Interaction between a Novel F9-Specific Factor and Octamer-Binding ...

MOLECULAR AND CELLULAR BIOLOGY, Dec. 1994, p. 7758-7769 0270-7306/94/$04.00+0 Copyright © 1994, American Society for Microbiology

Vol. 14, No. 12

Interaction between a Novel F9-Specific Factor and Octamer-Binding Proteins Is Required for Cell-Type-Restricted Activity of the Fibroblast Growth Factor 4 Enhancer LISA 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 is fundamental question that underlies much of developmental and cellular biology. One example is provided by the fibroblast growth factor 4 (FGF-4) gene, whose expression is restricted to specific embryonic tissues during development and to undifferentiated embryonal carcinoma cells in tissue culture. Analysis of the cisand trans-acting elements required for the activity of the previously identified FGF-4 enhancer in F9 embryonal carcinoma 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, which conform to a binding site for the high-mobility group (HMG) domain factor family, were also critical to enhancer function. We have identified a novel F9-specific factor, Fx, which specifically recognizes this motif. Fx formed complexes with either Oct-i or Oct-3 in a template-dependent manner. The ability of different enhancer variants to form the Oct-Fx complexes correlated with enhancer activity, indicating that these complexes play an essential role in transcriptional activation of the FGF-4 gene. Thus, while FGF-4 enhancer function is octamer site dependent, its developmentally restricted activity is determined by the interaction of octamerbinding proteins with the tissue-specific factor Fx. a

Embryonic development is determined by a complex series of intercellular interactions that include direct cell-cell contacts as well as responses to signaling molecules. Among the signaling molecules identified to date, members of the mammalian fibroblast growth factor (FGF) family have been shown to play an integral part in both early and late stages of embryogenesis (1, 16). Although the members of the FGF family share several overlapping activities in tissue culture assays, the distinct pattern of expression of each gene in the developing embryo suggests that individual FGFs play unique roles in vivo. It is thus implied that specific developmental events require the temporally and spatially regulated expression 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 later embryonic tissues (45). It has previously been shown that in tissue culture FGF-4 gene transcription is restricted to undifferentiated embryonal carcinoma (EC) cell lines and that differential expression of both the murine and the human FGF-4 genes is dependent on an enhancer element located in the untranslated portion of the third exon (4, 73). This enhancer efficiently stimulates transcription from both homologous and heterologous promoters in undifferentiated F9 EC cells but is inactive in differentiated F9, HeLa, or NIH 3T3 cells (4). In that study, it was also noted that both the murine and the human enhancers harbored consensus octamer motifs. This was of particular interest since undifferentiated EC cells * Corresponding authors. Mailing address (L.D.): The Rockefeller University, 1230 York Ave., New York, NY 10021. Phone (L.D.): (212) 327-7961. Fax (L.D.): (212) 327-7878. Mailing address (C.B.): Department of Microbiology, New York University School of Medicine, 550 First 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 (also called Oct-4 and NFA3) (33, 47, 56) and Oct-6 (56), in addition to the ubiquitously expressed Oct-i protein, suggesting that one or both of these EC-specific factors could be directly responsible for the restricted activation of the FGF-4 gene (35,

57). Octamer-binding proteins are members of the POU family of 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 is composed of two subregions that are jointly required for avid and 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), this element is also involved in the regulation of a variety of genes which are expressed in a tissue- or development-specific manner (6, 9, 36, 39, 40, 48). One of the central questions that emerges from these studies is how such a diversity of expression patterns can be achieved through a common DNA binding element. One possibility is that this reflects the differential activities of distinct proteins that recognize the octamer motif (54). However, octamer-binding protein(s) frequently requires additional factors in order to potentiate transcriptional activation (26, 43, 63, 69), suggesting that specific recognition of target genes may be determined by particular combinations of octamer-binding protein with such factors. More recently, a new family of development-regulatory DNA-binding proteins has been described. The prototype of this family, the Sry gene, encodes the male-sex-determining factor (7, 59), which contains a single 80-amino-acid highmobility group (HMG)-like domain (11, 23). This HMG motif is shared among the family members, which have therefore been designated Sox (Sry-HMG-box) factors (17). The Sox factors and the closely related HMG domain proteins, such as 7758

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LEF-1/TCF-1lo, can bind a common specific sequence [CTT TG(T/A)(T/A)] (8, 18, 44, 65, 68, 71, 72, 75) through an interaction within the minor groove (13, 14, 70) and exhibit distinct tissue-specific patterns of expression (17, 59, 68, 72). Thus, many of the questions regarding target gene selection and activation by the octamer factors are also relevant to genes whose transcription is regulated by HMG domain proteins. In this study, we have undertaken a detailed examination of the cis- and trans-acting elements required for transcription of the murine FGF-4 gene in F9 cells. A combination of in vitro DNA binding analyses and transfection assays using a series of FGF-4 enhancer-CAT gene reporter constructs has revealed that full enhancer activity requires DNA sequences that bind octamer, Sox, and SpI transcription factors. In particular, we have identified an F9-specific protein which binds to a Sox recognition motif located immediately upstream of a consensus octamer site and which is required for enhancer activity. This factor, designated Fx, forms complexes with both Oct-1 and Oct-3 on DNA fragments containing both the Fx and the octamer binding sites, suggesting that Fx plays a key role in the activation of FGF-4 gene transcription through its interaction with octamer-binding proteins. Although the activity of the FGF-4 enhancer is octamer sequence dependent, the results suggest that the F9-specific Fx factor, rather than Oct-3 or Oct-6, may be the critical regulator of FGF-4 expression in embryogenesis and that Fx can perform its function in conjunction with the ubiquitously expressed Oct-1 protein. These results not only indicate that the FGF-4 gene is a specific target gene for a Sox or other HMG domain factor but also provide new insights concerning both octamer-binding protein- and HMG domain factor-dependent regulation of developmentand tissue-specific gene expression.

MATERLALS AND METHODS Cell culture and nuclear extract preparation. F9 and HeLa cell cultures were grown in suspension in Joklik's medium (JRH Biosciences) supplemented with 10 or 5% fetal bovine serum, respectively. Cells were harvested at approximately 3 X 105/ml, and nuclear extracts were prepared as described previously (5). Plasmids and oligonucleotides. The pKfgfCAT construct contains approximately 1 kb of human FGF-4 promoter sequence upstream of the chloramphenicol acetyltransferase (CAT) gene as described previously (4). All enhancer constructs were derived from pKfgfCAT by insertion of enhancer fragments at the BamHI site downstream of the CAT gene sequences (4). Plasmids pFGF1A and pFGF1B were constructed by insertion of the 380-bp murine FGF-4 enhancer DraI fragment into the SmaI site of pUC18 or pGEM-3, respectively. Oligonucleotides were synthesized by the Protein Sequencing Facility at The Rockefeller University or by H. Yuan on an Applied Biosystems DNA synthesizer. The octamer site oligonucleotide competitor is derived from the octamer binding site of the H2B promoter (28). Sequences of other oligonucleotides used are depicted in the figures. Transfection and CAT assays. DNA was transfected into F9 or HeLa cells by the calcium phosphate precipitation method as described previously (4), with the following modifications: cells were split 4 to 6 h before the addition of DNA precipitates, and cell lysates were prepared 36 h after transfection. As an internal control, pCH110, a plasmid containing the 3-galactosidase gene fused to the simian virus 40 promoter, was cotransfected with CAT constructs. 13-Galactosidase activity was measured and used to normalize for transfection varia-

7759

tions. P-Galactosidase and CAT activities were measured as described by Curatola and Basilico (4). Mutagenesis. The 380-bp DraI-DraI murine enhancer DNA fragment was inserted into M13mpl8. Uracil-substituted single-stranded DNA was generated, and site-specific mutagenesis was performed. Mutagenic oligonucleotides were synthesized and purified by Sephadex G-25 chromatography. These oligonucleotides were designed to alter 6 to 12 consecutive bp of the enhancer sequence by changing, in most cases, pyrimidine to purine or vice versa. After synthesis, the doublestranded DNA hybrid was transformed into ung' Escherichia coli XL1-blue cells and mutant phages derived from the uracil-free DNA strand were selected. Three to five putative mutant phages from each mutagenesis were screened by dideoxy DNA sequencing (56), and those with the desired mutations were used as templates for amplifying the enhancer fragment by PCR. The amplified DNA fragment was digested with 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, TTAAGACTCTGCTGGtctcaggaatAGCAACCTCCCGA AT; m2, GCTGGGAGACTTCTGctaccaagaaCGAAI7AAC TFl7TATG; m3, CTTCTGAGCAACCTCCCGccggccagggcgG AGGCTACAGACAGCA; m4, CGAATTAAC7fITATGttct tagcacGACAGCAAGACTGGA; m5, AC7FITATGGGAGG CTACAttactacctcagGGAAAATCTCATTGGCAT; m6, CAG CAAGACTGGcttcaaTCATTGGCATFl7T; m7, AGACTGGA GT; m8, AAAATCTCAT AAATCTCcaattatg'lFF 'l'l'l'll TGGCATaaaggaggTTTl'GTC'lTl'CACATTCCT; m9, GGCA I'I'l'I'I'1''FIl'gtgagggaACATYCCTI-rAGAAA; mlO, TI[ J'lT'GTC'Tl'CACcggaagggAGAAAACTCThlGTT; ml 1, ACATTCFI'CFl7AGccccggCTFl7TGTITGGAT; m12, TTAGA AAACTCTggcgggGGATGCTAATGG; m13, AAACTC'ITT G'll7GGggtaacccGGGATACTTAAAATAC; m14, TTGGA TGCTAATtttcatcTTAAAATACTA; mi5, GCTAATGGGA TAaaattcATACTATTCTGT; m16, TGGGATACTTAAAAT cagcggagtgACCACAGCCCAAGAT; m17, CAAGATGGAA GAAGCacacaaaaccAGCTGAGGTGGGAGC; ml8, TACCA CAGCCCAAGAgttcctcctaCACACCCCAAAGCTG; m19, CAAGATGGAAGAAGCacacaaaaccAGCTGAGGTGGG AGGAGC; m20, AGCCACACCCCAAAGagtcttgtttAGCTC CTCCCAAACT; m21, AAGCTGAGGTGGGAGaataaaaacc ACfTTCCTFITCTGTCT; m22, GAGCTCCTCCCAAACagttg ggaatTCTGGTGGCTCACAGGAC; and m23, TCCCAAACT

TCC'Fl'lCTGgagttgttagaCCAGGACAATAAGA'l'lllG. Preparation of DNA probes. The Hinfl-RsaI or Hinfl-SacI probe was obtained by Hinfl digestion of the isolated EcoRIHindIII insert fragment from pFGFlA DNA followed by labelling with [a-32P]dATP (New England Nuclear) and E. coli Klenow DNA polymerase and subsequent digestion with RsaI or Sad. The RsaI-DraI fragment probe was generated by primary digestion of pFGFlB DNA with either HindIII (which cuts within the vector polylinker downstream of the FGF Dral site) or RsaI and labelled at the 5' end with [y-32P]ATP and T4 polynucleotide kinase after phosphatase treatment. Subsequent digestion with RsaI or HindIII was followed by isolation of the labelled fragments from a native 5% polyacrylamide gel in 0.25 x Tris-borate-EDTA (TBE). The specific activities of these 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 primers complementary to FGF-4 enhancer sequences from nucleotides 93 to 110 and 214 to 231. The PCR product was gel purified, and 0.5 to 1 ,ug was labelled with [-y_32P]ATP and T4 polynucleotide kinase. After digestion with Sad (see Fig. 3A),

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the 116-bp fragment probe was isolated by electroelution from a 5% native gel in 0.25X TBE. Oligonucleotide probes were prepared by labelling with T4 polynucleotide kinase and

PLASMID

[y-32P]ATP. DNA binding assays. The electrophoretic mobility shift assay (EMSA) was performed as described previously (5) by incubating 0.5 to 1 ng of fragment or oligonucleotide DNA probe 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 RI of polyclonal or monoclonal antibody preparations was added either before or after probe addition. Samples were incubated for 15 to 30 min at room temperature and resolved by electrophoresis on a 4% polyacrylamide gel containing 0.25X TBE and 0.1% Nonidet P-40. DNase I footprinting experiments using purified protein were performed by incubating 0.5 to 1 ng of DNA fragment probe with 10 to 40 ng of purified Oct-1 or recombinant Oct-3 (rOct-3) protein for 15 min at room temperature, after which CaCl2 and MgCl2 were added (10 and 1 mM final concentrations, respectively) and the mixtures were digested for 30 s with 0.5 Vg of DNase I (Sigma) per ml at room temperature. The reactions were stopped by the addition of EDTA, phenol extracted, and ethanol precipitated, and the reaction products were resolved in a 6% polyacrylamide-50% urea sequencing gel in 0.5 X TBE. DNase I footprinting and methylation interference analysis from complexes resolved on native gels were done as described previously (5). Antibody preparation. The anti-Oct-1 monoclonal antibody (5G5) recognizes an epitope within the N-terminal portion of Oct-1 (57a) and, together with an anti-Oct-1 antiserum, was a gift from Neil Segil at Rockefeller University. N. Segil also generously provided the recombinant construct pGEXOct-3, which contains the complete cDNA of murine Oct-3 inserted in the pGEX-2T vector (Pharmacia). The production of rOct-3 in transformed bacteria was induced by IPTG (isopropyl-P-Dthiogalactopyranoside), and lysates were prepared by sonication in BC1OON buffer (20 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid] [pH 7.9], 100 mM KCl, 20% glycerol, and 0.02% Nonidet P-40) supplemented with 0.5 mM phenylmethylsulfonyl fluoride and 0.5 mM dithiothreitol. rOct-3 protein was purified by passage of the cell lysate over an octamer site oligonucleotide column in BC1OON buffer and recovery of rOct-3 by elution with BCSOON. A 30-t>g sample of rOct-3 was incubated with aluminum hydroxide (Alu-gels; Serva) overnight at 4°C, and this preparation was injected into a New Zealand White rabbit at 1-month intervals. Serum was prepared 2 weeks after each injection. Chromatography. Approximately 80 mg of F9 or HeLa cell nuclear extract was applied to a 0.5-ml wheat germ agglutininagarose (WGA) (Vector Laboratories) column in BC1OON buffer and washed with 3 column volumes of BC1OON buffer, and the bound proteins were eluted with 0.3 M N-acetylglucosamine in BC100N (22). Active fractions from the WGA step were pooled and applied to a 0.5-ml oligoaffinity column (Doct column) which contains a DNA oligonucleotide with two octamer binding sites (42). After washes with 3 column volumes of BC1OON, bound proteins were step eluted with 0.2 and 0.5 M KCl BC buffer. Fx activity is distributed in several of these fractions, while Oct-3 is recovered in the WGA flowthrough (FT) and Oct-1 is recovered in the Doct 0.5 M KCl fraction. RESULTS The murine FGF-4 enhancer binds Spl as well as ubiquitous and F9-specific octamer proteins. The FGF-4 enhancer

RELATIVE CAT ACTIVITY

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

was previously mapped to a 700-bp fragment within the third exon of the human FGF-4 gene and to a homologous 380-bp region of the murine gene (4). Further analysis demonstrated that the 270-bp region between the Hinfl and DraI sites of the originally defined murine enhancer segment is sufficient for full F9-specific transcriptional activation (Fig. 1). In order to identify potential F9-specific transcriptional activators, the protein-DNA interactions throughout this entire region were analyzed. Two enhancer DNA subfragments (Hinfl-RsaI or RsaI-DraI; Fig. 1) were utilized in DNA binding assays, and the protein-DNA complexes formed when F9 nuclear extract was used were compared with those formed when HeLa cell nuclear extract was used. As shown in Fig. 2A, methylation interference analysis with the RsaI-DraI DNA probe and F9 nuclear extract demonstrated that G residues essential for factor binding are located within the two regions designated sites A (positions 184 to 193; Fig. 3A) and B (positions 209 to 217; Fig. 3A). These sites resemble Spi recognition motifs (24, 25), and Spl binding at each was confirmed by competition with Spl consensus, site A, or site B oligonucleotides in EMSAs (data not shown). Similar results were obtained with HeLa 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-4 enhancer sequences located between the Hinfl and RsaI sites (Fig. 2B). Two complexes detected in F9 nuclear extract (Oct-1 and Oct-3) were abolished by the inclusion of octamer oligonucleotide competitor (Fig. 2B, lane 2), and the identity of the protein component of each complex as Oct-1 or Oct-3 was confirmed by using antisera specific for each factor (data not shown). Formation of the protein-DNA complex of interme-

FACTORS REGULATING ACTIVITY OF FGF-4 ENHANCER

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FIG. 2. Analysis of protein-DNA interactions on the FGF-4 enhancer. (A) Methylation interference. The RsaI-DraI fragment probe, labelled either the coding or the noncoding strand, was partially methylated by treatment with dimethyl sulfate (38). Each probe was incubated with F9 nuclear extract and poly(dI-dC), and the reaction products were resolved on a native polyacrylamide gel. Bound and free probes were eluted and cleaved with piperidine, and the reaction products were resolved in a sequencing gel. Reaction products derived from unbound probe (Free) and those derived from probe bound to protein (Bound) and the regions showing the absence of bands in the bound-probe sample compared with the free 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 sample of radiolabelled Hinfl-RsaI FGF-4 enhancer fragment (Fig. 1) was incubated with F9 or HeLa nuclear extract and poly(dI-dC) in either the absence or the presence of 10 ng of octamer (lanes 2 and 5) or Spl (lanes 3 and 6) competitor oligonucleotides as indicated. The reaction products were resolved on a 4% native polyacrylamide gel and visualized by autoradiography. The positions of the Oct-i, Spl, and Oct-3 protein-DNA complexes are 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 of radiolabelled 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 partially purified recombinant mouse Oct-3 (lanes 5 to 7) and subjected to mild DNase I digestion as described in Materials and Methods. The samples in 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 sites within the FGF-4 enhancer are indicated in Fig. 3A. Lane 1 shows G residues of the probe DNA included as a marker. on

diate mobility was specifically inhibited by addition of an Spl oligonucleotide (Fig. 2B, lane 3), thus identifying the factor in this complex as Spl. Both Spl and Oct-i complexes were, as expected, also present in HeLa cell nuclear extract (Fig. 2B, lanes 4 to 6), whereas the Oct-3 protein-DNA complex 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 of either purified human Oct-i or recombinant mouse Oct-3 and the resultant complexes were analyzed by DNase I footprinting as shown in Fig. 2C. Both octamer-binding proteins protected two regions of the FGF-4 enhancer corresponding to the octamer binding sites noted by Curatola and Basilico (4) and were designated OCTA 1 (positions 76 to 83; Fig. 3A) and OCTA 2 (positions 130 to 137; Fig. 3A). Together, the results of the DNA binding analyses indicate that the FGF-4 enhancer contains recognition sequences for both Spl and octamer-binding protein families of transcriptional activators. Only one of these factors, Oct-3, is specific to F9 cells, while Oct-i and Spl are present in both F9 and HeLa cell extracts. consensus

Mutation analysis of the FGF-4 enhancer reveals regions critical to enhancer function that center around OCTA 2. To assess the relative contributions of the factor binding sites as well as other elements to FGF-4 enhancer activity, a comprehensive series of mutants was created. As summarized in Fig. 3, mutant enhancers containing substitutions of a unique set of 6 to 12 consecutive bp were inserted downstream of the FGF-4 promoter-CAT gene in the pKfgf plasmid (4) and tested for their ability to activate CAT gene expression after transfection into F9 cells. The results of this analysis, shown in Fig. 3B, revealed that most of the mutants displayed some variation of CAT expression levels with respect to the wild-type (wt) enhancer. Although some of the mutations reproducibly increased enhancer activity by about 30% (m5 and m1O) and thus could be affecting sequences involved in transcriptional repression, none of the mutations conferred activity in differentiated F9 or HeLa cells (data not shown). On the other hand, severe negative effects on enhancer activity were observed in mutants containing 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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TTTGTTTGG ggtaaccc GGGATACTT AAACAAACC ccattggg CCCTATGAA FIG. 4. Mutation of OCTA 2 in ml3 compromises octamer-protein binding. Oligonucleotide probes that contain the wt or the m13 mutated (mt) sequence at the OCTA 2 site were tested for their abilities to bind octamer-binding proteins by an EMSA using F9 or HeLa cell nuclear extracts. In competition assays, 100-fold molar excesses of unlabelled wt or mutant oligonucleotides were added to the reaction mixtures as indicated above the lanes. mt Probe

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FIG. 3. Mutation analysis of sequences within the FGF-4 enhancer. (A) Definition of the sequences altered for FGF-4 enhancer mutants 1 to 23. The DNA sequence of the entire FGF-4 enhancer between the Hinfl and DraI sites is shown. Binding sites for the octamer proteins (OCTA 1 and OCTA 2) and the Spl protein binding sites as defined in Fig. 2 (shaded residues) are indicated. The data defining the Spl site at positions 42 to 48 were not presented. Underlined sequences indicate the residues changed within each mutant. (B) Relative CAT activities of FGF-4 enhancer mutants 1 to 23. Each of the mutant enhancer fragments shown in panel A was cloned downstream of the CAT gene in the pKfgfCAT vector, and the ability to induce CAT gene expression was compared with that of the wt enhancer after transfection into F9 cells. The CAT activity of the wt enhancer construct is 100%. The results for each of the 23 mutant enhancer constructs are averages of two independent experiments. The entire length of the FGF-4 enhancer shown in panel A and the positions of the octamerbinding protein (white boxes) and Spl (shaded boxes) binding sites are represented schematically beneath the histogram.

tial role of this sequence in enhancer function. As expected, binding of neither Oct-1 nor Oct-3 was observed in F9 or HeLa cell extracts when an oligonucleotide containing the m13 mutant sequence was used (Fig. 4). Somewhat surprisingly, mutation of the OCTA 1 site (m7) had no appreciable effect on enhancer function, and an intact OCTA 1 element could not compensate 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 of the enhancer. It is therefore interesting that mutation of DNA sequences located either immediately upstream (m12) or downstream (m14 and miS) of the OCTA 2 site also had a severe 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 enhancer consists of Spl site A, the OCTA 2 site, and DNA sequences flanking OCTA 2. Since the Spl activity binding to site A was not F9 specific, we concentrated our analysis on OCTA 2 and its flanking regions. Additional DNA binding activities are revealed in the absence of poly(dI-dC). Several mechanisms could account for the importance of the DNA sequences flanking OCTA 2 for

VOL. 14, 1994

FACTORS REGULATING ACTIVITY OF FGF-4 ENHANCER

enhancer activity. The topology of these sequences might directly affect the association of octamer protein with OCTA 2 or induce a particular conformation of octamer protein bound at this site. If this was the case, the differential activity of the FGF-4 enhancer in F9 cells might be explained if the effects of the 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 plasmids with a plasmid expressing Oct-3 in either HeLa or differentiated F9 cells failed to activate the FGF-4 enhancer (data not shown), indicating that Oct-3 alone is not sufficient for enhancer function. We therefore considered the possibility that the DNA sequences flanking OCTA 2 may bind an additional factor(s) that was not identified in the DNA binding assays described above. Several recent reports have described proteins whose interaction with DNA occurs primarily through dA-dT residues within the minor groove (13, 31, 60, 61, 70). Since poly(dI-dC), the standard "nonspecific" competitor used in the binding assays, resembles dA-dT within the minor groove, it was possible that the presence of this polymer prevented the detection of analogous activities. To investigate this possibility, we substituted poly(dG-dC) for poly(dI-dC) as the nonspecific DNA competitor in the EMSA. In these experiments, we employed a probe that spans positions 113 to 149 within the FGF-4 enhancer (Fig. 5A; also Fig. 3A) and thus encompasses OCTA 2 and a portion of its flanking sequences. As shown in Fig. SB, lanes 12 to 14, incubation of the oligonucleotide probe from positions 113 to 149 with F9 nuclear extract in the presence of poly(dI-dC) resulted in the formation of two specific protein-DNA complexes that were sensitive to the addition of octamer site competitor oligonucleotide and thus represent binding by Oct-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 same oligonucleotide probe was incubated with F9 nuclear extract in the presence of poly(dG-dC). As shown in Fig. SB, lane 1, the Oct-1 and Oct-3 complexes that had been observed in the presence of poly(dI-dC) were also detectable in the presence of poly(dG-dC). However, three additional complexes, designated Oct-i*, Oct-3*, and Fx, were observed exclusively in reaction mixtures containing poly(dG-dC). Importantly, these additional complexes were not apparent in HeLa cell nuclear extract (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 upon addition of octamer site competitor oligonucleotide, indicating that they contained octamer-binding proteins (Fig. 5B, lane 4). Incubation of the reaction mixture with a monoclonal antibody generated against Oct-1 resulted in the supershift of the two bands 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 polyclonal antiserum raised against Oct-3 specifically blocked formation of the complexes Oct-3 and Oct-3* (Fig. SB, lane 3), indicating that 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 suggested that they also contain an F9-specific factor. As described above, the Fx complex detected in the presence of poly(dG-dC) was specific to F9 nuclear extract (compare lanes 11 and 15 in Fig. SB). Formation of the Fx complex was not sensitive to the addition of either octamer or Spl competitor oligonucleotides (Fig. 5B, lanes 4 and 11) but was effectively inhibited by an oligonucleotide, FxO-, containing DNA se-

7763

quences upstream of OCTA 2 (Fig. SB, lanes 5 to 7). These sequences contain the consensus binding motif (CTlTGT1T) of the tissue-specific and developmentally regulated Sox factor family. We thus investigated whether these residues provide a recognition site for Fx. As shown in Fig. SB, lanes 8 to 10, an oligonucleotide 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 Fx interaction with the FGF-4 enhancer. Significantly, both the Oct-1* and the Oct-3* complexes were also eliminated by incubation with the FxO- oligonucleotide, suggesting that these complexes contain both Fx and the octamer-binding proteins (Fig. 5B, lanes 5 to 7). This conclusion was further supported by the observation that formation of both Oct-1* and Oct-3* was unaffected by competition with the Fx-Ooligonucleotide (Fig. 5B, lanes 8 to 10). Together, these results indicate that a Sox consensus sequence located immediately upstream of the critical OCTA 2 site in the FGF-4 enhancer is recognized by an F9-specific factor, Fx, and that Fx can interact with octamer-binding protein bound at OCTA 2 to form the Oct-1* or Oct-3* complex. The formation of Oct* correlates with the presence of Fx in chromatographic fractions. To facilitate further analysis of the Oct-1* and Oct-3* complexes, Fx and the octamer proteins were partially purified from F9 nuclear extract by using a WGA column as described in Materials and Methods. DNA binding activities present in the input nuclear extract, FT, and step fractions were monitored by EMSA with the oligonucleotide probe spanning positions 113 to 149 as shown in Fig. SC. Fx activity present in F9 nuclear extract cofractionated with both Oct-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 the Oct-3* complexes were still observed in fractions containing both 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). Proteins bound to the affinity resin were eluted in two steps with 0.2 and 0.5 M KCl, and the activities present in each fraction were determined by EMSA. As shown in Fig. SC, lanes 4 to 6, a portion of the input Fx activity cofractionated with Oct-1 and was eluted from the DNA column by 0.5 M KCl; again, the Oct-1* complex was observed. However, Fx activity was also detected in the FT from this column, which was relatively free of Oct-1 and Oct-3 (Fig. SC, lane 4). We tested whether addition of the Doct FT fraction to either Oct-1 or Oct-3 could generate the Oct-1* and Oct-3* complexes. Incubation of Oct-1 protein purified from HeLa cell nuclear extract with the oligonucleotide probe spanning positions 113 to 149 resulted in formation of the single Oct-1 complex (Fig. SD, lane 3). However, addition of increasing quantities of the Doct FT fraction to HeLa Oct-1 resulted in the generation of the Oct-1 * complex (Fig. SD, lanes 3 to 5). Similarly, addition of the Doct FT fraction to the WGA FT fraction that contains Oct-3 (and Fx) resulted in a progressive increase in Oct-3* complexes (Fig. SD, lanes 6 to 9). These results further support the notion that the Fx factor, together with Oct-1 or Oct-3, is present in the 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 extracts were prepared from a number of mammalian tissue culture cell lines and incubated with a DNA probe that contains a single Fx binding site (FXO-; Fig. 5A). As shown in Fig. 6, Fx binding activity was detected only in nuclear extracts derived from the EC cell lines F9 and P19. Comparable Fx activity was not observed in any of the other cell extracts tested. This result indicates that Fx is present in cell lines that express FGF-4 (57,

7764

DAILEY ET AL.

MOL. CELL. BIOL.

A

OCT

SOX

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

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1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 the of poly(dI-dC). (A) DNA sequences of probe and competitor formed in absence 5. of the FIG. Analysis protein-DNA complexes oligonucleotides. The wt oligonucleotide spans positions 113 to 149 of the FGF-4 enhancer shown in Fig. 3A. The octamer-binding protein binding site 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 with those 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 reaction mixtures are indicated above the lanes, as are the quantities of each added. ct1 and c3, reaction mixtures for which monoclonal antibody raised against Oct-1 protein or polyclonal antiserum raised against Oct-3 protein, respectively, was used; Oct-i*, Oct-i, Oct-3*, Fx, and Oct-3, specific protein-DNA complexes observed as described in the text. A broad band that migrates in a position similar to that of Fx is sometimes observed when HeLa nuclear extracts are used (lane 15). This activity is nonspecific and distinct from Fx as determined by oligonucleotide competition (data not shown). (C) EMSA using chromatographic fractions derived from F9 nuclear extract. F9 nuclear extract was fractionated by WGA and DNA affinity column chromatography as detailed in Materials and Methods. Aliquots (1 ±1) from input nuclear extract and each of the chromatographic fractions were analyzed by EMSA using the wt oligonucleotide probe in the presence of poly(dG-dC), and the reaction products were resolved on a native polyacrylamide gel. NE, input nuclear extract; WFT, WGA FT fraction; W STEP, WGA step fraction; DOCT, fractions derived from the Doct 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 were added 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 the presence 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 transcribe FGF-4 (73) and is consistent with the notion that Fx is required for 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. 4 demonstrated the functional importance of OCTA 2 and its

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

FACTORS REGULATING ACTIVITY OF FGF-4 ENHANCER

VOL. 14, 1994

EXTRACT:

Fx

r9

-

Q

lb

B

FIG. 6. Fx binding activity is EC cell specific. Samples (3 pg) of nuclear extracts prepared from the various mammalian cell lines indicated above the lanes were analyzed for Fx binding activity by EMSA using 0.2 ng of FxO- probe DNA (Fig. 5A). The specificity of this complex was confirmed by competition experiments (data not shown). For clarity, 10 ng of an Spl binding site oligonucleotide was included in the reaction mixtures to reduce nonspecific protein-DNA complexes.

complexes observed when the Doct 0.5 M KCl fraction and the wt oligonucleotide probe spanning positions 113 to 149 (Fig.

5A) were compared with those obtained when analogous probes derived from enhancer mutants containing base substitutions 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-1 complexes, as well as Oct-i*. In contrast to the distinct band produced by Fx binding to the wt probe, a fainter cluster of nonspecific 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 probe was unable to bind Oct-i, and the Oct-1* complex was not observed (Fig. 7A, lane 7). These results indicate that formation of Oct-1* is template dependent and requires both the Fx and the octamer-binding protein binding sites. Interestingly, the intensity of the band corresponding to the Fx complex when the m13 probe was used appeared to be greater than that observed for the wt probe (compare lanes 1 and 7 of Fig. 7A). This suggested that whereas Fx was bound independently to the m13 probe, most of the Fx bound to the wt probe was present in the Oct-1* complex. To test this hypothesis, the Doct 0.5 M KCl fraction was preincubated with a polyclonal antiserum raised against purified Oct-i protein (58). While this treatment eliminated both the Oct-I and the Oct-i* complexes (Fig. 7A, lane 2), the intensity of the band corresponding to the Fx complex increased for the wt probe (compare lanes 1 and 2 of Fig. 7A). In contrast, no change in the number of independently bound Fx complexes was noted upon treatment with preimmune serum (compare lanes 1 and 3 of Fig. 7A), and anti-Oct-1 antibody pretreatment did not alter the amount of Fx bound to the mi3 probe (Fig. 7A, compare lanes 7 and 8). The correlation between the loss of Oct-1* and the concomitant increase in the number of independent Fx complexes bound to the wt probe in the presence of the anti-Oct-1

7765

antiserum indicates that the majority of Fx activity in this fraction is present in the Oct-i * complex when both factor sites (and both factors) are present. Together, these results demonstrate that the inability of the mi2 and m13 FGF-4 enhancer constructs to efficiently activate transcription directly correlates with their inability to direct the formation of Oct-l* and further establishes that binding of Fx or octamer-binding protein 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 footprinting assay was employed. Samples containing wt enhancer fragment probe and the Doct 0.5 M KC1 fraction were subjected to mild DNase I digestion, the protein-DNA complexes were resolved in a native gel, and the Oct-1*, Oct-i, and Fx complexes were eluted and analyzed in a denaturing gel. As shown in Fig. 7B, the region of the probe protected from nuclease digestion in the Oct-1* complex (lane 5) spans the regions protected by both Oct-i (lane 3) and Fx (lane 6). DISCUSSION In this report, we present evidence that interactions between a novel F9-specific factor and octamer-binding proteins are essential to FGF-4 enhancer function in F9 cells. These results identify FGF-4 as a specific target gene for an embryonically expressed factor, Fx, that is most likely a member of the HMG domain factor family and also demonstrate that this factor potentiates transcriptional activation through its interaction with an octamer-binding protein. Although both of these factor families have been implicated in the regulation of specific gene subsets during development, the mechanism of their selective interactions with their target genes has remained elusive. These conclusions thus have implications with respect to tissue-specific and development-specific regulation of both octamer and Sox site-dependent gene transcription. FGF-4 enhancer activity requires the interaction of several factors. We have analyzed the cis- and trans-acting elements required for activation of gene transcription by the FGF-4 enhancer in F9 cells. Since F9 cells, like other EC cell lines, resemble cells of the blastocyst inner cell mass, the components which we have identified as important to FGF-4 enhancer function are likely to be operative in the embryonic environment. DNA binding analyses have revealed that multiple proteins of the octamer and Spl factor families bind to several sites within the FGF-4 enhancer. The critical cis-acting elements, however, are confined to a relatively small region that includes Spi site A and octamer binding site OCTA 2. We have shown that OCTA 2 can bind the ubiquitous Oct-i protein as well as the F9-specific factor Oct-3, while Spl site A binds Spl, which is present in both F9 and HeLa cells. However, the mutagenesis studies emphasize that complete elucidation of this enhancer function also requires a definition of the contribution of the regions flanking the OCTA 2 site, since alteration of these DNA sequences is severely deleterious to enhancer activity. Accordingly, we have demonstrated that EMSAs in which poly(dI-dC) is replaced by poly(dG-dC) permit detection of an additional F9-specific factor, Fx, whose binding site lies within the functionally important sequences immediately upstream of OCTA 2. The role of the sequences immediately downstream of OCTA 2 is under investigation. Interaction of these sequences with an additional, F9-specific activity has not been observed (4b), and this region is therefore likely to contribute to overall enhancer activity. Taken together, these observations indicate that FGF-4 enhancer activity requires coordi-

7766

DAILEY ET AL.

MOL. CELL. BIOL.

B

A PROBE:

wt

m12

m13

d

Ab: OCT-1i OCT-1 -

Fx

G

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