Proc. Nati. Acad. Sci. USAVol. 86, pp. 840-844, February 1989Biochemistry

A single DNA response element can confer inducibility by botha- and y-interferons

(deletion analysis/gene expression/promoter)

LAURENCE E. REID, ADRIAN H. BRASNETT, CHRISTOPHER S. GILBERT, ANDREW C. G. PORTER,DIRK R. GEWERT, GEORGE R. STARK, AND IAN M. KERRImperial Cancer Research Fund Laboratories, P.O. Box 123, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

Contributed by George R. Stark, November 2, 1988

ABSTRACT Genomic and cDNA clones corresponding to9-27, a member of the human 1-8 gene family highly inducibleby a- and y-interferons (IFNs), have been isolated andcharacterized. A 1.7-kilobase genomic clone contains a com-plete functional gene with two exons, encoding a 125-aminoacid polypeptide of unknown function. The 5' flanking regionof the gene contains a 13-base-pair IFN-stimulable responseelement (ISRE), homologous to the ISREs of the 6-16, ISG 15,and ISG 54 genes, which are predominantly inducible byIFN-a,4. Analysis ofconstructs containing native and mutatedISREs suggests that this motif is essential for the response of9-27 to IFN-y as well as IFN-a. Furthermore, the 9-27(GGAAATAGAAACT) and 6-16 (GGGAAAATGAAACT)ISREs can each confer a response to both types of IFN whenplaced on the 5' side of a marker gene. Since the 6-16 gene doesnot normally respond to IFN-y, the context of the ISRE mustdetermine the specificity of the response.

Highly homologous response elements for a- and ,B-inter-ferons (IFNs) have been identified in the 5' flanking regionsof several IFN-inducible genes (1-6). They have partialhomology to other enhancer elements and bind at least threeIFN-modulated factors and a constitutive factor(s). Theformer appear with different kinetics after IFN treatment andare probably involved not only in the induction of transcrip-tion but also in its down-regulation and subsequent refractorystate (refs. 1-4 and T. C. Dale, A. M. A. Imam, I.M.K., andG.R.S., unpublished results).

Strongly differential responses in the induction of genes byIFN-a,,8 and -y have been observed in several instances. Forexample, the 6-16 gene (1, 7) and the ISG 15 and 54 genes (2,8) are highly induced by IFN-a and -,8 but not by IFN-y. Themajor histocompatibility complex class II genes and a num-ber of macrophage-specific genes are induced preferentiallyby IFN-y while the 1-8 gene family, the 2-5A synthetasegene, and the major histocompatibility complex class I genesare induced well by IFN-a,4 and -y (7, 9-12). The basis forthese differential responses is not understood.The 1-8 gene family was first identified through homology

to a cDNA corresponding to an IFN-inducible mRNA (13). Itis strongly induced by IFN-a,p and -y in all human cell linesstudied to date (7). To understand the regulation of the 1-8gene family in more detail and to compare it with theexamples cited above, we have isolated and sequenced agenomic clone for 9-27,* one member of the family, and haveanalyzed the differential effects of 5' deletions and of alter-ations in the IFN-stimulable response element (ISRE) uponinduction by IFN-a and -y.

MATERIALS AND METHODSGrowth of Cells, Transfections, Chloramphenicol Acetyl-

transferase (CAT) Assays, and IFN Treatment. These proce-dures were carried out as described (1). Human IFN-a, ahighly purified mixture [108 international units/ mg of protein(14)] was from Wellcome, U.K. Recombinant human IFN-aA/D (Bgl) hybrid, active on both human and mouse cells [2x 108 international units/mg of protein (15)], was fromSidney Pestka (Roche Institute of Molecular Biology). Re-combinant human and murine IFN-'y (1-4 x 107 internationalunits/mg of protein) were supplied by G. Adolf (ErnstBoehringer Institut ftir Arzneimittelforschung, Vienna).

Screening of Cosmid and cDNA Libraries. A library ofhuman lymphoid DNA cloned in the cosmid vector Lorist B(16) was plated on Pall Biodyne membranes that wereprocessed according to the manufacturer's instructions andscreened with a 9-27 3' cDNA fragment of250 base pairs (bp).AcDNA library made from mRNA ofHeLa cells treated withIFN-a for 10 hr was cloned in AgtlO and screened using the9-27 3' cDNA probe as above. The longest clone obtainedcorresponded to nucleotides (nt) -139 to +496 (numberedrelative to the initiator ATG; see Fig. 2).Mapping of Transcripts. The probe (Pst I-Bcl I fragment;

see Fig. 1A; 300,000 cpm by Cerenkov counting) was copre-cipitated with each RNA sample, resuspended in a solutionof80% (vol/vol) deionized formamide, 40mM Pipes (pH 6.4),400mM NaCl, and 1 mM EDTA and denatured at 85°C for 10min. Hybridization was at 52°C overnight. Digestion withribonuclease S1 (400 units/ml) was at 37°C for 30 min in asolution of 0.28 M NaCl, 4.5 mM ZnSO4, and 30 mM NaOAc(pH 4.4). The samples were extracted twice with phenol/chloroform, precipitated with ethanol, and electrophoresedin a 6% denaturing polyacrylamide gel. Plasmid pBR322, cutwith Hpa II and end-labeled by using the Klenow fragment ofDNA polymerase I, provided size markers.RNA Preparation and Northern Analysis of RNAs. All

cytoplasmic RNA samples were prepared by the phenol/chloroform method and analyzed by Northern transfer asdescribed (1). DNA probes were labeled by random-priming(17) to specific activities of 109 cpm/,ug and were used atconcentrations >106 cpm/ml.

Plasmid Constructions. Whole-gene constructs (Fig. JA). A3.4-kilobase (kb) HindIII-Pst I fragment and a 2-kb Pst Ifragment were subcloned from the cosmid into pUC19 toform pHP3.4 and pPP2.0, respectively (Fig. 1A). PlasmidpSBl.OpUC19 was generated by cloning the 1.0-kb Spe I-BamHI fragment (Fig. 1A) into pUC19 cut with Xba I and

Abbreviations: IFN, interferon; ISRE, interferon-stimulable re-sponse element; tk, thymidine kinase; nt, nucleotide(s); CAT,chloramphenicol acetyltransferase.*The sequence reported in this paper is being deposited in theEMBL/GenBank data base (accession no. J04164).

840

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Oct

ober

20,

202

0

Proc. Natl. Acad. Sci. USA 86 (1989) 841

A cn -aa. co

Si PROBE

0 a._I cn m)LC.

0.5kb

Em

CL.n a.

pHP3.4pPP2.0pSP1.7

LtC Ltk LtKB + + + Ltk-

pHP3.4 pPP2 C pSP1.7

_ ( u/ - V - Cyy - c 7

kb

4.0

2.0

1.0 mm *

FIG. 1. Structure and analysis of 9-27 genomic clones. (A) Mapof the 9-27 gene showing the position of the exons (solid boxes), thesubclones used in transfection assays in mouse Ltk- cells, and theprobe used in the analyses with ribonuclease S1. The positions oftherestriction sites used in generating the subclones are indicated. (B)Northern analysis ofRNA isolated from mouse Ltk- cells and Ltk-cells stably transfected with the 9-27 subclones indicated in A andgrown in the absence (lanes -) or presence of recombinant humanIFN-a A/D (Bgl) hybrid (500 international units/ml) (lanes a) orrecombinant murine IFN-y (1000 international units/ml) (lanes y) for18 hr.

BamHI. Plasmid pSP1.7 was constructed by ligation of the1.0-kb HindIII-BamHI fragment from pSB1.OpUC19 withthe 0.7-kb BamHI-Pst I fragment from pPP2.0, into pGEM-4linearized with HindIII and Pst I.CAT constructs (see Fig. 4). pHB1.8CATh' was derived

from pHP3.4 by digestion with BspMII, treatment with theKlenow fragment, and digestion with HindIII to release a1.8-kb fragment that was cloned into pCATh' that had beenlinearized at its unique BamHI site, treated with the Klenow

fragment, and cut with HindIll. Plasmid pPBO.4CATb' wasderived from pPP2.0 in an identical fashion. Plasmid pCATh'is very similar to pSVOCAT (18) but with the introduction ofa polylinker just on the 5' side of the CAT gene. PlasmidpSBO.2pUC19 was derived from pSB1.OpUC19 by digestionwith BspMII and BamHI, treatment with the Klenow frag-ment, and ligation at low concentration to delete the 800-bpBspMII-BamHI fragment (regenerating the BamHI site).Plasmid pSBO.2CATh' was generated by cloning the 230-bpHindIII-BamHI fragment (equivalent to the Spe I-BspMIIfragment, Fig. LA) from pSBO.2pUC19 into pCATh'. PlasmidpPAWpUC19 was generated by cloning a synthetic double-stranded oligonucleotide (nt -213 to nt -161 of the 9-27 genewith Pst I and Afl II complementary overhanging ends) intopSBO.2pUC19 that had been linearized with these enzymes todelete the fragment from nt -316 to nt -160. Thus, pPAW-pUC19 is identical to pSBO.2pUC19 except for a deletionfrom the Pst I site in the polylinker to the cytidine at nt -213in the 5' end of 9-27. A second plasmid, pPAMpUC19,constructed similarly, is identical to pPAWpUC19 except fortwo A -+ C mutations at nt -163 and nt -169 in the 5' endof 9-27. pPAWCATb' and pPAMCATh' were derived frompPAWpUC19 and pPAMpUC19, respectively, by isolation ofthe HindIII-BamHI fragment (equivalent to nt -213 to nt-86, Fig. 2) and cloning into pCATh' linearized with thoseenzymes. Plasmid p6-16CAT (see Fig. 5E) was generated bycloning a 1041-bp Bgl II-BamHI fragment, derived from the5' end of the 6-16 gene into pSVOCAT (1).

RESULTSIsolation and Transfection of 9-27 Genomic Clones. A

cosmid library ofDNA from human lymphoid cells (16) wasscreened using a 250-bp 9-27 cDNA probe corresponding tothe 3' end of the mRNA. This probe hybridizes to a uniquefragment on a Southern transfer ofgenomic DNA (13). From500,000 cosmids screened, four identical positive clones wereisolated; their restriction maps agree with previous data fromgenomic Southern transfers (13).The cosmid and subclones of the region spanning the 9-27

gene (Fig. 1A) were cotransfected into mouse Ltk- cells witha thymidine kinase (tk) expression plasmid [pAGO (20)] andstable populations were selected. RNA was isolated from thetransfected populations and the parental Ltk- line, with or

SpeICTAGTCCTGA CTTCACTTCT GATGAGGAAG CCTCTCTCCT TAGCCTTCAG CCTTTCCTCC CACCCTGCCA TAAGTAATTT GATCCTCAAG AAGTTAAACC-216 AflII vACACCTCATTGLGTCCCTGGC TAATTCACCAATTACAAAC AGCAGGAAAT AGAAACTTAA GAGAAATACA CACTTCTGAG AAACTGAAAC GACAGGGGAA

ISRE-116 BspMIIAGGAGGTCTC ACTGAGCACC GTCCCAGCAT CCGGACACCA CAGCGGCCCT TCGCTCCACG CAGAAAACCA CACTTCTCAA ACCTTCACTC AACACTTCCT

-16 -1 1TCCCCAAAGC CAGAAG ATG CACAAG GAG GAA CAT GAG GTG GCT GTG CTG GGG GCA CCC CCC AGC ACC ATC CTT CCA AGG TCC ACC

M H K E E H E V A V L G A P p S T I L P R S T+70 BclIGTG ATC AAC ATC CAC AGC GAG ACC TCC GTG CCC GAC CAT GTC GTC TGG TCC CTG TTC AAC ACC CTC TTC TTG AAC TGG TGC TGTV I N I H S E T S V P D H V V W S L F N T L F L N W C C

+154 vCTG GGC TTC ATA GCA TTC GCC TAC TCC GTG AAG TCT AGG GAC AGG AAG ATG GTT GGC GAC GTG ACC GGG GCC CAG GCC TAT GCCL G F I A F A Y S V K S R D R K M V G D V T G A Q A Y A

+238TCC ACC GCC AAG TGC CTG AAC ATC TGG GCC CTG ATT CTG GGC ATC CTC ATG ACC ATT GGA TTC ATC CTG TCA CTG GTA TTC GGCS T A K C L N I W A L I L G I L M T I G F I L S L V F G

+322 SpeITCT GTG ACA GTC TAC CAT ATT ATG TTA CAG ATA ATA CAG GAA AAA CGG GGT TAC TAG TAG CCGCCCATAG CCTGCAACCTS V T V Y H I M L Q I I Q E K R G Y

+412TTGCACTCCA CTGTGCAATG CTGGCCCTGC ACGCTGGGGC TGTTGCCCCT GCCCCCTTGG TCCTGCCCCT AGATACAGCA GTTTATACCC ACACACCTGT

+512CTACAGTGTC ATTCAATAAA GTGCACGTGC TTGTGA(n)

FIG. 2. Sequence of the 9-27 genomic 5' flanking region and cDNA. The ISRE, the two CCAAT boxes (one inverted), and thepolyadenylylation signal are underlined. The positions of the intron (v), the two major transcription initiation sites (v), and the 5' end of thelongest available cDNA are shown. The sequence ends with the first adenosine of the poly(A) tail, indicated as A(n).

Biochemistry: Reid et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

20,

202

0

Proc. Natl. Acad. Sci. USA 86 (1989)

without treatment for 16 hr with recombinant human IFN-aA/D (Bgl) hybrid (highly active on mouse as well as humancells) or recombinant murine IFN-y. Northern transfers oftheRNAs were analyzed using the 9-27 3' cDNA as a probe.Mouse cells transfected with the cosmid express the humangene in response to both IFNs to levels comparable to thoseobserved in parallel experiments with HeLa cells (cf. Fig. 3).The subclones all respond to both A/D hybrid IFN and murineIFN-y (Fig. 1B). There was some diminution of expressionwith the decreasing size of the subclones, but it is clear thatsequences governing the basic response of9-27 to both IFN-aand - y reside on the 3' side of the Spe I site (Fig. 1).

Sequence Analysis of a Full-Length 9-27 cDNA and the Ge-nomic Clone. An essentially full-length cDNA for 9-27 wasobtained and sequenced. It contains an open reading frame of125 amino acids, corresponding to a polypeptide with a pre-dicted molecular mass of 13.9 kDa (Fig. 2). An anti-peptideantibody recognizes IFN-inducible polypeptides of this size(L.E.R., urnpublished results), strongly implying that the openreading frame deduced from the cDNA, on which the peptideis based, is correct. Comparison of genomic and cDNA se-quences shows that the gene has only two exons (Fig. 1A). Theexon and cDNA sequences agree exactly. As initiation oftranscription is heterogeneous (see below), the sequence isnumbered from the initiator ATG in the first exon (Fig. 2).

Important features of the 5' flanking region are two CCAATboxes (nt -185 to nt -189 and nt -205 to nt -209, Fig. 2),inverted in orientation with respect to each other, and a 13-bpsequence (GGAAATAGAAACT, nt -160 to nt -172) highlyhomologous to the ISREs identified in 6-16 and other IFN-inducible genes (Table 1). The ISRE is positioned 18 bpupstream ofthe more 5' ofthe two major transcription initiationsites determined by analysis with ribonuclease S1 (see below).

Initiation of Transcription. RNA prepared from HeLa cellsand from Ltk- cells stably transfected with the 9-27 cosmid,with or without treatment with IFN-a or -y (Fig. 3), wasanalyzed with ribonuclease Sl. The results, in agreement withdata from primer extension experiments (A.H.B., unpublishedresults), showed that initiation oftranscription ofthe 9-27 geneis heterogeneous over a region of -30 bp, with two major startsites at approximately nt -140 and nt -112 (211 and 183 bpupstream of the Bcl I site at nt +71, Fig. 2). The pattern ofinitiation is identical in response to IFN-a and -y and appearsto be reproduced faithfully by the transfected gene (Fig. 3).

Sequences Governing the Transcriptional Response of 9-27to IFN-a and -y. To study the ability of 5' flanking regions of

Ltk- +cos 9-27

1- (f.'I

FIG. 3. Initiation of tran-scription of the 9-27 gene. RNA

238 was isolated from HeLa cells

217 and from mouse Ltk- cells sta-201 bly transfected with the 9-27

-1 90 cosmid, grown in the absence1 80 (lanes -) or presence of recom-

binant human IFN-a A/D (Bgt)hybrid (500 international units/ml) (lanes a) or recombinant mu-rine IFN-y (1000 internationalunits/ml) (lanes y) for 18 hr. TheRNA was analyzed with ribonu-clease S1 using the probe shown

- 1 10 in Fig. 1A labeled at the Bcl Isite. The products of S1 diges-tion were electrophoresed in a6% denaturing polyacrylamidegel with labeled DNA markers

9 0 (pBR322 cut with Hpa II), whichare shown on the right.

Table 1. ISREs of human and murine genesGene Sequence Location, nt

Human9-27 AGGAAATAGAAACT - 172 to - 1606-16 (1) G*****AT****** -112 to -99

-153 to -140ISG 54 (2, 5) G*****GT****** -87 to - 100ISG 15 (2) G*****CC****** - 108 to -952-5A syn-

thetase (3, 4) A*****C-*****C -100 to -87MurineMx (6) CA****C-****** -120 to -1312-5A synthetase (3) G*****TG****** -73 to -59

Consensus GGAAAN(N)GAAACTHomologous

region ofFriedman-Starkconsensus (21) AGAAGA-GAAACT

Sequences are all in 5' flanking regions of the genes and have beendemonstrated to be important for induction of transcription inresponse to IFN-a. Positions are given relative to sites of initiationof transcription except for 9-27 and the 2-5A synthetase genes, wherethe position is relative to the adenosine residue of the initiationcodon. Refs. are given in parentheses. * indicates identity.

9-27 to confer IFN inducibility upon a heterologous gene, aseries of fragments from these regions were cloned into thevector pCATb' that contains the CAT gene but no promoteror enhancer sequences (Fig. 4A). Lysates prepared fromtransiently transfected HeLa cells, with or without IFN-a or-y treatment, were assayed for CAT activity (Fig. 4B).Control experiments established that the IFN concentrationsand time points chosen were within the optimal range for eachtype of IFN.DNA from the BspMII site at nt -86 to nt -213, just on the

5' side of the upstream CCAAT box, is sufficient to conferinducibility in response to each type ofIFN (pPAWCATh', Fig.4 A and B). The maximum response to IFN-y is -60%o of theresponse to IFN-a for all of the constructs, in good agreementwith the levels of induction ofmRNA observed for the endog-enous 9-27 gene and for whole gene transfectants (Fig. 1B). Incontrast, CAT constructs usingDNA from the 5' flanking regionof the 6-16 gene show a response to IFN-y that is <10%o of theresponse to IFN-a (e.g., Fig. 5C) as is the case for the intact 6-16gene (7). The basis for the gradual decrease in the levels ofexpression upon deletion through the 5' flanking region of 9-27(Figs. 1 and 4 A and B) is not understood. The increased signalobserved with pPAWCATh' compared to pSBO.2CATh' isreproducible and may reflect negative regulation between nt-213 and nt -317. It seems clear however, that the sequencesessential for regulation of9-27 by IFN are located 3' ofthe distalCCAAT box.

Mutation of the ISRE. pPAMCATh' is identical to pPAW-CATh' except for two C-* A point mutations in the ISRE givingGGACATAGA-CACT in place of GGAAATAGAAACT be-tween residues -160 and -172. These mutations abolish theresponse to both types of IFN (Fig. 4C) confirming that theISRE is essential for the response to both IFN-a and -y.The 9-27 and 6-16 ISREs Can Confer Responsiveness to Both

IFN-a and -y. Oligonucleotides corresponding to the 6-16(GGGAAAATGAAACT) and 9-27 (GGAAATAGAAACT)ISREs and a mutant 6-16-(GGGAAAATGACACT) ISREwere cloned into the BamHI site of pBLCAT2 (19) immedi-ately upstream ofan intact tk promoter fused to the CAT gene(Fig. SD). (The 9-27 oligonucleotide was also cloned into theHindIII site with no evident difference in the resultsobtained.) These constructs were assayed for their ability torespond to IFN as described above. The ISREs from 9-27 and6-16, but not the mutant ISRE, are capable of conferring a

Hela

[-7- ../

I..

842 Biochemistry: Reid et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

20,

202

0

Proc. Natl. Acad. Sci. USA 86 (1989) 843

Hindell Pstl Spiel ISRE BspMII-I

-1 800

-51 0

-31 6

-21 3

0 0 0 23 h

1200 0 6 <I m m L

1000

800

01. l

1200-I: C

A~~~~~~~~~~~~a

C

400

300

E

0

200

100

0-

H H

a-)

0r.

FIG. 4. Response of 9-27 CAT

pHB 1.8 CATb' constructs to IFN-a and -y. (A)Map of the 9-27 5' flanking region

pPB 0.4 CATb' nt -1800 to nt +1, including theCCAAT boxes (inverted arrow-

pSB 0.2 CATb' heads), major transcription initia-

pPAW CATb' tion sites (arrows), and the ISRE.Fragments were generated withthe restriction enzymes shown or,for pPAWCATh' and pPAMCAT-b', constructed with a combina-tion of synthetic oligonucleotidesand restriction fragments andcloned into the vector pCATh'. (Band C) CAT activity was assayedin lysates from HeLa cells tran-siently transfected with the con-structs shown in A, pCATh', orpPAMCATh', which is identicalto pPAWCATh' except for twomutations in the ISRE. Lysateswere from control cells (openbars) or cells treated with IFN-a(solid bars) or IFN-y (stripedbars), each at 1000 internationalunits/ml for 30 hr.

comparable response to IFN-a and -y upon a heterologouspromoter (Fig. 5 A and B). This is in contrast with the resultsobtained for the 6-16 ISRE within the context of its own

promoter where there is a strongly preferential response toIFN-a (e.g., Fig. 5C).

DISCUSSIONA functional 9-27 gene has been isolated that, after stabletransfection into mouse Ltk- cells, is inducible by bothIFN-a and -y in a manner apparently identical to that of the

A

20

CV)0

0

0

B

49-27 MUTANT

pBL ISRE ISRE

endogenous human gene (Fig. 3). A 1.7-kb genomic fragmentcovers the entire gene including all the information essentialfor induction by the IFNs (Fig. 1). A full-length 9-27 cDNAencodes a polypeptide of 125 amino acids (Fig. 2). Neither theamino acid nor the nucleotide sequence shows any extensivehomology with sequences in the EMBL or GenBank databases (Release 16, 57 PIR 17, October 1988), nor does theamino acid sequence provide any obvious clue to likelyfunction. The 9-27 gene has two exons. The sequences at theexon-intron boundaries closely resemble those of the con-

C0_ 4

20,

30

20

1 0 -

0

6-1 6pBL ISRE

nn

1 0

0

p6-16 CAT

nSf

D-105 +57 CAT

ISRE

E

-603

FIG. 5. Response ofISRE-CAT and 6-16-CAT constructs to IFN-a and -y. (A-C) CAT activity in lysates from HeLa cells. (D and E) Mapsof the constructs. The cells were transfected transiently with 9-27 ISRE-CAT or a mutant ISRE-CAT (A), 6-16 ISRE-CAT (B), or p6-16CAT(C). Treatment with IFN-a (solid bars) or IFN-y (striped bars) at 1000 international units/ml was for 48 hr (A and C) or 24 hr (B). (Similar resultshave been obtained between 24 and 48 hr for all of the constructs.) For the ISRE constructs double-stranded oligonucleotides correspondingto the 9-27 (GGAAATAGAAACT), the 6-16 (GGGAAAATGAAACT), or a mutant 6-16 (GGGAAAATGACACT) ISRE were inserted into theBamHI site of the polylinker upstream of the tk promoter driving CAT in pBLCAT2. For p6-16CAT, a 5' fragment (nt -603 to +437) spanningthe promoter region of the 6-16 gene was inserted into pSVOCAT (1, 18). A similar differential response to IFN-a and -y was obtained withconstructs (1) in which shorter 5' fragments of 6-16-i.e., nt -603 to +42 and nt -603 to -40-were inserted into pSVOCAT (18) and pAjoCAT2(22), respectively (data not presented).

A

I

I

Biochemistry: Reid et al.

00. CAT

+437

Dow

nloa

ded

by g

uest

on

Oct

ober

20,

202

0

Proc. Natl. Acad. Sci. USA 86 (1989)

sensus sequences (23, 24) for such sites (data not presented).There is virtually no homology with the Kozak consensussequence (25) in the nucleotides surrounding the initiatingATG. Consistent with the absence of a TATA box initiationof transcription is heterogeneous, for both the endogenousand the transfected genes (Fig. 3). Initiation is from the sameset of sites in response to both IFN-a and --y but, in contrastto the mouse 202 gene (26), IFN treatment does not appearto stimulate transcription preferentially at one particular site.

Fragments from the 5' flanking region of the 9-27 gene andthe 9-27 or 6-16 ISREs alone can confer inducibility inresponse to IFN-a and -y (Figs. 4 and 5). In the experimentswith pBLCAT2 a small response (<2-fold) of the vector aloneto each type of IFN was frequently observed (Fig. 5). Thesignificance of this is not clear, but in the light of the muchgreater stimulation observed (>10-fold) with the constructscontaining the ISRIE, and the additional evidence highlightingthe importance of the ISRE, we do not believe that the overallinterpretation of the results is affected. Consistent with theimportance of the ISREs for the IFN response, they interactwith IFN-a and -y-modulated protein factors in gel retarda-tion assays (refs. 1-4; A. M. A. Imam, T. C. Dale, I.M.K.,and G.R.S., unpublished results). More conclusively, twopoint mutations in the 9-27 ISRE abolish its ability to conferinducibility by either IFN-a or -y (Fig. 4C). Accordingly, therole, if any, of other elements in the 5' region of the sequencewith partial homology to the ISRE (e.g., GAAACT atresidues -137 to -132 and GGAAA at residues -120 to-116) can only be secondary and dependent on the ISRE. Itis reasonable to conclude that the ISRE is the major elementgoverning the response of the 9-27 gene to IFN-a and -y.Others (3, 4) have demonstrated the ability of a relatedsequence in the IFN-a and -^y-responsive 2-5A synthetasegene (Table 1) to confer a response to IFN-a but did notaddress its possible role in the response ofthat gene to IFN-y.The IP10 gene responds preferentially to IFN-y in macro-phages (10) and its 5' flanking region (27) contains an ISREhomology, but this has not been shown to play a role inregulation by IFN. As discussed for the 6-16 ISRE (1), the9-27 ISRE has good homology with part of the Friedman-Stark consensus sequence (Table 1 and ref. 21) analyzed inmost detail for the class I major histocompatibility complexgenes (28-30). Recent results indicate that it is this part of theconsensus that is most important in governing regulation oftranscription by IFN-a (31).The IFN-a and -y-responsive 9-27 ISRE is highly homol-

ogous to the ISRE of the predominantly IFN-a-inducible 6-16gene (Table 1). It is interesting that the latter is also capableof conferring responsiveness to IFN-y upon a CAT genedriven by the tk promoter (Fig. 5) although it does not do soin its natural context within the 6-16 gene in human cells (7).The basis for this is not clear, but from a deletion analysis itdoes not appear to reflect a negative element upstream of thetandem repeat containing the ISREs in 6-16 (A.C.G.P. andI.M.K., unpublished results). Moreover, when stably trans-fected into mouse L cells, the human 6-16 gene respondsalmost as well to IFN-y as to -a (A.C.G.P. and I.M.K.,unpublished results). A more detailed analysis of the func-tional elements in the 5' flanking region of the 6-16 gene andof the proteins interacting with them will be required to definethe basis for this context effect.The fact that a single ISRE appears to govern the response

to both IFN-a and -y for the 9-27 gene does not exclude theinvolvement of a quite separate response element for othergenes regulated by IFN-y, as indeed has been suggested forthe class II HLAs (32-34). The IFN-y response is biphasic(35); for some genes it is primary-i.e., not dependent onprotein synthesis (e.g., ref. 11)-and for others it is second-

ary (e.g., ref. 36). It is not unreasonable to suggest thatdifferent response elements may be involved. Overall, theresults are consistent with ISREs of the type described beingbasic IFN response elements upon which additional controlscan be superimposed.

We thank Drs. Martin McMahon and Susan Manly for theircontributions to early stages of this work, Dr. Peter Little for thecosmid library, Dr. Nicholas Jones for pCATh', lain Goldsmith forsynthesizing oligonucleotides, and Drs. Stephen Goodbourn andMalcolm Parker for advice and comments on the manuscript.1. Porter, A. C. G., Chemajovsky, Y., Dale, T. C., Gilbert, C. S.,

Stark, G. R. & Kerr, I. M. (1988) EMBO J. 7, 85-92.2. Levy, D. E., Kessler, D. S., Pine, R., Reich, N. & Darnell, J. E.,

Jr. (1988) Genes Dev. 2, 383-393.3. Cohen, B., Peretz, D., Vaiman, D., Benech, P. & Chebath, J. (1988)

EMBO J. 7, 1411-1419.4. Rutherford, M. N., Hannigan, G. E. & Williams, B. R. G. (1988)

EMBO J. 7, 751-759.5. Wathelet, M. G., Clauss, I. M., Content, J. & Huez, G. A. (1988)

Eur. J. Biochem. 174, 323-329.6. Hug, H., Costas, M., Staeheli, P., Aebi, M. & Weissmann, C. (1988)

Mol. Cell. Biol. 8, 3065-3079.7. Kelly, J. M., Gilbert, C. S., Stark, G. R. & Kerr, I. M. (1985) Eur.

J. Biochem. 153, 370-371.8. Larner, A. C., Jonak, G., Cheng, Y.-S. E., Korant, B., Knight, E.

& Darnell, J. E., Jr. (1984) Proc. NatI. Acad. Sci. USA 81, 6733-6737.

9. Revel, M. & Chebath, J. (1986) Trends Biochem. Sci. 11, 166-170.10. Luster, A. D., Unkeless, J. C. & Ravetch, J. V. (1985) Nature

(London) 315, 672-676.11. Fan, X., Goldberg, M. & Bloom, B. R. (1988) Proc. Natl. Acad. Sci.

USA 85, 5122-5125.12. Newburger, P. E., Ezekowitz, R. A. B., Whitney, C., Wright, J. &

Orkin, S. H. (1988) Proc. Nati. Acad. Sci. USA 85, 5215-5219.13. Friedman, R. L., Manly, S. P., McMahon, M., Kerr, I. M. & Stark,

G. R. (1984) Cell 38, 745-755.14. Allen, G., Fantes, K. H., Burke, D. C. & Morser, J. (1982) J. Gen.

Virol. 63, 207-212.15. Rehberg, E., Kelder, B., Hoal, E. G. & Pestka, S. (1982) J. Biol.

Chem. 257, 11497-11502.16. Cross, S. H. & Little, P. F. R. (1986) Gene 49, 9-22.17. Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem. 132, 6-13.18. Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982) Mol. Cell.

Biol. 2, 1044-1051.19. Luckow, B. & Schutz, G. (1987) Nucleic Acids Res. 15, 5490.20. Colbere-Garapin, F., Chousterman, S., Horodniceanu, F., Kouril-

sky, P. & Garapin, A.-C. (1979) Proc. Natl. Acad. Sci. USA 76,3755-3759.

21. Friedman, R. L. & Stark, G. R. (1985) Nature (London) 314, 637-639.

22. Rosenthal, N., Kress, M., Gruss, P. & Khoury, G. (1983) Science222, 749-755.

23. Mount, S. M. (1982) Nucleic Acids Res. 10, 459-472.24. Shapiro, M. B. & Senapathy, P. (1987) Nucleic Acids Res. 15, 7155-

7174.25. Kozak, M. (1987) J. Mol. Biol. 196, 947-950.26. Samanta, H., Engel, D. A., Chao, H. M., Thakur, A., Garcia-

Blanco, M. A. & Lengyel, P. (1986) J. Biol. Chem. 261, 11849-11858.

27. Luster, A. D. & Ravetch, J. V. (1987) Mol. Cell. Biol. 7, 3723-3731.28. Israel, A., Kimura, A., Fournier, A., Fellous, M. & Kourilsky, P.

(1986) Nature (London) 322, 743-746.29. Sugita, K., Miyazaki, J., Appella, E. & Ozato, K. (1987) Mol. Cell.

Biol. 7, 2625-2630.30. Korber, B., Mermod, N., Hood, L. & Stroynowski, I. (1988)

Science 239, 1302-1306.31. Shirayoshi, Y., Burke, P. A., Appella, E. & Ozato, K. (1988) Proc.

Natl. Acad. Sci. USA 85, 5884-5888.32. Boss, J. M. & Strominger, J. L. (1986) Proc. Natl. Acad. Sci. USA

83, 9139-9143.33. Miwa, K., Doyle, C. & Strominger, J. L. (1987) Proc. Natl. Acad.

Sci. USA 84, 4939-4943.34. Thanos, D., Mavrothalassitis, G. & Papamatheakis, J. (1988) Proc.

NatI. Acad. Sci. USA 85, 3075-3079.35. Dianzani, F., Zucca, M., Scupham, A. & Georgiades, J. A. (1980)

Nature (London) 283, 400-402.36. Blanar, M. A., Boettger, E. C. & Flavell, R. A. (1988) Proc. NatI.

Acad. Sci. USA 85, 4672-4676.

844 Biochemistry: Reid et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

20,

202

0