E1A-binding domain of the retinoblastoma product can interact selectively with a sequence-specific DNA-binding protein

Cell, Vol. 65, 1073-1082,

June

14, 1991, Copyright

0 1991 by Cell Press

The T/ElA-Binding Domain of the Retinoblastoma Product Can Interact Selectively with a Sequence-Specific DNA-Binding Protein Thomas Chittenden, David M. Livingston, and William G. Kaelin, Jr. The Dana-Farber Cancer Institute and the Departments of Pathology and Medicine Harvard Medical School Boston, Massachusetts 02115

Summary A DNA-binding site selection and enrichment procedure revealed a sequence-specific DNA-binding activity selectively associated with glutathione S-transferase-retinoblastoma protein chimeras (GST-RB) that had been incubated with a human cell extract. Appropriate mutant forms of GST-RB, incubated in equivalent extracts, did not associate with this specific DNAbinding activity, and a peptide replica of the HPV E7 RB-binding segment selectively inhibited the association of GST-RB with the sequence-specific DNAbinding protein(s). Sequence analysis of oligonucleotides with high affinity for GST-RB complexes, as well as the results of competition binding studies, strongly suggest that RB can associate specifically with the transcription factor E2F or with a protein having closely related DNA-binding properties. Introduction The retinoblastoma susceptibility gene encodes a 928 amino acid nuclear protein (Lee et al., 1987; Friend et al., 1986) that can suppress and, possibly, otherwise regulate cell growth (Huang et al., 1988; Bookstein et al., 1990). Accumulated evidence of the past few years strongly suggests that at least part of its regulatory function is manifest through control of at least one key event in the cell cycle, transit from GO/G1 into S (Buchkovich et al., 1989; DeCaprio et al., 1989). Phosphorylation and dephosphorylation may help to regulate the expression of its biochemical function, and recent results suggest that the former is a multistep process, while the latter begins at a specific point in mitosis and is catalyzed by a member of the protein phosphatase 1 family (J. Ludlow, J. DeCaprio, and D. M. Livingston, unpublished data). How RB, the product of this gene, operates in vivo is not clear. However, its TIE1 A-binding domain can form stable complexes, in vitro, with three different transforming products of DNA tumor viruses (Hu et al., 1990; Huang et al., 1990; Kaelin et al., 1990) and with at least seven specific cell proteins (Kaelin et al., 1991). RB binding probably constitutes part of the mechanism used by the viral proteins to deliver neoplastic signals to target cells, and each is believed to modulate, perhaps by inhibiting, one or more aspects of the RB growth regulatory function (DeCaprio et al., 1988; Dyson et al., 1990; Whyte et al., 1989). The RB structure-function relationships that govern its interactions with viral and cellular proteins are similar, and

mutations that inactivate RB biological function simultaneously inactivate its protein-binding property (Kaelin et al., 1991; Hu et al., 1990). These results imply that the two are linked and, therefore, that the binding of one or more of the above-noted cellular proteins is an important part of the mechanism(s) of RB function. An RB segment extending from residue 379 to 792, alone, can serve as the minimal T/ElA-binding domain (Hu et al., 1990; Huang et al., 1990; Kaelin et al., 1990). In addition, this polypeptide can bind specifically to the cellular proteins identified above (Kaelin et al., 1991). It will be referred to as the “pocket” (Kaelin et al., 1991), because it serves as a receptor for proteins of a specific class. It can be excised from its surrounding sequence, fused to foreign protein sequence, and still retain its specific protein-binding function (Kaelin et al., 1991). Naturally occurring loss-of-function RB mutations that do not destabilize the protein regularly map to the pocket, suggesting that binding of proteins to this domain contributes to RB growth regulatory function (Bookstein et al., 1990; Horowitz et al., 1989,199O; Kaelin et al., 1991; Shew et al., 1990a, 1990b). A prime question at hand is how this domain and its abilitytoform stable complexeswithforeign proteins are translated into the cell cycle regulatory behavior of RB. One way to approach this question is to learn the functions of the cellular proteins that can interact specifically with the pocket. The obvious proteins, detected to date, range in size from ~1.50 kd to -26 kd (Kaelin et al., 1991; Huang et al., 1991). All appear to coextract with nuclei from various cell lysates, and their binding to the pocket can be effectively competed either by SV40 large T antigen or by a short peptide sequence of T or human papillomavirus (HPV) E7 that constitutes its core RB-binding domain (Kaelin et al., 1991; W. G. Kaelin, Jr. and D. M. Livingston, unpublished data). Therefore, an in vitro binding assay can select for proteins with specific pocket affinity. RB has been noted to exert significant effects on the transcription of the c-myc gene (Kim et al., 1991; Pientenpol et al., 1990) and can modulate expression of the c-fos gene (Kim et al., 1991; Robbins et al., 1990) in cotransfection experiments. These effects correlate with the presence of intact pocket-binding function (Kim et al., 1991; Robbins et al., 1990). The question, then, of whether pocket and/or one or more of its binding proteins have transcriptional regulatory function arises. A number of transcription factors have now been studied in detail, and those that do not perform core functions typically display an ability to recognize a specific, canonical DNA sequence of <20 nucleotides (Johnson and McKnight, 1989). With this in mind, we have queried, in a generic sense, whether RB itself, and/or any pocketbinding protein(s), has such a property. To investigate this question, we have used an approach described earlier for the detection of specific DNA sequences recognized by proteins suspected or known to play a role in transcription regulation (Kinzler and Vo-

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A

Figure 1. Experimental Approach to Select for DNA Sequences with High Affinity for RE3 Pocket or Pocket-Associated Cellular Proteins

Oligonucleotides

24mer primer

5’ CTCGGTACCTCGAGTGAAGCrrGA

3

Xhol

B

Selectloll strategy: Binding

Purification

Incubate 62bp align. with GST-RB bead complexes

-

wash beads, pwfy bound oligo. DNA

Amplification

-

(A) DNA sequences of synthetic oligonucleotides. The 62 base oligonucleotide contains a central 16 base core segment of random nucleotide incorporation (N), flanked by defined terminal ends of 24 (left) and 22 (right) bases. Also shown are the oligonucleotide primers used to amplify the 62 base oligonucleotide by PCR. (B) Outline of the binding site selection procedure. Each round of selection consists of incubation of 62 bp oligonucleotide DNA with GST-RB * CP complexes, with subsequent purification and amplification of bound DNA for use in the next round of selection.

PCR amplify ollgo. DNA and gel purify 62bp product

Repeat selection cycle

gelstein, 1989,199O; Tuerk and Gold, 1990; Blackwell and Weintraub, 1990; Blackwell et al., 1990; Perkins et al., 1991). The methodology is based on the expectation that, if a protein bearing specific DNA recognition properties is present in a given fraction, it might be able to select an oligonucleotide of defined sequence from awholly random mixture (Kinzler and Vogelstein, 1989). With the assistance of the polymerase chain reaction (PCR), an extraordinarily rare oligonucleotide sequence, captured by a protein of interest, could be amplified and sequenced after purification. In this report, we describe the use of this approach to demonstrate that at least one pocket-binding protein has sequence-specific DNA-binding activity. Moreover, it can recognize two sets of consensus sequences that are closely related, but not identical, to the currently known consensus recognition site for a well-described transcription factor, E2F (Bagchi et al., 1990; Kovesdi et al., 1986; Yee et al., 1987) Results Isolation of DNA Sequences That Bind Selectively to RB Pocket-Protein Complexes To identify specific DNA sequences that might be recognized by RB or RB pocket-bound proteins, we employed a bacterially synthesized chimeric protein consisting of glutathione S-transferase (GST) fused to RB residues 379-928 (referred to as GST-RB). This segment includes a functioning pocket domain, capable of binding specifically to T and El A, as well as a set of previously described cellular proteins (Kaelin et al., 1991). GST-RB and certain mutant derivatives were readily purified from bacterial extracts by incubation with glutathione-Sepharose beads (Kaelin et al., 1991). GST-RB bound to glutathione-sepharose beads can function as an affinity reagent to bind the aforementioned proteins from human cell extracts that

specifically interact with the RB pocket (Kaelin et al., 1991). It was previously shown that at least seven human cell proteins bind selectively to wild-type, but not various mutant, forms of GST-RB, suggesting that one or more of these pocket-binding proteins may be important targets for RB function. In a search for the identity of these proteins, we attempted to determine whether any RB pocket-binding cellular protein has a sequence-specific DNA-binding activity. By detecting such an activity and elucidating the nature of its cognate DNA recognition sequence, one might learn whether the protein(s) in question behaves, at least in part, like certain proteins of this class, e.g., transcription factors. Our strategy for isolating potential specific DNA-binding sites was similar to the experimental approaches used by others to identify the recognition sites of c-myc, Gil, and Evi-1 (Blackwell et al., 1990; Kinzler and Vogelstein, 1990; Perkinset al., 1991).A62 baseoligonucleotide, containing a central 16 base core of completely random DNA sequence, was synthesized. The wholly degenerate central region was flanked by terminal segments of defined sequence (Figure 1A). Oligonucleotides of 24 and 22 bases were also synthesized to serve as primers (Figure 1A) for amplification of the 62-mer by PCR. Figure 1 B depicts the procedure used to enrich for any 62 bp oligonucleotide molecules from the random pool that are capable of interacting with the protein target(s) of interest. Each round of selection consisted of several steps. First, double-stranded 62 bp oligonucleotide DNA was mixed with GST-RB-Sepharose beads that had been loaded with RB pocket-binding proteins (hereafter called GST-RB+CP) by prior incubation with an extract of the human retinoblastoma line WERI-Rb27, which does not produce detectable amounts of RB (Huang et al., 1988). The binding reaction was carried out in buffer containing 100 mM NaCl and 4 pglml poly(dl-dC) as competitor. The beads were washed to remove 62-mer molecules that did

RB Interacts 1075

with a Sequence-Specific

DNA-Binding

Protein

otides isolated after one, two, three, and four rounds of selection were 32P end labeled and incubated with GSTRB+CP, as described above. After washing the beads, the amount of 62-mer DNA that remained bound to the beads was quantitated by Cerenkov counting (Figure 2A). This assay revealed that the 62-mer DNA populations, derived from repeated rounds of selection, exhibited progressively more efficient binding to GST-RB+CP bead complexes than the starting mixture of 62-mer oligonucleotides (Figure 2A, solid bars). Importantly, 62-mer molecules from multiple rounds of selection did not show increased binding to control bead preparations containing GST alone, which either had (GST+CP) or had not (GST) been preincubated with aliquots of a WERI-Rb27 cell extract (Figure 2A). In addition, the selected 62-mer populations did not demonstrate increased binding to GST-RB beads alone (no prior loading of pocket-binding proteins; Figure 2A). The results of additional rounds of binding site selection suggested that maximal binding activity had been achieved after four to five rounds (data not shown). These results indicate that the selection procedure enriched for 62-mer DNA sequences that bind selectively to GSTRB+CP complexes. Furthermore, selective DNA-binding activity appears to reside in one or more RB-associated cellular proteins, since GST-RB alone did not exhibit appreciable affinity for the selected 62-mer DNA molecules.

1

Figure 2. Binding of Selected Various Bead Complexes

62 bp Oligonucleotide

Populations

to

(A) DNA-binding assay using 62 bp probes derived from multiple rounds of selection with GST-RB loaded with cellular proteins. The 62 bp DNA populations isolated from the indicated rounds of selection were 32P end labeled, as described in Experimental Procedures, and 5 x 1O5 cpm of each probe was incubated with aliquots of either GST or the GST-RB bead complexes. The RB sequence present in the chimera extended from residue 379 to 928. In some cases, beads had been preloaded with cellular proteins by prior incubation in WERI-Rb27 cell extract (+CP). Bound DNA was quantitated by Cerenkov counting. (B) Binding of 82 bp oligonucleotide populations derived from multiple rounds of attempted selection with GST-RB alone (no prior loading of cellular proteins). The binding assay was performed as described in (A).

not tightly associate with the GST-RB bead complexes, and any DNA that remained bound to the beads was then eluted and amplified by PCR. The amplified 62 bp material was then used for the next round of selection with GSTRB+CP. Finally, after a number of serial selectionlamplification steps, the amplified product(s) was assayed for binding to the RB pocket in the presence (to GST-RB+CP) and absence (to GST-RB) of bound cellular proteins. One would predict that if this strategy were successful, 62:mer oligonucleotide populations isolated after three or four rounds of selection should bind to GST-RB+CP substantially better than the starting population of 62-mer molecules (round 0). To test this possibility, 62-meroligonucle-

Attempted Selection of Specific Oligonucleotides That Can Bind Directly to the RB Pocket In a separate experiment, we attempted to select for 62-mer DNA sequences that might bind directly to the RB pocket (GST-RB). The same selection procedure as before was used (Figure l), but the selective agent in each round of attempted selection was GST-RB-Sepharose beads that had not been exposed to the WERI-Rb27 cell extract. After three rounds of selection, a binding assay was performed as described above. In this case, there was no enrichment for specific oligonucleotides that bound efficiently to GST-RB beads or to any of the various bead complexes tested, including GST-RB+CP (Figure 2B). The 62-mer pool isolated after three rounds of attempted binding selection, for example, did not interact significantly better with GST-RB beads than with the starting population of oligonucleotides (round 0). This result suggests that the RB pocket, at least in the form of the GSTRB bacterial fusion protein, cannot directly interact with a specific DNA sequence of 616 nucleotides and, again, implies that only when the pocket is specifically filled with cellular proteins can it select for specific sequences from the wholly random oligonucleotide mixture. Selective DNA-Binding Activity Requires One or More RB Pocket-Associated Cellular Proteins The simplest interpretation of the results presented in Figure 2 is that the selection and binding of oligonucleotide sequences were mediated by one or more RB pocketassociated cellular proteins. It was previously shown that mutations in the pocket segment of GST-RB fusion proteins impaired cellular protein binding (Kaelin et al., 1991). Some of the mutations tested corresponded to naturally

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PS Pwde

Figure 3. Role of RB Pocket-Associated DNA Binding by GST-RB Complexes

Cellular

Proteins

in Selective

(A) DNA-binding capacity of mutant GST-RB complexes. 9 endlabeled 62 bp probes from zero, four, or five rounds of selection with GST-RB+CP (as described in Figure 2A) were incubated with either mutant (GST-RB(706, C+F) and GST-RB(Aexon 21)) or wild-type GST-RE complexes, pretreated with cell extract (+CP). The DNAbinding assay was carried out as in Figure 2. (6) DNA-binding assay with E7 peptide-treated GST-RB complexes. Aliquots of GST-RB beads were mixed with the indicated amounts of either the wild-type E7 peptide, or the point mutant E7 peptide, followed by incubation with WERLRb27 cell extract. The peptide-treated GSTRB bead complexes were then mixed with a 62 bp oligonucleotide 32P-labeled probe from round 5 of binding selection, and the amount of bound probe was measured by Cerenkov counting.

occurring loss-of-function mutations. If the selective DNAbinding activity apparent in GST-RB+CP complexes was contributed by a specific pocket-binding protein, one would predict that appropriate mutant GST-RB+CP complexes, which cannot efficiently bind to cell proteins or T and EIA (Kaelin et al., 1991), should display greatly reduced affinity for the selected oligonucleotide populations. To test this possibility, we analyzed the binding capacity of two mutant RB pocket species: GST-RB(Aexon 2i), which contains the 379-928 RB segment with a deletion of exon 21 (residues 703 to 737), and GST-RB(706, C-F), which contains the 379-928 region bearing a substitution mutation at residue 706 (Figure 3A) (Kaelin et al., 1991).

GST-RB(Aexon 21), GST-RB(706, C-F), and GST-RB (contains the wild-type 379-928 segment) bound to glutathione-Sepharose beads were each preincubated with WERI-Rb27 cell extract. These bead preparations were then mixed with 3zP-labeled oligonucleotide probes isolated after zero, four, and five rounds of binding selection to GST-RB+CP complexes. Both mutant RB bead complexes were substantially impaired, relative to wild-type GST-RB, in their capacity to bind the labeled 62-mer oligonucleotides that had been serially selected by GSTRB+CP complexes (Figure 3A). Therefore, selective binding of 62-mer DNA probes to GST-RB+CP complexes requires a wild-type pocket structure. The association of pocket-binding cellular proteins with GST-RB can be competitively inhibited by coincubation with short, synthetic peptide replicas of the RB pocketbinding segments of either the SV40 large T antigen (Kaelin et al., 1991) or HPV type 16 E7 protein (Jones et al., 1990; W. G. Kaelin, Jr. and D. M. Livingston, unpublished data). We tested whether the E7 peptidecould significantly inhibit the 62-mer DNA binding to GST-RB beads in reactions containing a WERI-Rb27 cell extract. Aliquots of GST-RB beads were mixed with 0, 1, 5, or 10 pg of a 17 residue synthetic peptide replica of the pocket-binding domain (amino acids 16 to 32) of HPV E7 protein (Jones et al., 1990). Another aliquot of GST-RB beads was mixed with a mutant E7 peptide, identical to the wild-type peptide but containing a Glu to Gln substitution at residue 26. In prior experiments, this mutant E7 peptide did not bind to RB and failed to block cellular protein binding to the pocket (Jones et al., 1990; W. G. Kaelin, Jr. and D. M. Livingston, unpublished data). The GST-RB bead preparations, exposed to peptide, were then incubated with extracts of WERI-Rb27 cells, followed by incubation with 32P-labeled 62-mer DNA probe derived from the fifth round of binding selection on GST-RB+CP bead complexes. The results indicated that wild-type E7 peptide progressively inhibited oligonucleotide binding to GST-RB complexes (Figure 38). By contrast, the mutant peptide failed to inhibit significantly. From these results, we conclude that selective DNA binding is a function of one or more pocket-binding cellular proteins. DNA Sequence Analysis Reveals an E2F-like Recognition Site Shared by 62-mer Molecules Isolated after Multiple Rounds of Selection and Amplification GST-RB complexes, loaded with cellular proteins, have an apparently high affinity for certain oligonucleotides isolated after several rounds of selection, but not the starting oligonucleotide population, strongly suggesting that binding results from a sequence-specific interaction. To test this, it was necessary to determine the primary sequences of DNA molecules that showed increased affinity for GSTRB+CP complexes. Thus, oligonucleotides, isolated after five or six rounds of binding selection, were digested with Xhol and EcoRI, which cleave the right and left ends shared by all of the 62 bp molecules (Figure lA), and the products were cloned into a plasmid suitable for rapid, direct DNA sequencing of inserted DNA. The DNA se-

RB Interacts 1077

with a Sequence-Specific

DNA-Binding

Protein

Figure 4. DNA Sequences of Oligonucleotides Isolated after Five or Six Rounds of Binding Site Selection The 62 bp molecules from round 5 or 6 of selection with GST-RB+CP complexes were cloned into Bluescript II SK. Colonies were chosen at random for DNA sequence analysis. The sequences numbered 5.1 through 5.18 correspond to isolates from round 5 of the selection; 6.1 through 8.18 are isolates from round 6. The topmost sequence shows the structure of the starting (unselected) oligonucleotide population, where N represents random nucleotide incorporation. Capital letters denote the nucleotide positions corresponding to the original 16 base region of random sequence. Lowercase letters indicate the regions of DNA immediately flanking the 5’ and 3’ ends of the random core element that are shared by all 62 bp oligonucleotides (see Figure 1). The sequences are grouped according to their similarity (underlined regions) to the deduced consensus sequence classes (1 or 2). The EPF-binding sites present in certain viral and cellular genes are listed at the bottom left.

quences of 46 independent clones were determined and are summarized in Figure 4. It is clear from this analysis that the majority (37 of 46) of oligonucleotides derived from five or six rounds of binding selection share a consensus DNA sequence represented by TTTTGGCGGG (class 1 consensus). Seven additional isolates bear a related consensus sequence, ATTTGCGCGGG (class 2 consensus). The two remaining sequences do not show an obvious similarity to the two major classes of consensus DNA sequences, but share the sequence GGCGGG. These shared consensus DNA sequences represent potential DNA recognition sites of RB-associated sequence-specific DNA-binding protein(s). The original 62 bp oligonucleotide population (i.e., zero rounds of selection) was completely degenerate within the central 16 bp span of random DNA sequence. This was confirmed by direct DNA sequencing of the original 62 bp oligonucleotide DNA preparation (data not shown) by the method of Blackwell et al. (1990). However, the consensus sequences shared by independent isolates are located almost exclusively at the right end of the degenerate 16 bp core region (Figure 4). The consistently “right-justified” location of these sequences indicates that the 3’portion of the consensus binding sites may be fortuitously provided by the constant DNA sequences immediately adjacent (3’) to the 16 bp random core sequence (most likely the sequence GGG). This possibility is supported by the DNA sequence of the isolate 5.108, containing the sequence

ATTTGGC, which is not “right justified” yet is followed by GGG (Figure 4). While we can not precisely define the 3’ limits of the consensus binding site from these results, we infer that the constant GGG sequence bordering the 16 bp random core of the 62 bp oligonucleotide constitutes part of the selected binding site. A comparison of the deduced consensus sequences with known DNA regulatory sites revealed a strong similarity to the binding site of E2F, a known cellular transcription factor of an as yet undefined sequence (Kovesdi et al., 1986; Figure 4). The predominant (class 1) consensus sequence, TTTTGGCGGG, closely resembles a previously described consensus EPF-binding site, TTTTCGCGC (Kovesdi et al., 1986; Mudryj et al., 1990), in that both consist of four tandem T bases followed one base later by a GCG element (Figure 4). The GCG component of the EPF-binding site (indicated by the dots in Figure 4) has been shown to be critical for E2F binding (Yee et al., 1987), and these particular bases are shared by the class 1 consensus sequence. In contrast to the consensus E2F recognition site, the consensus binding sites selected in this experiment (both class 1 and class 2) contain a G (instead of C) immediately 3’to the TTTT stretch. There is a clear preference for G at this position in the FIB pocket binding/ selection experiment, since none of the isolated oligonucleotidescontained aCfollowingtheTTTTelement. While it is not known precisely how substitution of a G at this position affects E2F binding, most of the E2F recognition

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sites identified previously in viral and cellular promoteis harbor a C at this position (Kovesdi et al., 1987; Hiebert et al., 1989; Mudryj et al., 1990). However, there are at least two documented exceptions (Figure 4, bottom). The E2Fbinding site upstream of the murine c-myb promoter (Mudryj et al., 1990) is identical to the RB pocket-selected class 1 consensus oligonucleotide, TTTGGCGG. Moreover, an E2F-bound sequence upstream of the human N-myc promoter (Mudryj et al., 1990) is identical to the class 1 consensus sequence, except for a single C for G change (N-myc TTTGGCGC vs. TTTGGCGG; see Figure 4). The two isolates that do not match either the E2F-like class 1 or class 2 consensus sequences share the sequence GGCGGG in common, which loosely resembles the recognition site for the Spl cellular transcription factor (Kadonaga et al., 1986). Clearly, without additional sequence data, it is not yet possible to say whether these represent an additional consensus class of selected DNA sequences. The Adenoviral E2 Promoter E2F Recognition Site Also Binds to GST-RB Complexes, but with Lower Affinity than the Class 1 Consensus Sequence One interpretation of these results, given the similarity of the consensus class 1 and 2 sequences to the canonical E2F recognition site, is that E2F binds specifically to the RB pocket and functions as the prime oligonucleotide enrichment agent during the binding/selection procedure. Therefore, we wished to determine whether GST-RB+CP complexes bound both the well-characterized E2F recognition site present in the adenovirus E2 promoter (Yee et al., 1987; Kovesdi et al., 1986) and the newly defined consensus class 1 and 2 sequences. A series of oligonucleotides representing the adenoviral E2F-binding site (E2 and E22), the deduced consensus class 1 recognition site (C9 = isolate 6.9 in Figure 4), and the consensus class 2 sequence (Cl8 = 6.18 in Figure 4) was studied as binding targets for GST-RB+CP complexes. A mutant C9 oligonucleotide (CM), which corresponded to the class 1 sequence with a point mutation (C to A) within the GCG component of the consensus sequence, was also synthesized. The analogous mutation within the adenoviral E2 promoter-binding site was shown to greatly reduce E2Fbinding activity (Hiebert et al., 1989). An oligonucleotide (BK) of sequence wholly unrelated to E2F was also employed as a control in these binding studies. To test the relative affinities of the different consensus binding sequences for protein-loaded GST-RB complexes, each oligonucleotide was3*P end labeled and incubated with GST-RB beads that had previously been exposed to WERI-Rb27 cell extract (GST-RB+CP). In parallel, each probe was incubated with identically prepared GST-RB(Aexon 21)+CP complexes. The results indicate that the C9 oligonucleotide, which corresponds to the abundant consensus class 1 sequence, exhibited the highest affinity for GST-RB complexes (Figure 5A). The C to A point mutation abolished binding (Figure 5A, left, oligonucleotide CM). The consensus class 2 oligonucleotide, C18, showed specific but reduced binding to GST-

RB complexes, in keeping with its clear minority status among the 46 isolates. The adenoviral E2F oligonucleotide (E2) also showed low level but selective binding to GST-RB compared with GST-RB(Aexon 21) bead complexes in this assay. Indeed, the magnitude of differential E2 binding was comparable to that of C18. When this experiment was repeated, once again Cl8 and an adenoviral E2F recognition site oligonucleotide (E22) both showed, in replicate assay points, a relatively small but specific difference in binding to the wild-type over the mutant RB pocket+CP complexes (Figure 5A, right). The nonspecific control oligonucleotide (BK) and the consensus class 1 mutant oligonucleotide (CM) did not exhibit selective binding to wild-type GST-RB+CP complexes. We next performed competition assays to determine whether the same factor(s) in GST-RB complexes interacts with both class 1 and class 2 consensus sequences. GST-RB bead complexes were incubated with 3zP-labeled consensus class 1 oligonucleotide (C9) in the presence of either a 50- or 500-fold molar excess of unlabeled competitor oligonucleotide DNA. As expected, excess unlabeled C9 oligonucleotide effectively competed for its own binding (Figure 58, solid bars). The class 2 consensus oligonucleotide, Cl 8, also competed well for C9 binding to GSTRB bead complexes, although less effectively than C9 at the lower concentration (Figure 5A, left, hatched bars). The BK oligonucleotide (shaded bars) served as the nonspecific binding control in these assays. The ability of Cl 8 to compete with C9 for binding suggests that both classes of consensus sequences were recognized and selected by the same protein(s) in the GST-RB+CP complexes. Like C9 and Cl 8, an adenoviral E2F consensus oligonucleotide (E22) also competed with C9 for binding to GSTRB+CP complexes, although at each point in the titration, there was less competition than was noted with unlabeled C9 (Figure 5B, right). Nevertheless, there was clearly greater competition at both unlabeled E22 DNA input levels than was noted with the BK control oligonucleotide tested in parallel. These results, and those of the direct binding assays described above, strongly imply that the viral EPF-binding site also binds specifically, albeit more weakly than a class 1 consensus sequence, to a protein(s) that selectively binds to the RB pocket. Discussion An oligonucleotide enrichment procedure, using a wholly degenerate 16 bp sequence, was employed to search for DNA sequences that bound with high affinity to the RB pocket and/or to specific pocket-associated cellular proteins (Kaelin et al., 1991). The major conclusion of this work is that the pocket segment of RB interacts with a sequence-specific DNA-binding protein(s) with DNA recognition properties characteristic of the transcription factor E2F (Kovesdi et al., 1986; Yee et al., 1987). This protein(s) associated with a chimeric pocket-containing fusion protein during preincubation with an RB-‘- cell extract. By contrast, mutant RB pockets harboring naturally occurring loss-of-function mutations lacked this E2F-like DNAbinding activity after preincubation with the same cell ex-

y;7meracts

Figure

with a Sequence-Specific

5. Oligonucleotide-Binding

DNA-Binding

and Competition

Protein

Assays

21)) complexes, preloaded with cellular proteins (A) Binding of 32P end-labeled oligonucleotides to wild-type (GST-RB) or mutant (GST-RB(Aexon (+CP). Equal amounts of 32P-labeled oligonucleotide probes were incubated with the indicated GST-RB complexes. The C9 and Cl8 oligonucleotides are consensus class 1 and 2 sequences, respectively. CM contains a point (C+A) mutation within the GCG segment of the consensus class 1 (C9) sequence. E2 and E22 oligonucleotides encode the adenovirus E2 promoter E2F-binding sequence. BK is a nonspecific control oligonucleotide of unrelated DNA sequence. The results of two independent experiments are shown. Each value shown in experiment 2 represents the average of two data points generated in parallel. (B) Competition for binding of the consensus class 1 sequence (C9) to GST-RB+CP bead complexes. 32P-labeled C9 oligonucleotide was incubated with GST-RB complexes in the presence of a 50- or 500-fold molar excess of unlabeled competitor oligonucleotides (described above). Binding is expressed as the percentage of bound 32P-labeled C9, relative to the amount bound in the absence of any oligonucleotide competitor (defined as 100%). Two independent experiments, employing either Cl8 (left) or E22 (right) as competitor DNA, are shown.

tract. In addition, a synthetic peptide replica of a known RB-binding/viral protein-transforming domain inhibited the interaction between the pocket and the cellular protein(s) that was responsible for the specific DNA-binding activity. Taken together, these results imply that the interaction of FIB with this factor(s) may have important consequences for the growth control function of RB. Since E2F cDNA has not been cloned, we cannot be certain that the RB-associated factor is what has been heretofore defined as E2F (Yee et al., 1987; Kovesdi et al., 1986). However, the most abundant (class 1) consensus sequence isolated in this study, TTTTGGCGGG, is identical to the canonical (adenovirus E2 promoter) E2F-binding site, TTTTCGCGC, at seven of nine nucleotide positions (see Figure 4). Interestingly, the two nonidentical positions among these sequences (C to G substitutions) have been noted to vary among E2F-binding sites defined upstream of certain viral and cellular promoters, to which E2F can

specifically bind (Mudryj et al., 1990). E2F-binding sites flanking the N-myc and c-myb genes have the sequences TTTGGCGC and TTTGGCGG, respectively (see Figure 4) and the sequence TTTCGCGG is present within the adenovirus ElA promoter (Mudryj et al., 1990). All three of these E2F oligonucleotide sequences, one of which (the c-myb sequence) is identical to the core of the class 1 consensus sequence defined here, were reportedly equivalent to the consensus E2F sequence (TTTCGCGC) in their E2F-binding capacity. The latter was measured by the ability of each oligonucleotide to compete with sites located in the adenoviral E2 promoter for binding to E2F (Mudryjetal., 1990).Thus, theexistenceofasmallnumber of nucleotide differences between the class 1 consensus sequence and the known, canonical EPF-binding sites is not inconsistent with the possibility that the RB-associated binding activity is E2F. In addition, binding and competition experiments in this study demonstrated that the au-

Cdl 1080

thentic adenoviral EPF-binding site present in the E2 promoter interacted, albeit less efficiently, with the same RB-associated cellular factor(s) that bound consensus class 1 oligonucleotides. Therefore, we consider it highly likely that the RB pocket binds E2F, or a protein with DNAbinding properties closely related to E2F. Solely for the sake of simplicity, this element will be referred to as E2F in the remainder of this discussion. If E2F indeed bound to the RB pocket and functioned as the sequence-specific DNA-binding agent during the binding site selection/enrichment procedure, then why do none of the selected oligonucleotide isolates (Figure 4) contain the adenoviral consensus E2F-binding sequence? One possibility is that the classically defined E2F consensus motif does not represent the site with the greatest potential affinity for E2F. It is conceivable that the consensus class 1 sequence, in fact, provides an optimized DNA recognition site for E2F, at least under the binding conditions used in this particular assay. The selection procedure employed here requires high affinity interaction via a single recognition site, whereas E2F-binding sites defined in promoters generally occur in pairs, with the potential for cooperative binding (Mudryj et al., 1990). It is also possible that the DNA recognition properties of E2F are subtly altered by virtue of its interaction with the RB pocket, so that the class 1 type sequence may now be preferred over the “authentic” E2F recognition site. In this regard, it is interesting to note that the pX protein of human hepatitis B virus associates with, and subtly changes the DNA-binding specificity of, the cellular transcription factors ATF-2 and CREB (Maguire et al., 1991). Conceivably, RB alters the binding specificity of E2F, or limits its highest affinity to a certain subset of recognition sites. With highly purified E2F, it should be possible to test this possibility by performing in vitro E2F-binding assays using the class 1 and 2 consensus sequences and heretofore known E2F recognition sequences in the presence or absence of the RB pocket domain. It is also possible that there are multiple E2F species with subtly different DNA-binding properties. If so, one might speculate that a different subset binds to the RB pocket than has been recognized before. What are the biological consequences of an interaction between the RB pocket and E2F? As noted above, the results in this study suggest that the FIB-EPF complex interacts relatively poorly with the canonical E2F recognition sites located in the adenoviral E2 promoter. Earlier, Bagchi et al. (1990) found E2F from mouse L cell extracts to exist in a complex with a cellular protein of approximately 110 kd. The E2F-cell protein complex showed relatively low affinity for the adenoviral E2 promoter E2Fbinding site (see Figure 4). E2F readily dissociated from this complex following exposure of the complex to ElA protein, provided ElA domain 2 was intact. The result was coordinate restoration of strong binding to the viral E2binding site, once the 19 kd E4 product was added to the assay mixture (Bagchi et al., 1990). Given that domain 2 represents the high affinity RB-binding sequence within ElA and that pRB is ~110 kd, as defined by SDS-gel electrophoresis, it was suggested that an ElA domain 2binding protein (e.g., murine RB) represents an attractive candidate for the identified cellular EPF-binding protein

(Bagchi et al., 1990). That hypothesis is entirely compatible with the findings presented here. Although E2F, bound to the RB pocket, apparently binds less efficiently to the E2F recognition sites within the adenoviral E2 promoter than to the consensus class 1 sequence, it is not necessarily the case that RB simply inhibits E2F activity by associating with it. As noted by Bagchi et al. (1 QQO), it is possible that the adenoviral E2F recognition sites do not properly represent the normal cellular targets for the E2F-cell protein complex. Even if similar sites are important E2F cognate sequences upstream of certain cellular genes, it is also possible that, complexed with RB, E2F gains a new regulatory function, manifest in part by a preference for the class 1 consensus sequence described here. The RB pocket can associate with at least seven cellular proteins that, like the E2F-like binding activity identified in this study, fail to interact with mutant RB proteins bearing defective pockets (Kaelin et al., 1991). The binding of all of these species was specifically competed by T or E7 peptide replicas of the RB-binding domain. Given the fact that multiple proteins can associate with the RB pocket, it is formally possible that RB interacts with other sequencespecific DNA-binding proteins/transcription factors in addition to E2F. It is noteworthy that the two oligonucleotide isolates that did not fit the E2F-like consensus classes shared a sequence similar to the Spl-binding site (Kadonaga et al., 1986). The significance of this finding is now under investigation. Moreover, in the binding site enrichment strategy used in this study, we may have selected sequences recognized by only the most abundant or tightest binding of the RB-associated sequence-specific DNAbinding proteins. It may be possible to detect less abundant (or lower affinity) binding activities by repeating the selection protocol described here while selectively blocking EPF-like DNA-binding activity of GST-RB complexes by addition of competitor oligonucleotide DNA (e.g., the consensus class 1 sequence). It is also possible that additional sequence-specific DNA-binding activities might have been missed, if the assay was biased toward detecting E2F recognition sequences by virtue of the linkers employed in this study. Previous experiments in which wild-type or mutant RB pocket-encoding DNAs were cotransfected with appropriate reporter plasmids have suggested that RB might serve, directly or indirectly, to regulate transcription. In particular, Robbins et al. (1990) and Kim et al. (1991) have suggested that RB can regulate the c-fos and c-myc promoters. In this regard, Pietenpol et al. (1990) demonstrated a link between the action of RB and the suppression of c-myc transcription. Thus far, there is no evidence indicating that this regulation is mediated by E2F binding to the relevant promoters. However, if RB regulates the behavior of more than one transcription factor (see above), the aforementioned effects might result from effects of RB separate from its interaction with E2F. RB is not the only pocket-containing protein. There are at least two such proteins, with the recently cloned ~107 T/El A-binding protein representing the second (M. Ewen et al., submitted; Dyson et al., 1989; Ewen et al., 1989). The ~107 sequence contains a ~600 residue colinear seg-

RB Interacts 1081

with a Sequence-Specific

DNA-Binding

Protein

ment bearing major homology to the RB pocket and capable of performing the specific T/ElA/E-/-binding function of this protein. Recent evidence strongly suggests that the ~107 pocket can specifically recognize a family of about seven proteins that also bind to the RB pocket (M. Ewen, W. G. Kaelin, Jr., and D. M. Livingston, unpublished data). A priori, given these clear similarities of structure and function, it seems reasonable to question whether FIB is the sole E2F-binding target, or whether it is the physiological target. Conceivably, ~107 or yet another member of this pocket protein family may constitute the real target. This possibility would not be inconsistent with the evidence that E2F, as defined in adenovirus-infected cells, is activated through an interaction between El A and one or more cellular proteins (Bagchi et al., 1990). This interaction depends on the integrity of EIA domain 2, a segment of the viral protein that interacts specifically with both the RB and ~107 pockets (Dyson et al., 1989; Ewen et al., 1989; M. Ewen et al., submitted). In summary, we have demonstrated that a cellular protein(s) capable of binding specifically to the RB T/ElAbinding region (pocket) possesses sequence-specific DNA-binding activity. Analysis of the preferred recognition sequence for this protein(s) suggests that it is E2F or a protein that has a closely related DNA-binding property. To date, all spontaneously occurring loss-of-function RB mutations compatible with stable protein expression map to the TIElA-binding region (Kaelin et al., 1991). It seems logical, therefore, to propose that in the course of governing cell growth, RB directly modulates the activity of E2F or a related protein. However, we also cannot exclude the possibility that the regulation goes in the opposite direction, with E2F or its relative contributing to the modulation of RB function. Experimental Procedures Binding Site Selection GST-RB fusion proteins were expressed in bacteria transformed with recombinant plasmids generated in a previous study (Kaelin at al., 1991) and purified by incubation with glutathione-Sepharose beads as described previously(Kaelin et al., 1991; Smith and Johnson, 1988). Typically, 200 pl of beads, 1 :l (v/v) in NET-N (20 mM Tris [pH 8.0], 100 mM NaCI. 1 mM EDTA, 0.5% NP-40) supplemented with 0.5% nonfat powdered milk, was used to recover the fusion protein produced from a 200 ml bacterial culture. The beads were washed three times with NET-N and then mixed with whole-cell lysates prepared from WERIFib27 cells as described by Kaelin et al. (1991). The bead complexes were then washed three times with NET-N, resuspended in an equal volume of NET-N (typically 200 PI), and aliquoted (usually 30 to 50 ul) prior to use in binding experiments. For each round of binding site selection, GST-RB beads were prepared as described above and incubated with 4 Bg of poly(dl-dC) and approximately 50 ng of purified 62 bp oligonucleotide DNA in 250 ul of NET-N for 30 min at 4OC with gentle rocking. The beads were then washed four times with 1 ml of ice-cold NET-N buffer. DNA that remained bound to the beads was eluted by incubation for 3 hr at 5O’C in 400 ~1 of proteinase K digestion buffer (500 mM Tris [pH 8.8],20 mM EDTA, 10 mM NaCI, 1% SDS, 200 @g/ml proteinase K), followed by phenol extraction and ethanol precipitation. The DNA was then resuspended iri 20 ~1 of water, and IO PI of this material was used to amplify the 62 bp oligonucleotide by PCR (as described below). Amplified DNA was resolved in 12% TBE-polyacrylamide gels, and the 62 bp reaction product was isolated by excision of the appropriate gel slice. The DNA was then transferred to DEAE-cellulose membranes (Schleicher and Schuell) byelectrophoresis, eluted, and ethanol precipitated. This puri-

fied DNA fragment selection.

was then used

in a subsequent

round of binding/

PCR Amplification of the 62 bp Oligonucleotide PCR was carried out using a Gene-Amp kit (Perkin-Elmer Cetus) following the manufacturer’s procedure. Because of the internal degeneracy of the 62 bp template DNA, an additional step was performed to ensure that the majority of the amplified 62 bp oligonucleotides were perfectly complementary double-stranded molecules (and not heteroduplexes containing mismatched sequences at their random 18 bp core seg ments). Following 30 cycles of amplification, 2 ~1 of a 70 NM solution of each primer and 2.5 U of fresh Taq polymerase were added to the reaction. The reaction mixture was then subjected to one additional denaturationlannealinglextension cycle to permit synthesisof complementary strands. The 62 bp oligonucleotide amplification product was purified on 12% polyacrylamide gels as described above. DNA-Binding and Competition Assays Oligonucleotides were labeled with [y-32P]ATP and T4 polynucleotide kinase to a specific activity of approximately 1 x IO* cpmlwg (Maniatis et al., 1982). GST-RB bead complexes (either with or without prior incubation in WERI-Rb27 cell extract) were prepared as described above. Aliquots (50 ~1) of bead complexes were incubated in 250 pl of NET-N containing 4 Kg/ml poly(dl-dC) and the relevant 32P-labeled oligonucleotide probe (typically 5 x 1 O5 cpm, or about 5 ng). In some cases, unlabeled competitor oligonucleotide DNA was added to the binding reactions just prior to the addition of probe DNA. The binding mixtures were rocked for 30 min at 4“C and then washed four times with 1 ml of cold NET-N. The beads were transferred to clean tubes, and the bound 32P-labeled DNA was measured by Cerenkov counting. Oligonucleotides The sequences of the 62 bp oligonucleotide and the primers used for PCR amplification are shown in Figure I. The additional oligonucleotides used in binding and competition experiments were produced by annealing equimolar amounts of complementary single-stranded synthetic oligonucleotides. The sequence of the plus strand of each oligonucleotide is as follows: C9: 5’-CCATTTTGGCGGGAACTG-3’ Ci8: 5”CATTTGCGCGGGAACT-3’ CM: 5’~CCATTTTGGAGGGAACTG-3’ E2: 5’~CTAGTTTTCGCGCTTAG-3’ E22: 5’7CGTAGTTTTCGCGCTTAAGG-3’ BK: 5’GATCTCGTCGTGCATCTGTTGGATCCCCGGG-3’. DNA Sequencing Oligonucleotide DNA, digested with EcoRl and Xhol, was purified and cloned into EcoRI- and Xhol-digested Bluescript II SK (Stratagene). DNA sequencing of oligonucleotide inserts was performed with a Sequenase 2.0 kit (US Biochemical Corp.) using double-stranded miniprep DNA templates, the T7 sequencing primer, and the protocol provided by the manufacturer. Acknowledgments We are indebted to the members of our research group for many helpful suggestions and discussions. This work was supported by awards to three of us (T. C., W. G. K., and D. M. L.) from the National Cancer Institute. 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 USC Section 1734 solely to indicate this fact. Received

May 21, 1991

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