enhancer activity present upstream of the c-myc gene

The EMBO Journal vol.7 no.10 pp.3135-3142, 1988

An initiation site of DNA replication with transcriptionalenhancer activity present upstream of the c-myc gene

Sanae M.M.Iguchi-Arigal'3, Takahiro Okazaki1,Teru Itanil, Masaaki Ogata2, Yoshinari Sato2and Hiroyoshi Arigal'The Institute of Medical Science, The University of Tokyo, 4-6-1Shirokanedai, Minato-ku, Tokyo 108 and 2Research Institute, DaiichiSeiyaku Co. Ltd, 1-16-13 Kita-kasai, Edogawa-ku, Tokyo 134, Japan

3Present address: Institut fir Molekularbiologie II der UniversitiitZurich, CH-8093 Zurich, Switzerland

Communicated by A.Kornberg

We have previously reported that c-myc protein maypromote cellular DNA replication by binding to initiationsites of replication. Here we report that a putative originof human cellular DNA replication (on) is present at

- 2 kb upstream of the coding region of the c-myc geneitself. The c-myc protein, or protein(s) complexed withc-myc protein, bind to the upstream region (- 200 bp inlength) which has transcriptional enhancer activity as wellas autonomously replicating activity in human cells,suggesting that the c-myc protein may be an enhancerbinding protein as well as a DNA replication protein.Results with deletion mutants suggest that the sequenceessential to the origin ofDNA replication may be adjacentto, but cannot be clearly separated from, the sequenceresponsible for enhancer activity. Furthermore, whencloned DNA containing putative c-myc protein bindingsequences was transfected as competitor into HL-60 cells,expression of c-myc was inhibited, suggesting that c-mycprotein itself may be necessary for c-myc expression.Key words: DNA replication/c-myc protein/enhancer

and Bradley, 1986). We have also shown that c-myc proteincan substitute for SV40 T antigen in SV40 DNA replication(Iguchi-Ariga et al., 1987b). The coding genes for these viralT antigens are located in adjacent regions of SV40 orpolyoma ori respectively. These T antigens not only promotereplication but also regulate their own transcription (Tooze,1980; DePamphilis and Bradley, 1986). Considering thesimilarity between viral T antigens and the c-myc protein,it was suggested that c-myc protein-binding sequences whichinvolve an origin of replication may exist upstream of thec-myc gene, and that these sequences may also have atranscriptional regulatory function.Here we report that c-myc protein binding sequences

exist - 2 kb upstream of the human c-myc coding gene and

r

A

IntroductionThe function of c-myc protein, the product of the proto-oncogene c-myc (Sheiness and Bishop, 1979), has not yetbeen clarified, although it has been suggested that the proteinis localized in cell nuclei and is closely related to cellproliferation (Bishop, 1983). We have recently cloned anautonomously replicating sequence (ARS) from mouse cellswhich can replicate episomally in transfected mouse andhuman cells (Ariga et al., 1987). We have shown that thec-myc protein binds to this ARS and promotes its replicationin transfected cells (Iguchi-Ariga et al., 1987a). It wassuggested, therefore, that the c-myc protein may promotecellular DNA replication by binding to replication origins.This idea was supported by the cloning of an ARS fromhuman DNA as a sequence which bound the c-myc protein(Iguchi-Ariga et al., 1987a).

This function of c-myc protein in DNA replication isreminiscent of similar functions of SV40 T antigen and ofpolyomavirus T antigen. Both of these proteins bind to theorigin of DNA replication (SV40 ori or polyomavirus ori)and promote DNA replication (Tooze, 1980; DePamphilis

©IRL Press Limited, Oxford, England

-_ we

Fig. 1. Binding of c-myc product of HL-60 cells to sequences in thec-myc gene. A physical map of the human c-myc gene is shown in theupper part of the figure. Plasmids used in this study are shown on theleft and the carrying c-myc regions are indicated as black bars.O, non-coding exon; *, coding exon; PI, P2, promoters; B, BamHI;H, HindIII; P, PstI; K, KpnI; S, SnaI; X, XbaI; C, Clal; R, EcoRI.(A) 32P-labelled fragments were incubated with 5 A1 (lanes 1 and 3) or10 jd (lanes 2 and 4) of HL-60 nuclear extract and then precipitated asa complex with 2 jg of anti-human IgG (lanes 1 and 2) or anti-humanc-myc antibody (lanes 3 and 4). (B) The 32P-labelled fragments (laneM) were incubated with HL-60 nuclear extract in addition to non-labelled fragment from HindIll to KpnI (lanes 2 and 3), from HindIlIto PstI (lanes 4 and 5), from PstI to KpnI (lanes 6 and 7), from KpnIto SnaI (lanes 8 and 9) of c-myc gene and pUCl9 fragment fromHindIII to PstI (lanes 10 and 11). The amounts of DNA in thereactions were 0 jig (lane 1), 1 yg (lanes 2, 4, 6, 8 and 10) and 5 jig(lanes 3, 5, 7, 9 and 11). Bands in lane M represent the position ofpUC19 HindIII-PstI fragment, and c-myc gene upstream fragments ofHindRIl-KpnI region, KpnI-SmaI region and HindIII-PstI regionrespectively from the top. The equimolar fragments were mixed inlane M.

3135

_: :9 tl.

S.M.M.Iguchi-Ariga et al.

B

I,!i

Fig. 2. Replication of the upstream region of the c-myc gene in HL-60 cells. (A) Replication of the upstream regions of the c-myc gene in HL-60

cells. HL-60 cells were transfected with pmyc(H-P), pmyc(H-K), pmyc(K-S) and pUC19. Low mol. wt DNA was extracted 40 h after transfection,

digested with EcoRl and DpnI or MboI and analysed by the method of Southern (1975) using 32P-labelled pUC19 as a probe. (B) Inhibition of

replication of pmyc(H-P) in HL-60 cells by co-transfection with anti-human c-myc antibody. The DNAs were analysed by digestion with DpnI as in

A. Lane 1, pmyc(H-P) only; lane 2, pmyc(H-P) with 1 Ag of anti-human c-myc antibody; lane 3, pmyc(H-P) with 2 yg of anti-human c-mycantibody; lane 4, pmyc(H-P) with 2 yg of anti-human non-specific IgG. The M indicates the position of input EcoRI digested plasmid DNAs used

for transfection. C HL-60 cells transfected with pmyc(H-P) were passaged every 3 days and DNA was analysed using the DpnI assay at various daysafter transfection. M indicates the pmyc(H-P) digested with HindIII.

that these sequences have both autonomously replicatingactivity and transcriptional enhancer activity.

ResultsA c-myc protein binding region exists upstream of thec-myc geneWe first examined the binding of c-myc protein to the c-mycgene itself by an immunobinding assay. Upstream regionsof the human c-myc gene were subcloned into pUC19 (Figure1). Fragments of the c-myc gene spanning from HindIll toKpnI, KpnI to SmaI, and HindlIl to PstI (see Figure 1) anda vector pUC19 fragment from HindmI to PstI were selected,and their 3' ends were labelled with [ca-32P]dCTP. Resultsof immunobinding assays showed that two fragmentsincluding the region from HindIII to KpnI and from HindIlIto PstI were specifically precipitated with anti-human c-myc

antibody (Figure lA). These results suggest that c-myc

protein, or proteins complexed with c-myc protein, bind toa sequence in the HindIII-KpnI region upstream of thec-myc gene. To confirm these results, the same immuno-binding assay was carried out using various amounts ofnon-labelled DNA fragment as a competitor (Figure 1B).Precipitation of the fragments from HindmI to KpnI and fromHindUI to PstI with anti-c-myc antibody was inhibited bythe addition of unlabelled fragments into the immunobindingreaction, while neither the PstI to KpnI nor the KpnI to SmaIfragments could inhibit the precipitation. These results

suggest that the c-myc protein may bind to the HindIII-PstIregion.

ARS activity of the myc(H-P) regionWe next transfected HL-60 cells with cloned DNAcontaining the upstream region of the c-myc gene. In HL-60cells, a human promyelocytic leukaemia cell line, the c-mycgene is amplified to > 10 copies and is expressed vigorously.Two days after transfection, low mol. wt DNA was

extracted, analysed on agarose gels and blotted by the methodof Southern (1975) (Figure 2A). To quantitate the amountof plasmid actually replicated in the transfected cells, therecovered DNA was digested with DpnI to eliminateunreplicated DNA used for transfection and also with EcoRlto linearize the plasmid DNA before electrophoresis. Allplasmids used for transfection were grown in a dam+Escherichia coli strain, and adenine residues were methylatedat GATC sites. The plasmids are sensitive to cleavage byDpnI. The plasmid DNAs replicated in mammalian cells,however, are hemimethylated or unmethylated and are

insensitive to DpnI digestion, since dam methylase is absentin mammlian cells. The results (see Figure 2A) clearlyshowed that both pmyc(H-P) and pmyc(H-K) (see Figure1) replicated in HL-60 cells but pmyc(K-S) and pUC19 didnot. To confirm that DNA was replicated in the cells, DNAwas digested with MboI, which cuts unmethylated DNA. Theresults again showed that pmyc(H-P) and pmyc(H-K)replicated in HL-60 cells (Figure 2A). Replication of

3136

I.

l: = l.

F- F'f1 r-- :r.~-

%-"40L

,Y.

7",., 771- .,- C'

F.- l- -." Cl-

.I

Origin and enhancer of c-myc gene

A

1AA(;CTTGTTT (;GCCCTTTTA GGGTTTGTTG (;AATTT'rTTT TTCGTCTArG91TA('TT(;T(CAA TTATTTCACG TT'IGCCATfA CCGGTTCTCC ATAGGGTGAT

I10(;TTCATTA(;C AGTGGTCATA CCTTAATTTT CACATCTCTT ATGCGGTTGA

151ATi%(;TCACCT CTCAACCAAT T1TTCCTCCA GTAACTCCTC TTTCTTCGGA

201 210CCTTCTGCAG

IrA _-

E

B

Fig. 3. Transcriptional enhancer or promoter activity of the c-myc

upstream region with ARS activity. CAT assays were done withlysates of mouse L cells transfected with pPCAT(H-P)(+) [pSVPCATwith myc(H-P) fragment inserted downstream of the CAT gene],pPCAT(H-P)(-) [pSVPCAT with myc(H-P) fragment inserteddownstream of the CAT gene in the reverse orientation of pPCAT(H-P)(+)], p(H-P)(+)PCAT [pSVPCAT with myc(H-P) fragment insertedupstream of the promoter), p(H-P)(-)PCAT [pSVPCAT with myc(H-P) fragment inserted upstream of the promoter in a reverse orientationof p(H-P)(+)PCAT], p(H-P)CAT [pUCCAT with myc(H-P) fragmentinserted upstream of the CAT gene], pSV2CAT (pSVPCAT withSV40 enhancer inserted upstream of the promoter), pSVPCAT[pUCCAT with SV40 promoter (NsiI-HindIII site of SV40 genome),inserted upstream of the CAT gene] and pUCCAT (pUCl9 carryingCAT gene inserted into HindIlI site), from the left respectively. Ch,chloramphenicol; Ac-Cm, acetylated chloramphenicol. Numbers underthe figure show the average value of enhancer activity after scanningthe autoradiogram.

pmyc(H-P) was inhibited by addition of anti-human c-myc

antibody, but not by addition of nonspecific anti-human IgG(Figure 2B). These results indicate that the region upstreamof the c-myc gene [myc(H-P) region] contains an ARS andthat its replication depends upon c-myc protein. The plasmidwas stably maintained in the cells in the absence of selectionat least for 90 days (Figure 2C). To quantify the precisestability of transfected pmyc(H-P), the cells were furthercloned, and we measured what fraction of the cells in thepopulations contained the pmyc(H-P). All the populationsthat had been grown in the absence of selection containedthe pmyc(H-P). Furthermore, when pmyc(H-P) was injectedinto fertilized eggs to make transgenic mice, pmyc(H-P)could replicate in an episomal state in Fo mice and wastransmitted to at least F4 mice in an episomal state. Of> 100 mice including FO-F4 mice tested, all containedpmyc(H-P) in an intact form (K.Sudo, M.Ogata, T.Okazaki,Y.Sato, S.M.M.Iguchi-Ariga and H.Ariga, in preparation).These experiments clearly indicate that pmyc(H-P) can stablyreplicate in the cells, and the sequences necessary for stabilityof the plasmid in the cells may be present in the HindHI -PstI region of the c-myc gene.

Transcriptional enhancer activity of the myc(H-P)regionTo examine whether the c-myc upstream ARS region has

Tc

140T T A

TcC

T GA G

TT C A: IF

C C-90 C:GT A 130-T:A -150

T T TIAG A TG G TIC :G AC:G AI

80-A:T TATT G TT:A GIEA:T-100 C.:CC:G 1i0-A:T-160

TTTCACG TTTGC TTCATTAGC AGTGGTUAT CT(;AACCAAT TTTTCC70 110 l 170

A A1 70T T

C TC T

A TA CC. cT Tc C

160-T CC A-180T: cA:TC ATI: AC; C'A:TT CA C

150 A TC C-i90

CTT AT(.CCG TTTCTT

C

PARSh5

pmvc (H-IP)

pH I,mvc -I

1160 1770 i7809' ATGA1AACTCTTATATTGUT CTICAATAATTAAT 3'

* * * i**,.***x* *w* *** *.** * *

5 TTCACATCTCTTATA TGGCGGTTGAATAGTCAC% '129 * ^* ******* *** **

: CTACCA CTCTTATGC CCATTGA6CAGT ACG 3'15 2 5 4U 50

Fig. 4. Sequence and possible secondary structure of pmyc(H-P).(A) The nucleotide sequence of the fragment from HindIII to PstI ofhuman c-myc gene was determined by the dideoxy chain terminationmethod (Messing, 1983). (B) A possible secondary structure of thefragment was based on computer-assisted sequence analysis.(C) Comparison of the sequence among human ARS, pHLmycl(Iguchi-Ariga et al., 1987a), mouse ARS, pARS65 (Ariga et al.,1987) and pmyc(H-P). Stars indicate matched sequences andunderlined sequences are possible stems of hairpin loops ofpmyc(H-P).

some role in transcription, plasmids containing this regionand the bacterial chloramphenicol acetyltransferase (CAT)gene were constructed, and CAT enzyme activity wasassayed after the plasmids were transfected into mouse Lcells. The results, shown in Figure 3, suggest that the c-mycupstream ARS region [myc(H-P) region] has transcriptionalenhancer activity but not promoter activity. The enhanceractivity of the region was almost as strong as that of the SV40

3137


"e.i

.LL-~~~ ~ ~ ~ ~ ~ ~ * !p * ! 4FSf _

Fig. 5. Autonomously replicating activity and transcriptional enhancer activity of deletion mutants from PstI site of myc(H-P) region. (A) Deletionmutants of myc(H-P) region. Arrows indicate the inverted repeats which can possibly form stem and loop structures. (B) Autonomously replicatingactivity of deletion mutants of myc(H-P) region. Activity was assayed as described in the legend to Figure 2. Arrow indicates the band of replicatedDNA. (C) Transcriptional enhancer activity of deletion mutants of myc(H-P) region. The deleted fragments were inserted upstream of the promoterof pSVPCAT. Activity was assayed as described in the legend to Figure 3. Cm, chloramphenicol; Ac-Cm, acetylated chloramphenicol. Numbersunder the figure show the average value of enhancer activity after scanning the autoradiogram.

enhancer. The same results were also obtained after theplasmids were transfected into HL-60 cells (data not shown).

Sequence analysis of the myc(H-P) regionThe nucleotide sequence of the HindIII-PstI fragmentlocated upstream of the human c-myc gene [myc(H-P) region]has been determined by the dideoxy chain terminationmethod (Messing, 1983) (Figure 4A). The total number ofnucleotides is 210 and potential secondary structures areshown in Figure 4B. The sequence of the myc(H-P) regionis unique and without significant homology to sequencesreported to date for bacterial and viral DNA replicationorigins or ARSs in yeast. The sequence, however, possessespossible secondary structures which are observed in a varietyof replication origins from bacteria to animal viruses (Tooze,1980; Kornberg, 1982; DePamphilis and Bradley, 1986).Two hairpin loops can be constructed in the regions fromnucleotide 76 to 101 and from 120 to 160 (Figure 4B). Inthe latter region, two different loops can be constructedusing TGAATAGTCAC (nt 148-158). To determine thesequence essential for ARS activity, several deletion mutantsof pmyc(H-P) were constructed. pdl(H-P)-l carries thesequence from nucleotides 1-188 of the myc(H-P) fragment(see Figure SA), while pdl(H-P)-2 carries number 1-178;pdl(H-P)-3 carries number 1-120; pdl(H-P)-4 carriesnumber 1-114; pdl(H-P)-5 carries number 1-104 andpdl(H-P)-6 carries number 1-43 respectively. These mutant

3138

clones were transfected into HL-60 cells and examined forARS activity. The results are shown in Figure SB. Of thesemutants, only pdl(H-P)-1 had ARS activity in HL-60 as wellas pmyc(H-P). This suggests that the stem and loop structureat nucleotides 150-190 may be necessary for ARS activity.The deleted myc(H-P) fragments of the pmyc(H-P)

deletion mutants described above were also examined fortranscriptional enhancer activity by the CAT assay. Thedeleted fragments were inserted upstream of the promoterof pSVPCAT. As shown in Figure SC, pdl(H-P)-1-PCATand pdl(H-P)-2-PCAT showed strong enhancer activity, alittle higher than that of p(H-P)(+)PCAT, and pdl(H-P)-3-PCAT showed activity almost as high as p(H-P)(+)PCAT.pdl(H-P)-4-PCAT had weak activity while pdl(H-P)-5-PCAThad no detectable activity. However, pdl(H-P)-6-PCAT againgave strong CAT activity. To determine the sequencesessential for ARS activity, deletion mutants from the otherside of pmyc(H-P) were also constructed and ARS andenhancer activities were examined (Figure 6). Of thesemutants, pdlH-1 containing nucleotides 97-210 of themyc(H-P) had both ARS and enhancer activities, thestrengths of which were similar to those of pmyc(H-P).pdlH-2 containing nucleotides 118-210 had weak ARSactivity and no enhancer activity. Other mutants had neitherARS nor enhancer activities. It is therefore suggested that thesequence around nucleotides 114-125 may be responsiblefor transcriptional enhancer activity of the myc(H-P) region

... -


A

I.-. I

* I

* a* a

. -. H

* a

I I

Fig. 6. Autonomously replicating activity and transcriptional enhancer activity of deletion mutants from HindlIl site of myc(H-P) region. (A) Deletion

mutants of myc(H-P) region. Arrows indicate the inverted repeats which can possibly form stem and loop structures. (B) Autonomously replicating

activity of deletion mutants of myc(H-P) region. Activity was assayed as described in the legend to Figure 2. Arrow indicates the band of replicated

DNA. (C) Transcriptional enhancer activity of deletion mutants of myc(H-P) region. The deleted fragments were inserted upstream of the promoter

of pSVPCAT. Activity was assayed as described in the legend to Figure 3. Cm, chloramphenicol; Ac-Cm, acetylated chloramphenicol. Numbers

under the figure show the average value of enhancer activity after scanning the autoradiogram.

and that the sequence essential for initiation of DNAreplication is adjacent to, but cannot be separated from, thesequence with enhancer activity. pdl(H-P)-6-PCAT showedas strong an activity as pdl(H-P)-l-PCAT or pdl(H-P)-2-PCAT. Another enhancer sequence may, therefore, existin the dl(H-P)-6 fragment, and some negative regulatorysequences may be involved in the region of nucleotides43-104 of myc(H-P). So, it seems that there exist at leastthree regulatory regions in myc(H-P), spanning fromnucleotide 1 to 43, from 43 to 97 and from 97 to 188, thatwork positively, negatively and positively, respectively.

c-myc protein binding in the deletion mutantsThe immunobinding assay was carried out using variousdeletion mutants described above (Figure 7). Of the deletionmutants from the PstI site [dl(H-P) series], the fragmentsof dl(H-P)-1, -2 and -3 were precipitated, and a quite smallamount of the dl(H-P)4 fragment was precipitated by thec-myc protein antibody (Figure 7A). Of the deletion mutantsfrom the HindIll site (dlH series), the fragments dlH-1 and-2 were precipitated and those of dlH-3 and -4 were weaklyprecipitated (Figure 7B). Precipitation of the fragments ofdl(H-P)-5 and dlH-5 could not be observed. These resultssuggest that the c-myc protein binding region may extendfrom nucleotide 104 to 188 of myc(H-P) that contains bothARS and enhancer activities.

Transfection of the upstream sequences into HL-60cellsOur results suggested that the c-myc protein itself may bindto upstream regions of the human c-myc gene. To discoverhow this protein is concerned in the regulation of c-myc

expression, pmyc(H-P), pmyc(H-K), pmyc(P-K) andpmyc(K-S) were introduced into HL-60 cells as competitorsof binding to these proteins. HL-60 cells were transfectedwith the plasmid clones by a liposonie-mediated DNAtransfection technique (Itani et al., 1987). One day aftertransfection, the cells were harvested and total RNA was

extracted. The results of Northern blot hybridization are

shown in Figure 8. The amount of c-myc RNA markedlydecreased in the cells transfected with pmyc(H-P) or

pmyc(H-K), while no decrease was observed in cellstransfected with pmyc(K-S) or pmyc(P-K). This suggests thatexpression of c-myc RNA was affected by the transfectedplasmids which bind c-myc protein and, therefore, that thec-myc protein itself has some positive effect upon expressionof the c-myc gene. This idea is supported by results showingthat the amount of c-myc RNA also decreased in cellstransfected'with pARS65, which can autonomously replicatein human and mouse cells and may be bound by c-myc

protein (Ariga et al., 1987; Iguchi-Ariga et al., 1987a) (datanot shown) or by anti-c-myc antibody. Expression of theHGPRT gene was not affected by any DNA transfected(Figure 8A). The increasing amounts of transfected pmyc

3139

.-- I

L.

B ~

-Ai

-10,I

IMA. I-4-0 Im

I

iI

W..l


_ - m

Fig. 7. Binding of c-myc protein of HL-60 cells to sequences in the deletion mutants. Immunobinding assay was carried out as in Materials andmethods and in Figure 1. The plasmid DNA was digested with HindIH and EcoRI and 5' ends of resultant fragments were labelled with Klenowfragment. M indicates the DNA fragments used for reaction. The reactions of deletion mutants from PstI site [dl(H-P) series] and from HindIlI site(dlH series) were shown A and B respectively.

AY.- .Z.

.s.z.2... . i

Bc-myc 30i(1

('1 P-) ! -l t;L I( tq

9 : 'I () '3 (} ;>) I () '3 ( Il ?7'

HPRT m S(I] -)r - 1. (

1..f I i 3( i10 3 0}

S_u

Fig. 8. Expression of c-myc in HL-60 cells before and after transfection with upstream sequences. (A) HL-60 cells (1 x 107) were transfected with10 Ag of pmyc(H-P), pmyc(H-K), pmyc(P-K), pmyc(K-S), 2 pig of c-myc antibody or IgG. One day after transfection, total RNA was extracted fromthe cells and analysed by Northern blot hybridization. (-) shows the RNA of the HL-60 cells mock-transfected with control liposomes. The c-myc

fragments and human HGPRT gene were used as probes in the upper and lower figures respectively. Numbers under the figure show the average

value of c-myc or HGPRT expression after scanning the autoradiogram. (B) HL-60 cells were transfected with the various amounts of pmyc(H-P) or

pUC19, and the expression levels of c-myc and HGPRT were analysed as in A.

(H-P) inhibited c-myc expression in HL-60 cells (Figure 8B),suggesting that the c-myc protein may positively regulate itstranscription.3140

DiscussionThe results described here show that the HindIII-PstIfragment upstream of the c-myc gene contains a sequence

A B

-NW

.I - V, ".


bound by c-myc protein, or by protein(s) complexed withc-myc protein; this sequence can also function as ARS inhuman cells. We have cloned ARSs from mouse (Ariga et

al., 1987) and human (Iguchi-Ariga et al., 1987a) cells.Since these ARSs replicate depending upon c-myc protein,some consensus sequences necessary for recognition byc-myc protein or protein(s) complexed with c-myc proteinmay be present among these sequences. Homology among

these three ARSs is shown in Figure 4C: pHLmycl, a humanARS (Iguchi-Ariga et al., 1987a), and pARS65, a mouse

ARS (Ariga et al., 1987), all contain sequences homologousto TGAATAGTCA present in the stem of possible alternativeloops of pmyc(H-P) (Figure 4B). Furthermore, thesesequences homologous to pHLmyc are located in a putativehairpin loop of pHLmycl itself. Thus, TGAATAGTCAmay be quite important and may be a recognition site forc-myc protein or protein(s) complexed with it. The resultsof experiments with deletion mutants of pmyc(H-P) (Figure5A) also suggest that the stem and loop structure withTGAATAGTCA at the stem is necessary for ARS activityof pmyc(H-P). The myc(H-P) fragment has transcriptionalenhancer activity as well as ARS activity, suggesting thatthe c-myc protein may be an enhancer binding protein.Results with deletion mutants also suggest that the sequence

essential to ARS activity is adjacent to, but cannot beseparated from, the sequence responsible for enhanceractivity. The regions bound by c-myc protein, or protein(s)complexed with c-myc protein, cover the regions essentialfor ARS and enhancer activity. The strength of bindingwas different among such regions. pdl(H-P)-4, for instance,had no ARS activity, but weak enhancer activity. The regionof pdl(H-P)-4 had also quite weak binding activity. It maybe possible that the binding affinity of c-myc protein or

protein(s) complexed with c-myc protein, is different betweenARS and enhancer. Thus, enhancer function may be necess-

ary for ARS activity. This relationship is quite similar tothat of polyoma virus, in which DNA replication requiresenhancer in cis (de Villiers et al., 1984). Enhancer activityin the c-myc gene reported so far was in the region 500 nt(Chung et al., 1986) and 2.3 kb (Hay et al., 1987) upstreamof the first exon. The region described in this study is coveredby the latter case. These results suggest that expression ofthe c-myc gene is regulated by many elements.

Materials and methods

PlasmidsThe region of the c-myc gene described in Figure 1 was cloned into pUC 19;pmyc(H-K), for instance, myc(HindIII-KpnI) region was inserted intoHindI-KpnI sites of pUC19. The deletion mutants of pmyc(H-P) were

constructed as follows: pdl(H-P) series: pmyc(H-P) was digested with Sacdand BamHI present in the polylinker region of pUC 19, and this DNA was

further treated with exonuclese III followed by mung-bean nuclease. Aftertreatment of DNA with Klenow DNA polymerase, the DNA was self-ligated.pdlH series: after digestion of pmyc(H-P) with Hindf, the DNA was treatedwith Ba131 exonuclease. The digested DNA was treated with Klenow DNApolymerase and the HindIH linker was added to the end of DNA. The DNAwas digested again with HindIII and PstI and the resultant HindllI-PstIfragment of c-myc gene was inserted into the HindlIl -PstI site of pUC 19.

/mmunobinding assaypmyc(H-K), pmyc(K-S), pmyc(H-P) and pUC19 were digested with HindImand KpnI, KpnI and SmaI, HindI and PstI and HindIII and PstI respectively.The 3' ends of resultant fragments of the c-myc gene and pUC19(HindIII-PstI) were labelled with T4 DNA polymerase, and equimolar

amounts of labelled fragments were mixed together for immunobindingassays. The 32P-labelled DNA fragments were incubated with S or 10 1d1of HL-60 nuclear extract, prepared as described previously (Ariga andSugano, 1983) at 0°C for 1 h in 25 mM Tris-acetate buffer solution (pH6.5) containing 150 mM NaCl and 0.5% Nonidet P40, and then incubatedwith anti-human c-myc monoclonal antibody IF7 (Miyamoto et al., 1985)or anti-human nonspecific IgG (2 yg protein) at 0°C for 30 min. Thefollowing procedures were the same as described previously (Iguchi-Arigaet al., 1987a). For immunobinding assay using deletion mutants, 1.4%agarose gels were run in Tris-borate buffer (89 mM Tris, 89 mM boricacid, 2 mM EDTA).

Replication of plasmid DNA in HL-60 cellsTo examine replication of plasmid DNA, HL-60 cells were transfected withDNA or with DNA plus antibody by a liposome-mediated gene transfertechnique (Itani et al., 1987). 1 x 106 HL-60 cells grown in RPMI 1640medium supplemented with 10% fetal calf serum were transfected with 8 ugof DNA or 8 4g of DNA plus 1 or 2 Ag of antibody encapsulated in aliposome composed of 1 zmol of phosphatidylserine and cultured. Fortyhours after transfection, low mol. wt DNAs were extracted by the Hirtprocedure (Hirt, 1967), and digested with EcoRI to linearize the plasmidand with DpnI to eliminate the input plasmid DNA used for transfection,or with MboI to digest the replicated DNA. The digested DNA from theHirt supernatant was then electrophoresed on a 1.2% agarose gel, blottedby the method of Southern (1975) and hybridized with 3"P-labelled pUC19.Hybridization of the blotted filter with labelled probe was carried out asdescribed previously (Ariga et al., 1987).

CAT assayPlasmid DNAs were transfected into mouse L cells by the calcium phosphatemethod (Graham and van der Eb, 1973). Two micrograms of DNA wereused for each 106 cells. Two days after transfection, the cells wereharvested, suspended in 200 ul of 0.25 M Tris-HCI (pH 7.8), and disruptedby freeze-thawing three times prior to sonication. CAT assays were carriedout with the above cell lysate as described previously (Scholer and Gruss,1984).

Northern blot analysisTotal cellular RNA was extracted and purified by the method of Glisin etal. (1974) using guanidine isothiocyanate. RNA samples (20 sg), afterdenaturing by incubation at 65°C for 5 min in 10 mM sodium phosphatebuffer (pH 7.4) containing 2.2 M formaldehyde, 0.5 M EDTA and 50%formamide, were electrophoresed on 1% agarose gels containing 1.1 Mformaldehyde (Gonda et al., 1982), transferred onto nitrocellulose filtersand hybridized as described above. The probe DNAs used were aClaI-EcoRI fragment (1.5 kb) which contained almost all of the third exonand the contiguous non-coding region of the c-myc gene cloned from a humangastric carcinoma (Shibuya et al., 1985) or a human hypoxanthine-guaninephosphoribosyl transferase (HGPRT) gene fragment from pHPT4 (Brennandet al., 1982) with [32P]dCTP by nick translation.

AcknowledgementsWe gratefully acknowledge Yoshito Kaziro and Ken-ichi Arai for criticalreview of the manuscript and valuable suggestions. We also thank NobuoYamaguchi for valuable discussion. We are indebted to Toshio Ikeda, KohichiKawakami and Toshihiko Moritsuka for computer-assisted analysis. Thiswork was supported by grants from the Ministry of Education, Science andCulture in Japan and from the Princess Takamatsunomiya Cancer ResearchFund.

ReferencesAriga,H. and Sugano,S. (1983) J. Virol., 48, 481-491.Ariga,H., Itani,T. and Iguchi-Ariga,S.M.M. (1987) Mol. Cell. Biol., 7,

1-6.Bishop,J.M. (1983) Annu. Rev. Biochem., 52, 301-310.Brennand,J., Chinault,A.C., Konecki,D.S., Melton,D.W. and Caskey,C.T.

(1982) Proc. Natl. Acad. Sci. USA, 79, 1950-1954.Chung,J., Sinn,E., Reed,R.R. and Leder,P. (1986) Proc. Natl. Acad. Sci.

USA, 83, 7918-7922.DePamphilis,M.L. and Bradley,M.K. (1986) The Papovaviridae. Plenum,New York, Vol. 1, pp. 99-246.

de Villiers,J., Schaffner,W., Tyndall,C., Lupton,S. and Kaman,R. (1984)Nature, 312, 242-246.

Glisin,V., Crkvenjakov,R. and Byus,C. (1974) Biochemistry, 13,

3141

S.M.M.Iguchi-Ariga et a!.

2633-2637.Gonda,T.J., Sheiness,D.D. and Bishop,J.M. (1982) Mol. Cell. Biol., 2,617-624.

Graham,F.L. and van der Eb,A.J. (1973) Virology, 52, 456-467.Hay,N., Bishop,J.M. and Levens,D. (1987) Genes Dev., 1, 659-671.Hirt,J.B. (1967) J. Mol. Biol., 26, 365-369.Iguchi-Ariga,S.M.M., Itgani,T., Kiji,Y. and Ariga,H. (1987a) EMBO J.,

6, 2365-2371.Iguchi-Ariga,S.M.M., Itani,T., Yamaguchi,M. and Ariga,H. (1987b)

Nucleic Acids Res., 15, 4889-4899.Itani,T., Ariga,H., Yamaguchi,N., Tadakuma,T. and Yasuda,T. (1987)

Gene, 56, 267-276.Kornberg,A. (1982) Supplement to DNA Replication. W.H.Freeman, San

Francisco, CA.Messing,J. (1983) Methods Enzymol., 101, 20-73.Miyamoto,C., Chizzonite,R., Crowl,R., Rupprecht,K., Kramer,R.,

Schaber,M., Kumar,G., Poonian,M. and Ju,G. (1985) Proc. Natl. Acad.Sci. USA, 82, 7232-7236.

Scholer,H.R. and Gruss,P. (1984) Cell, 36, 403-411.Sheiness,D. and Bishop,J.M. (1979) J. Virol., 31, 514-521.Shibuya,M., Yokota,J. and Ueyama,Y. (1985) Mol. Cell. Biol., 5,414-418.

Southern,E.M. (1975) J. Mol. Biol., 93, 503-5 17.Tooze,J. (1980) DNA Turnor Viruses: Molecular Biology of Tumor Viruses,

Part 2, 2nd edn. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY.

Received on May 9, 1988; revised on June 27, 1988

3142

Documents

enhancer activity present upstream of the c-myc gene