INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Differential gene expression is achieved by the assembly of multicomponent complexes containing precise combinations and arrangements of individual transcription factors. On certain cis-acting promoter sequences, the activity of specific factors within these complexes can be further modulated in a combinatorial fashion by site-specific regulatory proteins. One example, the Drosophila homeobox protein Deformed, binds weakly to low-complexity DNA sequences present in many homeotically regulated promoters but specifically upregulates transcription at a subset of promoters which harbor additional binding sites for the Deformed activator molecule DEAF-1 (10). The effects of such activator molecules in transient transcriptional regulation are less well documented than in homeotic control, and few candidate complexes have been identified. We have isolated and cloned a previously undescribed but widely expressed heterodimer from HeLa cells, called parvovirus initiation factor (PIF), which resembles DEAF-1 in a number of critical ways. We suggest that both are members of the same emerging family of transcriptional modulators characterized by conservation of a domain containing the signature amino acid motif KDWK.

PIF was first identified as an essential ancillary factor for DNA replication initiation from the minimal left-end origin of the parvovirus minute virus of mice (MVM) (4). In this and comparable viral systems, specific cellular transcription factors stimulate in vitro initiation up to a 1,000-fold, so that successful initiation can often provide a sensitive indicator for monitoring how such factors cooperate with other proteins and interact with their DNA substrates (23, 24). The MVM origin is approximately 50 bp long (7) and contains sequences which play a dual role both in initiation and as part of the early viral transcriptional promoter (Fig. 1). MVM replicates by a rolling-hairpin mechanism which resembles prokaryotic rolling-circle replication, so that DNA synthesis is initiated by the introduction of a site-specific single-strand nick, mediated by the virally encoded nickase NS1. NS1 is itself a DNA-binding protein which recognizes an ACCA repeat motif in the origin, as shown in Fig. 1, and it is oriented in such a way that its footprint lies over the nick site (6). However, by itself, NS1 is in a catalytically inactive state. Before it can nick the origin, a cellular factor must become bound to one end of the origin sequence, at a site which overlaps a consensus activated transcription factor (ATF) binding motif. This site is known to bind ATF and is involved in regulation of the promoter during the cell cycle and in response to cellular transformation (8, 18). However, ATF/cyclic AMP response element-binding protein (CREB) family members inhibit, rather than activate, the viral replication origin, since they compete for their binding site with PIF, the authentic activator complex (3). PIF comprises related polypeptides denoted p96 and p79 and, as depicted in Fig. 1, binds coordinately to the ACGT motif within the ATF site and to an identical motif located 5 bp away, proximal to the NS1 footprint (3). In this study, we examined the remarkably flexible way in which the PIF complex interacts with its bipartite recognition sequence and showed that its individual component polypeptides are capable of activating transcription in transfected cells. Finally, we investigated the possible relationship between PIF and other previously described activities which modulate transcription at the tyrosine aminotransferase and transferrin receptor promoters.

View larger version (35K): [in this window] [in a new window]   FIG. 1.   The left end of the MVM genome. The left (3') hairpin of the MVM genome contains both the upstream regulatory region of the P4 promoter and sequences that give rise to the left-end DNA replication origin. This origin, part of which is shown in expanded form, is a duplex copy of sequences from the top strand of the hairpin, which is formed in dimeric replication intermediates. The PIF half-sites (spaced ACGT motifs) overlapping a consensus ATF site are indicated and marked proximal (P) and distal (D) to denote their positions with respect to the NS1 binding site. The dinucleotide "bubble" sequence is a critical spacer element lying between the proximal ACGT and the NS1 binding site. The box which starts at the bubble sequence and extends rightwards toward the other end of the minimal origin indicates the sequences protected from DNase I digestion by NS1. Within this box are the NS1-binding motif, which overlaps the E box and NF-Y sites of the promoter, and the nick site, where NS1 initiates replication by nicking the DNA to liberate a base-paired 3' nucleotide capable of priming DNA synthesis.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Purification of HeLa PIF complexes. Nuclear extracts from HeLa S3 cells were prepared and fractionated on Q-Sepharose and Zn²⁺-metal chelate Sepharose as described previously (3). The Zn²⁺-metal chelate Sepharose eluate was diluted fivefold in buffer A (25 mM Tris-HCl [pH 7.8], 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 0.01% Nonidet P-40, 1 mM dithiothreitol, 100 mM NaCl, 10% glycerol) supplemented with sonicated salmon sperm DNA (200 µg/ml) and 60 µg of a nonspecific, duplex oligonucleotide (SCRAM; described previously [3]) per ml and absorbed for 1 h on nonspecific DNA-Sepharose coupled with duplex SCRAM DNA. The extract was then applied serially to two 1-ml DNA-Sepharose columns coupled with a duplex copy of the TR56 oligonucleotide 5'-GATCGATCTGTCAGAGCACCTCGCGAGCGTACGTGCCTCAGGAAGTGACGCACAGC-3', representing nucleotides 61 through 115 from the transferrin receptor promoter (19), equilibrated in buffer A. The columns were washed with 50 volumes of buffer A, and bound proteins were eluted with buffer A adjusted to 300 mM NaCl. Fractions containing PIF activity were identified by gel shift and nicking assays and were flash-frozen in liquid N₂ before being stored at 80°C. Selected fractions were concentrated by ultrafiltration on Centricon 30 filters and separated by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). PIF bands were localized by staining with Coomassie brilliant blue, excised, and submitted to the Keck Biotechnology Center for peptide sequence analysis.

cDNA cloning of PIF subunits. Predicted nucleotide sequences for peptides MMDSGQIDFYQHDK from p96 and IMDSGELDFYQHDK from p79 were used to design the degenerate oligonucleotides 5'-ATGGATTCIGGICAIATIGATTTCTACCAICATGATAA-3' and 5'-ATIATGGATGGITCIGAICTIGATTTCTACCAICATGATAA-3', respectively. These were used simultaneously to screen a lambda gt11 HeLaS3 cDNA library (Clontech, Palo Alto, Calif.) and a Uni-Zap XR HeLa cDNA library (Stratagene, San Diego, Calif.). Probes were incubated with duplicate filters overnight at 45°C in 1× SSC (0.15 M NaCl, 0.015 M sodium citrate)-20 mM NaH₂PO₄-0.4% SDS containing denatured salmon sperm DNA (500 µg/ml) and ³²P-labeled probe (2 × 10⁵ cpm/ml) and washed twice for 20 min in 3× SSC-0.1% SDS at room temperature and once for 10 min at 45°C. Four clones were identified out of 3 × 10⁶ plaques screened, all of which hybridized exclusively to the p96 probe. Inserts derived from the lambda gt11 library were excised with EcoRI and subcloned into pGem4Z (Promega, Madison, Wis.) before analysis. Those from the Uni-Zap library were excised in vivo as specified by the manufacturer. All the clones had open reading frames (ORFs) containing the probe peptide and were identical in their overlapping sequence, but only one clone contained the full p96 ORF.

Recombinant baculoviruses. To optimize the expression of wild-type p96 and p79, PCR fragments which started 3 nucleotides upstream of the ORF were cloned into the baculovirus transfer vector pVL1393 (Invitrogen, Carlsbad, Calif.) to generate pVL1393-p96 and pVL1393-p79, respectively. Recombinant baculoviruses were recovered, grown, and assayed as previously described (2).

Purification and analysis of recombinant proteins. Recombinant PIF p96-p79 heterodimers were generated by coinfecting High Five insect cells at high multiplicity (~10 PFU per cell) with both recombinant baculoviruses expressing p79 and p96. Nuclear extracts were prepared essentially as described previously (2) and purified using by fast protein liquid chromatography on a MonoQ (HR5/5) column equilibrated in 25 mM Tris-HCl (pH 7.8)-1 mM EDTA-0.1 mM phenylmethylsulfonyl fluoride-0.01% Nonidet P-40-1.0 mM dithiothreitol-10% glycerol, containing 150 mM NaCl. Bound proteins were eluted with a 40-ml linear NaCl gradient (0.05 to 0.7 M) in the same buffer.

Transcription vector construction and assays. Vectors used in this study were part of a mammalian two-hybrid system purchased from Clontech. To generate pGal-p96 and pVP16-p96 vectors expressing p96 molecules with amino-terminal yeast GAL4 DNA-binding domains or herpes simplex virus VP16 transcriptional activator domains, respectively, SmaI-SalI or EcoRI-SalI fragments containing the full p96 ORF were inserted, in frame with the relevant domains, in the vectors pM and pVP16, respectively. Similarly, the full p79 ORF was cloned into the EcoRI site of pM and pVP16, resulting in the vectors pGal-p79 and pVP16-p79.

Nucleotide sequence accession number. The sequence of p79^pif and p96^pif have been deposited with GenBank under accession no. AF173867 and AF173868, respectively.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Two polypeptides copurify with PIF activity. The PIF complex was purified from HeLa S3 nuclear extracts (from ~10¹⁰ cells) by the scheme depicted in Fig. 2A. For the final site-specific DNA-binding column, we initially used an oligomerized form of a 23-mer containing the PIF-binding site from the MVM origin, but were unable to release the active complex from this substrate. Instead we substituted a nonoligomerized duplex 53-mer sequence, derived from the transferrin receptor promoter, which Roberts et al. (20) had previously shown bound and eluted a factor that we believed was identical to PIF (3). Purification was monitored at each step by measuring site-specific DNA binding activity in EMSA, in the presence and absence of the duplex alternating copolymer poly(dI-dC)-poly(dI-dC), which effectively competes for PIF but not for most other DNA-binding proteins (3).

View larger version (45K): [in this window] [in a new window]   FIG. 2.   Purification and analysis of the HeLa PIF complex. (A) Fractionation scheme used to purify PIF from HeLa nuclear extracts. (B) Silver-stained SDS-PAGE gel showing fractions 1, 2, 3, 4, and 5 eluted from the site-specific DNA affinity chromatography. Lane M contains molecular mass markers. (C) EMSA analysis of fractions. The input is a PIF fraction from a Zn2+-metal chelate Sepharose column which was loaded on to the site-specific DNA affinity column; run thru is the unbound material; wash is the last column wash before elution; lanes 1, 2, 3, 4, and 5 are serial fractions obtained by elution with 300 mM NaCl. (D) Eluted fractions catalyze nicking and covalent attachment of NS1 to the minimal MVM origin. Nicking assay mixtures contained 100 ng of GST-NS1 and ATP; samples in lanes labeled 1 through 5 also received fractions eluted from the DNA affinity column. The nicking assay in the last lane was performed in the presence of fraction 2, GST-NS1, and ATP but using a control substrate (ori), in which a single extra nucleotide inserted into the bubble sequence inactivates viral replication origin function (7).

Isolation of PIF cDNAs. The longest peptides obtained from each molecule were identical at 11 of 14 residues, and their sequences were used to design separate degenerate oligonucleotide probes for screening HeLa-derived lambda phage cDNA libraries. Screening ~3 × 10⁶ plaques gave four cDNA clones which hybridized to the p96-based probe but none that hybridized to p79. The most extensive p96 insert was 1,862 nucleotides long and contained an ORF capable of encoding a protein of 563 amino acids, with a predicted molecular mass of 61,379 Da, which contained all three of the p96 peptide sequences, as shown in Fig. 3A. The discrepancy between the migration rate of p96 on SDS-PAGE and the calculated molecular weight of the protein prompted us to check the sequence in HeLa cDNA by using 5' RACE, but all RACE clones gave the same 5' sequence. Although the p96 gene is not present in the standard GenBank database, the dbEST database contains many sequences related to it, derived from both human and murine tissues, representing various cell types including colon, liver, embryonic stem cells, neuroepithelium, and placenta, suggesting that p96 is expressed in a broad range of tissues. One insert, from human colon (AA148980), contains a predicted single insertion of 10 amino acids, which is suggested by genome sequencing to be the product of alternate splicing (22a).

View larger version (59K): [in this window] [in a new window]   FIG. 3.   Polypeptide sequence analysis of the PIF subunits. (A) Alignment of the amino acid sequences predicted from the ORFs of cloned cDNAs encoding p79pif and p96pif, aligned by using the GCG BESTFIT program. The highly conserved region which contains the KDWK motif discussed in the text and panel B is boxed. (B) Alignment of sequences from the most highly conserved domain of human p96pif and p79pif, with individual proteins or representative members of protein groups, obtained from the GenBank database. The alignment, obtained by using the Genetics Computer Group Pile-Up program, was refined manually. Residues identical in at least half of the compared sequences are shaded. The oval box indicates the highly conserved KDWK motif, and the smaller boxes indicate extended relationships between pairs of compared molecules. Full names, GenBank accession numbers, and references for these proteins are cited in the text.

PIF polypeptides p96 and p79 are related and share a conserved KDWK domain with several known proteins. The deduced amino acid sequences of p96 and p79 (Fig. 3A) show an overall identity of 40 to 42% (for p96 and p79, respectively), which fluctuates along the length of the polypeptide chains but is most evident between residues 82 and 174 in p96 and residues 89 and 182 in p79, where the two chains show 80% identity. When these highly conserved sequences were used to search available databases, a group of protein and expressed sequence tag sequences were found with peptide homologies that stretched through most of the 94 residues of the conserved domain. This region includes a KDWK motif first noticed by Gross and McGinnis (10) in the Drosophila melanogaster DEAF-1 protein (U46686), which itself shows 31% identity to p96 and 28% identity to p79 over this entire region. Other KDWK domain proteins recognized in the search included two human interferon-induced nuclear phosphoproteins (L22342 and L22343) of unknown function (represented by HNP-1 in Fig. 3B), a human leukocyte-specific SP140 protein (U63420), and human lymphoid cell-specific SP100 homologues (U36499 to U36501), which are tissue-specific components of dot-like, subnuclear bodies often described as PML/SP100 bodies. The search also identified various rat and human forms (AF007165, AF068892 to AF068895, and U59659) of a protein initially named "suppressin" (12, 13). Recently a monkey cDNA encoding a very closely related molecule, dubbed NUDR (for "nuclear DEAF-1 related") transcription factor, was identified by its ability to bind, albeit weakly, to a synthetic retinoic acid response element (AF049461) (11). Significantly, recombinant NUDR recognizes a bipartite DNA sequence in which both half-sites contain CpG dinucleotides, and it is able to enhance transcription from a promoter containing such elements, both properties described in this paper for PIF. NUDR-homologous cDNAs were also isolated from human (AF04959) and rat (AF04960) libraries (11). The alignment between the PIF polypeptides and one member from each of these groups is shown in Fig. 3B. Over this region, identity scores between PIF and these polypeptides are all about 30% and include some broadly scattered positions where sequence is absolutely conserved throughout all members of the family and others where one or two particular residues are clearly favored. Boxed sequences at the C terminus of this region are conserved in a pairwise fashion between protein classes, suggesting that they might characterize distinct subsets of the KDWK family. Multiple cosmids from Caenorhabditis elegans also have this pattern of conserved residues, including one (Z81089) which shows 40% identity to the p79 sequence across this region. Significantly, no matches were found in the genomes of prokaryotes or eukaryotic unicellular organisms, suggesting that this gene family is a feature of metazoans.

Recombinant PIF heterodimers bind to the ACGT motifs that overlap the ATF/CREB site in the viral origin. Cloning and DNA sequencing revealed p96 and p79 to be proteins of 563 and 530 amino acids, with predicted molecular masses of 61,379 and 56,397 Da, respectively. These values are both significantly lower than expected from their mobilities measured in denaturing gels. When recombinant baculoviruses were used to coexpress p96 and p79 in insect cells, the purified products comigrated on denaturing gels with their authentic HeLa counterparts (Fig. 4A). Each of the PIF cDNAs also generated proteins with mobilities that matched those of the HeLa cell-derived proteins when translated in vitro (data not shown), indicating that each cloned cDNA contains the entire coding sequence of its respective protein. This also suggests that the discrepancies between their calculated and observed molecular weights are intrinsic properties of the polypeptide chains themselves rather than the result of posttranslational modification.

View larger version (53K): [in this window] [in a new window]   FIG. 4.   Characterization of recombinant PIF subunits expressed from separate baculovirus vectors. (A) SDS-PAGE analysis of purified recombinant PIF. DNA-affinity purified PIF from HeLaS3 cells was compared to purified recombinant PIF p96-p79, expressed in insect cells by using baculovirus vectors. The positions of the p96 and p79 subunits are indicated by arrows. (B) DNase I protection and methylation interference patterns of rPIF binding to a duplex DNA fragment containing the minimal replication origin, in which the top strand, as represented in Fig. 1, was 32P labeled at its 3' end. The sequence through this part of the origin is indicated on the right. Lowercase letters indicate flanking vector DNA sequence. PIF half-sites are shaded, the consensus ATF site is boxed, bubble nucleotides are shown in outline, and the open-ended box indicates the start of the NS1 footprint, in which the first TGGT motif is stippled. Sequences protected by rPIF from DNase I digestion are shown in the left lanes. The right three lanes, labeled MI, show methylation interference profiles: input indicates piperidine cleavage products of the methylated DNA sample used in the binding assay; bound indicates cleaved DNA from the retarded rPIF-DNA complex; and free indicates free probe which was not shifted by the binding reaction. Methylated residues which impair rPIF binding are indicated by arrows.

View larger version (60K): [in this window] [in a new window]   FIG. 5.   Recombinant PIF heterodimers bind spaced, bipartite ACGT motifs. Sequences of double-stranded oligonucleotides, representing wild-type and mutant forms of the PIF-binding region in the MVM 3' origin that were used as competitors in the EMSAs, are shown at the top. Positions of wild-type proximal (P) and distal (D) ACGT half-sites are boxed, and the ATF/CREB-binding site is shaded. Individually mutated nucleotides are indicated in bold type. The lower three panels compare rPIF-, rATF1-, and rCREB-binding specificities on these sequences by competitive EMSA with 100-fold molar excesses of the wild-type or mutant competitor oligonucleotide. Assay mixtures contained the same 32P-labeled PIF wild-type oligonucleotide probe and constant amounts of rPIF, rATF1, or rCREB, as indicated in each panel.

The spacing between the two half-sites can be highly flexible. Although the PIF recognition sequence in the MVM origin involves two ACGT motifs spaced by 5 nucleotides, insertions of a single extra nucleotide between the half-sites had little effect on binding (Fig. 5). This prompted us to ask if PIF would tolerate more substantial changes in the length of this spacer element. Accordingly, we constructed a series of oligonucleotides with insertions or deletions at this position (Fig. 6), end labeled them with ³²P, and compared their ability to bind to both authentic, affinity-purified HeLa PIF and the recombinant p96-p79 complex in direct binding assays. Both complexes bound sequences which had 1, 3, or 10 additional nucleotides inserted into the spacer region (Fig. 6, mutants In2, Three-In, and Ten-In, respectively), although comparing the proportion of bound to unbound probe in each reaction made it clear that as the inserts became longer the mutants bound progressively less well than wild-type molecules. For comparison, mutant In1, which has only a single intact ACGT site, failed to bind either of the protein complexes. Mutant sequences with deletions of 1 or 4 nucleotides in the normal spacer region (Fig. 6, One-del and Four-del, respectively) also bound both forms of PIF, although, once again, binding efficiency progressively diminished relative to the wild type. Thus, PIF heterodimers can bind sequences in which the spacing element varies in length from 1 to 15 nucleotides, although spacers of around 5 nucleotides may be optimal. Methylation interference data for the mutant with a 15-nucleotide spacer confirmed that PIF was binding to the two spaced ACGT motifs in this sequence, ruling out the possibility that we had created alternate binding sites (data not shown).

View larger version (56K): [in this window] [in a new window]   FIG. 6.   Both HeLa PIF and rPIF recognize flexibly spaced bipartite sites. Sequences of the probe oligonucleotides, with differently spaced ACGT motifs, that were used as substrates, are shown at the top. The proximal and distal PIF half-sites are boxed, deletions are indicated by thin lines connecting adjacent nucleotides, and inserted nucleotides are shown in bold type. The bottom panel shows direct EMSAs with 32P-labeled, double-stranded wild-type and mutant oligonucleotides and either DNA affinity-purified PIF from HeLa cells or purified recombinant p96-p79 heterodimers.

p96 and p79 function as transcriptional activators. Although we initially identified PIF because it is an essential cofactor for viral replication initiation, in the host cell it is most likely to function as a transcriptional regulator. Because its binding site in the viral origin/promoter overlaps binding sites for other transcription factors, using this sequence to assess its effects on transcription proved problematic. To circumvent these difficulties, we fused p79 and p96 coding sequences to the yeast GAL4 DNA binding domain and cotransfected the resulting constructs into 293 cells along with pG5CAT, in which the CAT reporter gene is driven by the adenovirus EIB minimal promoter fused to a tandem array of five GAL4-binding sites (Fig. 7). In this context, both Gal-p79 and Gal-p96 activated the transcription of the reporter gene between 5- and 20-fold in several independent experiments (Fig. 7). Although these experiments indicate that p79 and p96 may individually activate transcription, it is also possible that the observed activity results from heterodimerization with an endogenous partner polypeptide in the transfected cell. Cotransfection of Gal-p79 or Gal-p96 with p96 or p79 fused to the herpes simplex virus transcriptional activator VP16 (VP16-p96 and VP16-p79, respectively) increased the activity of the GAL4 fusion constructs approximately fourfold (Fig. 7), indicating that the products of the transfected genes probably assemble into heterodimers in the transfected cell.

View larger version (26K): [in this window] [in a new window]   FIG. 7.   PIF activates transcription from a model reporter construct in vivo. The pG5CAT reporter plasmid contains the CAT gene driven by the adenovirus E1B minimal promoter (TATA) fused to five upstream GAL4-binding sites. N-terminal fusions of p96pif (hatched box) or p79pif (open box) to amino acids 1 to 147 of the GAL4 DNA-binding domain (shaded box) or with amino acids 411 through 455 of HSV VP16 (solid box) were constructed in vectors driven by the simian virus 40 early promoter, as described in Materials and Methods. 293 cells were mock transfected or transfected with pG5CAT alone or with various combinations of the fusion proteins, as indicated below the bar graph. A 5-µg portion of each plasmid was transfected. Transfection efficiency was monitored by measuring -galactosidase activity generated by cotransfected pCH110, and CAT activity is represented as the mean of duplicate samples, which differed by less than 10%.

p96-p79 heterodimers bind to regulatory elements within the tyrosine aminotransferase and transferrin receptor promoters. During these studies, we found two reports of other DNA-binding activities which showed similar physical characteristics. The first of these, mentioned above, is the ubiquitously expressed GMEB heterodimer, which binds to a regulatory element in the rat tyrosine aminotransferase promoter and makes it sensitive to induction by low concentrations of glucocorticoids (17). The sequences of six tryptic peptides from the rat complex all corresponded to sequences present in the human p96 and p79 genes, and subsequent cloning and DNA sequencing strongly indicate that one of the GMEB polypeptides, GMEB-2, is the rat homolog of p79^pif (26). Surprisingly, the element which responds to this complex, GME (Fig. 8A), is very different from the sequence in the MVM origin but does contain two spaced CpG dinucleotides within the sequence 5'-GCGTCAGCGC-3'. When used as substrate in an EMSA, the GME sequence was recognized and shifted efficiently by recombinant p96-p79 (Fig. 8A). Moreover, mutant oligonucleotides M1 through M3 competed for rPIF in exactly the way described previously for GMEB. Thus, M1, which carries a mutation upstream of the first CpG dinucleotide, competed as well as the wild-type oligonucleotide, whereas M2, which has the first CpG mutated, was unable to compete. A significant and initially confusing observation was that oligonucleotide M3, which has the second CpG mutated, was also able to compete. Although the activity of mutant M3 had led Oshima and Simons to conclude that GMEB bound to the first CGTC motif, boxed in Fig. 8A (16), its binding is quite consistent with the flexible bipartite nature of the PIF binding site as described in this paper, since M3 contains a new CpG dinucleotide, fortuitously introduced 3 bases downstream of the mutated CpG. To test whether this new CpG dinucleotide introduced into M3 was critical in allowing recombinant PIF to bind this site, mutant M5, which resembles M3 but no longer contains the new CpG, was constructed. As predicted, this single-base substitution drastically reduced recognition of the oligonucleotide by recombinant PIF (Fig. 8A).

View larger version (49K): [in this window] [in a new window]   FIG. 8.   rPIF binds to an element in the tyrosine aminotransferase promoter. (A) EMSAs of recombinant PIF binding to a double-stranded, 32P-labeled oligonucleotide representing the wild-type GME present in the rat tyrosine aminotransferase promoter. Samples in lanes labeled GME through M5 were incubated in the presence of a 100-fold molar excess of an unlabeled wild-type (GME) or mutant (M1 to M5) oligonucleotide competitor, as indicated. (B) Methylation interference analysis of recombinant PIF binding to the rat tyrosine aminotransferase promoter element. The double-stranded wild-type GME oligonucleotide was 5'-end labeled with 32P on either the top or bottom strand, partially methylated, incubated with rPIF, and gel purified following fractionation by EMSA. Methylated residues which impair rPIF binding are indicated in boldface, and residues which interfered with GMEB binding, as determined by Oshima et al. (17), are indicated by arrows.

View larger version (23K): [in this window] [in a new window]   FIG. 9.   PIF binding to regulatory elements within viral and cellular promoters. Comparison of the methylation interference pattern of rPIF binding to the MVM origin site, the GMEB site in the tyrosine aminotransferase promoter, and the TRA element of the TR promoter. Tetranucleotide motifs conforming to the PuCGPy consensus are shaded, and residues which, when methylated, interfere with rPIF binding are indicated by arrows.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

PIF binds variably spaced CpG motifs and has probably been encountered in previous studies of transcriptional activation. PIF has a bipartite recognition sequence, binding coordinately to two ACGT motifs spaced 5 nucleotides apart in the MVM origin, one of which also forms the core of an ATF site. However, PIF can accommodate linker sequences which vary in size from 1 to at least 15 nucleotides. In addition, some degeneracy in the first and fourth positions of each ACGT motif is tolerated, giving a consensus binding site of PuCGPy, with thymidine residues possibly being favored in the fourth position. Since the CpG dinucleotides at the centers of each motif are absolutely essential for binding, it is not surprising that synthetic polymers of alternating dI and dC residues were found to inhibit PIF binding (3). Historically, this is likely to be significant because ACGT is the core of consensus ATF/CREB binding sites and is also present in a subset of E-box motifs, comprising binding sites for members of the upstream stimulatory factor (USF) family and complexes containing c-Myc. PIF is expressed at around 2 × 10⁴ copies per HeLa cell, so that without widespread use of poly(dI-dC) as a nonspecific competitor, PIF-binding activity would probably have been noticed in previous analyses of sequences which contained a single ATF site or E-box but which had an additional PuCGPy tetranucleotide within 15 bp or so. The spacing criteria for PIF appear so flexible that high-affinity binding sites for it are likely to be present in many human promoters, particularly those which incorporate ATF/CREB-, USF-, and c-Myc-binding sites.

p96 and p79 belong to an emerging family of polypeptides which share a KDWK domain. Sequence analysis shows that p96 and p79 have calculated molecular masses of 61 and 56 kDa, respectively, and are highly related but quite distinct, with only 40 to 42% identity overall. This homology is most evident in a 94-amino-acid domain, which shows 80% identity, beginning 80 to 90 residues from the amino termini of both chains. Recent studies with progressive truncations of both PIF subunits indicate that this region contains the DNA-binding domain. Searches involving this highly conserved sequence identified a small group of nuclear proteins which show conspicuous homology throughout most of this domain but not elsewhere. Initially recognized in the Drosophila homeobox modulator DEAF-1, this domain was designated KDWK, after one highly conserved cluster of residues (10). Another transcriptionally active nuclear protein, NUDR, which also shares this motif, was recently cloned from human, monkey, and rat cDNA libraries (11), while the SP100 group of nuclear dot antigens, which have also been implicated in transcriptional regulation (25), all contain a related domain. Significantly, most of these proteins have been reported to exhibit aberrantly low migration rates when analyzed by SDS-PAGE (1, 9, 21), suggesting that the KDWK domain itself may well be responsible for this anomaly. To date, no definitive binding specificities have been published for any of these proteins, but available data appear consistent with the novel mode of DNA binding described here for PIF. It thus seems likely that these molecules and, by analogy, all members of the KDWK family may bind DNA by engaging low-complexity, flexibly spaced half-sites containing a CpG dinucleotide core.

Factors which recognize flexibly spaced half-sites are rare. Dimeric DNA-binding proteins generally recognize both the sequence and spacing of their two half-sites, but a few exceptions to this rule are currently recognized. For example, a subfamily of nuclear receptors which bind nonsteroid ligands, including vitamin D, thyroid hormone, and retinoic acid, all recognize variously spaced copies of the same minimal hexad half-site, AGGTCA. However, to activate transcription, these all associate with a second molecule, the retinoid X receptor (RXR), and interactions within the heterodimers cause them to have precise spacing requirements, so that vitamin D receptor-RXR heterodimers bind direct repeats spaced 3 nucleotides apart, thyroid hormone receptor-RXR heterodimers require a 4-nucleotide spacing, etc., in accordance with a model called the "1-to-5 rule" (reviewed in reference 14). Thus, this flexibility is more apparent than real and actually results from the use of different combinations of binding modules.