MOLECULAR AND CELLULAR BIOLOGY, Dec. 1994, p. 7758-77690270-7306/94/$04.00+0Copyright © 1994, American Society for Microbiology

Interaction between a Novel F9-Specific Factor and Octamer-BindingProteins Is Required for Cell-Type-Restricted Activity of the

Fibroblast Growth Factor 4 EnhancerLISA DAILEY,1* HUABING YUAN,2 AND CLAUDIO BASILICO2*

The Rockefeller University, New York New York 10021,1 and Department of Microbiology,New York University School of Medicine, New York, New York 100162

Received 5 July 1994/Returned for modification 30 August 1994/Accepted 9 September 1994

Understanding how diverse transcription patterns are achieved through common factor binding elements isa fundamental question that underlies much of developmental and cellular biology. One example is providedby the fibroblast growth factor 4 (FGF-4) gene, whose expression is restricted to specific embryonic tissuesduring development and to undifferentiated embryonal carcinoma cells in tissue culture. Analysis of the cis-and trans-acting elements required for the activity of the previously identified FGF-4 enhancer in F9 embryonalcarcinoma cells showed that enhancer function depends on sequences that bind Spl and ubiquitous as well as

F9-specific octamer-binding proteins. However, sequences immediately upstream of the octamer motif, whichconform to a binding site for the high-mobility group (HMG) domain factor family, were also critical toenhancer function. We have identified a novel F9-specific factor, Fx, which specifically recognizes this motif. Fxformed complexes with either Oct-i or Oct-3 in a template-dependent manner. The ability of different enhancervariants to form the Oct-Fx complexes correlated with enhancer activity, indicating that these complexes playan essential role in transcriptional activation of the FGF-4 gene. Thus, while FGF-4 enhancer function isoctamer site dependent, its developmentally restricted activity is determined by the interaction of octamer-binding proteins with the tissue-specific factor Fx.

Embryonic development is determined by a complex seriesof intercellular interactions that include direct cell-cell con-

tacts as well as responses to signaling molecules. Among thesignaling molecules identified to date, members of the mam-malian fibroblast growth factor (FGF) family have been shownto play an integral part in both early and late stages ofembryogenesis (1, 16). Although the members of the FGFfamily share several overlapping activities in tissue cultureassays, the distinct pattern of expression of each gene in thedeveloping embryo suggests that individual FGFs play uniqueroles in vivo. It is thus implied that specific developmentalevents require the temporally and spatially regulated expres-sion of particular FGF genes during embryogenesis.The FGF-4 gene (originally called kFGF or hst) (7, 67) is

expressed in all cells of the blastocyst inner cell mass as well as

in a subset of cells within the primitive streak and laterembryonic tissues (45). It has previously been shown that intissue culture FGF-4 gene transcription is restricted to undif-ferentiated embryonal carcinoma (EC) cell lines and thatdifferential expression of both the murine and the humanFGF-4 genes is dependent on an enhancer element located inthe untranslated portion of the third exon (4, 73). Thisenhancer efficiently stimulates transcription from both homol-ogous and heterologous promoters in undifferentiated F9 ECcells but is inactive in differentiated F9, HeLa, or NIH 3T3cells (4). In that study, it was also noted that both the murineand the human enhancers harbored consensus octamer motifs.This was of particular interest since undifferentiated EC cells

* Corresponding authors. Mailing address (L.D.): The RockefellerUniversity, 1230 York Ave., New York, NY 10021. Phone (L.D.): (212)327-7961. Fax (L.D.): (212) 327-7878. Mailing address (C.B.): Depart-ment of Microbiology, New York University School of Medicine, 550First Ave., New York, NY 10016. Phone (C.B.): (212) 263-5431. Fax

(C.B.): (212) 263-8276.

contain cell-type-specific octamer-binding proteins, Oct-3 (alsocalled Oct-4 and NFA3) (33, 47, 56) and Oct-6 (56), in additionto the ubiquitously expressed Oct-i protein, suggesting thatone or both of these EC-specific factors could be directlyresponsible for the restricted activation of the FGF-4 gene (35,57).

Octamer-binding proteins are members of the POU familyof transcription factors that are believed to play key roles as

developmental regulators (20, 50). These factors share an

evolutionarily conserved motif, the POU domain, which iscomposed of two subregions that are jointly required for avidand specific binding of the factor to its target DNA sequence(21, 27, 64, 74). Although the octamer consensus motif,ATGCAAAT (9, 48), is an essential promoter element of a

number of ubiquitously expressed genes (2, 19, 28, 37, 42), thiselement is also involved in the regulation of a variety of geneswhich are expressed in a tissue- or development-specific man-

ner (6, 9, 36, 39, 40, 48). One of the central questions thatemerges from these studies is how such a diversity of expres-sion patterns can be achieved through a common DNA bindingelement. One possibility is that this reflects the differentialactivities of distinct proteins that recognize the octamer motif(54). However, octamer-binding protein(s) frequently requiresadditional factors in order to potentiate transcriptional activa-tion (26, 43, 63, 69), suggesting that specific recognition oftarget genes may be determined by particular combinations ofoctamer-binding protein with such factors.More recently, a new family of development-regulatory

DNA-binding proteins has been described. The prototype ofthis family, the Sry gene, encodes the male-sex-determiningfactor (7, 59), which contains a single 80-amino-acid high-mobility group (HMG)-like domain (11, 23). This HMG motifis shared among the family members, which have thereforebeen designated Sox (Sry-HMG-box) factors (17). The Soxfactors and the closely related HMG domain proteins, such as

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LEF-1/TCF-1lo, can bind a common specific sequence [CTTTG(T/A)(T/A)] (8, 18, 44, 65, 68, 71, 72, 75) through aninteraction within the minor groove (13, 14, 70) and exhibitdistinct tissue-specific patterns of expression (17, 59, 68, 72).Thus, many of the questions regarding target gene selectionand activation by the octamer factors are also relevant to geneswhose transcription is regulated by HMG domain proteins.

In this study, we have undertaken a detailed examination ofthe cis- and trans-acting elements required for transcription ofthe murine FGF-4 gene in F9 cells. A combination of in vitroDNA binding analyses and transfection assays using a series ofFGF-4 enhancer-CAT gene reporter constructs has revealedthat full enhancer activity requires DNA sequences that bindoctamer, Sox, and SpI transcription factors. In particular, wehave identified an F9-specific protein which binds to a Soxrecognition motif located immediately upstream of a consen-sus octamer site and which is required for enhancer activity.This factor, designated Fx, forms complexes with both Oct-1and Oct-3 on DNA fragments containing both the Fx and theoctamer binding sites, suggesting that Fx plays a key role in theactivation of FGF-4 gene transcription through its interactionwith octamer-binding proteins. Although the activity of theFGF-4 enhancer is octamer sequence dependent, the resultssuggest that the F9-specific Fx factor, rather than Oct-3 orOct-6, may be the critical regulator of FGF-4 expression inembryogenesis and that Fx can perform its function in con-junction with the ubiquitously expressed Oct-1 protein. Theseresults not only indicate that the FGF-4 gene is a specific targetgene for a Sox or other HMG domain factor but also providenew insights concerning both octamer-binding protein- andHMG domain factor-dependent regulation of development-and tissue-specific gene expression.

MATERLALS AND METHODS

Cell culture and nuclear extract preparation. F9 and HeLacell cultures were grown in suspension in Joklik's medium(JRH Biosciences) supplemented with 10 or 5% fetal bovineserum, respectively. Cells were harvested at approximately 3 X105/ml, and nuclear extracts were prepared as described pre-viously (5).

Plasmids and oligonucleotides. The pKfgfCAT constructcontains approximately 1 kb of human FGF-4 promoter se-quence upstream of the chloramphenicol acetyltransferase(CAT) gene as described previously (4). All enhancer con-structs were derived from pKfgfCAT by insertion of enhancerfragments at the BamHI site downstream of the CAT genesequences (4). Plasmids pFGF1A and pFGF1B were con-structed by insertion of the 380-bp murine FGF-4 enhancerDraI fragment into the SmaI site of pUC18 or pGEM-3,respectively. Oligonucleotides were synthesized by the ProteinSequencing Facility at The Rockefeller University or by H.Yuan on an Applied Biosystems DNA synthesizer. The oc-tamer site oligonucleotide competitor is derived from theoctamer binding site of the H2B promoter (28). Sequences ofother oligonucleotides used are depicted in the figures.

Transfection and CAT assays. DNA was transfected into F9or HeLa cells by the calcium phosphate precipitation methodas described previously (4), with the following modifications:cells were split 4 to 6 h before the addition of DNA precipi-tates, and cell lysates were prepared 36 h after transfection. Asan internal control, pCH110, a plasmid containing the 3-ga-lactosidase gene fused to the simian virus 40 promoter, wascotransfected with CAT constructs. 13-Galactosidase activitywas measured and used to normalize for transfection varia-

tions. P-Galactosidase and CAT activities were measured asdescribed by Curatola and Basilico (4).

Mutagenesis. The 380-bp DraI-DraI murine enhancer DNAfragment was inserted into M13mpl8. Uracil-substituted sin-gle-stranded DNA was generated, and site-specific mutagene-sis was performed. Mutagenic oligonucleotides were synthe-sized and purified by Sephadex G-25 chromatography. Theseoligonucleotides were designed to alter 6 to 12 consecutive bpof the enhancer sequence by changing, in most cases, pyrimi-dine to purine or vice versa. After synthesis, the double-stranded DNA hybrid was transformed into ung' Escherichiacoli XL1-blue cells and mutant phages derived from theuracil-free DNA strand were selected. Three to five putativemutant phages from each mutagenesis were screened bydideoxy DNA sequencing (56), and those with the desiredmutations were used as templates for amplifying the enhancerfragment by PCR. The amplified DNA fragment was digestedwith BamHI and cloned into the pKfgfCAT reporter plasmid(4).The sequences of the oligonucleotides used for mutagenesis

are as follows (lowercase letters denote mutant substitutions):ml, TTAAGACTCTGCTGGtctcaggaatAGCAACCTCCCGAAT; m2, GCTGGGAGACTTCTGctaccaagaaCGAAI7AACTFl7TATG; m3, CTTCTGAGCAACCTCCCGccggccagggcgGAGGCTACAGACAGCA; m4, CGAATTAAC7fITATGttcttagcacGACAGCAAGACTGGA; m5, AC7FITATGGGAGGCTACAttactacctcagGGAAAATCTCATTGGCAT; m6, CAGCAAGACTGGcttcaaTCATTGGCATFl7T; m7, AGACTGGAAAATCTCcaattatg'lFF 'l'l'l'll GT; m8, AAAATCTCATTGGCATaaaggaggTTTl'GTC'lTl'CACATTCCT; m9, GGCAI'I'l'I'I'1''FIl'gtgagggaACATYCCTI-rAGAAA; mlO, TI[J'lT'GTC'Tl'CACcggaagggAGAAAACTCThlGTT; ml 1,ACATTCFI'CFl7AGccccggCTFl7TGTITGGAT; m12, TTAGAAAACTCTggcgggGGATGCTAATGG; m13, AAACTC'ITTG'll7GGggtaacccGGGATACTTAAAATAC; m14, TTGGATGCTAATtttcatcTTAAAATACTA; mi5, GCTAATGGGATAaaattcATACTATTCTGT; m16, TGGGATACTTAAAATcagcggagtgACCACAGCCCAAGAT; m17, CAAGATGGAAGAAGCacacaaaaccAGCTGAGGTGGGAGC; ml8, TACCACAGCCCAAGAgttcctcctaCACACCCCAAAGCTG; m19,CAAGATGGAAGAAGCacacaaaaccAGCTGAGGTGGGAGGAGC; m20, AGCCACACCCCAAAGagtcttgtttAGCTCCTCCCAAACT; m21, AAGCTGAGGTGGGAGaataaaaaccACfTTCCTFITCTGTCT; m22, GAGCTCCTCCCAAACagttgggaatTCTGGTGGCTCACAGGAC; and m23, TCCCAAACTTCC'Fl'lCTGgagttgttagaCCAGGACAATAAGA'l'lllG.

Preparation of DNA probes. The Hinfl-RsaI or Hinfl-SacIprobe was obtained by Hinfl digestion of the isolated EcoRI-HindIII insert fragment from pFGFlA DNA followed bylabelling with [a-32P]dATP (New England Nuclear) and E. coliKlenow DNA polymerase and subsequent digestion with RsaIor Sad. The RsaI-DraI fragment probe was generated byprimary digestion of pFGFlB DNA with either HindIII (whichcuts within the vector polylinker downstream of the FGF Dralsite) or RsaI and labelled at the 5' end with [y-32P]ATP and T4polynucleotide kinase after phosphatase treatment. Subse-quent digestion with RsaI or HindIII was followed by isolationof the labelled fragments from a native 5% polyacrylamide gelin 0.25 x Tris-borate-EDTA (TBE). The specific activities ofthese probes were generally 30 x 103 to 50 x 103 cpm/ng.The 116-bp DNA fragment used as a probe in Fig. 7B was

generated by PCR using a pFGFlA template and primerscomplementary to FGF-4 enhancer sequences from nucleo-tides 93 to 110 and 214 to 231. The PCR product was gelpurified, and 0.5 to 1 ,ug was labelled with [-y_32P]ATP and T4polynucleotide kinase. After digestion with Sad (see Fig. 3A),

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7760 DAILEY ET AL.

the 116-bp fragment probe was isolated by electroelution froma 5% native gel in 0.25X TBE. Oligonucleotide probes wereprepared by labelling with T4 polynucleotide kinase and[y-32P]ATP.DNA binding assays. The electrophoretic mobility shift

assay (EMSA) was performed as described previously (5) byincubating 0.5 to 1 ng of fragment or oligonucleotide DNAprobe and protein extract (5 to 10 gug) or fraction with 2 to 8[Lg of poly(dI-dC) or poly(dG-dC). Where indicated, 1.5 to 2 RIof polyclonal or monoclonal antibody preparations was addedeither before or after probe addition. Samples were incubatedfor 15 to 30 min at room temperature and resolved byelectrophoresis on a 4% polyacrylamide gel containing 0.25XTBE and 0.1% Nonidet P-40. DNase I footprinting experi-ments using purified protein were performed by incubating 0.5to 1 ng of DNA fragment probe with 10 to 40 ng of purifiedOct-1 or recombinant Oct-3 (rOct-3) protein for 15 min atroom temperature, after which CaCl2 and MgCl2 were added(10 and 1 mM final concentrations, respectively) and themixtures were digested for 30 s with 0.5 Vg of DNase I (Sigma)per ml at room temperature. The reactions were stopped bythe addition of EDTA, phenol extracted, and ethanol precip-itated, and the reaction products were resolved in a 6%polyacrylamide-50% urea sequencing gel in 0.5 X TBE. DNaseI footprinting and methylation interference analysis fromcomplexes resolved on native gels were done as describedpreviously (5).Antibody preparation. The anti-Oct-1 monoclonal antibody

(5G5) recognizes an epitope within the N-terminal portion ofOct-1 (57a) and, together with an anti-Oct-1 antiserum, was agift from Neil Segil at Rockefeller University. N. Segil alsogenerously provided the recombinant construct pGEXOct-3,which contains the complete cDNA of murine Oct-3 insertedin the pGEX-2T vector (Pharmacia). The production of rOct-3in transformed bacteria was induced by IPTG (isopropyl-P-D-thiogalactopyranoside), and lysates were prepared by sonica-tion in BC1OON buffer (20 mM HEPES [N-2-hydroxyeth-ylpiperazine-N'-2-ethanesulfonic acid] [pH 7.9], 100 mM KCl,20% glycerol, and 0.02% Nonidet P-40) supplemented with 0.5mM phenylmethylsulfonyl fluoride and 0.5 mM dithiothreitol.rOct-3 protein was purified by passage of the cell lysate over anoctamer site oligonucleotide column in BC1OON buffer andrecovery of rOct-3 by elution with BCSOON. A 30-t>g sample ofrOct-3 was incubated with aluminum hydroxide (Alu-gels;Serva) overnight at 4°C, and this preparation was injected intoa New Zealand White rabbit at 1-month intervals. Serum wasprepared 2 weeks after each injection.

Chromatography. Approximately 80 mg of F9 or HeLa cellnuclear extract was applied to a 0.5-ml wheat germ agglutinin-agarose (WGA) (Vector Laboratories) column in BC1OONbuffer and washed with 3 column volumes of BC1OON buffer,and the bound proteins were eluted with 0.3 M N-acetylglu-cosamine in BC100N (22). Active fractions from the WGAstep were pooled and applied to a 0.5-ml oligoaffinity column(Doct column) which contains a DNA oligonucleotide with twooctamer binding sites (42). After washes with 3 column vol-umes of BC1OON, bound proteins were step eluted with 0.2 and0.5 M KCl BC buffer. Fx activity is distributed in several ofthese fractions, while Oct-3 is recovered in the WGAflowthrough (FT) and Oct-1 is recovered in the Doct 0.5 MKCl fraction.

RESULTS

The murine FGF-4 enhancer binds Spl as well as ubiqui-tous and F9-specific octamer proteins. The FGF-4 enhancer

PLASMID

5,Dr

pM-380DrDr [

MURINE DNA INSERT

3.ral Hinfl Sfanl Rsal Sad Dral

3,71 5,

pM-1O9DrHi H I

pM-27OHiDr

RELATIVE CATACTIVITY

Ea HaLa

225 6.2

237 nd

16 0.8

-d 218 5.0I-pM-320DrSc -

pM-230DrSf

pM-150SfDr V

-I

H-lpM-58ScDr

pKfgfCAT

143 3.0

43 7.0

47 3.0

19 1.0

7.0 4.5

100 100pRSVCAT

FIG. 1. The 270-bp Hinfl-DraI fragment of the third exon of themurine FGF-4 gene contains full enhancer activity. The enhanceractivities of pKfgfCAT plasmids containing different segments ofFGF-4 exon 3 were measured by CAT assay after transfection intoundifferentiated F9 or HeLa cells. pM-38ODrDr and pM-38ODrDr(B)were derived from pKfgfCAT by insertion of the 380-bp DraI-DraIfragment in the sense and antisense orientations, respectively, into theBamHI site downstream of the CAT gene. Subfragments of theDraI-DraI region were generated by digestion at the indicated restric-tion sites and cloned into pKfgfCAT at the BamHI site. The results foreach construct are the average of at least three independent experi-ments and are expressed as percentages of pRSVCAT activity.

was previously mapped to a 700-bp fragment within the thirdexon of the human FGF-4 gene and to a homologous 380-bpregion of the murine gene (4). Further analysis demonstratedthat the 270-bp region between the Hinfl and DraI sites of theoriginally defined murine enhancer segment is sufficient for fullF9-specific transcriptional activation (Fig. 1). In order toidentify potential F9-specific transcriptional activators, theprotein-DNA interactions throughout this entire region wereanalyzed. Two enhancer DNA subfragments (Hinfl-RsaI orRsaI-DraI; Fig. 1) were utilized in DNA binding assays, and theprotein-DNA complexes formed when F9 nuclear extract wasused were compared with those formed when HeLa cellnuclear extract was used. As shown in Fig. 2A, methylationinterference analysis with the RsaI-DraI DNA probe and F9nuclear extract demonstrated that G residues essential forfactor binding are located within the two regions designatedsites A (positions 184 to 193; Fig. 3A) and B (positions 209 to217; Fig. 3A). These sites resemble Spi recognition motifs (24,25), and Spl binding at each was confirmed by competitionwith Spl consensus, site A, or site B oligonucleotides inEMSAs (data not shown). Similar results were obtained withHeLa cell nuclear extracts (data not shown).We next compared the protein-DNA complexes formed in

F9 and HeLa cell nuclear extracts by EMSA using FGF-4enhancer sequences located between the Hinfl and RsaI sites(Fig. 2B). Two complexes detected in F9 nuclear extract (Oct-1and Oct-3) were abolished by the inclusion of octamer oligo-nucleotide competitor (Fig. 2B, lane 2), and the identity of theprotein component of each complex as Oct-1 or Oct-3 wasconfirmed by using antisera specific for each factor (data notshown). Formation of the protein-DNA complex of interme-

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FACTORS REGULATING ACTIVITY OF FGF-4 ENHANCER

B C

Protein:

Extract: F9 m HeLa

Oligo

competitor: -

OCT-1

OCT-3 _

OCT-I OCT-3

oligo competitor: ck

Protein: + + +

2J_ _ _~~

*I**qpk-O94_0 4

1 2 3 4 5 6

2 3 4

coding non coding

* al1 2 3

4 5 6 7

FIG. 2. Analysis of protein-DNA interactions on the FGF-4 enhancer. (A) Methylation interference. The RsaI-DraI fragment probe, labelledon either the coding or the noncoding strand, was partially methylated by treatment with dimethyl sulfate (38). Each probe was incubated with F9nuclear extract and poly(dI-dC), and the reaction products were resolved on a native polyacrylamide gel. Bound and free probes were eluted andcleaved with piperidine, and the reaction products were resolved in a sequencing gel. Reaction products derived from unbound probe (Free) andthose derived from probe bound to protein (Bound) and the regions showing the absence of bands in the bound-probe sample compared with thefree probe (regions A and B, corresponding to sites A and B within the enhancer as indicated in Fig. 3A) are indicated. (B) EMSA. A 1-ng sampleof radiolabelled Hinfl-RsaI FGF-4 enhancer fragment (Fig. 1) was incubated with F9 or HeLa nuclear extract and poly(dI-dC) in either the absenceor the presence of 10 ng of octamer (lanes 2 and 5) or Spl (lanes 3 and 6) competitor oligonucleotides as indicated. The reaction products wereresolved on a 4% native polyacrylamide gel and visualized by autoradiography. The positions of the Oct-i, Spl, and Oct-3 protein-DNA complexesare indicated on the left. (C) DNase I footprinting analysis of the interaction of Oct-i and Oct-3 with the FGF-4 enhancer. A 1-ng sample ofradiolabelled Hinfl-SacI (Fig. 1) probe was incubated in the presence of 10 to 20 ng of either Oct-i purified from HeLa cells (lane 3) or partiallypurified recombinant mouse Oct-3 (lanes 5 to 7) and subjected to mild DNase I digestion as described in Materials and Methods. The samplesin lanes 6 and 7 also contained 50 ng of Spl and octamer oligonucleotide competitor, respectively. The reaction products were resolved on a 6%polyacrylamide sequencing gel. The two regions of protection, OCTA 1 and OCTA 2, are shown (brackets 1 and 2). The locations of these siteswithin the FGF-4 enhancer are indicated in Fig. 3A. Lane 1 shows G residues of the probe DNA included as a marker.

diate mobility was specifically inhibited by addition of an Splconsensus oligonucleotide (Fig. 2B, lane 3), thus identifyingthe factor in this complex as Spl. Both Spl and Oct-icomplexes were, as expected, also present in HeLa cell nuclearextract (Fig. 2B, lanes 4 to 6), whereas the Oct-3 protein-DNAcomplex was observed exclusively in F9 nuclear extract.To define the octamer protein binding regions within the

FGF-4 enhancer, probe DNA was incubated with an excess ofeither purified human Oct-i or recombinant mouse Oct-3 andthe resultant complexes were analyzed by DNase I footprintingas shown in Fig. 2C. Both octamer-binding proteins protectedtwo regions of the FGF-4 enhancer corresponding to theoctamer binding sites noted by Curatola and Basilico (4) andwere designated OCTA 1 (positions 76 to 83; Fig. 3A) andOCTA 2 (positions 130 to 137; Fig. 3A).

Together, the results of the DNA binding analyses indicatethat the FGF-4 enhancer contains recognition sequences forboth Spl and octamer-binding protein families of transcrip-tional activators. Only one of these factors, Oct-3, is specific toF9 cells, while Oct-i and Spl are present in both F9 and HeLacell extracts.

Mutation analysis of the FGF-4 enhancer reveals regionscritical to enhancer function that center around OCTA 2. Toassess the relative contributions of the factor binding sites aswell as other elements to FGF-4 enhancer activity, a compre-hensive series of mutants was created. As summarized in Fig.3, mutant enhancers containing substitutions of a unique set of6 to 12 consecutive bp were inserted downstream of the FGF-4promoter-CAT gene in the pKfgf plasmid (4) and tested fortheir ability to activate CAT gene expression after transfectioninto F9 cells. The results of this analysis, shown in Fig. 3B,revealed that most of the mutants displayed some variation ofCAT expression levels with respect to the wild-type (wt)enhancer. Although some of the mutations reproducibly in-creased enhancer activity by about 30% (m5 and m1O) and thuscould be affecting sequences involved in transcriptional repres-sion, none of the mutations conferred activity in differentiatedF9 or HeLa cells (data not shown). On the other hand, severenegative effects on enhancer activity were observed in mutantscontaining sequence alterations within the OCTA 2 site (m13)or Spl binding site A (m19). Mutation of OCTA 2 (m13)nearly abolished enhancer activity, clearly indicating an essen-

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At HlnfI SPiTCTGCTGGGA GACTTCTGAG CAA CCTCCC G AATTAACTTTAT C CT

2 OCTRl 3 4

51 ACAGACAGCA AGACTGGAAAATCTC kTTGG CAT VTTTTTT TTTTGTCTTT

4 5 6 7 8 9y sranl OCTR2

10 1 CACATTCCTT TAGAAAACTC TTTGTTTGG KTGCTAAT]GGGATACTTAAAA10 11 12 3 14 15

t Rsal R

151 TACTATTCTG TACCACAGCC CAAGATGGAAGAA _ AAAGCTG

1 6 Sacld B 1 7 18 19 20

201 AGGTGGGA AA CTTCCTTTCT GTCTGGTGGC TCACAGGACA

20 21 22222251 ATAAGATTTT GTGTTTTTT

WT Probe +

Mt Probe100 X WT Competitor100 X mt Competltor

OCT I A

F9 Cell HeLa Cell

+ + - + ,

- - + -

i .-

- - +

OCT3

B

140%

100%

80%-

60%/6

40%

20%/

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 WT

1 2 A B

FIG. 3. Mutation analysis of sequences within the FGF-4 enhancer.(A) Definition of the sequences altered for FGF-4 enhancer mutants 1to 23. The DNA sequence of the entire FGF-4 enhancer between theHinfl and DraI sites is shown. Binding sites for the octamer proteins(OCTA 1 and OCTA 2) and the Spl protein binding sites as definedin Fig. 2 (shaded residues) are indicated. The data defining the Spl siteat positions 42 to 48 were not presented. Underlined sequencesindicate the residues changed within each mutant. (B) Relative CATactivities of FGF-4 enhancer mutants 1 to 23. Each of the mutantenhancer fragments shown in panel A was cloned downstream of theCAT gene in the pKfgfCAT vector, and the ability to induce CAT geneexpression was compared with that of the wt enhancer after transfec-tion into F9 cells. The CAT activity of the wt enhancer construct is100%. The results for each of the 23 mutant enhancer constructs areaverages of two independent experiments. The entire length of theFGF-4 enhancer shown in panel A and the positions of the octamer-binding protein (white boxes) and Spl (shaded boxes) binding sites arerepresented schematically beneath the histogram.

TTTGTTTGG ATGCTAAT GGGATACTTWT Probe AAACAAACC TACGATTA CCCTATGAA

mt Probe TTTGTTTGG ggtaaccc GGGATACTTAAACAAACC ccattggg CCCTATGAA

FIG. 4. Mutation of OCTA 2 in ml3 compromises octamer-proteinbinding. Oligonucleotide probes that contain the wt or the m13mutated (mt) sequence at the OCTA 2 site were tested for theirabilities to bind octamer-binding proteins by an EMSA using F9 orHeLa cell nuclear extracts. In competition assays, 100-fold molarexcesses of unlabelled wt or mutant oligonucleotides were added to thereaction mixtures as indicated above the lanes.

tial role of this sequence in enhancer function. As expected,binding of neither Oct-1 nor Oct-3 was observed in F9 or HeLacell extracts when an oligonucleotide containing the m13mutant sequence was used (Fig. 4). Somewhat surprisingly,mutation of the OCTA 1 site (m7) had no appreciable effect onenhancer function, and an intact OCTA 1 element could notcompensate for the loss of OCTA 2 in the m13 construct. Thus,while both OCTA 1 and OCTA 2 can bind octamer proteins(Fig. 2C), they are not functionally equivalent in the context ofthe enhancer. It is therefore interesting that mutation of DNAsequences located either immediately upstream (m12) ordownstream (m14 and miS) of the OCTA 2 site also had asevere negative effect on FGF-4 enhancer function in F9 cells,reducing CAT levels to approximately 20, 35, and 30% of wt,respectively. Clearly, the activating region of the FGF-4 en-hancer consists of Spl site A, the OCTA 2 site, and DNAsequences flanking OCTA 2. Since the Spl activity binding tosite A was not F9 specific, we concentrated our analysis onOCTA 2 and its flanking regions.

Additional DNA binding activities are revealed in the ab-sence of poly(dI-dC). Several mechanisms could account forthe importance of the DNA sequences flanking OCTA 2 for

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FACTORS REGULATING ACTIVITY OF FGF-4 ENHANCER 7763

enhancer activity. The topology of these sequences mightdirectly affect the association of octamer protein with OCTA 2or induce a particular conformation of octamer protein boundat this site. If this was the case, the differential activity of theFGF-4 enhancer in F9 cells might be explained if the effects ofthe flanking sequences were specific or preferential for Oct-3,whose expression is restricted to undifferentiated F9 cells (54).However, cotransfection of our CAT-FGF-4 enhancer plas-mids with a plasmid expressing Oct-3 in either HeLa ordifferentiated F9 cells failed to activate the FGF-4 enhancer(data not shown), indicating that Oct-3 alone is not sufficientfor enhancer function. We therefore considered the possibilitythat the DNA sequences flanking OCTA 2 may bind anadditional factor(s) that was not identified in the DNA bindingassays described above. Several recent reports have describedproteins whose interaction with DNA occurs primarily throughdA-dT residues within the minor groove (13, 31, 60, 61, 70).Since poly(dI-dC), the standard "nonspecific" competitor usedin the binding assays, resembles dA-dT within the minorgroove, it was possible that the presence of this polymerprevented the detection of analogous activities.To investigate this possibility, we substituted poly(dG-dC)

for poly(dI-dC) as the nonspecific DNA competitor in theEMSA. In these experiments, we employed a probe that spanspositions 113 to 149 within the FGF-4 enhancer (Fig. 5A; alsoFig. 3A) and thus encompasses OCTA 2 and a portion of itsflanking sequences. As shown in Fig. SB, lanes 12 to 14,incubation of the oligonucleotide probe from positions 113 to149 with F9 nuclear extract in the presence of poly(dI-dC)resulted in the formation of two specific protein-DNA com-plexes that were sensitive to the addition of octamer sitecompetitor oligonucleotide and thus represent binding byOct-1 and Oct-3 as described above.To determine whether additional protein-DNA complexes

could be detected in the absence of poly(dI-dC), the sameoligonucleotide probe was incubated with F9 nuclear extract inthe presence of poly(dG-dC). As shown in Fig. SB, lane 1, theOct-1 and Oct-3 complexes that had been observed in thepresence of poly(dI-dC) were also detectable in the presenceof poly(dG-dC). However, three additional complexes, desig-nated Oct-i*, Oct-3*, and Fx, were observed exclusively inreaction mixtures containing poly(dG-dC). Importantly, theseadditional complexes were not apparent in HeLa cell nuclearextract (Fig. 5B, lanes 15 and 16) and are therefore F9 specific.Two of the poly(dl-dC)-sensitive complexes observed in F9

nuclear extract, Oct-1* and Oct-3*, were eliminated uponaddition of octamer site competitor oligonucleotide, indicatingthat they contained octamer-binding proteins (Fig. 5B, lane 4).Incubation of the reaction mixture with a monoclonal antibodygenerated against Oct-1 resulted in the supershift of the twobands with the slowest mobilities, Oct-1 and Oct-1* (Fig. 5B,lane 2), showing that each of these complexes contained Oct-1.Similarly, incubation of the reaction mixture with polyclonalantiserum raised against Oct-3 specifically blocked formationof the complexes Oct-3 and Oct-3* (Fig. SB, lane 3), indicatingthat they both contain Oct-3 protein.Fx binds to a site adjacent to OCTA 2. The observations that

the Oct-I* and Oct-3* complexes contain Oct-1 and Oct-3,respectively, and are not detected in HeLa cell extracts sug-gested that they also contain an F9-specific factor. As de-scribed above, the Fx complex detected in the presence ofpoly(dG-dC) was specific to F9 nuclear extract (compare lanes11 and 15 in Fig. SB). Formation of the Fx complex was notsensitive to the addition of either octamer or Spl competitoroligonucleotides (Fig. 5B, lanes 4 and 11) but was effectivelyinhibited by an oligonucleotide, FxO-, containing DNA se-

quences upstream of OCTA 2 (Fig. SB, lanes 5 to 7). Thesesequences contain the consensus binding motif (CTlTGT1T) ofthe tissue-specific and developmentally regulated Sox factorfamily. We thus investigated whether these residues provide arecognition site for Fx. As shown in Fig. SB, lanes 8 to 10, anoligonucleotide containing the specific mutation of the Sox site(Fx-O-) failed to compete for Fx binding to the wt probe,confirming that the Sox consensus sequence is required for Fxinteraction with the FGF-4 enhancer. Significantly, both theOct-1* and the Oct-3* complexes were also eliminated byincubation with the FxO- oligonucleotide, suggesting thatthese complexes contain both Fx and the octamer-bindingproteins (Fig. 5B, lanes 5 to 7). This conclusion was furthersupported by the observation that formation of both Oct-1*and Oct-3* was unaffected by competition with the Fx-O-oligonucleotide (Fig. 5B, lanes 8 to 10). Together, these resultsindicate that a Sox consensus sequence located immediatelyupstream of the critical OCTA 2 site in the FGF-4 enhancer isrecognized by an F9-specific factor, Fx, and that Fx can interactwith octamer-binding protein bound at OCTA 2 to form theOct-1* or Oct-3* complex.The formation of Oct* correlates with the presence of Fx in

chromatographic fractions. To facilitate further analysis of theOct-1* and Oct-3* complexes, Fx and the octamer proteinswere partially purified from F9 nuclear extract by using a WGAcolumn as described in Materials and Methods. DNA bindingactivities present in the input nuclear extract, FT, and stepfractions were monitored by EMSA with the oligonucleotideprobe spanning positions 113 to 149 as shown in Fig. SC. Fxactivity present in F9 nuclear extract cofractionated with bothOct-1 in the WGA step fraction and Oct-3 in the FT fraction(Fig. SC, lanes 1 to 3). Significantly, both the Oct-1* and theOct-3* complexes were still observed in fractions containingboth Fx and Oct-1 or Oct-3 proteins, respectively.The WGA step fraction was further fractionated by using an

octamer oligonucleotide affinity column (Doct column). Pro-teins bound to the affinity resin were eluted in two steps with0.2 and 0.5 M KCl, and the activities present in each fractionwere determined by EMSA. As shown in Fig. SC, lanes 4 to 6,a portion of the input Fx activity cofractionated with Oct-1 andwas eluted from the DNA column by 0.5 M KCl; again, theOct-1* complex was observed. However, Fx activity was alsodetected in the FT from this column, which was relatively freeof Oct-1 and Oct-3 (Fig. SC, lane 4). We tested whetheraddition of the Doct FT fraction to either Oct-1 or Oct-3 couldgenerate the Oct-1* and Oct-3* complexes. Incubation ofOct-1 protein purified from HeLa cell nuclear extract with theoligonucleotide probe spanning positions 113 to 149 resulted information of the single Oct-1 complex (Fig. SD, lane 3).However, addition of increasing quantities of the Doct FTfraction to HeLa Oct-1 resulted in the generation of the Oct-1 *complex (Fig. SD, lanes 3 to 5). Similarly, addition of the DoctFT fraction to the WGA FT fraction that contains Oct-3 (andFx) resulted in a progressive increase in Oct-3* complexes(Fig. SD, lanes 6 to 9). These results further support the notionthat the Fx factor, together with Oct-1 or Oct-3, is present inthe Oct-1* and Oct-3* complexes.Fx is not detected in nuclear extracts from cell lines that do

not express FGF-4. To further characterize Fx, nuclear extractswere prepared from a number of mammalian tissue culture celllines and incubated with a DNA probe that contains a single Fxbinding site (FXO-; Fig. 5A). As shown in Fig. 6, Fx bindingactivity was detected only in nuclear extracts derived from theEC cell lines F9 and P19. Comparable Fx activity was notobserved in any of the other cell extracts tested. This resultindicates that Fx is present in cell lines that express FGF-4 (57,

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SOX OCT

wt 5'-GAA.MCTCTTTGITTGGATGCTAATGGGATACTTAAA-3'FxO- TA CCATGTCGACTGFx-0- TA GCACTGAC-CCATGTCGACTG

F9 r HeLai

_. dGdC -dl-dCGG C *C

OCTr- FxO- r Fx-O- SpI OCTr Spial cz3 10 25 50 10 25 50 100 10100100100 ng

OCTOCT- 1 * eli * * _

OCT3:Fx - * ^ ::.

OCT-3

.A -A

........ i ...N

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*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

S1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

C rDOCT-i

OCT- 1 *

OCT-1 4i

OCT-3* - %...-.j

D Hr

r1 OCT- 1 W FT

MAil DOCT FT: 1 2 - 1 2 - 1 2 3

OCT-1 *- 4OCT-1t

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FIG. 5. Analysis of the protein-DNA complexes formed in the absence of poly(dI-dC). (A) DNA sequences of probe and competitoroligonucleotides. The wt oligonucleotide spans positions 113 to 149 of the FGF-4 enhancer shown in Fig. 3A. The octamer-binding protein bindingsite OCTA 2 and the Sox consensus binding sequence are indicated. Sequences changed in the FxO- and Fx-O- oligonucleotides are indicated(unchanged residues are shown as a solid line). (B) EMSA of protein-DNA complexes observed in the presence of poly(dG-dC) compared withthose observed for poly(dI-dC). A 1-ng sample of radiolabelled wt oligonucleotide probe (panel A) was incubated with F9 (lanes 1 to 14) or HeLa(lanes 15 and 16) cell nuclear extract and either poly(dG-dC) or poly(dI-dC) as indicated. Competitor oligonucleotides included in some reactionmixtures are indicated above the lanes, as are the quantities of each added. ct1 and c3, reaction mixtures for which monoclonal antibody raisedagainst Oct-1 protein or polyclonal antiserum raised against Oct-3 protein, respectively, was used; Oct-i*, Oct-i, Oct-3*, Fx, and Oct-3, specificprotein-DNA complexes observed as described in the text. A broad band that migrates in a position similar to that of Fx is sometimes observedwhen HeLa nuclear extracts are used (lane 15). This activity is nonspecific and distinct from Fx as determined by oligonucleotide competition (datanot shown). (C) EMSA using chromatographic fractions derived from F9 nuclear extract. F9 nuclear extract was fractionated by WGA and DNAaffinity column chromatography as detailed in Materials and Methods. Aliquots (1 ±1) from input nuclear extract and each of the chromatographicfractions were analyzed by EMSA using the wt oligonucleotide probe in the presence of poly(dG-dC), and the reaction products were resolved ona native polyacrylamide gel. NE, input nuclear extract; WFT, WGA FT fraction; W STEP, WGA step fraction; DOCT, fractions derived from theDoct column (FT and 0.2 and 0.5 M KCl steps). (D) Addition of Fx-containing Doct FT fraction to Oct-i and Oct-3. Samples of FT fraction wereadded either to Oct-i purified from HeLa cell nuclear extract (lanes 3 to 5) or to the WGA FT fraction that contains Oct-3 (lanes 6 to 9) in thepresence of the wt oligonucleotide probe and poly(dG-dC). The reaction products were analyzed by EMSA.

73) while cell lines that do not contain Fx also do not transcribeFGF-4 (73) and is consistent with the notion that Fx is requiredfor FGF-4 gene transcription.The activities of the FGF-4 enhancer mutants correlate with

their ability to form Oct-l*. The mutational analysis of Fig. 4demonstrated the functional importance of OCTA 2 and its

flanking sequences for FGF-4 enhancer activity. The Fx bind-ing site described in the previous paragraph could be critical toenhancer function, since it is included in the sequences alteredin the enhancer mutant m12. To establish a correlation be-tween the ability of different enhancer fragments to form Oct*and their ability to activate transcription in F9 cells, the

A

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EXTRACT: r9 Q lb

Fx - B

FIG. 6. Fx binding activity is EC cell specific. Samples (3 pg) ofnuclear extracts prepared from the various mammalian cell linesindicated above the lanes were analyzed for Fx binding activity byEMSA using 0.2 ng of FxO- probe DNA (Fig. 5A). The specificity ofthis complex was confirmed by competition experiments (data notshown). For clarity, 10 ng of an Spl binding site oligonucleotide was

included in the reaction mixtures to reduce nonspecific protein-DNAcomplexes.

complexes observed when the Doct 0.5 M KCl fraction and thewt oligonucleotide probe spanning positions 113 to 149 (Fig.5A) were compared with those obtained when analogousprobes derived from enhancer mutants containing base substi-tutions within either the Fx binding sequence (mi2; Fig. 3) or

the OCTA 2 site (m13; Fig. 3) were used. As shown in Fig. 7A,lane 1, the wt probe supported formation of Fx and Oct-1complexes, as well as Oct-i*. In contrast to the distinct bandproduced by Fx binding to the wt probe, a fainter cluster ofnonspecific binding proteins with slightly faster mobilities was

observed when the m12 probe was used (Fig. 7A, lanes 4 to 6).Furthermore, while the binding of Oct-i to the m12 probe was

comparable to that observed with the wt probe, the Oct-1*complex was not apparent (Fig. 7A, lane 2). The mi3 probewas unable to bind Oct-i, and the Oct-1* complex was notobserved (Fig. 7A, lane 7). These results indicate that forma-tion of Oct-1* is template dependent and requires both the Fxand the octamer-binding protein binding sites. Interestingly,the intensity of the band corresponding to the Fx complexwhen the m13 probe was used appeared to be greater than thatobserved for the wt probe (compare lanes 1 and 7 of Fig. 7A).This suggested that whereas Fx was bound independently tothe m13 probe, most of the Fx bound to the wt probe was

present in the Oct-1* complex. To test this hypothesis, theDoct 0.5 M KCl fraction was preincubated with a polyclonalantiserum raised against purified Oct-i protein (58). While thistreatment eliminated both the Oct-I and the Oct-i* complexes(Fig. 7A, lane 2), the intensity of the band corresponding to theFx complex increased for the wt probe (compare lanes 1 and 2of Fig. 7A). In contrast, no change in the number of indepen-dently bound Fx complexes was noted upon treatment withpreimmune serum (compare lanes 1 and 3 of Fig. 7A), andanti-Oct-1 antibody pretreatment did not alter the amount ofFx bound to the mi3 probe (Fig. 7A, compare lanes 7 and 8).The correlation between the loss of Oct-1* and the concomi-tant increase in the number of independent Fx complexesbound to the wt probe in the presence of the anti-Oct-1

antiserum indicates that the majority of Fx activity in thisfraction is present in the Oct-i * complex when both factor sites(and both factors) are present. Together, these results demon-strate that the inability of the mi2 and m13 FGF-4 enhancerconstructs to efficiently activate transcription directly corre-lates with their inability to direct the formation of Oct-l* andfurther establishes that binding of Fx or octamer-bindingprotein alone is not sufficient for enhancer activity.To further analyze the DNA sequences that interact with the

factor components of Oct-1*, a modified DNase I footprintingassay was employed. Samples containing wt enhancer fragmentprobe and the Doct 0.5 M KC1 fraction were subjected to mildDNase I digestion, the protein-DNA complexes were resolvedin a native gel, and the Oct-1*, Oct-i, and Fx complexes wereeluted and analyzed in a denaturing gel. As shown in Fig. 7B,the region of the probe protected from nuclease digestion inthe Oct-1* complex (lane 5) spans the regions protected byboth Oct-i (lane 3) and Fx (lane 6).

DISCUSSION

In this report, we present evidence that interactions betweena novel F9-specific factor and octamer-binding proteins areessential to FGF-4 enhancer function in F9 cells. These resultsidentify FGF-4 as a specific target gene for an embryonicallyexpressed factor, Fx, that is most likely a member of the HMGdomain factor family and also demonstrate that this factorpotentiates transcriptional activation through its interactionwith an octamer-binding protein. Although both of these factorfamilies have been implicated in the regulation of specific genesubsets during development, the mechanism of their selectiveinteractions with their target genes has remained elusive.These conclusions thus have implications with respect totissue-specific and development-specific regulation of bothoctamer and Sox site-dependent gene transcription.FGF-4 enhancer activity requires the interaction of several

factors. We have analyzed the cis- and trans-acting elementsrequired for activation of gene transcription by the FGF-4enhancer in F9 cells. Since F9 cells, like other EC cell lines,resemble cells of the blastocyst inner cell mass, the compo-nents which we have identified as important to FGF-4 en-hancer function are likely to be operative in the embryonicenvironment.DNA binding analyses have revealed that multiple proteins

of the octamer and Spl factor families bind to several siteswithin the FGF-4 enhancer. The critical cis-acting elements,however, are confined to a relatively small region that includesSpi site A and octamer binding site OCTA 2. We have shownthat OCTA 2 can bind the ubiquitous Oct-i protein as well asthe F9-specific factor Oct-3, while Spl site A binds Spl, whichis present in both F9 and HeLa cells. However, the mutagen-esis studies emphasize that complete elucidation of this en-hancer function also requires a definition of the contribution ofthe regions flanking the OCTA 2 site, since alteration of theseDNA sequences is severely deleterious to enhancer activity.Accordingly, we have demonstrated that EMSAs in whichpoly(dI-dC) is replaced by poly(dG-dC) permit detection of anadditional F9-specific factor, Fx, whose binding site lies withinthe functionally important sequences immediately upstream ofOCTA 2. The role of the sequences immediately downstreamof OCTA 2 is under investigation. Interaction of these se-quences with an additional, F9-specific activity has not beenobserved (4b), and this region is therefore likely to contributeto overall enhancer activity. Taken together, these observa-tions indicate that FGF-4 enhancer activity requires coordi-

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Ab: d

B

G @&<Si9 ((4sI

OCT-1i - k.aOCT-1 -

Fx -

12345 6 78 9

l 2 3 4 5 6FIG. 7. Analysis of the protein-DNA complexes formed on wt and mutant FGF-4 enhancer oligonucleotides. (A) Enhancer DNA

oligonucleotide probes spanning positions 113 to 149 (Fig. 3) were synthesized to contain the DNA sequence of the wt, m12, or m13 FGF-4enhancer (Fig. 3). A 0.2-ng sample of each probe was incubated with 1 RI of Doct 0.5 M KCl fraction in the presence of poly(dG-dC). Whereindicated above the lanes, CL-Oct-1 polyclonal or preimmune antiserum (PRE) was preincubated with the Doct 0.5 M KCl fraction for 10 min priorto probe addition. Samples were analyzed by native gel electrophoresis. Ab, antibody. (B) DNase I footprinting analysis of Fx, Oct-i, and Oct-1*on the FGF-4 enhancer. The wtl16 enhancer fragment probe, which spans positions 95 to 211 (Fig. 3A), was generated by PCR as described inMaterials and Methods. Probe DNA was incubated with the Doct 0.5 M KCl fraction and subjected to mild DNase I digestion, and the reactionproducts were resolved on a native polyacrylamide gel. Free DNA, Fx, Oct-i, and Oct-1*-DNA complexes were each eluted, denatured, andsubjected to electrophoresis in a denaturing polyacrylamide gel. A G ladder of the probe (38) was also included as a position marker (lane 1). Thedigestion pattern of the unbound probe (FREE) and those derived from each of the complexes (Oct-i, Fx, and Oct-i*) and the regions ofprotection of the individual Oct-i and Fx protein-DNA complexes (brackets) are indicated.

nated interactions between the Spl, octamer-binding, and Fxproteins bound to their critical sites.

Oct-3 or Oct-6 alone is not sufficient for FGF-4 enhanceractivity. The octamer-binding proteins Oct-3 and Oct-6 are

each EC cell specific in that they are not detectable in anyother tissue culture cell types (although our nuclear extractpreparations did not permit detection of Oct-6). Thus, thesimple correlation between octamer site-dependent FGF-4enhancer activity in F9 cells and the presence of Oct-3 andOct-6 in these cells suggests that one of these proteins, ratherthan Oct-i, which is also present in cells that do not expressFGF-4, is the activator of FGF-4 gene transcription.There are several considerations, however, that make Oct-3

and Oct-6 unlikely candidates as the key activators of theFGF-4 gene. Cotransfection of the FGF-4 reporter-CAT con-

struct with an Oct-3 expression plasmid is unable to activateCAT gene expression in HeLa cells, despite the fact that Oct-3is clearly produced in the transfected cells (4a). Scholer et al.(55) were able to demonstrate that Oct-3-mediated activationof an analogous CAT construct containing six tandem repeatsof an immunoglobulin (Ig) gene enhancer fragment could beachieved by the additional expression of adenovirus ElAprotein in transfected HeLa cells. However, we were unable todetect activation of transcription from the FGF-4-CAT genereporter construct even when it was cotransfected with bothElA and Oct-3 expression plasmids in HeLa cells, or with

Oct-3 in 293 cells, which constitutively express ElA (4a).Furthermore, no clear correlation between FGF-4 expressionand that of either Oct-3 or Oct-6 is observed by comparison ofin situ hybridization data for FGF-4, Oct-3, and Oct-6 tran-script distribution in the developing embryo. FGF-4 transcriptshave been detected in the blastocyst inner cell mass, cellswithin the primitive streak, the bronchial arch, and tooth bud,and within the developing limb bud (45). Significantly, the onlycommon sites for the localization of Oct-3 and Oct-6 tran-scripts and of FGF-4 in the embryo are the inner cell mass and,for Oct-3, the primitive streak (51, 66). Therefore, while Oct-3or Oct-6 may be important for FGF-4 gene activity, theapparent uncoupling of FGF-4 transcription and the expres-sion of these factors implies that they alone cannot account forthe cell type expression pattern of this gene.FGF-4 enhancer activity correlates with the ability to form

the Oct* complex. The novel factor, Fx, which we haveidentified in this study displays several interesting properties.Fx is EC cell specific and binds to sequences essential forFGF-4 enhancer activity that are located immediately up-stream of the critical octamer binding element OCTA 2. TheFx binding site and its location relative to OCTA 2 areconserved in the human FGF-4 enhancer (4), suggesting thatthe activation of the human gene depends on the sameelements as those we have defined for the murine enhancer.Using a combination of EMSA and DNase I footprinting, we

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have demonstrated that Fx and either the ubiquitously ex-

pressed Oct-1 or the F9-specific Oct-3 protein can togetherbind the FGF-4 enhancer to form the Oct-1* and Oct-3*complexes, respectively. Furthermore, comparison of differentFGF-4 enhancer mutant probes shows a correlation betweenformation of the Oct* complex and enhancer activity. Muta-tions that impair binding of either Fx (m12) or Oct-1 (m13)inhibit the formation of the Oct* complex and compromiseenhancer function. In this light, it is interesting that theinability of OCTA 1 to compensate for mutations of OCTA 2in the transfection assays may reflect the facts that there is noFx binding site upstream of OCTA 1 and that, like the mi2mutant, OCTA 1 can bind octamer-binding protein but isunable to form the critical Oct* complex.Taken together, these results strongly suggest that Fx is a key

regulator of FGF-4 gene activation in F9 cells and that itsactivity is mediated via interaction with octamer-binding pro-

teins on the FGF-4 enhancer. In line with this conclusion,preliminary experiments indicate that a six-copy concatamer ofthe DNA sequence including OCTA2 and the Fx binding sitecan mimic the behavior of the FGF-4 enhancer on heterolo-gous promoters, i.e., it will stimulate gene expression inundifferentiated F9 cells but not in differentiated F9 or HeLacells (data not shown).

Since the Oct* complex can be formed with either Oct-1 or

Oct-3, it is possible that there is no need for a tissue- or

development-specific octamer-binding protein as the activatorat OCTA 2. FGF-4 transcription could occur in those cells thatexpress Fx or a functionally analogous factor and the ubiqui-tously expressed Oct-1. This hypothesis, which does not pre-

clude the possibility that Fx can also act in conjunction withOct-3 (and indeed, Fx can form the Oct-3* complex), will beinvestigated as soon as the cloning and characterization of Fxare completed.Fx displays characteristics of an HMG domain factor. The

site of Fx interaction with the FGF-4 enhancer corresponds to

the consensus Sox or LEF-1/TCF-lot factor binding site [CTTTG(T/A)(T/A)], suggesting that Fx is a member of the HMGdomain factor family. Although this point cannot be unequiv-ocally proven at this time, it is consistent with the followingobservations. (i) The hydrogen bond donors and acceptors

between base pairs within poly(dI-dC) are analogous to thoseof dA-dT within the minor groove. The binding of Fx to theFGF-4 enhancer is sensitive to the presence of poly(dI-dC) butnot poly(dG-dC), suggesting that its interaction with DNAoccurs primarily at dA-dT residues within the minor groove, a

feature well documented for these factors (13, 70). (ii) Factorrecognition within the minor groove can be sequence specific(13, 31, 61, 70, 76) or relatively nonspecific, the latter illus-trated by HMGI/Y, which will bind any combination of six or

more dA-dT residues (60). The conclusion that Fx specificallyrecognizes the Sox factor site is evidenced by the facts thattargeted alteration of this sequence eliminates Fx binding andthat DNase I footprinting analysis demonstrates protection byFx only over the Sox binding site and not simply at dA-dTclusters. Why then is poly(dI-dC) able to compete for Fxbinding? It is relevant that LEF-1 binds to the Sox site withhigh affinity but with specificity that exceeds its interaction withrandom DNA sequences by only 20- to 50-fold (13). Thus, if Fxwere to exhibit a similar narrow differential between specificand nonspecific sequence recognition, the presence of therelatively large quantities of poly(dI-dC) necessary for analysisin crude extract could preclude specific interaction of Fx withenhancer DNA probe. Together, the binding properties of Fxand its restricted cell type expression strongly support the

argument that Fx is an HMG domain protein of the Sox orLEF-1 type. Cloning of the cDNA for Fx is in progress.Fx may act as a tissue-specific coactivator of octamer-

binding proteins. The findings presented here parallel those ofoctamer site-dependent Ig gene expression in several respects.B cells contain a cell-type-specific octamer-binding protein,Oct-2, suggesting that this protein was responsible for theB-cell-specific transcriptional activation of the Ig genes (29, 32,41, 53, 62). However, mice in which both allelles of the Oct-2gene have been disrupted still produce Ig transcripts at appar-ently normal levels in pre-B cells (3). In addition, analogousOct-2 gene disruption in tissue culture cells revealed theuncoupling of Oct-2 and Ig gene expression (10). Consistentwith these findings, transcription analysis of Ig promoterfunction in vitro has demonstrated that either Oct-1 or Oct-2can activate Ig gene transcription in the presence of anadditional, B-cell-specific coactivator, OCA-B (30, 34, 49).OCA-B can specifically activate transcription from the Igpromoter through interaction with either Oct-1 or Oct-2 andthus offers an explanation for the transcriptional activity ofthese genes in the absence of Oct-2. Like OCA-B, Fx is atissue-specific activity that is required for transcriptional acti-vation by octamer-binding proteins. We have not yet deter-mined the nature of transcriptional potentiation by Fx (e.g.,protein-protein interaction and/or cooperative binding withoctamer-binding proteins) and thus cannot formally designateFx an octamer-binding protein coactivator. However, the de-pendence on these activities for FGF-4 enhancer functionsuggests that Fx, like OCA-B, may be a member of a generalclass of tissue-specific factors which determine the restrictedexpression of specific gene subsets through interaction withproteins bound to octamer elements.One obvious difference between these two factors is that

OCA-B apparently does not bind DNA while Fx does. In thisrespect, Fx resembles more the viral coactivator VP16, whichinteracts with DNA sequences adjacent to an octamer bindingsite in the herpes simplex virus immediate-early gene enhancerto activate Oct-1-dependent transcription in conjunction withan additional cellular factor (12, 15, 26, 46, 63). Therefore,although there seems to be no general rule concerning theinitial mode of octamer-coactivator association, it will beinteresting to determine whether these different strategieselicit similar consequences on the molecular properties ofoctamer factors or distinct effects that are factor and/ortemplate specific.The role of the tissue-specific octamer-binding proteins in

relation to that of Oct-1 remains to be elucidated. It is possiblethat they may modulate the overall activity of these octamer-dependent genes by activities that are either redundant orantagonistic to those of Oct-1, or they may interact with anentirely separate set of target genes. However, it is likely thatthe key to understanding tissue-specific octamer-dependenttranscription for many genes will rely on the identification ofadditional cell-restricted activities, such as OCA-B and Fx, thatcan act in conjunction with octamer-binding proteins.

ACKNOWLEDGMENTS

We thank Anna Maria Curatola and Yehia Daaka for contributionsto some earlier studies. We also thank T. Gerster and H. Hamada forthe Oct-3 clones that were used to generate constructs for antibodyproduction, Neil Segil for the polyclonal and monoclonal antibodiesraised against Oct-1, and F. LaBella for generously providing non-ECnuclear extracts. We are grateful to N. Heintz, M. Konarska, D. Levy,B. Moorefield, N. Segil, and N. Tanese for helpful comments on themanuscript and to Connie Cheung and Eva Deutsch for experttechnical assistance.

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This investigation was supported by PHS grants CA45689 andCA49472 from the National Cancer Institute to C.B. and L.D.,respectively.

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3. Corcoran, L. M., M. Karvelas, G. J. V. Nossal, Z.-S. Ye, Y. Jacks,and D. Baltimore. 1993. Oct-2, although not required for earlyB-cell development, is critical for later B-cell maturation and forpostnatal survival. Genes Dev. 7:570-582.

4. Curatola, A. M., and C. Basilico. 1990. Expression of the K-fgfproto-oncogene is controlled by 3' regulatory elements which arespecific for embryonal carcinoma cells. Mol. Cell. Biol. 10:2475-2484.

4a.Curatola, A. M., and L. Dailey. Unpublished results.4b.Dailey, L. Unpublished observation.5. Dailey, L., S. B. Roberts, and N. H. Heintz. 1988. Purification of

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