E2F-mediated transcription is essential for S phase entry. Therefore, it is important to identify a critical target(s) of E2F for S phase induction to understand the mechanism of G₁/S transition in mammalian cells. Thus far, among E2F-regulated genes, only the cyclin E gene, expression of which is both growth- and cell cycle-regulated, can partially replace E2F activity. Overexpression of cyclin E can drive cells into S phase in the absence of measurable E2F activity (33), and microinjection of active Cdk2-cyclin E kinase complex can induce S phase in quiescent fibroblasts (34). Conversely, however, overexpression of E2F1 was found to drive cells into S phase without significant activation of Cdk2-cyclin E kinase (35, 36). These contradictory results suggest the existence of (a) common target(s) of E2F and Cdk2-cyclin E, the activity of which can be independently stimulated by E2F and Cdk2-cyclin E. Alternatively, S phase may be induced by redundant pathways, each of which can be activated by either E2F or Cdk2-cyclin E (11).

Genetic studies in S. cerevisiae have indicated an essential role for Cdc7, another class of serine/threonine kinase, in initiation of S phase (37-39). Recent studies indicate that Cdc7 kinase activity is required not only for initiation of DNA replication, but also for origin firing throughout the S phase (40, 41). Its kinase activity, which depends on the regulatory subunit Dbf4, increases at the G₁/S boundary. This oscillation of Cdc7 kinase activity is caused mainly by fluctuation of the levels of Dbf4 mRNA and protein. In contrast to CDC7 gene, whose transcript level is constant throughout the cell cycle, DBF4 gene is transcriptionally regulated, reaching a maximum at the G₁/S boundary (42), whereas its protein level increases at G₁/S and stays high throughout the S phase.

Our group and others isolated cDNAs encoding Cdc7-related kinase catalytic subunits and their regulatory subunits from S. pombe (43-45), human (46-49), Xenopus (46), mouse (50, 51), and Chinese hamster (52), indicating that the regulatory mechanisms of initiation of DNA replication by Cdc7-related kinases are conserved among eukaryotes. Human ASK (for activator of S phase kinase, also known as HsDbf4), has been isolated using yeast two-hybrid screening with huCdc7 as a bait (48). Transcription of ASK is repressed in quiescent cells and is induced by serum stimulation during the G₀ to S phase progression. The ASK mRNA and protein levels fluctuate during the proliferating cell cycle, whereas huCdc7 is relatively constant. The ASK protein level is low in G₁ phase, increases as cells progress into S phase, and is maintained at a high level throughout the S phase. Accordingly, the huCdc7 kinase activity shows similar cell cycle oscillation. Microinjection of ASK-specific antibodies into human cells inhibited DNA replication (48). These results suggest that ASK plays a crucial role in cell proliferation, especially in S phase regulation, through up-regulation of huCdc7 kinase activity. Therefore, it is important to understand the mechanisms of regulation of ASK expression during cell cycle.

For this purpose, we isolated the promoter region of human ASK gene and investigated the cis-elements and trans-acting factors involved in regulation of ASK gene expression. We show here that a 63-bp segment of the human ASK promoter containing a typical Sp1 binding site but no canonical E2F binding site can confer growth regulation and stimulation by E2F transcription factor.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Northern Blot Analysis-- Total RNA was extracted from synchronized mouse interleukin-3 (muIL-3)-dependent pro-B cell line Ba/F3 cells using TRIzol reagent (Invitrogen) according to the manufacturer's recommendations. The total RNA was run on a 1% agarose-formaldehyde gel and was transferred to Hybond-N+, positively charged nylon membranes (Amersham Biosciences). The membranes were hybridized with the full-length mouse ASK cDNA probe, and the results were analyzed on Fuji imaging plates.

Cell Synchronization and Cell Cycle Analysis by Flow Cytometry-- Ba/F3 cells were arrested by depletion of muIL-3 for 12 h, and were restimulated by addition of 2.5 ng/ml muIL-3. Cells were harvested every 4 h. Harvested cells were washed once with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄ (pH 7.5)) and were fixed in 75% ethanol overnight. The cells were stained with 50 µg/ml propidium iodide and 250 µg/ml RNase A at room temperature for 1 h. DNA content was analyzed by fluorescence-activated cell sorting (Becton-Dickinson). Cell cycle profile was calculated using a program, ModFit.

Isolation of the 5' Region of Human ASK Gene-- A human BAC genomic clone containing a partial ASK cDNA sequence (accession no. AC003083) was obtained and was digested with HindIII. The digest was ligated into the HindIII site of pBluescript II KS vector (Stratagene), and clones containing the promoter region were screened by colony hybridization with a radiolabeled human ASK cDNA probe (0.4-kb EcoRI-EcoRV fragment). In the same way, EcoRI-NotI fragment containing the promoter region was subcloned into the KS vector (designated as KS-EN) using the 1.5-kb HindIII fragment, which was identified in the above screening, as a probe.

Primer Extension-- Total RNA was extracted from HeLa and WI-38 cells using TRIzol reagent (Invitrogen) according to the manufacturer's recommendations. An end-labeled antisense primer (p476; 2 × 10⁵ cpm) and 5 µg of total RNA were mixed and heated at 65 °C for 90 min and then slowly cooled to room temperature. cDNA was synthesized by reverse transcriptase (Expand reverse transcriptase, Roche Molecular Biochemicals) at 42 °C for 1 h, followed by incubation with 20 µg/ml RNase A and ethanol precipitation. Elongated DNA fragments were separated by electrophoresis on a 8% polyacrylamide gel containing 8 M urea. Signals were visualized by autoradiography using a Fuji imaging plate.

S1 Mapping Analysis-- A 251-bp human ASK 5' DNA fragment containing the putative transcription start sites, generated by PstI digestion (see Fig. 2D), was subcloned into pBluescript II KS (KS-PST). A 232-bp DNA fragment was isolated by digesting the KS-PST with PstI and BamHI. This fragment was dephosphorylated and end-labeled at the BamHI site with T4 polynucleotide kinase and [-³²P]ATP. Eighty µg of HeLa total RNA, isolated as above, and the DNA probe (5 × 10⁴ cpm) were mixed and ethanol-precipitated. The pellet was dissolved in 20 µl of hybridization buffer containing 80% of formamide (provided in RiboQuant^TM from PharMingen). The hybridization mixture was denatured at 90 °C for 5 min, then slowly cooled down to 48 °C, after which hybridization was continued for 16 h. The DNA-RNA hybrids were digested with 200 units/ml S1 nuclease at 37 °C for 1 h. The products of S1 nuclease digestion were recovered by ethanol precipitation and were analyzed by electrophoresis on a 5% (19:1 acrylamide/bisacrylamide) polyacrylamide gel containing 8 M urea. Signals were visualized by autoradiography using a Fuji imaging plate.

Construction of Reporter Plasmids-- The 1.5-kbp BglII-BamHI fragment containing the ASK promoter segment was subcloned into the BglII site of a luciferase reporter plasmid pGL2-Basic vector (Promega) to generate pAK-1. pAK-2 was generated by digesting pAK-1 with SacI and AatII and re-ligating after blunt-ending with T4 DNA polymerase. pAK-3 was constructed by digesting pAK-1 with SmaI and self-ligation. pAK-6 was constructed by digesting pAK-1 with BglII and NotI and re-ligation after blunt-ending with the Klenow fragment. pAK-4 and pAK-5 were generated by PCR using the following primers, 5'-GAA GAT CTA GGA CGG CGG CGT GAG GG-3' and 5'-GAA GAT CTA GGC AGG CAC GAG GGG CG-3', respectively, in combination with a pGL2 reverse primer, 5'-CTT TAT GTT TTT GGC GTC TTC CA-3'. These PCR products were digested with BglII and NotI and were subsequently cloned into the BglII-NotI vector fragment derived from pAK-1. pAK-0 was generated by inserting a SalI-NotI fragment of the KS-EN into the XhoI-NotI vector fragment from pAK-1. D-1, D-2, and D-3 were generated by PCR using the following primers, 5'-GAA GAT CTC GCG AGG CGG GGC ACG GC-3', 5'-GAA GAT CTA CGG GGC GGG GCG CGC GT-3' and 5'-GAA GAT CTG CGC GTA TCG GCG CCG CG-3', respectively, in combination with a pGL2 reverse primer, 5'-CTT TAT GTT TTT GGC GTC TTC CA-3'. These PCR products were digested with BglII and HindIII and were subsequently cloned into pGL2-Basic digested with the same enzymes. D-4 was generated by PCR using the primer set, 5'-GAA GAT CTA GGC AGG CAC GAG GGG CG-3' and 5'-CGG GAT CCA CAA ACG AGT GGG CTG CG-3', and cloning of the resulting DNA fragment digested by BglII and BamHI at the BglII site of pGL2-Basic. Mutations were introduced into the putative E2F and Sp1 recognition sites in the human ASK promoter region by PCR-mediated site-directed mutagenesis as follows: E2F motif 1, GCGCGAGA to GATCGAGA; E2F motif 2, GCGCGAGA to GATCGAGA; E2F motif 3, GCGCCAAG to GATCCAAG; E2F motif 4, GCGCCAAC to GATCCAAC; E2F motif 5, GCGGGAAA to GATGGAAA; Sp1 motif 1, GGGGCGGCGGGGC to GGGAATGGGC; Sp1 motif 2, GAGGCGGGGC to GAGGAATTCC; Sp1 motif 3, GGGGCGGGGC to GGGGAATTCC.

The presence of the expected mutations in the amplified PCR fragment was confirmed by dideoxy sequencing using an automated sequencer (Applied Biosystems Inc.). The 63-bp minimal promoter DNA segment (MP) was generated by DNA chain elongation with the Klenow fragment on the following pair of annealed oligonucleotides; 5'-GGG GCG GGG CGC GCG TAT CGG CGC CGC GGC CGC GTG ACG CGT TTT CAA ATC TTC AAC CGC CGC AGC T-3' and 5'-GCG GCG GTT GAA GAT TTG AAA-3' (RP). The product was cloned into pGL2-Basic vector digested with SmaI and SacI, resulting in pGL2-MP. MPM-1-6 were similarly generated using the following pair of annealed oligonucleotides. MPM-1, 5'-GGG AAT TCG CGC GCG TAT CGG CGC CGC GGC CGC GTG ACG CGT TTT CAA ATC TTC AAC CGC CGC AGC T-3' and RP; MPM-2, 5'-GGG GCG GGG CGC GAA TTC CGG CGC CGC GGC CGC GTG ACG CGT TTT CAA ATC TTC AAC CGC CGC AGC T-3' and RP; MPM-3, 5'-GGG GCG GGG CGC GCG TAT CGG CGA ATT CGC CGC GTG ACG CGT TTT CAA ATC TTC AAC CGC CGC AGC T-3' and RP; MPM-4, 5'-GGG GCG GGG CGC GCG TAT CGG CGC CGC GGC CGG AAT TCG CGT TTT CAA ATC TTC AAC CGC CGC AGC T-3' and RP; MPM-5, 5'-GGG GCG GGG CGC GCG TAT CGG CGC CGC GGC CGC GTG ACG CGT ACG CGT; ATC TTC AAC CGC CGC AGC T-3' and 5'-GCG GCG GTT GAA GAT ACG CGT-3'; MPM-6, 5'-GGG GCG GGG CGC GCG TAT CGG CGC CGC GGC CGC GTG ACG CGT TTT CAA ATC TAC GCG TGC CGC AGC T-3' and 5'-GCG GCA CGC GTA GAT TTG AAA-3'.

Cell Culture and Transient Transfections-- NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS). Transient transfection was performed by electroporation (300 V, 125 µF or 250 V, 125 µF) using Gene Pulser (Bio-Rad). 1.5 × 10⁶ cells were transfected with 2 µg of various ASK promoter reporter constructs together with 0.5 µg of pCMV-alkaline phosphatase (pCMV-AP) DNA as an internal control. To measure growth-dependent induction of the huASK promoter activity, transfected NIH3T3 cells were arrested in G₀ phase by incubation in the presence of 0.1% FCS for 24 h, and then released into the cell cycle by readdition of 15% FCS. Cells were harvested at various times after serum stimulation and were assayed for luciferase and alkaline phosphatase activities. HeLa, WI-38, and REF52 cells were also maintained in DMEM containing 10% FCS. Ba/F3 was grown in RPMI 1640 medium containing 5% FCS and 0.25 ng/ml muIL-3 in a humidified atmosphere containing 5% CO₂ at 37 °C. To measure responses of the ASK promoter to E2F, Ba/F3 cells (2-3 × 10⁶) were transiently transfected by electroporation (200 V, 960 µF) with an ASK promoter reporter construct, E2F expression vector and pCMV-AP of indicated amount. After a 12-h incubation in muIL-3 () medium, cells were harvested, extracts were prepared, and luciferase and alkaline phosphatase activities were measured. Luciferase activity was always normalized to alkaline phosphatase activity, all the values presented in the figures are the averages of three experiments, and standard deviations are shown as error bars.

Luciferase and Alkaline Phosphatase Assays-- Harvested cells were washed once with PBS, resuspended in 100 µl of 250 mM Tris-HCl (pH 7.8) and were lysed by three cycles of freeze and thaw. Cell debris was spun down, and supernatant was used for assays. Luciferase activity was measured using a luciferase assay substrate (Promega) on the luminometer (model LB9501; Berthold Lumat Co. Ltd. Japan). Alkaline phosphatase activity was determined by using Phospha-Light^TM (Tropix, Inc.) according to the manufacturer's instructions.

Infection with Recombinant Adenoviruses-- The recombinant adenoviruses for expression of E2F1, Ad-E2F1, and control virus, Ad-Con, were described previously (53). Infection of REF52 cells with Ad-Con or Ad-E2F1 was performed as described previously (53). Briefly, quiescent REF52 cells cultured in DMEM containing 0.1% FCS for 48 h were infected with Ad-E2F1 or Ad-Con at a multiplicity of infection of 300 plaque-forming units/cell in 2 ml/150-mm plate for 1 h at 37 °C. Cells were further cultured in DMEM containing 0.1% FCS for 21 h and harvested for RNA isolation.

Gel Shift Assays-- For preparing whole cell extracts from HeLa, harvested cells were washed once with ice-cold PBS, lysed by incubation in the lysis buffer (10 volumes of the packed cells; 50 mM Hepes-KOH (pH 7.9), 250 mM KCl, 0.1% Nonidet P-40, 10% glycerol, 0.4 mM NaF, 0.4 mM sodium orthovanadate, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 1 µg/ml pepstatin, and 2 µg/ml leupeptin) for 30 min on ice, and then centrifuged at 15,000 rpm for 10 min. Supernatant was transferred to a new tube and protein concentration was determined by the Bio-Rad Protein Assay kit (Bio-Rad) using bovine serum albumin as a standard. The ASK minimal promoter fragment isolated by digesting pGL2-MP with SmaI and XhoI was labeled by filling in the XhoI terminus with the Klenow fragment of DNA polymerase I and [-³²P]dTTP. The DHFR promoter region isolated by digesting a DHFR-chloramphenicol acetyltransferase plasmid (54) with EcoRI and HindIII was labeled at both ends in a similar manner. The whole cell extract (3 µg of total protein) and a probe (1-2 × 10⁴ cpm) were incubated in 10 µl of binding buffer (20 mM Hepes-KOH (pH 7.9), 40 mM KCl, 6 mM MgCl₂, 1 mM EGTA, 0.1% Nonidet P-40, 10% glycerol, 300 µg/ml bovine serum albumin, and 50 µg/ml salmon sperm DNA) for 30 min at room temperature. The reaction mixture was run on a 5% polyacrylamide gel (acrylamide/bisacrylamide ratio being 29:1 and 75:1 for ASK MP and DHFR, respectively) containing 5% glycerol in 0.25 × Tris borate-EGTA buffer at 4 °C (DHFR) or at room temperature (ASK MP). For competition experiments, unlabeled probes were added at 100-fold molar excess at 10 min prior to addition of the labeled probes. The double-stranded oligonucleotides, 5'-ATT TAA GTT TCG CGC CCT TCC TCA A-3', 5'-ATT CGA TCG GGG CGG GGC GAG C-3', and 5'-CGA GCG CGA GGA ATT CCA CGG CGC GT-3' were used as specific E2F and Sp1 site competitors, and a nonspecific competitor, respectively. For supershift experiments, 3 µg of antibody against E2F1, -2, -3, -4, or Sp1 (Santa Cruz; sc-193X, sc-633X, sc-878X, sc-512X, or sc-420X, respectively) was added to the reaction mixture and was incubated for 1 h at 4 °C prior to the addition of the probe.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Up-regulation of ASK Gene Expression after Growth Stimulation-- We have examined the growth-dependent regulation of the ASK gene in muIL-3-dependent pro-B cell line Ba/F3. Ba/F3 cells were arrested by depletion of muIL-3 for 12 h and were restimulated by addition of 2.5 ng/ml muIL-3. Fluorescence-activated cell sorting analysis indicates that Ba/F3 cells enter S phase at 8-12 h after muIL-3 stimulation (Fig. 1A). Total RNA was extracted from the cells harvested at quiescence and at various time intervals after stimulation. Northern blot was probed with radiolabeled mouse ASK cDNA.² Transcription of the murine ASK gene increased after muIL-3 stimulation in Ba/F3 cells (Fig. 1B), reaching maximum at 12 h after stimulation and staying at a high level thereafter, consistent with the previous results of ours and others (48, 51, 52) on human ASK.

View larger version (42K): [in this window] [in a new window]   Fig. 1.   Growth-regulated expression of ASK mRNA. A, muIL-3 dependent pro-B cells, Ba/F3, were arrested at the quiescent state by depleting muIL-3 for 12 h and restimulated to enter the cell cycle by addition of 2.5 ng/ml muIL-3. Cell cycle profile of muIL-3-stimulated Ba/F3 cells was measured by fluorescence-activated cell sorting, as described under "Materials and Methods." B, Northern analysis of muASK gene expression. Total RNA was extracted from the cells harvested at each time point after addition of muIL3 and was analyzed by Northern blotting using muASK cDNA as a probe. The lower panel shows ethidium bromide staining of the same gel as a loading control.

Isolation of the 5' Promoter Region of Human ASK Gene-- We identified in GenBank^TM a human BAC genomic clone (CTB-60N22) derived from 7q21 containing a portion of the human ASK cDNA sequence (accession no. AC003083). The genomic sequences carried on this BAC clone perfectly matched with the reported human ASK cDNA sequences. To isolate the 5' region of the ASK gene, this BAC DNA was digested with HindIII, and the resulting fragments were subcloned into pBluescript II KS. Clones containing the 5' region of the human ASK gene were selected by colony hybridization using radiolabeled human ASK cDNA (0.4-kb EcoRI-EcoRV fragment) as a probe. A 4.6-kb EcoRI-NotI fragment containing the promoter region was also subcloned into pBluescript II KS.

Genomic Structure of the Human ASK Gene-- We also found another human BAC genomic clone, CTB-135C18, from 7q21 in GenBank^TM containing a portion of the human ASK cDNA sequence (accession no. AC005164). Alignment of human ASK cDNA and these two BAC clones allowed us to deduce the exon-intron organization of the gene, as shown in Fig. 2A. The human ASK gene consists of 12 exons and 11 introns that extend over a region of approximately 32 kb. All the exon-intron boundaries adhere to the consensus sequence (Table I). Exons 4-8 are relatively small (less than 100 bp), whereas exon 12, encoding a long C-terminal tail unique to mammalian Dbf4-related molecules, is quite large (1188 bp). Human ASK protein carries three motifs conserved in Dbf4-related molecules, namely Dbf4-motif-N, -M, and -C (55). Exon 2 and 3 contain motif-N, which is related to BRCT motif and was suggested to be involved in interaction with chromatin and in checkpoint pathway (45). Exons 8/9 and exons 10/11 contain motif-M, a proline-rich motif, and zinc finger-like motif-C, respectively, both of which are required for association with and activation of the catalytic subunit, and for mitotic functions in S. pombe (56).³ Table I shows the summary of analyses of the exon-intron structure. This exon-intron structure is highly conserved in mouse.⁴

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Genomic organization, transcription start sites, and nucleotide sequences of the human ASK gene. A, exon-intron structure of the human ASK gene. Boxes represent exons. Black and white regions indicate coding and noncoding exons, respectively. The exons encoding the three conserved Dbf4 motifs (55) are indicated. B, primer extension. 5 µg of total RNA extracted from asynchronously growing HeLa (lane 1) or WI-38 (lane 2) cells was mixed with the 5' end-labeled oligonucleotide p476 (indicated in D), and cDNA was syn- thesized by reverse transcriptase. Lane 3 is a negative control (no RNA). The extension products were separated on a gel along with a sequencing ladder using the same primer and the cloned human ASK 5' region as a size marker. C, S1 mapping analysis. S1 mapping was conducted with yeast tRNA (lane 4) or HeLa total RNA (lane 5), as described under "Materials and Methods." The digestion products were electrophoresed on a 5% polyacrylamide gel containing 8 M urea. Lanes 1 and 2, are labeled HaeIII-digested X174 DNA and a mixture of labeled NotI-BamHI, and MluI-BamHI DNA fragments (derived from the promoter region), respectively. The latter two represent 73- and 83-nucleotide fragments, respectively. D, DNA sequence of the 5' region of the human ASK gene. The potential E2F and Sp1 sites, major restriction enzyme sites, and translation initiation site (ATG) are indicated. One of the major transcription start sites, indicated by a vertical black arrow, is taken as +1, and other start sites are also indicated by gray vertical arrows. The two PstI sites used for subcloning into KS vector and the BamHI site labeled for generation of an S1 mapping probe are indicated along with other restriction sites.

Mapping of Transcription Start Sites-- To determine the positions of transcription start sites, we conducted primer extension analyses. As shown in Fig. 2B, primer extension using an antisense oligonucleotide derived from the first exon of ASK (p476, Fig. 2D), and total RNA extracted from HeLa cells or WI-38 cells yielded DNA bands of identical patterns. We observed three clusters of major potential initiation sites (Fig. 2B). To confirm the result of primer extension assays, we also performed S1 mapping analysis. In Fig. 2C, two major bands, which were 54 and 62 nucleotides long, were observed. These two bands exactly correspond to the two proximal bands defined in primer extension analysis. The band corresponding to the most distal site was also detected in S1 analysis, but its intensity was weaker than the proximal sites. The results indicate the presence of multiple initiation sites clustered in the close vicinity of the MluI site (Fig. 2D). The presence of multiple transcription initiation sites was previously reported for other TATA-less promoters (23, 57). We have taken an adenine residue in the middle cluster as +1 (Fig. 2, C and D).

As described below, these transcription initiation sites are present in a highly conserved segment found in both human and mouse. Computer analysis revealed that the 3.4-kb HindIII fragment lacked a consensus TATA box but contained a cluster of E2F and Sp1 binding motifs near the transcription start sites (Fig. 2D). Presence of multiple E2F and Sp1 sites has been noted in other growth-regulated genes such as cyclin E (19), E2F1 (21), and MCM5 and MCM6 (27). It should also be noted that the putative E2F binding motifs in the huASK 5' region are diverged from the consensus E2F binding sequence (Table II) and all are located in the opposite direction relative to the transcription direction.

Functional Analyses of the Promoter of huASK Gene-- To analyze the promoter activity of the huASK gene, a reporter plasmid, pAK-1, was constructed by inserting the 1.5-kb BglII-BamHI genomic fragment containing the human ASK promoter upstream of the firefly luciferase cDNA in pGL2-Basic vector (Fig. 3A). Upon transient transfection into asynchronously growing HeLa, NIH3T3, and Ba/F3 cells, this reporter plasmid exhibited a significant level of luciferase activity compared with the pGL2-Basic vector alone (Fig. 3B and data not shown). To further characterize the cis-elements of the huASK promoter, six additional reporter plasmids containing the promoter of varied lengths (pAK-0, pAK-2, pAK-3, pAK4, pAK-5, and pAK-6) were constructed and the promoter activities were measured (Fig. 3, A and B; data not shown). The results indicate that pAK-5, containing the sequences downstream of 106, still retains strong promoter activity, whereas the promoter activity is significantly reduced in pAK-6, lacking upstream of position 27, indicating the presence of an important positive regulatory element(s) between 106 and 27, which contains two putative Sp1 binding motifs.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Activities of the human ASK promoter-driven reporter constructs. A, schematic representation of the human ASK promoter-luciferase plasmid and its deletion derivatives. Putative transcription factor binding sites are indicated. B, human ASK promoter activities in asynchronously growing cells. NIH3T3 cells were transfected with 2 µg of a reporter construct together with 0.5 µg of pCMV-AP as described under "Materials and Methods." At 36 h after transfection, cells were harvested, and extracts were prepared to measure luciferase and alkaline phosphatase activities. Ba/F3 cells were transfected with 2 µg of a reporter construct together with 0.1 µg of a pCMV-AP. At 12 h after transfection, cells were treated as above. Values are presented as relative luciferase activities, with that of pAK-0 taken as 100.

To characterize the cis-elements required for growth-dependent regulation of the huASK gene expression, pAK-1 was transiently transfected into NIH3T3 cells. Transfected cells were serum-starved and released into cell cycle by addition of serum. Cells were harvested at various times after serum stimulation and were assayed for luciferase activity. Luciferase activity increased after serum stimulation as cells entered cell cycle, and reached maximum at 16 h after stimulation when almost all the cells had entered S phase (Fig. 4A and data not shown). The extent of induction of the ASK promoter after serum stimulation in transient transfection assays is comparable with that observed with other serum-responsive promoters assayed under similar conditions (24, 58). The kinetics of induction of luciferase activity is similar to that of endogenous ASK mRNA expression (Fig. 1B), suggesting that this 1.5-kb DNA fragment contains all the regulatory cis-elements required for growth-regulated activation of the ASK transcription.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Characterization of the human ASK promoter. A, growth-dependent induction of the huASK promoter activity. NIH3T3 cells were transfected with 2 µg of the pAK-1 construct together with 0.5 µg of pCMV-AP, followed by serum starvation and restimulation as described under "Materials and Methods." Values are presented as relative luciferase activities, with that of time 0 taken as 1. B, activation of the huASK promoter by members of the E2F family. Ba/F3 cells were transfected with 3 µg of the pAK-1 and 0.2 µg of CMV promoter-driven expression vectors for E2F1, E2F2, or E2F3 together with 0.1 µg of pCMV-AP. A parental CMV vector, pcDNA1, was used as a negative control. Transfected cells were treated as described under "Materials and Methods" to determine the extent of stimulation by E2F. Values are presented as relative luciferase activities, with that of control vector pcDNA1 taken as 1. C, endogenous ASK mRNA is induced by E2F1. Quiescent, serum-deprived REF52 cells were infected with recombinant adenoviruses indicated and were harvested for Northern blot analysis. The blot was hybridized with a mouse ASK probe or with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe as a control for RNA loading.

Activation of ASK Gene by E2F Transcription Factors-- To investigate the role of E2F proteins in activation of the huASK promoter, pAK-1 was transiently transfected into Ba/F3 cells together with a vector expressing E2F1, E2F2, or E2F3 protein. After transfection, the cells were cultured in medium lacking muIL3 for 12 h. Cell extracts were prepared and assayed for luciferase activity. Ectopic co-expression of E2F1, E2F2, or E2F3 caused approximately 35-, 25-, or 10-fold increase of the huASK promoter activity, respectively (Fig. 4B). These results strongly suggest that E2F family plays a critical role in activation of the huASK promoter. We also observed the huASK promoter activation by E2F transcription factor in NIH3T3 cells, albeit to a lesser extent (data not shown). To examine the role of E2F in expression of the ASK gene under more physiological conditions, we examined the expression of endogenous ASK gene in response to the recombinant adenovirus expressing E2F1 (Ad-E2F1). Quiescent rat embryonic fibroblast cell line REF52 cells were infected with Ad-E2F1 and were harvested at 21 h after infection, and RNA was prepared for Northern blot analysis. Induction of the endogenous ASK gene was observed after infection with Ad-E2F1 (Fig. 4C). These results indicate that overexpression of E2F1 can activate the endogenous ASK gene. Although overexpression of E2F1 was reported to induce S phase in quiescent cells, S phase cannot be induced under the conditions employed (21 h after infection of the recombinant adenovirus), and, in fact, we did not detect any significant effect on S phase population at the time of cell harvest in this experiment (data not shown). Therefore, the activation of the endogenous ASK gene by Ad-E2F1 infection is not a consequence of S phase entry of REF52 cells but is caused by direct activation of its promoter by E2F1.

Fine Mapping of the Human ASK Promoter to Identify cis-Elements Required for the Responses to Growth Stimulation and E2F-- The above results indicate that the human ASK promoter is growth-regulated and can be activated by E2F. We then determined in more detail cis-elements essential for responses to growth stimulation and E2F. Various deletion reporter constructs (Fig. 5A) were transiently transfected into NIH3T3 cells, and promoter activation by serum was examined as above. Cells were harvested immediately before serum stimulation (0 h), as well as at 16 h after serum addition, and were assayed for luciferase activity (Fig. 5B). Interestingly, pAK-5, which lacked all the putative E2F binding motifs but retained two Sp1 sites, could respond to serum stimulation. D-2, which lost the second proximal Sp1 site, was also activated by growth stimulation, although the overall level of the promoter activity was significantly reduced. In contrast, D-3, lacking both of these proximal Sp1 sites, exhibited very little response to growth stimulation. In addition, D-4, missing the segment between +22 and +61 containing an E2F-like motif (TTTGCGCC), could also respond to growth stimulation. These results suggest that the proximal Sp1 sites play critical roles in the level of the human ASK promoter activity as well as in its response to serum stimulation.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Deletion analyses of the huASK promoter to identify cis-elements required for the responses to growth stimulation and E2F. A, schematic drawing of deletion mutants constructed. B, activation of deletion constructs by serum stimulation. NIH3T3 cells were transfected with 2 µg of the reporter plasmid indicated together with 0.5 µg of pCMV-AP, followed by serum starvation and restimulation as described under "Materials and Methods." Cells were harvested immediately before serum stimulation (0 h) and at 16 h after serum addition, and were assayed for luciferase and alkaline phosphatase activities. Values (striped and black bars) are presented as relative luciferase activities, with that of pAK-1 at 0 h taken as 1. The gray bars represent ratios of the value at 16 h to that at 0 h to indicate -fold induction. C, activation of deletion constructs by E2F1. Ba/F3 cells were transfected with 3 µg of the reporter plasmid indicated and 0.2 µg of E2F1 expression vector or the parental CMV vector, pcDNA1 (control), together with 0.1 µg of pCMV-AP. Transfected cells were treated as described under "Materials and Methods." Values are presented as relative luciferase activities, with that of pAK-1 with pcDNA1 taken as 1.

Next, responses to E2F1 were examined with some of the above deletion constructs containing different regions of the ASK promoter fragments (Fig. 5C). E2F1 could transactivate pAK-5, which lost all the putative E2F binding sites but contained the two Sp1 sites. Furthermore, D-2, containing only the most proximal Sp1 but lacking the other Sp1 site, could still be activated by E2F1, whereas D-3, lacking both Sp1 sites, showed greatly diminished response to E2F1. These results suggest that the promoter proximal Sp1 sites contribute also to activation of the huASK promoter by E2F1.

Mutational Analyses of E2F Binding Motifs-- The deletion analyses above suggest that the five putative E2F binding sites do not play essential roles in transcriptional regulation of the huASK gene. To examine more precisely whether these E2F sites are functional or dispensable, we have generated mutants in which all the putative E2F sites are mutated on the pAK-1 and pAK-3 backbone (pAK-1-Mall and pAK-3-M1,2, respectively; Fig. 6A). These mutants were transiently transfected into Ba/F3 cells together with E2F1-expressing vector. The Mall mutant still responded to E2F1, although the extent of activation was reduced by 30% (Fig. 6B). We also investigated the role of E2F sites in induction of the huASK gene by growth stimulation. The wild type and mutant reporter constructs were transfected into NIH3T3 cells, and luciferase activities were measured before and after serum stimulation (Fig. 6C). The response to serum stimulation was only slightly reduced in the two mutants. These results suggest that contribution of the E2F binding motifs in responses to growth stimulation and to E2F is limited, consistent with the conclusion drawn from the results of deletion analyses. In addition, the E2F sites in the huASK promoter seem to function as positive elements for transcription, rather than as negative elements repressing the promoter activity in the quiescence, because introduction of mutations into the E2F binding motifs did not result in constitutive activation of the promoter, which is often observed with other E2F-regulated promoters such as those for E2F1 (21), HsOrc1 (23), and HsCdc6 (24-26).

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Mutational analyses of E2F binding motifs. A, schematic drawing of E2F site mutants. Crosses indicate the mutation sites. B, responses of the E2F mutants to exogenous E2F1. Ba/F3 cells were transfected with 2 µg of the reporter plasmid indicated, 0.2 µg of E2F1 expression vector or empty vector, and 0.1 µg of pCMV-AP. Transfected cells were treated as described under "Materials and Methods" to determine the extent of stimulation by E2F1. Values are presented as relative luciferase activities, with that of pAK-1 with pcDNA1 taken as 1. C, responses of the E2F mutants to serum stimulation. NIH3T3 cells were transfected with 2 µg of the reporter plasmid indicated together with 0.5 µg of pCMV-AP. Transfected cells were treated as described under "Materials and Methods." Values (striped and black bars) are presented for each pair (pAK-1 and pAK-3) as relative luciferase activities, with that of pAK-1 or pAK-3 at time 0 taken as 1. The gray bars represent ratios of the value at 16 h to that at 0 h to indicate -fold induction.

Mutational Analyses of Sp1 Binding Motifs-- Deletion analyses indicated that the most proximal Sp1 site plays important roles in responses to serum stimulation and E2F. Therefore, we introduced mutations into three proximal Sp1 sites on pAK-3 (Fig. 7A), which are conserved in human and mouse, and examined their responses to serum stimulation and to E2F1 (Fig. 8A and data not shown; see below). The mutants Sp1-1, Sp1-2, and Sp1-3, in which each one of these Sp1 sites were mutated, still responded to growth stimulation, although the basal promoter activity as well as the extent of activation was slightly reduced. In contrast, TM and DM mutants, derived from pAK-3 and D-1, respectively, and lacking all the putative proximal Sp1 sites, displayed the promoter activity close to the background level and did not respond to serum stimulation (Fig. 7B). However, the TM mutant, showing reduced basal promoter activity, still responded to E2F1, albeit to a reduced extent (Fig. 7C). These results indicate that the Sp1 sites on the ASK promoter play essential roles in both basal promoter activity and in up-regulation in response to serum but not in its response to E2F1.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Mutational analyses of Sp1 binding motifs. A, schematic drawing of Sp1 site mutants. Crosses indicate the mutation sites. B, responses of the mutants to serum stimulation. NIH3T3 cells were transfected with 2 µg of the reporter plasmid indicated together with 0.5 µg of pCMV-AP as an internal control. Transfected cells were treated as described under "Materials and Methods." Values (striped and black bars) are presented as relative luciferase activities, with that of pAK-3 at time 0 taken as 1. The gray bars represent ratios of the value at 16 h to that at 0 h to indicate -fold induction. C, responses of the mutants to exogenous E2F1. Ba/F3 cells were transfected with 2 µg of the reporter plasmid indicated together with 0.2 µg of E2F1 expression vector or empty vector and 0.1 µg of pCMV-AP. Transfected cells were treated as described under "Materials and Methods" to determine the extent of stimulation by E2F1. Values are presented as relative luciferase activities, with that of pAK-3 with pcDNA1 taken as 1.

View larger version (46K): [in this window] [in a new window]   Fig. 8.   The 63-bp segment conserved between human and mouse is sufficient to confer responses to both serum stimulation and ectopic E2F1 expression. A, alignment of the conserved 63-bp segment of the human and mouse ASK promoters, and lists of linker scanning mutants of the minimal promoter segment constructed. Sp1 site, an AT-rich segment, and restriction sites are indicated. Bent arrows indicate transcription initiation sites on huASK. The GAATTC (MPM-1 to -4) and ACGCGT (MPM-5 and -6) sequences inserted in the MP fragment are indicated by underlined uppercase letters. B, responses of MP and its mutants to serum stimulation. NIH3T3 cells were transfected with 2 µg of the reporter plasmid indicated together with 0.5 µg of pCMV-AP. Transfected cells were treated as in described under "Materials and Methods." Values (striped and black bars) are presented as relative luciferase activities, with that of MP at time 0 taken as 1. The gray bars represent ratios of the value at 16 h to that at 0 h to indicate -fold induction. C, response of pAK-3delMB, lacking the putative AT-rich repressor segment, to serum stimulation. NIH3T3 cells were transfected with 2 µg of the reporter plasmid together with 0.5 µg of pCMV-AP. Transfected cells were treated as described under "Materials and Methods." Values are presented as relative luciferase activities, with that of pAK-3 at time 0 taken as 1. D, responses of MP and its mutants to exogenous E2F1. Ba/F3 cells were transfected with 2 µg of a minimal promoter mutant, 0.2 µg of E2F1 expression vector, or empty vector, in combination with 0.1 µg of pCMV-AP. Transfected cells were treated as described under "Materials and Methods" to determine the extent of stimulation by E2F1. Values (striped and black bars) are presented as relative luciferase activities, with that of MP with pcDNA1 taken as 1. The gray bars represent ratios of the value with E2F1 to that with pcDNA1 to indicate the relative level of activation.

A 63-bp Promoter DNA Segment Highly Conserved between Human and Mouse Can Act as a Minimal Promoter-- Data base search led us to identify the genomic clone containing murine ASK gene (muASK) in GenBank^TM (accession no. AC074175). Alignment of the 5' region of the huASK gene and that of the muASK gene revealed that the three proximal Sp1 sites, but not the E2F sites, are conserved in mouse (Fig. 8A and data not shown). We found a highly conserved segment of 63 bp containing the most proximal Sp1 site and the major transcription start sites of the huASK gene (Fig. 8A). We speculated that the conserved 63-bp segment may constitute a minimal ASK promoter element (MP), which can respond to serum stimulation and E2F1. This prediction was confirmed by the results of luciferase assays conducted with pGL2-MP containing only this 63-bp segment in front of the luciferase gene. The promoter was activated by both serum and E2F1, although the overall level of the promoter activity was approximately 10% of pAK-3 containing the 292-bp promoter segment (Fig. 8, B and D; data not shown). We then further conducted more detailed mutational analyses of this MP element to identify the cis-elements essential for regulation of the ASK gene expression (Fig. 8, A, B, and D). Mutations in the putative Sp1 site almost completely abolished the response to growth stimulation (MPM-1), as expected from the results of the previous sections. MPM-1, -3, -4, and -6 also displayed significantly reduced response to serum, suggesting the presence of additional factors involved in growth regulation of the ASK promoter. Interestingly, mutations in an AT-rich region in the conserved 63-bp segment (MPM-5) resulted in up-regulation of the promoter activity in quiescence, suggesting that this AT-rich region functions as an element involved in repression of the promoter in quiescence. This proposal was supported by the result of an internal deletion construct, pAK-3delMB, which lost the AT-rich region on the pAK-3 backbone (Fig. 8C). Luciferase activity of this deletion construct in quiescence (0 h) was the same as that of the parent pAK-3 after serum stimulation. Serum stimulation did not further increase the level of expression in pAK-3delMB. These results indicate that multiple positive and negative factors that interact with the conserved 63-bp segment regulate the ASK promoter activity during the cell cycle. Finally, the wild-type MP responded to E2F1 (Fig. 8D), indicating that the 63-bp segment can fully respond to E2F1 despite the fact that it does not contain any typical E2F sites. MPM-1 (Sp1 site) and MPM-2 still responded to E2F to a significant level, whereas MPM-3, -4, and -6 showed much reduced response. Thus, E2F-mediated activation may not absolutely require the presence of an Sp1 binding site, but requires the sequences proximal to the coding region within MP.

E2F Binds to the ASK Minimal Promoter in Vitro-- To clarify whether activation of the ASK MP by E2F1 results from binding of E2F1 to the promoter, we performed gel mobility shift assays. Three bands were observed when labeled MP fragment was incubated with HeLa total cell extract and was run on a polyacrylamide gel. Addition of anti-E2F1 antibody retarded the mobility of the top band (Fig. 9A, lane 3), showing the presence of E2F1 in this complex. No significant effect was observed on the patterns of gel shift by addition of anti-E2F2, anti-E2F3, anti-E2F4, or a control antibody (anti-Flag antibody). A DNA fragment containing the DHFR promoter known to contain typical overlapping E2F binding sites generated three shifted bands, the top of which disappeared and supershifted upon addition of anti-E2F4 antibody, as expected (Fig. 9A, lane 10). The above results indicate that protein-DNA complexes formed on MP contain at least E2F1 protein.

View larger version (47K): [in this window] [in a new window]   Fig. 9.   Gel shift assays with MP: supershift with the E2F1 antibody and competition assays. A, effect of addition of antibodies against E2F proteins on mobility shift of the MP fragment (lanes 1-7) and the DHFR promoter fragment (lanes 8-11) by HeLa cell extract. Antibodies added were as follows: lane 3, anti-E2F1; lane 4, anti-E2F2; lane 5, anti-E2F3; lane 6 and 10, anti-E2F4; lanes 7 and 11, anti-Flag (negative control). Lanes 1 and 8, no extract added. B, competition assays by competitor DNAs, as indicated, were conducted as described under "Materials and Methods." Probes used were MP (lanes 1-6) and the DHFR promoter (lanes 7-12). Lanes 1 and 7, no extract added. C, competition assays by linker scanning mutant fragments, as indicated. Lane 1, no competitor added. D, gel shift assays with labeled linker scanning mutant fragments. E, binding of Sp1 to ASK MP. D-2 fragment, illustrated in the diagram below the panel, was used as a probe in the presence of various competitor DNAs or antibodies, as indicated. Lane 1, no extract added. In all the supershift experiments, 3 µg of antibodies were added. In all the competition experiments, 100-fold excess of cold competitors were added.

We then performed competition assays with various DNA fragments on the MP-protein complexes. Whereas the same unlabeled MP fragment itself completely competed out the complex formation, the DHFR fragment or consensus E2F site oligonucleotide did not show any competition. On the other hand, the E2F site oligonucleotide efficiently competed the formation of the DHFR-E2F complexes, but the ASK MP did not (Fig. 9B). These results suggest that a mode of interaction of E2F with MP is distinct from typical E2F binding to a canonical site.

Identification of Sequences Critical for Complex Formation on MP-- To more precisely localize the critical region on MP for the complex formation, we conducted competition assays using the MP fragments bearing linker scanning mutations. Among MPM-1 through MPM-6, all the mutants except for MPM-4 exhibited competition as efficient as the wild type (Fig. 9C). Conversely, the gel shift assays using the labeled mutant MP indicate that only MP-4 is deficient in the complex formation (Fig. 9D). Thus, a pentanucleotide sequence, CGTGA, present between the NotI and MluI sites of MP, is critical for the formation of the protein-DNA complexes observed on MP under the current experimental condition.

The results of transient transfection assays indicate that responses to both serum and E2F are reduced in MPM-4, consistent with the functional significance of this sequence. MPM-3 and MPM-6 also poorly responded to serum and E2F (Fig. 8, B and D), indicating that sequences on MP mutated in these mutants are also functionally important for regulation of the ASK promoter. MPM-5, with mutations in an AT-rich segment, exhibited higher basal promoter activity and thus the extent of stimulation was lower than other mutants.

Although the putative Sp1 site on MP is critical for serum activation of the promoter (Figs. 7B and 8B), we did not observe mobility shift of the MP-protein complexes by anti-Sp1 antibody or competition by an Sp1 oligonucleotide (data not shown). We then used a D-2 fragment containing the Sp1 site along with 10 bp of extra nucleotides adjacent to this site as a probe (Fig. 9E). This generated a single mobility-shifted band, which was efficiently competed by the Sp1 oligonucleotide and was supershifted by anti-Sp1 antibody, demonstrating that Sp1 indeed binds to MP. This complex is not affected by the presence of anti-E2F antibody, consistent with the lack of the critical pentanucleotide sequence on D-2. The lack of Sp1 binding to MP may be because of its location on the fragment. Although the Sp1 site is present on MP, it may not form a stable complex in gel shift assays because the site is at the very end of the fragment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ASK: A Critical Regulator of S Phase Initiation and Progression-- ASK is the functional homologue of Dbf4 protein, an activator of Cdc7 kinase in S. cerevisiae, whose kinase activity is essential for initiation of chromosomal DNA replication. Cdc7-Dbf4 kinase complex is known to phosphorylate MCM protein complex, a component of a pre-RC generated at each replication origin. Cdc7 kinase is likely to fire each origin by activating an essential component(s) of pre-RC. Therefore, the activity of Cdc7 kinase needs to be strictly regulated so that origins are fired with the correct timing and in a coordinated manner. Because Cdc7 kinase activity in mammals totally depends on availability of ASK protein, the regulation of ASK expression is critical for control of initiation by Cdc7 kinase. In S. cerevisiae, transcription of DBF4 gene is likely to be regulated by the transcription factor, MBF (MCB binding factor), since the promoter region of DBF4 contains a consensus motif called MCB (MluI cell cycle box) to which MBF is known to bind in a cell cycle-dependent manner (59, 60). ASK gene expression is also growth- and cell cycle-regulated and appears to be up-regulated in many transformed cell lines (48). Therefore, analysis of transcriptional regulation of the ASK gene should contribute not only to understanding the mechanism of regulation of origin firing but also to determining the correlation between deregulated ASK gene activation and tumorigenesis.

Growth and E2F Regulation of ASK Transcription-- In this study, we have isolated the promoter region of the human ASK gene and analyzed the mechanism of growth-dependent gene activation. In transient transfection assays, the ASK promoter is repressed in quiescence and responds to growth stimulation. In the same assays, it is strongly activated by E2F transcription factor. The endogenous promoter is also activated by ectopic E2F1 expression. The results indicate that the ASK promoter is another target of the E2F transcription factor. However, mutations of all five putative E2F sites present in the human ASK promoter region did not significantly affect the basal promoter activity or response to growth stimulation and E2F, suggesting that they may have redundant roles or there may be an additional non-canonical E2F site(s). The fact that these putative E2F sites in the human ASK promoter are not conserved in mouse also supports the prediction that they may not play significant roles in regulation of the human ASK gene. During the course of this study, we identified a novel gene encoding a putative mitochondria carrier protein (GenBank^TM accession no. AF125531), which is transcribed in the opposite direction at approximately 160 bp upstream of the major transcription start site of the human ASK gene. We have detected the promoter activity into the opposite direction in the isolated promoter fragment,⁵ suggesting that this DNA segment functions as a bidirectional promoter. The diverging gene is not conserved in mice, raising a possibility that the E2F binding sites in human may be involved in transcriptional control of this gene.

The 63-bp Minimal ASK Promoter Responds to Serum and E2F-- We have identified putative Sp1 sites in the human ASK promoter and have shown that they play essential roles in growth-dependent activation of the promoter as well as in basal promoter activity, because mutations of the proximal Sp1 sites decreased responses both to serum stimulation and to E2F1. These Sp1 sites are conserved in mice, supporting the importance of Sp1 protein for ASK gene activation. The 63-bp segment containing the major transcription start site of the huASK gene is highly conserved between human and mouse. This segment contains the conserved, gene-proximal Sp1 site and is sufficient for responses to growth stimulation and to E2F1, suggesting that it constitutes a minimal promoter for the ASK gene.

Interestingly, mutations in AT-rich sequences present in the 63 bp proximal to the gene up-regulated the promoter activity in quiescence. The promoter organization of ASK may share some similarity with that of cyclin A, cdc25C, cdc2, or cyclin E, in which CDE (cell cycle-dependent element)-CHR (cell cycle gene homology region) module (cyclin A, cdc25C, and cdc2) (61, 62) or CERM module (cyclin E; 63), composed of an AT-rich region and neighboring GC-rich region, is located near the major transcription start sites and represses transcription in quiescence. However, the repressor element in the ASK gene does not share any sequence homology with these AT-rich repressor modules, suggesting that growth-dependent regulation of the ASK gene expression may be controlled by a distinct factor. It should be noted that, at present, we cannot rule out the possibility that the mutations in the AT-rich region, which lies between the two clusters of transcription initiation sites, may create a better initiator sequences.

Association of E2F with MP Lacking Canonical E2F Binding Site-- Gel shift assays in the presence of a specific antibody indicated the presence of E2F1 protein in the complex on MP (Fig. 9A). The extent of supershift by the antibody on MP was partial, suggesting that the association of E2F with the MP fragment in gel shift assays under the present condition may not be as efficient as that with the DHFR and other canonical E2F binding sites. Alternatively, the E2F in the complex on MP may not be recognized by the antibody as efficiently as one on the DHFR fragment. Nevertheless, the supershift observed with anti-E2F1 antibody is specific, because we did not observe any effect of addition of the same antibody on mobility shift of the D-2 fragment (Fig. 9E). Because E2F2 and E2F3 can activate the ASK promoter in transient transfection assays, they are also likely to bind to MP in vivo. The absence of significant effect of anti-E2F2 or anti-E2F3 antibody on the mobility shift may be because of their inefficient association with MP or to instability of the complex in the gel shift assays.

However, there are no canonical E2F binding sites within the 63-bp segment. We did not observe any mutual competition between ASK MP and DHFR promoter fragment. A consensus E2F oligonucleotide competed the DHFR complexes but not the ASK MP complex. These results indicate a possibility that E2F1 recognizes a non-canonical sequence on MP through a domain distinct from its known DNA binding domain or that it may associate with MP without involving its direct binding to the target site. However, we think that E2F1 is likely to make contact with the target sequence, because a DNA binding mutant of E2F1 cannot activate transcription of MP.⁵ Mutational and deletion analyses indicated the E2F binding to MP requires the 10-bp NotI-MluI segment (data not shown). This segment contains a sequence, 5'-CCGCGTGA-3', which resembles the consensus E2F binding site, 5'-GCGCGAAA-3'. The MPM-4, deficient in forming complexes in gel shift assays, carries a linker scanning mutation that replaces the CGTGA sequence with GAATT. The results with other linker scanning mutants indicate that the coding proximal segment as well as the sequences overlapping with NotI also play essential roles in promoter activation by serum and E2F. These results suggest that generation of a functional transcriptional complex on MP involves factors other than E2F1 and additional sequences on MP. The lack of competition between MP and a typical E2F site may be because the incorporation of E2F1 in a larger transcriptional complex alters the target specificity of E2F. Alternatively, E2F may act on MP in a complex with a factor other than DP1, which is a known partner of E2F, resulting in novel specificity.

Recently, Weinmann et al. have identified novel E2F target sites using chromatin immunoprecipitation assays. Among them, ChET4 (chromatin-precipitated E2F target 4) and ChET8 were strongly bound by E2Fs in vivo but did not contain a consensus E2F binding site (64). Cyclin E promoter was also shown to be regulated by E2F by non-consensus E2F site (63). Thus, recruitment of E2F onto promoters lacking consensus E2F site may not be unique to the ASK promoter. Further studies on ASK promoter activation will clarify the nature of E2F-mediated transcriptional activation on those promoters without apparent E2F binding sites. It should be noted, however, that, at present, contribution of an E2F-activated second factor to ASK promoter activation cannot be formally ruled out.

Essential Role of Sp1 in Activation of ASK Promoter-- We have shown that a putative Sp1 site in MP plays a crucial role in basal promoter activity. It is also essential for responses to serum and to E2F. Gel shift assays demonstrated Sp1 binding to this Sp1 site. Sp1 was previously reported to form a complex with E2F1, -2, and -3 (15, 65). Therefore, we consider a possibility that Sp1 may be one of the factors in the E2F complex generated on MP, facilitating transcription in the basal state as well as after stimulation. Possible interaction between E2F and Sp1 may contribute to the sustained expression of ASK through S to G₂ phase. The Cdk2-cyclin A kinase complex is known to bind and phosphorylate E2F1 in S phase and down-regulate its DNA binding activity (66, 67). Because the Sp1 binding domain of E2F overlaps with cyclin A binding domain, the E2F-Sp1 complex may be sequestered from phosphorylation and consequently from inactivation, thus permitting high level expression throughout S phase.

Overexpression of ASK, a Novel Target of E2F, and Cancer-- Our results demonstrated that ASK is another target of E2F protein. Although overexpression of E2F1 can induce S phase in quiescent cells, the critical targets of E2F in S phase induction are still unclear. We have shown that overexpression of ASK, a novel target of E2F, could partially bypass the mitogen requirement for S phase in Ba/F3 cells (data not shown). Therefore, ASK may play crucial roles in E2F-mediated induction of S phase.

Disruption of cell cycle control mechanism is one of the key features of human cancer. The p16-Cdk4-Rb pathway, which plays a critical role in regulation of G₁/S transition, is frequently mutated in tumors. E2F is the most important downstream target of Rb protein. In this report, we show that overexpression of E2F causes up-regulation of the ASK promoter activity, suggesting that disruption of the Rb-E2F pathway can lead to overexpression of ASK protein. Because Cdc7 kinase is the critical determinant regulating the firing individual replication origins, deregulated Cdc7 kinase activity resulting from overexpression of ASK protein may lead to overfiring of origins or loss of ordered activation of origins. This may be followed by gene amplification, gene deletion, chromosomal instability, or polyploidy.

Williams et al. (68) reported that Cdc6 and Mcm5 could be used as useful markers of cell proliferation and suggested a major diagnostic potential of the antibodies against these proteins for detecting abnormal cells in cervical smears and biopsies. Human Cdc7 is overexpressed in many transformed cell lines as well as in many tumor specimens, but this overexpression does not always correlate with hyperproliferation (69). It is possible that huCdc7 overexpression is associated with neoplastic transformation. Because huCdc7 kinase activity is mostly determined by the level of ASK protein, examination of the expression levels of ASK protein in tumor specimens would provide a novel insight into how aberration of huCdc7 kinase may contribute to development of various cancers.

	ACKNOWLEDGEMENTS

We thank Jung-Min Kim, Min-Kwon Cho, and Hiroyuki Kumagai for useful advice on experiments. We thank Teruko Kameyama, Chikara Jin, Masanori Takahashi, and Miyuki Kawashima for laboratory maintenance. We also thank all the members of our laboratory for helpful discussion.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Cell Biology, Tokyo Metropolitan Inst. of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. Tel.: 81-3-5685-2264; Fax: 81-3-5685-2932; E-mail: hmasai@rinshoken.or.jp.

Published, JBC Papers in Press, May 15, 2002, DOI 10.1074/jbc.M202884200

² J.-M. Kim and H. Masai, manuscript in preparation.

³ N. Sato, K. Arai, and H. Masai, unpublished results.

⁴ N. Yamashita, J. M. Kim, and H. Masai, unpublished data.

⁵ M. Yamada, K. Ohtani, and H. Masai, unpublished data.

	ABBREVIATIONS

The abbreviations used are: pre-RC, pre-replicative complex; MCM, minichromosome maintenance; ORC, origin recognition complex; DHFR, dihydrofolate reductase; MP, minimal promoter segment; muIL, murine interleukin; Rb, retinoblastoma protein; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; FCS, fetal calf serum; µF, microfarad(s).

	REFERENCES

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