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

Received for publication, March 25, 2002, and in revised form, May 8, 2002Published, JBC Papers in Press, May 15, 2002, DOI 10.1074/jbc.M202884200

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 §Departmentof 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 DentalUniversity, Bunkyo-ku, Tokyo 113-8510, Japan

Cdc7-Dbf4 kinase complexes, conserved widely in eu-karyotes, play essential roles in initiation and progres-sion of the S phase. Cdc7 kinase activity fluctuates dur-ing cell cycle, and this is mainly the result of oscillationof expression of the Dbf4 subunit. Therefore, it is crucialto understand the mechanisms of regulation of Dbf4expression. We have isolated and characterized the pro-moter region of the human ASK gene encoding Dbf4-related regulatory subunit for human Cdc7 kinase. Wehave identified a 63-base pair ASK promoter segment,which is sufficient for mediating growth stimulation.This minimal promoter segment (MP), containing anSp1 site but no canonical E2F site, can be activated byectopic E2F expression as well. Within the 63-base pairregion, the Sp1 site as well as other elements are essen-tial for stimulation by growth signals and by E2F,whereas an AT-rich sequence proximal to the codingregion may serve as an element required for suppres-sion in quiescence. Gel shift assays in the presence of anantibody demonstrate the presence of E2F1 in the pro-tein-DNA complexes generated on the MP segment.However, the complex formation on MP was not com-peted by a DHFR promoter fragment, known to bind toE2F, nor by a consensus E2F binding oligonucleotide.Gel shift assays with point mutant MP fragments indi-cate that a non-canonical E2F site in the middle of thissegment is critical for generation of the E2F complex.Our results suggest that E2F regulates the ASK pro-moter through an atypical mode of recognition of thetarget site.

One of the key problems in cell proliferation is to achievefaithful and coordinated replication of their genomes. The rep-lication of chromosomal DNA of eukaryotic cells is initiatedfrom multiple replication origins. Thus, precise regulation offiring individual origins during S phase is critical for regulatedgrowth of eukaryotic cells. Mechanisms of the initiation of DNAreplication in eukaryotic cells are best understood in the bud-

ding yeast Saccharomyces cerevisiae (1–4). Initiation of DNAreplication is controlled by regulated assembly of a pre-repli-cative complex (pre-RC)1 at each replication origin. The pre-RCincludes at least two protein complexes, the minichromosomemaintenance (MCM) complex and the origin recognition com-plex (ORC), together with Cdc6 proteins (4). The six-subunitORC binds specifically to S. cerevisiae replication originsthroughout the cell cycle. ORC recruits Cdc6, which in turnpromotes loading of the MCM complex onto chromatin. Associ-ation of these proteins with chromatin during G1 renders itcompetent to respond to the second set of factors, the activationof which in late G1 is thought to trigger initiation. This secondset of proteins includes a family of cyclin-dependent kinase(Cdk) and the Cdc7 kinase, the activities of which depend onG1/S-specific cyclin regulatory subunits (Clb5 and Clb6) and onthe regulatory subunit called Dbf4, respectively (5–7).

It is well established that Cdks play critical roles in cell cycleprogression. G1 Cdk activity is essential for progression fromG0 or G1 to S phase. In mammalian cells, inhibition of eithercyclin D- or cyclin E-dependent kinase activity prevents Sphase induction (8, 9). At least part of the activities of thesekinases is involved in the phosphorylation of retinoblastoma(Rb) protein, a tumor suppressor. The major role of Rb and Rbfamily members (p107 and p130) in cell growth control is theregulation of E2F transcription factors. It is now well knownthat E2F is essential for coordinating transcription during themammalian cell cycle (10–12). A number of genes have beenfound 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), Hs-Orc1 (23), HsCdc6 (24–26), and MCM5/6 (27). The strikingproperty of E2F proteins is that they can drive quiescent cellsinto S phase (28–32). Because mutational analyses have shownthat the ability of E2F proteins to drive cells into S phaserequires their DNA binding and transactivation domains, andthere is strong correlation between the ability of E2F to acti-vate transcription of target genes and that to promote DNAsynthesis (28, 29), it has been assumed that the activation ofE2F-mediated transcription is essential for S phase entry.

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

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

1 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’smodified Eagle’s medium; PBS, phosphate-buffered saline; FCS, fetalcalf serum; �F, microfarad(s).

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 31, Issue of August 2, pp. 27668–27681, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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Therefore, it is important to identify a critical target(s) of E2Ffor S phase induction to understand the mechanism of G1/Stransition in mammalian cells. Thus far, among E2F-regulatedgenes, only the cyclin E gene, expression of which is bothgrowth- and cell cycle-regulated, can partially replace E2Factivity. Overexpression of cyclin E can drive cells into S phasein the absence of measurable E2F activity (33), and microin-jection of active Cdk2-cyclin E kinase complex can induce Sphase in quiescent fibroblasts (34). Conversely, however, over-expression of E2F1 was found to drive cells into S phase with-out significant activation of Cdk2-cyclin E kinase (35, 36).These contradictory results suggest the existence of (a) com-mon target(s) of E2F and Cdk2-cyclin E, the activity of whichcan 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 essentialrole for Cdc7, another class of serine/threonine kinase, in ini-tiation of S phase (37–39). Recent studies indicate that Cdc7kinase activity is required not only for initiation of DNA rep-lication, but also for origin firing throughout the S phase (40,41). Its kinase activity, which depends on the regulatory sub-unit Dbf4, increases at the G1/S boundary. This oscillation ofCdc7 kinase activity is caused mainly by fluctuation of thelevels 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 maximumat the G1/S boundary (42), whereas its protein level increasesat G1/S and stays high throughout the S phase.

Our group and others isolated cDNAs encoding Cdc7-relatedkinase catalytic subunits and their regulatory subunits fromS. pombe (43–45), human (46–49), Xenopus (46), mouse (50,51), and Chinese hamster (52), indicating that the regulatorymechanisms of initiation of DNA replication by Cdc7-relatedkinases are conserved among eukaryotes. Human ASK (foractivator of S phase kinase, also known as HsDbf4), has beenisolated using yeast two-hybrid screening with huCdc7 as abait (48). Transcription of ASK is repressed in quiescent cellsand is induced by serum stimulation during the G0 to S phaseprogression. The ASK mRNA and protein levels fluctuate dur-ing the proliferating cell cycle, whereas huCdc7 is relativelyconstant. The ASK protein level is low in G1 phase, increases ascells progress into S phase, and is maintained at a high levelthroughout the S phase. Accordingly, the huCdc7 kinase activ-ity 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 cellproliferation, especially in S phase regulation, through up-regulation of huCdc7 kinase activity. Therefore, it is importantto understand the mechanisms of regulation of ASK expressionduring cell cycle.

For this purpose, we isolated the promoter region of humanASK gene and investigated the cis-elements and trans-actingfactors involved in regulation of ASK gene expression. We showhere that a 63-bp segment of the human ASK promoter con-taining a typical Sp1 binding site but no canonical E2F bindingsite can confer growth regulation and stimulation by E2F tran-scription factor.

MATERIALS AND METHODS

Northern Blot Analysis—Total RNA was extracted from synchro-nized mouse interleukin-3 (muIL-3)-dependent pro-B cell line Ba/F3cells using TRIzol reagent (Invitrogen) according to the manufacturer’srecommendations. The total RNA was run on a 1% agarose-formalde-hyde gel and was transferred to Hybond-N�, positively charged nylonmembranes (Amersham Biosciences). The membranes were hybridized

with the full-length mouse ASK cDNA probe, and the results wereanalyzed 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 wererestimulated by addition of 2.5 ng/ml muIL-3. Cells were harvestedevery 4 h. Harvested cells were washed once with phosphate-bufferedsaline (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 cellswere stained with 50 �g/ml propidium iodide and 250 �g/ml RNase A atroom temperature for 1 h. DNA content was analyzed by fluorescence-activated cell sorting (Becton-Dickinson). Cell cycle profile was calcu-lated using a program, ModFit.

Isolation of the 5� Region of Human ASK Gene—A human BACgenomic clone containing a partial ASK cDNA sequence (accession no.AC003083) was obtained and was digested with HindIII. The digest wasligated into the HindIII site of pBluescript II KS vector (Stratagene),and clones containing the promoter region were screened by colonyhybridization with a radiolabeled human ASK cDNA probe (0.4-kbEcoRI-EcoRV fragment). In the same way, EcoRI-NotI fragment con-taining the promoter region was subcloned into the KS vector (desig-nated as KS-EN) using the 1.5-kb HindIII fragment, which was iden-tified in the above screening, as a probe.

Primer Extension—Total RNA was extracted from HeLa and WI-38cells using TRIzol reagent (Invitrogen) according to the manufacturer’srecommendations. An end-labeled antisense primer (p476; 2 � 105 cpm)and 5 �g of total RNA were mixed and heated at 65 °C for 90 min andthen slowly cooled to room temperature. cDNA was synthesized byreverse transcriptase (Expand reverse transcriptase, Roche MolecularBiochemicals) at 42 °C for 1 h, followed by incubation with 20 �g/mlRNase A and ethanol precipitation. Elongated DNA fragments wereseparated by electrophoresis on a 8% polyacrylamide gel containing 8 M

urea. Signals were visualized by autoradiography using a Fuji imagingplate.

S1 Mapping Analysis—A 251-bp human ASK 5� DNA fragment con-taining the putative transcription start sites, generated by PstI diges-tion (see Fig. 2D), was subcloned into pBluescript II KS (KS-PST). A232-bp DNA fragment was isolated by digesting the KS-PST with PstIand BamHI. This fragment was dephosphorylated and end-labeled atthe BamHI site with T4 polynucleotide kinase and [�-32P]ATP. Eighty�g of HeLa total RNA, isolated as above, and the DNA probe (5 � 104

cpm) were mixed and ethanol-precipitated. The pellet was dissolved in20 �l of hybridization buffer containing 80% of formamide (provided inRiboQuantTM from PharMingen). The hybridization mixture was dena-tured at 90 °C for 5 min, then slowly cooled down to 48 °C, after whichhybridization was continued for 16 h. The DNA-RNA hybrids weredigested with 200 units/ml S1 nuclease at 37 °C for 1 h. The products ofS1 nuclease digestion were recovered by ethanol precipitation and wereanalyzed by electrophoresis on a 5% (19:1 acrylamide/bisacrylamide)polyacrylamide gel containing 8 M urea. Signals were visualized byautoradiography using a Fuji imaging plate.

Construction of Reporter Plasmids—The 1.5-kbp BglII-BamHI frag-ment containing the ASK promoter segment was subcloned into theBglII site of a luciferase reporter plasmid pGL2-Basic vector (Promega)to generate pAK-1. pAK-2 was generated by digesting pAK-1 with SacIand 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 andre-ligation after blunt-ending with the Klenow fragment. pAK-4 andpAK-5 were generated by PCR using the following primers, 5�-GAAGAT CTA GGA CGG CGG CGT GAG GG-3� and 5�-GAA GAT CTA GGCAGG CAC GAG GGG CG-3�, respectively, in combination with a pGL2reverse primer, 5�-CTT TAT GTT TTT GGC GTC TTC CA-3�. ThesePCR products were digested with BglII and NotI and were subsequentlycloned into the BglII-NotI vector fragment derived from pAK-1. pAK-0was generated by inserting a SalI-NotI fragment of the KS-EN into theXhoI-NotI vector fragment from pAK-1. D-1, D-2, and D-3 were gener-ated by PCR using the following primers, 5�-GAA GAT CTC GCG AGGCGG GGC ACG GC-3�, 5�-GAA GAT CTA CGG GGC GGG GCG CGCGT-3� and 5�-GAA GAT CTG CGC GTA TCG GCG CCG CG-3�, respec-tively, in combination with a pGL2 reverse primer, 5�-CTT TAT GTTTTT GGC GTC TTC CA-3�. These PCR products were digested withBglII and HindIII and were subsequently cloned into pGL2-Basic di-gested with the same enzymes. D-4 was generated by PCR using theprimer set, 5�-GAA GAT CTA GGC AGG CAC GAG GGG CG-3� and5�-CGG GAT CCA CAA ACG AGT GGG CTG CG-3�, and cloning of theresulting DNA fragment digested by BglII and BamHI at the BglII siteof pGL2-Basic. Mutations were introduced into the putative E2F andSp1 recognition sites in the human ASK promoter region by PCR-

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mediated site-directed mutagenesis as follows: E2F motif 1, GCGC-GAGA to GATCGAGA; E2F motif 2, GCGCGAGA to GATCGAGA; E2Fmotif 3, GCGCCAAG to GATCCAAG; E2F motif 4, GCGCCAAC toGATCCAAC; E2F motif 5, GCGGGAAA to GATGGAAA; Sp1 motif 1,GGGGCGGCGGGGC to GGGAATGGGC; Sp1 motif 2, GAGGCGGGGCto GAGGAATTCC; Sp1 motif 3, GGGGCGGGGC to GGGGAATTCC.

The presence of the expected mutations in the amplified PCR frag-ment was confirmed by dideoxy sequencing using an automated se-quencer (Applied Biosystems Inc.). The 63-bp minimal promoter DNAsegment (MP) was generated by DNA chain elongation with the Klenowfragment on the following pair of annealed oligonucleotides; 5�-GGGGCG GGG CGC GCG TAT CGG CGC CGC GGC CGC GTG ACG CGTTTT CAA ATC TTC AAC CGC CGC AGC T-3� and 5�-GCG GCG GTTGAA GAT TTG AAA-3� (RP). The product was cloned into pGL2-Basicvector digested with SmaI and SacI, resulting in pGL2-MP. MPM-1–6were similarly generated using the following pair of annealed oligonu-cleotides. MPM-1, 5�-GGG AAT TCG CGC GCG TAT CGG CGC CGCGGC CGC GTG ACG CGT TTT CAA ATC TTC AAC CGC CGC AGCT-3� and RP; MPM-2, 5�-GGG GCG GGG CGC GAA TTC CGG CGCCGC GGC CGC GTG ACG CGT TTT CAA ATC TTC AAC CGC CGCAGC T-3� and RP; MPM-3, 5�-GGG GCG GGG CGC GCG TAT CGGCGA ATT CGC CGC GTG ACG CGT TTT CAA ATC TTC AAC CGCCGC AGC T-3� and RP; MPM-4, 5�-GGG GCG GGG CGC GCG TATCGG CGC CGC GGC CGG AAT TCG CGT TTT CAA ATC TTC AACCGC CGC AGC T-3� and RP; MPM-5, 5�-GGG GCG GGG CGC GCGTAT CGG CGC CGC GGC CGC GTG ACG CGT ACG CGT; ATC TTCAAC CGC CGC AGC T-3� and 5�-GCG GCG GTT GAA GAT ACGCGT-3�; MPM-6, 5�-GGG GCG GGG CGC GCG TAT CGG CGC CGCGGC CGC GTG ACG CGT TTT CAA ATC TAC GCG TGC CGC AGCT-3� and 5�-GCG GCA CGC GTA GAT TTG AAA-3�.

Cell Culture and Transient Transfections—NIH3T3 cells were main-tained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10%fetal calf serum (FCS). Transient transfection was performed by elec-troporation (300 V, 125 �F or 250 V, 125 �F) using Gene Pulser(Bio-Rad). 1.5 � 106 cells were transfected with 2 �g of various ASKpromoter reporter constructs together with 0.5 �g of pCMV-alkalinephosphatase (pCMV-AP)DNAasan internal control.Tomeasuregrowth-dependent induction of the huASK promoter activity, transfectedNIH3T3 cells were arrested in G0 phase by incubation in the presenceof 0.1% FCS for 24 h, and then released into the cell cycle by readditionof 15% FCS. Cells were harvested at various times after serum stimu-lation and were assayed for luciferase and alkaline phosphatase activ-ities. HeLa, WI-38, and REF52 cells were also maintained in DMEMcontaining 10% FCS. Ba/F3 was grown in RPMI 1640 medium contain-ing 5% FCS and 0.25 ng/ml muIL-3 in a humidified atmosphere con-taining 5% CO2 at 37 °C. To measure responses of the ASK promoter toE2F, Ba/F3 cells (2–3 � 106) were transiently transfected by electropo-ration (200 V, 960 �F) with an ASK promoter reporter construct, E2Fexpression vector and pCMV-AP of indicated amount. After a 12-hincubation in muIL-3 (�) medium, cells were harvested, extracts wereprepared, and luciferase and alkaline phosphatase activities weremeasured. Luciferase activity was always normalized to alkaline phos-phatase activity, all the values presented in the figures are the averagesof three experiments, and standard deviations are shown as error bars.

Luciferase and Alkaline Phosphatase Assays—Harvested cells werewashed once with PBS, resuspended in 100 �l of 250 mM Tris-HCl (pH7.8) and were lysed by three cycles of freeze and thaw. Cell debris wasspun down, and supernatant was used for assays. Luciferase activitywas measured using a luciferase assay substrate (Promega) on theluminometer (model LB9501; Berthold Lumat Co. Ltd. Japan). Alkalinephosphatase activity was determined by using Phospha-LightTM

(Tropix, Inc.) according to the manufacturer’s instructions.Infection with Recombinant Adenoviruses—The recombinant adeno-

viruses for expression of E2F1, Ad-E2F1, and control virus, Ad-Con,were described previously (53). Infection of REF52 cells with Ad-Con orAd-E2F1 was performed as described previously (53). Briefly, quiescentREF52 cells cultured in DMEM containing 0.1% FCS for 48 h wereinfected with Ad-E2F1 or Ad-Con at a multiplicity of infection of 300plaque-forming units/cell in 2 ml/150-mm plate for 1 h at 37 °C. Cellswere further cultured in DMEM containing 0.1% FCS for 21 h andharvested for RNA isolation.

Gel Shift Assays—For preparing whole cell extracts from HeLa,harvested cells were washed once with ice-cold PBS, lysed by incubationin 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, 0.4mM sodium orthovanadate, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithio-threitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 �g/ml aprotinin, 1�g/ml pepstatin, and 2 �g/ml leupeptin) for 30 min on ice, and then

centrifuged at 15,000 rpm for 10 min. Supernatant was transferred to anew tube and protein concentration was determined by the Bio-RadProtein Assay kit (Bio-Rad) using bovine serum albumin as a standard.The ASK minimal promoter fragment isolated by digesting pGL2-MPwith SmaI and XhoI was labeled by filling in the XhoI terminus withthe Klenow fragment of DNA polymerase I and [�-32P]dTTP. The DHFRpromoter region isolated by digesting a DHFR-chloramphenicol acetyl-transferase plasmid (54) with EcoRI and HindIII was labeled at bothends in a similar manner. The whole cell extract (3 �g of total protein)and a probe (1–2 � 104 cpm) were incubated in 10 �l 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 �g/ml bovine serum albumin, and50 �g/ml salmon sperm DNA) for 30 min at room temperature. Thereaction mixture was run on a 5% polyacrylamide gel (acrylamide/bisacrylamide ratio being 29:1 and 75:1 for ASK MP and DHFR, respec-tively) containing 5% glycerol in 0.25 � Tris borate-EGTA buffer at 4 °C(DHFR) or at room temperature (ASK MP). For competition experi-ments, unlabeled probes were added at 100-fold molar excess at 10 minprior to addition of the labeled probes. The double-stranded oligonu-cleotides, 5�-ATT TAA GTT TCG CGC CCT TCC TCA A-3�, 5�-ATT CGATCG GGG CGG GGC GAG C-3�, and 5�-CGA GCG CGA GGA ATT CCACGG CGC GT-3� were used as specific E2F and Sp1 site competitors,and a nonspecific competitor, respectively. For supershift experiments,3 �g 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 thereaction mixture and was incubated for 1 h at 4 °C prior to the additionof the probe.

RESULTS

Up-regulation of ASK Gene Expression after Growth Stimu-lation—We have examined the growth-dependent regulation ofthe 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 andwere restimulated by addition of 2.5 ng/ml muIL-3. Fluores-cence-activated cell sorting analysis indicates that Ba/F3 cellsenter S phase at 8–12 h after muIL-3 stimulation (Fig. 1A).Total RNA was extracted from the cells harvested at quies-cence and at various time intervals after stimulation. Northern

FIG. 1. Growth-regulated expression of ASK mRNA. A, muIL-3dependent pro-B cells, Ba/F3, were arrested at the quiescent state bydepleting muIL-3 for 12 h and restimulated to enter the cell cycle byaddition of 2.5 ng/ml muIL-3. Cell cycle profile of muIL-3-stimulatedBa/F3 cells was measured by fluorescence-activated cell sorting, asdescribed under “Materials and Methods.” B, Northern analysis ofmuASK gene expression. Total RNA was extracted from the cells har-vested at each time point after addition of muIL3 and was analyzed byNorthern blotting using muASK cDNA as a probe. The lower panelshows ethidium bromide staining of the same gel as a loading control.

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blot was probed with radiolabeled mouse ASK cDNA.2 Tran-scription of the murine ASK gene increased after muIL-3 stim-ulation in Ba/F3 cells (Fig. 1B), reaching maximum at 12 hafter stimulation and staying at a high level thereafter, con-sistent with the previous results of ours and others (48, 51, 52)on human ASK.

Isolation of the 5� Promoter Region of Human ASK Gene—Weidentified in GenBankTM a human BAC genomic clone (CTB-60N22) derived from 7q21 containing a portion of the humanASK cDNA sequence (accession no. AC003083). The genomicsequences carried on this BAC clone perfectly matched with thereported human ASK cDNA sequences. To isolate the 5� regionof the ASK gene, this BAC DNA was digested with HindIII, andthe resulting fragments were subcloned into pBluescript II KS.Clones containing the 5� region of the human ASK gene wereselected by colony hybridization using radiolabeled humanASK cDNA (0.4-kb EcoRI-EcoRV fragment) as a probe. A 4.6-kbEcoRI-NotI fragment containing the promoter region was alsosubcloned into pBluescript II KS.

Genomic Structure of the Human ASK Gene—We also foundanother human BAC genomic clone, CTB-135C18, from 7q21 inGenBankTM containing a portion of the human ASK cDNAsequence (accession no. AC005164). Alignment of human ASKcDNA and these two BAC clones allowed us to deduce theexon-intron organization of the gene, as shown in Fig. 2A. Thehuman ASK gene consists of 12 exons and 11 introns thatextend 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), whereasexon 12, encoding a long C-terminal tail unique to mammalianDbf4-related molecules, is quite large (1188 bp). Human ASKprotein carries three motifs conserved in Dbf4-related mole-cules, namely Dbf4-motif-N, -M, and -C (55). Exon 2 and 3contain motif-N, which is related to BRCT motif and was sug-gested to be involved in interaction with chromatin and incheckpoint pathway (45). Exons 8/9 and exons 10/11 containmotif-M, a proline-rich motif, and zinc finger-like motif-C, re-spectively, both of which are required for association with andactivation of the catalytic subunit, and for mitotic functions inS. pombe (56).3 Table I shows the summary of analyses of theexon-intron structure. This exon-intron structure is highly con-served in mouse.4

Mapping of Transcription Start Sites—To determine the po-sitions of transcription start sites, we conducted primer exten-sion analyses. As shown in Fig. 2B, primer extension using anantisense oligonucleotide derived from the first exon of ASK

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.

FIG. 2. Genomic organization, transcription start sites, andnucleotide sequences of the human ASK gene. A, exon-intronstructure of the human ASK gene. Boxes represent exons. Black andwhite regions indicate coding and noncoding exons, respectively. Theexons encoding the three conserved Dbf4 motifs (55) are indicated. B,primer extension. 5 �g of total RNA extracted from asynchronouslygrowing 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 sequencingladder using the same primer and the cloned human ASK 5� region asa size marker. C, S1 mapping analysis. S1 mapping was conducted withyeast tRNA (lane 4) or HeLa total RNA (lane 5), as described under“Materials and Methods.” The digestion products were electrophoresedon a 5% polyacrylamide gel containing 8 M urea. Lanes 1 and 2, arelabeled HaeIII-digested �X174 DNA and a mixture of labeled NotI-BamHI, and MluI-BamHI DNA fragments (derived from the promoterregion), respectively. The latter two represent 73- and 83-nucleotidefragments, respectively. D, DNA sequence of the 5� region of the humanASK gene. The potential E2F and Sp1 sites, major restriction enzymesites, and translation initiation site (ATG) are indicated. One of themajor transcription start sites, indicated by a vertical black arrow, istaken as �1, and other start sites are also indicated by gray verticalarrows. The two PstI sites used for subcloning into KS vector and theBamHI site labeled for generation of an S1 mapping probe are indicatedalong with other restriction sites.

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(p476, Fig. 2D), and total RNA extracted from HeLa cells orWI-38 cells yielded DNA bands of identical patterns. We ob-served three clusters of major potential initiation sites (Fig.2B). To confirm the result of primer extension assays, we alsoperformed S1 mapping analysis. In Fig. 2C, two major bands,which were 54 and 62 nucleotides long, were observed. Thesetwo bands exactly correspond to the two proximal bands de-fined in primer extension analysis. The band corresponding tothe most distal site was also detected in S1 analysis, but itsintensity was weaker than the proximal sites. The resultsindicate the presence of multiple initiation sites clustered inthe close vicinity of the MluI site (Fig. 2D). The presence ofmultiple transcription initiation sites was previously reportedfor other TATA-less promoters (23, 57). We have taken anadenine residue in the middle cluster as �1 (Fig. 2, C and D).

As described below, these transcription initiation sites arepresent in a highly conserved segment found in both humanand mouse. Computer analysis revealed that the 3.4-kbHindIII fragment lacked a consensus TATA box but containeda cluster of E2F and Sp1 binding motifs near the transcriptionstart sites (Fig. 2D). Presence of multiple E2F and Sp1 sites hasbeen noted in other growth-regulated genes such as cyclin E(19), E2F1 (21), and MCM5 and MCM6 (27). It should also benoted 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 relativeto the transcription direction.

Functional Analyses of the Promoter of huASK Gene—Toanalyze the promoter activity of the huASK gene, a reporterplasmid, pAK-1, was constructed by inserting the 1.5-kb BglII-BamHI genomic fragment containing the human ASK pro-moter upstream of the firefly luciferase cDNA in pGL2-Basicvector (Fig. 3A). Upon transient transfection into asynchro-nously growing HeLa, NIH3T3, and Ba/F3 cells, this reporterplasmid exhibited a significant level of luciferase activity com-pared with the pGL2-Basic vector alone (Fig. 3B and data notshown). To further characterize the cis-elements of the huASKpromoter, six additional reporter plasmids containing the pro-moter of varied lengths (pAK-0, pAK-2, pAK-3, pAK4, pAK-5,and pAK-6) were constructed and the promoter activities weremeasured (Fig. 3, A and B; data not shown). The results indi-cate that pAK-5, containing the sequences downstream of�106, still retains strong promoter activity, whereas the pro-moter activity is significantly reduced in pAK-6, lacking up-stream of position �27, indicating the presence of an importantpositive regulatory element(s) between �106 and �27, whichcontains two putative Sp1 binding motifs.

To characterize the cis-elements required for growth-depend-ent regulation of the huASK gene expression, pAK-1 was tran-siently transfected into NIH3T3 cells. Transfected cells wereserum-starved and released into cell cycle by addition of serum.Cells were harvested at various times after serum stimulationand were assayed for luciferase activity. Luciferase activityincreased 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 stim-ulation in transient transfection assays is comparable withthat observed with other serum-responsive promoters assayedunder similar conditions (24, 58). The kinetics of induction ofluciferase activity is similar to that of endogenous ASK mRNAexpression (Fig. 1B), suggesting that this 1.5-kb DNA fragmentcontains all the regulatory cis-elements required for growth-regulated activation of the ASK transcription.

Activation of ASK Gene by E2F Transcription Factors—Toinvestigate the role of E2F proteins in activation of the huASKpromoter, pAK-1 was transiently transfected into Ba/F3 cellstogether with a vector expressing E2F1, E2F2, or E2F3 protein.After transfection, the cells were cultured in medium lackingmuIL3 for 12 h. Cell extracts were prepared and assayed forluciferase activity. Ectopic co-expression of E2F1, E2F2, orE2F3 caused approximately 35-, 25-, or 10-fold increase of thehuASK promoter activity, respectively (Fig. 4B). These resultsstrongly suggest that E2F family plays a critical role in acti-vation of the huASK promoter. We also observed the huASKpromoter activation by E2F transcription factor in NIH3T3cells, albeit to a lesser extent (data not shown). To examine therole of E2F in expression of the ASK gene under more physio-logical conditions, we examined the expression of endogenousASK gene in response to the recombinant adenovirus express-ing E2F1 (Ad-E2F1). Quiescent rat embryonic fibroblast cellline REF52 cells were infected with Ad-E2F1 and were har-vested at 21 h after infection, and RNA was prepared forNorthern blot analysis. Induction of the endogenous ASK genewas observed after infection with Ad-E2F1 (Fig. 4C). Theseresults indicate that overexpression of E2F1 can activate theendogenous ASK gene. Although overexpression of E2F1 wasreported to induce S phase in quiescent cells, S phase cannot beinduced under the conditions employed (21 h after infection ofthe recombinant adenovirus), and, in fact, we did not detect anysignificant effect on S phase population at the time of cellharvest in this experiment (data not shown). Therefore, theactivation of the endogenous ASK gene by Ad-E2F1 infection isnot a consequence of S phase entry of REF52 cells but is causedby direct activation of its promoter by E2F1.

Fine Mapping of the Human ASK Promoter to Identify cis-Elements Required for the Responses to Growth Stimulationand E2F—The above results indicate that the human ASKpromoter is growth-regulated and can be activated by E2F. Wethen determined in more detail cis-elements essential for re-sponses to growth stimulation and E2F. Various deletion re-porter constructs (Fig. 5A) were transiently transfected intoNIH3T3 cells, and promoter activation by serum was examinedas above. Cells were harvested immediately before serum stim-ulation (0 h), as well as at 16 h after serum addition, and wereassayed for luciferase activity (Fig. 5B). Interestingly, pAK-5,which lacked all the putative E2F binding motifs but retainedtwo Sp1 sites, could respond to serum stimulation. D-2, whichlost the second proximal Sp1 site, was also activated by growthstimulation, although the overall level of the promoter activity

TABLE IExon-intron structure and splice sites of the human ASK gene

TABLE IIComparison between E2F consensus sequence and putative E2F

binding motifs in the human ASK promoter region

E2F consensus sequence GCG (G/C)(G/C) AAAAdenovirus E2 promoter GCGCGAAA

Human ASK promoterE2F motif 1 GCGCGAGAE2F motif 2 GCGCGAGAE2F motif 3 GCGCCAAGE2F motif 4 GCGCCAACE2F motif 5 GCGGGAAA

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was significantly reduced. In contrast, D-3, lacking both ofthese proximal Sp1 sites, exhibited very little response togrowth stimulation. In addition, D-4, missing the segment be-tween �22 and �61 containing an E2F-like motif (TTT-GCGCC), could also respond to growth stimulation. These re-sults suggest that the proximal Sp1 sites play critical roles inthe level of the human ASK promoter activity as well as in itsresponse to serum stimulation.

Next, responses to E2F1 were examined with some of theabove deletion constructs containing different regions of theASK promoter fragments (Fig. 5C). E2F1 could transactivatepAK-5, which lost all the putative E2F binding sites but con-tained the two Sp1 sites. Furthermore, D-2, containing only themost proximal Sp1 but lacking the other Sp1 site, could still beactivated by E2F1, whereas D-3, lacking both Sp1 sites, showedgreatly diminished response to E2F1. These results suggestthat the promoter proximal Sp1 sites contribute also to activa-tion of the huASK promoter by E2F1.

Mutational Analyses of E2F Binding Motifs—The deletionanalyses above suggest that the five putative E2F binding sitesdo not play essential roles in transcriptional regulation of thehuASK gene. To examine more precisely whether these E2Fsites are functional or dispensable, we have generated mutantsin which all the putative E2F sites are mutated on the pAK-1and pAK-3 backbone (pAK-1-Mall and pAK-3-M1,2, respec-tively; Fig. 6A). These mutants were transiently transfectedinto Ba/F3 cells together with E2F1-expressing vector. TheMall mutant still responded to E2F1, although the extent ofactivation was reduced by 30% (Fig. 6B). We also investigatedthe role of E2F sites in induction of the huASK gene by growthstimulation. The wild type and mutant reporter constructswere transfected into NIH3T3 cells, and luciferase activitieswere measured before and after serum stimulation (Fig. 6C).The response to serum stimulation was only slightly reduced inthe two mutants. These results suggest that contribution of theE2F binding motifs in responses to growth stimulation and toE2F is limited, consistent with the conclusion drawn from theresults of deletion analyses. In addition, the E2F sites in thehuASK promoter seem to function as positive elements for

transcription, rather than as negative elements repressing thepromoter activity in the quiescence, because introduction ofmutations into the E2F binding motifs did not result in consti-tutive activation of the promoter, which is often observed withother E2F-regulated promoters such as those for E2F1 (21),HsOrc1 (23), and HsCdc6 (24–26).

Mutational Analyses of Sp1 Binding Motifs—Deletion anal-yses indicated that the most proximal Sp1 site plays importantroles in responses to serum stimulation and E2F. Therefore, weintroduced mutations into three proximal Sp1 sites on pAK-3(Fig. 7A), which are conserved in human and mouse, and ex-amined 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 pro-moter activity as well as the extent of activation was slightlyreduced. In contrast, TM and DM mutants, derived from pAK-3and D-1, respectively, and lacking all the putative proximalSp1 sites, displayed the promoter activity close to the back-ground level and did not respond to serum stimulation (Fig.7B). However, the TM mutant, showing reduced basal pro-moter activity, still responded to E2F1, albeit to a reducedextent (Fig. 7C). These results indicate that the Sp1 sites on theASK promoter play essential roles in both basal promoter ac-tivity and in up-regulation in response to serum but not in itsresponse to E2F1.

A 63-bp Promoter DNA Segment Highly Conserved betweenHuman and Mouse Can Act as a Minimal Promoter—Data basesearch led us to identify the genomic clone containing murineASK gene (muASK) in GenBankTM (accession no. AC074175).Alignment of the 5� region of the huASK gene and that of themuASK gene revealed that the three proximal Sp1 sites, butnot the E2F sites, are conserved in mouse (Fig. 8A and data notshown). We found a highly conserved segment of 63 bp contain-ing the most proximal Sp1 site and the major transcriptionstart sites of the huASK gene (Fig. 8A). We speculated that theconserved 63-bp segment may constitute a minimal ASK pro-moter element (MP), which can respond to serum stimulationand E2F1. This prediction was confirmed by the results of

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 inasynchronously growing cells. NIH3T3 cells were transfected with 2 �g of a reporter construct together with 0.5 �g 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 alkalinephosphatase activities. Ba/F3 cells were transfected with 2 �g of a reporter construct together with 0.1 �g 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.

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luciferase assays conducted with pGL2-MP containing only this63-bp segment in front of the luciferase gene. The promoter wasactivated by both serum and E2F1, although the overall level ofthe promoter activity was approximately 10% of pAK-3 con-taining the 292-bp promoter segment (Fig. 8, B and D; data notshown). We then further conducted more detailed mutationalanalyses of this MP element to identify the cis-elements essen-tial for regulation of the ASK gene expression (Fig. 8, A, B, and

D). Mutations in the putative Sp1 site almost completely abol-ished the response to growth stimulation (MPM-1), as expectedfrom the results of the previous sections. MPM-1, -3, -4, and -6also displayed significantly reduced response to serum, sug-gesting the presence of additional factors involved in growthregulation of the ASK promoter. Interestingly, mutations in anAT-rich region in the conserved 63-bp segment (MPM-5) re-sulted in up-regulation of the promoter activity in quiescence,

FIG. 4. Characterization of the human ASK promoter. A, growth-dependent induction of the huASK promoter activity. NIH3T3 cells weretransfected with 2 �g of the pAK-1 construct together with 0.5 �g 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 promoterby members of the E2F family. Ba/F3 cells were transfected with 3 �g of the pAK-1 and 0.2 �g of CMV promoter-driven expression vectors for E2F1,E2F2, or E2F3 together with 0.1 �g of pCMV-AP. A parental CMV vector, pcDNA1, was used as a negative control. Transfected cells were treatedas 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 wereinfected with recombinant adenoviruses indicated and were harvested for Northern blot analysis. The blot was hybridized with a mouse ASK probeor with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe as a control for RNA loading.

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suggesting that this AT-rich region functions as an elementinvolved in repression of the promoter in quiescence. This pro-posal was supported by the result of an internal deletion con-struct, pAK-3delMB, which lost the AT-rich region on thepAK-3 backbone (Fig. 8C). Luciferase activity of this deletionconstruct in quiescence (0 h) was the same as that of the parentpAK-3 after serum stimulation. Serum stimulation did notfurther increase the level of expression in pAK-3delMB. Theseresults indicate that multiple positive and negative factors thatinteract with the conserved 63-bp segment regulate the ASKpromoter activity during the cell cycle. Finally, the wild-typeMP responded to E2F1 (Fig. 8D), indicating that the 63-bpsegment can fully respond to E2F1 despite the fact that it doesnot contain any typical E2F sites. MPM-1 (Sp1 site) andMPM-2 still responded to E2F to a significant level, whereasMPM-3, -4, and -6 showed much reduced response. Thus, E2F-mediated activation may not absolutely require the presence ofan Sp1 binding site, but requires the sequences proximal to thecoding region within MP.

E2F Binds to the ASK Minimal Promoter in Vitro—To clarifywhether activation of the ASK MP by E2F1 results from bind-

ing of E2F1 to the promoter, we performed gel mobility shiftassays. Three bands were observed when labeled MP fragmentwas incubated with HeLa total cell extract and was run on apolyacrylamide gel. Addition of anti-E2F1 antibody retardedthe mobility of the top band (Fig. 9A, lane 3), showing thepresence of E2F1 in this complex. No significant effect wasobserved on the patterns of gel shift by addition of anti-E2F2,anti-E2F3, anti-E2F4, or a control antibody (anti-Flag anti-body). A DNA fragment containing the DHFR promoter knownto contain typical overlapping E2F binding sites generatedthree shifted bands, the top of which disappeared and super-shifted upon addition of anti-E2F4 antibody, as expected (Fig.9A, lane 10). The above results indicate that protein-DNAcomplexes formed on MP contain at least E2F1 protein.

We then performed competition assays with various DNAfragments on the MP-protein complexes. Whereas the sameunlabeled MP fragment itself completely competed out thecomplex formation, the DHFR fragment or consensus E2F siteoligonucleotide did not show any competition. On the otherhand, the E2F site oligonucleotide efficiently competed theformation of the DHFR-E2F complexes, but the ASK MP did

FIG. 5. Deletion analyses of the huASK promoter to identify cis-elements required for the responses to growth stimulation andE2F. A, schematic drawing of deletion mutants constructed. B, activation of deletion constructs by serum stimulation. NIH3T3 cells weretransfected with 2 �g of the reporter plasmid indicated together with 0.5 �g of pCMV-AP, followed by serum starvation and restimulation asdescribed under “Materials and Methods.” Cells were harvested immediately before serum stimulation (0 h) and at 16 h after serum addition, andwere assayed for luciferase and alkaline phosphatase activities. Values (striped and black bars) are presented as relative luciferase activities, withthat 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 deletionconstructs by E2F1. Ba/F3 cells were transfected with 3 �g of the reporter plasmid indicated and 0.2 �g of E2F1 expression vector or the parentalCMV vector, pcDNA1 (control), together with 0.1 �g 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.

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not (Fig. 9B). These results suggest that a mode of interactionof E2F with MP is distinct from typical E2F binding to acanonical site.

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

The results of transient transfection assays indicate thatresponses to both serum and E2F are reduced in MPM-4, con-sistent 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 inthese mutants are also functionally important for regulation ofthe ASK promoter. MPM-5, with mutations in an AT-rich seg-ment, exhibited higher basal promoter activity and thus theextent of stimulation was lower than other mutants.

Although the putative Sp1 site on MP is critical for serumactivation of the promoter (Figs. 7B and 8B), we did not observemobility shift of the MP-protein complexes by anti-Sp1 anti-body or competition by an Sp1 oligonucleotide (data not shown).We then used a D-2� fragment containing the Sp1 site alongwith 10 bp of extra nucleotides adjacent to this site as a probe(Fig. 9E). This generated a single mobility-shifted band, whichwas efficiently competed by the Sp1 oligonucleotide and wassupershifted by anti-Sp1 antibody, demonstrating that Sp1indeed binds to MP. This complex is not affected by the pres-ence of anti-E2F antibody, consistent with the lack of thecritical pentanucleotide sequence on D-2�. The lack of Sp1binding to MP may be because of its location on the fragment.Although the Sp1 site is present on MP, it may not form astable complex in gel shift assays because the site is at the veryend of the fragment.

DISCUSSION

ASK: A Critical Regulator of S Phase Initiation and Progres-sion—ASK is the functional homologue of Dbf4 protein, anactivator of Cdc7 kinase in S. cerevisiae, whose kinase activityis essential for initiation of chromosomal DNA replication.Cdc7-Dbf4 kinase complex is known to phosphorylate MCMprotein complex, a component of a pre-RC generated at each

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 �g of the reporter plasmid indicated, 0.2 �g of E2F1expression vector or empty vector, and 0.1 �g of pCMV-AP. Transfected cells were treated as described under “Materials and Methods” to determinethe extent of stimulation by E2F1. Values are presented as relative luciferase activities, with that of pAK-1 with pcDNA1 taken as 1. C, responsesof the E2F mutants to serum stimulation. NIH3T3 cells were transfected with 2 �g of the reporter plasmid indicated together with 0.5 �g ofpCMV-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 valueat 16 h to that at 0 h to indicate -fold induction.

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replication origin. Cdc7 kinase is likely to fire each origin byactivating an essential component(s) of pre-RC. Therefore, theactivity of Cdc7 kinase needs to be strictly regulated so thatorigins are fired with the correct timing and in a coordinatedmanner. Because Cdc7 kinase activity in mammals totally de-pends on availability of ASK protein, the regulation of ASKexpression is critical for control of initiation by Cdc7 kinase. InS. cerevisiae, transcription of DBF4 gene is likely to be regu-lated by the transcription factor, MBF (MCB binding factor),since the promoter region of DBF4 contains a consensus motifcalled MCB (MluI cell cycle box) to which MBF is known to bindin a cell cycle-dependent manner (59, 60). ASK gene expressionis also growth- and cell cycle-regulated and appears to beup-regulated in many transformed cell lines (48). Therefore,analysis of transcriptional regulation of the ASK gene shouldcontribute not only to understanding the mechanism of regu-lation of origin firing but also to determining the correlationbetween deregulated ASK gene activation and tumorigenesis.

Growth and E2F Regulation of ASK Transcription—In thisstudy, we have isolated the promoter region of the human ASKgene and analyzed the mechanism of growth-dependent geneactivation. 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 transcrip-tion factor. The endogenous promoter is also activated by ec-topic E2F1 expression. The results indicate that the ASK pro-moter is another target of the E2F transcription factor.However, mutations of all five putative E2F sites present in thehuman ASK promoter region did not significantly affect thebasal promoter activity or response to growth stimulation andE2F, suggesting that they may have redundant roles or theremay be an additional non-canonical E2F site(s). The fact thatthese putative E2F sites in the human ASK promoter are notconserved in mouse also supports the prediction that they maynot play significant roles in regulation of the human ASK gene.During the course of this study, we identified a novel gene en-coding a putative mitochondria carrier protein (GenBankTM

accession no. AF125531), which is transcribed in the oppositedirection at approximately 160 bp upstream of the major tran-scription start site of the human ASK gene. We have detected thepromoter activity into the opposite direction in the isolated pro-moter fragment,5 suggesting that this DNA segment functions as

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

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 �g of the reporter plasmid indicated together with 0.5 �gof 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 thatat 0 h to indicate -fold induction. C, responses of the mutants to exogenous E2F1. Ba/F3 cells were transfected with 2 �g of the reporter plasmidindicated together with 0.2 �g of E2F1 expression vector or empty vector and 0.1 �g 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-3with pcDNA1 taken as 1.

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a bidirectional promoter. The diverging gene is not conserved inmice, raising a possibility that the E2F binding sites in humanmay be involved in transcriptional control of this gene.

The 63-bp Minimal ASK Promoter Responds to Serum andE2F—We have identified putative Sp1 sites in the human ASKpromoter and have shown that they play essential roles in

FIG. 8. The 63-bp segment conserved between human and mouse is sufficient to confer responses to both serum stimulation andectopic E2F1 expression. A, alignment of the conserved 63-bp segment of the human and mouse ASK promoters, and lists of linker scanningmutants of the minimal promoter segment constructed. Sp1 site, an AT-rich segment, and restriction sites are indicated. Bent arrows indicatetranscription initiation sites on huASK. The GAATTC (MPM-1 to -4) and ACGCGT (MPM-5 and -6) sequences inserted in the MP fragment areindicated by underlined uppercase letters. B, responses of MP and its mutants to serum stimulation. NIH3T3 cells were transfected with 2 �g ofthe reporter plasmid indicated together with 0.5 �g 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 ratiosof 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 serumstimulation. NIH3T3 cells were transfected with 2 �g of the reporter plasmid together with 0.5 �g of pCMV-AP. Transfected cells were treated asdescribed 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 �g of a minimal promoter mutant, 0.2 �g of E2F1expression vector, or empty vector, in combination with 0.1 �g of pCMV-AP. Transfected cells were treated as described under “Materials andMethods” to determine the extent of stimulation by E2F1. Values (striped and black bars) are presented as relative luciferase activities, with thatof 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 ofactivation.

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FIG. 9. Gel shift assays with MP: supershift with the E2F1 antibody and competition assays. A, effect of addition of antibodies againstE2F proteins on mobility shift of the MP fragment (lanes 1–7) and the DHFR promoter fragment (lanes 8–11) by HeLa cell extract. Antibodiesadded 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 andMethods.” 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 linkerscanning 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 competitorDNAs or antibodies, as indicated. Lane 1, no extract added. In all the supershift experiments, 3 �g of antibodies were added. In all the competitionexperiments, 100-fold excess of cold competitors were added.

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growth-dependent activation of the promoter as well as in basalpromoter activity, because mutations of the proximal Sp1 sitesdecreased responses both to serum stimulation and to E2F1.These Sp1 sites are conserved in mice, supporting the impor-tance of Sp1 protein for ASK gene activation. The 63-bp seg-ment containing the major transcription start site of thehuASK gene is highly conserved between human and mouse.This segment contains the conserved, gene-proximal Sp1 siteand is sufficient for responses to growth stimulation and toE2F1, suggesting that it constitutes a minimal promoter for theASK gene.

Interestingly, mutations in AT-rich sequences present in the63 bp proximal to the gene up-regulated the promoter activityin quiescence. The promoter organization of ASK may sharesome similarity with that of cyclin A, cdc25C, cdc2, or cyclin E,in which CDE (cell cycle-dependent element)-CHR (cell cyclegene homology region) module (cyclin A, cdc25C, and cdc2) (61,62) or CERM module (cyclin E; 63), composed of an AT-richregion and neighboring GC-rich region, is located near themajor transcription start sites and represses transcription inquiescence. However, the repressor element in the ASK genedoes not share any sequence homology with these AT-richrepressor modules, suggesting that growth-dependent regula-tion of the ASK gene expression may be controlled by a distinctfactor. It should be noted that, at present, we cannot rule outthe possibility that the mutations in the AT-rich region, whichlies between the two clusters of transcription initiation sites,may create a better initiator sequences.

Association of E2F with MP Lacking Canonical E2F BindingSite—Gel shift assays in the presence of a specific antibodyindicated the presence of E2F1 protein in the complex on MP(Fig. 9A). The extent of supershift by the antibody on MP waspartial, suggesting that the association of E2F with the MPfragment in gel shift assays under the present condition maynot be as efficient as that with the DHFR and other canonicalE2F binding sites. Alternatively, the E2F in the complex on MPmay not be recognized by the antibody as efficiently as one onthe DHFR fragment. Nevertheless, the supershift observedwith anti-E2F1 antibody is specific, because we did not observeany effect of addition of the same antibody on mobility shift ofthe D-2� fragment (Fig. 9E). Because E2F2 and E2F3 canactivate the ASK promoter in transient transfection assays,they are also likely to bind to MP in vivo. The absence ofsignificant effect of anti-E2F2 or anti-E2F3 antibody on themobility shift may be because of their inefficient associationwith MP or to instability of the complex in the gel shift assays.

However, there are no canonical E2F binding sites within the63-bp segment. We did not observe any mutual competitionbetween ASK MP and DHFR promoter fragment. A consensusE2F oligonucleotide competed the DHFR complexes but not theASK MP complex. These results indicate a possibility thatE2F1 recognizes a non-canonical sequence on MP through adomain distinct from its known DNA binding domain or that itmay associate with MP without involving its direct binding tothe target site. However, we think that E2F1 is likely to makecontact with the target sequence, because a DNA binding mu-tant of E2F1 cannot activate transcription of MP.5 Mutationaland deletion analyses indicated the E2F binding to MP re-quires the 10-bp NotI-MluI segment (data not shown). Thissegment contains a sequence, 5�-CCGCGTGA-3�, which resem-bles the consensus E2F binding site, 5�-GCGCGAAA-3�. TheMPM-4, deficient in forming complexes in gel shift assays,carries a linker scanning mutation that replaces the CGTGAsequence with GAATT. The results with other linker scanningmutants indicate that the coding proximal segment as well asthe sequences overlapping with NotI also play essential roles in

promoter activation by serum and E2F. These results suggestthat generation of a functional transcriptional complex on MPinvolves factors other than E2F1 and additional sequences onMP. The lack of competition between MP and a typical E2F sitemay be because the incorporation of E2F1 in a larger transcrip-tional complex alters the target specificity of E2F. Alterna-tively, E2F may act on MP in a complex with a factor other thanDP1, which is a known partner of E2F, resulting in novelspecificity.

Recently, Weinmann et al. have identified novel E2F targetsites using chromatin immunoprecipitation assays. Amongthem, ChET4 (chromatin-precipitated E2F target 4) andChET8 were strongly bound by E2Fs in vivo but did not containa consensus E2F binding site (64). Cyclin E promoter was alsoshown to be regulated by E2F by non-consensus E2F site (63).Thus, recruitment of E2F onto promoters lacking consensusE2F site may not be unique to the ASK promoter. Furtherstudies on ASK promoter activation will clarify the nature ofE2F-mediated transcriptional activation on those promoterswithout apparent E2F binding sites. It should be noted, how-ever, that, at present, contribution of an E2F-activated secondfactor to ASK promoter activation cannot be formally ruled out.

Essential Role of Sp1 in Activation of ASK Promoter—Wehave shown that a putative Sp1 site in MP plays a crucial rolein basal promoter activity. It is also essential for responses toserum and to E2F. Gel shift assays demonstrated Sp1 bindingto this Sp1 site. Sp1 was previously reported to form a complexwith E2F1, -2, and -3 (15, 65). Therefore, we consider a possi-bility that Sp1 may be one of the factors in the E2F complexgenerated on MP, facilitating transcription in the basal state aswell as after stimulation. Possible interaction between E2F andSp1 may contribute to the sustained expression of ASK throughS to G2 phase. The Cdk2-cyclin A kinase complex is known tobind and phosphorylate E2F1 in S phase and down-regulate itsDNA binding activity (66, 67). Because the Sp1 binding domainof E2F overlaps with cyclin A binding domain, the E2F-Sp1complex may be sequestered from phosphorylation and conse-quently from inactivation, thus permitting high level expres-sion throughout S phase.

Overexpression of ASK, a Novel Target of E2F, and Cancer—Our results demonstrated that ASK is another target of E2Fprotein. Although overexpression of E2F1 can induce S phasein quiescent cells, the critical targets of E2F in S phase induc-tion are still unclear. We have shown that overexpression ofASK, a novel target of E2F, could partially bypass the mitogenrequirement for S phase in Ba/F3 cells (data not shown). There-fore, ASK may play crucial roles in E2F-mediated induction ofS phase.

Disruption of cell cycle control mechanism is one of the keyfeatures of human cancer. The p16-Cdk4-Rb pathway, whichplays a critical role in regulation of G1/S transition, is fre-quently mutated in tumors. E2F is the most important down-stream target of Rb protein. In this report, we show that over-expression of E2F causes up-regulation of the ASK promoteractivity, suggesting that disruption of the Rb-E2F pathway canlead to overexpression of ASK protein. Because Cdc7 kinase isthe critical determinant regulating the firing individual repli-cation origins, deregulated Cdc7 kinase activity resulting fromoverexpression of ASK protein may lead to overfiring of originsor loss of ordered activation of origins. This may be followed bygene amplification, gene deletion, chromosomal instability, orpolyploidy.

Williams et al. (68) reported that Cdc6 and Mcm5 could beused as useful markers of cell proliferation and suggested amajor diagnostic potential of the antibodies against these pro-teins for detecting abnormal cells in cervical smears and biop-

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sies. Human Cdc7 is overexpressed in many transformed celllines as well as in many tumor specimens, but this overexpres-sion does not always correlate with hyperproliferation (69). It ispossible that huCdc7 overexpression is associated with neo-plastic transformation. Because huCdc7 kinase activity ismostly determined by the level of ASK protein, examination ofthe expression levels of ASK protein in tumor specimens wouldprovide a novel insight into how aberration of huCdc7 kinasemay contribute to development of various cancers.

Acknowledgments—We thank Jung-Min Kim, Min-Kwon Cho, andHiroyuki Kumagai for useful advice on experiments. We thank TerukoKameyama, Chikara Jin, Masanori Takahashi, and Miyuki Kawashimafor laboratory maintenance. We also thank all the members of ourlaboratory for helpful discussion.

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A Novel Mode of E2F Action on Human ASK Promoter 27681

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Hisao MasaiMasayuki Yamada, Noriko Sato, Chika Taniyama, Kiyoshi Ohtani, Ken-ichi Arai and

Kinase Gene Encoding the Regulatory Subunit for Human Cdc7-relatedASKthe Human

Can Confer Growth-dependent and E2F-mediated Transcriptional Stimulation of A 63-Base Pair DNA Segment Containing an Sp1 Site but Not a Canonical E2F Site

doi: 10.1074/jbc.M202884200 originally published online May 15, 20022002, 277:27668-27681.J. Biol. Chem. 

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