Regulation of the DNA-Binding and Transcriptional Activities of ...

MOLECULAR AND CELLULAR BIOLOGY, Oct. 1995, p. 5552–5562 0270-7306/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 15, No. 10

Regulation of the DNA-Binding and Transcriptional Activities of Xenopus laevis NFI-X by a Novel C-Terminal Domain ´ RE ` SE ARMENTERO,1 GRIGORIOS KREY,1 EMMANUELLE ROULET,1 MARIE-THE 1 ´ BLAISE CORTHESY, CHRISTINE DREYER,2 NICOLAS MERMOD,1 1,3 AND WALTER WAHLI * Institut de Biologie Animale, Universite´ de Lausanne, CH-1015 Lausanne,1 and Glaxo Institute for Molecular Biology, CH-1228 Plan-les-Ouates, Geneva,3 Switzerland, and Max-Planck Institut fu ¨r Entwicklungsbiologie, D-72011 Tu ¨bingen, Germany2 Received 17 May 1995/Accepted 29 June 1995

The nuclear factor I (NFI) family consists of sequence-specific DNA-binding proteins that activate both transcription and adenovirus DNA replication. We have characterized three new members of the NFI family that belong to the Xenopus laevis NFI-X subtype and differ in their C-termini. We show that these polypeptides can activate transcription in HeLa and Drosophila Schneider line 2 cells, using an activation domain that is subdivided into adjacent variable and subtype-specific domains each having independent activation properties in chimeric proteins. Together, these two domains constitute the full NFI-X transactivation potential. In addition, we find that the X. laevis NFI-X proteins are capable of activating adenovirus DNA replication through their conserved N-terminal DNA-binding domains. Surprisingly, their in vitro DNA-binding activities are specifically inhibited by a novel repressor domain contained within the C-terminal part, while the dimerization and replication functions per se are not affected. However, inhibition of DNA-binding activity in vitro is relieved within the cell, as transcriptional activation occurs irrespective of the presence of the repressor domain. Moreover, the region comprising the repressor domain participates in transactivation. Mechanisms that may allow the relief of DNA-binding inhibition in vivo and trigger transcriptional activation are discussed. Here we report on the cloning and functional characterization of three Xenopus laevis NFI-X (xNFI-X) members of the NFI family. We show that, on one hand, the DNA replication function is conserved in the N-terminal region of the xNFI-X subtype. The C-terminal region, on the other hand, can be divided into the subtype-specific domain (SSD), identical in all three proteins, and the variable domain that is specific for each isoform. The three xNFI-X full-length proteins can activate transcription in insect cells, devoid of endogenous NFI, and differences observed in the efficiency of transcriptional activation can be attributed to their variable domains at the C terminus. Moreover, GAL4 fusion chimeras with different portions of the C-terminal region show that the SSD participates with the variable domain in transactivation, in contrast to the xNFI-C subtype, in which case the activation domain is restricted to the proline-rich domain. Interestingly, xNFI-X proteins contain a region in the SSD that represses DNA-binding activity in vitro. Mechanisms that might explain how this in vitro DNA-binding inhibition is relieved within the cells, leading to efficient transcriptional activation, are discussed.

Transcription factors have been shown to possess a modular organization typically consisting of two functionally distinct domains implicated in sequence-specific DNA-binding and transcriptional activities. DNA-binding domains are often found adjacent to regions required for protein oligomerization, and in most cases the latter activity is concomitant with or required for DNA recognition. Dimerization and DNA-binding modules are believed to adopt highly ordered and welldefined structural configurations. In contrast, domains responsible for transcriptional activity may not possess such a welldefined organization. It has been proposed that transcriptional activation domains become structured only upon interaction with basal transcription factors (8, 17, 43). A typical example of the modular organization of transcription factors is the cellular nuclear factor I (NFI) family of proteins. These proteins have been shown to be implicated in the activation of many cellular and viral genes (1, 21, 23, 37) as well as in the activation of adenovirus DNA replication (33). In vertebrates, NFI family members consist of four different subtypes, called NFI-A, NFI-B, NFI-C, and NFI-X, each encoded by a different gene (25, 38). In addition, each subtype possesses different isoforms generated by alternative splicing. All NFI polypeptides identified so far display a conserved N-terminal domain and can be distinguished through their divergent Cterminal regions (38). Although numerous NFI proteins have been identified, only the NFI-C subtype has been characterized to some extent. The N-terminal part of the NFI-C proteins contains the DNA-binding, dimerization, and replication functions, while the C-terminal domain is involved in the activation of transcription (20, 32).

MATERIALS AND METHODS Screening and sequencing. Approximately 5 3 105 phages of an X. laevis XTC cell line cDNA library in lgt11 (kindly provided by I. Dawid, National Institutes of Health, Bethesda, Md.) were screened, using as a probe the NcoI-SacI fragment of human NFI-C1 (hNFI-C1), corresponding to the N-terminal portion of hNFI-C1 (32), under low-stringency conditions (63 SSC [13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate]–0.5% sodium dodecyl sulfate [SDS]–53 Denhardt’s solution–100 mg of sonicated salmon sperm DNA per ml–4 3 106 cpm of 32 P-labeled probe at 558C for 12 h). Filters were washed three times at 608C in 33 SSC–0.5% SDS. Two clones, which contained xNFI-C1 and a partial NFI-X subtype sequence, were isolated. A second screening of the same library was performed, using as a probe a fragment containing the C-terminal part of this NFI-X clone. Three of seven clones, xNFI-X1, xNFI-X2, and xNFI-X3, were further analyzed by restriction mapping, Southern blotting, and DNA sequencing together with the xNFI-C1 clone.

* Corresponding author. Mailing address: Institut de Biologie Animale, Baˆtiment de Biologie, Universite ´ de Lausanne, CH-1015 Lausanne, Switzerland. Phone: (41 21) 692-4110. Fax: (41 21) 692-4105. 5552

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PCR analysis. Total RNA from X. laevis tissues and embryos at different developmental stages was extracted as described previously (12, 16). Two micrograms of total RNA was used as a template for reverse transcription. Two xNFI-X-specific primers were designed to analyze the expression of the xNFI-X mRNA species. Primer 1 is 24 nucleotides long and corresponds to a DNA fragment between nucleotides 1151 and 1175 present in all three xNFI-X coding sequences. Primer 2 is 24 nucleotides long and corresponds to a common sequence present in the 39 untranslated regions of all three xNFI-X sequences. PCRs were performed with radioactive deoxynucleoside triphosphates as described previously (41). The labeled products were analyzed by electrophoresis on a 5% denaturing polyacrylamide gel. Plasmid construction. For in vitro expression of xNFI-X polypeptides, the PflMI site positioned at the initiation codon of all three cDNAs was changed to an NcoI site by Klenow filling and ligation. The NcoI-EcoRI fragments containing the entire coding sequences of pxNFI-X1, pxNFI-X2, and pxNFI-X3 were inserted into the corresponding sites of the transcription vector pT7bSal (35), resulting in plasmids pT7xNFI-X1, pT7xNFI-X2, and pT7xNFI-X3. The NcoIEcoRI fragment of pxNFI-C1, containing the entire cDNA sequence, was cloned into the corresponding sites in pT7bSal to generate pT7xNFI-C1. For transfection experiments in Drosophila Schneider line 2 (SL-2) cells, plasmids pT7xNFI-X1, pT7xNFI-X2, pT7xNFI-X3, and pT7xNFI-C1 were linearized with PvuII and recircularized in the presence of an octameric XhoI linker (New England Biolabs). NcoI-XhoI fragments were cloned into the corresponding sites of the pPADH vector (14), resulting in pPADH-xNFI-X1, pPADH-xNFI-X2, pPADH-xNFI-X3, and pPADH-xNFI-C1. The xNFI-X3 39-truncated derivatives xNFI-X3D92, xNFI-X3D176, xNFI-X3D209, and xNFI-X3D277 were constructed by digestion of pPADH-xNFI-X3 with BalI, EcoRI, SmaI, and RsaI, respectively, and recircularization of the plasmids in the presence of an SpeI linker (New England Biolabs) containing a translation stop codon in all three reading frames. The xNFI-C1 39-truncated derivatives xNFI-C1D98 and xNFI-C1D246 were obtained by double digestion with EcoRV and NdeI and recircularization of the plasmids as described above. Internal deletions of xNFI-X2 were recovered from pT7xNFI-X2 by excision of a BalI fragment. The plasmid was recircularized in the presence of an octameric XhoI linker that restored the proper reading frame and resulted in the insertion of three amino acids, Pro, Arg, and Gly. To generate pPADH-xNFI-X2D102, an NcoI-SalI fragment of plasmid pT7xNFI-X2D102 was excised and cloned into the NcoI-XhoI sites of the pPADH vector. pPADHxNFI-X3D312-411 was generated by digestion of pPADH-xNFI-X3 with BalIBamHI, which removes the entire C-terminal part, and by addition of the variable domain generated by PCR amplification, using as oligonucleotides 59ATATAACAGGTCCGATATCCCAGCAGCCGGGCC-39 and 59-ATATAAG GATCCTCAAAGGAACCAGGATTG-39. The amplified fragment was digested by PvuII and BamHI and inserted into pPADH-xNFI-X3 digested with BalI and BamHI. The chimeric plasmid pPADH-xNFI-C1D98/X3 407-502 was constructed by using the same PCR fragment as specified above but digested with EcoRV BamHI and inserted into pPADH-xNFI-C1 digested with the corresponding enzymes, which remove the entire Pro-rich C-terminal domain. GAL4/xNFI-X1 chimeric sequences were constructed by inserting the BalISacI, SmaI-SacI, and RsaI-SacI fragments of pT7xNFI-X1 into the SmaI-SacI sites of plasmid pSG424 (39), resulting in pGal 405-414, pGal 287-414, and pGal 219-414, respectively, in which the coding sequence for the N-terminal 147 amino acids of GAL4 (GAL4 1-147) is fused to that of the indicated NFI-X1 sequence amino acid coordinates. GAL4/xNFI-X2 chimeric sequences were constructed by inserting the same fragments of pT7xNFI-X2 into the SmaI-SacI sites of plasmid pSG424, resulting in pGal 405-433, pGal 287-433, and pGal 219-433, respectively. GAL4/xNFI-X3 chimeric sequences were constructed with the same fragments from pT7xNFI-X3 into pSG424, resulting in pGal 405-487, pGal 287-497, and pGal 219-497. For the internal deletion of pGal 287-433 and pGal 219-433, the SmaI-SacI and RsaI-SacI fragments of pPADH-xNFI-X2D102 were inserted into the SmaI-SacI sites of pSG424, resulting in pGal 287-433(D102) and pGal 219-433(D102). pGal 363-407 was constructed by PCR cloning using the oligonucleotide primers 59-AATATAGGATCCCAAATGATGACAC-39 and 59-AA TATATCTAGACTAGCCAGAGGTGTCTGAGCA-39. The resulting fragment coding for the inhibitory domain was digested with BamHI and XbaI and inserted into pSG424 digested with the corresponding enzymes. pGal 312-411 was generated by PCR cloning using the oligonucleotide primers 59-ATATTAGGATC CGCGTGTCCCAGACACC-39 and 59-ATATTATCTAGAGCC-AGAGGTGT CTGAG-39. The resulting fragment coding for the SSD was digested with BamHI and XbaI and inserted into pSG424 digested with the corresponding enzymes. All constructs were verified by sequencing. For the preparation of vaccinia virus recombinants, the NcoI-EcoRI fragments of pT7xNFI-X3 and pT7xNFI-X3D209 were inserted into the EcoRI site of plasmid pHGS-1 (6) in the presence of a 32-bp NcoI-EcoRI oligonucleotide coding for 12 additional amino acids including a six-histidine tag, resulting in p6H-xNFI-X3 and p6H-xNFI-X3D209. Reporter plasmids were paCAT-D87-33Ad, containing the chloramphenicol acetyltransferase (CAT) enzyme coding sequence linked to the human a-globin promoter and three copies of the CTF/NFI-binding sites from the adenovirus origin of replication; paCAT-D55, containing only a truncated form of the a-globin promoter (32); and pG5BCAT, containing five GAL4-binding sites inserted upstream of the adenovirus E1b TATA box (27).

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In vitro expression of NFI protein derivatives. xNFI RNAs were transcribed with T7 RNA polymerase in vitro from plasmids pT7xNFI-X1, pT7xNFI-X2, pT7xNFI-X3, and pT7xNFI-C1 linearized with EcoRI (xNFI-X1, xNFI-X2, and xNFI-X3), BalI (xNFI-X3D92), SmaI (xNFI-X3D176), RsaI (xNFI-X3D209), EcoRI (xNFI-X3D277), EcoRV (xNFI-X3D304 and xNFI-C1D98), or NdeI (xNFI-C1D246), while RNAs for hNFI-C or its C-terminally truncated derivative were transcribed in vitro from pT7-CTF1 and pT7-CTF1D230 as described previously (3). RNAs were translated by using rabbit reticulocyte lysate in the presence of [35S]methionine as instructed by the manufacturer (Promega). Translation products were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), and the amount of proteins was normalized by densitometric scanning of the autoradiograms. Transfections and CAT assays. Drosophila SL-2 cells or HeLa cells were transfected as described previously (14, 31). For CAT assays, 107 SL-2 cells or 106 HeLa cells were transfected with 1.5 to 4 mg of the reporter plasmid, 2 to 8 mg of the expression vector, and 3 mg of pRSV-Luc. The total amount of DNA was adjusted to 20 mg, using pBluescript KS1 (Stratagene). Cell extracts were prepared, and the CAT activity was measured and normalized to the luciferase activity as described previously (15, 19). For preparative transfections, cells were transfected as before with 15 mg of the indicated expression vector and 3 to 4 mg of pRSV-Luc along with pBluescript KS1 to a total of 20 mg. At 48 h posttransfection, whole cell extracts were prepared (26). The amount of total proteins in whole cell extracts was measured with a Bio-Rad assay kit, and equal amounts of total proteins were used for gel retardation assays. Gel retardation assays. The DNA-binding activities of equal amounts of wild-type and mutant xNFI proteins expressed in rabbit reticulocyte lysate or purified from recombinant vaccinia virus-infected cells were tested by a gel retardation assay (22), using the TD15 DNA probe, which contains the first 50 bp of the adenovirus replication origin (24). The DNA-binding activity of the GAL4/ xNFI-X chimeric polypeptides was tested by using a 23-bp oligonucleotide containing a consensus binding site of GAL4 (9). All reaction mixtures were resolved on a 5% polyacrylamide gel in 0.253 TBE (22.5 mM Tris-boric acid, 0.5 mM EDTA [pH 8.0]). Immunoprecipitation. The NFI-X3 antibody was produced against the variable part of the protein by using the glutathione S-transferase fusion protein system. Five microliters of in vitro-translated or cotranslated [35S]Met-labeled proteins was incubated with 3 ml of rabbit polyclonal antiserum directed against either the NFI-X3 variable domain or the hNFI-C C terminus and precipitated with protein-A Sepharose as described previously (3). The samples were resolved by SDS-PAGE, and the proteins were detected by autoradiography. Preparation, expression, and purification of vaccinia virus recombinant proteins. p6H-xNFI-X3 and p6H-xNFI-X3D209 were used for vaccinia virus genome homologous recombination as described previously (3). HeLa S3 cell infection with recombinant vaccinia viruses, nuclear extract preparation, and protein purification by Ni21-nitrilotriacetic acid-agarose column (Qiagen) chromatography were as described previously (3). The purified proteins (approximately 80% purity as determined by Coomassie blue staining) were dialyzed against buffer D* (20 mM N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid [HEPES]NaOH [pH 7.5], 2 mM MgCl2, 20% [vol/vol] glycerol, 0.5 mM EDTA, 0.01% Nonidet P-40, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 100 mM KCl) and stored in aliquots at 2708C. Replication proteins and DNA replication in vitro. The viral preterminal protein (pTP) and DNA polymerase (Pol) were overexpressed in Spodoptera frugiperda (Sf9) cells by using the recombinant baculovirus system and purified by single-stranded DNA affinity chromatography as described previously (42). pTP and Pol concentrations and purity (approximately 95%) were estimated by Coomassie blue staining of SDS-polyacrylamide gels. The pTP-Pol complex was formed after mixing equal molar amounts of each protein and incubation at 208C for 15 min. The initiation reaction for adenovirus DNA replication was performed with purified proteins essentially as described previously (10, 11). The reaction mixture (15 ml) contained 3 mCi of [a-32P]dCTP (3,000 Ci/mmol), 50 ng of purified adenovirus genome DNA-protein complex, approximately 3 ng of Pol and 1.6 ng of pTP, and an increasing amount of histidine-tagged purified NFI derivatives as indicated for the relevant figures. The labeled pTP-dCMP complexes were resolved by SDS-PAGE after immunoprecipitation with an anti-pTP antibody as described previously (3) and detected by autoradiography. Western blotting (immunoblotting). Whole cell extracts from HeLa cells transfected with pGal 405-433 or pGal 219-433 were prepared as described above. Total proteins (100 mg) were separated by SDS-PAGE and transferred to nitrocellulose membranes. GAL4 fusion proteins were detected by using a mixture of two monoclonal antibodies, 2GV10 and 3GV2, directed against the GAL4 DNA-binding domain (44). Purified xNFI-X3 and xNFI-X3D209 proteins were detected by using a polyclonal antibody directed against the NFI DNAbinding domain (7). The nitrocellulose membranes were developed with an Immune-Lite chemiluminescence kit (Bio-Rad).

RESULTS Isolation and characterization of NFI-encoding cDNAs. A lgt11 cDNA library derived from the X. laevis XTC cell line was screened with a DNA fragment encoding the N-terminal

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MOL. CELL. BIOL. FIG. 1. Structures of X. laevis NFI proteins. (A) Alignment of the deduced amino acid sequences of four Xenopus NFI proteins. Regions of amino acid identity are indicated by gray boxes, and the divergent amino acids are depicted within white boxes. (B) Schematic representation of the Xenopus NFI-X and xNFI-C1 proteins. The total length in amino acids (a.a.) and the different domains of each protein are indicated. The domains are not drawn to scale. The NFI-specific domain (180 amino acids) and the xNFI-X SSD (230 amino acids) are as indicated by the white boxes. The xNFI-C SSD is indicated by a gray box. The variable C-terminal ends of xNFI-X1, xNFI-X2, and xNFI-X3, generated by alternative splicing, are indicated by the black, gray, and hatched boxes, which depict conditional exons present in the individual xNFI-X isoforms. The xNFI-C1 proline-rich region at the C-terminal end is indicated by the checkered box. The percentage of amino acid identity between the NFI-specific domains and SSDs of the xNFI-X and xNFI-C1 proteins is given. (C) Amino acid sequences of the variable domains of xNFI-X1, xNFI-X2, and xNFI-X3. The four-amino-acid motif common to the C termini of xNFI-X1 and xNFI-X3 is shaded.

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conserved part of the human transcription factor NFI-C1 (CTF-1 [32]). Eight independent clones were isolated and analyzed by restriction endonuclease mapping and Southern blotting. Four distinct clones were analyzed further. The first clone contains an insert of 1.8 kb with an open reading frame of 502 amino acids, which is highly homologous to the human, pig, and rat NFI-C subtype (36, 40) and was named xNFI-C1 (Fig. 1A). Sequence analysis of the three other cDNAs revealed that they code for three distinct proteins that are more related to the previously identified NFI-X subtype (18, 25) and thus were called xNFI-X1, xNFI-X2, and xNFI-X3 (Fig. 1A). These three cDNAs have in common a 1,230-bp fragment encoding 410 N-terminal amino acids. However, the very 39 ends of the xNFI-X cDNAs are distinct and encode variable C-terminal domains (Fig. 1B). Analysis of the nucleotide sequences at the 39 extremity of the coding region indicated that the different subtypes most likely result from alternative splicing events of a common pre-mRNA molecule. In agreement with this proposal, the nucleotide sequence corresponding to the 39 untranslated region was found to be identical in all three clones. xNFI-X proteins can be subdivided into three domains. The NFI-specific domain consists of 180 N-terminal amino acids that are highly conserved among all known members of the NFI family (38) (Fig. 1B). The central region, of 230 amino acids and referred to as the SSD, contains amino acid sequences that are specific for the NFI-X subtype. The C-terminal amino acid sequence is different in all three xNFI-X polypeptides and forms the variable domain. In the shortest polypeptide, xNFI-X1, this domain is composed of just four amino acids (Fig. 1C), while the xNFI-X2-encoding cDNA contains at least an additional exon. The variable domain of the largest protein, xNFI-X3, is encoded by two or more additional exons, whose fusion after splicing results in a frameshift in the exon that xNFI-X3 shares with xNFI-X2 (exon b in Fig. 2A). Expression of the three different xNFI-X mRNAs. To assess whether all three cloned xNFI-X cDNAs correspond to expressed xNFI-X mRNA species, reverse transcription-PCR (RT-PCR) was carried out with primers complementary to a segment in the 39 portion of the SSD and to a segment in the common 39-end untranslated region (Fig. 2A). The presence of the three distinct mRNA species encoding xNFI-X1, xNFI-X2, and xNFI-X3 variable domains was expected to yield amplified fragments of 260, 320, and 420 bp, respectively (Fig. 2A). The L32 ribosomal protein mRNA (4) was amplified as a control. Reactions were performed with equal amounts of total RNA isolated from X. laevis oocytes and embryos at different stages of development as well as from different tissues of adult animals. Figure 2B shows that amplification of all RNA samples generated the expected fragments corresponding to the presence of transcripts for xNFI-X1, xNFI-X2, and xNFI-X3. The same three fragments were also obtained from XTC cell RNA from which the library was prepared (data not shown). These results indicate that the three isolated cDNAs correspond to actual xNFI-X isoforms. In embryos, xNFI-X1 and xNFI-X2 transcripts are detected in high amounts compared with xNFIX3. On the other hand, xNFI-X2 transcripts are in general more abundant than xNFI-X1 and xNFI-X3 transcripts in adult tissues. Different levels of isoform expression might suggest specific functional roles for each of the three proteins during development and in differentiated tissues. In vitro DNA binding of the xNFI-X polypeptides is inhibited by a repressor function located in the SSD. To characterize the DNA-binding properties of the newly identified NFI polypeptides, a series of xNFI-X and xNFI-C1 deletions was generated from the C terminus of the coding region. Full-

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FIG. 2. Tissue and developmental stage-dependent expression of the different xNFI-X mRNAs. (A) Schematic representation of the localization of the primers used for RT-PCR on the different xNFI-X mRNAs. Primers 1 and 2 are depicted by arrows and were the same for all reactions (see Materials and Methods). As indicated, the expected sizes of the amplified fragments are 260, 350, and 420 bp for xNFI-X1, xNFI-X2, and xNFI-X3, respectively. The thick black bars indicate the xNFI-X coding sequences. Additional exons a and b, present in xNFI-X2 and xNFI-X3, are depicted by the gray and hatched boxes as in Fig. 1B; splice sites are shown by vertical arrowheads. (B) Top, autoradiogram displaying the products of RT-PCRs performed on X. laevis total RNA from oocytes, different adult tissues, and embryos of different stages in early development as indicated. Embryos were staged according to Nieuwkoop and Faber as described in reference 16: fertilized egg (E1), early blastula (E6), early gastrula (E10), and neurula (E19). Lane C represents the control without added RNA. The positions of the expected amplified fragments are indicated by arrows. Bottom, products obtained by RT-PCR amplification of the same RNAs with primers specific for the X. laevis ribosomal protein L32 as a control. Note that the levels of L32 transcripts increase significantly between E10 and E19, in agreement with results of Bagni et al. (4). Asterisks indicate PCR artifactual products.

length and truncated polypeptides, represented in Fig. 3A, were expressed by in vitro transcription-translation in rabbit reticulocyte lysate in the presence of [35S]methionine. SDSPAGE analysis revealed products of the expected size that were synthesized in comparable amounts (Fig. 3B). Equal amounts of each protein were tested for sequence-specific DNA-binding activity by a gel retardation assay using the NFIbinding site from the adenovirus type 2 replication origin as a probe (Fig. 3C). All three full-length xNFI-Xs displayed a very low DNA-binding activity (lanes 1 to 3) compared with xNFI-C1 (lane 9). Removal of the 92 C-terminal amino acids of xNFI-X3, which correspond to the entire variable domain and the four last amino acids of the SSD (xNFI-X3D92), did not modify the DNA-binding activity of the protein (lane 4). However, further C-terminal deletions that removed the entire xNFI-X3 variable domain together with 89 amino acids (xNFIX3D176; lane 5) or 102 amino acids (xNFI-X3D209; lane 6) of

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FIG. 3. DNA-binding properties of xNFI proteins. (A) Schematic representation of the full-length and C-terminal deletions of xNFI-X and NFI-C polypeptides. Proteins are drawn as indicated in the legend to Fig. 1B; the names of the C-terminal deletions are, for example, xNFI-X3DY or xNFI-C1DY, where Y represents the number of residues removed starting from the last C-terminal amino acid. DNA-binding activities of the different polypeptides were determined by gel retardation assays as shown in panel C. These activities are indicated by 11, 1, 1/2, and 2, which indicate maximal, intermediate, low (detectable after overexposure of the autoradiogram), and undetectable binding activities, respectively. (B) Full-length and truncated proteins were translated in vitro in the presence of [35S]methionine, and 2 ml of extract was resolved by SDS-PAGE and autoradiographed. Lane numbers correspond to the numbering of individual xNFI-X and xNFI-C1 derivatives as shown in panel A. (C) DNAbinding analysis of the full-length and truncated xNFI polypeptides expressed in

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the xNFI-X SSD resulted in a significant increase in the DNAbinding activity. Conversely, deletions that further truncated part of the conserved NFI-specific resulted in a weak (xNFIX3D277) [lane 7] and data not shown) or undetectable (xNFIX3D304; lane 8) DNA binding. These results indicate that a region comprising the C-terminal end of the SSD common to all xNFI-X polypeptides (dotted region in Fig. 3A) is responsible for the low DNA-binding activities observed for the fulllength proteins. Therefore, it was termed the repressor domain. The DNA-binding-inhibitory effect of this domain is specific to the xNFI-X proteins, as C-terminal deletions of the corresponding xNFI-C1 sequences do not affect the DNAbinding activity in the same way (Fig. 3C). Indeed, the fulllength xNFI-C1 displayed high sequence-specific binding (Fig. 3C, lane 10), while deletion of 98 (xNFI-C1D98) and 246 (xNFI-C1D246) C-terminal amino acids resulted in similar and reduced DNA-binding activities, respectively (Fig. 3C, lanes 10 and 11). xNFI-X1, xNFI-X2, and xNFI-X3 form dimers in vitro. Mammalian NFI-C proteins form dimers in solution and bind their cognate DNA recognition sites as dimers (20, 32). Thus, the impaired in vitro DNA-binding activity observed for the full-length xNFI-X proteins might reflect a failure to dimerize. The oligomeric nature of the polypeptides in solution was assessed by using a coimmunoprecipitation assay of in vitrotranslated proteins. Full-length xNFI-X1 was expressed alone or cotranslated with full-length xNFI-X3 (Fig. 4A, lanes 1 to 3). The complexes formed were then immunoprecipitated with an antiserum directed against the variable domain of the xNFI-X3 protein and resolved by SDS-PAGE. xNFI-X1 alone did not precipitate, as it is not recognized by the antibody because it lacks most of the variable domain, but was precipitated when coexpressed with xNFI-X3 (compare lanes 2 and 3 with lanes 5 and 6), indicating the formation of a heterodimeric complex between the two proteins. Similarly, xNFI-X3 was able to dimerize with versions of xNFI-X deleted of the repressor domain (data not shown). The human NFI-C protein was used as a control, since members of this subtype do not possess a repressor domain. hNFI-C1 was cotranslated with a C-terminally truncated version of its own and immunoprecipitated with an antipeptide antibody recognizing only the full-length protein. Again, the formation of heterodimers was detected by the coimmunoprecipitation of the truncated protein (Fig. 4A, lane 12). Comparison of the results obtained with the two subtypes indicates that full-length xNFI-X proteins are able to form dimers in solution apparently as efficiently as the hNFI-C proteins do. Thus, the impaired DNA-binding activity observed for the full-length xNFI-X does not result from a deficient dimerization function. To further address the DNA-binding potential of the heteromeric complexes, dimers of full-length and truncated proteins were tested in a gel retardation assay. As observed previously, the homodimers of the truncated form of xNFI-X3 display a high DNA-binding activity, compared with the very low DNA-binding capacity of the full-length proteins (Fig. 4B; compare lane 1 with lanes 2, 5, and 8). When full-length xNFI-X species were cotranslated with xNFI-X3D209, complexes of slower mobility were observed (lanes 4, 7, and 10), indicating the formation of a heterodimer between the full-

vitro. Extracts (2 ml) were incubated with the radiolabeled TD15 DNA probe as indicated in Materials and Methods. Lanes are numbered as in panels A and B; lane R indicates the reaction performed with unprogrammed rabbit reticulocyte lysate. The asterisk indicates a nonspecific complex produced by the unprogrammed rabbit reticulocyte lysate.

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FIG. 4. xNFI-X and xNFI-C form dimeric complexes. (A) Dimer formation by xNFI polypeptides. xNFI-X3 and xNFI-X1 were in vitro translated separately (lanes 1 and 4 and lanes 2 and 5, respectively) or cotranslated (lanes 3 and 6) and resolved by SDS-PAGE before (TR; lanes 1 to 3) or after (IP; lanes 4 to 6) immunoprecipitation with an xNFI-X3-specific antiserum and autoradiographed. Lanes 7 to 12 display a similar experiment performed with hNFI-C1 and hNFIC1D230, using an antiserum that recognizes hNFI-C1 only (2). (B) DNA-binding activities of xNFI dimers. Equivalent molar amounts of in vitro-translated proteins, as determined by SDS-PAGE, were tested for DNA-binding activity in a gel retardation assay using the labeled TD15 DNA probe. xNFI-X1, xNFI-X2, and xNFI-X3 polypeptides were translated separately (2; lanes 2, 5, and 8, respectively), were cotranslated with xNFI-X3D209 (Cotrsl.; lanes 4, 7, and 10), or were mixed with xNFI-X3D209 after separate translation (M; lanes 3, 6, and 9). In lane 1, xNFI-X3D209 was translated alone. xNFI-X3 was translated alone (lane 13), cotranslated with xNFI-C1D246 (lane 15), or mixed with xNFI-C1D246 after separate translation (lane 14). R (lanes 11 and 16) and P (lane 12) indicate that the gel retardation assay reaction was performed with unprogrammed rabbit reticulocyte lysate and in the absence of extract, respectively. The homo- and heterodimers as well as the free probe are as indicated; the open and filled circles indicate xNFI-X and xNFI-C1, respectively. The asterisks indicate nonspecific complexes.

length and truncated proteins. Furthermore, xNFI-X3, when cotranslated with a truncated protein of the xNFI-C subtype, xNFI-CD246, also yielded a complex indicative of the formation of a heteromeric structure (lane 15). Taken together, these results indicate that the DNA-binding repressor domain of full-length xNFI-X proteins does not affect their potential to form dimers with themselves or with other NFI subtypes. xNFI-X polypeptides stimulate adenovirus DNA replication. NFI proteins have been shown to activate the initiation of adenovirus DNA replication (33). The first step in the replication of adenovirus is the formation of an 80-kDa complex between the first nucleotide of the nascent DNA strand and the pTP that serves as a primer for DNA synthesis. This step is stimulated up to 50-fold upon addition of hNFI-C (40). The replication-activating function of hNFI-C has been localized within the NFI-specific domain of the protein (20, 32), and it requires a functional DNA-binding activity as well as interaction with adenovirus Pol (3). However, the potential of other NFI subtypes to activate replication is unknown. The ability of adenovirus to infect both frog and human cells (13) would be consistent with the conservation of NFI DNA replication function from amphibians to humans. This observation prompted us to determine whether xNFI-X proteins have the potential to activate adenovirus replication and what might be the function of the repressor domain in this context. Previous studies had indicated that an N-terminal hexahistidine tag does not alter the DNA-binding, dimer formation, and replication properties of NFI proteins (3). Therefore, histidine-tagged xNFI-X3 and xNFI-X3D209 coding sequences were overexpressed in HeLa cells by using two vaccinia virus recombinants and subsequently purified by Ni21 chelate affinity chromatography. Western blot analysis using an antiserum that recognizes the

highly conserved N-terminal domain common to all the NFI proteins indicated that similar amounts of native and deleted xNFI-X proteins were obtained (Fig. 5A). As a control, hNFI-C1 polypeptide was expressed, purified, and assayed in parallel. In a gel retardation assay, the truncated xNFI-X3D209 (lanes 6 to 8) bound with similar efficiency as hNFI-C1 (lane 2), while full-length xNFI-X3 displayed barely detectable DNAbinding activity (lanes 3 to 5). These results for DNA-binding activity correlate with those obtained with in vitro-expressed proteins (Fig. 3), therefore indicating that the in vitro repressor domain function is independent of the source of the proteins. Interestingly, addition of a 20-fold excess of xNFI-X3 to the binding reaction mixture resulted in the complete shift of the DNA probe (Fig. 5C), suggesting that the repressor domain actually decreases rather than abolishes the affinity of xNFI-X polypeptides for DNA in vitro. Purified xNFI-X derivatives were then assayed for the ability to activate DNA replication in vitro. Low amounts of fulllength xNFI-X3 that show negligible DNA-binding activity was unable to activate adenovirus DNA replication in vitro (Fig. 5D, lanes 3 to 5). In contrast, xNFI-X3D209, which showed high DNA-binding activity, enhanced DNA replication (lanes 6 to 8) to levels similar to those obtained with comparable amounts of hNFI-C1 (lane 2). These results indicate that the xNFI-X polypeptides exhibit the structural requirements to potentiate DNA replication in vitro after deletion of their repressor domains. In a second assay, we tested whether high levels of xNFI-X3 would result in the stimulation of DNA synthesis. As shown in Fig. 5E, saturating amounts of xNFI-X3 corresponding to a 20-fold excess compared with the amounts of xNFI-X3D209 and hNFI-C1 yielded significant activation of adenovirus DNA synthesis (lane 3). These results indicate that

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FIG. 5. xNFI-X proteins activate adenovirus DNA replication. (A) Western blot analysis of 6 ng of purified xNFI-X3, xNFI-X3D209, and hNFI-C1. (B) DNA-binding activities of the purified proteins were tested by a gel retardation assay using the radiolabeled TD15 DNA probe. Lane 1, probe alone; lane 2, 3 ng hNFI-C1; lanes 3 to 5, 1.5, 3, and 6 ng of xNFI-X3, respectively; lane 6 to 8, 1.5, 3, and 6 ng of xNFI-X3D209, respectively. Complexes resulting from the DNA-protein interactions and the free probe are as indicated. (C) Same as for panel B except that 3 ng of hNFI-C1 (lane 2), 60 ng of xNFI-X3 (lane 3), and 3 ng of xNFI-X3D209 (lane 4) were used in the gel retardation assay. (D) The initiation of adenovirus DNA replication was assayed in vitro as described in Materials and Methods. Lane 1, no NFI proteins; lane 2, 6 ng of hNFI-C1; lanes 3 to 5, 1.5, 3, and 6 ng of xNFI-X3, respectively; lanes 6 to 8, 1.5, 3, and 6 ng of xNFI-X3D209, respectively. The pTP-dCMP complex which results from the first step of adenovirus DNA replication is indicated by the arrow. (E) Same as for panel D except that 3 ng of hNFI-C1 (lane 2) and xNFI-X3D209 (lane 4) or 60 ng of xNFI-X3 (lane 3) were used in the reactions.

the reduced replication activity of xNFI-X3 is mainly a consequence of its low in vitro DNA-binding affinity. The variable domains of the xNFI-Xs mediate transcriptional activation. The C-terminal proline-rich domain of hNFI-C1 has been shown to be important for transcriptional activation (32). Our results indicate that the C-terminal halves of full-length NFI-X proteins contain a novel function that, at least in vitro, inhibits their DNA-binding activities. Thus, it was of interest to investigate the transcriptional potential of the Xenopus proteins and examine how it might be influenced by the DNA-binding repressor domain. Full-length cDNAs coding for the xNFI-X and xNFI-C1 proteins were transfected into Drosophila SL-2 cells, which lack endogenous NFI-binding activity, together with a reporter plasmid containing three NFIbinding sites (40). As shown in Fig. 6A, the expression of the four full-length xNFI-X proteins resulted in a significant stimulation of the reporter gene, with a sixfold activation (5100%) obtained with xNFI-X3 and about half that level obtained with xNFI-X1 and xNFI-X2. Activation was dependent on the presence of NFI-binding sites in the reporter promoter (data not shown). To determine whether differences in activation potential result from the distinct variable domains of the xNFI proteins, 39-deletion mutants of clones xNFI-X3 and xNFI-C1 were tested similarly. Deletion of the C-terminal variable domain of xNFI-X3 (xNFI-X3D92) significantly diminished the transactivation, and further deletions (xNFI-X3D176, xNFIX3D209, and xNFI-X3D277) essentially abolished activation.

Additionally, an internal deletion removing part of the SSD containing the repressor domain of xNFI-X3 (xNFI-X3D312411) diminished transcriptional activity as well. These data suggested that the xNFI-X variable domains contribute to but are not sufficient for full transcriptional activity in which the SSD takes also an active part. In comparison, removal of the entire Pro-rich region of xNFI-C1 (xNFI-C1D98) prevented transcriptional activation, as shown previously for its human homolog (32). Fusion of the C-terminal variable part of xNFI-X3 to the inactive xNFI-C1D98 did not restore maximal activity, again suggesting that the entire xNFI-X C-terminal part including both the SSD and the variable domain is needed for full transcriptional activity. As full-length xNFI-X proteins activate transcription in insect cells, it was important to assess their DNA-binding activities when expressed in these cells. Whole cell extracts of transfected cells were tested for DNAbinding activity by a gel retardation assay using a probe containing the NFI-binding site. As shown in Fig. 6B, full-length xNFI-X and truncated xNFI-X3D92 proteins failed to bind efficiently to their cognate DNA recognition sites (lanes 3 to 6). Conversely, expression of proteins truncated in the SSD, xNFIX3D176 and xNFI-X3D209, resulted in significant levels of DNA-binding activity (lanes 7 and 8). As a control, the same experiment was performed with the hNFI-C1 subtype. Both the full-length hNFI-C1 and the truncated hNFI-C1D98 also displayed high DNA-binding activities (lanes 10 and 11). These results indicate that the repressor domain of xNFI-X also in-

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FIG. 6. Transcriptional and DNA-binding activities of the wild-type and mutant xNFIs. (A) xNFI-Xs and xNFI-C1 derivative expression vectors were transfected in SL-2 cells with a reporter plasmid containing three NFI-binding sites (paCAT-D87-33Ad). Mean CAT activities of three to five independent experiments were normalized to the activation conferred by xNFI-X3, which was assigned an arbitrary value of 100. A value of ,10 indicates that no significant stimulation was observed. DNA-binding activities are evaluated and displayed as in Fig. 3A. nt, not tested. (B) One hundred-microgram aliquots of proteins of whole cell extracts from SL-2 cells transfected with the xNFI-X- and xNFI-C1containing pPADH expression vectors, as indicated above the lanes, were tested for DNA-binding activities in a gel retardation assay. C (lane 1), a reaction performed with extracts from nontransfected cells; pPADH (lane 2), the reaction performed with extracts transfected with the pPADH expression vector alone; 2 (lane 12), probe alone.

hibits the DNA-binding activities in vitro of full-length proteins produced in insect cells. The SSD of xNFI-X participates in transcriptional activation. We have shown that the SSD of the xNFI-X proteins possesses a region that represses DNA binding in vitro, and transfection experiments with SL-2 cells suggest that the SSD may participate with the variable domain in transcriptional activation. However, a role of the SSD in xNFI-X transcriptional activity has not been demonstrated. To define such a possible role, the GAL4/xNFI-X fusion constructs shown in Fig. 7A were assayed in cotransfection experiments of HeLa and SL-2 cells in the presence of a reporter promoter containing five GAL4-binding sites. Nearly identical results were ob-

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tained with the two cell lines, and thus only the results obtained with HeLa cells are presented below. As expected, the expression of an unfused xNFI-X or GAL4 DNA-binding domain did not increase the level of CAT expression (Fig. 7A and data not shown). Enhancement of transcription was observed with all chimeras containing the entire C-terminal part of the xNFI-X polypeptides, including the SSD and the variable domain (Gal 219-497, Gal 219-433, and Gal 219-414). However, the induction by the chimeric polypeptide containing the xNFI-X1 Cterminal part was consistently lower than that mediated by xNFI-X2 or xNFI-X3. This result suggested that the C-terminal variable domains modulate the efficiency of transcription. Removal of part of the SSD (Gal 287-497, Gal 287-433, and Gal 287-414) decreased the activation of transcription. Further deletions of the entire SSD diminished (xNFI-X3 Gal 405-497) or nearly abolished (xNFI-X1 Gal 405-414 and xNFI-X2 Gal 405-433) the activation of CAT expression. In addition, internal deletions that remove part of the SSD including the repressor domain of xNFI-X2 [Gal 287-433(D102) and Gal 219433(D102)] also reduced the activation to low but detectable levels. All of these data suggested that the SSD in itself contributes to transactivation. This possibility was confirmed by using the fusion constructs containing the entire SSD (Gal 219-407) or part of it (Gal 304-406), which showed an activity similar to that of the complete C-terminal portion of xNFI-X1 (Gal 219414) (Fig. 7A). Thus, as already suggested by the SL-2 transfection experiments shown in Fig. 3A, the entire SSD, including the repressor domain, has an autonomous activity and contributes to transcriptional enhancement. In fusion proteins, the variable domain of xNFI-X3 is itself a stronger activator than the SSD. In contrast, the SSD appears to play a more important role in the xNFI-X1 and xNFI-X2 proteins, in which the variable domain has a lower transactivation ability in itself. Similar results were obtained upon cotransfection of the GAL4 fusions into SL-2 cells (data not shown). The xNFI-X repressor domain inhibits GAL4 DNA binding. We next wanted to assess whether the DNA-binding repressor domain present in the xNFI-X proteins is capable of inhibiting the heterologous GAL4 DNA-binding activity in vitro. The DNA-binding activities of the GAL4/xNFI-X chimeras expressed in HeLa cells were analyzed by gel retardation (Fig. 7B) and are summarized in Fig. 7A. The chimeric proteins containing both the SSD and variable domains of xNFI-X1, xNFI-X2, and xNFI-X3 (Gal 219-497, Gal 219-433, and Gal 219-414, respectively) bound DNA very weakly, as revealed by overexposure of the gel (Fig. 7B, lanes 5, 8, and 11, and data not shown), while GAL4 1-147 bound efficiently to its cognate DNA element (lane 2). Deletion of the N-terminal portion of the SSD (Gal 287-497, Gal 287-433, and Gal 287-414) did not result in efficient DNA recognition (lanes 4, 7, and 10). In contrast, further deletions into the SSD (Gal 405-497, Gal 405-433, and Gal 405-414) restored GAL4 DNA-binding properties (lanes 3, 6, and 9). Consistently, internal deletions removing part of the repressor domain [Gal 287-433(D102) and Gal 219-433(D102)] restored significant DNA-binding activity (lanes 12 and 13). As a control, we show that Gal 405-433, which strongly binds DNA, and Gal 219-433, which does not bind DNA efficiently, were expressed at comparable levels (Fig. 7C; compare lanes 2 and 3), indicating that differences in binding activity reflect intrinsic properties of the proteins and do not result from variations in the level of fusion protein expression. We conclude that the DNA-binding repressor domain of the xNFI-X proteins represents an independent protein structure that, in vitro, is capable of blocking GAL4 interaction with DNA in fusion proteins. However, we cannot

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FIG. 7. Transcriptional and DNA-binding activities of chimeric GAL4/xNFI-X fusion proteins. (A) Expression vectors for xNFI-X1, xNFI-X2, xNFI-X3, and chimeric derivatives were constructed as described in Materials and Methods. The names of the fusion proteins are Gal X-Y, where X and Y correspond to the amino acid (a.a.) positions at the two extremities of xNFI-X fragments fused to the 147-amino-acid-long DNA-binding domain of the yeast GAL4 protein. In addition, the lengths of the xNFI-X fragments fused to the GAL4 DNA-binding domain are indicated. The variable domains of the individual xNFI-X polypeptides are as depicted in Fig. 1B, and the DNA-binding-inhibitory domain is shown by the dotted box. DNA-binding activities are represented as described in the legend to Fig. 3 and are taken from experiments such the one presented in panel B. Cotransfection experiments were performed in HeLa cells with a reporter plasmid containing five GAL4 DNA-binding sites. Fold induction (ind.) was calculated as the ratio of CAT activity obtained in presence of the different chimeric proteins relative to the basal activity conferred by the GAL4 DNA-binding domain alone. Reported values are the averages of three independent experiments and were normalized to the 80-fold induction conferred by the chimera Gal 219-497, which was assigned the arbitrary value of 100. nt, not tested. (B) HeLa cells were transfected with plasmids expressing the chimeric proteins as described for panel A. Whole cell extracts were prepared, and 15-mg aliquots of total proteins were assayed for DNA-binding activities in a gel retardation experiment using a radiolabeled DNA probe containing the GAL4 DNA-binding site (8). Expressed proteins are as indicated above each lane. C (lane 1) and Gal 1-147 (lane 2), experiments performed with whole cell extracts from nontransfected cells and from cells transfected with the expression vector for the GAL4 DNA-binding domain alone, respectively; 2 (lane 15), probe alone. (C) HeLa cells were transfected with an expression vector for the Gal 405-433 or Gal 219-433 fusion protein. Expression of each protein was analyzed by SDS-PAGE and Western blotting of whole cell extracts, using GAL4-specific monoclonal antibodies as described in Materials and Methods. C (lane 1) indicates an experiment performed with the extract from nontransfected cells. The migration of molecular weight (mw) markers is indicated on the right.

conclude from these experiments that the repressor mechanism is identical in xNFI-X and Gal-fusion proteins. DISCUSSION This study reports on the cloning and functional characterization of four new members of the NFI family of proteins from X. laevis. One protein, called xNFI-C1, is the amphibian homolog of the hNFI-C1 protein. xNFI-C1 possesses all of the sequences and functional characteristics of its human counterpart, namely, an N-terminal domain containing the dimerization and DNA-binding functions and a C-terminal proline-rich domain involved in transcriptional activation. The other three polypeptides, xNFI-X1, xNFI-X2, and xNFI-X3, belong to the NFI-X subtype previously identified in chicken and hamster cells (18, 25) and recently in human cells (2). The NFI-specific domain of the xNFI-X polypeptides contains the minimal DNA-binding domain shared by all members of the NFI family. On the basis of the amino acid sequence, we divided the C-terminal part of each of the proteins into two main domains:

the SSD conserved in all NFI-X polypeptides and an alternatively spliced variable domain specific for each isoform. In xNFI-X, the region containing the transcriptional activity has been localized within the entire C-terminal domain of the proteins. This C-terminal part has all of the characteristics of a typical transcriptional activator, that is, a Pro-, Ser-, and Thrrich region similar to that of the hNFI-C subtype. Two independent transactivation domains were mapped: the SSD of about 230 amino acids, which contains the in vitro repressor domain, and the variable domain, which varies in length. Differences in the transactivation properties of the three xNFI-X isoforms can be directly correlated with their distinct variable domains. Indeed, the transcriptional activation property of each of the three isoforms is enhanced by their distinct variable regions, which act in synergy or additively with the SSD for maximal activity. The adenovirus DNA replication-stimulatory function was initially identified in hNFI-C polypeptides (40). The activation of DNA replication requires sequence-specific DNA-binding and dimerization activities as well as an interaction with the

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viral Pol (3, 11). We have shown that the DNA replication function is conserved in the xNFI-X proteins. In full-length xNFI-X, the DNA replication function is intrinsic to the protein, but in vitro, it is modulated by the repressor domain that decreases the DNA binding. Indeed, saturating amounts of xNFI-X, which result in full occupancy of the adenovirus replication origin, activate DNA replication as efficiently as the protein devoid of the repressor domain. Despite extensive conservation of the xNFI-X DNA-binding domain with those of other NFI proteins, we have found that full-length xNFI-X proteins are unable to specifically bind DNA with high affinity in vitro. However, deletions that eliminate an 84-amino-acid repressor region of the SSD result in the release of the DNA-binding function. All xNFI-X polypeptides that contain the xNFI-X repressor domain, irrespective of the source of the proteins (in vitro translated or expressed in SL-2 cells or HeLa cells), exhibit similarly reduced DNA-binding functions in vitro. Thus, inhibition of DNA binding in vitro is an intrinsic property of the xNFI-X repressor domain and does not result from expression in a particular system. In addition, this function can be transferred to a heterologous protein, GAL4, indicating that the inhibitory property is specific to this region, as it functions in a different protein context, although it remains to be demonstrated that the inhibitory mechanism is identical in both situations. Furthermore, the inhibition of xNFI-X DNA-binding activity is unlikely to be accounted for by improper protein folding as a result of overexpression, as the full-length and truncated proteins retain their dimerization functions. These in vitro experiments, thus, define a DNA-binding-inhibitory domain that is specific to the xNFI-X subtype of the NFI protein family. One possible mechanism that may explain this in vitro repression of xNFI-X DNA binding is the masking of the DNAbinding domain by the repressor domain, through a specific configuration of the protein. For instance, the inhibitory domain might mediate the formation of unproductive multimers, as proposed for a recently identified domain that inhibits the Zid zinc finger DNA-binding proteins (5). In this model, the repressor domain would direct protein multimerization that perturbs proper arrangement of the DNA-binding domain. Interestingly, the xNFI-X repressor domain also represses the heterologous GAL4 DNA-binding domain in fusion proteins despite the fact that the NFI DNA-binding domain does not present any obvious amino acid sequence similarity with that of GAL4 (30). However, the two proteins are similar in N-terminal organization, having adjacent DNA-binding and dimerization domains. Analysis of the structures of many different types of DNA-binding domains has demonstrated that these domains often contact the DNA major groove with a positively charged amphipathic alpha helix which is adjacent to the dimerization determinants. An interesting possibility would be that the repressor region specifically interacts with and/or masks such a structure, both in xNFI-X and in GAL4/xNFI-X chimeras. Equally likely, however, is that unproductive multimers of GAL4 may arise in vitro as proposed above for full-length xNFI-X proteins. Interestingly, the region containing the DNA-binding repressor domain contributes to the transactivation properties of the xNFI-X proteins. Furthermore, within the cell, the repressor domain in various xNFI-Xs and chimeric proteins does not provoke a loss of transcriptional activation that might result from the inhibition of DNA binding. This finding implies that the DNA-binding function is preserved in the cell and that the effects of the repressor domain seen in vitro are suppressed in vivo. Within the cells, the repressor domain may associate with a regulatory factor that releases DNA-binding inhibition, lead-

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ing to full transactivation. A related mechanism has been proposed for the Ets proteins, whose cellular but not oncogenic form carries a DNA-binding inhibitory activity, although both c-Ets and v-Ets function as transcriptional activators in transient-transfection assays (28). The repressor activity may not be detected in transient-transfection experiments if it is part of a fine tuning mechanism that might be operating in response to specific stimuli and in a cell-type-specific manner. Along this line of thought, NFI transcriptional activity has been proposed to be controlled by transforming growth factor b (37) as well as by the Ha-v-ras (34) and myc (45) oncogenes. In this context, the identification of the xNFI-X repressor domain suggests novel regulatory possibilities. The biological relevance of the existence of different isotypes of NFI proteins has yet to be elucidated. All proteins from the different subtypes analyzed to date display indistinguishable DNA-binding specificities mediated by their conserved N-terminal regions. Thus, functional differences between distinct subtypes must depend upon their distinct C-terminal portions. Here, we show striking functional differences between the xNFI-C and xNFI-X subtypes, as the latter possesses a specific domain with the dual role of inhibiting in vitro DNA binding and enhancing transcription to different levels in vivo, depending upon the variable domain. Combining activation and inhibition functions in one domain might represent a very efficient manner of modulating the activity of transcription factors, as the relief of inhibition would simultaneously enhance DNA binding and transactivation. Furthermore, this overlap might direct xNFI-X polypeptides to transcriptionally active promoter-binding sites. At responsive promoters, the activation domain should mediate productive interactions with other component of the transcription machinery and may no longer be available to inhibit DNA binding.

ACKNOWLEDGMENTS We thank N. Pellegrinelli for technical assistance, M. Horwitz for providing adenovirus genome, R. Hay for recombinant baculoviruses and the NFI-directed polyclonal antibody, P. Chambon for GAL4directed monoclonal antibodies, and I. B. Dawid for the XTC cell line cDNA library. We are grateful to B. Desvergne for reading the manuscript. This work was supported by the Etat de Vaud and by grants from the Swiss National Science Foundation to W.W. and N.M. The first two authors contributed equally to this work. REFERENCES 1. Apt, D., T. Chong, Y. Liu, and H. U. Bernard. 1993. Nuclear factor I and epithelial cell-specific transcription of human papillomavirus type 16. J. Virol. 67:4455–4463. 2. Apt, D., Y. Liu, and H. U. Bernard. 1994. Cloning and functional analysis of spliced isoforms of human nuclear factor I-X: interference with transcriptional activation by NFI/CTF in a cell type specific manner. Nucleic Acids Res. 22:3825–3833. 3. Armentero, M.-T., M. S. Horwitz, and N. Mermod. 1994. Targeting of DNA polymerase to the adenovirus origin of DNA replication by interaction with nuclear factor I. Proc. Natl. Acad. Sci. USA 91:11537–11541. 4. Bagni, C., P. Mariottini, F. Annesi, and F. Amaldi. 1990. Structure of Xenopus laevis ribosomal protein L32 and its expression during development. Nucleic Acids Res. 18:4423–4426. 5. Bardwell, V. J., and R. Treisman. 1994. The POZ domain: a conserved protein-protein interaction motif. Genes Dev. 8:1664–1677. 6. Bertholet, C., R. Drillien, and R. Wittek. 1985. One hundred base pairs of 59 flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription. Proc. Natl. Acad. Sci. USA 82:2096–2100. 7. Bosher, J., A. Dawson, and R. T. Hay. 1992. Nuclear factor I is specifically targeted to discrete subnuclear sites in adenovirus type 2-infected cells. J. Virol. 65:3140–3150. 8. Carey, M. 1991. Mechanism advances in eukaryotic gene activation. Curr. Opin. Cell Biol. 3:452–460.

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