Two consecutive zinc fingers in Sp1 and in MAZ are essential for interactions with cis-elements.

The zinc finger proteins Sp1 and Myc-associated zinc finger protein (MAZ) are transcription factors that control the expression of various genes. Regulation of transcription by these factors is based on interactions between GC-rich DNA-binding sites (GGGCGG for Sp1 and GGGAGGG for MAZ) and the carboxyl-terminal zinc finger motifs of the two proteins. Sp1 and MAZ have three and six zinc fingers, respectively, and the details of their interactions with cis-elements remain to be clarified. We demonstrate here that Sp1 and MAZ interact with the same GC-rich DNA-binding sites, apparently sharing DNA-binding sites with each other. We found that the DNA binding activities of Sp1 and MAZ depended mainly on consecutive zinc fingers, namely the second and third zinc fingers in Sp1 and the third and fourth zinc fingers in MAZ. Furthermore, the interactions of the zinc finger proteins with the same cis-elements appear to play a critical role in the regulation of gene expression. It seems plausible that two consecutive zinc finger motifs in a zinc finger protein might be essential for interaction of the protein with DNA.

The regulation of gene expression is mediated by the binding of transcription factors to cis-elements in promoter regions. The promoter regions of many eukaryotic genes contain GCrich sequences (1), and some of the most widely distributed promoter elements are GC boxes and related motifs. The zinc finger proteins Sp1 and Myc-associated zinc finger protein (MAZ) 1 are transcription factors that bind to GC-rich sequences, namely GGGCGG and GGGAGGG, respectively, to regulate the expression of various target genes.
Sp1 was originally characterized as a ubiquitous transcription factor of 778 amino acids that recognized GC-rich sequences in the early promoter of simian virus 40 (2,3). The DNA-binding domain of Sp1 consists of three contiguous C2H2-type zinc fingers (4). The amino-terminal region contains two serine-and threonine-rich domains and two glutamine-rich domains, which are essential for the transcriptional activity of Sp1 (5). The carboxyl-terminal domain of Sp1 is involved in synergistic activation and interactions with other transcription factors. Sp1 is considered to be a constitutively expressed transcription factor and has been implicated in the regulation of a wide variety of housekeeping genes, tissue-specific genes, and genes involved in the regulation of growth (6). It interacts with many factors, such as YY1 (7), E2F (8), and p300 (9). Moreover, Sp1-null mouse embryos exhibit severely retarded growth and die within 10 days after displaying a wide range of abnormalities (10).
MAZ was first identified as a transcription factor that bound to a GA box (GGGAGGG) at the ME1a1 site of the c-myc promoter and to the CT-element of the c-myc gene (11). It is a zinc finger protein with six C2H2-type zinc fingers at the carboxyl terminus, a proline-rich region, and three alanine repeats. It is expressed ubiquitously, albeit at different levels in different tissues (12). It can regulate the expression of numerous genes, such as c-myc (11,13), genes for insulin I and II (14), the gene for CD4 (15), the gene for the serotonin receptor (16), and the gene for nitric oxide synthase (17).
The sequences of the binding sites for Sp1 and MAZ are very similar, and they are often present in the same gene. However, the details of the interactions of Sp1 and MAZ with GC-rich cis-elements remain unknown. We report here that Sp1 and MAZ share DNA-binding sites with each other and that the specific binding of Sp1 and of MAZ to the DNA-binding sites depends on pairs of consecutive zinc fingers.
Cell Culture, Transfection, and Assay of Chloramphenicol O-acetyltransferase (CAT) Activity-HeLa cells, 293 cells, and NIH3T3 cells were grown in Dulbecco's modified Eagle's medium, and NCI-H460 cells were grown in RPMI 1640 medium that contained 10% fetal bovine serum (Life Technologies, Inc.). Cells were transfected with plasmid DNA using the FuGENE TM 6 transfection reagent (Roche Molecular Biochemicals) according to the protocol provided by the manufacturer. The assay of CAT activity was performed as described elsewhere (12,19).
DNase I Footprinting and Gel Shift Assay-The DNA probes (nt Ϫ383 to Ϫ70) were radiolabeled by the Klenow large fragment of DNA polymerase (New England BioLabs, Inc., Beverly, MA) using [␣-32 P]dCTP. The reaction was performed in 50 l of buffer that contained 25 mM Tris-HCl (pH 8.0), 6 mM MgCl 2 , 0.5 mM EDTA, 10% glycerol, 0.5 mM dithiothreitol, 50 mM KCl, and a radiolabeled DNA probe with an extract of HeLa cells, GST-Sp1, GST-MAZ, and GST (glutathione S-transferase), respectively. The reaction mixture was first chilled on ice for 30 min and then 0.1 unit of DNase I (TAKARA, Kyoto, Japan) was added. The incubation was continued on ice for 4 min. The reaction was stopped by the addition of 25 l of buffer that contained 20 mM EDTA, 0.5% SDS, and 250 g/ml tRNA as a carrier. The reaction products were purified by phenol-chloroform extraction and ethanol precipitation and were then subjected to electrophoresis on a DNA sequencing gel. Gel shift assays were performed as described elsewhere (6,20).

RESULTS
Sp1 and MAZ Share DNA-binding Sites-The sequences of the DNA-binding sites of Sp1 and MAZ are very similar. To investigate whether Sp1 and MAZ might bind to the same cis-elements, we prepared DNA probes from the GC-rich promoter of the human gene for MAZ (from nt Ϫ383 to Ϫ70), which contains multiple putative binding sites for both Sp1 and MAZ, for gel shift assays using the nuclear extract of HeLa cells (12). We prepared three probes: the SM probe, nt Ϫ313 to Ϫ284, which contained one putative Sp1-binding site and one putative MAZ-binding site that partially overlapped (Fig. 1A); the M probe, nt Ϫ232 to Ϫ216, which contained one putative MAZbinding site (Fig. 1B); and the S probe, nt Ϫ153 to Ϫ137, which contained one putative Sp1-binding site (Fig. 1C). We detected one major and one minor DNA-protein complex (B1 and B2) with the SM probe that included the overlapping binding sites for Sp1 and MAZ (Fig. 1A). Addition of unlabeled oligodeoxynucleotides to reaction mixtures revealed competition by the unlabeled wild-type SM probe (lanes 5-7), but not by the mutant SM probe, for binding to MAZ and Sp1 (lanes 2-4). As shown in Fig. 1A, the retarded bands corresponding to B1 and B2 were shifted still further upon addition of the antibodies against Sp1 and MAZ (lanes 9 and 11). Control antibodies did not affect the mobilities of the complexes (lanes 12 and 13). These results indicated that Sp1 and MAZ specifically recognized overlapping sites in the same cis-element. Similarly, we detected supershifted bands when antibodies against MAZ or Sp1 were added to reaction mixtures with the M probe or S probe (Figs. 1, B and C, lanes 2 and 4). The results indicated that both Sp1 and MAZ interacted with the putative MAZbinding sites and the putative Sp1-binding sites in all three probes. We also examined other GC-rich cis-elements in the MAZ promoter and found that the DNA-binding sites were recognized similarly by Sp1 or by MAZ or by both (data not shown).
We next performed DNase I footprinting assays to determine whether the factors did, in fact, bind to these putative ciselements in cells. We used a nuclear extract of HeLa cells for the assays. All of the putative binding sites for Sp1 and MAZ were protected from nucleolytic digestion ( Fig. 2A). In an attempt to identify whether Sp1 and MAZ could bind to these sites, we used purified GST-Sp1 and GST-MAZ fusion proteins in DNase I footprinting assays. As shown in Fig. 2B, all of the sixteen putative binding sites for Sp1 and MAZ were protected from nucleolytic digestion by GST-MAZ (lanes 8 -10), and 13 of the 16 putative binding sites for Sp1 and MAZ were protected by GST-Sp1 (lanes 3-5). No cis-elements were protected when we used GST alone as a negative control (Fig. 2C). The patterns of protection obtained with purified GST-Sp1 were almost the same as those obtained with purified GST-MAZ. Thus, both fusion proteins bound to almost the same cis-elements as the respective binding proteins in a nuclear extract of HeLa cells. These results indicated that Sp1 and MAZ could bind to the same cis-elements and suggested, moreover, that Sp1 and MAZ might share DNA-binding sites.
Pairs of Consecutive Zinc Finger Motifs in Sp1 and MAZ Are Essential for DNA Binding Activity-We found that the second and third zinc fingers of Sp1 are strongly homologous to the fourth and third zinc fingers of MAZ, respectively (Fig. 3A). To identify whether these zinc fingers are necessary for interaction with GC-rich sequences, we generated mutant GST-Sp1 and GST-MAZ fusion proteins with deletions of the zinc finger motifs (Figs. 3, B and C), and we examined the DNA binding abilities of these fusion proteins in gel shift assays. The DNA binding activity of the GST-Sp1 fusion protein decreased significantly when either the second or the third zinc finger motif had been deleted (GST-Sp1-⌬F2 and GST-Sp1-⌬F3; Fig. 3B).  No DNA binding activity of the GST-Sp1 fusion protein was detected when both zinc fingers had been deleted (GST-Sp1-⌬F23; Fig. 3B). These results clearly indicated that the second and third zinc fingers of Sp1 were essential for its DNA binding ability. Similarly, the DNA binding activity of the GST-MAZ fusion protein decreased when the third and/or the fourth zinc finger of MAZ had been deleted (GST-MAZ-⌬F3, GST-MAZ-⌬F4, and GST-MAZ-⌬F34; Fig. 3C). The DNA binding ability of MAZ was not affected when the first two zinc fingers or the last two zinc fingers had been deleted (GST-MAZ-⌬F12 and GST-MAZ-⌬F56, Fig. 3C). These results indicated that the third and fourth zinc fingers of MAZ were essential for its DNA binding activity.
Because two consecutive zinc fingers in both Sp1 and MAZ were required for binding to GC-rich DNA sequences, we exchanged the two pairs of consecutive zinc fingers between GST-Sp1 and GST-MAZ (Fig. 3D). The chimeric fusion proteins bound both to Sp1-and to MAZ-binding sites after the pairs of consecutive zinc fingers had been exchanged with one another (GST-Sp1-X and GST-MAZ-X, Fig. 3D). This result confirmed the involvement of the pairs of consecutive zinc fingers in Sp1 and MAZ in the binding to GC-rich sequences in vitro.

Sp1 and MAZ Repress Expression of the Gene for MAZ-We transfected HeLa cells with MAZ-CAT reporter constructs in the presence or absence of an Sp1-or a MAZ-expression vector.
The CAT reporter activity was inhibited significantly in the presence of Sp1 and of MAZ (Fig. 4A), whereas the ectopic expression of Sp1 and MAZ had no effect on the transcription of pRSVCAT, the control plasmid. These results indicated that both Sp1 and MAZ repressed transcription of the MAZ gene. We also examined the inhibitory effects of Sp1 and MAZ on the MAZ promoter in other cell lines, namely 293 cells, NCI-H460 cells, and NIH3T3 cells. Both Sp1 and MAZ repressed the activity of the MAZ promoter in these cell lines, albeit to different extents in different cell lines (Fig. 4B). The effects of repression by Sp1 and MAZ were clearly in a dose-dependent fashion (Fig. 4C).
Using the minimum effective dose of ectopically expressed Sp1, we examined the effect of increasing amounts of the MAZ expression vector and found that MAZ can replace Sp1 to repress the reporter activity of MAZ-CAT (Fig. 4D). This repression was also dose-dependent. Similar repression was detected when we examined the ability of Sp1 to replace MAZ in transcriptional repression of the MAZ-CAT reporter gene (data not shown). These results indicate that repression of transcription by Sp1 and by MAZ does not involve cooperation between the two proteins. The binding of Sp1 and MAZ to the GC-rich elements seemed to be independent since we failed to detect any cooperation between Sp1 and MAZ in the repression of gene expression. These observations might reflect the competition for DNA-binding sites.
We next studied the effects of the two consecutive zinc fingers of Sp1 and MAZ on the expression of MAZ. Plasmids expressing the wild-type protein, the deletion mutant that lacked the two consecutive zinc fingers, and a chimera with consecutive zinc fingers from MAZ or Sp1 were constructed and used to transfect HeLa cells. Repression of the MAZ promoter was reversed in the absence of the two consecutive zinc fingers of Sp1 or of MAZ (pCMV-MAZ-⌬F34 and pCMV-Sp1-⌬F23 in Fig. 4E). Furthermore, the dose-dependent repression by the chimera encoded by pCMV-Sp1-X, in which two consecutive zinc fingers of Sp1 had been replaced by those of MAZ, was similar to that by wild-type Sp1 (Fig. 4F). We observed similar repression in the case of pCMV-MAZ-X (Fig. 4F). These data indicate that the respective pairs of consecutive zinc fingers can substitute for one another. DISCUSSION Many transcription factors, including Sp1 and MAZ, have zinc finger motifs and can bind to the GC-rich cis-elements that are widely distributed in the promoters, enhancers, and locuscontrol regions of housekeeping genes as well as tissue-specific genes (21). The consensus sequence of Sp1-binding sites is very similar to that of MAZ-binding sites, and both types of sites are often present in the same gene. We examined the DNA binding activities of the two factors in gel shift assays (Fig. 1) and found that Sp1 bound to consensus Sp1-binding sites as well as to consensus MAZ-binding sites. Similarly, MAZ bound to the consensus binding sites for both MAZ and Sp1. The results of the gel shift assays indicated that both Sp1 and MAZ recognized the same cis-elements. We also performed DNase I footprinting assays to determine whether Sp1 and MAZ might share DNA-binding sites. We used a nuclear extract of HeLa cells, purified GST-Sp1, and purified GST-MAZ for these assays. All of the sixteen putative binding sites for Sp1 and MAZ in the probe were protected from nucleolytic digestion by the GST-MAZ fusion protein, and 13 of the 16 putative binding sites for Sp1 and MAZ were protected by the GST-Sp1 fusion protein (Fig. 2B). The patterns of protection obtained with purified GST-Sp1 and GST-MAZ were almost the same as that obtained with a nuclear extract of HeLa cells ( Fig. 2A), indicating that Sp1 and MAZ shared the same DNA-binding sites.
It has been reported that a number of transcription factors can bind to the same GC-rich DNA-binding sites, and functional interference by Sp1 with NF-B at the same DNA-binding site has been reported (22). It has also been reported that Sp1 binds to a variety of GC-rich nucleotide sequences as well as to the consensus Sp1-binding site (23). Our observations that MAZ and Sp1 share binding sites indicate that the regulatory activity associated with some GC-rich elements is consistent with cooperative interactions by multiple transcription factors, such as zinc finger proteins, at the same or overlapping DNA motifs.
The numbers of zinc finger motifs in zinc finger proteins vary from only two to more than a dozen (24 -27). It is possible that some but not all of the multiple zinc fingers are required for the binding to cis-elements. In view of the physical space available at cis-elements and the size of a zinc finger motif, it seems reasonable to postulate that two consecutive zinc fingers might be essential for direct interaction with a cis-element while additional zinc fingers might play an auxiliary role in the interaction or might be involved in cooperation with other factors. For example, the Ikaros protein contains six zinc fingers; the first four zinc fingers are involved in interactions with a cis-element, while the two carboxyl-terminal zinc fingers are involved in homodimerization of the protein itself (24,28). We observed similar results for transcription factors Sas3 (29) and RIP60 (30).
There are three and six C2H2-type zinc finger motifs in the carboxyl-terminal regions of Sp1 and MAZ, respectively. Despite the difference in the number of zinc finger motifs between Sp1 and MAZ, the DNA binding abilities of the two proteins are very similar. The second and third zinc fingers of Sp1 are strongly homologous to the fourth and third zinc fingers of MAZ, respectively (Fig. 3A). In our analysis by gel shift assays of mutant GST-Sp1 and GST-MAZ fusion proteins with deletions of zinc fingers, we found that GST-Sp1 no longer bound to the Sp1-binding site or to the MAZ-binding site when the second and third zinc fingers had been deleted (Fig. 3B). Similarly, the DNA binding activity of the GST-MAZ fusion protein decreased when the third and/or the fourth zinc finger of MAZ had been deleted (Fig. 3C). These results indicated that the second and third zinc fingers of Sp1 and the third and fourth zinc fingers of MAZ were necessary for the DNA binding activity of the respective proteins. Because two adjacent zinc fingers in both Sp1 and MAZ were required for binding to GC-rich DNA sequences, we exchanged the pairs of consecutive zinc fingers between GST-Sp1 and GST-MAZ (Figs. 3D, 4, E and F). The binding of the chimera GST-Sp1/MAZ to both Sp1-and MAZ-binding sites was unchanged after the pairs of zinc fingers had been exchanged with one another, indicating that the pairs of consecutive zinc finger motifs could substitute for one another. Moreover, the extent of the repression of the MAZ-CAT reporter was similar with the reciprocal combination of Sp1 and MAZ expression vectors (Fig. 4D). These results confirmed that the pairs of adjacent zinc fingers in Sp1 and MAZ were essential for the binding to GC-rich sequences. Our data suggest that two consecutive zinc fingers might be essential for interactions with cis-elements and might play a critical role in the regulation of expression of genes with GC-rich elements.
The high resolution structures of complexes of the zinc finger proteins Zif268 and GLI with DNA provide a basis for the modeling of other zinc finger proteins (31,32). Computer analysis revealed that Sp1 and MAZ most closely resemble Zif268 and GLI, respectively, so we generated models of Sp1-DNA and MAZ-DNA complexes that were based on the structures of the Zif268-DNA and GLI-DNA complexes (Fig. 5). The model of the Sp1-DNA complex suggests that the Sp1 protein has at least seven residues, namely Lys-550, Ser-552, Arg-580, Glu-583, Arg-586, Asp-610, and His-611, that make direct contact with bases in the DNA. Furthermore, Lys-535, Arg-555, His-557, Arg-559, Arg-565, Lys-576, Thr-579, Gln-585, Lys-595, and Lys-604 appear to make contact with the backbone of the DNA (Figs. 5, A-C). In the model of the MAZ-DNA complex, Arg-318, Arg-321, Arg-357, Lys-378, Arg-380, Arg-382, Tyr-405, and Asp-408 make contact with bases, whereas Lys-317, Lys-319, Asp-320, Lys-344, Ser-347, Arg-348, His-351, Gln-358, Arg-364, His-384, Arg-387, Lys-391, Lys-399, and Lys-411 make contact with the backbone of the DNA (Figs. 5, D-F). The model structures indicate, furthermore, that the second and third zinc fingers of Sp1 and the third and fourth zinc fingers of MAZ are more important than the other zinc fingers in recognition of the DNA-binding site. We also examined mutant Sp1 and MAZ proteins, in which pairs of zinc fingers had been exchanged with another, to determine whether the details of resultant contacts with DNA might differ from those of the wild-type proteins. The models indicated that the DNA binding features of the chimeric constructs, namely Sp1-X (Fig. 5A) and MAZ-X (Fig. 5D), were not significantly different from those of the wild-type proteins. These observations are supported by our studies of DNA binding activities (Fig. 3D) and the regulation of gene expression (Figs. 4, D and F).
Although the amino-terminal transactivation domains of the two proteins exhibit less extensive homology, the DNA binding abilities of Sp1 and MAZ are very similar. In fact, two consec-utive zinc fingers of Sp1 and MAZ can be interchanged without loss of the ability to repress transcription (Figs. 4, E and F). We do not know the exact roles in vivo of Sp1 and MAZ in the control of transcription of the target genes with GC-rich elements. Our transcription experiments indicated that the extent of regulation of expression of a target gene with GC-rich elements by Sp1 and MAZ is dose-dependent. We cannot rule out the possibility that each factor might function at different stages of the cell cycle and might recruit different cofactors to the same cis-elements. Further studies are required for a full understanding the molecular mechanism of action of Sp1 and MAZ in vivo, but it is clear that two consecutive zinc finger motifs in Sp1 and in MAZ are essential for the binding of these proteins to DNA and for the coordinated repression of transcription.