©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Retinoblastoma Susceptibility Gene Product Represses Transcription When Directly Bound to the Promoter (*)

Jalila Adnane (§) , Zhaohui Shao , Paul D. Robbins (¶)

From the (1) Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Rb represses E2F-mediated transcription in part by blocking the trans-activation domain of E2F. In addition, Rb can convert an E2F binding site from a positive to a negative element. To examine the effect of a Rb-DNA-bound complex on transcription, full-length Rb was fused to the DNA binding domain of GAL4. Here, we report that GAL4-Rb can repress transcription mediated by either Sp1, AP-1, or p53, dependent upon the presence of both the GAL4 DNA binding domain and GAL4 binding sites. Moreover, GAL4-Rb inhibited the activity of the herpes simplex virus tk promoter from GAL4 binding sites located at a distance from the promoter. In contrast, GAL4-Rb was unable to repress basal transcription. Cotransfection of specific cyclins and cyclin-dependent kinases or SV40 T-antigen abolished the repressive activity of GAL4-Rb. The domains of Rb involved in mediating the repression of transcription were mapped to regions that are overlapping, but not identical, to those required for the interaction with E2F. We propose that Rb can function as a general repressor of transcription when bound to the promoter region.


INTRODUCTION

The protein product of the retinoblastoma susceptibility gene, Rb, plays a key role in the regulation of cell growth and differentiation (1) . Rb appears to control cell growth, in part, by regulating gene expression (for review see Ref. 2). Indeed, Rb is able to regulate the transcription of a variety of genes encoding growth-regulatory factors, such as c- fos (3) , c- myc (4) , TGF-1 (5) , TGF-2 (6) , IGF-II (7) , IL-6 (8) , and neu (9) . It appears that Rb regulates transcription by interacting with specific transcription factors and modulating their activity. Rb can interact in vivo with E2F (10, 11, 12) , MyoD (13) , and Elf-1 (14) and in vitro with ATF-2 (6) , N-Myc (15) , c-Myc (15) , PU.1 (16) , and UBF (17) .

Rb is believed to repress transcription by interacting directly with transcription factors, such as E2F, and blocking their trans-activation domains (18) . In addition, Rb has been shown to convert an E2F site from a positive to a negative regulatory sequence, suggesting an additional role for E2F-bound Rb besides blocking the trans-activation domain of E2F (19) . To examine the effect of a Rb-DNA-bound complex on transcription, full-length Rb was fused to the DNA binding domain of GAL4. In this manner, Rb can be brought to the promoter through the interaction with GAL4 binding sites without having to interact with specific transcription factors. Our results demonstrate that GAL4-Rb, when bound to DNA through GAL4 binding sites, can repress transcription mediated by several transcription factors. An interaction with SV40 T-antigen or phosphorylation by specific cyclins prevented GAL4-Rb from repressing transcription. Moreover, GAL4-Rb proteins with deletions or point mutations within domains A and B of the Rb pocket were deficient in their ability to repress transcription. Our results suggest that Rb can function as a general repressor of activated transcription when bound to the promoter region.


EXPERIMENTAL PROCEDURES

Plasmid Constructs

The plasmids G5BCAT, pBLCAT2, and pSG424 have been previously described (3, 7, 20) . E1b-CAT was provided by Dr. M. Green (21) . The GAL4-TK-CAT, GAL4-UAS, and GAL4-UASreporter plasmids were provided by Dr. F. Rauscher (22) . GAL4-Sp1-CAT, Sp1-CAT, GAL4-AP1-CAT, AP1-CAT, and GAL4-p53-CAT were constructed using Sp1 (CTAGGGGGCGGGGC), AP-1 (CTAGGTGACTCAGCGCG), and p53 (CTAGAGGCATGTCT) double-stranded oligonucleotides. Briefly, single-stranded oligonucleotides were annealed, and phosphorylated for 1 h at 37 °C in presence of 10 m M ATP and 18 units of T4 bacteriophage kinase and then partially ligated using T4 DNA ligase. The ligation products were separated by an agarose gel (SeaKem 1% + NuSieve 2%), and fragments of different sizes were isolated. The fragments with three binding sites of a specific transcription factor were purified and ligated to G5-E1b-CAT (for the construction of GAL4-Sp1-CAT, GAL4-AP1-CAT, and GAL4-p53-CAT) and to E1b-CAT (for the construction of Sp1-CAT and AP1-CAT) at the XbaI site. SVE, a SV40 early promoter-based expression vector, and the human Rb expression plasmid were provided by Dr. D. Templeton (23) . The 1137 plasmid encodes for a truncated T-antigen (amino acids 1-121) containing the Rb/p107 binding domain (24) . The 1137rb plasmid harbors a single amino acid change Glu-107 to Lys that abolishes the Rb/p107 binding ability of 1137. GAL4-Rb was constructed by inserting the 4.5-kilobase EagI-filled in- SacI fragment from the human Rb expression plasmid into SmaI and SacI sites of pSG424. The GAL4-Rb plasmid contains almost the entire region of Rb (10-928 amino acids) fused in-frame to the GAL4 (1-147) DNA binding domain. All of the GAL4-Rb deletion mutants were constructed by replacing the wild-type sequences in GAL4-Rb with the mutated sequences, individually, from their expression plasmids, which were provided by Dr. D. Templeton (23) . GAL4-Rb (706) and GAL-Rb (592) encodes for a GAL4-Rb fusion protein with a single amino acid change at amino acids 706 of Rb and a deletion of exon 22 (amino acids 738-775), respectively. The 706 and 592 expression plasmids were provided by Dr. J. M. Horowitz (25, 26) . The GAL-Rb (295-928) was provided by Dr. M. Green (27) .

Cell Culture and Transfection

CCL-64 and COS-7 cells were maintained in Dulbecco's modified Eagle's medium plus 5 and 10% fetal calf serum, respectively. CCL-64 and COS-7 cells were transfected using a calcium phosphate protocol (28) . 48 h post-transfection, the protein extracts were prepared as previously described (6) and used for chloramphenicol acetyltransferase (CAT)() assay. The acetylated and nonacetylated forms of [C]chloramphenicol were separated by thin-layer chromatography and counted directly on a Betagen counter. The level of CAT activity was calculated as the percentage of the two acetylated forms of chloramphenicol relative to the total amount of [C]chloramphenicol. For normalization of transfection efficiencies, a luciferase expression plasmid pSV-luc was included in each transfection as an internal control. The transfection experiments were repeated at least three times.

Western Blotting

2 10COS-7 cells were transfected with 5 µg of each expression plasmid as described above. 48 h post-transfection, the cells were collected and resuspended in phosphate-buffered saline and then lysed by mixing with equal volume of 2 Laemmli sample buffer. Equal amounts of total proteins from each sample were separated on 8% SDS-polyacrylamide gels and transferred to nitrocellulose membranes by standard techniques (29) . The membranes were then probed with a rabbit polyclonal antibody to GAL4 (kindly provided by I. Sadowski) followed by incubating with a horseradish peroxidase-conjugated anti-rabbit antibody (Sigma). Cross-reactive proteins were detected by the ECL system (Amersham Corp.).


RESULTS

GAL4-Rb Represses Sp1-, AP-1-, and p53-mediated Transcription from Adjacent GAL4 Binding Sites

Rb protein regulates the expression of a number of genes encoding growth-regulatory factors by interacting with specific transcription factors and modulating their activity (3, 4, 5, 6, 7, 8, 9) . To better understand the mechanism through which Rb protein regulates transcription when it is a part of a DNA-bound complex, Rb was targeted to the promoter by fusing Rb to the heterologous GAL4 DNA binding domain. A GAL4-Rb expression vector was constructed by inserting the Rb cDNA in-frame downstream of the yeast transcription factor GAL4 DNA binding domain (GAL4 1-147) in the plasmid pSG424. G5BCAT, containing 5 GAL4 binding sites upstream of E1b TATA box, was used as a basal promoter reporter plasmid. Cotransfection of the GAL4-Rb expression vector with the G5BCAT reporter plasmid into CCL-64 cells did not affect transcription of G5BCAT (data not shown), suggesting that GAL4-Rb is unable to regulate basal transcription.

To examine the effect of GAL4-Rb on activated transcription, multiple copies of the consensus binding sites for Sp1, AP-1, and p53 were inserted between the GAL4 binding sites and the E1b TATA box in G5BCAT. Cotransfection of GAL4-Rb and Sp1, AP-1, and p53 reporters into CCL-64 cells resulted in a significant repression of transcription in a dose-dependent manner (Fig. 1). However, GAL4-Rb was not able to repress transcription from the Sp1-CAT and AP1-CAT reporters lacking GAL4 binding sites (Fig. 1, B and E). The GAL4 DNA binding domain alone, expressed from the pSG147 plasmid, and Rb protein were unable to repress transcription from the GAL4-Sp1-CAT reporter (Fig. 1 A). The levels of Rb and GAL4-Rb proteins expressed in the transfected cells were shown to be similar by Western blot analysis using an anti-RB antibody (data not shown). Thus, the repression of transcription mediated by GAL4-Rb requires both Rb to be fused to the GAL4 DNA binding domain and for GAL4 binding sites to be present in the reporter plasmid. These results also demonstrate the ability of GAL4-Rb to repress transcription mediated by three distinct transcription factors, Sp1, AP-1, and p53.


Figure 1: GAL4-Rb represses transcription mediated by Sp1, AP-1, and p53. CCL-64 cells were transfected with SVE (an SV40 expression plasmid), pSG147 (a control plasmid encoding for GAL4 DNA binding domain), or GAL4-Rb expression plasmid and 1.5 µg of the following reporter plasmids: GAL4-Sp1-CAT ( panel A), Sp1-CAT ( panel B), GAL4-p53-CAT ( panel C), GAL4-AP1-CAT ( panel D), or AP1-CAT ( panel E). The results shown are representative of at least three separate experiments. The results are presented as relative CAT activity with SVE given a value of 1. The structure of the reporters are shown below the corresponding graphs.



GAL4-Rb Represses Transcription from a Distance

To examine whether GAL4-Rb was able to repress transcription from a distance, reporter plasmids carrying GAL4 binding sites adjacent to the HSV tk promoter (GAL4-TK-CAT) and either 760 bp upstream (GAL4-UAS) or 1000 bp downstream (GAL4-UAS) of the transcription initiation site were tested. The results of the cotransfection experiments with the different reporters showed that the repression of the activity of HSV tk promoter by GAL4-Rb was dependent upon the presence of the GAL4 binding sites but was independent of their position (Fig. 2). Again, repression of transcription was observed only with GAL4-Rb and not with Rb (Fig. 2 A). These results demonstrate the ability of GAL4-Rb to repress transcription from GAL4 binding sites positioned at a distance from HSV tk promoter. These results also rule out the possibility that GAL4-Rb was inhibiting transcription by preventing the binding of Sp1, AP-1, or p53 to their sequence-specific binding sites.


Figure 2: GAL4-Rb represses the transcription from GAL4 binding sites located at a distance from the transcription initiation site. CCL-64 cells were transfected with SVE, pSG147, GAL4-Rb, or Rb expression plasmid and 1.5 µg of the following reporter plasmids: GAL4-TK-CAT ( panel A), pBLCAT2 ( panel B), GAL4-UAS( panel C), and GAL4-UAS( panel D). The results shown are representative of at least three separate experiments. The results are presented as relative CAT activity with SVE given a value of 1. The structure of the reporters are shown below the corresponding graphs.



SV40 T-antigen Prevents GAL4-Rb From Repressing the Transcription

Rb protein interacts with several viral oncoproteins such as SV40 T-antigen (30) , human papillomavirus (HPV), E7 protein (31, 32) , and adenovirus E1A (33) . The interaction of Rb with the viral oncoproteins alters normal Rb function, presumably by dissociating Rb complexes. To determine the effect of viral oncoproteins on the repression of transcription mediated by GAL4-Rb, a pair of truncated SV40 T-antigen mutants differing in their ability to bind to Rb were used in the transfection assay (Fig. 3). The truncated SV40 T-antigen dl1137 prevented GAL4-Rb from repressing transcription in a dose-dependent manner. In contrast, the mutant dl1137rb, deficient in its ability to interact with Rb, did not block repression by GAL4-Rb at all input doses. The reason for the marginal stimulation of GAL4-Tk-CAT by 1137 at the high input dose in the absence of GAL4-Rb is unclear but may reflect an interaction of 1137 with endogenous p107, p130, and/or Rb. Taken together, these results demonstrate the ability of SV40 T-antigen to at least partially block GAL4-Rb-mediated repression and suggest that the T-antigen binding pocket of Rb may be required for the observed repression.

Expression of Cyclins and cdks Abrogates GAL4-Rb-mediated Repression

Rb activity is regulated by serine/threonine phosphorylation during the cell cycle (34, 35, 36) . Several cdks and their regulatory subunits (cyclins) have been shown to interact with and to phosphorylate Rb (37, 38, 39, 40, 41, 42, 43, 44) . To determine the effect of Rb phosphorylation on GAL4-Rb-mediated repression, cdk4, cdk2, and their respective regulatory subunits, cyclin D1 and cyclin E, were cotransfected, either alone or in combination, with GAL4-Rb (Fig. 4). Cotransfection of GAL4-Rb with either cyclin D1 and cdk4 or cyclin E and cdk2 abrogated GAL4-Rb-mediated repression, restoring HSV tk promoter activity. In contrast, no significant effect on GAL4-Rb-mediated repression was observed when cyclin D1, cyclin E, cdk4, and cdk2 were cotransfected separately with GAL4-Rb. Moreover, in the absence of GAL4-Rb, the overexpression of cyclin D1, cyclin E, cdk4, and cdk2, either alone or in combination, did not affect the activity of the GAL4-TK-CAT reporter. Thus, the abrogation of GAL4-Rb-mediated repression by cyclin D1-cdk4 and cyclin E-cdk2 complexes may be the result of the phosphorylation of GAL4-Rb.

Mapping the Domains Required for GAL4-Rb-mediated Repression

To map the domain(s) of GAL4-Rb involved in the repression of transcription, a panel of GAL4-Rb deletion mutants were tested (Fig. 5). The levels of expression of the GAL4-Rb mutants were determined by Western blot analysis using an anti-GAL4 antibody (Fig. 5 C). Except for dl37-89 and dl309-343 (deleted for the amino acids indicated), the GAL4-Rb mutants were expressed at a similar level in COS-7 cells and in CCL-64 cells. The GAL4-Rb mutants were also tested for their ability to interact in vivo with adenovirus E1A protein. E1A has been shown to activate transcription when brought to the promoter through an interaction with GAL4-Rb (27) . Therefore, the ability of E1A to activate transcription from G5BCAT when brought to the promoter through an interaction with the GAL4-Rb mutants was determined. E1A was able to activate transcription in the presence of the full-length GAL4-Rb and from the majority of the GAL4-Rb mutants with an intact pocket domain (Fig. 5 C). In contrast, deletions or point mutations within domain A and domain B resulted in an inability of E1A to activate transcription. However, four deletion mutants outside the pocket region failed to support E1A activation, two of which were expressed at a lower level (dl37-89 and dl309-343). The reason of the decrease of the ability of E1A to activate transcription from the mutants dl202-248 and dl248-309 is not clear.


Figure 5: Rb ``small pocket'' is required for GAL4-Rb-mediated repression. A, structure and the amino acid positions of the mutant GAL4-Rb proteins. B, effect of GAL4-Rb and GAL4-Rb mutants on HSV tk promoter activity. CCL-64 cells were transfected with 1.5 µg of GAL4-TK-CAT reporter and 2 µg of SVE, GAL4-Rb, or GAL4-Rb mutant expression plasmid. The GAL4-Rb proteins designated by the annotation dl are deleted for the amino acids indicated. The GAL4-Rb mutants 295-928 and 706* encode for Rb ``large pocket'' and a naturally occurring mutation at position 706. The results are presented as an average of four separate experiments. C, top: level of expression of GAL4-Rb and GAL4-Rb mutants. COS7 cells were transfected with 5 µg of each of the GAL4-Rb plasmids. 2 days post-transfection, the cells were lysed with RIPA buffer, and equal amounts of total proteins from each sample were separated on 8% SDS-polyacrylamide gels. The nitrocellulose membrane was probed with an anti-GAL4 antibody. Bottom, activation of transcription by E1A through its interaction with GAL4-Rb and GAL4-Rb mutants. NIH3T3 cells were cotransfected with 4 µg of G5BCAT reporter plasmid, 2 µg of SVE, GAL4-Rb, or GAL4-Rb mutant expression plasmid, and 4 µg of E1A expression plasmid. The results are presented as relative -fold activation with SVE given a value of 1.



The ability of the GAL4-Rb deletion mutants to repress the transcription from GAL4-TK-CAT reporter was examined in CCL-64 cells (Fig. 5 B). GAL4-Rb proteins with deletions within the amino-terminal domain of Rb did not alter GAL4-Rb-mediated repression. The reduction in the repressive activity of GAL4-Rb dl37-89 and dl309-343 may be the result of the low cellular level of the proteins (Fig. 5 C). Deletion of domain A (mutant dl389-580) resulted in a significant reduction in GAL4-Rb-mediated repression. In addition, deletion or point mutation within domain B (dl614-662, dl662-775, and the naturally occurring mutants 592 (dl738-775) and 706) resulted in the abrogation of GAL4-Rb-mediated repression. Deletion of 34 amino acids in the spacer region (dl580-614) did not affect the repressive activity of GAL4-Rb. Furthermore, the GAL4-Rb mutants truncated in the carboxyl-terminal region of Rb (dl775-817, dl817-839, and dl839-892) retained their ability to repress transcription. These results demonstrate the requirement for domains A and B, but not for the amino- and carboxyl-terminal regions of Rb, for full inhibition of HSV tk promoter activity. Interestingly, the mutants dl202-248 and dl248-309, which supported only weak E1A activation, were able to repress tk promoter activity to the same level as with the full-length GAL4-Rb.


DISCUSSION

Rb appears to regulate gene expression by interacting and modulating the activity of specific transcription factors (6, 7, 10, 13, 14, 16) . It has been suggested that Rb suppresses E2F-mediated transcription through direct physical association with E2F trans-activation domain. However, it also has been shown that the Rb-E2F complex, when bound to an E2F site, can inhibit the activity of adjacent promoter elements and repress transcription (19) . This result suggested that Rb may regulate transcription through an additional mechanism(s) besides simply blocking the trans-activation domain of E2F.

To determine the function of Rb when it is a part of a DNA-bound complex, we have bypassed the requirement of specific transcription factors for bringing Rb to the DNA by fusing Rb to the DNA binding domain of the yeast transcription factor GAL4. The cotransfection experiments have shown that GAL4-Rb, when bound to GAL4 sites, was able to repress Sp1-, AP-1-, and p53-mediated transcription as well as transcription mediated by the HSV tk promoter. The inhibition of transcription by GAL4-Rb was not the consequence of steric hindrance since GAL4-Rb repressed the activity of the HSV tk promoter from GAL4 binding sites located either 760 bp upstream or 1000 bp downstream from the site of transcriptional initiation. This effect was specific to GAL4-Rb in that wild-type Rb protein was unable to repress transcription, and GAL4 binding sites in the reporter plasmid were required for GAL4-Rb-mediated transcription. Thus, GAL4-Rb can function as a general repressor of transcription when bound to the DNA, independent of an interaction with a specific transcription factor.

We demonstrated that the binding of SV40 T-antigen to GAL4-Rb was able to at least partially abrogate GAL4-Rb-mediated repression. The interaction of SV40 T-antigen with GAL4-Rb may prevent Rb from interacting with a cellular factor(s) involved in conferring the repressive effect of GAL4-Rb. The ability of SV40 T-antigen to abrogate the repressive effect of GAL4-Rb suggested that the ``small pocket'' of Rb was involved in repressing transcription. In addition, we demonstrated that GAL4-Rb-mediated repression was abolished when cyclin D1-cdk4 or cyclin E-cdk2 were coexpressed with GAL4-Rb. Thus, the abrogation of GAL4-Rb-mediated repression by cyclin-cdk complexes could be the result of the phosphorylation of GAL4-Rb.

Deletion analyses have identified the domains of Rb important for binding to certain viral oncoproteins such as adenovirus E1A, SV40 T-antigen, and HPV E7 (45, 46) . These domains extend from amino acid 379 to 571 (domain A) and from 649 to 792 (domain B). The E2F binding region in Rb coincides with the large pocket extending from residues 379 to 928 excluding the spacer region located between amino acids 571 and 649 (47, 48) . Deletion of domain A of the pocket partially abolished GAL4-Rb-mediated repression, whereas deletions or point mutations within domain B were defective in repressing transcription. However, an intact spacer region was not required. These results demonstrate the importance of domains A and B for GAL4-Rb-mediated repression, consistent with the ability of SV40 T-antigen to block repression. It is important to note that GAL4-Rb proteins with deletions in the carboxyl-terminal domain of Rb within the E2F binding domain also were able to inhibit the activity of HSV tk promoter. Thus, the sequences of Rb involved in mediating the repression of transcription map to ``small pocket'' and are overlapping but not identical to those required for the interaction with E2F.

Our results demonstrate that Rb, when brought to the promoter by a GAL4 DNA binding domain, can significantly repress transcription. Normally during the cell cycle, Rb is brought to the promoter region through interactions with specific transcription factors such as E2F. The interaction of Rb with E2F appears to block its ability to activate transcription, in part, by blocking its trans-activation domain. We propose that besides blocking the trans-activation domain of E2F, Rb can negatively regulate transcription through an alternate mechanism. Given that GAL4-Rb represses activated but not basal transcription, we have examined the ability of Rb to interact with a panel of different coactivator proteins or TAFs (TBP-associated protein) including TAF250, TAF150, TAF110, TAF70, TAF32, TAF30-, and TAF30-. TAF250 was able to bind with high affinity to Rb and to GAL4-Rb in vivo and in vitro (49) and in the yeast two-hybrid system (data not shown). Thus, Rb can bind to TFIID through an association with TAF250.

Two models for how GAL4-Rb can regulate transcription are shown in Fig. 6. Rb can be brought to the promoter by the GAL4 DNA binding domain or through an interaction with a specific transcription factor. Rb then can interact either with other transcription factors to block their interaction with TFIID or interact with TFIID directly through binding to a coactivator(s) such as TAF250. In the first model, Rb might interact with other factors involved in transcription such as adjacently bound transcription factors. In the second model, Rb may function as an adapter-like molecule that can regulate transcription through protein-protein interactions, bridging specific transcription factors such as E2F with coactivators such as TAF250. Since our reporter plasmid contains only a binding site for a single transcription factor, such as Sp1, AP-1, or p53, that has not been shown to bind directly to Rb, we favor the latter model.


FOOTNOTES

*
This work was supported by Public Health Service Award CA55227 from NCI, National Institutes of Health (to P. D. R.). 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.

§
Supported by a postdoctoral fellowship from the Association pour la Recherche contre le Cancer.

To whom correspondence should be addressed. Tel.: 412-648-9268; Fax: 412-624-1401.

The abbreviations used are: CAT, chloramphenicol acetyltransferase; TAF, TATA binding protein-associated factor; HSV, herpes simplex virus; bp, pase pair(s); cdk, cyclin-dependent kinase.


ACKNOWLEDGEMENTS

We thank Drs. Martin Schmidt and Steve Phillips for critically reviewing the manuscript. We also thank Di Jiang, Dennis Templeton, Ivan Sadowski, Charles Sherr, and Steve Reed for providing important reagents.


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