Several Regions of P53 Are Involved in Repression of RNA Polymerase III Transcription

Several regions of p53 are involved in repression of RNA polymerase III transcription

Torsten Stein1, Diane Crighton1, Lorna J Warnock2, Jo Milner2 and Robert J White*,1

1Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, UK; 2YCR P53 Research Group, Department of Biology, University of York, York YO10 5DD, UK

The tumour suppressor p53 has been shown to regulate for ensuring that damage is not perpetuated amongst RNA polymerase (pol) III transcription both in vitro and cellular progeny. Most tumours evade these defences in vivo. We have characterized the regions of p53 that by inactivating p53 (Cox and Lane, 1995; Hollstein et contribute to this effect. Repression of pol III transcrip- al., 1991, 1994; Ko and Prives, 1996; Levine, 1997; tion in vivo does not require residues 13 – 19 near the N- Sionov and Haupt, 1999; Vogelstein et al., 2000; terminus of p53 that are highly conserved through Vousden, 2000). evolution. However, amino acids 22 and 23 in the The effectiveness of p53 in this protective role stems adjacent transactivation domain do contribute to the largely from its ability to regulate batteries of genes inhibition of pol III activity. Deletions within the central (Crook et al., 1994; Ko and Prives, 1996; Pietenpol et DNA-binding core domain (residues 102 – 292) of p53 al., 1994). A well-characterized example is the p21/ can entirely abolish the repression function in these WAF1/CIP1 gene, which is activated by p53 and plays assays, despite the fact that pol III templates contain no an important role in G1 arrest (El-Deiry et al., 1993). recognized p53 binding site. Deletion or substitution The transcriptional activation function of p53 involves within the C-terminal domain of p53 can also compro- sequence-specific binding to promoter DNA (Farmer et mise its ability to repress pol III activity in vitro and in al., 1992; Kern et al., 1991; Zambetti et al., 1992). In transfected cells. These observations reveal that repres- addition, p53 can repress certain genes that lack its sion of pol III transcription is a complex function DNA recognition site (Avantaggiati et al., 1997; Cairns involving multiple regions of p53 extending throughout and White, 1998; Chen et al., 1992; Chesnokov et al., much of the protein. 1996; Crook et al., 1994; Ginsberg et al., 1991; Oncogene (2002) 21, 5540 – 5547. doi:10.1038/sj.onc. Horikoshi et al., 1995; Mack et al., 1993; Murphy et 1205739 al., 1996, 1999; Ragimov et al., 1993; Seto et al., 1992; Subler et al., 1994; Yu et al., 1999; Zhao et al., 2000). Keywords: p53; repression; RNA polymerase III; It is sometimes argued that this repression is relatively TFIIIB; transcription non-specific, since it occurs in the absence of a p53- binding DNA sequence; however, a microarray analysis found that only 0.9% of the *6000 genes Introduction examined were inhibited by p53, whereas 1.8% were induced (Zhao et al., 2000). Since some of the genes The ability of cells to reduce macromolecular synthesis that are repressed by p53 encode growth-promoting whilst concurrently activating stress-response genes products, it has been suggested that the transcriptional provides an essential defence against environmental repression activity may contribute towards the anti- insults. The p53 protein is a key player in this proliferative properties of p53 (Cox and Lane, 1995; protective response in higher organisms (Cox and Ko and Prives, 1996; Neufeld and Edgar, 1998). This Lane, 1995; Ko and Prives, 1996; Levine, 1997; Sionov possibility is supported by the fact that many p53 point and Haupt, 1999; Vogelstein et al., 2000; Vousden, mutants which are unable to inhibit transcription have 2000). Under a range of physiological stress conditions, also lost the capacity to suppress cell growth (Chen et p53 can induce either cell cycle arrest or apoptosis, al., 1992; Crook et al., 1994; Ginsberg et al., 1991; depending on circumstances (Cox and Lane, 1995; Ko Ragimov et al., 1993; Seto et al., 1992). Furthermore, and Prives, 1996; Levine, 1997; Sionov and Haupt, two oncoproteins were found to block specifically the 1999; Vogelstein et al., 2000; Vousden, 2000). The repression function of p53, without affecting its ability arrest function provides an opportunity to repair to activate expression (Shen and Shenk, 1994). damaged DNA before it is replicated and passed on Genes transcribed by RNA polymerase (pol) III to daughter cells. Apoptosis offers an alternative route provide an abundant class of template that is repressed by p53 both in vitro and in vivo (Cairns and White, 1998; Chesnokov et al., 1996). Overexpression of p53 *Correspondence: RJ White; E-mail: [email protected] can repress pol III transcription in transfected cells Received 20 March 2001; revised 16 May 2002; accepted 7 June (Chesnokov et al., 1996), whilst synthesis of tRNA and 2002 5S rRNA by pol III is elevated significantly in Domains used by p53 to repress pol III transcription T Stein et al 5541 fibroblasts derived from p53 knockout mice (Cairns and White, 1998). These effects result from the ability of p53 to bind directly to the pol III-specific factor TFIIIB and suppress its activity (Cairns and White, 1998; Chesnokov et al., 1996). Thus, a stable interaction between TFIIIB and recombinant or endogenous p53 has been demonstrated using cofrac- tionation, coimmunoprecipitation and pull-down assays (Cairns and White, 1998; Chesnokov et al., 1996). In reconstituted transcription experiments, recombinant p53 inactivates TFIIIB with a high degree of specificity (Cairns and White, 1998). Furthermore, p53 knockout fibroblasts display abnormally elevated TFIIIB activity when compared with matched wild- type controls (Cairns and White, 1998). Since sustained growth requires high levels of pol III products, such as tRNA and 5S rRNA, the ability of p53 to repress TFIIIB may contribute towards its growth-restraining capacity. The current study has extended the previous analyses in order to characterize which regions of p53 contribute to its ability to regulate the pol III system. We demonstrate that the central DNA-binding domain is necessary, despite the fact that genes transcribed by pol III rarely contain sequences that resemble the p53 Figure 1 Deletion of conserved region II, III, IV or V prevents recognition motif. Regions both N- and C-terminal to p53 from repressing pol III transcription. (a) Schematic diagram the central core domain are also involved in repression of the organization of p53, illustrating the positions of conserved of pol III transcription, both in vitro and in vivo. regions I – V and the transactivation, specific DNA-binding and oligomerization domains. Numbers indicate amino acid residues. Residues near the N-terminus of p53 that are (b) SAOS2 cells were cotransfected with pVA1 (3 mg), HSV- important for controlling pol III coincide with the CAT (3 mg) and 2 mg of empty vector (lane 1), or vector encoding region that is known to mediate transactivation or wild-type p53 (lane 2) or p53DI (lane 3), p53DII (lane 4), p53DIII repression of pol II templates. Deletion of the C- (lane 5), p53DIV (lane 6) or p53DV (lane 7). RNA and protein were isolated 48 h after transfection. The upper and second panels terminal domain abolishes the ability of p53 to inhibit show primer extension analyses of extracted RNA using primers pol III transcription. The repression function can also specific for VA1 (upper panel) and CAT (middle panel). The third be compromised by substitution within this region. We panel shows a Western blot of extracted protein using anti-p53 conclude that at least three distinct regions of p53 antibody DO-1; the p53DI mutant is not detected because this de- contribute to its capacity to regulate transcription by letion disrupts the epitope recognized by DO-1. Expression of p53DI is therefore confirmed in the bottom panel using antibody pol III. PAb421

Results SAOS2 cells (Kubbutat et al., 1998). As a reporter for The central domain of p53 is required for repression of pol pol III activity we transfected the adenovirus VA1 III transcription gene, as this has an internal promoter which is p53 is generally well conserved through vertebrate extremely well characterized and is representative of evolution, but this is especially true of five regions that the majority of pol III templates, including tRNA are almost identical from trout to man (Soussi et al., genes (Paule and White, 2000; White, 1998a). As an 1990). Of these, region I lies near the N-terminus, internal control for transfection efficiency and RNA whilst regions II – V are all located within the central recovery, we cotransfected a reporter carrying HSV – core domain (residues 102 – 292) that is essential for CAT, in which the chloramphenicol acetyltransferase specific DNA binding (Figure 1a). Since the conserved gene is transcribed from the basal thymidine kinase regions are most likely to be responsible for the promoter of herpes simplex virus. Production of RNA important functions of p53, we tested whether they from the VA1 and HSV – CAT reporters was then contribute to its ability to repress pol III transcription determined by quantitative primer extension assays. A in vivo. We made use of a series of five mutant representative example of such experiments is shown in expression constructs in which the conserved regions Figure 1b. It is clear that wild-type p53 represses the had been deleted individually; these were transfected pol III-dependent expression of VA1, when compared into the p53-negative osteosarcoma cell line SAOS2, as with the empty vector (Figure 1b, top panel); this effect were the equivalent construct expressing wild-type p53 is specific, since the pol II reporter HSV – CAT is not and empty vector, as positive and negative controls, inhibited (Figure 1b, middle panel). Efficient and respectively. Each of the mutants is stably expressed in specific repression of VA1 is also obtained using the

Oncogene Domains used by p53 to repress pol III transcription T Stein et al 5542 DI mutant, in which the N-terminal conserved region has been deleted. Although the VA1 signal with the DI mutant is slightly higher in this experiment than that obtained with the wild-type, this is also true of the HSV – CAT control, suggesting sample overloading; in several repeats, we observe no consistent diﬀerence between the DI mutant and wild-type p53 in these assays. In contrast, deletion of any one of conserved regions II – V prevents p53 from repressing VA1. Western blot analysis of lysates from transfected cells conﬁrms that the inactivity of these mutants is not due to lack of expression, since each is expressed at a comparable level to the wild-type p53 (Figure 1b, bottom two panels). These data demonstrate that the central core domain of p53 is necessary for it to regulate a pol III promoter.

The N-terminal region of p53 contributes to the control of pol III transcription Having established that the central region of p53 is required for it to repress the VA1 gene, we next asked whether sequences outside this core domain are also involved. To begin to address this, we used recombinant p53 protein that was expressed in Sf9 cells using a baculovirus vector. The recombinant p53 carries a polyhistidine tag, which allowed its affinity-purification to near homogeneity (Figure 2a). When titrated into a nuclear extract, wild-type p53 produced a clear repression of VA1 transcription (Figure 2b, lanes 1 – Figure 2 The N-terminal 23 residues of p53 are important for re- 3). In contrast, no inhibition was observed using an pression of pol III transcription, whereas C-terminal 29 residues equal amount of mutant p53(67 – 363), which is missing are not. (a) Two hundred ng of p53(24 – 392) (lane 1), p53(1 – the N-terminal 66 residues and the C-terminal 29 363) (lane 2), p53(67 – 363) (lane 3) and wild-type p53 (lane 4) were electrophoresed on a 10% SDS-polyacrylamide gel and then residues (Figure 2b, lanes 4 – 6). This demonstrates that visualized by staining with Coomassie Blue. (b) pVA1 (250 ng) the regulation obtained with full-length p53 is a specific was transcribed using HeLa nuclear extract (15 mg) that had been effect and further suggests that control of pol III preincubated for 10 min at 308C without addition (lanes 1, 4, 7, activity requires sequences outside the central core 10 and 13), or with the addition of 0.6 mg or 1.2 mg of wild-type p53 (lanes 2 and 3, respectively), 0.6 and 1.2 mg of p53(67 – 363) domain. The mutant p53(1 – 363), in which just the C- (lanes 5 and 6, respectively), 0.6 and 1.2 mg of p53(1 – 363) (lanes terminal 29 residues are deleted, was found to retain 8 and 9, respectively), or 0.6 and 1.2 mg of p53(24 – 392) (lanes 11 the ability to repress VA1 gene transcription (Figure and 12, respectively) 2b, lanes 7 – 9). In contrast, deletion of residues 1 – 23 in the mutant p53(24 – 392) was found to be sufficient to inactivate p53 in this assay (Figure 2b, lanes 10 – 13). It is important to note that the central core involved in regulating VA1 expression. In support of domain remains intact and in the wild-type conforma- this, we found that full-length p53 carrying a double tion in all of the mutants tested in this experiment; this substitution at positions 22 and 23 was less efficient at was confirmed by immunoprecipitation with conforma- VA1 inhibition than the wild-type protein when tion-sensitive monoclonal antibodies (data not shown). transfected into SAOS2 cells (Figure 3). Western This result suggests that sequences near the N-terminus analysis confirmed that this double substitution did contribute to pol III repression by p53. not compromise expression of the mutant in the Figure 1 showed that VA1 regulation does not transfected cells. We conclude that efficient repression require conserved region I, which consists of residues by p53 of pol III transcription from the VA1 promoter 13 – 19. However, the adjacent sequence has been requires an N-terminal sequence that includes amino shown to be important for transcriptional control of acids 22 and 23 but excludes residues 13 – 19. a variety of pol II promoters (Fields and Jang, 1990; Raycroft et al., 1990). Indeed, residues 22 and 23 are The C-terminal domain of p53 contributes to the control important for binding TFIID, a function that of pol III transcription contributes to the regulation of pol II templates (Chang et al., 1995; Farmer et al., 1996a,b; Lin et al., We have shown previously that the ability to regulate 1994; Lu and Levine, 1995; Thut et al., 1995). We pol III transcription can be blocked specifically if p53 therefore examined whether these residues are also is preincubated with the monoclonal antibody DO-1

Oncogene Domains used by p53 to repress pol III transcription T Stein et al 5543

Figure 3 Substitution of residues 22 and 23 compromises the ability of p53 to repress pol III transcription. SAOS2 cells were Figure 4 Repression of pol III transcription by p53 can be cotransfected with pVA1 (3 mg), HSV – CAT (3 mg) and 2 mgof blocked speciﬁcally using antibodies against the central core or empty vector (lane 1), or vector encoding wild-type p53 (lane 2) C-terminal domains. pVA1 (250 ng) was transcribed using HeLa or pRC/CMV-SN22/23 (lane 3). RNA and protein were harvested nuclear extract (15 mg) that had been preincubated for 10 min 48 h after transfection. The upper and middle panels show primer at 308C in the presence of buffer (lane 1) or 1 mg of wild-type extension analyses of extracted RNA using primers speciﬁc for p53 (lanes 2 – 5). Prior to use, the p53 was incubated at 48C for VA1 (upper panel) and CAT (middle panel). The lower panel 2 h in the presence of buffer (lane 2), or 0.5 mg of pAb1801 (lane shows a Western blot of extracted protein using anti-p53 antibody 3), pAb421 (lane 4) or pAb1620 (lane 5) PAb421

(Cairns and White, 1998). This is consistent with our region, that does not require residues 364 – 392. To test conclusion that the N-terminal domain of p53 is for such an activity, we investigated the effect on VA1 required for pol III repression, since DO-I recognizes expression of an extensive truncation that removes all residues 11 – 25. Figure 4 shows an experiment in which p53 sequence between residue 291 and the C-terminus, a similar approach was taken using antibodies that but leaves the core domain intact. The truncated bind to either the central core (pAb1620) or the C- mutant is expressed efficiently in SAOS2 cells, but terminal region of p53 (pAb421). In both cases, was found to have lost the ability to repress a preincubation with the antibody prevented repression cotransfected VA1 gene (Figure 5). This observation of pol III transcription. As a control for specificity, we suggests that the C-terminal domain of p53 does tested an equal amount of monoclonal antibody indeed contribute to pol III regulation. pAb1801, which binds human p53 but does not To test further for an involvement of the C- recognize the murine p53 used in this experiment; terminal domain in pol III repression in vivo,we repression was not blocked by this negative control. examined the effect of a quadruple substitution The effect of pAb1620 is consistent with the deletion (ALAL) in residues 365, 372, 379 and 387. This analysis described in Figure 1 and suggests that the mutant has constitutive DNA-binding activity and central core of p53 is required for pol III regulation in can efficiently transactivate pol II transcription and vitro,asitisin vivo. induce G1 arrest or apoptosis (Marston et al., 1998). Since residues 371 – 380 are recognized by pAb421, Flow cytometry of propidium iodide-stained cells the ability of this antibody to prevent repression confirmed that the ALAL mutant can indeed provoke suggests that the C-terminal domain of p53 may be G1 arrest in the SAOS2 cells used in our experiments involved in controlling pol III activity. However, this (Figure 6a). Nevertheless, the ALAL mutation was function is not prevented by deletion of residues 364 – found to compromise the ability of p53 to repress the 392 (Figure 2b). A possible explanation for this VA1 promoter (Figure 6b). This result provides apparent contradiction might be that pAb421 induces further evidence that the C-terminal domain of p53 a conformational change elsewhere in p53 that contributes to pol III regulation. In addition, it influences its ability to regulate pol III. Indeed, binding provides an important demonstration that the repres- of pAb421 is known to affect the function of the sion observed in vivo is not an indirect effect of cell central core domain (Hupp et al., 1992). Alternatively, cycle arrest. We conclude that the pol III control the bound antibody might interfere with some activity function of p53 requires distinct domains that span performed by the adjacent part of the C-terminal much of the molecule.

Oncogene Domains used by p53 to repress pol III transcription T Stein et al 5544

Figure 5 Deletion of the C-terminal 102 residues of p53 abolishes its ability to repress pol III transcription. SAOS2 cells were cotransfected with pVA1 (3 mg), HSV – CAT (3 mg) and 2 mg of empty vector (lane 1), or vector encoding wild-type p53 (lane 2) or p53D291 (lane 3). RNA and protein were isolated 48 h after transfection. The upper and middle panels show primer extension analyses of extracted RNA using primers speciﬁc for VA1 (upper panel) and CAT (middle panel). The lower panel shows a Western blot of extracted protein using anti-p53 antibody DO-1

Discussion

Our data indicate that at least three regions of p53 are involved in the efficient repression of pol III transcription; N-terminal sequences that include residues 22 and 23, the central core domain, and a region towards the C-terminus of the protein. However, residues 13 – 19 and 364 – 392 can be dispensed with. It is therefore apparent that the repression of pol III transcription involves much, but not all, of the p53 molecule. The consensus DNA sequence that is recognized by p53 is not found associated with most of the genes that it inhibits. Although it is difficult to exclude the possibility of low-affinity non-consensus binding sites, Figure 6 A C-terminal domain p53 substitution mutant retains the ability to induce G1 arrest but cannot repress pol III tran- it is likely that p53 regulates such genes by protein – scription. SAOS2 cells were cotransfected with pVA1 (3 mg), protein interactions, rather than direct recognition of HSV – CAT (3 mg) and 2 mg of empty vector (lane 1), or vector DNA. We believe this to be the case for pol III encoding wild-type p53 (lane 2) or p53ALAL (lane 3). The bar transcription, since p53 can repress every pol III chart reveals the G0/G1 phase content after 30 h as revealed by flow cytometry after propidium iodide staining. RNA and protein template tested, although the only sequences shared were isolated 48 h after transfection. The upper and middle panels by all these genes are the promoter and terminator show primer extension analyses of extracted RNA using primers elements that are recognized by the basal pol III specific for VA1 (upper panel) and CAT (middle panel). The low- machinery. In these cases, p53 appears to influence er panel shows a Western blot of extracted protein using anti-p53 expression by binding to TFIIIB, a factor that is antibody DO-1 essential to recruit pol III to its templates (Cairns and White, 1998; Chesnokov et al., 1996). It is therefore perhaps unexpected that the sequence-specific DNA- blocked by antibody pAb1620, which binds to the p53 binding domain of p53 is required for repression of pol core. It will be interesting to establish the role played III transcription. Nevertheless, our data show clearly by this region in controlling pol III activity. An that deletions within this central core domain abolish obvious possibility is that central deletions or antibody completely the ability to regulate VA1 expression in binding interfere with repression by disrupting the vivo (Figure 1). Furthermore, repression in vitro can be conformation of the p53 molecule. Alternatively, this

Oncogene Domains used by p53 to repress pol III transcription T Stein et al 5545 domain might provide a docking site for TFIIIB or a map to the same region of this complex molecule. All hypothetical cofactor that may contribute to regula- we can conclude with confidence at present is that VA1 tion. Further experiments will be carried out to repression requires sequence that lies between residues distinguish these possibilities. 291 and 364. The ability of p53 to inhibit VA1 is compromised by It might be argued that some of the effects we have deletions at either end of the protein. This is observed on VA1 expression in transient transfections reminiscent of the situation for pol II transcription, could be an indirect response to cell cycle arrest. It is where both the N- and C-terminal regions of p53 have certainly the case that pol III transcriptional activity in been shown to function in transcriptional repression mammalian cells is repressed during G0 and G1 phases (Horikoshi et al., 1995; Sang et al., 1994; Subler et al., (Abelson et al., 1974; Brown et al., 2000; Mauck and 1994). A potential explanation is offered by the fact Green, 1974; Scott, 2001; White et al., 1995a). We have that the N- and C-terminal domains of p53 both shown recently that this is primarily due to the action interact with TBP, the TATA-binding protein (Hori- of RB. Thus, fibroblasts from RB-knockout mice are koshi et al., 1995). TBP is an essential component of severely defective in their ability to down-regulate pol the TFIIIB complex (Kassavetis et al., 1992; Lobo et III activity following G0/G1 arrest (Scott et al., 2001). al., 1992; Simmen et al., 1992; Taggart et al., 1992; Since the current study has used SAOS2 cells, which White and Jackson, 1992). Furthermore, functional lack functional RB (Shew et al., 1990), the G1 arrest TBP is required for titration of excess TFIIIB to relieve that is induced by p53 might have a minimal effect on repression of VA1 by p53 in vitro (Cairns and White, the pol III machinery. Indeed, the ALAL mutant 1998). It is therefore plausible that p53 binds and induces G1 arrest in SAOS2 cells and yet is unable to represses TFIIIB by interacting directly with its TBP repress VA1 effectively. These observations demon- subunit; this might require the TBP-binding functions strate that G1 arrest in these RB-negative cells is located in the N- and C-terminal domains of p53 insufficient to inhibit pol III transcription and eliminate (Horikoshi et al., 1995). An alternative explanation is the suspicion that p53 is working indirectly through an suggested by the finding that both these regions also effect on the cell cycle. We believe that p53 exerts its bind to the corepressor mSin3a, which can inhibit pol effect by interacting directly with TFIIIB; a direct effect II transcription by recruiting histone deacetylases is supported strongly by the fact that p53 also represses (Murphy et al., 1999). Expression of pol III-transcribed pol III activity in vitro. genes in vivo can be influenced strongly by histone It is very well documented that a wide range of deacetylase activity (Sutcliffe et al., 2000). These transformed and tumour cell types display abnormally observations raise the possibility that p53 might repress high levels of pol III products (reviewed in Brown et pol III templates by binding to TFIIIB and recruiting al., 2000; White, 1998b). This phenomenon may be histone deacetylases via mSin3a. partially explained by our unexpected finding that the An apparent anomaly exists between the various DNA-binding core domain is essential for repression of lines of evidence that implicate the C-terminal domain pol III transcription in vivo, even though this of p53 in pol III control. Transcriptional repression can regulation appears not to involve sequence-specific be blocked by pAb421 antibody binding to residues DNA recognition. Mutations in p53 are found in 371 – 380 (Figure 4) or by the ALAL quadruple about half of all human tumours and in most of these substitution at residues 365, 372, 379 and 387 (Figure cases the mutations localize to the central core domain 6b), but it is not impaired by deletion of residues 364 – of the protein (Hollstein et al., 1994). This suggests 392 (Figure 2b). Both pAb421 and the ALAL mutation that TFIIIB will be released from repression in many have been shown to induce a conformational change cancers carrying p53 mutations, just as we have shown that activates DNA binding by p53 (Hupp et al., 1992; previously in fibroblasts derived from p53-knockout Marston et al., 1998). It is therefore possible that this mice (Cairns and White, 1998). Indeed, we have structural rearrangement is preventing the pol III recently obtained evidence that the pol III control repression function. Nevertheless, this function is also function can be severely compromised by tumour- abolished by deletion of residues 291 – 329; this derived core domain substitutions, such as the R175H suggests that the C-terminal domain does contribute ‘hot-spot’ mutation (Stein et al., 2002). Such effects are to pol III control and that the effects of pAb421 and likely to contribute significantly to the abnormal ALAL are not simply due to rearrangements elsewhere elevation of pol III activity that is so frequently in the molecule. Residues 339 – 346 were reported to observed in human cancers (Brown et al., 2000; White, possess autonomous transrepression activity with 1998b). respect to pol II reporters (Hong et al., 2001) and it is possible that this contributes to the pol III response. Furthermore, an autonomous tetramerization domain Materials and methods is located at residues 325 – 356 (Jeffrey et al., 1995; Sturzbecher et al., 1992) and the ALAL mutant is Plasmids unable to form tetramers. We have attempted to The pVA1 plasmid contains the adenovirus VA1 gene (Dean determine the role of oligomerization in the pol III and Berk, 1988). HSV – CAT contains the CAT gene driven repression function of p53, but have yet to obtain a by the thymidine kinase promoter of herpes simplex virus. All clear answer, perhaps because several distinct activities p53 expression constructs have been described previously.

Oncogene Domains used by p53 to repress pol III transcription T Stein et al 5546 The p53DI – p53DV series have each had a conserved region 50% ethanol/PBS. Cells were washed again and treated with deleted, as follows: p53DI, residues 13 – 19 deleted, p53DII, RNaseA (25 U/ml) and propidium iodide solution (10 ng/ml) residues 117 – 142 deleted; p53DIII, residues 171 – 181 deleted; for 20 min at 48C. Cells were sorted on a FACScan cell sorter p53DIV, residues 234 – 258 deleted; p53DV, residues 270 – 286 (Becton Dickinson) and analysed using the Cell Quest deleted (Crook et al., 1994). pRC/CMV – SN22/23 carries a Software package. Approximately 105 cells expressing CD20 double substitution at residues 22 and 23 (Lin et al., 1994). were counted. p53D291 encodes p53 with the C-terminal 102 residues deleted (Crook et al., 1994). Mutant p53ALAL carries Preparation of proteins and transcription assays substitutions in residues 365, 372, 379 and 387 (Sturzbecher et al., 1992). Construction of recombinant baculovirus encoding His- tagged versions of p53 has been described (Okorokov and Milner, 1999). Recombinant p53 was expressed in Sf9 insect Cell culture and transfection cells and then affinity-purified as previously described The human osteosarcoma cell line SAOS2 was cultured in (Molinari et al., 1996; Okorokov et al., 1997). Transcription Dulbecco’s modified Eagle medium (DMEM) with 10% reactions were carried out as previously (White et al., 1989), foetal calf serum. Cells were transiently transfected using the except that pBR322 was not included and the incubations calcium-phosphate precipitation method. DNA precipitates were for 60 min at 308C. HeLa nuclear extract was purchased were left on the plates overnight and then the cells were from the Computer Cell Culture Center (Mons, Belgium). washed with PBS and cultured for 48 h before harvesting. Total RNA was extracted using TRI reagent (Sigma), Antibodies and Western blotting according to the manufacturer’s instructions. It was then analysed by primer extension using both VA1-specific (5’- Western immunoblot analysis was performed as previously CACGCGGGCGGTAACCGCATG-3’) and CAT-specific described (White et al., 1995b) using anti-p53 antibody DO-1 (5’-CGATGCCATTGGGATATATCA-3’) labelled primers. (Santa Cruz) or PAb421 (Calbiochem). pAb1801 and Primer extension reactions were conducted as previously pAb1620 were obtained from Calbiochem. described (White et al., 1996).

FACS analysis Acknowledgements SAOS2 cells (106) seeded in a 100 mm dish were transfected We wish to thank Arnold Berk, Sheila Graham, Arnold with 10 mg of the indicated p53 expression vectors or empty Levine and Karen Vousden for plasmids. This work was vector together with 8 mg of pCMV – CD20. After 30 h, cells funded by Yorkshire Cancer Research to J Milner and by were harvested in cell dissociation buﬀer (Sigma) and project grant SP2314/0102 to RJ White from Cancer incubated with FITC-conjugated CD20 antibody (Becton Research UK (formerly the Cancer Research Campaign). Dickinson) to identify the transfected cell population. The RJ White is a Jenner Research Fellow of the Lister cells were then washed in PBS and ﬁxed overnight at 48Cin Institute of Preventive Medicine.

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