Flexible DNA Binding of the BTB/POZ-domain Protein FBI-1*

Frank Pessler {ddagger} § || and Nouria Hernandez {ddagger} ¶

From the {ddagger}Cold Spring Harbor Laboratory and Howard Hughes Medical Institute, Cold Spring Harbor, New York 11724

Received for publication, March 24, 2003 , and in revised form, May 14, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
POZ-domain transcription factors are characterized by the presence of a protein-protein interaction domain called the POZ or BTB domain at their N terminus and zinc fingers at their C terminus. Despite the large number of POZ-domain transcription factors that have been identified to date and the significant insights that have been gained into their cellular functions, relatively little is known about their DNA binding properties. FBI-1 is a BTB/POZ-domain protein that has been shown to modulate HIV-1 Tat trans-activation and to repress transcription of some cellular genes. We have used various viral and cellular FBI-1 binding sites to characterize the interaction of a POZ-domain protein with DNA in detail. We find that FBI-1 binds to inverted sequence repeats downstream of the HIV-1 transcription start site. Remarkably, it binds efficiently to probes carrying these repeats in various orientations and spacings with no particular rotational alignment, indicating that its interaction with DNA is highly flexible. Indeed, FBI-1 binding sites in the adenovirus 2 major late promoter, the c-fos gene, and the c-myc P1 and P2 promoters reveal variously spaced direct, inverted, and everted sequence repeats with the consensus sequence G(A/G)GGG(T/C)(C/T)(T/C)(C/T) for each repeat.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Five to ten percent of zinc finger proteins contain at their N terminus a conserved 120-amino acid motif called the BTB/POZ domain (for broad-complex, tramtrack, and bric-a-brac/pox virus and zinc finger), from hereon referred to as the POZ domain. This domain is also found in some actin-binding proteins, ion channels, and other proteins of various functions (13), recently reviewed in Ref. 4. Its primary function in transcription appears to be the mediation of protein-protein interactions involved in combinatorial regulation, transcription repression, and chromatin remodeling. Significant insight has been gained into the protein-protein interactions undergone by POZ-domain zinc finger proteins and the resulting functional implications. In contrast, much less is known about their DNA binding properties, and DNA binding sites are known for only about 10% of the ~140 POZ-domain zinc finger proteins listed in the SMART data base (5).

The known POZ-domain protein DNA binding sites display considerable sequence heterogeneity. Two POZ-domain zinc finger proteins whose DNA binding properties have been characterized in some detail, i.e. the GAGA factor and Kaiso, bind DNA with surprising flexibility (68). The GAGA factor can bind variable numbers of sites with flexible spacing, and Kaiso recognizes distinct binding sites of different sequences. It is not known whether this flexibility is a frequent feature of POZ-domain proteins, but at least in these cases it is consistent with these proteins playing a role in the regulation of a variety of genes.

We have previously described FBI-1 (factor binding to IST) (9), a human protein that binds specifically to an unusual human immunodeficiency virus, type 1 (HIV-1)1 promoter element, the inducer of short transcripts (IST) (10, 11). Molecular cloning revealed FBI-1 to be a 61.5-kDa Krüppel-type zinc finger protein with a POZ domain at the N terminus and four C2-H2 zinc fingers at the C terminus (12). FBI-1 can self-associate via both the POZ and zinc finger domains (12). In addition, it associates via its POZ domain with activation competent, but not with activation-deficient, HIV-1 Tat protein (13). Indeed, overexpressed FBI-1 stimulates Tat trans-activation in transient transfection assays and partially co-localizes with Tat and the cellular Tat co-factor P-TEFb in the splicing factor-rich nuclear speckles (13). Consistent with playing a role in cellular transcription regulation, a less soluble fraction of FBI-1 also localizes to an unusual subnuclear domain where it appears to associate with active chromatin (13). Indeed, FBI-1 has been shown to repress transcription of some extracellular matrix genes (14) (these authors refer to FBI-1 as hcKrox-{beta}) and of the ADH5/FDH promoter (15). In the latter case, the proposed mechanism of repression is interference by FBI-1 with the binding of SP-1 to a GC-box adjacent to an FBI-1 binding site. Most recently, FBI-1 was identified as one of several genes overexpressed in precursor B-leukemia cells protected from apoptosis by integrin stimulation (16).

Two mammalian homologues of FBI-1, whose DNA binding sites are unknown, have been isolated. The mouse homologue, leukemia/lymphoma-related factor, is a developmentally highly regulated nuclear protein that associates with the POZ-domain protein BCL-6 (17). The rat homologue, osteoclast-derived zinc finger protein, has been shown to play a role in osteoclast differentiation (18). Together, these observations suggest that FBI-1 is a transcription factor involved in diverse aspects of development and differentiation.

Considering the paucity of information on DNA binding of POZ-domain proteins, we have used the binding site of FBI-1 in the HIV-1 promoter, as well as other viral and cellular FBI-1 binding sites of unknown function, to characterize the DNA binding properties of a POZ-domain protein in detail. We find that high affinity FBI-1 binding sites consist of either a single guanine-rich site or two half-sites each with the consensus sequence G(A/G)GGG(T/C)(C/T)(T/C)(C/T), whose relative orientation and spacing vary greatly. Taken together, our results demonstrate that FBI-1 binds DNA with remarkable flexibility. Consistent with it playing a role in diverse biological processes, FBI-1 thus has the potential to act at a variety of cellular sequences.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—Relevant sequences of all constructs are shown in Fig. 1 and Fig. 5A. The constructs DD, DD{Delta}30, UD and UD{Delta}25 were generated as follows. Oligonucleotides with complementary sequences of ~30 nucleotides at their 3' ends were annealed and the 3' recesses were then filled in with the large fragment of DNA polymerase 1 (Klenow fragment). The resulting double-stranded DNA fragments carried the desired sequence changes, as well as BamHI restriction sites at both ends. After digestion with BamHI, the fragments were ligated into pUC119 cleaved with BamHI. These constructs carry the following sequence changes. In DD and DD{Delta}30, the HIV-1 sequence segment from +49 to +84 is everted. In addition, in DD{Delta}30 the segment from +15 to +44 is deleted. In UD and UD{Delta}25, the segments comprising –22 to +12 and +49 to +84 are everted. In addition, in UD{Delta}25 the sequence from +19 to +43 is deleted.



View larger version (72K):
[in this window]
[in a new window]
 
FIG. 1.
A, location of the IST 5' and 3' half-elements and the FBI-1 5' and 3' half-sites within the HIV-1 long terminal repeat. The start site of transcription is at +1. The brackets above the sequence indicate the location of the IST element; the thick bracket delineates the IST 5' half-element, which contributes most to IST activity. The brackets below the sequence indicate the location of the FBI-1 binding site; the thick bracket delineates the 5' half-site, which is the most important for efficient FBI-1 binding. The contributions of various sequences to efficient FBI-1 binding are further indicated by various shades of gray, with the darkest shade (region between +1 to +8) contributing most to binding. The dotted box and bracket indicate nonspecific sequences that are required for efficient FBI-1 binding. The arrows indicate the location of an imperfect inverted repeat; the 5' repeat is indicated by a solid arrow, and the 3' repeat, which lacks one nucleotide as compared with the 5' repeat, is indicated by a dotted arrow. B, sequences of the wild-type and mutant constructs used in this work. In msABC, pIST, and pISTBu–, the underlined nucleotides indicate point mutations (nucleotide changes or insertions); in DD/pUC, DD{Delta}30/pUC, UD/pUC, and UD{Delta}25/pUC, the underlined nucleotides indicate sequences that were inverted. The dashes indicate deletions. The asterisks indicate polylinker sequences. The arrows indicate the locations of the sequence repeats.

 


View larger version (29K):
[in this window]
[in a new window]
 
FIG. 5.
A, methylation interference analysis of FBI-1 binding to the HIV-1 promoter. The probe contained HIV-1 sequences from –45 to +82 and was labeled at its 5' end either on the upper strand or lower strand, as indicated. Lanes 1 and 3 show the free probe (F) whereas lanes2 and 4 show the probe bound to FBI-1. The location of the start site of transcription (+1) and of the TATA box is indicated. The locations of guanine residues which, when methylated, interfere with binding of FBI-1, are indicated by arrowheads, with the size of the arrowhead reflecting the degree of interference. In the lower panel, the HIV-1 sequences around the regions of interference are shown. The solid and dotted arrows indicate the 5' and 3' repeats, respectively. B, C, and D, methylation interference analyses of the Ad2 MLP, c-fos intron 1, and c-myc P1 promoter, respectively. Symbols are as in A.

 

pHIV-1/RpUC119 was constructed by ligating the PvuII/BamHI fragment from pHIV-1/R containing HIV-1 sequences –19 to +82 into pUC119 cleaved with HincII and BamHI. 3'pIST/pUC was constructed by ligating the BglII/BamHI fragment from pIST, containing HIV-1 sequences +21 to +82, into pUC119 cleaved with BamHI. 5'IST/pUC was generated by ligating the PvuII/BglII fragment from pHIV-1/R, comprising HIV-1 sequences –19 to +24, into pUC119 cleaved with BamHI and HincII. These constructs allowed for the amplification of probe fragments containing the respective HIV-1 sequences with universal primers hybridizing on either side of the pUC119 polylinker. 5{Delta}2/R was constructed by oligonucleotide-mediated site-directed mutagenesis (19).

The deletion mutants D1 through D8 were constructed by ligating double-stranded oligonucleotides carrying the appropriate deletions into the vector pHIV-1/R cleaved with XhoI and AflII. The latter, as well as plasmids pIST and pIST/Bu-, are described in Ref. 11. WT[ABC]WT is identical to WT[WT]WT described in Ref. 11, except that the insert between positions +24 and +40 corresponds to the +1 to +59 fragment from msABC (3).

Electrophoretic Mobility Shift Assays—Electrophoretic mobility shift assays (EMSA) were performed with end-labeled DNA probes generated by PCR amplification as described previously (9). The use of a common radiolabeled PCR primer assured equal specific activity of all probes. The sources of FBI-1 were either heparin-Sepharose or hydroxy apatite column fractions from HeLa cell nuclear extract (9). In Fig. 4C, 2 µl of heparin-Sepharose column fraction (20-fold purified in FBI-1 binding activity as compared with the nuclear extract, containing 3.5 µg/µl total protein and 5 ng/µl FBI-1 protein (9, 20)) were used per binding reaction. In all other experiments, 0.5 to 1 µl of hydroxy apatite column fraction (54-fold purified in FBI-1 binding activity, containing 3.9 µg/µl total protein and 15 ng/µl FBI-1 protein (9, 20)) was used per binding reaction. FBI-1 contained in nuclear extract or the column fractions of different degrees of purification binds to DNA in the same sequence-specific manner (9).



View larger version (66K):
[in this window]
[in a new window]
 
FIG. 4.
FBI-1 interacts with DNA in a flexible manner. A, sequences of the probes used in B. The shadings are as in Fig. 1A. The 5' and 3' repeats are indicated with solid and dotted arrows, respectively. The brackets on top of the sequences indicate the location of clustered point mutations analyzed previously that defined the 3' border of the 5' half-site (+4 to +8) and the 5' border of the 3' half-site (+49 to +56) (9). Below the sequences, +7 and the bracket +53 +56 indicate the refined inward borders of the half-sites, as deduced from the EMSAs shown in B. B, EMSA with the same protein fraction as in Fig. 2A, and the probes as indicated above the lanes. The sequences of the probes are shown in A and in Fig. 1B. The numbers above the lanes indicate the distance in base pairs between the guanine at position +1, near the 5' end of the 5' repeat, and the corresponding guanine on the lower strand at position +59, near the 5' end (on the lower strand) of the 3' inverted repeat (spacing (bp)), and the rotational displacement between these same guanine residues in each half-site, calculated using the distances above and a 36° rotation/bp (rotation (°)). C, EMSA performed with 2 µl per reaction of heparin-Sepharose protein fraction (see Ref. 9 and "Experimental Procedures"), and the probes as indicated above the lanes. The sequences of the probes are shown in Fig. 1B.

 



View larger version (47K):
[in this window]
[in a new window]
 
FIG. 2.
Each half-site can bind FBI-1, although with reduced affinity. A, EMSA with a column fraction containing partially purified FBI-1 (hydroxy apatite fraction; see Ref. 9 and "Experimental Procedures") and the probes as indicated above the lanes. The shadings in the schematics above the lanes are as in Fig. 1A. The sequences of the various probes are shown in Fig. 1B. The exact identity of the complexes of intermediate mobility is unknown, but they likely result from partially degraded FBI-1 protein. B, EMSA with the same protein fraction as in A, the HIV-1/RpUC probe, and the competitors as indicated above the lanes. The sequences of the various competitors are shown in Fig. 1B. The crosses in the msABC schematic represent six double point mutations. The competitors were added at a 30-fold molar excess. C, EMSA with the same protein fraction as in A, and the probes and competitors as indicated above the lanes. The sequences of the various probes and competitors are shown in Fig. 1B. The competitors were added at a 100-fold (lanes 2, 7, 12, and 17), 50-fold (lanes 3, 8, 13, and 18), 25-fold (lanes 4, 9, 14, and 19), and 6.25-fold (lanes 5, 10, 15, and 20) molar excess.

 
Methylation Interference Assays—Methylation interference assays with dimethyl sulfate were carried out essentially as described (21). DNA-protein binding reactions were identical to those employed for EMSAs (see above), except that they contained 300,000 cpm of end-labeled probe and 2–3 µl of hydroxy apatite column fraction containing partially purified FBI-1 (9) in a total volume of 50 µl. After electrophoretic separation of bound from unbound DNA, 100,000 cpm were typically detected in the band corresponding to the FBI-1·DNA complex.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
FBI-1 recognizes an unusually large bipartite binding site in the HIV-1 promoter, which is depicted in Fig. 1A. It spans ~100 base pairs between positions –23 and +78 with respect to the transcription start site and includes the IST promoter element (9). This binding site is composed of two separate half-sites: the 5' half-site, which is the most important for binding, consists of specific sequences from position +1 to +8, as well as nonspecific sequences from position –22 to –1. The 3' half-site, which is less important but nevertheless contributes to efficient binding, consists of sequences from position +49 to +78, with the sequences from +56 to +66 contributing most to the affinity of this half-site. The 5' and 3' half-sites are largely contained within the previously described IST 5' and 3' functional half-elements (11). We characterized the interaction of FBI-1 with the HIV-1 promoter in detail by testing a number of mutations, which are depicted in Fig. 1B. We also used the methylation interference assay to identify guanine residues in close proximity to FBI-1 in the HIV-1 promoter, several cellular promoters, and the Ad2 major late promoter.

Each HIV-1 Half-site Is Capable of Binding FBI-1—To define further the role of each HIV-1 half-site in FBI-1 binding, we generated probes containing HIV-1 sequences from –18 to +24 and thus including the specific sequences constituting the 5' half-site or HIV-1 sequences from +21 to +82 (and thus including the 3' half-site) and tested them in an EMSA with partially purified FBI-1 [hydroxy apatite column fraction (see Ref. 9); this partially purified FBI-1 forms the same complex in an EMSA as recombinant FBI-1 expressed in Escherichia coli (see Ref. 12).2 The results are shown in Fig. 2A. As expected, the FBI-1·DNA complex formed efficiently on the wild-type probe (Fig. 2A, lane 1, arrow). Signals of decreasing intensities were observed with probes containing the 5' (lane 2) and 3' (lane 3) half-sites, respectively, whereas only a background signal was obtained with a probe containing just the polylinker (lane 4). The signals observed were of the sizes expected for FBI-1·DNA complexes considering the different lengths of the probes. In Fig. 2B, the same probes were used as unlabeled competitors against the labeled wild-type probe. Consistent with the results in Fig. 2A, the complete FBI-1 binding site was an efficient competitor, the 3' half-site was poor, and the 5' half-site was intermediate (compare lanes 24 with lane 1). However, even the 3' half-site was a better competitor than the full FBI-1 binding site debilitated by point mutations (msABC, lane 5; see Fig. 1B for the sequence of this mutant) or a DNA fragment carrying just the polylinker (lane 6).

We then used each half-site as unlabeled competitor against the other half-site and against itself. As expected, each unlabeled half-site competed efficiently against itself (Fig. 2C, lanes 25 and 1720). In addition, each half-site competed against the other half-site for formation of the complex, and the 5' half-site competed more efficiently than the 3' half-site against both a labeled 5' half-site (compare lanes 25 with lanes 710) and a labeled 3' half-site (compare lanes 1215 with lanes 1720). This was similar to the pattern observed above with the labeled wild-type probe, where the 5' half-site was also a better competitor than the 3' half-site (Fig. 2B).

The binding of FBI-1 to an isolated 3' half-site was unexpected, because we observed previously (9) that FBI-1 did not bind detectably to a mutant probe (msABC(5')) carrying an intact 3' FBI-1 half-site and a mutated 5' half-site. This discrepancy probably results from the higher concentration of nonspecific competitor nucleic acids used in our previous experiments. In addition, it is possible that the mutated 5' half-site, which is not present in the probes used here (Fig. 2), in some way interfered with FBI-1 binding to the 3' half-site. At any rate, the present findings indicate that complexes with similar binding specificities form on each half-site and on the complete FBI-1 binding site. Together with our previous observation that on probes containing the entire FBI-1 binding site, point mutations in either FBI-1 half-site weaken the FBI-1·DNA complex but do not change its mobility (9), the results suggest that each individual half-site can bind FBI-1 but with lower affinity than the complete wild-type binding site.

The Half-sites Contain an Imperfect Palindrome—As indicated by the arrows in Fig. 1A, the sequences most important for FBI-1 binding in each half-site constitute a pair of imperfect inverted repeats, which corresponds to the sequences encoding the base of the TAR stem-and-loop structure. This suggests that each half-site may be recognized similarly by FBI-1. If this is the case, making the 5' half-site more similar to the weaker 3' half-site should decrease binding of FBI-1, whereas making the 3' half-site more similar to the stronger 5' half-site should increase binding of FBI-1. To test this hypothesis, we introduced the mutations shown in Fig. 3B and tested the resulting probes in the EMSA shown in Fig. 3A. Deleting the C-G base pair at position +5 and thus making the 5' repeat more similar to the 3' repeat reduced FBI-1 binding (mutant 5'{Delta}2; compare lanes 3 and 2). Strikingly, however, inserting a G-C base pair after position +55 and thus making the 3' repeat more similar to the 5' repeat enhanced FBI-1 binding (mutant pISTBu–, compare lanes 5 and 4). Taken together, these results suggest that FBI-1 recognizes sequences in each half-sites with the same specificity and that the 3' repeat is "imperfect" with respect to the 5' repeat.



View larger version (20K):
[in this window]
[in a new window]
 
FIG. 3.
The half-sites contain an imperfect inverted sequence repeat. A, EMSA with the same protein fraction as in Fig. 2A, and the probes as indicated above the lanes. In the schematics above the lanes, the 5' "perfect" repeat is indicated by a solid arrow, whereas the 3' "imperfect" repeat is indicated by a dotted arrow. B, the sequence changes in the 5'{Delta}2 and pISTBu– probes are indicated. For 5'{Delta}2, the upper strand is shown; deletion of a C renders the 5' repeat identical to the 3' repeat. Note that the 5'{Delta}2 mutant also lacks an A-T base pair at position +17, outside of the FBI-1 binding site (see Fig. 1B). For pISTBu–, the lower strand is shown; insertion of a C renders the 3' repeat identical to the 5' repeat. Note that this mutant also differs from pIST by an inserted T-A base pair outside of the binding site at position +44 (see Fig. 1B). 0.5 µl of protein fraction was used in each binding reaction.

 

FBI-1 Binds to DNA in a Flexible Manner—The segments of the FBI-1 half-sites most critical for binding are located in transcribed DNA that encodes the base of the TAR RNA stem-and-loop structure. It is therefore possible that the structure of TAR, e.g. the binding sites for Tat and its co-factors, imposes a suboptimal spacing of the FBI-1 half-sites. To test this hypothesis, we altered the spacing between the half-sites by insertions or deletions and determined the relative affinities of the resulting probes for FBI-1 by EMSA. Fig. 4A shows the 5' and 3' half-sites (shaded sequences) as determined previously by clustered point mutations, as well as the locations of the clustered point mutations (brackets above sequences) that delineated the internal borders of the 5' and 3' half-sites (constructs ms153 and ms2–3/fp in Ref. 9). The spacing between the two half-sites in the various constructs, calculated between the guanine at position +1, near the 5' end of the 5' repeat, and the corresponding guanine on the lower strand at position +59, near the 5' end (on the lower strand) of the 3' inverted repeat, is indicated above the lanes in Fig. 4B, as well as the rotation angle between these same guanine residues.

As shown in Fig. 4B, increasing the spacing between the half-sites by 44 bp reduced the binding efficiency by ~50% (construct WT[ABC]WT; compare lanes 2 and 3). In contrast, progressive deletions between the half-sites initially either enhanced binding in a minor way (mutants pIST, D1, and D5; compare lane 3 with lanes 4, 5, and 9) or had no effect (mutants D3, D4, and D6; compare lane 3 with lanes 7, 8, and 10). Thus, D6, which lacked the sequences from +8 to +52, bound FBI-1 with wild-type efficiency. In contrast, the next deletion, D7, which lacked sequences from +6 to +55, abolished detectable binding (compare lanes 3, 10, and 11), either because it removed sequences required for binding, or because it gave rise to a suboptimal spacing between the half-sites. The D6 mutant thus localizes the maximal extent of the "inward" borders of the 5' and 3' half-sites more precisely: +7 for the 3' border of the 5' half-site, and +53 for the 5' border of the 3' half-site. As described below (see Fig. 5A and Fig. 6A), methylation of the Gly residue at position +7 interfered with binding of FBI-1, thus localizing the 3' border of the 5' half-site at +7. Moreover, our previous analysis of clustered point mutations mapped the 5' border of the 3' half-site between +49 and +56 (see bracket on top in Fig. 4A). Thus, we can now localize the 5' border of the 3' half-site between +53 and +56. These new maximal inward borders of the 5' and 3' half-sites are indicated below the sequences in Fig. 4A.



View larger version (34K):
[in this window]
[in a new window]
 
FIG. 6.
Summary of FBI-1 binding sites in cellular and viral genes. A, schematic summaries of the methylation interference analyses depicted in Fig. 5, AD and of identical analyses of the c-myc P2 and c-fos exon 1 FBI-1 binding sites. The numbering is relative to the transcription start site (position +1). The dots indicate positions of methylation interference. The binding sites are depicted from top to bottom with decreasing affinity for FBI-1, as estimated from Fig. 10 in Ref. 9. B, alignment of the FBI-1 binding sites. The underlined nucleotides indicate positions of methylation interference (if the residue is a Cys, interference was observed on the Gly in the other strand). The asterisks indicate that the lower strand is shown. All sequences are shown 5' to 3'. In the consensus sequence, residues in bold appear in at least six of the eleven binding sites aligned.

 

As shown in Fig. 4B, the deletions rotated the half-sites with respect to each other to different extents (labeled in degrees above the lanes), but there was no correlation between the rotational displacement and formation of the FBI-1·DNA complex. Taken together, the above results indicate that (i) any distance between the half-sites from the natural spacing of 58 bp in HIV-1 to near juxtaposition is compatible with efficient binding of FBI-1, and (ii) there is no requirement for a particular rotational alignment of the half-sites on the DNA surface.

We then tested whether the flexible binding of FBI-1 to DNA would extend to variations in the orientation of the half-sites with respect to each other. As outlined above, FBI-1 recognizes an imperfect inverted DNA sequence repeat downstream of the HIV-1 transcription start site. In mutant DD/pUC, sequences containing the 3' half-site are flipped such that the 5' and 3' half-sites now form a direct repeat. In mutant UD/pUC, both half-sites are flipped, resulting in an everted repeat. In DD{Delta}30/pUC and UD{Delta}25/pUC, the spacing between the flipped half-sites is reduced by 30 and 25 bp, respectively, by deletion of non-binding sequences between the half-sites (see Fig. 1B, and see diagram above Fig. 4C). As shown in Fig. 4C, the FBI-1·DNA complex formed efficiently on all probes but was slightly enhanced on probes containing direct repeats and slightly reduced on probes containing everted repeats (probes UD, lane 4 and UD{Delta}25, lane 5) as compared with the wild-type HIV-1 sequences (lane 1). Quantification with a PhosphorImager of the shifted complexes in this and two replicate experiments revealed the following signals with respect to the wild-type probe: DD, 108–123%; DD{Delta}30, 115–122%; UD, 83–91%; UD{Delta}25, 73–80%. Thus, although small differences in FBI-1 binding efficiency were observed, no particular orientation of the half-sites was required, suggesting that FBI-1 can adopt a variety of conformations on the DNA.

FBI-1 Contacts Guanine Residues in Each Half-site—To identify nucleotides in close contact with FBI-1, we used a methylation interference assay. Probes carrying HIV-1 sequences from –45 to +82 were modified with dimethyl sulfate, which methylates guanine residues at the N7 position in the major groove of the DNA, and were then used in an EMSA. Free DNA and DNA complexed with FBI-1 were then cleaved with piperidine and analyzed on a sequencing gel. The results are shown in Fig. 5. On the upper strand, methylation of three guanine residues at positions +1, +2, and +3 interfered significantly with formation of the FBI-1·DNA complex, with minor interference observed at position –10 (compare lanes 1 and 2; the wedges mark residues where interference was observed, and the size of the wedges correlates with the degree of interference). On the lower strand, methylation of two guanine residues at +5 and +7, as well as three guanine residues at +57, +58, and +59, interfered the most with FBI-1 binding, with less significant interference also observed at positions +61, +71, and +73 (compare lanes 3 and 4). Together with the deletion analysis described above (Fig. 4B), the observation that methylation of guanine +7 interferes strongly with binding suggests that the 3' border of the 5' half-site is at this position (see Fig. 4A).

In summary, the methylation interference assay identified two clusters of guanine residues whose chemical modification within the major groove of the DNA interfered severely with FBI-1 binding: one located between +1 and +7, and one located between +57 and +59 (see bottom panel of Fig. 6A). These two clusters reside within the two regions in the HIV-1 promoter that were identified previously (9) by EMSA as contributing most to FBI-1 binding (see also Fig. 1A).

Establishment of an FBI-1 Consensus Binding Site—We have shown previously (9) that FBI-1 binds to a variety of cellular promoters and the Ad2 MLP. These promoters do not, however, contain any obvious sequence similarities to the HIV-1 IST, and the exact locations of the FBI-1 binding sites are unknown. We therefore used the dimethyl sulfate methylation interference assay to identify FBI-1 binding sites in several cellular genes and the Ad2 MLP and then aligned the identified half-sites to derive a consensus sequence. The results for the Ad2 MLP, c-fos intron 1, and c-myc P1 promoter are shown in Fig. 5, B, C, and D, respectively. All the results are summarized schematically in Fig. 6A, where the binding sites are depicted from top to bottom with decreasing affinity for FBI-1.

In the Ad2 MLP, methylation of three clusters of guanine residues flanking the TATA box interfered severely with binding, revealing a curious guanine-rich binding site, where the 5' cluster and the most 3' cluster are followed by a cytosine and resemble a pair of direct repeats (Fig. 5B). In the first intron of the c-fos gene, the FBI-1 binding site consisted of two direct repeats (Fig. 5C). In the c-myc P1 promoter, we identified an FBI-1 binding site consisting of two inverted repeats reminiscent of the site in the HIV-1 promoter, except that the distance between the half-sites was significantly shorter (Fig. 5D). Moreover, in the c-myc P2 promoter, we found a binding site consisting of a single half-site, and in the first exon of the c-fos gene, we found a site consisting of two abutting everted repeats (data not shown but summarized in Fig. 6A). The number of guanines whose methylation interfered with binding varied from 5 (c-myc P2) to 17 (MLP), and the distance between the half-sites (calculated between the first of the three strictly conserved guanines identified in the consensus sequences in Fig. 6B) varied between 7 (c-fos, exon 1) and 51 bp (c-fos, intron 1).

This analysis revealed no correlation between the affinity for FBI-1 and the distance between the half-sites or the orientation of the half-sites with respect to each other. Moreover, as exemplified by the c-myc P2 promoter binding site, a single site was sufficient for binding. These results agree well with the above observations that each HIV-1 half-site can bind FBI-1 on its own (see Fig. 2) and that the relative orientation and spacing of the HIV-1 half-sites can be varied (see Fig. 4, B and C). Thus, even though the functional significance of FBI-1 binding to these sites is unknown, these findings provide further evidence that FBI-1 binds DNA with remarkable flexibility.

From the half-sites identified in HIV-1 and the cellular promoters, the single site found in the c-myc P2 promoter, and the 5' and 3' Ad2 MLP sites, we then derived the consensus FBI-1 half-site shown in Fig. 6B. It consists of two well but not absolutely conserved guanines followed by a core of three strictly conserved guanines and four well conserved pyrimidines, with a preference for thymine at the first and cytosine at the second position after the three guanines. This consensus sequence resembles the FBI-1 binding site in the ADH5/FDH promoter (15), which is depicted on the bottom of Fig. 6B. FBI-1 binds to this site in vivo (15), suggesting that the consensus sequence reflects the properties of natural FBI-1 binding sites. The consensus sequence is also reminiscent of the guanine-rich binding sites of some other Krüppel-type zinc finger and POZ-domain proteins (see "Discussion").


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The FBI-1 Binding Site in the HIV-1 Promoter—Our previous characterization (9) of the FBI-1 binding site in the HIV-1 promoter localized the specific sequences constituting the two half-sites as extending from position +1 to +8 and from position +49 to +78. The deletion analysis and methylation interference assay described here, together with our previous analysis of cluster point mutations (9), allow us to localize the inward borders of the FBI-1 half-sites more accurately to +7(3' border of the 5' half-site) and between +53 and +56 (5' border of the 3' half-site). Remarkably, these borders are close to or coincide with the ends of the imperfect repeats present in each half-site. Outside of the imperfect repeats, however, the two half-sites display considerable sequence dissimilarity. Indeed, the sequences forming the upper part of the 3' half-site (5' of the guanine triplet on the lower strand, i.e. positions +78 to +60), contribute to FBI-1 binding in a sequence-specific manner, whereas FBI-1 makes no sequence-specific contacts in the corresponding segment of the 5' half-site (5' of the guanine triplet on the upper strand, i.e. positions –22 to –1) (9). The observation that mutating the 3' repeat to render it identical to the 5' repeat increases the affinity of the probes for FBI-1 suggests, however, that both half-sites are recognized by identical DNA binding domains.

What is the stoichiometry of FBI-1 on DNA? Probes carrying mutations in one half-site form FBI-1·DNA complexes of the same electrophoretic mobilities as wild-type probes of the same length (9). Moreover, the FBI-1·DNA complexes that form on probes carrying a single binding site migrate only slightly faster than those formed on full-length probes carrying two half-sites (see Fig. 2A), consistent with these complexes differing only by the size of the probe. This suggests that FBI-1 binds to single sites with the same stoichiometry as to duplicated sites. This could be a monomer, with similar or identical DNA binding domains recognizing each half-site, or a homodimer (or multiple thereof), with each FBI-1 molecule contacting one half-site. Using glycerol gradient equilibrium centrifugation, we have previously estimated the molecular mass of native FBI-1 to be 140 kDa (20), which is close to the predicted molecular mass of a homodimer, 123 kDa. This result and the observations that (i) FBI-1 does not contain a duplicated DNA binding domain but rather four different zinc fingers (12), (ii) recombinant FBI-1 can self-associate in solution via both the POZ domain and the zinc fingers (12), and (iii) the crystal structure of the POZ domain of the related POZ-domain zinc finger transcription factor PLZF reveals a tightly intertwined dimer with an extensive dimerization surface (22) all strongly suggest that, prior to binding to DNA, FBI-1 exists as a preformed dimer in which each FBI-1 molecule contributes one DNA binding domain. Because mutation of the first or second (but not the third or fourth) zinc fingers severely reduces FBI-1 binding (12), this binding domain includes, minimally, the first two FBI-1 zinc fingers. The ability of FBI-1 to bind to very differently spaced and oriented half-sites suggests that the zinc finger DNA binding domains are separated from the dimerized POZ domains by flexible linkers. It also suggests that self-association of the FBI-1 zinc finger domains on DNA may occur only on DNA binding sites with appropriately spaced and oriented half-sites.

Our results reveal a remarkable coincidence between the HIV-1 FBI-1 binding site and the IST promoter element. Consistent with this, the affinities of various IST mutants for FBI-1 and their abilities to support the synthesis of short transcripts correlated extensively in previous experiments (9, 23). Nevertheless, transient transfection and cell-free transcription assays have not revealed a specific effect of FBI-1 on IST function.3 Rather, overexpressed FBI-1 stimulates Tat trans-activation in transient transfection assays (13). Interestingly, mutations that debilitate the IST and abrogate FBI-1 binding magnify this effect, indicating that endogenous FBI-1 binds to the IST and may actually function as a repressor of HIV-1 transcription.4 Conceivably, this latter effect may relate to a role of FBI-1 in the synthesis of the short transcripts.

Flexible Binding of FBI-1 to DNA—The known DNA binding sites of other POZ-domain proteins display considerable sequence heterogeneity. Some of them, however, resemble the FBI-1 binding sites. For instance, Egr-1 and MAZR recognize single guanine-rich sites (24, 25), and the c-Krox protein binds alternatively to a single guanine-rich site (26) or to tandem repeats of the sequence 5'-GGAGGG-3', separated by nine base pairs (18). Consistent with these similarities, the rat homologue of FBI-1, osteoclast-derived zinc finger protein, binds to both the c-Krox tandem site and the Egr-1 single site, albeit with weaker affinity than their cognate proteins (18). Moreover, at least two other POZ-domain zinc finger proteins interact with DNA in a flexible manner; the Kaiso protein recognizes the specific consensus sequence TCCTGCNA, as well as methyl-CpG dinucleotides (8), and the GAGA factor binds cooperatively to repeats of the sequence motif GAGA that can vary in number and spacing (6, 7).

Our analysis of FBI-1 binding to various permutations of its HIV-1 binding site and to other cellular and viral binding sites reveals that FBI-1 binds efficiently to a variety of sequences with different spacings and orientations. This flexibility is reminiscent of the versatile interactions of other proteins with DNA. For example, the yeast homeodomain protein {alpha}2 binds as a dimer to variously spaced half-sites forming inverted, direct, or everted repeats, because the relative orientations of the two homeodomains in the dimer are unconstrained (27). The POU domain proteins constitute another example of highly flexible binding to DNA. The POU domain consists of two DNA binding domains, the POU-specific (POUS) domain and the POU homeo (POUH) domain, joined together by a flexible linker. Oct-1, a broadly expressed transcription factor, is capable of binding to a wide variety of sequences (21). Such flexibility derives in part from the ability of the POUS and POUH domains to contact DNA in different orientations and different positions relative to one another (28, 29). In the above cases, DNA binding flexibility is achieved in part through the presence of two DNA binding domains, which can be carried either on two polypeptides or on a single polypeptide, whose orientation and relative position on the DNA are unconstrained. In the case of FBI-1, it probably results from the interaction between two monomers via their POZ domains and, perhaps on some binding sites, zinc finger domains. This flexibility may allow FBI-1 to recognize a variety of cellular binding sites. The nature of the binding site (e.g. single versus bipartite) and the orientation of the half-sites with respect to each other may allow an additional level of transcriptional regulation, for instance by exposing different surfaces of FBI-1 to the transcriptional machinery or by allowing it to engage in different protein-protein interactions.


    FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Div. of Rheumatology, Children's Hospital of Philadelphia, 3516 Civic Center Blvd./1102 ARC, Philadelphia, PA 19104. Back

|| To whom correspondence should be addressed. Tel.: 215-590-7180; Fax: 215-590-1258; E-mail: pessler{at}email.chop.edu.

1 The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; EMSA, electrophoretic mobility shift assay; Ad2, adenovirus 2; MLP, major late promoter; IST, inducer of short transcripts; p, plasmid; WT, wild-type. Back

2 D. J. Morrison and N. Hernandez, unpublished data. Back

3 F. Pessler and P. S. Pendergrast, unpublished results. Back

4 P. S. Pendergrast and N. Hernandez, unpublished data. Back


    ACKNOWLEDGMENTS
 
We thank B. Ma for DNA sequencing, S. Teplin for expert synthesis of oligonucleotides, M. Sheldon for providing plasmid WT[ABC]WT, P. S. Pendergrast for helpful discussion and critical reading of the manuscript, A. Stenlund and V. Mittal for valuable discussion, and J. Duffy and P. Renna for artwork and photography. We are grateful to V. Mittal and Kevin Pessler for assistance with graphics and T. Kuhlman and E. Ford for support.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Albagli, O., Dhordain, P., Deweindt, C., Lecocq, G., and Leprince, D. (1995) Cell Growth Differ. 6, 1193–1198[Abstract]
  2. Bardwell, V. J., and Treisman, R. (1994) Genes Dev. 8, 1664–1677[Abstract]
  3. Zollman, S., Godt, D., Prive, G. G., Couderc, J. L., and Laski, F. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10717–10721[Abstract/Free Full Text]
  4. Collins, T., Stone, J. R., and Williams, A. J. (2001) Mol. Cell. Biol. 21, 3609–3615[Free Full Text]
  5. Schultz, J., Copley, R. R., Doerks, T., Ponting, C. P., and Bork, P. (2000) Nucleic Acids Res. 28, 231–234[Abstract/Free Full Text]
  6. Espinas, M. L., Jimenez-Garcia, E., Vaquero, A., Canudas, S., Bernues, J., and Azorin, F. (1999) J. Biol. Chem. 274, 16461–16469[Abstract/Free Full Text]
  7. Katsani, K. R., Hajibagheri, M. A., and Verrijzer, C. P. (1999) EMBO J. 18, 698–708[Abstract/Free Full Text]
  8. Daniel, J. M., Spring, C. M., Crawford, H. C., Reynolds, A. B., and Baig, A. (2002) Nucleic Acids Res. 30, 2911–2919[Abstract/Free Full Text]
  9. Pessler, F., Pendergrast, P. S., and Hernandez, N. (1997) Mol. Cell. Biol. 17, 3786–3798[Abstract]
  10. Ratnasabapathy, R., Sheldon, M., Johal, L., and Hernandez, N. (1990) Genes Dev. 4, 2061–2074[Abstract]
  11. Sheldon, M., Ratnasabapathy, R., and Hernandez, N. (1993) Mol. Cell. Biol. 13, 1251–1263[Abstract]
  12. Morrison, D. J., Pendergrast, P. S., Stavropoulos, P., Colmenares, S. U., Kobayashi, R., and Hernandez, N. (1999) Nucleic Acids Res. 27, 1251–1262[Abstract/Free Full Text]
  13. Pendergrast, P. S., Wang, C., Hernandez, N., and Huang, S. (2002) Mol. Biol. Cell 13, 915–929[Abstract/Free Full Text]
  14. Widom, R. L., Lee, J. Y., Joseph, C., Gordon-Froome, I., and Korn, J. H. (2001) Matrix Biol. 20, 451–462[CrossRef][Medline] [Order article via Infotrieve]
  15. Lee, D. K., Suh, D., Edenberg, H. J., and Hur, M. W. (2002) J. Biol. Chem. 277, 26761–26768[Abstract/Free Full Text]
  16. Astier, A. L., Xu, R., Svoboda, M., Hinds, E., Munoz, O., de Beaumont, R., Crean, C. D., Gabig, T., and Freedman, A. S. (2003) Blood 101, 1118–1127[Abstract/Free Full Text]
  17. Davies, J. M., Hawe, N., Kabarowski, J., Huang, Q. H., Zhu, J., Brand, N. J., Leprince, D., Dhordain, P., Cook, M., Morriss-Kay, G., and Zelent, A. (1999) Oncogene 18, 365–375[CrossRef][Medline] [Order article via Infotrieve]
  18. Kukita, A., Kukita, T., Ouchida, M., Maeda, H., Yatsuki, H., and Kohashi, O. (1999) Blood 94, 1987–1997[Abstract/Free Full Text]
  19. Zoller, M. J., and Smith, M. (1983) Methods Enzymol. 100, 468–500[Medline] [Order article via Infotrieve]
  20. Pessler, F. (1994) Protein Complexex on the HIV-1 Promoter. Ph.D. dissertation, State University of New York, Stony Brook, NY
  21. Sturm, R., Baumruker, T., Franza, B. R., Jr., and Herr, W. (1987) Genes Dev. 1, 1147–1160[Abstract]
  22. Ahmad, K. F., Engel, C. K., and Prive, G. G. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12123–12128[Abstract/Free Full Text]
  23. Pessler, F., and Hernandez, N. (1998) J. Biol. Chem. 273, 5375–5384[Abstract/Free Full Text]
  24. Cao, X., Mahendran, R., Guy, G. R., and Tan, Y. H. (1993) J. Biol. Chem. 268, 16949–16957[Abstract/Free Full Text]
  25. Kobayashi, A., Yamagiwa, H., Hoshino, H., Muto, A., Sato, K., Morita, M., Hayashi, N., Yamamoto, M., and Igarashi, K. (2000) Mol. Cell. Biol. 20, 1733–1746[Abstract/Free Full Text]
  26. Galera, P., Musso, M., Ducy, P., and Karsenty, G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9372–9376[Abstract/Free Full Text]
  27. Smith, D. L., and Johnson, A. D. (1992) Cell 68, 133–142[Medline] [Order article via Infotrieve]
  28. Cleary, M. A., and Herr, W. (1995) Mol. Cell. Biol. 15, 2090–2100[Abstract]
  29. Cleary, M. A., Pendergrast, P. S., and Herr, W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8450–8455[Abstract/Free Full Text]