From the Department of Molecular Genetics,
Biochemistry and Microbiology, University of Cincinnati College of
Medicine, Cincinnati, Ohio 45267 and the § Department of
Biochemistry, Case Western Reserve University,
Cleveland, Ohio 44106
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ABSTRACT |
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The Ski oncoprotein has been shown to bind DNA and activate transcription in conjunction with other cellular factors. Because tumor cells or myogenic cells were used for those studies, it is not clear that those activities of Ski are related to its transforming ability. In this study, we use a nuclear extract of c-ski-transformed cells to identify a specific DNA binding site for Ski with the consensus sequence GTCTAGAC. We demonstrate that both c-Ski and v-Ski in nuclear extracts are components of complexes that bind specifically to this site. By evaluating the features of the sequence that are critical for binding, we show that binding is cooperative. Although Ski cannot bind to this sequence on its own, we use cross-linking with ultraviolet light to show that Ski binds to this site along with several unidentified cellular proteins. Furthermore, we find that Ski represses transcription either through upstream copies of this element or when brought to the promoter by a heterologous DNA binding domain. This is the first demonstration that Ski acts as a repressor rather than an activator and could provide new insights into regulation of gene expression by Ski.
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INTRODUCTION |
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c-Ski is an 84-kDa nuclear protein that has been shown to bind DNA when a complementing activity is supplied by a nuclear extract (1). v-Ski is a truncated form of the c-Ski protein that is missing 20 amino acids from the amino terminus and 292 amino acids from the carboxyl terminus (2-4). This truncation, which removes a carboxyl-terminal dimerization domain, plays no role in the activation of ski as an oncogene (5, 6). Overexpression of either c-ski or v-ski induces transformation in chicken embryo fibroblasts (CEFs)1 and either muscle differentiation or transformation in cultured quail embryo fibroblasts (QEFs), depending on the growth conditions (7, 8). The ability of ski to induce both transformation and muscle differentiation in the same cells (QEFs) is an intriguing paradox and suggests that ski plays a pivotal role in regulating cell growth and differentiation. That such a role for ski is conserved in other organisms is demonstrated by the phenotype of v-ski transgenic mice, which have increased muscle mass caused by hypertrophy of type II fast muscle fibers (9). Ski has been shown to affect the proliferation and differentiation of cells outside of the myogenic lineage as well. For example, v-ski transforms a myeloid-erythroid hematopoietic multipotential progenitor cell from avian bone marrow (10, 11). Recently, mice that are homozygous for a null mutation in the c-ski gene have been generated. These mice die at birth and show a variety of developmental defects, including defective closure of the rostral neural tube and decrease in skeletal muscle mass (12).
Because of its effect on muscle differentiation, upstream regulatory regions of muscle-specific genes were used in some initial studies of Ski's transcriptional regulatory properties. One such study showed that Ski stimulates transcription from the enhancers of both myosin light chain 1/3 and muscle creatine kinase by 2-3-fold in myoblasts in an E-box-dependent fashion (13). However, given the diverse biological consequences of Ski overexpression and loss of function, it is likely that Ski also regulates genes outside the myogenic lineage. Kelder and co-workers (14) have shown that v-Ski, overexpressed in mouse L-cells, activates the SV40, human cytomegalovirus immediate early, and RSV long terminal repeat enhancers (14). In addition, we have shown that Ski can interact with the DNA binding site for the nuclear factor I (NFI) transcription factor family by protein-protein interaction with the NFI protein and potentiate NFI-stimulated transcription of a reporter that has multimerized NFI binding sites upstream of a TATA box element (15).2
Recently, it has become clear that some transcription factors can regulate gene expression by interacting with multicomponent protein complexes (16-18). This allows a single transcription factor to interact with different binding sites, depending on the cellular context. The ability of Ski to affect transcription through E-box elements and to interact with the NFI binding site through interaction with the NFI protein suggests such a mechanism for Ski transcriptional regulation. The interaction of Ski with the NFI binding site was identified by cyclic amplification and selection of targets (CASTing), using a nuclear extract from v-ski-transduced mouse L-cells as the source of Ski protein (15, 19). Those cells are highly transformed, and v-ski overexpression appears to suppress their transformed phenotype.3 In the present work, we describe the identification of a second DNA binding site for the Ski protein that is more likely to be relevant to the process of transformation. For this purpose, we used a nuclear extract from c-ski-transformed CEFs (c-ski-CEFs), because CEFs, unlike L-cells, are transformed by overexpression of c-ski or v-ski. The binding site we have identified using this strategy has the sequence GTCTAGAC. There are no known cellular factors that have been previously characterized as binding to this sequence. We show that both v-Ski and c-Ski bind this element, and we examine the affinity of the interaction. Here we also show that Ski is able to repress transcription through multimerized copies of the GTCTAGAC binding site cloned upstream of a minimal promoter. This is the first demonstration of Ski acting as a repressor rather than an activator and could provide insight into the ability of Ski to induce both transformation and differentiation in cells depending on the cellular environment.
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MATERIALS AND METHODS |
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Cell Culture, Preparation of Nuclear Extracts, and Western Blotting-- Culture and infection of chicken embryo fibroblasts (CEFs) with the replication-competent avian retrovirus, RCASBP, carrying c-ski or v-ski was performed as described previously (7, 20). Nuclear extracts were prepared from normal CEFs or CEFs infected with RCASBPc-ski or RCASBPv-ski retroviruses using the method of Dignam (21). Nuclear extracts were analyzed for expression level and integrity of Ski proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with the anti-Ski monoclonal antibody (mAb), G8 (7), and the chemiluminescent detection system of Tropix. The relative amounts of exogenous or endogenous Ski in the extracts was determined by densitometry of exposed film.
Oligonucleotides--
The oligodeoxynucleotide used for
identification of the c-Ski binding site (CASTing oligonucleotide)
contains a central 22 bases of random sequence flanked by nonrandom
sequence on both sides. The 16-base nonrandom sequence at the 5-end
contains a recognition site for the restriction enzyme
BamHI, and the 14-base nonrandom sequence at the 3
-end
contains a recognition site for PstI. The double-stranded
CASTing oligonucleotide that was used in the first round of binding and
selection was prepared by annealing a 16-base pair primer (RO16-2) to
the 3
-end of the CASTing oligonucleotide and extending with the Klenow
fragment of DNA polymerase I. Oligomers that were selected after each
round of CASTing were amplified using primers RO16 and RO16-2. The
sequences of these oligonucleotides are as follows: CASTing
oligonucleotide, ggcggatccacctaca ... N22 ... tgtgcactgcagtg; RO16, ggcggatccacctaca; RO16-2: gccactgcagtgcaca. The
sequences of oligonucleotides used as probes and competitors in
electrophoretic mobility shift assays (EMSAs) are given in the
figures.
Selection of Ski-bound Oligonucleotides--
Anti-Ski mAb beads
were produced by overnight incubation of Dynal sheep anti-mouse
IgG-coated magnetic beads with either G8 or M6 mAb in antibody buffer
(1 × phosphate-buffered saline, 0.1% Nonidet P-40, 0.02% sodium
azide, and 100 µg/ml bovine serum albumin). Following incubation, the
beads were washed with antibody buffer and resuspended (6.7 × 108 beads/ml) in antibody buffer without Nonidet P-40.
c-ski-CEF nuclear extract (20 µg) was incubated for 20 min
at room temperature with 5 µg of double-stranded CASTing
oligonucleotide and 5 µg of poly(dI-dC) in CASTing buffer (25 mM Hepes, pH 7.5, 100 mM NaCl, 10% glycerol,
0.2 mM EDTA, 0.1% Nonidet P-40, 2 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and
0.5 mM Pefabloc from Boehringer Mannheim). Anti-Ski
mAb-coated beads (200 µl), or sheep -mouse IgG-coated beads (200 µl) for the mock reaction, were equilibrated in CASTing buffer and
then resuspended in a 10-µl volume of this same buffer. The DNA
binding reaction was then added to the beads, and the mixture was
incubated for 1 h at 4 °C on a rotating platform. The beads
were then washed six times with 250 µl of CASTing buffer and
resuspended in 100 µl of PCR buffer. A portion of this suspension (20 µl) was added to a PCR reaction with a total volume of 30 µl and
amplified using Boehringer Mannheim Taq polymerase and the
primers RO16 and RO16-2. To avoid overamplification, triplicate
reactions were prepared and amplified for 10, 15, or 20 cycles (19).
One-third of the amplified material (10 µl) from each reaction was
run on a 12% polyacrylamide gel. The reaction in which amplified
material was first visible on the ethidium bromide-stained gel was
determined, and the remaining 20 µl from this reaction was used as
the starting material for the next round of CASTing. Five additional
rounds of binding, washing, and amplification were performed.
Cloning and Sequencing of Selected Oligonucleotides--
After
the sixth round of CASTing, the amplified oligomers were digested with
BamHI and PstI and cloned into a modified pUC18 vector (15). This modification caused a disruption of the translational reading frame, which rendered the pUC-encoded -peptide of
-galactosidase nonfunctional. Insertion of a single oligomer
fragment restores the reading frame, so that recombinant plasmids
produce blue bacterial colonies on plates containing
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside and
isopropyl
-thiogalactoside (22). Positive (blue) colonies were
picked at random, and the inserted DNA sequenced by the dideoxy chain
termination method (23).
EMSA and Competition Analysis--
32P-Labeled probe
was prepared according to the method of Mertz and Rashtchian (24).
Briefly, oligomer inserts were amplified from pUC18 clones using the
RO16 and RO16-2 primers in the presence of 6 mM dCTP,
dGTP, and dTTP, and 50 µCi of 3000 Ci/mmol
[-32P]dATP in a 20-µl PCR reaction. For the GTCT/2
probe, clone number 6-6 was amplified. A single copy insert was
produced by recombining clones 6-6 and 6-9 at the XbaI site,
which comprises the central six base pairs of the binding site. The
GTCT/1 probe was made by PCR amplification of the resulting single copy
insert. GTCT/2 and GTCT/1 probe sequences are shown in Fig.
2A. GTCT/1.5 probes were made by PCR amplification of the
pUC clones, numbers 6-7 and 6-15, and the sequences are shown in Fig.
2C. PCR labeling reactions were electrophoresed on a 7%
polyacrylamide gel. Probe bands were cut out, and probe was eluted by
soaking in CASTing buffer without glycerol overnight. Final probe
concentration was approximately 2 fmol/µl with a specific activity of
5 × 107 cpm/pmol.
Plasmid Construction--
The tkCAT reporter construct was a
gift from H. L. Grimes and P. N. Tsichlis (25) and contains
the fragment of the herpes simplex virus thymidine kinase promoter from
105 to +51. Oligonucleotides complementary to the GTCT/2, GTCT/1, and
mutGC/2 oligonucleotides shown in Fig. 4D were synthesized
such that the annealed oligonucleotides would have a
BamHI-compatible overhang at one end and a
BglII-compatible overhang at the other end. The
double-stranded binding site oligonucleotides were then cloned into the
BamHI to BglII-digested tkCAT reporter plasmid,
placing the binding site immediately upstream of the TK promoter.
Reporter Gene Assays-- The UMN-SAH/DF#1 chicken fibroblast cell line was a gift from D. Foster of the University of Minnesota. These cells were seeded at a density of 2.5 × 105 cells/35-mm plate in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Cells were cultured overnight prior to transfection. Triplicate or duplicate plates were co-transfected with each combination of CAT reporter (600 ng) and the indicated amounts of ski expression plasmid. RSVPL or pUC18 plasmid DNA was added, as indicated in the figure legends, so that the total amount of ski-containing plus empty vector DNA was always 600 ng. The DOTAP liposomal transfection reagent was used according to the manufacturer's directions (Boehringer Mannheim). The DOTAP-DNA mixture was left on the cells for 12 h, at which time they were washed and refed. Cells were harvested 60-72 h after transfection. For CAT assays, cells were harvested and lysates were prepared as described previously (15). CAT assays were performed by the liquid scintillation method as described previously (15, 30). For luciferase assays, lysates were prepared and assayed for luciferase activity using the Promega luciferase assay system according to the manufacturer's directions. The protein content of each lysate was determined by the Bio-Rad protein assay, and these values were used to normalize the CAT and luciferase activity data. In addition, at least two different DNA preparations were tested for all expression plasmids and reporters. We did not employ an internal control for transfection efficiency, because our earlier work had shown that Ski activates expression of all the commonly used control plasmids.
Cross-linking and Immunoprecipitation of Ski DNA Binding Complex-- The probe for DNA-protein cross-linking was prepared by annealing an excess of GTCTX primer (TGCTAGTCTAGAC) to 5 pmol of GTCT/2 binding site oligonucleotide (Fig. 5A) and extending with the Klenow fragment of DNA polymerase I for 30 min at room temperature in the presence of 20 µM BrdUTP, 20 µM dTTP, 200 µM dGTP, 200 µM dCTP, and 85 µCi of 3000 Ci/mmol [32P]dATP. Because the GTCTX primer anneals to one of the GTCTAGAC elements, bromodeoxyuridine (BrdUrd) is incorporated into the complementary strand of only a single GTCT element. The filled in probe was purified by electrophoresis on a 7% polyacrylamide gel as described above for EMSA probes. The specific activity of the resulting probe is approximately 1 × 107 cpm/pmol.
Probe binding reactions were carried out the same as for EMSA except that final probe concentration was 2.5 nM. Samples were transferred to a 96-U-bottom-well microassay plate for incubation. After incubation, this plate was placed on ice and irradiated with ultraviolet light at a distance of 10 cm for 5 min. in a UV Stratalinker 1800 (Stratagene). SDS-loading buffer was added to some samples, which were boiled and loaded directly onto a 7% SDS-polyacrylamide gel. For immunoprecipitations, five 20-µl cross-linking reactions were pooled, diluted 1:3, and adjusted to either high stringency buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.5% SDS, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride) or low stringency buffer (20 mM Hepes, pH 7.9, 100 mM NaCl, 0.5% Nonidet P-40, 10% glycerol). 1 µl of G8 or M6 mAb ascites or 1 µl of the polyclonal antibody 32360 (a gift from S. Hughes at NCI-Frederick Cancer Research and Development Center), which reacts with full-length c-Ski but not with v-Ski4 was added to the diluted cross-linking reaction. These reactions were incubated on ice for 1 h, 50 µl of a 1:1 protein A-agarose slurry was added, and incubation was continued for an additional 1 h at 4° on a rotating platform. The beads were then washed 5 times with 1 ml of high stringency buffer or low stringency buffer and resuspended in 60 µl of SDS-loading buffer. Samples were boiled and centrifuged, and the bead supernatant was loaded onto the same gel along with the samples that were not immunoprecipitated. ![]() |
RESULTS |
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In Vitro Selection of a DNA Binding Site Using a Nuclear Extract of c-ski-CEFs-- Nuclear extracts from CEFs infected by a c-ski retrovirus (RCASc-ski29) were used as the source of Ski protein for binding site selection. DNA oligomers bound by Ski-containing complexes were purified using magnetic beads coated with the anti-Ski mAbs, G8 and M6. As a negative control, magnetic beads coated with sheep anti-mouse IgG were used in a parallel selection.
Starting with a 54-base pair double-stranded oligomer containing a central 22 base pairs of degenerate sequence (see "Materials and Methods"), six cycles of DNA binding, antibody purification, and amplification were carried out. The population of oligomers purified with G8 or M6 mAbs was cloned, and randomly selected clones were sequenced. The resulting sequences (Fig. 1, A and B, indicated by 6-N) revealed a very strong consensus for the dyad symmetrical sequence GTCTAGAC; 24 out of 29 selected oligomers contain this sequence, and among these, 18 carry two tandem repeats of the sequence (Fig. 1A). This 16-base consensus can be extended further to include an A at the 3
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Protein Complexes in the c-ski-CEF Extract Show Specific Interaction with the GTCTAGAC Binding Site-- The complexes that form with the GTCT/2 and GTCT/1 probes and c-ski-CEF nuclear extract are compared by EMSA in Fig. 2A. The GTCT/2 probe yields four shifted bands (Fig. 2A, lanes 1-8). Two of these (labeled 1 and 2) can be competed efficiently with an excess of unlabeled GTCT/2 (Fig. 2A, lanes 3-5). A third band (complex X), which is also competed efficiently, overlaps with a nonspecific complex on this gel (Fig. 2A, lanes 3-5). The fourth and fastest migrating band (complex Y) is competed with equal efficiency by both specific and nonspecific competitor and is not reproducible (Fig. 2A and data not shown). Unlabeled GTCT/1 also competes for the formation of complexes 1, 2, and X although less efficiently than GTCT/2 (Fig. 2A, lanes 6-8). The GTCT/1 probe yields only one specific shifted band, which corresponds in mobility to complex 1 produced by the GTCT/2 probe (Fig. 2A, lanes 9-16). This suggests that complex 1 formed with the GTCT/2 probe results from binding of a protein complex to only one of the two tandem 8-base pair elements, whereas complex 2 results from the binding of a protein complex to both copies of the GTCT element. The fact that the GTCT/1 band and complex 1 with GTCT/2 are much fainter than the complex 2 band with the GTCT/2 probe suggests that cooperativity plays a role in the binding of this complex to the tandem GTCTAGAC element.
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Ski is Part of a Complex That Binds to the GTCTAGAC Element-- To determine if Ski protein participates in the specific GTCTAGAC binding, antibody supershifts were performed (Fig. 2, B, C, and D). Both complexes 1 and 2 produced by nuclear extract from c-ski-CEFs are supershifted by anti-Ski mAbs, G8 or M6 (Fig. 2B, lanes 1-3). On this gel, and most others, complex X is resolved into at least two bands (Fig. 2B, lanes 1-9) that are specifically competed by cold GTCT competitor (data not shown). Complex X (seen more clearly in lanes 4-9) is not supershifted by the anti-Ski mAbs and must contain proteins other than Ski that bind specifically to the GTCTAGAC sequence.
To determine whether endogenous c-Ski binds the GTCT element, EMSAs were performed with a nuclear extract from normal CEFs. In this case, the GTCT/2 probe produces a single complex that is supershifted by both anti-Ski monoclonal antibodies (Fig. 2B, lanes 4-6 and lanes 7-9). This complex is much fainter than either of the complexes formed with extract from RCASc-ski-transformed CEFs and can be seen more clearly after a longer exposure (Fig. 2B, lanes 7-9). The endogenous complex has a mobility that is intermediate between the two c-ski-CEF complexes (compare complex 1 and the location of asterisks). Endogenous Ski from mouse and human cell lines also binds the GTCTAGAC element (data not shown). The specificity of the G8 mAb supershift is demonstrated in Fig. 2D. These results show that the supershift formed with G8 mAb is eliminated by adding GST-
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Competition Analysis with GTCT/1.5 Sites and GTCT/2 Site Mutants-- The results presented above indicate that binding to the GTCT element is dependent on the number of sites and their divergence from the consensus. To obtain a more quantitative measurement of the effects of these variables on binding, we performed competition experiments (Fig. 4, A-D). In the first experiment, EMSAs were performed using unlabeled GTCT/2, GTCT/1, or GTCT/1.5 oligomers to compete for binding to GTCT/2 (Fig. 4, A and B). The amount of complexes 1 and 2 was quantitated and is presented graphically in Fig. 4A and summarized as relative amount required for 50% competition (I0.5) in Fig. 4B. The results indicate that GTCT/1 competes approximately 100-fold less efficiently for binding than GTCT/2. Among the GTCT/1.5 elements, the two with the partial site sequence TGTCTGGNC (6-7 and 6-12) are similar to GTCT/2 in their competition efficiencies. The loss of a single match relative to this sequence (the C8 residue) produces oligonucleotides that match the partial site consensus TGTCTGG (6-9 and 6-21) but results in a 5-fold decrease in competition efficiency. As indicated by comparisons of 6-9 with 6-21 and of 6-12 with 6-7, oligonucleotides with one-nucleotide or three-nucleotide spacers between the two copies of the binding site have the same relative binding affinity.
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Ski Can Be Cross-linked to the GTCT Binding Site Along with Two Other Proteins-- Irradiation of protein-DNA complexes with UV light causes covalent bonds to form between the DNA and proteins that are in close contact with the DNA (31). This reaction occurs more efficiently in the presence of halogenated analogs of thymidine, such as BrdUrd. If the DNA probe is labeled with 32P, the label will be covalently attached to the cross-linked protein, so that components of the binding complex can then be separated by SDS-PAGE, and cross-linked proteins can be visualized by autoradiography (32, 33). We used this method to analyze the components of the Ski-GTCT binding complex in c-ski-CEFs and to determine if Ski contributes directly to DNA binding.
The probe used for this experiment was produced by priming DNA synthesis from a GTCT/2 template using a complementary 32P-labeled GTCT/1 primer and a dNTP mix containing equal concentrations of dTTP and BrdUTP (Fig. 5A). Because there are only two positions for T or BrdUrd in the single synthesized GTCT, this method provides for the incorporation of one BrdUrd per GTCT/2 probe molecule. Consequently, only one polypeptide should be cross-linked per probe molecule, thereby allowing us to enumerate the polypeptides bound to the GTCT element. After binding and UV irradiation, the cross-linked products were analyzed by SDS-PAGE. At least five proteins appear to cross-link specifically to the probe, as shown by their disappearance in the presence of unlabeled specific competitor, but not in the presence of mutant competitor (Fig. 5B, lanes 5-7). The largest cross-linked species, which is marked by a closed arrow in Fig. 5B, migrates more slowly than in vitro translated c-Ski (Fig. 5B, lane 3) but could represent c-Ski protein, which has reduced mobility due to the added mass of the cross-linked probe. This is apparently the case, because this is the only protein detected following immunoprecipitation of the cross-linked complexes in high stringency buffer (see "Materials and Methods") with three different Ski-specific antibodies (Fig. 5B, lanes 8, 10, and 12) but not with preimmune serum or protein A-agarose alone (Fig. 5B, lanes 14 and 16).
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The GTCTAGAC Element Mediates Transcriptional Repression by Ski-- To determine if Ski can regulate transcription through the GTCTAGAC binding site, reporters were made in which copies of the binding site were cloned upstream of the herpes simplex virus thymidine kinase promoter (tkCAT). Co-transfection with the RSVc-ski expression plasmid into the chicken fibroblast cell line, UMN-SAH/DF#1, represses these reporters in proportion to the number of GTCT binding sites cloned upstream of the promoter (Fig. 6). Reporters with one, two, and four GTCT/1 sites are repressed 8-, 10-, and 12-fold respectively, and a reporter with two GTCT/2 sites is repressed 19-fold. There was some variation in the basal activity of the different reporters from which the values of -fold repression were calculated, but these differences do not correlate with the number of binding sites or the degree of repression observed in the presence of Ski. In addition, we consistently observe that c-Ski represses reporters with no binding sites or with mutant binding sites no more than 3-4-fold. This same experiment was done in primary CEFs and in the liver hepatoma cell line, HepG2, with similar results.
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DISCUSSION |
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It is now clear that protein-protein interactions among DNA binding transcription factors play important roles in modulating both the DNA binding properties and transcriptional regulatory properties of such factors (16-18). Carrying out binding site selection using a nuclear extract as the source of protein has the advantage that the transcription factor can interact with a given DNA binding site while maintaining many of the same protein-protein interactions that would occur inside the cell. Using this approach, we have identified a novel DNA binding site for the Ski protein with the consensus sequence GTCTAGAC. Exogenous c-Ski in CEF nuclear extracts binds to this sequence with high affinity and specificity. v-Ski also binds to this binding site, but with lower affinity than c-Ski, probably due to the loss of the carboxyl-terminal dimerization domain. Furthermore, endogenous Ski proteins from chicken, mouse, and human all bind to this GTCT element, suggesting that binding to this site may be important for the normal function of Ski.
Structure of Binding Site and Analysis of Binding Site Mutants-- The GTCT consensus is extraordinarily conserved in the oligomers that we identified by CASTing. There is only a single base change in one of the 20 GTCT/2 oligomers, and none of the GTCT/1.5 oligomers had any substitutions in the first copy of the consensus. We were surprised that none of the binding sites contained two imperfect copies of the GTCT element. The reason for this observation became apparent when we analyzed binding affinities by competition. All of the symmetrical single base substitutions that we made in the GTCT/2 binding site dramatically reduced the binding affinity, such that it was equivalent to or less than that of a single copy binding site. On the other hand, GTCT/1.5 sites with 0-3 base pairs between GTCT elements and two or more base substitutions in one element have only slightly lower binding affinity than GTCT/2. This demonstrates that at least one perfect copy of the binding site is essential for stable Ski complex formation and provides strong evidence for cooperative binding to multiple GTCT elements.
Composition of the Two Ski DNA Binding Complexes-- Neither purified bacterially produced Ski nor in vitro translated Ski binds the GTCT element. Possible explanations for this failure include the following: 1) bacterially produced and in vitro translated Ski protein may not fold properly; 2) Ski may contain an autoinhibitory domain that prevents DNA binding; 3) post-translational modification such as phosphorylation may be required; 4) a protein cofactor may be required to stabilize Ski's interaction with the DNA. The first explanation is unlikely because bacterially produced and in vitro translated Ski proteins have been shown to dimerize efficiently, and analysis of in vitro translated Ski by partial proteolytic digestion indicates that it is correctly folded (29). Autoinhibitory domains have been shown to regulate DNA binding of such transcription factors as ETS-1 and p53; however, in these cases the inhibition of DNA binding is relieved by deletion of the autoinhibitory domains (36-38). We have performed EMSAs with various deleted forms of in vitro translated Ski, but no deletions have been identified that enable the Ski protein to bind the GTCT element (data not shown). Previous work by Sutrave et al. (20) has shown that chicken c-Ski is a phosphoprotein, whereas v-Ski is not. Because both c-Ski and v-Ski can bind to DNA, it is unlikely that phosphorylation is required for DNA binding of chicken Ski proteins. Ishii and co-workers (1) have presented evidence indicating that Ski requires a cofactor present in a nuclear extract from Molt 4 cells to bind DNA. The Ski homologue, SnoN, which has been shown to heterodimerize efficiently with Ski (5, 6) is an obvious candidate for a Ski-GTCT co-binding partner. For this reason, we performed EMSAs with Ski and Sno heterodimers formed by co-in vitro translation, but no binding to the GTCT site was detected (data not shown).
In this study, we provide evidence for additional factors that interact specifically with the GTCT element. The non-Ski-containing complex X, which binds GTCT/2 specifically, migrates faster than Ski-containing complexes in EMSAs. It is possible that Ski associates with the components of this complex to form a ternary complex with further reduced mobility. This would account for the reduced level of complex X in c-ski-CEFs relative to normal CEFs. Interestingly, there is more complex X visible in gel shifts with v-ski-CEF extract than with c-ski-CEF extract, suggesting that v-Ski might form an unstable association with complex X. It is possible that we have identified the components of complex X as the four species (two sets of doublets) that cross-link specifically to the BrdUrd-substituted probe in addition to Ski. The fact that Ski can be UV-cross-linked to a GTCT probe indicates that it probably contains a domain that makes contacts within the Van der Waals radius of the DNA (31). There are a number of mechanisms by which Ski could co-bind to the GTCT site along with other proteins and still play a role in specific sequence recognition. For example, another protein could bind in an overlapping complex with the Ski protein and stabilize Ski binding by cooperative interaction. The yeast proteins Mcm1 and MATRole of Ski Dimerization in Cooperative Binding to the GTCT Element-- Exogenous c-Ski forms two complexes with GTCT/2 and GTCT1.5 probes (complex 1 and complex 2). The faster migrating form has the same mobility as the single complex that forms with the GTCT/1 probe. Since Ski is present in both complexes 1 and 2, the simplest model for the composition of these complexes is that complex 1 represents a Ski monomer plus associated proteins bound to a single GTCT site and complex 2 is a dimer of these elements. This suggestion fits nicely with the observations that binding of Ski to the GTCT/2 element is highly cooperative and that c-Ski dimerizes efficiently through a carboxyl-terminal dimerization domain (5, 6). This model also agrees with the observation that v-Ski, which lacks this high affinity dimerization domain, binds to the GTCT/2 site much less efficiently than c-Ski. On the other hand, it predicts that the two Ski forms should be very similar in binding to GTCT/1, because in this case dimers would not be required. However, we investigated binding of v-Ski to the GTCT/1 element and were unable to detect the formation of a shifted band by EMSA.
The weak binding of v-Ski to GTCT/2 and its failure to bind GTCT/1 suggest that dimerization of Ski is important for binding to both single copy and double copy binding sites. This conclusion is contrary to the model described above and provides support for an alternative model in which a Ski dimer and associated proteins bind to each copy of the GTCT element. Cooperative binding to the GTCT/2 site then occurs by interaction between adjacent Ski dimers bound to DNA. Because the rate-limiting step in cooperative binding is the first step, the carboxyl-terminal dimerization domain of c-Ski greatly enhances cooperative binding by stabilizing dimers so that they can bind effectively to a single element. Accordingly, v-Ski is impaired in binding GTCT/1 because its dimers are unstable, but it is able to bind GTCT/2 because the cooperative interaction on DNA stabilizes two weakly associated dimers. This model is consistent with our previous data that demonstrated low affinity multimer formation by v-Ski (5).Ski Represses Transcription through the GTCT Binding Site-- Here we demonstrate for the first time that both c-Ski and v-Ski can repress transcription. c-Ski is shown to be a stronger repressor than v-Ski, and this difference is consistent with their relative abilities to bind the GTCT element and is mirrored by their relative transforming activity. Repression by Ski cannot be explained by competition with an activator for binding to the GTCT site, because c-Ski linked to an unrelated DNA binding domain is able to repress transcription of a cognate reporter. Therefore, Ski can be classified as an active repressor (35). Mechanisms of active repression include interfering with the activity of a DNA-bound activator (44) and interacting with the basal transcription machinery (45) or with histone deacetylases (46). Often these interactions are mediated by corepressors, as in the case of thyroid hormone receptor and the Krüppel-associated box domain-containing proteins (17, 47). Since the minimum transforming region of Ski has been localized to the N-terminal 304 residues (29), it will be interesting to determine whether this region contains the repression domain.
In light of the fact that Ski can activate transcription from some muscle-specific and viral enhancers and promoters, it was surprising to find that Ski represses transcription through the GTCT binding site. However, there are now numerous examples of transcription factors that can act as either activators or repressors depending on protein-protein interactions dictated by the promoter and physiological context (48). Although Ski appears to activate transcription by interacting with other DNA-bound transcription factors, including the myogenic regulatory factors (13) and members of the NFI family (15),2 we have been unable to identify an activation domain by fusing various regions of Ski with an independent DNA binding domain (data not shown). In the case of repression through binding to the GTCT binding site, Ski's action appears to be different. Although Ski requires cofactors to bind to this sequence, it appears to possess a repression domain that can function independently of these proteins. However, further characterization of the factors that co-bind to the GTCT site along with Ski will be required to determine the role that Ski plays in repressing transcription through this binding site. The ability of Ski to cause oncogenic transformation and induce terminal muscle differentiation in the same cells has been a paradox. Previous results showing transcriptional activation by Ski are now contrasted by our findings that it can act as a transcriptional repressor. It is interesting that the duality inherent in the biological activities of the Ski protein is thereby reflected at the transcriptional level. This makes it tempting to speculate that the opposite biological activities are coupled to the opposite transcriptional activities. However, this will remain only an attractive speculation until the key genes activated and repressed by Ski are identified. ![]() |
ACKNOWLEDGEMENTS |
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We thank P. Tarapore for the contribution of the plasmids RSVPL, RSVc-ski, RSVv-ski, and SG424c-ski and S. B. Cohen for the plasmid pGL3.2G5TKLuc. We are grateful to D. Foster of the University of Minnesota for the UMN-SAH/DF#1 chicken fibroblast cell line, to H. L. Grimes and P. N. Tsichlis of Fox Chase Cancer Center for the TKCAT reporter plasmid, and to S. H. Hughes of NCI-Frederick Cancer Research and Development Center for the anti-Ski polyclonal antibody 32360. We also thank G. Zheng and S. B. Cohen for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by NCI, National Institutes of Health, Grant CA-43600 (to E. S.) and by a fellowship from the Albert J. Ryan Foundation (to R. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: 10900 Euclid Ave., Cleveland OH, 44106-4935. Tel.: 216-368-3353; Fax: 216-368-3419; E-mail: exs44{at}po.cwru.edu.
1 The abbreviations used are: CEF, chicken embryo fibroblast; QEF, quail embryo fibroblast; RCASBP, replication-competent avian retrovirus containing Bryan high titer polymerase; RSV, Rous sarcoma virus; TK, thymidine kinase; NFI, nuclear factor I; CASTing, cyclic amplification and selection of targets; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; BrdUrd, bromodeoxyuridine; PCR, polymerase chain reaction.
2 P. Tarapore, H. C. Heyman, R. A. W. Rupp, U. Kruse, A. E. Sippel, and E. Stavnezer, manuscript in preparation.
3 B. Kelder and J. Kopchick, manuscript in preparation.
4 D. Shardy and E. Stavnezer, unpublished results.
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