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Volume 270, Number 26, Issue of June 30, pp. 15815-15820, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A cis-Acting Element in Rous Sarcoma Virus Long Terminal Repeat Required for Promoter Repression by HeLa Nuclear Protein p21 (*)

Chen-Hsiung Yeh , Aaron J. Shatkin (§)

From the (1)Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

HeLa cell basic nuclear protein (p21), which represses Rous sarcoma virus long terminal repeat (RSV LTR) promoter activity, diminished v-src expression and the appearance at permissive temperature of the transformed phenotype in tsRSVLA23 Rat-1, a cell line transformed with a temperature-sensitive mutant of RSV. Nuclear run-on analyses using COS-1 cells cotransfected with p21 cDNA and chloramphenicol acetyltransferase reporter indicated that p21 inhibits transcription initiation by targeting a region in the RSV LTR promoter between positions -108 and -85 upstream of the cap site. Insertion of this 24-base pair sequence in place of one of the 72-base pair enhancers in the SV40 early promoter rendered it sensitive to p21 repression. Electrophoretic mobility shift assays using a synthetic oligomer corresponding to the 24-base pair LTR promoter element revealed that p21 altered the pattern of proteinDNA complex formation apparently without binding DNA directly. Complex formation assayed by UV cross-linking and DNA affinity chromatography indicated further that a cellular factor which can interact with this element was decreased in cells transfected with p21 expression plasmid. The results indicate that p21 repression of RSV LTR is mediated by a cis-acting element and may occur by alteration of protein complexes formed on this promoter element.

INTRODUCTION

Rous sarcoma virus (RSV)()has become adept at utilizing the cellular regulatory machinery for viral gene expression. The long terminal repeat (LTR) contains an array of strong enhancers that are used for high-level expression from the viral promoter. The LTR can also augment transcription from a number of heterologous promoters in a wide variety of cell types(1, 2, 3) , suggesting that the molecular mechanisms responsible for LTR-mediated transcription activation apply generally.

Results from previous studies have identified cis-acting elements in the RSV LTR that are important for up-regulating the level of viral transcription, including at least two enhancer domains that can interact with several cellular factors(4, 5, 6) . One of these domains lies between the LTR 5` end and nucleotide -137 (transcription start site at +1), whereas the second enhancer is between nucleotides -137 and -54(7) . Regulatory DNA elements within these regions include CCAAT motifs, two CArG boxes and sequences homologous to the SV40 and adenovirus E1A enhancer cores(4, 5, 6, 7, 8) . Enhancer activity apparently requires binding of cellular trans-acting proteins to specific domains(9) . Several of these activating proteins have been identified, purified and, in some cases, cloned(10) . Although both cis-acting functional elements and corresponding DNA binding factors have been identified, the precise molecular mechanisms of regulated RSV LTR promoter activity have not been fully characterized.

Our earlier results, based on transient cotransfection assays, indicated that HeLa cells code for a TFIIS-related nuclear protein, p21 which can repress RSV LTR promoter activity(11) . The regions in the 157-amino acid polypeptide necessary for this activity were mapped to an Arg/Ser-rich domain (amino acids 12-49) and a zinc finger-like motif within amino acids 50-100(12) , but the mechanism of p21 repression of RSV LTR-directed transcription was not explored. To gain a better insight into the molecular basis of p21 function and to define the contribution of specific LTR region(s) to the repression effects of p21, a series of 5`-truncation mutants of the RSV LTR have been tested for expression in cotransfection experiments. A 24-base pair cis-acting element required for p21 inhibition has been identified in the LTR between positions -108 and -85. Although p21 apparently does not bind DNA directly, a cellular factor which binds to the corresponding synthetic 24-mer has been detected in COS cell nuclear extracts and is decreased by p21 expression. The results suggest that p21 represses transcription from the RSV LTR by targeting the cellular factor(s) that interact with a cis-acting element in the promoter.

MATERIALS AND METHODS

Cell Culture, DNA Transfection, and CAT Assay

Rat-1 tsRSVLA23 (13) and COS-1 cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal calf serum. Transfection of Rat-1 tsRSVLA23 cells was performed by a calcium phosphate precipitation technique(14) . DNA mixtures in 0.5 ml of 0.125 M CaCl containing 1 µg of pcDNA3/neo and either 10 µg of parental vector pBC12BI or 10 µg of plasmid for expression of HA-tagged p21(11) , referred to as p21, were added dropwise to 0.5 ml of 2 HBS buffer (280 mM NaCl, 1.5 mM NaHPO7HO, 50 mM HEPES) and incubated for 30 min at room temperature. The resulting calcium phosphate-DNA precipitates were then added directly to 100-mm tissue culture dishes containing Rat-1 tsRSVLA23 cells that had been plated at 1 10 cells/dish in Dulbecco's modified Eagle's medium 24 h earlier. After 2 weeks of drug selection at 37 °C with G418 (200 µg/ml), clonal lines were isolated and expanded.

CAT reporter plasmids pR-CAT and pSV2CAT and their mutant derivatives were cotransfected into COS-1 cells with the indicated amounts of pBC12BI or p21 expression plasmid by the DEAE-dextran method. Cell extracts prepared 48 h later were assayed for CAT protein levels, all as described previously(11) .

Plasmid Constructions

Parental vector pBC12BI and plasmids for expression of p21 and a p21 deletion mutant pd50-100 (both HA-tagged) have been described(11) . Reporter plasmid pR-CAT/-141 was constructed by SphI digestion and religation of pR-CAT. A set of 5` end-nested deletion derivatives (pR-CAT/-120, pR-CAT/-108, and pR-CAT/-85) was also constructed using SphI-digested pR-CAT that was treated with Bal-31 nuclease before religation. Deletion breakpoints of these derivatives were determined by nucleotide sequencing to be at positions -141, -120, -108, and -85.

pSV2CAT/d72, made by digestion of pSV2CAT with SphI and religation, was used to construct pSV2CAT/RSV-108/-851 and pSV2CAT/RSV-108/-853. They contain blunt-ended single copy and three head-to-tail copies, respectively, of chemically synthesized RSV-108/-85 oligodeoxynucleotide inserted into the blunt-ended SphI site of pSV2CAT/d72. The number and orientation of the oligonucleotide copies were determined by sequencing.

Nuclear Run-on Analysis

COS-1 cells transfected with parental vector pBC12BI or p21 expression plasmid were collected 48 h post-transfection, washed two times, and harvested in ice-cold phosphate-buffered saline. After centrifugation, cell pellets were resuspended in lysis buffer (10 mM Tris, pH 7.4, 3 mM CaCl, 2 mM MgCl and 0.5% Nonidet P-40), disrupted in a Dounce homogenizer, and nuclei were sedimented for 10 min at 1,000 g. Approximately 5 10 nuclei were resuspended in 0.2 ml of 50 mM Tris-HCl, pH 8.3, containing 40% glycerol, 5 mM MgCl, and 0.1 mM EDTA and stored at -70 °C. Transcription reactions were started by thawing 0.2 ml of frozen nuclei and mixing with 0.2 ml of 2 reaction buffer containing 10 mM Tris-HCl, pH 8.0, 5 mM MgCl, 0.3 M KCl, 5 mM dithiothreitol, 1 mM each of ATP, GTP, and CTP, and 10 µl of [-P]UTP (3,000 Ci/mmol, Amersham Corp.)(15) . Radiolabeled RNA products were isolated and hybridized for 36 h at 65 °C to dot blots containing 5 µg of the indicated denatured plasmid DNAs, followed by sequential washing with 2 SSC for 1 h at 65 °C, 2 SSC in the presence of RNase A (10 mg/ml, Sigma) for 30 min at 37 °C, and 2 SSC for 1 h at 37 °C before autoradiography.

Mobility Shift DNA Binding Assay

Two complementary overlapping synthetic oligonucleotides 5`-CGTGCCTTATTAGGAAGGCAACAG-3` and 5`-CACGCTGTTGCCTTCCTAATAAGG-3` corresponding to position -108 to -85 upstream of the RSV LTR transcription start site were annealed (RSV-108/-85). The double-stranded probe was end-labeled with Klenow polymerase and [-P]dCTP (3,000 Ci/mmol; Amersham). Nuclear extracts (10 µg of protein) prepared as described (16) from pBC12BI- or p21 expression plasmid-transfected COS-1 cells were incubated with 10,000 cpm (1 ng) of P-labeled oligonucleotide probe and 1 µg of poly(dI-dC)poly(dI-dC) in 15 µl of binding buffer (10 mM HEPES, pH 7.9, 5 mM MgCl, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.05% Nonidet P-40, 12% glycerol) for 30 min at room temperature. To assay for specificity, unlabeled RSV-108/-85 or the unrelated double-stranded oligonucleotide (5`-ACCAACAAGCGCACCCGCGGCCCC-3`) was included in binding reactions. ProteinDNA complexes were resolved by electrophoresis for 2.5 h on a 4% polyacrylamide gel in Tris glycine high ionic strength buffer(17) . Under these conditions designed to increase complex resolution, free probe migrated out of the gel.

UV Cross-linking

ProteinDNA binding reactions were performed in 96-well plates under the same conditions used above for gel shift assays. Immediately following the 30-min incubation, the plates were UV-irradiated with 0.8 J/s for 10 min in a Stratalinker (Stratagene). Samples were mixed with an equal volume of 2 SDS loading buffer, boiled, and applied on a 10% polyacrylamide-SDS gel. UV-cross-linked proteinDNA complexes were detected by autoradiography and quantitated by PhosphorImager (Molecular Dynamics).

DNA Affinity Chromatography

Chemically synthesized complementary oligodeoxynucleotides were annealed, 5`-phosphorylated, and ligated as described(18) . The ligated DNA was added to CNBr-activated Sepharose 4B (Pharmacia Biotech Inc.) that had been extensively prewashed with 1 mM HCl and resuspended in coupling buffer containing 0.1 M NaHCO, pH 8.3. The coupling reaction was carried out at room temperature for 16 h on a rotary shaker, and the resin was then washed with at least 5 bed volumes of coupling buffer to remove excess DNA. The remaining active groups were blocked by transferring the gel to 1 M ethanolamine, pH 8.0, and letting it stand for 2 h. The resin was then washed sequentially with 10 ml each of 1 M potassium phosphate, pH 8.0; 1 M KCl; HO; 10 mM Tris-HCl, pH 7.6, containing 0.3 M NaCl, 1 mM EDTA, and 0.02% (w/v) NaN before storage at 4 °C in the same Tris buffer. The efficiency of DNA attachment to the resin was 40-60%, and the concentration of covalently bound DNA measured by A was 20-30 µg/ml of resin.

DNA affinity resin (0.2 ml) was equilibrated in a Bio-Rad Econo-Column with DNA binding buffer consisting of 25 mM HEPES, pH 7.8, 60 mM KCl, 1 mM EDTA, 12.5 mM MgCl 1 mM dithiothreitol, 20% glycerol, and 0.05% Nonidet P-40. Nuclear extract (0.8-1.0 mg of total protein) prepared from approximately 10 COS-1 cells transfected with parental vector or p21 expression plasmid was mixed with 40-60 µg of double-stranded poly(dI-dC)poly(dI-dC) in a total volume of 0.5 ml of DNA binding buffer and allowed to stand for 20 min to reduce nonspecific DNA binding. The proteinDNA solution was loaded onto the affinity resin which was then mixed and incubated for another 20 min at room temperature before gravity flow through the column. The column was then washed three times with 0.5 ml of DNA binding buffer containing 0.1 M KCl. Finally, DNA binding buffer containing 1.0 M KCl was added to the column, and the resin was thoroughly mixed and allowed to stand for 15 min before collecting the eluate for analysis by 12% SDS-PAGE and silver staining.

RESULTS

p21 Expression Reverses the RSV-mediated Transformed Phenotype in Rat-1 Cells

Expression of HeLa cell protein p21 represses RSV LTR promoter activity and also inhibits RSV transformation of primary chick embryo fibroblasts(11) . To test the effect of p21 on mammalian cells, we used a Rat-1 cell line (LA23) which is stably transfected with a temperature-sensitive (ts) RSV mutant. Shift from the restrictive (39.5 °C) to the permissive (34 °C) temperature results in the appearance of the transformed phenotype due to activation of v-src(13) . LA23 cells were cotransfected with a neomycin resistance gene and p21 expression plasmid or the corresponding parental vector pBC12BI, and lines were selected and grown in the presence of G418. Analysis of the resulting neo-resistant lines at 34 °C demonstrated that parental vector-transfected cells were refractile and formed disordered multiple cell layers characteristic of the transformed phenotype (Fig. 1). By contrast, p21-transfected cells grew as a more flat, contact-inhibited monolayer. At 37 °C, pBC12BI-transfected cells tended to form foci at a subconfluent density, whereas p21-expressing cells grew as a flat monolayer. Both cell lines displayed similar adherent, flat morphology when shifted to the restrictive temperature of 39.5 °C. Western blot analyses demonstrated p21 expression in p21-transfected cells grown at all three temperatures (Fig. 1, p21, lanes 4-6), although partially decreased (60%) at 39.5 °C (lane 6). v-src was expressed at 34 °C and 37 °C (v-src, lanes 1 and 2) but was almost undetectable at the restrictive temperature (lane 3) in pBC12BI-transfected cells and in p21-expressing cells at all three temperatures (lanes 4-6). These results demonstrate that overexpression of p21 in mammalian cells inhibits v-src expression and suppresses the RSV-transformed phenotype, consistent with inhibition by p21 of avian cell transformation by RSV(11) .

Figure 1: Effect of p21 expression on the morphology of Rat-1 cells transformed by a temperature-sensitive mutant of RSV. The (ts)RSVLA23 Rat-1 cell line was transfected with p21 expression plasmid (right) or parental vector pBC12BI (left), and stable cell lines were obtained as described under ``Materials and Methods.'' Cells were plated at 5 10/60-mm dish and examined after 2 days at 39.5 °C or 1 day at 37 or 34 °C. p21 and v-src expression was assayed in extracts prepared from the cells grown at 34 °C (lanes 1 and 4), 37 °C (lanes 2 and 5), or 39.5 °C (lanes 3 and 6). Equal amounts of protein (10 or 20 µg) were loaded and analyzed by 12% SDS-PAGE followed by Western blotting with anti-HA (10 µg) or anti-v-src (20 µg) monoclonal antibody (38). Lane 7 contained extract (20 µg) from parental (ts)RSVLA23 Rat-1 cells grown at 34 °C as a positive control for v-src.


Repression of RSV LTR by p21 Occurs at the Initiation Stage of Transcription

Results of Northern analyses indicated that CAT reporter gene expression was decreased in a dose-dependent manner by p21 coexpression(11) . To test if the lower steady state levels of CAT RNA resulted from an effect of p21 on transcription, nuclear run-on analyses were done using cells cotransfected with CAT reporter and either p21 expression plasmid or parental vector pBC12BI. In cells cotransfected with p21 cDNA, there was 5.1-fold less CAT RNA synthesized as compared with cells cotransfected with pBC12BI, after normalizing for -actin mRNA synthesis (Fig. 2A). This result agrees well with the 4-6-fold decrease in steady state levels of CAT RNA in p21-expressing cells (11) and suggests that p21 inhibition of RSV LTR-directed CAT expression occurs mainly at the transcriptional level.

Figure 2: p21-mediated repression of transcription initiation from RSV LTR. Nuclear run-on assays were used to measure the levels of newly synthesized CAT transcripts in COS-1 cells cotransfected with pR-CAT and either p21 expression plasmid or parental vector pBC12BI. A, in the presence of p21, CAT expression was decreased by 5.1-fold after normalization based on the -actin internal control. B, the ratio of labeled transcripts bound to 5`-terminal CAT and 3`-terminal CAT probes was close to 1 in nuclei from both p21- and pBC12BI-transfected cells.


Since p21 possesses significant sequence homology to eukaryotic transcription elongation factor TFIIS (12) and promoter context has been implicated in the control of transcriptional elongation in several reports(19, 20) , we investigated the possibility that p21 represses RSV LTR-driven transcription by decreasing elongation. pR-CAT was transiently transfected into COS-1 cells with p21 expression plasmid or pBC12BI, and nuclear run-on assays were done using denatured probes corresponding to the 5`-terminal 285-base pair (5`-CAT) and 3`-terminal 544-base pair segments (3`-CAT) of CAT cDNA, with parental plasmid pBC12BI as a control. Data were quantified by PhosphorImager, and values obtained with the 5`-CAT and 3`-CAT probes were corrected for sequence differences by dividing the signals by the number of U residues in the corresponding complementary sequences. If p21 expression inhibits elongation and thus results in an increased number of incomplete labeled nascent chains, an elevated ratio of 5`- to 3`-signal, as detected with the 5`-CAT and 3`-CAT probes, would be expected. As shown in Fig. 2B, no such increase was observed. Instead, the signals obtained with the promoter-proximal and promoter-distal probes were essentially equal (5`-CAT-to-3`-CAT ratio close to 1) in nuclei from either parental vector pBC12BI- or p21-transfected cells, indicating that elongation was not decreased when transcription was repressed by p21. Taken together, the results suggest that p21 inhibition of RSV LTR occurs at the initiation stage of transcription.

A 24-Base Pair Element of RSV LTR Promoter Is Required for p21 Repression

In order to localize the region(s) in RSV LTR necessary for p21 inhibition, a series of 5` end-nested deletions were constructed in pR-CAT and cotransfected into COS-1 cells with the p21 expression plasmid or pBC12BI. Deletion of increasing portions of promoter sequences from -256 to -85 (relative to the start site) resulted in corresponding decreases in basal activity (Fig. 3A). pR-CAT and three of the truncation constructs, pR-CAT/-141, pR-CAT/-120, and pR-CAT/-108, were each inhibited by more than 10-fold by p21 expression (Fig. 3B). However, extending the deletion from -108 to -85 resulted in a construct with low promoter activity that was no longer affected by p21 (Fig. 3B). These results indicate that the region in RSV LTR between -108 and -85 contains a p21 test-responsive element.

Figure 3: Localization of a p21-responsive element in the RSV LTR promoter. A, 0.5 µg of pR-CAT or 5`-deletion constructs pR-CAT/-141, pR-CAT/-120, pR-CAT/-108, and pR-CAT/-85 were cotransfected into COS-1 cells with 5 µg of either p21 expression plasmid or parental vector pBC12BI. CAT synthesis was measured by enzyme-linked immunosorbent assay 48 h later. The left scale refers to CAT levels obtained with pR-CAT and pR-CAT/-141 and the right scale to pR-CAT/-120, pR-CAT/-108, and pR-CAT/-85 cotransfections. B, schematic map of RSV LTR promoter fused to the CAT gene and 5`-deletion mutants, all in pR-CAT. CAT levels in A were converted to repression values by comparison of p21- and pBC12BI-cotransfected cells.


To test whether the -108 to -85 region of RSV LTR promoter can confer sensitivity to p21 repression on a heterologous promoter, chimeras were made by subcloning the corresponding synthetic 24-mer into pSV2CAT. The 24-base pair oligomer was inserted into pSV2CAT in one or three copies after removal of the SV40 72-base pair inverted repeat enhancer, thus positioning the p21-responsive element(s), relative to the TATA box, as in the RSV LTR. As shown in Fig. 4and observed previously(11) , in pSV2CAT-transfected COS-1 cells the activity of wild type SV40 early promoter was partially inhibited by cotransfection of p21 expression plasmid (average of three experiments, 27% decrease relative to basal level in pBC12BI cotransfected cells). Deletion of the SV40 72-base pair inverted repeat enhancer reduced the basal activity of pSV2CAT/d72 from 7,500 to 3,900 pg of CAT/mg of protein (48%), but p21 expression had no effect on the level of CAT produced (Fig. 4). However, the presence of one copy of RSV-108/-85 inserted in the pSV2CAT/d72 construct restored basal promoter activity, suggesting that the RSV LTR sequences between -108 and -85 contain binding site(s) for positive-acting factor(s) that are functional in the context of SV40 early promoter. In addition, expression of CAT from this chimeric construct was 4.6-fold repressed by p21 coexpression. Although introduction of one copy of the RSV-108/-85 element into the pSV2CAT/d72 construct conferred p21 sensitivity, three copies eliminated p21 susceptibility, possibly due to a limiting amount of p21 relative to positive factor(s) under these circumstances.

Figure 4: p21 repression of SV40 early promoter containing one copy of RSV-108/-85 sequence. 0.5 µg of chimeric reporter constructs pSV2CAT, pSV2CAT/d72 (72 base pair inverted repeat enhancer deleted), pSV2CAT/RSV-108/-851 (1 copy of -108/-85 inserted) or pSV2CAT/RSV-108/-853 (3 copies of -108/-85 inserted) were cotransfected into COS-1 cells with 5 µg of parental vector pBC12BI (basal) or p21 expression plasmid (p21). CAT levels were measured by enzyme-linked immunosorbent assay after 48 h. Values are the average of three independent experiments ± S.D.


Alteration of Protein Complex Formation on RSV-108/-85 Element by p21 Expression

Nuclear extracts of COS-1 cells transfected with the p21 expression plasmid or pBC12BI were incubated with synthetic RSV-108/-85 sequence as radiolabeled probe. Analyses by electrophoretic mobility shift assay indicated that a high molecular weight complex detected in pBC12BI-transfected cells was absent in nuclei of cells transfected with p21 cDNA (Fig. 5, upper arrow, compare lanes 2 with 3 or 6 with 7). In addition, p21 transfection resulted in an increase in the level of a lower molecular weight complex which migrated slightly slower than another major complex present in both extracts (Fig. 5, lower arrow, compare lanes 2 with 3 or 6 with 7). Complex formation apparently was specific, since 100-fold molar excess of unlabeled RSV-108/-85 eliminated the radiolabeled bands (lanes 4 and 5), whereas the same excess of the unrelated oligonucleotide decreased the signal only partially (lanes 6 and 7). Although addition of HA-specific monoclonal antibody, which detects p21HA by Western immunoblot or immunoprecipitation assay(11) , decreased the amount of the lower molecular weight complex, it did not result in a supershifted complex (lanes 8 and 9). The antibody also did not immunoprecipitate the RSV-108/-85 probe (data not shown). These data suggest that p21 does not bind DNA directly but may modulate RSV LTR promoter activity by altering protein complex formation on the p21-responsive element.

Figure 5: Effects of p21 expression on proteinDNA complex formation with RSV-108/-85. Nuclear extracts (10 µg) from COS-1 cells transfected with 5 µg of p21 expression plasmid (lanes 2, 4, 6, and 8) or parental vector pBC12BI (lanes 3, 5, 7, and 9) were incubated with P-labeled double-stranded oligodeoxynucleotide corresponding to RSV LTR sequences -108 to -85. Unlabeled RSV-108/-85 oligonucleotide (lanes 4 and 5) or the unrelated 24-mer (lanes 6 and 7) were included in the binding reaction at 100-fold molar excess. Anti-HA monoclonal antibody (1 µl, 1:50 dilution) was also included as indicated (lanes 8 and 9). Resulting proteinDNA complexes were analyzed by nondenaturing polyacrylamide gel electrophoresis and autoradiography as described under ``Materials and Methods.'' The sample in lane 1 contained no protein. Arrows indicate positions of complexes increased (lower) or decreased (upper) by p21 expression.


p21 Expression Decreases Interaction of a COS-1 Cell Protein with the RSV-108/-85 Element

UV cross-linking was used to assay for protein components which bind to the RSV-108/-85 DNA element. Nuclear extracts of COS-1 cells that had been transfected with pBC12BI or plasmids for expression of p21 or p21 mutant pd50-100 were incubated with P-labeled RSV-108/-85 oligomer, UV-irradiated, and analyzed by SDS-PAGE. Three high molecular weight cross-linked bands were obtained at positions corresponding to 145, 95, and 85 kDa in each of the extracts (Fig. 6A, lanes 1-3). The relative intensity of the three bands was similar in pBC12BI extract (lane 3) and in the extract of cells transfected with the p21 pd50-100 construct (lane 2), a mutant almost devoid of RSV LTR repression activity(11) . However, labeling of the 145-kDa band in nuclear extract from p21-transfected cells was decreased 6.3-fold relative to the two faster migrating bands as determined by PhosphorImager (Fig. 6A, lane 1, arrow). The specificity of these complexes was tested in competition experiments using unlabeled RSV-108/-85 or the unrelated oligonucleotide. Addition of 50-fold molar excess of unlabeled RSV-108/-85 oligomer essentially abolished DNA cross-linking of the three polypeptides (Fig. 6A, lanes 4-6), whereas the same molar excess of unrelated oligonucleotide competed well with the two faster migrating bands but not the one at 145 kDa (lanes 7-9).

Figure 6: Reduced binding of a COS-1 cell protein to the RSV-108/-85 element after p21 transfection. A, nuclear extracts of COS-1 cells transfected with plasmid for expression of p21 (lanes 1, 4, and 7) or p21 mutant pd50-100 (lanes 2, 5, and 8) or with parental vector pBC12BI (lanes 3, 6, and 9) were incubated with P-labeled RSV-108/-85 probe. DNA-bound proteins were cross-linked to the labeled oligonucleotide by UV irradiation, and products were analyzed by 10% SDS-PAGE and autoradiography. For the samples in lanes 4-6, a 50-fold molar excess of unlabeled RSV-108/-85 oligonucleotide was present, and for lanes 7-9, a 50-fold molar excess of unrelated oligomer was included. The sample shown in lane 10 contained no nuclear extract. B, cross-linking of a 145-kDa polypeptide to P-labeled RSV-108/-85 oligomer in the absence (lanes 1-3) or presence of 25 (lanes 4-6)-, 50 (lanes 7-9)-, or 75-fold (lanes 10-12) molar excess of the unrelated competitor 24-mer. Relative intensities of the 145-kDa protein band after UV cross-linking are plotted for nuclear extracts from COS-1 cells transfected with p21 expression plasmid (lanes 1, 4, 7, and 10), pd50-100 (lanes 2, 5, 8, and 11) or pBC12BI (lanes 3, 6, 9, and 12). C, loss of affinity purification of a 145-kDa protein on RSV-108/-85 oligonucleotide in extracts of p21-transfected cells. Nuclear extracts of COS-1 cells transfected with pBC12BI (lanes 2 and 4) or p21 expression plasmid (lanes 3 and 5) were subjected to RSV-108/-85 DNA affinity chromatography and analyzed by 12% SDS-PAGE followed by silver staining as described under ``Materials and Methods.'' Lane 1, molecular weight markers; lanes 2 and 3, total nuclear extract loaded onto the column; lanes 4 and 5, proteins bound and eluted with 1 M KCl. Arrow indicates position of a 145-kDa band present in the bound fraction from parental vector- but not p21 plasmid-transfected cells (lane 5).


Nuclear extracts from cells transfected with p21 expression plasmid consistently had less of the 145-kDa band (as measured by UV cross-linking) than extracts of cells transfected with parental vector or p21 mutant pd50-100. This difference persisted in the presence of up to 75-fold molar excess of nonspecific competitor, a concentration that decreased the intensity of the 145-kDa band by 2-4-fold (Fig. 6B). In cells transfected with plasmid for expression of p21 mutant pd50-100, a doublet that possibly included a modified form of the 145-kDa band was consistently obtained (Fig. 6, A: lanes 2 and 8, B: lanes 2, 5, 8, and 11).

In another approach to identify protein(s) that bind to RSV-108/-85 element, nuclear extracts from COS-1 cells transfected with either parental vector or p21 expression plasmid were analyzed by DNA affinity column followed by SDS-PAGE and silver staining. From the complex mixture of nuclear proteins added to the DNA column (Fig. 6C, lanes 2 and 3) only a small amount bound and eluted with 1 M KCl (lanes 4 and 5). Among the bound proteins, a prominent band at a position similar to the 145-kDa band detected by UV cross-linking (arrow) was present in the pBC12BI-transfected sample (lane 4), but not in the extract from p21-transfected cells (lane 5). Expression of p21 also resulted in decreased levels of several other lower molecular weight proteins bound to the column. Consistent with other results suggesting that p21 does not bind directly to the RSV-108/-85 DNA element, p21 was essentially all (>90%) in the flow-through fraction as determined by Western blot analysis (data not shown). Together with the UV cross-linking results, the data indicate that p21 expression decreased binding of a cellular factor to the RSV-108/-85 element.

DISCUSSION

In this report, we have shown that HeLa cell protein p21 represses RSV LTR at the transcription initiation level with no apparent effect on elongation. The 5-fold inhibition of newly transcribed CAT RNA by p21 can account for the previously reported 4-6-fold decrease in CAT steady state RNA levels(11) . Consistent with its negative regulatory effect on RSV LTR, p21 decreased v-src levels and antagonized the appearance of the transformed phenotype at permissive temperature in rat cells transformed with a temperature-sensitive mutant of RSV. These findings and the inhibition of chick embryo fibroblast transformation by cotransfection of RSV cDNA and p21 expression plasmid (11) indicate that p21 can function as a repressor of RSV LTR in cells of different species.

Repression by p21 required a 24-base pair element of the RSV LTR promoter located between positions -108 and -85 upstream of the transcription start site. This sequence also conferred p21 responsiveness to the SV40 early promoter, but inhibition was lower on the heterologous promoter as compared with RSV LTR. This observation implies that promoter context influences the effect of p21. Thus, p21 repressed transcription from the SV40 early/RSV LTR chimeric promoter (pSV2CAT/RSV-108/-851) by 4-5-fold as compared with 15-fold for RSV LTR, possibly indicating that p21 cannot fully counteract the effects of transcription activators driving the SV40 early promoter. Interestingly, the inhibitory effect of p21 on the SV40 promoter was lost when three copies of the RSV-108/-85 sequence were inserted, suggesting that p21 is limiting relative to putative activating factor(s) under these conditions.

The sequence in the RSV LTR between -108 and -85, which we identified as necessary for p21-mediated repression, contains a CArG box (CCTTATTAGG) overlapping by three residues (bold) an E1A enhancer core sequence (AGGAAGGCA) and an ETS binding site (AGGAA). CArG motifs occur upstream of several cellular genes, including the cardiac and skeletal actin genes(21) , and are required for their tissue-specific expression(22, 23) . A CArG motif is also present in the c-fos serum response element (SRE), which mediates both cycloheximide- and serum-inducible expression of c-fos(24, 25) as well as its subsequent repression(26) . Previous studies have revealed a region between positions -112 and -87 in RSV LTR that binds a cellular factor, EFIII(6) . The EFIII binding site, like other CArG motifs(27, 28) , can mediate a modest transcriptional stimulation in response to serum. It has been proposed (6) that EFIII may be the avian homolog of the serum response factor (SRF), a positive regulatory protein that interacts with the c-fos SRE(29) . It is noteworthy in this regard that the p21-responsive element in RSV LTR and the c-fos SRE both include a CArG box and that p21 exerts opposite effects on RSV LTR (repression) and the c-fos promoter (activation)(11) . Thus p21 may modulate the activity of transcription factor(s) that interact with the corresponding elements in RSV LTR and the c-fos promoter, with the dominant effect determined by the composition of regulatory protein complexes.

The c-fos SRE has been shown to be essential for serum induction of c-fos gene expression(30) . In the course of determining whether the RSV LTR is also serum inducible and if it can be repressed by p21 coexpression, we confirmed with COS-1 cells that RSV LTR promoter activity was induced 2.7-fold by addition of serum to depleted cell cultures(6) , and this effect was reversed by p21 expression.()Thus p21 may mediate repression of RSV LTR via the CArG box in the -108 and -85 region.

Although the molecular basis for p21 repression of RSV LTR is not clear, our data indicate that p21 does not bind DNA directly but decreases the binding of a cellular protein(s) to the RSV-108/-85 element. Several mechanisms have been described for regulating DNA binding and controlling the activity of transcription factors(31) . The DNA binding activity of some AP-1 proteins, including Jun and Fos, can be regulated by a redox (reduction-oxidation) reaction(32) . HTLV-1 Tax forms multimeric complexes with bZIP proteins, notably CREB, resulting in preferential DNA binding to Tax-responsive elements(33, 34) . The DNA binding of SRF is increased by Phox1 factor (paired-like homeobox) (35). Phox1 enhances both the association and dissociation rate of the SRFSRE complex, and it has been proposed that an increased exchange of SRF on its binding site could allow for a faster response to transient mitogenic signals(35) . p21 may alter posttranslational modification(s) of cellular LTR binding protein(s) or affect other proteins that interact with these factor(s). It will be of interest to determine if the high molecular weight cellular factor detected by UV cross-linking to the RSV-108/-85 element belongs to the ETS family of transcription factors (36) or to the recently recognized MADS family, a distinct group of DNA-binding proteins that have homology to SRF and share a conserved domain termed the MADS box(37) .

FOOTNOTES

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

§
To whom correspondence should be addressed: Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854. Tel.: 908-235-5311; Fax: 908-235-5318.

The abbreviations used are: RSV, Rous sarcoma virus; LTR, long terminal repeat; CAT, chloramphenicol acetyltransferase; SV40, simian virus 40; HA, hemagglutinin; pd, putative domain; ts, temperature-sensitive; PAGE, polyacrylamide gel electrophoresis; SRE, serum response element; SRF, serum response factor.

C. H. Yeh, unpublished results.

ACKNOWLEDGEMENTS

We thank Dr. Peter K. Vogt for providing the Rat-1 tsRSVLA23 cell line used in this study; Dr. Peter Lobel for the anti-HA monoclonal antibody 12CA5; Dr. Sarah Parsons for anti-v-src monoclonal antibody EB7; and Drs. Cory Abate, Celine Gelinas, and Arnold Rabson for critical reading of the manuscript and valuable discussion.

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C.-H. Yeh, W.-X. Zong, and A. J. Shatkin
The Ser[IMAGE]-Ser[IMAGE] Pair in HeLa Nuclear Protein p21/SIIR Mediates Ser/Thr Phosphorylation and Is Essential for Rous Sarcoma Virus Long Terminal Repeat Repression
J. Biol. Chem., October 27, 1995; 270(43): 25313 - 25315.
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