Identification of amino acid residues of transcription factor AP-2 involved in DNA binding1

doi:10.1006/jmbi.2000.4019 available online at http://www.idealibrary.com on

J. Mol. Biol. (2000) 301, 807±816

Identification of Amino Acid Residues of Transcription Factor AP-2 Involved in DNA Binding Miguel Angel GarcõÂa{{, MoÂnica Campillos{, Samuel Ogueta, Fernando Valdivieso and JesuÂs VaÂzquez* Centro de BiologõÂa Molecular Severo Ochoa, CSICUniversidad AutoÂnoma de Madrid, 28049, Madrid, Spain

AP-2 is a cell-type speci®c, developmentally regulated transcription factor which has been described as a critical regulator of gene expression during vertebrate development and embryogenesis. Although the overall domains of this factor necessary for their activity have been identi®ed, the exact identity of AP-2 amino acid residues responsible for its interaction with the DNA structure has not yet been described. Here, we describe the identi®cation of a region of AP-2 which was protected by an oligonucleotide probe containing its binding site from trypsin digestion, monitored by peptide mapping by MALDI-TOF mass spectrometry. Furthermore, we analyzed the relative in vitro DNA-binding activity, the stimulatory potency on the AP-2-dependent APOE promoter, as well as the ability to inhibit the effect of the wild-type protein of each one of a set of single-site substitution AP-2 mutants spanning the identi®ed region. Taken together, our data clearly demonstrate that the region between amino acid residues 252-260 of AP-2 is essential for its DNAbinding activity. Particularly, the individual substitution in any of the residues 253, 254, 255, 257 or 260 is suf®cient for completely abolishing the interaction with DNA and the stimulation of APOE promoter activity. These results indicate a crucial role of this region in the formation of an active DNA-binding domain and strongly suggest that these residues provide direct contacts with the DNA structure at the AP-2 binding site. # 2000 Academic Press

*Corresponding author

Keywords: transcription factor AP-2; DNA-binding activity; site-directed mutagenesis; APOE promoter; a-helix

Introduction Regulation of gene transcription is fundamental to the dynamic processes underlying cellular differentiation and development. Retinoic acid (RA) is a key morphogen in vertebrate development and a potent regulator of cell differentiation (Eichele, 1989; Chambon, 1994). cAMP and diacylglycerol act as second messengers in important signal transduction pathways regulating many functions, including gene expression (Kaczmarek & {Present address: M. A. GarcõÂa, Laboratory of Cell Regulation, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK. {These authors contributed equally to this work. Abbreviations used: RA, retinoic acid; cAMP, cyclic adenin-monophosphate; APOE, apolipoprotein E gene; AD, Alzheimer's disease; HSH, helix-span-helix; EMSA, electrophoretic mobility shift assay. E-mail address of the corresponding author: [email protected] 0022-2836/00/040807±10 $35.00/0

Kaminska, 1989). Analysis of a number of regions of different gene promoters controlled by RA reveals that the transcription factor AP-2 binds to these regions and this binding induces cell differentiation. RA treatment of teratocarcinoma cell lines results in a transient induction of AP-2 mRNA levels on a transcriptional level which correlates with cellular differentiation (LuÈscher et al., 1989). Phorbol esters along with cAMP induce AP2 activity independent of protein synthesis (Imagawa et al., 1987). AP-2a (AP-2a ˆ AP-2), a 52 kDa protein, which was ®rst puri®ed from HeLa cells and cloned from cDNA library of HeLa cells (Williams et al., 1988), is a cell-type speci®c developmentally regulated transcription factor which has been described as a critical regulator of gene expression during vertebrate development and embryogenesis (Mitchell et al., 1991; Meier et al., 1995). This transcription factor recognizes the palindromic sequence 50 -GCCNNNGGC-30 (Imagawa et al., # 2000 Academic Press

808 1987; Mitchell et al., 1987; Williams & Tjian, 1991a; Mohibullah et al., 1999). The AP-2a gene was mapped to a region on chromosome 6 near the HLA locus (Gaynor et al., 1991). Two additional AP-2-related genes have been described, AP-2b and AP-2g (Moser et al., 1995; Oulad-Abdelghani et al., 1996). The AP-2 protein sequence has no obvious resemblance to well-characterized DNAbinding motifs or to elements present in the DNAbinding domains of other transcription factors (Williams et al., 1988). Several studies using deletion mutants of AP-2 allowed the determination of the overall domains of the factor necessary for their activity (Williams & Tjian, 1991a,b). However, and in spite of the growing importance of this transcription factor, the exact identity of AP-2 amino acid residues responsible for its interaction with the DNA structure has not yet been described. We have recently described the presence of allelic polymorphisms in the transcriptional regulatory region of apolipoprotein E (ApoE), which produce variations in the transcriptional activity of ApoE gene (APOE) (Artiga et al., 1998a). Two of these common variants are associated with an increase in risk for developing late-onset Alzheimer's disease (AD) (Bullido et al., 1998; Artiga et al., 1998b). In this regard, we have found that the activity of the proximal APOE promoter is upregulated by cAMP and RA, in astrocytic but not hepatic cells (GarcõÂa et al., 1996). This regulatory process is mediated by interaction of AP-2 with two sites located in the proximal region of the promoter (GarcõÂa et al., 1996). We have also found that the activating effect of the factor on APOE promoter was modulated by phosphorylation at Ser239 by protein kinase A (GarcõÂa et al., 1999). In this work, we present experimental evidence of that the region located between the amino acid residues 252 and 260 is implicated in the binding of AP-2 to DNA and in the physiological action of this transcription factor on APOE promoter.

Results Identification of the region of AP-2 protected by DNA from trypsin digestion In order to obtain information about the region of AP-2 involved in DNA binding, we ®rstly analyzed whether in vitro binding to recombinant AP-2 of a small, double-stranded oligonucleotide, containing the AP-2 binding site, would produce differences in the pattern of peptides generated by trypsin digestion. For this purpose, a pure, bacterially expressed, polyhistidine-tagged form of AP-2, lacking the 122 N-terminal residues and having in vitro DNA-binding activity (GarcõÂa et al., 1999), which was termed AP-2r, was subjected to controlled digestion in solution using a wide range of trypsin concentrations, and the generated peptides were analyzed by mass spectrometry. The peptides resulting from partial digestion of AP-2r were clearly identi®ed by their characteristic

AP-2 Residues Involved in DNA-binding

masses by MALDI-TOF mass spectrometry (Figure 1(a) and (b)) or by their MS/MS fragmentation patterns by nanospray-ion trap mass spectrometry (not shown). When AP-2r was previously incubated with oligonucleotide CXX, containing the AP-2 binding site (GarcõÂa et al., 1999), the intensity of some peptide peaks diminished when the trypsin concentration used was in the range from 1:40 to 1:400 enzyme/substrate mass ratios (compare, for example, Figure 1(a) and (b)). As shown in Figure 1(c), the peptides whose generation was signi®cantly inhibited were precisely located in a de®ned region of the protein, spanning tryptic fragments 16 to 22, which corresponded to amino acid residues 237 to 268. These results strongly suggested that access of trypsin to basic residues 254, 255, 257, 259, 263 and 266 were somewhat hindered by the binding of CXX to AP-2r, thus de®ning a potential region of the protein which could make direct contact with the DNA probe. We also noticed that two ¯anking glycine residues at either side seemed to de®ne a region between amino acid residues 252-260 (Figure 1(c)), where all the possible tryptic sites were protected by DNA, and an a-helix of nine amino acid residues may be theoretically formed. We then decided to study whether this region contained amino acid residues critical for DNA binding. The 252-260 region of transcription factor AP-2 is a cluster of amino acid residues which are critical for DNA binding In order to explore the relevance of amino acid residues in this region on the DNA-binding properties of AP-2, a detailed mutational analysis was undertaken. A set of eight plasmids containing single-site alanine substitutions were constructed spanning the region from amino acid residues 252260 (Table 1). Alanine was chosen for being the most common amino acid in protein a-helices (Richardson & Richardson, 1988), thus minimizing the potential disruption of the a-helix structure. Similarly, alanine in position 256 was substituted by a methionine residue (Table 1). Theoretical predictions of secondary structure of each of the nine mutants suggested the maintenance of the a-helix structure. Polyhistidine-tagged forms of AP-2 (AP-2r), lacking the 122 N-terminal residues (GarcõÂa et al., 1999) and containing the set of mutations spanning the 252-260 region, were ef®ciently expressed in bacteria and puri®ed using Ni2‡ columns (Figure 2(a)). The in vitro DNA-binding activities of the nine AP-2r mutants were then analyzed by electrophoretic mobility shift assays (EMSA) using the AP-2speci®c oligonucleotide probe CXX and carefully adjusted, identical amounts of each of the expressed mutant proteins (Figure 2(a)). The DNAbinding activity of each mutant was found to be signi®cantly lower than that of AP-2r; in clear contrast, the S239A mutant, located outside this region, retained the binding activity of intact AP-2r

AP-2 Residues Involved in DNA-binding

809

Figure 1. Trypsin digestion protection assays. (a) and (b) Comparative analysis by MALDI-TOF mass spectrometry of peptides generated from AP-2r by digestion with trypsin at a 1:40 enzyme/substrate mass ratio, after a previous incubation in the presence (a) or the absence (b) of the oligonucleotide probe CXX, which contains the AP-2-binding site. The common tryptic fragments are indicated in (a); fragments whose generation is signi®cantly impeded by the presence of CXX are indicated in (b). Tryptic fragments are numbered according to their position in AP-2r sequence. (c) Scheme of all the identi®ed AP-2r peptides generated by digestion with trypsin at enzyme/substrate mass ratios ranging from 1:40 to 1:400. Peptides are represented by black horizontal bars; the tryptic peptides whose generation were impeded by preincubation of AP-2r with CXX at any trypsin concentration are drawn below the horizontal thin line (-DNA); the remainder of peptides are represented above the line (DNA). The numbers represent amino acid residue positions according to the sequence of AP-2, except for the numbers in italics, which correspond to the numeration of tryptic fragments of AP-2r. Horizontal bars are drawn to scale according to the number of amino acid residues. The shaded area corresponds to the region between amino acid residues 250-262 of AP-2.

810

AP-2 Residues Involved in DNA-binding

Table 1. Amino acid sequence of AP-2 mutants between residues 250 and 262 Sequence GGVLRRAKSKNGG GGALRRAKSKNGG GGVARRAKSKNGG GGVLARAKSKNGG GGVLRAAKSKNGG GGVLRRMKSKNGG GGVLRRAASKNGG GGVLRRAKAKNGG GGVLRRAKSANGG GGVLRRAKSKAGG

Denomination AP-2 V252A L253A R254A R255A A256 M K257A S258A K259A N260A

(Figure 2(b) and (c)). The inhibition of binding was almost complete when alanine substitution was performed at sites 253, 254, 255 and 257. A lesspronounced but still signi®cant reduction in activity was also observed in positions 252, 256, 258, 259 and 260 (Figure 2(b) and (c)). These results indicate that each one of the residues between 252 and 260 of AP-2 protein are critical for maintaining the in vitro DNA-binding activity of the transcription factor. Analysis of transcriptional activity of AP-2 mutants The physiological relevance of the results described above was analyzed in HepG2 cells by measuring the ability of the mutants to transactivate an APOE promoter-luciferase construct (GarcõÂa et al., 1996, 1999). For this purpose, the cells were cotransfected with the APOE promoter construct and with each one of the AP-2 mutant expression vectors, and the activity of the promoter was measured in each case. As shown in Figure 3, cotransfection of cells with plasmids coding for wild-type AP-2, as well as for each AP-2 mutant, results in the ef®cient nuclear expression of all the corresponding proteins, as judged by Western-blot analysis using an antibody directed towards the C-terminal end of AP-2 (Figure 3(a)). Overexpression of wild-type AP-2 resulted in more than tenfold activation of APOE promoter activity (Figure 3(b)), as expected (GarcõÂa et al., 1996). The activity of S239A mutant was indistinguishable from that of wild-type AP-2 (not shown), as described previously (GarcõÂa et al., 1999). In clear contrast, the activating effect of each AP-2 mutant was always lower than that of the wild-type AP-2 (Figure 3(b)). V252A, A256 M, S258A and K259A mutants partially maintained their capacity of transactivating the APOE promoter, whereas the activity of L253A, R254A, R255A, K257A and N260A mutants showed a very marked reduction (Figure 3(b)). This pattern of transcriptional activity exhibited by full-length AP-2 mutants was essentially identical to that of DNA-binding activity exhibited by the recombinant, mutant proteins in EMSA (compare Figure 2(c) with Figure 3(b)), suggesting that the loss in transcriptional activity

Figure 2. Analysis of the in vitro DNA-binding activity of AP-2 mutants. (a) Analysis by SDS-10 % PAGE followed by Coomassie staining of recombinant (r) AP-2 mutants puri®ed by Ni2‡ column af®nity chromatography. The positions of molecular mass markers are shown on the left. (b) DNA-binding activity of AP-2 mutants assayed by electrophoretic mobility shift assay (EMSA). The double-stranded, radioactively labeled oligonucleotide probe was incubated with identical amounts of each protein before being subjected to EMSA, as described in Materials and Methods. (c) Quanti®cation of DNA-binding activity of AP-2 mutants by densitometric analysis of gel-shift bands. DNA-binding activity is expressed in arbitrary units. These data are representative of six independent experiments.

was a consequence of a diminished capacity of binding to DNA. In another set of experiments, each mutant protein was expressed simultaneously with wild-type AP-2 and their overall effect on APOE promoter activity was analyzed. HepG2 cells were cotrans-

AP-2 Residues Involved in DNA-binding

811

Figure 3. Analysis of the transcriptional activity of AP-2 mutants. (a) Western blot analysis of expression of AP-2 mutants in HepG2 cells transfected with pcDNA3 alone (Control), or with expression vectors for wild-type AP-2 (AP-2 wt) or any of the AP-2 mutants (indicated by the corresponding mutation). The AP-2 protein band is indicated by an arrow. (b) Analysis of transcriptional transactivation of APOE promoter driven by AP-2 mutants. HepG2 cells were transiently cotransfected with the same constructs as in (a), and a construct containing the APOE promoter fused to a luciferase gene. (c), Competition experiments of wild-type AP-2 with each of the AP-2 mutants. HepG2 cells were transiently cotransfected with APOE-luciferase construct, a small amount of either pcDNA3 (ÿ) or AP-2 expression vector (‡) and a eightfold excess of either pcDNA3 (-) or each one of the indicated mutant expression vectors (‡). Data are expressed as the mean S.E.M. of three determinations, and are representative of three independent experiments.

fected with a low, constant, amount of AP-2 expression vector and a eightfold excess of each AP-2 mutant vector. Under these conditions, as shown in Figure 3(c), the transient expression of

wild-type AP-2 alone resulted in only a fourfold increase in APOE promoter activity. With the exception of A256 M, pairwise-cotransfection of mutant and wild-type AP-2 expression vectors

812 failed to demonstrate increased activity relative to the wild-type expression vector alone (Figure 3(c)); rather, most of the mutants produced an inhibitory effect. The inhibitory potency of each of the mutants correlated very well with their own effect on APOE promoter activity; thus, the mutants yielding strongest inhibition of wild-type AP-2 activity were those with lower intrinsic activity (compare Figure 3(b) and (c)). These results clearly discard the possibility that the differential activity of the mutants (Figure 3(b)) may be due to differences in their level of expression; rather, they support the notion that the mutants are expressed as active proteins capable of sequestering the activity of the wild-type AP-2 isoform. Since all the mutations were introduced in a region which is not needed to maintain the dimerization properties of AP-2 (Williams & Tjian, 1991a,b), the clear parallelism between the physiological data presented in Figure 3 and the in vitro DNA-binding activity data (Figure 2) is indicative of the formation of heterodimers between each mutant protein and the wild-type AP-2 which are de®cient in binding to DNA.

Discussion Taken together, the data presented here de®ne a region between amino acid residues 252-260 of AP-2 which are essential for formation of an active DNA-binding domain and may be directly involved in the interaction with its binding site at the APOE promoter. This conclusion was reached after a set of trypsin-protection experiments yielded preliminary evidence about the precise location of this zone, thus allowing the realization of more conclusive experiments by single sitedirected mutagenesis. The trypsin-protection experiments are conceptually equivalent to the DNase protection or ``footprinting'' assays widely used to determine the region of DNA which interacts with a protein. Although these experiments do not strictly exclude the possibility that the protein undergoes a conformational change upon DNA binding, and this new structure alters the accessibility of trypsin to particular residues, they provide compelling evidence that a speci®c region of the protein is being directly protected by DNA from trypsin attack, an idea which is further supported by the subsequent data. To design these experiments, we reasoned that complete digestion would have destroyed the structure of the protein, probably producing the dissociation of the protein-DNA complex. On the contrary, in a controlled, partial digestion the peptides located between the sites most exposed to trypsin would be preferentially generated; the presence of bound DNA would then partially or totally impede the access of protease to basic residues located in the neighborhood of the DNAbinding site, thus diminishing the generation of peptides in this region. A set of increasing trypsin

AP-2 Residues Involved in DNA-binding

concentrations was then used, and all the data generated was employed to construct a peptide map which de®ned the protected region. Within the 252-260 region, the individual substitution of anyone of residues 253, 254, 255, 257 or 260 was suf®cient for completely abolishing the interaction with DNA and the physiological action of the factor on APOE promoter. These results demonstrate that this region of the protein is essential for formation of an active DNA-binding domain. To the best of our knowledge, this is the ®rst report which addresses the identity of particular AP-2 amino acid residues implicated in DNA binding. The ®nding that the region protected by DNA from trypsin attack contains a set of amino acid residues which are essential for DNA binding is not satisfactorily explained assuming that a conformational change produced by DNA binding makes these amino acid residues less accessible to trypsin. Rather, it strongly suggests a crucial role of these residues in providing direct contacts with the DNA structure at the AP-2 binding site. In addition, (with the exception of the site located between tryptic fragments number 34 and 35 (see Figure 1), which falls within the region required for dimerization) we detected that trypsin was able to cut on all the other possible sites inside the DNA binding region. Therefore, the 252-260 region is the only one over which DNA exerts a protective effect. If this region is not the one which directly binds DNA, at least a partial protective effect of DNA over another region should have been detected. By using large deletion mutants of AP-2, previous studies have identi®ed the general domains of AP-2 necessary for their activity. AP-2 contains a large DNA binding domain whose C-terminal half contains a dimerization domain (Figure 1), which consists of two putative amphipathic a-helices separated by a large intervening span region (Williams & Tjian, 1991a,b); this hypothetical helix-span-helix-motif (HSH) mediates homodimer formation. The N-terminal part of DNA binding region, comprising amino acid residues 209-279, has a net basic charge (Williams & Tjian, 1991a), and is highly conserved (>90 % identity) in all known sequences of AP-2 proteins from different species (Williams et al., 1988; Moser et al., 1995; Oulad-Abdelghani et al., 1996; Winning et al., 1991; Shen et al., 1997; Bauer et al., 1998; Monge & Mitchell, 1998), as well as from the various AP-2 isoforms (Meier et al., 1995). The HSH domain is unable to bind DNA when is separated from the basic domain and dimer formation is an essential requirement for AP-2 protein-DNA interaction (Williams & Tjian, 1991a,b). The region identi®ed in this work is a stretch of nine amino acid residues ¯anked by two glycine residues in each side, located inside the basic domain, and, according to theoretical predictions, its sequence is consistent with the formation of an a-helix structure. In this helix, as shown in Figure 4(a), the amino acid residues essential for DNA binding would be ade-

AP-2 Residues Involved in DNA-binding

Figure 4. Helical wheel (a) and a-helical (b) projections of the putative DNA-binding domain of AP-2. AP-2 residues identi®ed in this work as being essential for activity are shaded. Protein sequence is plotted from right to left, as indicated.

quately oriented for making contacts with the DNA structure. This ®nding is consistent with the fact that the vast majority of transcription factors adopt an a-helix structure, which binds to the DNA major groove, for recognition (Suzuki et al., 1995). The a-helical projection of this putative a-helix is shown in Figure 4(b); residues 254, 257 and 260 fall along the same line, and seem to de®ne a DNA-interacting face of the a-helix which is ¯anked by the other essential residues, 253 and 255. The structure of this region and the location of the residues essential for DNA-binding activity are, therefore, consistent with a role of these residues in providing direct DNA contacts. All the known AP-2 variants display 100 % sequence identity in the region 250-262. However, the sequence identity remains very high in the region 209-279, suggesting that the amino acid residues in this region, in general, may be important for AP-2 activity, probably providing structural requirements for the formation of an active binding domain. In this regard, the protective effect of DNA upon trypsin attack was only localized over a discrete region of this domain, in agreement with the proposed role of this subregion in making DNA contacts. It should also be noted that our data do not argue against the possibility that the

813 region located between amino acid residues 242 and 249 could also make additional DNA contacts, since this sequence may also form an a-helix, and its possible implication in direct DNA binding is not ruled out by the DNA protection experiments (Figure 1(c)). In this work we have also found that almost all AP-2 mutants containing a residue modi®ed in the 252-260 region not only displayed a diminished activity, but were also capable of inhibiting the stimulatory effect of the wild-type protein on the APOE promoter. Since deletion studies have shown that this region is required for DNA binding and is not necessary for dimerization (Williams & Tjian, 1991a,b), it is expected that the dimerization of low-activity mutants with wild-type AP-2 protein produces heterodimers de®cient in binding to DNA. These results are in agreement with those reported by other authors, who describe the inhibition of AP-2 activity by competing with the N-terminally truncated AP-2 form
N278 (Bosher et al., 1995) or the splice variant AP-2B (Moser et al., 1997), which are de®cient in DNA-binding activity. The set of AP-2 mutants reported here could, therefore, be used as highly-speci®c negative regulators of AP-2 activity, and could be potentially used as a tools for the study of AP-2 functionality, as well as its implication in several pathogenic conditions, such as Alzheimer's disease. It has been described that each of the three AP-2 genes display a distinct expression pattern in mouse embryos (Moser et al., 1995; Chazaud et al., 1996), suggesting that each of the family members plays a different role during development. This non-redundancy argument is further strengthened by the ®nding that targeted disruption of the AP2a gene alone in mice has severe developmental consequences within tissues of ectodermal origin (Schorle et al., 1996; Zhang et al., 1996), whereas AP-2b -/- mice complete embryonic development and die at postnatal days 1 and 2 due to polycystic kidney disease (Moser et al., 1997). However, all the three AP-2 proteins have been demonstrated to be capable of forming heterodimers and activate the c-erbB-2 promoter by binding to the same DNA sequence (Bosher et al., 1996); in other recent report, the three AP-2 proteins have been shown to bind a common site at the Hoxa2 gene enhancer (Maconochie et al., 1999). Therefore, and in spite of their putative differential roles, it is probable that these three AP-2 proteins share a common molecular mechanism for binding to DNA. In addition, all the AP-2 proteins are known to bind to the consensus sequence 50 -GCCNNNGGC-30 (Imagawa et al., 1987; Mitchell et al., 1987; Williams & Tjian, 1991a,b). Hence, the structural predictions made in this work could be tentatively extrapolated to the family of AP-2-related proteins.

814

Materials and Methods Trypsin digestion protection assays Pure, recombinant AP-2 (AP-2r), prepared as described (GarcõÂa et al., 1999) at 20 ng/ml was incubated for 30 minutes at 37  C in 10 ml of a buffer containing 25 mM NH4HCO3 (pH 7.8), in the absence or the presence of 100 ng/ml double-stranded oligonucleotide CXX (50 -CTGTGCCTGGGGCAGGGGGAGAACA-30 ) (GarcõÂa et al., 1999), containing the AP-2 binding site in the APOE promoter. Trypsin (Promega), was then added at varios enzyme/substrate ratios, and the mixture was incubated for 30 minutes at 37  C. The reaction was stopped by the addition of 1 ml of 10 % (v/v) tri¯uoroacetic acid, and the samples were dried down.

AP-2 Residues Involved in DNA-binding L253A, R254A, R255A, A256 M, K257A, S258A, K259A and N260A, respectively, by exchanging the nucleotides underlined. All the synthetic oligonucleotides were obtained from Isogen Bioscience (Maarssen, The Netherlands). The introduced mutations were con®rmed by restriction enzyme analysis and DNA sequencing. The vectors for the expression of recombinant proteins were prepared from the corresponding mutant vectors following the same procedure for construction of the protein AP-2r. Expression and protein purification All the recombinant proteins used in this work were expressed and puri®ed as described (GarcõÂa et al., 1999).

Mass spectrometry

Electrophoretic mobility shift assays (EMSA)

MALDI-TOF mass spectrometry were performed using either a Kompact Probe instrument (Kratos-Shimazdu, Manchester, UK), equipped with delayed extraction and a 1.7 m extended ¯ight tube operating in linear mode or a Re¯ex III instrument (Bruker, Bremen, Germany), equipped with delayed extraction and operating in re¯ector mode. Dried samples were resuspended in 1 ml of 50 % methanol/water containing 0.1 % tri¯uoroacetic acid (MHF), and 0.5 ml of each were applied onto target and dried out. Then 0.5 ml of a saturated solution of a-hydroxycinnamic acid matrix in acetonitrile/water (1:1, v/v) containing 0.1 % tri¯uoroacetic acid was added and dried out. Calibration was performed internally, using as references matrix peaks and the most abundant AP-2r peptides, whose identity was known by ion-trap MS/MS analysis. The identity of some peptides was con®rmed by nanospray-ion trap mass spectrometry in a LCQ (Finningan, ThermoQuest, San JoseÂ, CA, USA), as described (Marina et al., 1999), by subjecting the corresponding ions to fragmentation in MS/MS mode, and checking the generation of fragments corresponding to the expected y00 or b series.

EMSA experiments were carried out as reported (Bullido et al., 1998) with minor modi®cations. Brie¯y, 300 ng of puri®ed protein were incubated with 40,000 cpm of the oligonucleotide CXX described previously (GarcõÂa et al., 1999) in buffer A (10 mM Tris-HCl (pH 7.6), 30 mM KCl, 5 % (v/v) glycerol, 4 mM MgCl2, 1 mM DTT, 0.5 mM EDTA) for 20 minutes at room temperature. Reactions were preincubated with 2 mg of poly(dIdC) for ten minutes on ice. The complexes were separated in a 7 % (w/v) non-denaturing polyacrylamide gel, which was dried and exposed to X-ray ®lm.

Plasmid constructions and site-directed mutagenesis For the expression of recombinant AP-2 transcription factor with a poly-histidine extension at its N-terminal end (AP-2r), an AP-2 expression vector (kindly provided by Dr Buettner) was digested with BamHI and HindIII to isolate a fragment containing the coding region of last 315 amino acid residues of the protein, which was then subcloned at the corresponding sites in pTrcHisB (Invitrogen Corporation). Site-directed mutagenesis was performed using the QuikchangeTM site-mutagenesis system kit from Stratagene, following the manufacturer's instructions. The AP-2 plasmid was used as template and the following oligonucleotides: 50 -TGCTGGGCGGGGCCCTCCGGAGGGCGAA-30 ; 50 -TGGGCGGAGTCGCGAGGAGGGCGAAGTCTA-30 ; 50 -CTGGGCGGAGTACTCGCGAGGGCGA-30 ; 50 - GCGGAGTGCTTAAGGCGGCGAAGTCT-30 ; 50 -GCTCCGGAGGATGAAGTCTAAAAATGGAG-30 ; 50 -CGGAGGGCGGCTAGCAAAAATGGAGGAAGAT-30 ; 50 -CTCCGGAGGGCGAAGGCTAAAAATGGAGGA-30 ; 50 -GCTCCGGAGGGCCAAGTCGGCCAATGGAGGAAGAT-30 ; 50 -AGGGCGAAGTCTAAAGCCGGCGGAAGATCTTT-30 ; together with their corresponding complementaries were used as primers to generate the substitutions V252A,

Cell culture and transfections Cell culture and transfections were performed as described (GarcõÂa et al., 1999). For competition experiments, each well containing cultured HepG2 cells were transiently transfected with 0.4 mg of the APOE promoter-luciferase construct and 50 ng of either pcDNA3 (Invitrogen Corporation) or AP-2 expression vector, and 400 ng of either pcDNA3 or each of the expression vectors for the AP-2 mutants. Luciferase assay The procedure for the measuring of luciferase activity of the transfection assays was described by GarcõÂa et al. (1999). Western blot analysis Equal amounts of cell lysate protein were fractionated by SDS-10 % PAGE and transferred to nitrocellulose membranes by electroblotting. The blots were probed with a antibody AP-2 (C-18), a rabbit polyclonal antibody made against a synthetic peptide corresponding to AP-2 C-terminal amino acid residues 420-437 (Santa Cruz, CA, USA).

Acknowledgments We thank Dr J.L. Castrillo and Dr J.M. Redondo for helpful comments and suggestions, I. Sastre for expert technical assistance, and M. Alonso for their help in MALDI-TOF mass spectrometry analysis. This work was supported by FundacioÂn RamoÂn Areces, Spanish Ministerio de EducacioÂn y Cultura (grant CICYT SAF97-

AP-2 Residues Involved in DNA-binding 0171) and Fondo de InvestigacioÂn Sanitaria (grant 950022).

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Edited by M. Yaniv (Received 15 February 2000; received in revised form 19 May 2000; accepted 6 July 2000)