Jun 13, 1996 - is by binding to the transcription factor E2F and inhibiting its transactivating function ... genes [e.g.dihydrofolate reductase(DHFR), cdc2, PCNA,.
The EMBO Journal vol.15 no.20 pp.5668-5678, 1996
Inhibition of E2F-mediated transcription by p202
Divaker Choubey1'2, Shyr-Jiann Li3, Bansidhar Datta, Jordan U.Gutterman4 and Peter Lengyel Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520 and 4Department of Molecular Oncology, Box 41, The University of Texas M.D.Anderson Cancer Center, Houston, TX 77030, USA 'Present address: Department of Molecular Oncology, Box 41, The University of Texas M.D.Anderson Cancer Center, Houston, TX 77030, USA 3Present address: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA 2Corresponding author
Many of the antimicrobial, immunomodulatory and cell growth inhibitory activities of the interferons are mediated by interferon-inducible proteins. Earlier we characterized an interferon-inducible murine protein, p202, whose expression in transfected cells inhibits cell proliferation and which can form a complex with retinoblastoma protein (pRb). Here we report that in transfected cells expression of p202 inhibits E2Fstimulated transcription of a reporter gene and of endogenous genes. Inhibition of the transcriptional activity of E2F by p202 does not depend on fully functional pRb and is correlated with inhibition of the sequence-specific DNA binding of E2F. p202 interacts with the transcription factor E2F (E2F-1/DP-1) in vitro and in vivo. Inhibition of E2F activity by p202 may contribute to growth inhibition by the interferons. Keywords: cell growth inhibition/E2F binding protein/ inhibition of E2F-mediated transcription/interferoninducible p202
Introduction One of the ways in which the retinoblastoma tumor suppressor protein (pRb) is thought to regulate cell growth is by binding to the transcription factor E2F and inhibiting its transactivating function (Bandara and La Thangue, 1991; Chellapan et al., 1991; Chittenden et al., 1991; Hamel et al., 1992; Helin et al., 1992, 1993; Hiebert et al., 1992; Kaelin et al., 1992; Shan et al., 1992; Flemington et al., 1993). The first E2F-type transcription factor (E2F-1) was identified as a cellular DNA binding protein involved in activation of the adenoviral E2a promoter (Kovesdi et al., 1986; Nevins, 1992). Subsequently E2F recognition sites have been found in the promoters of various growth-responsive and growth promoting cellular genes [e.g. dihydrofolate reductase (DHFR), cdc2, PCNA, b-myb and c-myc] and were shown to contribute to the transcriptional regulation of these genes (Blake and
Azizkhan, 1989; Thalmeier et al., 1989; Dalton, 1992; Adams and Kaelin, 1995; DeGregori et al., 1995). E2F-1 is a member of the E2F family of transcription factors that also includes E2F-2, E2F-3, E2F-4 and E2F-5 (Ivey-Hoyle et al., 1993; Lees et al., 1993; Beijersbergen et al., 1994; Ginsberg et al., 1994; La Thangue, 1994). All share several homologous domains, including, among others, a DNA binding domain embedded in the N-terminal half and a pRb family binding domain embedded in an acidic transactivation domain in the C-terminal region. The N-terminal region of E2F-1 is involved in binding cyclin A and cyclin-dependent kinase-2 and is also required for transcriptional activation of some promoters (i.e. of herpes simplex virus thymidine kinase) (Shin et al., 1996). The E2F proteins form heterodimers with the related DP family proteins (including DP-1, DP-2 and DP-3), thereby greatly enhancing their DNA binding and transactivating activity (Bandara et al., 1993; Helin et al., 1993; Krek et al., 1993; Ormondroyd et al., 1995). Activity of the E2F proteins is regulated, in part, by binding to the pRb, p107 and p130 'pocket' proteins (Cobrinik et al., 1993; Beijersbergen et al., 1994; Dynlacht et al., 1994; Ginsberg et al., 1994; Smith and Nevins 1995). pRb binds to some of the E2F proteins (E2F-1, E2F-2 and E2F-3) in complex with DP-1 and inhibits the transactivation function of the complexes (Weinberg, 1995). When pRb is phosphorylated by various cyclindependent kinases, it no longer binds E2F proteins (Chellapan et al., 1991; Hinds et al., 1992; Helin et al., 1993; Kato et al., 1993; Dynlacht et al., 1994). Similarly, p107 can form complexes with E2F-4 and thus inhibit its activity (Beijersbergen et al., 1994; Ginsberg et al., 1994). Moreover, the ability of p107 to bind E2F-4 can also be blocked by phosphorylation by cyclin-dependent kinases (Beijersbergen et al., 1995). Binding of pRb or p107 to E2F factors does not inhibit DNA binding of these factors: pRb was shown to repress E2F-mediated transcription by forming a complex with E2F specifically bound to DNA (Weintraub et al., 1992; Johnson et al., 1994; Sellers et al., 1995). However, pRb was also reported to be part of an inhibitory complex, E2F-I, that blocked the DNA binding activity of E2F in extracts from mouse L cells (Bagchi et al., 1991). E2F-1 binds cyclin A and cyclin-dependent kinase-2. This results in S phase in the phosphorylation of E2F-1 and its heterodimeric partner DP-1, inhibition of their DNA binding and their inactivation (Dynlacht et al., 1994; Krek et al., 1994; M.Xu et al., 1994). Mdm2 and p53 were also reported to modulate E2F-1 activity (Martin et al., 1995; O'Connor et al., 1995). Although the physiological role of the pRb-E2F pathway is unknown, the crucial role of the E2F family of transcription factors in cell proliferation is revealed by the findings that overexpression of E2F-1 in quiescent fibroblasts induces S phase entry and overexpression of
6 Oxford University 5668
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Inhibition of E2F activity by p202
E2F-4 together with an activated ras oncogene can cause oncogenic transformation of primary rodent fibroblasts (Johnson et al., 1993; Beijersbergen et al., 1994). Other E2F and DP family members have also been reported to have proto-oncogenic properties (Singh et al., 1994; Jooss et al., 1995; G.Xu et al., 1995). Mice homozygous for non-functional E2F- 1 alleles were reported recently to be viable and fertile, but to experience testicular atrophy and exocrine gland dysplasia, together with an excess of mature T cells (as a consequence of a defect in maturation stage-specific apoptosis) and also to develop a broad and unusual spectrum of tumors. Intriguingly, these results reveal that the E2F-1 gene may also function as a tumor suppressor gene (Field et al., 1996; Weinberg, 1996; Yamasaki et al., 1996). Many of the antimicrobial, immunomodulatory and cell growth inhibitory activities of the interferons (IFNs) are mediated by IFN-inducible proteins (De Maeyer and De Maeyer-Guignard, 1988; Sen and Lengyel, 1992; Gutterman, 1994; Lengyel et al., 1995). p202 is a murine, 52 kDa, primarily nuclear phosphoprotein whose level is increased in cultured cells 15- to 20-fold in response to treatment with IFN (Choubey and Lengyel, 1993). In vitro p202 binds to double- and single-stranded DNA nonspecifically (Choubey and Gutterman, 1996). Overexpression of p202 in transfected cells is growth inhibitory (Choubey and Lengyel, 1993, 1995; Min et al., 1996). p202 was shown to bind to pRb in vitro and in vivo (Choubey and Lengyel, 1995) as well as to the transcription factors AP- 1, c-Fos and c-Jun and NF-iKB p50 and p65 and to inhibit the transcriptional activity of these factors in vivo (Min et al., 1996). The treatment of IFN-responsive hematopoietic cells with IFN-a has been previously found to arrest these cells in the Gd/GI phase of the cell cycle and to inhibit DNA binding activity of the E2F transcription factor (Melamed et al., 1993). Since expression of p202 in transfected cells inhibited cell proliferation, we tested whether p202 affects E2Fmediated transcription. Here we report that in cells transfected with a p202 expression plasmid, E2F-mediated transcription of a reporter gene and of several endogenous genes was inhibited. This inhibition of E2F activity by p202 was correlated with inhibition of the sequencespecific binding of E2F to DNA. Furthermore, we observed that p202 interacted with E2F (E2F-1/DP-1) both in vitro and in vivo.
Results p202 inhibits E2F-mediated transcription We transfected C33-A human cervical carcinoma cells with an E2F4CAT reporter plasmid in which chloramphenicol acetyl transferase (CAT) expression was driven by four E2F-specific enhancers and tested the effect of the transfection of various amounts of plasmids encoding pRb or p202 on CAT activity in transient assays. As reported earlier (Bandara et al., 1993; Helin et al., 1993; Krek et al., 1993), transfected pRb inhibited CAT activity and the extent of inhibition increased with the amount of pRbencoding plasmid transfected (Figure IA). Transfected p202 also inhibited CAT activity in a concentrationdependent manner (Figure IA). Furthermore, in accord
with earlier observations (Bandara et al., 1993; Helin et al., 1993; Krek et al., 1993), the transfection of a plasmid encoding E2F-I increased CAT activity (-15fold) and co-transfection of plasmids encoding E2F-1 and DP-1 resulted in a further (altogether 19-fold) increase in CAT expression (Figure 1B). Transfection of plasmids encoding pRb or p202 inhibited CAT activity in cells transfected with an E2F-1 plasmid in a concentrationdependent manner (Figure 1B). Furthermore, transfection of a p202-specific plasmid also inhibited CAT expression in cells transfected with both E2F-1- and DP-1-specific plasmids. p202 did not non-specifically inhibit transcription, since the expression of a luciferase reporter gene driven by the SV40 enhancer, an internal control, was not affected reproducibly by the transfected p202. In transient expression assays the level of p202 protein encoded by the transfected expression plasmid might be unphysiologically high. Thus, we proceeded by testing whether constitutive overexpression of p202 in transfected stable cell lines would also inhibit the E2F-regulated transcription of endogenous genes. For this purpose we generated stable murine AKR-2B cell lines overexpressing p202 by transfecting cells with a p202 expression plasmid (pCMV-202) and selecting for G418-resistant colonies. In several independent cell lines obtained the constitutive level of p202 was 2- to 3-fold higher than in the control line, which had been transfected with the vector (pCMV) (data not shown). Two cell lines (CO-1 and CO-3) which were selected for further studies grew more slowly than the control cell line and their cells appeared flat and enlarged (Figure IC). These observations were consistent with earlier findings with murine L929 cells in which p202 was constitutively overexpressed (Min et al., 1996). We compared the mRNA levels of several E2F-regulated genes in control AKR-2B cultures (which had been transfected with the pCMV vector) without and after treatment with IFN (1000 U/ml for 48 h), as well as in two cultures (CO-1 and CO-3) constitutively overexpressing p202. To increase the proportion of cells in the S phase of the cell cycle (in which E2F-mediated transcription is enhanced), we first serum starved our cell cultures for 48 h (resulting in cultures with 80-85% of the cells in the GI phase) and thereafter serum stimulated them for 18 h (resulting in cultures with 75-80% of the cells in S phase). Total cytoplasmic RNA was isolated from the cultures and tested by Northern blotting with appropriate cDNA probes. The data in Figure 1D reveal that constitutive overexpression of p202 in the CO-1 and CO-3 cell lines led to a 6070% reduction in the steady-state levels of PCNA mRNA (lanes 3 and 4 respectively) and an -50% reduction in b-myb mRNA. The steady-state level of c-myc mRNA was, however, not altered. Furthermore, this reduction in steady-state levels of PCNA and b-myb mRNA, but not of c-myc mRNA, in cell lines expressing p202 was consistent with the reduction observed in AKR-2B cells treated with IFN (compare lane 2 with lanes 1, 3 and 4). We proceeded by comparing the rates of transcription of PCNA and of other E2F-regulated mRNAs by nuclear run-on assays in control cells and in one of the p202 overexpressing cell lines, CO-3. The cells were serum starved for 48 h and then serum stimulated for 18 h, as described above. Nuclei were prepared from equal numbers of cells and transcription in the presence of [32P]UTP was
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Fig. 1. Inhibition of E2F-mediated transcription by expression of p202 or pRb in transfected cells. (A) Inhibition of transcription mediated by endogenous E2F. Growing C33-A cells were transfected with E2F4 CAT reporter plasmid, pGL2 (encoding luciferase) as an internal control plasmid and, if so indicated, with plasmids encoding pRb (pCMV-Rb) or p202 (pCMV-202) in the amounts indicated. Thirty six hours after transfection the cell extracts were assayed for CAT and luciferase activities. The CAT activities were normalized to luciferase, whose activity varied in a 2-fold range. The normalized CAT activity in the culture not transfected with plasmids encoding pRb or p202 was taken as 1. The error bars represent standard deviation. (B) Inhibition of transcription boosted by transfected E2F-1 and DP-1. The conditions were as in (A), except that, if so indicated, the cultures were also transfected with plasmids encoding E2F-1 or DP-1. The CAT activities shown were normalized to luciferase activities. The normalized CAT activity in the culture not transfected with plasmids encoding E2F-1, DP-1, p202 or pRb was taken as 1. The luciferase activities were not affected reproducibly by transfection of the p202-specific plasmid. For further details see Materials and methods. (C) Altered morphology of AKR-2B cells constitutively overexpressing p202. Cells transfected with the plasmid vector (pCMV), serving as the control (A), or with a plasmid expressing p202 (pCMV-202) (B and C) were selected for G418 resistance to obtain the two stable cell lines CO-1 (B) and CO-3 (C). These grew more slowly than the control line and their cells were larger and flatter. (D) Treatment with IFN or constitutive overexpression of p202 in stable AKR-2B cell lines decreased the amount of mRNAs encoded by some of the E2F-regulated S phase genes. Northern blots were prepared using cytoplasmic RNA from control AKR-2B cultures without or after treatment with 1000 U/ml for 48 h (+IFN) and from cultures of AKR-2B cells constitutively overexpressing p202 (CO-1 and CO-3). Before extracting the RNA the cultures were serum starved for 48 h and then serum stimulated for 18 h. The blots were probed with 32P-labeled cDNA for the indicated genes and were visualized by radioautography. The RNA applied was visualized by staining with ethidium bromide. The blot was stripped prior to reprobing. (E) Constitutive overexpression of p202 in stable AKR-2B cell lines reduces the rate of transcription of some of the E2F-regulated S phase genes. Nuclear run-on assays were performed using nuclei from a control AKR-2B culture and from an AKR-2B culture constitutively overexpressing p202 (CO-3). Equal amounts (in c.p.m.) of 32P-labeled RNA samples were hybridized to the indicated cDNAs immobilized on a membrane filter, visualized by autoradiography and counted. pBluescript DNA served as a negative control. For further details see Materials and methods. 5670
Inhibition of E2F activity by p202
allowed to continue for 60 min. The incorporation of 32p into particular mRNAs was determined and used as a measure of transcription rate. The data in Figure lE reveal that the rates of transcription of the DHFR and PCNA genes were 60-80% diminished in the p202 overexpressing CO-3 line, whereas the rates of transcription of c-mnc and cyclin A were similar in the two cell lines. These results, which are in accord with the results of the Northern blotting (Figure ID), indicate that overexpression of p202 results in a decrease in the rate of transcription of several, but not all, of the E2F-regulated genes.
Interaction of p202 with E2F (E2F- 1/DP- 1) in vitro Exploring the basis of inhibition of the transcriptional activity of E2F by p202 we examined whether p202 interacts with E2F (E2F- 1/DP- 1) in vitro. For this purpose, we immobilized glutathione S-transferase-E2F-1 (GSTE2F- 1) or GST-DP- 1 fusion proteins on glutathioneSepharose beads and used the beads as affinity reagents for 35S-labeled p202 protein translated in vitro. The GSTE2F- 1- or GST-DP- 1-Sepharose beads retained p202 (Figure 2A, lanes 3 and 4), whereas the GST-Sepharose beads did not (lane 2). The p202-related protein p204 (Choubey et al., 1989; Choubey and Lengyel, 1992) (which contains two 200 amino acid repeat segments similar in sequence to two such segments in p202) was not retained by GST-E2F-1- and GST-DP-1-Sepharose (not shown), providing further evidence for the selectivity of the interaction between p202 and E2F-1. Immobilized, essentially full size p202 [GST-p202(19-445)] (Choubey and Lengyel, 1995) bound 35S-labeled E2F-1 (Figure 2B, lane 3), whereas immobilized GST did not (lane 2). Furthermore, GST-p202(19-445) failed to bind p204, luciferase and three brome mosaic virus-encoded proteins (of 109, 94 and 35 kDa) (data not shown), once again attesting to the selectivity of the interaction. To identify domains in p202 involved in binding to E2F- 1, we generated a series of GST-p202 deletion mutants. Labeled E2F- 1 did not bind to the GSTp202(295-445) segment (Figure 2B, lane 9), but did bind to various other p202 segments (lanes 4, 7 and 8), the shortest of which, GST-202(255-293) (lane 8), contained a 38 amino acid segment. Since E2F-1 (Krek et al., 1994; M.Xu et al., 1994) and p202 (Choubey and Lengyel, 1993) can be phosphorylated in vivo, which might in turn affect their interactions, we tested whether E2F-1 from extracts of murine AKR-2B cells or human HeLa cells would also bind to p202. E2F- 1 from either of these extracts bound to GST-p202(19-445) but not to GST (Figure 2C, compare lane 2 with 1 and lane 3 with 4). Furthermore, p202 from extracts of IFNtreated AKR-2B cells bound to GST-E2F-1 but not to GST (Figure 2D, compare lane 4 with 3). However, no p202 binding to GST-E2F-1 (or to GST) was detected in extracts from cells not exposed to IFN, likely because these cells express only low levels of p202 (Choubey and Lengyel, 1993). Since pRb, which can bind both E2F-1 (Weinberg, 1995) and p202 (Choubey and Lengyel, 1995), is present in reticulocyte lysates and in cell extracts and could act as an adaptor for the binding of p202 to GST-E2F-1, we tested in vitro whether p202 can bind to E2F- 1 directly. We incubated purified, recombinant GST-p202 with GST-
E2F- 1 and immunoprecipitated proteins from the reaction mixture with a monoclonal anti-E2F- 1 antibody. As revealed by SDS-PAGE and Western blotting with an immunopurified anti-p202 antibody, this resulted in the co-immunoprecipitation of p202 with E2F- 1 (Figure 2E, lane 5). This indicates that p202 binds E2F- 1 directly. To support the validity of this conclusion we verified that anti-E2F- 1 antibodies did not immunoprecipitate GSTp202 in the absence of GST-E2F- 1 (lane 2), anti-p202 antibodies did not cross-react with GST-E2F- 1 (lane 1) and GST, even in large excess, did not affect the binding of GST-p202 to GST-E2F- 1 (compare lanes 4 and 5).
Interaction of p202 with E2F- 1 in vivo Whether E2F-1 and p202 interact in vivo was first tested in the yeast two-hybrid system (Vojtek et al., 1993; Hollenberg et al., 1995). As revealed by comparing 3-galactosidase activities, the interaction between E2F- 1 and p202 was as pronounced as that of E2F- 1 with pRb, but weaker than the strong interaction between the MyoD and Da proteins used as positive control (not shown). The association of E2F- 1 with p202 in vil'o was next explored using extracts from AKR-2B cells which had been treated with IFN to increase the level of p202 and extracts from control AKR-2B cells. The extracts were processed by immunoprecipitation with monoclonal antiE2F- 1 antibodies linked to agarose beads. The immunoprecipitated proteins were analyzed by SDS-PAGE and Western blotting with immunoaffinity-purified anti-p202 antibodies. Immunocoprecipitation of p202 with E2F- 1 (Figure 3, lane 2) indicated that in IFN-treated cells p202 is associated with E2F- 1. No p202 was detected in the immunoprecipitate from the extracts of cells not exposed to IFN (lane 1). This was expected, in view of the low level of p202 in those cells which had not been exposed to IFN (Choubey and Lengyel, 1993). It should be noted that monoclonal anti-E2F- 1 antibodies did not cross-react with p202 and immunoprecipitated p202 only as a consequence of its association with E2F- 1 (not shown).
p202 and pRb can bind to two distinct, non-overlapping segments in E2F-1 pRb was shown to bind directly to an 18 amino acid segment of E2F- 1 located within the transactivation region close to the C-terminus of E2F- 1 (Figure 4A) (Helin et al., 1992; Kaelin et al., 1992; Shan et al., 1992). We established that E2F- 1 truncated at its C-terminus [E2F- 1(1-285)] and lacking the pRb binding segment did not bind pRb (Figure 4B, lane 8), but bound to p202 (lane 6). Furthermore, an even shorter E2F- 1 segment [E2F- 1 (88-241)] also bound to p202 (Figure 4C, lane 3). This indicated that amino acids 1-87 and 242-437 of E2F- 1 were dispensable for p202 binding and that p202 and pRb can bind two distinct, non-overlapping segments in E2F- 1. Inhibition of the specific DNA binding activity of E2F by IFN treatment and expression of p202 Since p202 bound a short E2F- 1 segment extending from amino acid 88 to 241 that included the DNA binding domain of E2F- 1 (Helin et al., 1992; Ivey-Hoyle et al., 1993; Jordan et al., 1994), we examined whether the binding of E2F- 1 to DNA was impaired in an extract from
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binding to E2F-I1 was tested. The thin vertical lines indicate the borders of the 200 amino acid positions of the conserved MFHATVAT sequences. The numbers of the N- and C-termninal aminoacyl residues of these sequences are indicated. +, binding; -, no detectable binding. (C) Binding of E2F- 1 in cell extracts to and 2) and HeLa cells (lanes 3 and 4) were incubated with glutathione-Sepharose beads GST-202. Extracts from confluent AKR-2B cells (lanes and 4) or GST-202 (lanes 2 and 3). After washing the beads, the bound proteins were analyzed by SDS-PAGE and loaded with GST (lanes Westemn blotting with an anti-E2F-1 monclonal antibody. The E2F-1 band is indicated by an arrow. (D) Binding of p202 in cell extracts to and 3) or after exposure to IFN (lanes 2 and 4) were incubated with glutathioneGST-E2F- 1. Extracts from growing AKR-2B cells without (lanes and 2) or GST-E2F-1 (lanes 3 and 4). After washing the beads, the bound proteins were eluted and Sepharose beads loaded with GST (lanes analyzed by SDS-PAGE followed by Western blotting using anti-p202 antiserum. Aliquots from total extracts of control and IFN-treated AKR-2B schematic
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Materials and methods.
IFN-treated AKR-2B cells in which levels of p202 are higher than in control cells (Choubey and Lengyel, 1993). In an electrophoretic mobility shift assay (EMSA) with an extract from control AKR-2B cells, we detected one E2F-specific band (Figure 5A, lane 1). This band was supershifted with monoclonal antibodies to human p107, suggesting that this E2F complex contained p107 (data not shown). Addition of deoxycholate (DOC), which is known to release proteins associated with E2F (Bagchi et al., 1990), gave rise to a new E2F-specific band ('free E2F'; Figure 5A, lane 4). This result indicated that extracts from AKR-2B cells did not contain detectable amounts 5672
of free E2F and was in accord with a previous report concerning mouse L cell extracts (Bagchi et al., 1990). IFN treatment of AKR-2B cells, which increases the level of p202 ~16-fold (Choubey and Lengyel, 1993), strongly inhibited the DNA binding activity of E2F in the cell extracts (Figure 5A, lane 6). Next we examined whether IFN-induced expression of p202 correlated with inhibition of the sequence-specific DNA binding of E2F. For this purpose AKR-2B cells were either exposed to increasing concentrations of IFN for 48 h (Figure 5B) or exposed to 1000 U/ml IFN for increasing lengths of time (Figure 5C). Whole cell extracts
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Fig. 3. Interaction between p202 and E2F- 1 in vivo. Immunoprecipitation of p202 with E2F- 1 from a cell extract. Extracts from serum starved control (lane 1) or IFN-treated AKR-2B cells (lane 2) were prepared and immunoprecipitated with monoclonal anti-E2F-l antibodies conjugated to agarose and analyzed by immunoblotting with anti-p202 antiserum. The p202 band is indicated by an arrow. For further details see Materials and methods.
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then analyzed for E2F DNA binding activity by EMSA (Figure SB and C, upper panels) and for p202 levels by Western blotting (lower panels). As seen in Figure 5B and C, in the extracts prepared from IFNtreated cells, inhibition of E2F DNA binding activity was correlated with the increase in the level of p202. It should be noted that the increase in inhibition in the DNA binding of E2F from 24 to 36 and 48 h of IFN treatment (Figure SC, compare lane 3 with lanes 4 and 5) may be the consequence, in part, of the slow translocation of p202 from the cytoplasm to nuclei. We tested, in transient assays, whether overexpression of p202 in transfected cells affects the DNA binding activity of E2F. We transfected a p202 expression plasmid (pFLAG-CMV-202) or, as a control, the pFLAG-CMV vector into growing AKR-2B cells. As in the previous experiment (Figure SA), the extract from control cells revealed only one E2F-specific band (Figure 6A, lane 1) and the addition of DOC gave rise to free E2F (lane 4). The expression of p202 in the transfected cells inhibited the DNA binding activity of E2F in the cell extract (lane 6). Although only 4-7% of cultured cells (estimates based on separate experiments) were transfected with the pFLAG-CMV-202 plasmid, the overexpression of p202 in transfected cells was apparently sufficient to inhibit in the cell extracts the bulk of DNA binding by E2F (see also Figure 6). The extent of inhibition in four separate experiments varied from 50 to 75% and the maximal inhibition was manifested 48 h after transfection. This was as expected, since we established earlier that the bulk of p202 is detected in the nucleus of AKR-2B cells only 35-40 h after IFN treatment (Choubey and Lengyel, 1993). The DNA binding activity of the transcription factors Oct- I and Sp- 1 was not inhibited significantly in these cell extracts (data not shown). Therefore, these observations suggested that p202 expression diminished the DNA binding of E2F selectively. Similar results were obtained after transfecting the p202 expression plasmid into human C33-A cells (data not shown). were
To examine whether the release of E2F-associated
proteins can alleviate inhibition of the DNA binding activity of E2F in extracts prepared from cells treated with IFN or transfected with p202, we treated the extracts with DOC or EDTA, a chelating agent shown to diminish ionic interactions between proteins (Montenarh and Quaiser, 1989; Bagchi et al., 1990). Treatment with EDTA
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Fig. 4. p202 and pRb can bind to non-overlapping segments in E2F- 1. (A) Schematic representation of E2F-1 (1-437, full-length) and two of its deletion mutants and their binding to p202 and pRb. Domains in E2F-1 and the numbering of their N- and C-terminal aminoacyl residues are indicated. NT, not tested. (B) Assay of the binding of E2F-1 and C-terminally truncated E2F-1 to p202 and to pRb linked to GST. 35S-Labeled E2F-1(1-437) or 35S-labeled E2F-1(1-285) (denoted AE2F-1) translated in vitro were incubated with glutathione-Sepharose beads loaded with GST (lanes 3 and 4 respectively) or GST-p202 (lanes 5 and 6 respectively) or GST-pRb (lanes 7 and 8 respectively). The bound proteins were eluted and analyzed by SDS-PAGE and fluorography. 35S-Labeled E2F-1(1-437) (IVT-E2F-l. lane 1) and E2F-1(1-285) (IVT-AE2F-1. lane 2) were also run and are indicated by arrows. (C) Assay of the binding of p202 to N- and C-terminally truncated E2F-1 linked to GST. 35S-Labeled p202 translated inl vitro was incubated with glutathione-Sepharose beads loaded with GST (lane 2) or GST-E2F-1(88-241) (lane 3). The bound proteins were eluted and analyzed by SDS-PAGE. An aliquot of 35S-labeled p202 was also run (IVT-202, lane 1). The p202 band is indicated by an arrow. For further details see Materials and methods.
did not appreciably increase DNA binding by E2F in the extract from control cells (Figures 5A and 6A, compare lanes 1 and 7). It did, however, overcome inhibition of the DNA binding activity of E2F in the extracts from cells treated with IFN (Figure 5A, compare lanes 6 and 8) or transfected with a plasmid encoding p202 (Figure 6A, compare lanes 6 and 8). Treatment with DOC did not overcome the inhibition. The level of p202 expression may be unphysiologically high in transient transfection assays. Consequently, we also tested whether the sequence-specific binding of E2F to DNA was inhibited in extracts from the two stable cell lines (CO- I and CO-3) in which the constitutive expression of p202 was 2- to 3-fold higher than in the control AKR2B cell line. As seen in the EMSA in Figure 6B, sequencespecific DNA binding of E2F was much lower in extracts from cells of the CO- 1 and CO-3 lines than in extracts from control cells (compare lanes 5 and 6 with lane 3). This reveals that the 2- to 3-fold overexpression of p202
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Fig. 5. Inhibition of the specific DNA binding activity of E2F in AKR-2B cells treated with IFN. EMSA with a labeled E2F-specific oligonucleotide. (A) EDTA overcomes inhibition by IFN in vitro. Complexes in extracts from control cells (lanes 1-5 and 7) and cells treated with 1000 U/ml IFN for 48 h (lane 6 and 8), no further treatment (lane 1, 5 and 6), addition of a 50-fold excess of unlabeled wild-type E2F oligonucleotide (lane 2), addition of a 100-fold excess of a mutant E2F oligonucleotide (lane 3), DOC treatment (lane 4) and EDTA treatment (lane 7 and 8). The position of the E2F and 'free' E2F bands are indicated by thick and thin arrows respectively. An E2F-specific band competed by only 100-fold excess or more of wild-type E2F oligonucleotide is indicated by a circle. The mutant E2F oligonucleotide did not compete for binding to E2F with the labeled wildtype oligonucleotide (lane 3), whereas the wild-type oligonucleotide did (lane 2). (B) Dependence on IFN concentration. (Upper panel) Complexes in extracts from control cells (lanes 1-3), no further treatment (lane 3), addition of a 50-fold excess of wild-type E2F oligonucleotide (lane 1), DOC treatment (lane 2), complexes in extracts from cells treated for 48 h with IFN at 250 (lane 4), 500 (lane 5) or 1000 U/ml (lane 6). The positions of the E2F and 'free' E2F bands are indicated by thick and thin arrows respectively. (Lower panel) Western blot assay of the levels of p202 in control cells (lane 1), cells exposed for 48 h to IFN at 250 (lane 2), 500 (lane 3) and 1000 U/ml (lane 4). The p202 band is indicated by an arrow. (C) Dependence on the length of the IFN treatment. (Upper panel) Complexes in extracts from control cells (lane 1), cells exposed to IFN at 1000 U/ml for 12 (lane 2), 24 (lane 3) or 48 h (lane 5). The position of the E2F bands is indicated by a thick arrow. (Lower panel) Western blot assay of the levels of p202 in control cells (lane 1), cells exposed to IFN at 1000 U/ml for 12 (lane 2), 24 (lane 3) or 48 h (lane 5). For further details see Materials and methods.
suffices to impair the specific DNA binding activity of E2F. As shown already in Figure 5, treatment with IFN diminished DNA binding of E2F in the cell extracts (Figure 6B, compare lane 4 with lane 3). Furthermore, the addition of an extract from IFN-treated cells to an extract from control cells (in a 1:1 ratio) resulted in a strong inhibition of DNA binding of E2F (compare lane 7 and lane 3). This reveals that p202 in the extracts from IFN-treated cells did inhibit DNA binding of E2F in the extracts from control cells (i.e. in trans). Similarly, the mixing of an extract from cells constitutively overexpressing p202 with an extract from control cells (in a 1:1 ratio) also resulted in a strong inhibition of the DNA binding activity of E2F in control extracts. These observations are consistent with a direct inhibitory interaction between p202 and E2F-1.
Discussion Transfection of a p202 expression plasmid into AKR-2B and L929 cells resulted in only a few clones (Choubey and Lengyel, 1995; Min et al., 1996) and those clones that constitutively overexpressed p202 (2- to 3-fold above the basal level) grew much more slowly than clones transfected with the control vector. Moreover, constitutive overexpression of p202 in transfected NIH 3T3 cells was reported to inhibit the exit of serum starved cells from
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GO/G1 phase after serum stimulation (Lembo et al., 1995). The results presented in this study reveal that p202 inhibits the activity of E2F and thereby shed light on a mechanism that is likely to contribute to growth inhibition by p202. In transient assays involving cells transfected with a p202 expression plasmid the transcriptional activities of the endogenous E2F transcription factors and of a transfected E2F- 1 transcription factor were found to be inhibited. Since the level of p202 might be unphysiologically high in transient transfection assays, we repeated the experiments using stable AKR-2B lines in which the constitutive level of p202 was 2- to 3-fold higher than in cells from the control line and which grew more slowly than the control line. Experiments involving Northern blotting revealed that the levels of mRNAs encoded by several E2F-regulated endogenous genes (i.e. PCNA and b-myb) were much lower in cells from the lines overexpressing p202 than in cells from the control line. Since p202 is inducible by IFN, it was expected that treatment of the control cells with IFN might also result in a decrease in the level of the PCNA and b-myb mRNAs. This expectation was verified. Further studies, involving nuclear run-on assays, established that the decrease in E2F-regulated mRNA levels in cells overexpressing p202 was a consequence, at least in part, of a decreased rate of transcription. The finding that constitutive overexpression of p202 or
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Fig. 6. Inhibition of the specific DNA binding activity of E2F in AKR-2B cells by p202. EMSA with a labeled E2F-specific oligonucleotide. (A) Inhibition by a transfected p202 expression plasmid. Complexes in extracts from cells transfected with the pFLAG-CMV vector (serving as control) (lanes 1-5 and 7), cells transfected with pFLAG-CMV-p202 expression plasmid (lanes 6 and 8). no further treatment (lanes 1 and 5), addition of a 50-fold excess of wild-type E2F oligonucleotide (lane 2), a 50-fold excess of mutant E2F oligonucleotide (lane 3). DOC treatment (lane 4). EDTA treatment (lanes 7 and 8). The E2F and 'free' E2F bands are indicated by thick and thin arrows respectively. (B) Inhibition in stable cell lines overexpressing p202. (Upper panel) Complexes in extracts from control cells transfected with the pCMV vector (lanes 1-3), with no further treatment (lane 3). a 50-fold excess of wild-type E2F oligonucleotide (lane 1), DOC treatment (lane 2). Complexes in extracts from cells transfected with the pCMV vector and treated with IFN at 1000 U/ml for 48 h (lane 4), complexes in extracts from cells constitutively overexpressing p202 from the CO-1 line (lane 5) and the CO-3 line (lane 6), complexes in a 1:1 mixture of extracts from control cells and cells treated with IFN (lane 7). The position of the E2F and 'free' E2F bands are indicated by thick and thin arrows respectively. (Lower panel) Western blotting of the levels of p202 in control cells (lane 1), cells treated with IFN (1000 U/mI for 48 h) (lane 2), cells constitutively overexpressing p202, the CO-1 line (lane 3) and CO-3 line (lane 4). For further details see Materials and methods.
IFN treatment retards cell proliferation made it conceivable that inhibition of the transcriptional activity of E2F by p202 might have been a consequence of the retarded cell proliferation of these cells. Several observations reported in this study make this possibility unlikely: (i) p202 bound E2F- 1 directly in vitro; (ii) in the yeast two-hybrid system, the interaction between E2F- 1 and p202 was as pronounced as that of E2F- I with pRb; (iii) p202 and E2F were immunocoprecipitated from the cell extracts, indicating an association in vivo; (iv) transfected or constitutively overexpressed p202 inhibited the specific DNA binding of E2F in cell extracts; (v) treatment of extracts from cells overexpressing p202 with EDTA released inhibition of the specific DNA binding of E2F by p202. All these observations are in line with a direct effect of p202 on E2F activity. Our experiments concerning the interaction between p202 and E2F in vitro were restricted to the E2F- 1 protein. However, the strong inhibition of E2F activity by p202 in vivo makes it probable that p202 may also affect the activity of other members of the E2F family. Indeed, data to be reported elsewhere indicate that binding of p202 to E2F-4 inhibits activity of the latter (Choubey et al., in
preparation). The finding that p202 inhibited the specific DNA binding of E2F is in accord with binding of p202 to the E2F-1 domain implicated in DNA binding (Helin et al., 1992; Jordan et al., 1994). The precise localization of the
p202 binding domains in E2F- 1 will require further studies involving deletions and mutagenesis. It may be relevant to note that the binding of p202 to the transcription factors c-Fos and c-Jun also takes place through the DNA binding domains of these proteins and that p202 also inhibits the specific DNA binding and transcriptional activities of these two transcription factors (Min et al., 1996). The observation that treatment with EDTA can release inhibition of the specific DNA binding of E2F by p202 is in accord with earlier observations concerning inhibition of the specific DNA binding of E2F by IFN (Melamed al., 1993). Our data are consistent with an inhibition of E2F activity by p202 that is based on a direct interaction between these two proteins. However, the fact that p202 can interact with other proteins (e.g. DP-1, pRb and p107) that are known to affect transcription by E2F makes it conceivable that modulation of E2F activity by p202 is more complex. Thus, the finding that p202 could bind pRb and E2F- 1, together with the fact that p202 and pRb bound to distinct and non-overlapping domains of E2F-1 make it conceivable that a complex including all three of these proteins (and possibly also a DP protein) may exist. The finding that p202 could inhibit E2F activity in cells from a line (C33-A) expressing a mutant pRb, defective in the pocket region, indicated that inhibition by p202 did not depend on fully functional pRb. However, the fact that p202 also bound to the N-terminal segment of pRb et
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(Choubey and Lengyel, 1995) leaves open the possibility that pRb with a defective pocket region may form a complex with p202 and might contribute to inhibition. It remains to be established whether an inhibitor (E2F-I) which was detected in mouse L cell extracts and inhibited DNA binding by E2F contains p202. In conclusion, the results presented indicate that the IFN-inducible p202 protein can inhibit E2F-mediated transcription. The inhibition of transcription of S phasespecific genes (e.g. PCNA, DHFR and b-myb) by p202 expression is likely to contribute to the antiproliferative activity of the IFNs. p202 joins a set of proteins (including, for example, pRb, p107, Mdm2 and p53) reported to interact with E2F proteins and modulate their transcriptional activity (Martin et al., 1995; O'Connor et al., 1995; Weinberg, 1995). Though p202 and pRb both inhibit transactivation by E2F- 1, p202 impairs the specific binding of E2F-1 to DNA, whereas pRb does not. Thus, p202 might inhibit both activation and repression of gene expression by E2F- 1. It remains to be established whether defects in the expression of functional p202 contribute to aberrant cell proliferation.
Materials and methods Plasmids The plasmids used for expressing labeled p202 in reticulocyte lysates and GST-p202 proteins in Escherichia coli have been described (Choubey and Lengyel, 1995). The 202 cDNA (Choubey et al., 1989) was cloned into the BamHI site of the plasmid pCMV to generate pCMV-202. Human E2F- 1 mRNA was transcribed and translated in vitro from the pCMVE2F-1 plasmid (Johnson et al., 1993) (a generous gift from J.Nevins). The GST-E2F-1(2-437) plasmid was constructed by ligating a StyI-BamHI DNA fragment from pCMV-E2F-1 (after blunt ending) into the SmaI site of the pGEX-2T vector (Pharmacia). The GST-E2F-1(1-285) plasmid was constructed by cleaving GST-E2F1(2-437) with AJIII and BamHI and eliminating the small fragment of E2F-1 before ligation. pCMV-E2F-1, pCMV-DP-1 and E2F4CAT plasmids were kindly provided by K.Helin and E.Harlow (Helin et al., 1992, 1993).
activity was in a 2-fold range. Protein concentration was determined with the BioRad protein assay kit.
Yeast two-hybrid assay The procedures of Vojtek et al. (1993) and Hollenberg et al. (1995) were followed for: (i) transforming the yeast strains L40 (MATa) and AMR70 (MATa) with the indicated plasmids; (ii) testing by f-galactosidase assay for pairwise interactions in yeast between proteins, one of which was fused to the LexA DNA binding domain and the other to the VP-16 transactivation domain. In each case three independent colonies were assayed. Vectors for expression in yeast were generously provided by S.Hollenberg: pBTM116 (constructed by P.Bartel and S.Fields) was used for linkage of E2F-1 to the LexA DNA binding domain and pVP16 was used for linkage of p202 and pRb to the VP16 transactivation domain. The constructs used as positive and negative controls were generously provided by S.Hollenberg. Plasmids encoding p202 linked to the transactivation domain of VP-16 and E2F-1 linked to the DNA binding domain from LexA were introduced into yeast. f-Galactosidase driven by a LexA-specific enhancer served as the reporter gene. The interactions between proteins encoded by the MyoD(A) and Da-(D) constructs and between proteins encoded by the pRb(A) and E2F-1-(D) constructs served as positive controls and the lack of interactions between the proteins encoded by the lamin-(D) and either the p202-(A) or the pRb-(A) constructs served as negative controls.
GST fusion proteins and affinity chromatography For expression of GST fusion proteins, the appropriate plasmids were introduced into Ecoli DHSa. The fusion proteins were affinity purified on glutathione-Sepharose beads as described (Kaelin et al., 1992) and their amounts were estimated by SDS-PAGE. The beads were loaded with fusion protein and extracts from AKR-2B cells or HeLa cells were prepared and subjected to affinity chromatography on the immobilized fusion proteins as reported earlier (Choubey and Lengyel, 1995).
lmmunoprecipitation and Western blotting Extracts from AKR-2B cells (control or treated with IFN) were prepared as described (Choubey and Lengyel, 1995). The extracts were incubated with monoclonal anti-E2F-1 antibodies (KH95), conjugated to agarose beads (Santa Cruz Biotechnology Inc.), at 4°C for 2 h. The beads were washed with extraction buffer five times and the bound proteins were eluted with extraction buffer supplemented with 500 mM NaCl and analyzed by SDS-PAGE and Western blotting with an anti-p202 antiserum (Choubey and Lengyel, 1995). Similarly, whole cell extracts prepared from control or p202 expressing cell lines (CO-l and CO-3) were analyzed by SDS-PAGE and Western blotting with an anti-p202 antiserum (Choubey and Lengyel, 1995).
Cell lines
Transcription and translation in vitro
Murine AKR-2B cloned embryo cells, human HeLa and C-33A cervical carcinoma cells (from the American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Choubey and Lengyel, 1992). When indicated, the cells were treated with recombinant human IFNa2/a1(1-83) (1000 U/ml for the time indicated). This IFN is highly active in murine cells (Weber et al., 1987). AKR-2B cells were transfected with the pCMV-202 expression plasmid or with the pCMV vector by the calcium phosphate precipitation method. Following G418 (450 jg/ml) selection 15 G418resistant colonies were obtained. Two of these (CO-1 and CO-3) expressed p202 at a level approximately 2- to 3-fold higher than that in the control cell lines (AKR-2B or AKR-2B carrying the pCMV vector). The CO-1 and CO-3 cell lines were grown and maintained in complete DME medium containing 250 gg/ml G418.
mRNAs for p202, pRb and pE2F-l were transcribed from pBluescript202, pGEM-Rb and pCMV-E2F-1 respectively and were translated in a rabbit reticulocyte lysate supplemented with [35S]methionine as described (Choubey and Lengyel, 1995).
Transfections and reporter assays C33-A cells grown on 100 mm culture dishes were transfected using the calcium phosphate precipitation method (Sambrook et al., 1989) with 4 ,ug E2F4CAT reporter plasmid (CAT expression driven by four E2F-specific sequences), 4 jg pGL2 (luciferase expression driven by SV40 enhancer and promoter) as the internal control plasmid and, if so indicated, with plasmid encoding E2F-1 (pCMV-E2F-1, 0.2 ,ug), DP-l (pCMV-DP- 1, 4 jig) as well as pRb (pCMV-Rb) and p202 (pCMV-202) in the amounts specified. pBluescript SK was used to adjust the total amount of DNA transfected to 20 jg. The cultures were harvested after 36-48 h and the extracts were assayed for CAT and luciferase activities (De Wet et al., 1987; Sambrook et al., 1989). In extracts from cells not transfected with pRb- or p202-specific plasmids 10-55% of the chloramphenicol was acetylated. The variation in luciferase specific
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RNA detection Cells were cultured in 150 mm dishes to 50% confluency and then incubated in DME medium containing 0.1 % fetal bovine serum for 48 h to increase cells in GI phase. Following serum starvation cells were incubated in DME medium supplemented with 20% fetal bovine serum for 18 h to increase cells in S phase. Cells were processed for propidium iodide staining and fluorescence activated cell sorting to determine cell cycle distribution. Cytoplasmic RNA was extracted and Northern blotting was performed according to Sambrook et al. (1989) or nuclei from 20 000 000 cells were processed for nuclear run-on analysis as described by Farrell (1993). Equal amounts (c.p.m.) of 32P-labeled transcripts were hybridized to membrane carrying immobilized cDNAs. The PCNA and murine c-myc cDNA used as probes for Northern or nuclear run-on analysis were kindly provided by Drs Rodrigo Bravo and Adrian Hayday respectively. Oligoprobes (40mer) complementary to murine b-myb and human cyclin A mRNA were synthesized. DHFR probe was purchased from the American Type Culture Collection. Hybridizations were performed for 1-1.5 h at the desired temperature using rapid hybridization buffer (Clontech Inc.) as suggested by the supplier.
Electrophoretic mobility shift assays
(EMSA)
To transfect growing AKR-2B or C33-A cells (in 150 mm dishes) calcium phosphate precipitates containing (50 ig) plasmid pFLAGCMV (from IBI, Kodak) or pFLAG-CMV-202 were incubated with the
Inhibition of E2F activity by p202 cells for 24 h prior to the medium change. For EMSA extracts from control, IFN-treated or transfected AKR-2B cells were prepared in buffer (400 mM KCI, 20 mM HEPES, pH 7.5, 20% glycerol, 1 mM DTT, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 50 tg/ml leupeptin, 5 ,ug/ml aprotinin) by freezing and thawing cells (three times) and centrifuging the lysate at 4°C for 10 min. The lysates were stored at -70°C until use. The binding conditions of E2F to 32P-labeled wild-type E2F oligonucleotide (5'-ATTTAAGTTTCGCGCCCTTTCTCA-3', 0.5 ng) were as described (Bagchi et al., 1990). The EDTA treatment was according to Melamed et al. (1993). The samples were run on a 4% acrylamide gel in 0.3X TBE (22.5 mM Tris-borate, 0.5 mM EDTA) at 4°C. The mutant E2F oligonucleotide (5'-ATTTAAGTTTCGATCCCTTTCTCA-3') was obtained from Santa Cruz Biotechnology Inc.
Acknowledgements We are grateful to K.Helin and E.Harlow for the pCMV-E2F-l, pCMVDP- I and E2F4CAT plasmids, to J.Nevins for the pCMV-E2F- I plasmid. to S.Hollenberg for the reagents and the protocol for a modified version of the yeast two-hybrid assay, to C.Weissmann and H.Weber for human IFNcx2/tx1(l-83), to David J.Hall and Kelly Jordan for GST-E2F-1(88241), to Rodrigo Bravo for the PCNA plasmid, to Adrian Hayday for the c-mvc plasmid, to G.B.Mills for helpful discussions and to Nessie Stewart for typing the manuscript. This work at the University of Texas M.D.Anderson Cancer Center was supported, in part, by a grant from the Biomedical Research Support Group (to D.C.) and, in part, by the Biomedical Research Foundation and, in part, by the Clayton Foundation for Research (to J.U.G.). At Yale University the work was supported by National Institutes of Health Research Grant R37-A12320 (to P.L.).
References Adams,P.D. and Kaelin,W.G.,Jr (1995) Transcriptional control by E2F. Serniui. Cancer Biol., 6, 99-108. Bagchi,S., Raychaudhuri,P. and Nevins,J.R. (1990) Adenovirus EIA proteins can dissociate heteromeric complexes involving the E2F transcription factor: a novel mechanism for ElA transactivation. Cell, 62, 659-669. Bagchi,S., Weinmann,R. and Raychaudhuri,P. (1991) The retinoblastoma protein copurifies with E2F-I, an EIA-regulated inhibitor of the transcription factor E2F. Cell, 65, 1063-1072. Bandara,L.R. and La Thangue,N.B. (1991) Adenovirus ElA prevents the retinoblastoma gene product from complexing with a cellular transcription factor. Natlure, 351, 494-497. Bandara,L.R., Buck,W.M., Zamanian,M., Johnston,L.H. and La Thangue,N.B. (1993) Functional synergy between DP-1 and E2F-1 in the cell cycle-regulating transcription factor DRTFl/E2F. EMBO J., 12, 4317-4324. Beijersbergen,R.L., Kerkhoven,R.M., Zhu,L., Calee,L., Voorhoven.P.M. and Bernards,R. (1994) E2F-4, a new member of the E2F gene family, has oncogenic activity and associates with p107 in vi4io. Genies Del., 8, 2680-2690. Beijersbergen,R.L., CarleeL., Kerkhoven,R.M. and Bernards.R. (1995) Regulation of the retinoblastoma protein-related p107 by GI cyclin complexes. Genes Dev., 9, 1340-1353. Blake.M.C. and Azizkhan,J.C. (1989) Transcription factor E2F is required for efficient expression of the hamster dihydrofolate reductase gene in vitro and in Oivo. Mol. Cell. Biol., 9, 4994-5002.
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