©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Targeted Melting and Binding of a DNA Regulatory Element by a Transactivator of c-myc(*)

(Received for publication, December 13, 1994)

Leonard Bazar (1)(§) Deborah Meighen (1)(§) Violaine Harris (1) Robert Duncan (2) David Levens (2) Mark Avigan (1)(§)(¶)

From the  (1)Departments of Pathology and Medicine, Georgetown University School of Medicine, Washington, D. C. 20007 and the (2)Laboratory of Pathology, NCI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A far upstream element (FUSE) of c-myc stimulates promoter activity when bound by a newly identified trans-acting protein, which is expressed in cycling cells. Since FUSE binding protein (FBP) binds only the noncoding strand (NCS) of its regulatory element in a sequence-specific manner, and not double-stranded (ds) DNA, formation of the proteinbulletDNA complex in vivo first requires unwinding of the DNA helix. In this report, we show evidence that FBP forces strand separation of short stretches of linear dsDNA. Because FUSE is contained within a region of helical instability that is partially unwound in negatively supercoiled DNA, it is a target for more extensive duplex strand separation by FBP, which first exposes and then selectively binds its NCS cognate sequence. In contrast, other single-stranded DNA binding proteins (SSBs) do not demonstrate this FUSE targeting activity. The novel linkage of regional dsDNA melting with cis-element binding by a transcriptional activator has broad implications in the regulation of eukaryotic gene expression.


INTRODUCTION

The assembly of trans-acting proteins on sequence-specific DNA cis-elements is crucial in the regulation of eukaryotic gene expression(1, 2) . We have purified and cloned a differentiation-regulated trans-acting protein which binds to a far upstream element (FUSE) (^1)in the c-myc proto-oncogene promoter and which stimulates expression in proliferating leukemia cells(3, 4) . FUSE-binding protein (FBP) mRNA and FBP binding activity disappear 24 h after induction of differentiation, coinciding with the irreversible down-regulation of c-myc transcription. Transfection of FBP into U-937 cells stimulates c-myc promoter-driven expression from a reporter plasmid in a FUSE-dependent manner(4) .

Although cellular FBP was initially purified on an affinity column which was linked to double-stranded oligonucleotides encompassing FUSE, it was surprising that both recombinant and purified, cellular FBP bind the noncoding strand (NCS) of FUSE in a single-stranded (ss) sequence-specific manner. No significant complex forms with dsFUSE, FUSE coding strand (CS), or other single-stranded oligonucleotides. This unusual pattern of in vitro binding parallels the observation in vivo that nonsynchronous proliferating HL60 cells contain a segment of genomic DNA with unwound bases at the FBP binding site(4) . The opened bases within and surrounding FUSE include a segment of A + T-rich DNA expected to melt when topologically constrained in negatively supercoiled DNA(5, 6, 7) . The presence of this site of potential helical instability in the region of the differentiation-regulated FUSE suggested that FBP might first locally promote the unwinding of supercoiled DNA and then bind its single-stranded cognate sequence, to transactivate the c-myc promoter.


EXPERIMENTAL PROCEDURES

DNA Strand Separation of Negatively Supercoiled and Relaxed Forms of FUSE in the Presence of Recombinant FBP and Other SSBs

Glutathione S-transferase (GST)-FBP was prepared and quantitated, as described previously(4) . An Afl3/Ava2 fragment encompassing -1430 to -1600 bp upstream from the P1 promoter (assigned a value of +1) of c-myc, which contains FUSE and flanking sequences, was blunt-ended and cloned into the SmaI site of pUC19 to generate pFUSE1. 10 µg of supercoiled (SC) pFUSE1 was converted to the relaxed form (ReF) by treatment with 160 units of topoisomerase I (Life Technologies, Inc.) for 2 h at 37 °C. The SC and ReF plasmids were isolated, as described previously(8) . 0.5 µg of SC or ReF pFUSE1 was incubated with GST, GST-FBP, T4 gene 32 protein (T4 gp32, Pharmacia Biotech Inc.), or Escherichia coli SSB (Pharmacia) in 15 µl of buffer containing 25 mM HEPES (pH 7.6), 0.25 mM EDTA, 100 µg/ml bovine serum albumin, 5 mM NaCl, 0.1 mM ZnCl(2), 0.001% Triton X-100, and 1 mM dithiothreitol for 2 h at 37 °C. Cleavage with mung bean nuclease (100 units, Life Technologies, Inc.) in a total volume of 25 µl was performed at 37 °C for 1 h, as described previously(7) . The mung bean nuclease cleavage products were purified with QIAEX (QIAGEN, Inc.) and digested with SalI or EcoRI to radiolabel the ends of the CS and NCS of c-myc sequences encompassing FUSE, respectively. To assign radiolabeled mung bean nuclease cleavage products to strands within either the vector or the c-myc sequence, a strategy employing restriction endonucleases to selectively eliminate radiolabeled products of one or the other strand was used. After mung bean nuclease cleavage of pFUSE1, the digestion products were cut in the polylinker with SalI, radiolabeled with [-P]ATP and T4 polynucleotide kinase (Life Technologies, Inc.), and then cleaved, either with HindIII to eliminate the digested vector strand products or with BamHI to eliminate the digested c-myc CS products. Radiolabeled EcoRI-cut DNA was cleaved with PvuII to eliminate the digested vector strand products in the size range mapping to FUSE or with TaqI to eliminate the digested c-myc NCS products. To mark the site of the inverted repeat within FUSE, radiolabeled products not digested with mung bean nuclease were cut with AvaI. To assign the mung bean nuclease digested products to sites of nucleotide cleavage, DNA sequencing reactions were performed by the method of Maxam and Gilbert (9) . The nuclease digested samples were analyzed on 8% polyacrylamide-urea sequencing gels.

Oligonucleotide Affinity Purification of FBP from HL60 Cells

Whole cell extracts prepared from proliferating HL60 cells (3) were fractionated by chromatography on a Bio-Rex 70 column (Bio-Rad). Briefly, the extracts, which contained 0.42 M KCl, were diluted with Buffer A (20 mM Hepes (pH 7.6), 1.5 mM MgCl(2), 0.2 mM EDTA, 20% glycerol) to a final concentration of 50 mM KCl and loaded onto a Bio-Rex 70 column, previously equilibrated with Buffer A that contained 50 mM KCl. The column was washed with 3-5 bed volumes of Buffer A containing 50 mM KCl and then eluted with a linear gradient from 50 mM to 600 mM KCl in Buffer A. Fractions were assayed for binding activity to a radiolabeled double-stranded FUSE probe in an electrophoretic mobility shift assay. Those fractions containing FUSE binding activity were pooled, assayed for protein content, and loaded onto a hydroxyapatite column (45 mg of protein/g of dry powder/column), previously equilibrated with Buffer A that contained 0.2 mM MgCl(2) and 50 mM KCl. The column was washed with 3 volumes of this buffer, followed by stepwise elution with Buffer A containing 50 mM, 100 mM, 200 mM, and 500 mM sodium phosphate (pH 7.5). Fractions were assayed for protein and FUSE binding activity as above. Complementary FUSE oligonucleotides were annealed and conjugated to CNBr-activated Sepharose 4B (Pharmacia), and the active fractions were pooled and applied to the FUSE affinity column (2.5 ml bed volume), previously equilibrated with Buffer A, that contained 50 mM NaCl. The column was washed with 20 bed volumes of equilibration buffer and then eluted stepwise with Buffer A containing 100 mM, 200 mM, 500 mM, and 1.0 M NaCl. Fractions containing FUSE binding activity were pooled, dialyzed against Buffer A containing 50 mM NaCl, and stored in 50-µl aliquots at -80 °C.

In Vitro Strand Displacement by Recombinant and Eukaryotic FBP

An Afl3/Ava2 fragment, between -1600 and -1430 bp 5` to the P1 promoter of c-myc, which contained FUSE and flanking sequences, was inserted into the SmaI site of M13mp18RF and M13mp19RF, generating M13mp18MYC and M13mp19MYC ssDNA, respectively. Oligonucleotides comprising FUSE CS (5`-ACAAAATAAAAAATCCCGAGGGAATATA-3`) or FUSE NCS (5`-TATATTCCCTCGGGATTTTTTATTTTGTA-3`), FUSE flanking sequences, and M13 vector control sequences were then hybridized to complementary M13mp18 or M13mp19 ssDNA and radiolabeled, as described previously (10) . Strand displacement reactions which contained 0.7-1 fmol of DNA template and 0.05-2.50 pmol of GST or GST-FBP were incubated for 1 h at 37 °C in 15 µl in a buffer that contained 20 mM HEPES (pH 7.6), 0.25 mM EDTA, 5 mM NaCl, 100 µg/ml bovine serum albumin, and 20% glycerol and analyzed, as described previously(10) . In some experiments, the addition of 2% SDS in the gel loading buffer was omitted to permit detection of DNAbulletprotein complexes. T4 gp32 (3-14 pmol) was added to reactions, where indicated.

Blocking of GST-FBP DNA strand displacement activity was performed with GST-FBP affinity-purified rabbit polyclonal anti-FBP antibodies. These antibodies were raised against a GST-FBP fusion protein encompassing FBP amino acids 145-511. GST-FBP was selectively removed from extracts with Protein A-agarose-conjugated affinity-purified anti-GST-FBP antibodies(11) . In some experiments, equivalent amounts of agarose-conjugated anti-bovine terminal deoxynucleotidyltransferase antibodies (TdT, Supertechs Inc., Gaithersburg, MD) were used as controls. In other experiments, soluble antibodies were preincubated with extracts for 4 h to block strand displacement.

Sequences of oligonucleotides shown in Table 1were tested for the presence or absence of GST-FBP binding activity and strand displacement from M13mp18MYC or M13mp19MYC. Binding by GST-FBP to oligonucleotides was measured by an electrophoretic mobility shift assay, as described previously(4) .



145-bp StyI/TaqI and 516-bp PvuII restriction fragments from pFUSE1 which encompass the FUSE site were blunt-ended with dNTPs and the Klenow fragment of E. coli DNA polymerase and then tested for GST-FBP-induced DNA melting. 140-bp and 180-bp blunt-ended restriction fragments from cDNAs encoding the kinase domain of GASK, a novel tyrosine kinase from murine gut epithelial cells(13) , and a 65-base oligonucleotide from the c-myc promoter NCS containing FUSE and 3`- and 5`-flanking sequences hybridized to M13mp19MYC were also tested (65-mer sequence: 5`-TGAAATGATCTATATTTAATATATAATGTATATTCCCTCGGGATTTTTTATTTTGTGTTATTCCA-3`). Analysis of DNA melting at 37 °C for 16 h was performed, as described(10) .


RESULTS

The genomic segment which encompasses FUSE contains a short inverted repeat contiguous with an A + T-rich region which is unwound in nonsynchronized cells in vivo. Because regions with similar A + T-rich sequences have been shown to be susceptible to strand separation in topologically constrained DNA(5, 6, 7) , we speculated that FUSE-targeted melting and binding activities by FBP in continuous dsDNA could be assisted by the torsional energy gained from negative supercoiling. To test this hypothesis, the negatively supercoiled and open circle forms of a plasmid which contains a 172-bp fragment encompassing FUSE and its flanking sequences were incubated with GST or GST-FBP. Unwound regions were cleaved with mung bean nuclease prior to digestion at a restriction enzyme site within the polylinker and strand-specific labeling of the cleavage products(8, 9, 12) . In the absence of added protein, mung bean nuclease generated a series of cleavages in the CS and NCS of the supercoiled plasmid spanning the A + T-rich region (Fig. 1), indicating torsion-induced melting of this sequence. The addition of GST-FBP generated a dominant cleavage site within the inverted repeat of the CS, thus demonstrating targeted separation of the single-stranded recognition element by FBP in supercoiled DNA. In addition, GST-FBP augmented a number of supercoil-associated cleavage sites within the A + T-rich melted region and generated a series of new major cleavage sites extending 18 bases 5` of the inverted repeat that were surrounded by minor sites, consistent with extensive FBP-induced melting of the CS of FUSE. This localized effect in over 2.8 kilobases of DNA sequence was not observed with GST alone. Although FBP augmented two cleavage sites within the A + T-rich segment and induced only minor cleavage sites in and near the inverted repeat on the NCS, it generated a major new cleavage site 26 bases 5` of the inverted repeat and a series of new sites as far as 40 bases upstream. Missing contact point analysis (13) has demonstrated that FBP contacts bases within the inverted repeat of the NCS of FUSE (data not shown). The asymmetry of cleavages between the CS and NCS is consistent both with the binding of FBP to the NCS of FUSE and the regional protection of the NCS from permanganate degradation in vivo(4) . Conversion of the negatively supercoiled plasmid, containing c-myc, to the relaxed form with topoisomerase I (8) , abolished all spontaneous and GST-FBP-induced melting of the strands (Fig. 1), as evidenced by the lack of any mung bean nuclease cleavage products. Taken together, these data support a model in which supercoil-induced distortion of the DNA helix provides FBP with a nidus from which it can further promote opening of the duplex and bind its single-stranded cognate sequence.


Figure 1: FUSE-targeted DNA strand separating and binding activity of GST-FBP in negatively supercoiled DNA. A, mung bean nuclease (MBN) cleavage patterns of supercoiled (SC) and relaxed form (ReF) FUSE CS in the presence of GST or GST-FBP. After mung bean nuclease cleavage of pFUSE1, the digestion products were cut with SalI and radiolabeled at the 5` ends of the linearized strands. In two lanes, further restriction endonuclease digestion of pFUSE1 at either side of the SalI site was performed to eliminate radiolabeled digestion products derived from the strand containing FUSE sequence or the strand comprised of vector sequences. This enabled strand-specific discrimination of the radiolabeled mung bean nuclease cleavage products generated from pFUSE1 and assignment to the FUSE CS (lane with products of the SC FUSE CS) or to background bands from the strand with vector sequences (lane with products of the SC VecS). The arrow indicates the major site of mung bean nuclease cleavage in the FUSE CS induced by GST-FBP. C + T and G + A DNA sequencing ladders of the FUSE CS cloned into pFUSE1 were run to map the sites of mung bean nuclease cleavages. The inverted repeat of FUSE was marked by AvaI cleavage. Cleavage of the pFUSE1 CS converted to a ReF with topoisomerase I was also analyzed in the presence of GST or GST-FBP. B, mung bean nuclease cleavage patterns of SC and ReF FUSE NCS in the presence of GST or GST-FBP. After mung bean nuclease cleavage, the digestion products of pFUSE1 were cut in the polylinker with EcoRI and radiolabeled. In the lane containing mung bean nuclease cleavage fragments of SC FUSE NCS, digestion at a restriction site within the vector generated a 90-bp fragment that contains VecS (bottom of lane), but eliminated radiolabeled VecS digestion products in the size range to which the c-myc regulatory element maps. In the adjacent lane, the contribution of background bands by cleavage products generated from the SC VecS is shown. The arrow indicates the major site of mung bean nuclease cleavage in the FUSE NCS induced by GST-FBP. DNA sequencing reactions of the FUSE NCS cloned into pFUSE1 were run to map the sites of nuclease cleavage. C, map of mung bean nuclease cleavage sites in the region of FUSE. The open arrows indicate sites susceptible to mung bean nuclease in the absence of added protein. The solid arrows indicate cleavage sites due to the addition of GST-FBP. The lengths of the arrows correlate with relative cleavage activities. The AvaI restriction site in the inverted repeat of FUSE is marked.



A number of prokaryotic SSBs, including T4 gp32 and E. coli SSB, show no sequence specificity in DNA strand separation or binding (14, 15) . To determine whether these SSBs selectively induce the separation of strands and bind DNA in the region of FUSE within negatively supercoiled DNA, mung bean nuclease cleavage analysis was performed. In contrast to the targeted strand separation and binding by FBP, T4 gp32 and E. coli SSB did not induce strand separation of either the FUSE CS (Fig. 2) or NCS (data not shown). Moreover, they caused helical stabilization within the A + T-rich region of this site, as evidenced by loss of all the nuclease cleavage sites in the region of FUSE. This difference in activities between FBP and the nonspecific SSBs was not titration-dependent and persisted over a broad range of concentrations. It can be inferred that the helical stabilizing effect on FUSE in negatively supercoiled DNA is a consequence of the formation of non-B conformations induced by these proteins at distant sites, contained within the same topologically constrained domain(5) . This process would relieve superhelical torsion at FUSE. In the supercoiled plasmids, both SSBs had a dominant effect on helical stabilization and caused the loss of FBP targeting to FUSE (data not shown). This is consistent with a required ``opened'' conformation of the A + T-rich region, which serves as a nidus for FBP entry, that is lost due to the action of either SSB. Thus, the ability to target strand separation of FUSE in topologically constrained DNA is singularly manifest by FBP and not by other SSBs.


Figure 2: Protein specificity of FUSE-targeted DNA strand separating activity in negatively supercoiled DNA demonstrated by gel analysis of FUSE CS mung bean nuclease cleavage products. In addition to GST and GST-FBP, pFUSE1 was incubated with the sequence nonspecific single-stranded DNA binding proteins E. coli SSB and T4 gp32. Amounts of protein added to each reaction are indicated. The arrow marks the major site of mung bean nuclease cleavage induced by GST-FBP but not by the other single-stranded polynucleotide binding proteins. The AvaI-cut fragment indicates a site within the inverted repeat of the FUSE CS.



The FUSE-targeted strand separating activity of FBP innegatively supercoiled DNA demonstrated that this protein forces strand separation of topologically constrained duplex DNA in a region of helical instability. To test whether FBP possesses intrinsic strand separating activity on linear dsDNA, FUSE and its flanking sequences from the upstream region of c-myc were cloned into M13 to generate ssDNA comprised of the CS or NCS of FUSE. Oligonucleotides homologous to FUSE CS and FUSE NCS were hybridized to complementary M13 ssDNA, and their 3`-ends were extended with a single alpha-P-nucleotide(10, 16) . Affinity-purified GST-FBP fusion protein, but not GST alone, displaced both the [P]FUSE CS and the [P]FUSE NCS (Fig. 3A) but formed an FBPbulletDNA complex only with [P]FUSE NCS (Fig. 3B). Consistent with the finding that FBP binds FUSE NCS but not FUSE CS, the dsDNA melting activity of the fusion protein was blocked by the addition of excess cold FUSE NCS but not by FUSE CS. Strand displacement by FBP is not magnesium- or ATP-dependent. With the apparent absence of an energy source for dsDNA unwinding, it is probable that FBP induces melting of dsDNA as a result of the trapping of regions of transiently localized instability to maintain a separation of strands and not as a consequence of enzymatic activity. Titration of melting activity on M13 oligonucleotide hybrid products indicated that strand displacement is highly cooperative, suggesting that the exposed strands become a substrate for protein filament formation, forcing the equilibrium to single strands(10) .


Figure 3: DNA strand displacement by recombinant GST-FBP and eukaryotic FBP. A, electrophoretic analysis after protein denaturation. Reactions consisted of 0.8 fmol of FUSE CS or NCS hybridized to single-stranded c-myc in M13 and 4 pmol of GST or GST-FBP. Where indicated, 100 fmol of FUSE CS or NCS were added as competitor. Following incubation for 1 h at 37 °C, the reactions were terminated by the addition of 1.6 µl of 2% SDS, 0.5% bromphenol blue, and the entire contents were loaded onto a 5% nondenaturing polyacrylamide gel. B, electrophoretic analysis without protein denaturation. Strand displacement reactions were performed as in Fig. 1A except that SDS was omitted from the loading buffer so as to preserve DNA-protein complexes. Where indicated, the templates were denatured by boiling and fast cooling. The arrow indicates the position of the FUSE NCSbulletGSTbulletFBP complex. C, strand displacement activity in the presence of T4 gp32. Reactions were performed as in Fig. 1A except that GST-FBP varied from 2.5 fmol to 2.5 pmol per reaction. T4 gp32 (14 pmol) was added where indicated. D, Blocking of GST-FBP induced strand displacement with anti-FBP antibodies (alphaFBP) in soluble form (sol) or linked to Protein A-agarose. Also shown are reactions with control antibodies to terminal deoxynucleotidyltransferase (alphaTdT). The strand displacement assay was performed in the presence of 14 pmol of T4 gp32 and 0.8 fmol of FUSE NCS hybridized to single-stranded c-myc in M13. E, strand displacement activity of eukaryotic FBP. The assay was performed as described in Fig. 1C with approximately 2.5 pmol of affinity-purified FBP from HL60 cells and 100 fmol of competitor, where indicated.



In vitro, dsDNA melting by FBP required an unusually high concentration of FBP (260 nM). Since cells have a low abundance of FBP, other conditions or factors may permit strand separation at lower FBP concentrations. As a substitute for such an effect, T4 gp32 (190 nM; assay concentration; Pharmacia) was added (Fig. 3C). This SSB nonspecifically binds ssDNA but does not melt natural dsDNAs(14) . Although, as shown above (Fig. 2), T4 gp32 caused helical stabilization in the negative supercoiled form of FUSE, it was predicted that the SSB would assist FBP in the melting of short linear stretches of duplex DNA containing FUSE, not topologically linked to other helically unstable sites. Addition of T4 gp32 caused a greater than 50-fold reduction in the amount of GST-FBP (5.3 nM; assay concentration) required to melt both FUSE CS and NCS (Fig. 3C). Strand displacement could be blocked by both soluble and Protein A-Sepharose-linked anti-FBP antibodies but not by antibodies raised to an unrelated antigen, confirming association of DNA melting activity with the FBP fusion protein (Fig. 3D). Under these conditions, oligonucleotide affinity-purified FBP from HL60 cells also displaced the FUSE NCS (Fig. 3E). As in experiments with recombinant GST-FBP (Fig. 3A), strand separation of DNA was blocked by the addition of excess cold FUSE NCS (which binds FBP) and not by excess FUSE CS (which does not bind FBP). Thus, dsDNA melting activity is an intrinsic property of FBP.

To determine whether FBP's DNA melting activity has the same DNA sequence specificity as its binding activity, 16 hybrid products of sequences that flank FUSE in c-myc and in the M13 vector, all with theoretical melting temperatures identical with the FUSE NCS and CS hybrid products, were tested for strand displacement and binding by GST-FBP. Both activities were simultaneously measured in the same reactions by gel electrophoresis performed under nondenaturing conditions (Table 1). Only six of the DNA hybrids were susceptible to GST-FBP-induced melting, both in the presence and absence of T4 gp32, while, in addition to FUSE NCS, only one of the unwound strands was stably bound by the fusion protein. Nonetheless, it can be inferred that low levels of FBP binding, not detected by the electrophoretic mobility shift assay, were sufficient to cause strand displacement in some of the reactions. Thus, FBP can promote the unwinding of DNA strands with which it does not form an easily measurable stable complex, suggesting that this activity occurs even in the presence of nonspecific DNA binding. However, the melting is non-random and restricted to a subset of DNA sequences.

FBP contains four repeating units, each composed of a repeated element followed by an amphipathic alpha helix(4) . In addition, FBP possesses a potent transcriptional activation domain in its C-terminal region. (^2)A GST-fusion protein comprised only of repeat-helix units 3 and 4 (FBP residues 278-511) is able to bind the NCS of FUSE in a sequence-specific manner, and mutational analysis has indicated that the structural integrity of these units is required for binding. This partial length FBP fusion protein is also able to melt FUSE (Fig. 4). To determine whether the structural elements of FBP that are required for helical destabilization are identical with those required for DNA sequence-specific binding, a series of truncation and insertion mutants of GST-FBP (FBP residues 278-511) (4) were tested for both strand displacement activity and binding to the NCS of FUSE (Fig. 4). Insertion mutants with disruptions in Repeat 3 and in the beginning of the adjacent alpha helix (mutants 2, 3, and 4) and an insertion mutant with a disruption in Repeat 4 (mutant 7) lost DNA melting activity. These changes correlated with loss of FUSE NCS binding. In contrast, an insertion mutant with disruption of the C-terminal end of the alpha helix in repeat-helix unit 3 (mutant 5) blocked stable binding to FUSE NCS but had no effect on the strand separation of DNA, both in the presence and absence of T4 gp32. Taken together, these results imply that the structural features of FBP that are associated with these two activities are related but that DNA melting caused by this protein occurs even when it does not form a stable complex with FUSE.


Figure 4: Mutational analysis of FBP strand separating activity. The diagrams symbolize the structures associated with the numbered amino acids of FBP contained in the GST-FBP fusion proteins: hatching indicates glycine-rich segments; solid boxes indicate FBP repeats; shaded boxes indicate amphipathic helices. Insertion and truncation mutants (0.5 pmol) of GST-FBP containing units 3 and 4 were assayed separately for binding and melting activity in the presence of 8.5 pmol of T4 gp32 (see Fig. 1C) and 1 fmol of FUSE NCS hybridized to single-stranded c-myc in M13.



To determine whether FBP induces strand separation on longer linear DNA duplexes which contain FUSE, a blunt-ended 145-bp restriction fragment of c-myc encompassing FUSE was tested for susceptibility to melting by GST-FBP. As a control, we included a 140-bp restriction fragment from a cDNA encoding a novel tyrosine kinase from gut epithelial cells(16) . In addition, the displacement of a 65-residue oligonucleotide containing the FUSE NCS and 3`- and 5`-flanking sequences, hybridized to M13 ssDNA, was tested. GST-FBP, but not GST, induced significant melting of the M13-65-mer and the 145-bp FUSE restriction fragment (Fig. 5). In contrast, under the same conditions, GST-FBP failed to melt the control restriction fragment and a significantly longer 516-bp restriction fragment encompassing FUSE and flanking c-myc sequences (data not shown). The structural characteristics of dsDNA that cause these differences in susceptibility to melting remain to be elucidated. Perhaps, in some cases, the protein is unable to destabilize internal stretches of dsDNA which have very stable duplex structures. In summary, FBP-induced melting of linear DNA that encompasses FUSE is both length-dependent and sequence-restricted. In negatively supercoiled DNA, the melting activity is sufficient to cause the targeted melting of FUSE with the consequent binding of FBP to its single-stranded cognate sequence.


Figure 5: GST-FBP-induced melting of linear dsDNA which encompasses FUSE. The electrophoretically analyzed samples include a 65-base oligonucleotide comprising FUSE NCS with 3`-and 5`-flanking sequences (FUSE 65-mer) hybridized to single-stranded c-myc in M13, a 145-bp restriction fragment (RF) encompassing FUSE and flanking c-myc sequences, and 140-bp and 180-bp control RFs. To identify the distance of migration of the melted strands, each DNA fragment was also heat-denatured and run with the other samples.




DISCUSSION

The c-myc promoter is complex, and its activity is a function of the integration of input from multiple elements(17, 18) . The transactivator-induced melting and binding of a defined site within the upstream region of c-myc implies a mechanism which links targeted DNA strand separation and transcriptional regulation. Proteins whose affinity for ssDNA or for unwound DNA is higher than for dsDNA may stabilize the melted forms of helically unstable sites that are generated as a consequence of superhelical torsion. Regions of such helical instability are often A + T-rich(5, 6, 7) . We have presented evidence that the A + T-rich region within the region of FUSE is open, both in vivo(4) and in negatively supercoiled plasmid DNA. In the context of a continuous sequence of topologically constrained DNA, this segment serves as a nucleation site for FBP-induced melting over three helical turns, leading to the sequence-selective binding of the FUSE NCS by this protein. The melting by FBP of short segments of relaxed dsDNA and the induction of nuclease cleavage sites by FBP in a FUSE-containing segment of continuous supercoiled upstream c-myc sequence strongly supports this model.

Although nonspecific contact between protein and DNA is necessary to force the separation of strands, FUSE recognition by FBP does not correlate with this activity. This was demonstrated in two ways. First, the range of dsDNA sequences that are destabilized by FBP, although limited, is broader than the highly selective NCS sequence required for stable binding. Second, disruption of the C-terminal end of the alpha helix in the repeat-helix unit 3 of FBP reduces DNA sequence-specific binding to the NCS, but does not perturb melting activity. Thus, a broad range of nonspecific DNA binding activities is capable of forcing strand separation of dsDNA by FBP. This phenomenon may enable this transactivator to target its sequence-specific single-stranded binding activity to multiple regulatory cis-elements within the genome, each of which possess distinct flanking sequences.

Previous studies have shown that non-B-DNA conformations such as melted DNA, Z-DNA, and cruciform structures form in a reversible manner as a consequence of transcriptionally induced supercoiling in vitro and in vivo, both in prokaryotes and eukaryotes (19, 20, 21, 22, 23, 24, 25) . This is because the free energy gained by the associated changes in supercoil density is sufficient to overcome the energy barrier that resists formation of these structures(26) . Since c-myc is transcribed continuously in cycling cells(17, 18) , mechanisms must exist to absorb torsional energy that will accumulate during nonreplicative phases of the cell cycle. These include changes of DNA conformation in the upstream region of this gene which have been observed both in vitro and in vivo, including the opening of DNA (4) and the formation of Z-DNA forms(27) . We have shown that the melting and binding of FUSE in continuous negatively supercoiled DNA is highly restricted to FBP. This activity is not observed with other SSBs. Both T4 gp32 and E. coli SSB demonstrated the paradoxical effect of stabilizing the A + T region of helical instability within FUSE, presumably by the preferred opening of other distant sites within the same topologically constrained domain, leading to a reduction of superhelical torsional stress(5) .

It is possible that the c-myc promoter contains a series of helically unstable sites whose individual conversion to non-B-DNA structures occurs in a hierarchical fashion and depends on the presence or absence of regulatory proteins with which they selectively bind. There are a growing number of nuclear proteins that have been found to bind non-B-DNA structures within or near upstream elements of c-myc(28, 29, 30, 31) . These have been implicated in the regulation of transcription. For example, expression of heterogeneous nuclear ribonucleoprotein K, when bound to the coding strand of a CT element, stimulates transcriptional activity(31) . The PUR factor binds a purine-rich consensus sequence present in both the human c-myc and dhfr genes and may affect transcription and/or replication(28, 29) . It is probable that, in addition to FBP, other proteins are able to conformationally alter sites of helical instability, in order to complement the activities of various transcription and replication factors which bind regulatory sites of this gene.

If a minimal amount of promoter activity is a prerequisite for negative supercoiling in the region of FUSE, then FBP, which attaches to its binding site in supercoiled DNA, may act as an amplifier of ongoing c-myc transcriptional activity. Evidence is mounting that mechanisms which affect superhelical density within regions of c-myc are differentiation-regulated. For example, a Z-DNA structure near FUSE has been observed which disappears upon cellular differentiation(27) . Site-specific-induced changes of DNA structures by topoisomerase II in intron I has also been found to be highly differentiation-regulated(32) . It has been shown that the in vitro activity of certain plasmid-containing promoters requires the presence of some degree of negative supercoiling(25) , presumably at the site of transcriptional initiation. Thus, the processes which regulate site-directed unwinding and transactivation upstream of the c-myc promoters are growth-related and may have multiple and sometimes subtle effects on c-myc transcription.

The dual site-specific melting and sequence-specific binding activities of FBP, a transcriptional transactivator, are novel. Although one consequence of these activities is the stabilization of a region of ``underwound'' DNA which is topologically less constrained than corresponding regions with fully wound strands, the potential for the formation of site-specific loops or bends (33) would also create strategically located ``hinges'' whose function might allow for the interaction of distal regulatory cis-elements with each other or with the promoter itself. This may explain our previous finding that the presence of both proximal and distal upstream elements are critical for the stimulatory effect of FUSE on c-myc(3, 4) . Elucidation of the contributions of transcriptional activators, such as FBP, on c-myc promoter activity must take into account the topographical context of each of their recognition elements within the framework of a larger genomic region.


FOOTNOTES

*
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§
Supported by National Cancer Institute Grant CA54818.

To whom correspondence and reprint requests should be addressed. Tel.: 202-687-1312; Fax: 202-687-2933.

(^1)
The abbreviations used are: FUSE, far upstream element; FBP, FUSE binding protein; NCS, noncoding strand; CS, coding strand; ss, single-stranded; ds, double-stranded; SSB, single-stranded DNA-binding protein; bp, base pair(s); SC, supercoiled; ReF, relaxed form(s); T4 gp32, T4 gene product 32; RF, replicating form; GST, glutathione S-transferase.

(^2)
R. Duncan and D. Levens, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank A. Reigle, E. Crooke, L. Liotta, A. Wellstein, U. Siebenlist, and S. Irving for many helpful discussions in the preparation of this manuscript.


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