Regulation of the Wilms' tumour suppressor protein ... - Nature

ã

Oncogene (1999) 18, 6546 ± 6554 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc

Regulation of the Wilms' tumour suppressor protein transcriptional activation domain Lynn M McKay1, Brian Carpenter1 and Stefan GE Roberts*,1 1

Division of Gene Expression, Department of Biochemistry, Wellcome Trust Building, University of Dundee, Dundee DD1 5EH, UK

The Wilms' tumour suppressor protein WT1 contains a transcriptional regulatory domain that can either activate or repress transcription depending upon its cellular environment. The mechanistic basis for this dichotomy is unclear however. Here, we dissect the transcriptional regulatory domains of WT1. We ®nd that a region within the domain of WT1 attributed to transcriptional repression is a potent suppressor of the activation domain at several promoters and in dierent cell types. In vitro transcription analysis suggests that the mechanism of suppression of the activation domain occurs at the level of transcription initiation. Furthermore we ®nd that the WT1 suppression domain is able to inhibit a heterologous activation domain when fused in cis. Dissection of this domain resulted in the delineation of a 30 amino acid region that was sucient to confer suppression of a transcriptional activation domain both in vivo and in vitro. Additionally, we ®nd that the WT1 transcriptional activation domain interacts with the general transcription factor TFIIB and that this interaction is not aected by the suppression domain. Taken together, these studies suggest that the suppression domain of WT1 interacts with a cosuppressor protein to mediate inhibition of the WT1 transcriptional activation domain. Keywords: Wilms' tumour; WT1; transcription

Introduction Wilms' tumour is a paediatric malignancy of the kidneys that aects 1 in 10 000 children and is also associated with other genitourinary disorders such as Denys-Drash and WAGR syndrome (reviewed in Hastie, 1994; Reddy and Licht, 1996; Englert, 1998). Predisposition to Wilms tumour arises from mutations in at least three genes, of which one has so far been identi®ed (WT1). WT1 is highly expressed in the developing kidneys and gonad and, consistent with a role in development, heterozygous mutations in WT1 lead to genitourinary disorders (Pritchard-Jones et al., 1990). Analysis of null mutant mice revealed that WT1 is essential for urogenital development and survival beyond 15 days gestation (Kreidberg et al., 1993). These WT1 knockout mice also exhibited problems with heart and lung development. Moreover, WT1 appears to be abnormally expressed in diverse

*Correspondence: SGE Roberts Received 9 February 1999; revised 22 June 1999; accepted 23 June 1999

malignancies including leukaemia and mesothelioma (Pritchard-Jones and King-Underwood, 1997; Englert, 1998). The primary sequence of WT1 contains a series of zinc ®ngers commonly found in DNA binding proteins, suggesting that WT1 was likely to be a transcriptional regulator. Transient transfection assays con®rmed this prediction with the ®nding that WT1 is a repressor of transcription (Madden et al., 1991, 1993). However, WT1 has since been found to activate in some instances and repress in others (Wang et al., 1993, 1995a,b; Reddy et al., 1995a; Cook et al., 1996; Nachtigal et al., 1998). WT1 is subject to alternative splicing (Reviewed in Reddy and Licht, 1996; Englert, 1998). One of the regions involved in this is the Zinc ®nger cluster at the C-terminus, producing a variant which has a three amino acid insertion (KTS) between zinc ®ngers 3 and 4. The +KTS form of WT1 has a much lower anity for DNA than the 7KTS form and has an altered sequence speci®city (Bickmore et al., 1992; Bardeesy and Pelletier, 1998). However, this +KTS form is able to bind to a speci®c recognition sequence present in the RNA transcript of the IGFII gene (Caricasole et al., 1996). Intriguingly, nuclear localization studies have found that the +KTS form of WT1 is associated with coiled bodies and can be immunoprecipiated with antibodies against mRNA splicing factors (Larsson et al., 1995; Davies et al., 1998). On the other hand, the 7KTS form colocalizes with transcription factors such as TFIIB. Additionally, structural modelling has identi®ed a potential RNA recognition motif at the N-terminus of WT1 (Kennedy et al., 1996). However, the function of WT1 in the spliceosome is yet to be determined. Thus, it appears that the two major splicing variants of WT1 may participate in dierent aspects of gene expression, namely, the 7KTS form in transcription and the +KTS form in RNA processing. The zinc ®ngers of WT1 are a major site for proteinprotein interactions as well as for nucleic acid binding. Several factors can interact with the Zinc ®nger region of WT1 including p53, E1b, Par4, Ciao 1 and U2AF65 (Maheswaran et al., 1995, 1998a; Johnstone et al., 1996, 1998; Davies et al., 1998). The interaction with U2AF65 is speci®c for the +KTS isoform, consistent with a role in RNA processing (Davies et al., 1998). Interactions with p53, Par4 and E1b may be important for the transcriptional repression function of WT1, although this is not yet proven. The other alternate splice site is a 17 amino acid region (exon V) between the transcriptional regulatory and DNA binding domains that is present only in mammalian WT1 (Kent et al., 1995). Furthermore, the proportion of exon V-containing transcripts can vary

Transcriptional regulation by WT1 LM McKay et al

between dierent cell types (Renshaw et al., 1997). The function of this 17 amino acid insertion is unclear. Several problems have hampered the study of WT1 in living cells. For example, the DNA binding speci®city of WT1 is similar to that of other GC-rich binding proteins, resulting in interference from these factors in transient transfection analysis. Moreover, the functional eects observed by WT1 in transient transfection studies is aected by the choice of promoter driving WT1 expression (Reddy et al., 1995a). Dissection of the WT1 transcriptional regulatory domains by using GAL4-fusion proteins has helped in this regard (Wang et al., 1995a,b; Madden et al., 1993). Such studies have identi®ed distinct domains responsible for either transcriptional activation or repression and cell-type speci®c eects in the function of the repression domain. In this study we directly analyse the WT1 transcriptional regulatory domain both in vivo and in vitro. Our ®ndings suggest that WT1 contains a potent transcriptional activation domain that is inhibited by a juxtaposed suppression domain. This suppression domain can also act on a heterologous activation domain suggesting that it functions by interacting with an intermediary protein. We discuss our observations in relation to the pleiotropic transcriptional eects previously attributed to WT1.

Results The WT1 transcriptional activation domain is regulated Previous studies of WT1 have identi®ed a cell-type speci®c transcriptional repression domain located between residues 70 and 180 and a transcriptional activation domain between residues 180 and 250

(Madden et al., 1993). In the following experiments these domains will be referred to as R (repression) and A (activation). We constructed fusions of WT1 RA, R and A to the DNA binding domain of GAL4 (residues 1 ± 93) under the control of a CMV promoter (Figure 1a). For comparison, we also fused the repression domain from the well characterized transcriptional repressor even skipped (Austin and Biggin, 1995; Um et al., 1995; Li and Manley, 1998) to the same DNA binding domain (GAL4-EVE). These constructs were transfected into human embryonic kidney 293 cells along with a CAT reporter containing the Adenovirus E4 minimal promoter downstream of ®ve GAL4 DNA binding sites. Figure 1b shows the relative CAT activity produced by the various constructs when increasing amounts of GAL4 expression plasmid were transfected. GAL4-RA and GAL4-R exhibited weak transcriptional repression when compared to the GAL4-EVE construct. However, GAL4-A exhibited a signi®cant degree of transcriptional activation at the minimal E4 promoter. Thus, despite the fact that this activation domain is present in the GAL4-RA construct, its activity is suppressed. To ensure that the lack of transcriptional activation by GAL4-RA was not due to dierences in the level of expression in 293 cells, we prepared extracts derived from cells that were transfected as in part B and immunoblotted them with an anti-GAL4 antibody. Figure 1c shows that all of the constructs are expressed to equivalent levels. Thus, the WT1 R-domain directly inhibits the function of the transcriptional activation domain. A possible mechanism for this inhibition is that the R-domain aects the ability of the GAL4 moiety to bind DNA. To address this we prepared nuclear extracts from cells that had been transfected with either empty expression vector, GAL4, GAL4-WT1RA or GAL4-WT1A. These extracts were used in

Figure 1 The WT1 transcriptional activation domain is regulated by a juxtaposed suppression domain. (a) A diagram of WT1 is shown indicating the DNA-binding and transcriptional regulatory domains. The R-domain (residues 71 ± 180) and A-domain (residues 180 ± 250) were fused together or independently to the GAL4 DNA binding domain (residues 1 ± 93). The construct GAL4 (1 ± 93) EVE (residues 89 ± 376) is also shown. The reporter G5E4CAT is diagrammed at the bottom. (b) A graph showing the relative CAT activities in embryonic 293 cells transfected with 0.5 mg G5E4CAT and 0.1 mg, 0.5 mg and 2.5 mg of the indicated constructs. (c) 293 cells transfected with 1 mg of each of the indicated expression constructs were used to prepare whole cell extracts, resolved by SDS ± PAGE and immunoblotted with anti-GAL4 antibody. Positions of the molecular weight markers (kDa) are shown at left. (d) 293 cells transfected with 1 mg of each of the indicated expression constructs were used to prepare nuclear extracts. Equal amounts of extract (10 mg) were then used in a bandshift assay with a 32P-labelled double stranded oligonucleotide containing a GAL4 binding site. Speci®c mobility shifts and the free probe are indicated

6547


6548

electrophoretic mobility shift assay with a 32P-labelled GAL4 DNA binding site (Figure 1d). The speci®c mobility shifts produced by the nuclear extracts expressing GAL4-WT1RA and GAL4-WT1A indicate that the R-domain does not prevent the interaction of the GAL4-fusion with DNA. Thus, WT1 contains a transcriptional activation domain that is inhibited by a juxtaposed suppressor region (R-domain). Characterization of suppression of the WT1 activation domain Transcriptional repression by WT1 has been shown to occur in a cell-type speci®c manner (Madden et al., 1993). We therefore considered the possibility that suppression of the WT1 activation domain by the Rdomain may show cell-type speci®city. NIH3T3 cells were transfected with constructs containing either GAL4, GAL4-RA or GAL4-A with the G5E4CAT reporter. Representative data are shown in Figure 2a. As seen in 293 cells, GAL4-A mediated activation of transcription, but GAL4-RA was without eect in NIH3T3 cells. Similar eects were also observed in Human Foreskin Fibroblasts (HFF cells; Figure 2b). Thus, inhibition of the WT1 transcriptional activation domain by the R-domain is not cell type speci®c. Core promoter sequences can aect the response to upstream regulatory proteins (Novina and Roy, 1996). We therefore tested whether the WT1 suppression domain can inhibit the activation domain at a dierent core promoter in 293 cells. Figure 2c shows the results of a transient transfection assay using the Adenovirus major late (Ad2ML) core promoter downstream of ®ve GAL4 sites and controlling the CAT gene. Again GAL4-WT1RA had no eect on transcription, but GAL4-WT1A was able to transactivate this reporter. To rule out the possibility that our results may be an unusual eect at viral core promoters we next tested a cellular core promoter. We chose the core promoter of the IGF II gene (P3 promoter) as it is a strong candidate gene for regulation by WT1 (Drummond et al., 1992). As seen with both the Major late and E4

core promoters of adenovirus, GAL4-WT1RA had no eect on the IGFII P3 promoter, but transfection of GAL4-A resulted in transcriptional activation (Figure 2d). The WT1 transcriptional activation domain functions in vitro The above transient transfection data have demonstrated that the WT1 transcriptional activation domain is inhibited by a juxtaposed suppressor region. However, these results do not rule out the possibility that the eects we observed were post-transcriptional. This is particularly important in light of the evidence that WT1 can associate with RNA processing factors. To rule out this possibility and directly test the eects of the WT1 suppression domain on transcription initiation, we performed in vitro transcription analysis. GAL4, GAL4-RA and GAL4-A were expressed in and puri®ed from E. coli (Figure 3a) and tested in an in vitro transcription system using HeLa cell nuclear extract and the G5E4T promoter. Correctly initiated transcripts were detected by primer extension (Figure 3b). As we observed in vivo, GAL4-A was a potent activator of transcription in vitro, but GAL4-RA was not. GAL4-R also had no eect on basal transcription of the E4 promoter in vitro (data not shown). Signi®cantly, we also did not observe transcriptional repression with GAL4-RA, in contrast to GAL4-EVE (Figure 3c), which is well documented to repress transcription in vitro (Austin and Biggin, 1995; Li and Manley, 1998). We next tested the GAL4-WT1 derivatives in nuclear extracts prepared from 293 cells (Figure 3d). Results comparable to those seen in HeLa nuclear extracts were obtained; GAL4-RA had no eect on transcription, but GAL4-A elicited transcriptional activation. This is the ®rst demonstration that WT1 contains a transcriptional activation domain that is functional in vitro. Furthermore, these data clearly show that the WT1 suppression domain has a direct eect on the stimulation of transcription initiation by the activation domain.

Figure 2 Inhibition of the WT1 transcriptional activation domain by the suppression domain is not cell-type or promoter-speci®c. (a) NIH3T3 cells were transfected with 0.5 mg of G5E4CAT and 1 mg of each of the constructs indicated. CAT activity relative to the empty expression vector is shown. (b) As in part a except that transfections were performed in human foreskin ®broblasts (HFF) cells. (c) 293 cells were transfected with 0.5 mg G5MLCAT (diagrammed at bottom) and 1 mg of the indicated GAL4 constructs. (d) As in part (c) except that the reporter was G5 IGFP3 CAT (diagrammed at bottom)


Figure 3 The WT1 transcriptional suppression domain functions at the level of transcription initiation in vitro. (a) Coomassie stained gel of the puri®ed GAL4-fusion proteins. A bacterial contaminant present in both samples is indicated (*). (b) 200 ng of each of the GAL4 fusion proteins indicated was used in an in vitro transcription assay with HeLa cell nuclear extract and the promoter G5E4T. Correctly initiated transcripts were detected by primer extension and denaturing electrophoresis. (7) indicates no addition of a GAL4 derivative. E4 transcripts and the free primer are indicated. (c) As in part (a) except that 200 ng of GAL4-EVE was used. (d) As in part (b) except that the nuclear extract was prepared from 293 cells

The WT1 suppression domain can inhibit a heterologous transcriptional activation domain In order to further dissect the WT1 suppression domain, we next tested if the context of the R-domain and A-domain was important in the suppression of the activation domain. We constructed a GAL4-fusion protein in which the activation domain and suppression domains were inverted (GAL4-AR; Figure 4a). This construct was tested alongside GAL4-RA and GAL4-A by transfection into 293 cells. All of the proteins were expressed to equivalent levels as assessed by immunoblotting (Figure 4a). As before, strong activation was observed by GAL4-A, but not by GAL4-RA. GAL4-AR also had no eect on transcription. Thus, the suppression domain acts upon the activation domain in a context independent manner. This suggests that the inhibitory function of the suppression domain is not dependent upon the WT1 activation domain. Therefore, we next tested if the WT1 R-domain could inhibit a heterologous activation domain. We used one of the activation domains from SP1, another Zinc ®nger transcription factor. A fragment from the SP1 activation domain A (residues 143 ± 230) of similar size to the WT1 activation domain was cloned in-frame to GAL4-WT1R to produce GAL4-WT1R-SP1 (Figure 4b). Transient transfection assays in 293 cells were performed comparing this construct to the same SP1 activation domain alone fused to GAL4. GAL4-SP1 activated transcription of the G5E4CAT reporter. However, when fused to the

WT1 R-domain transcriptional activation was completely abolished. The inactivity of GAL4-RSP1 was not due to a dierence in protein levels as shown by immunoblotting. Thus, the WT1 suppression domain resides entirely within residues 71 ± 180 and its function is not speci®c to the WT1 transcriptional activation domain. Given the ability of the WT1 R-domain to suppress a heterologous transcriptional activation domain in cis, we next tested the eect of GAL4-R on two dierent promoters that are intrinsically active due to the presence of DNA-binding sites for cellular activator proteins. Figure 4c shows transient transfection assays with the reporters HSV TKCAT and HIV LTRCAT either with or without GAL4-DNA binding sites. In agreement with previous results, cotransfection of GAL4-R speci®cally suppressed the TK promoter when GAL4 DNA-binding sites were present (Madden et al., 1993; Wang et al., 1995a). GAL4-R also suppressed the activity of the HIV LTR. Taken together these experiments suggest that the WT1 Rdomain can inhibit transcriptional activation domains in trans. Dissection of the WT1 suppression domain In order to further de®ne the suppression domain of WT1 we performed deletion mutagenesis (Figure 5). All of the deletion constructs were expressed at equivalent levels in 293 cells as demonstrated by immunoblotting (Figure 5a). N-terminal deletion constructs containing suppression domain residues 99 ± 180 or 120 ± 180 fused to the WT1 activation domain elicited levels of transcriptional activation close to that observed with the activation domain alone (Figure 5). Thus, amino acids 71 ± 99 are required for the function of the WT1 suppression domain. We next tested C-terminal deletion constructs of the WT1 suppression domain. GAL4-fusions containing suppression domain residues 71 ± 137 or 71 ± 101 fused to the WT1 transcription activation domain did not elicit transcriptional activation (Figure 5b). Thus, WT1 amino acids 71 ± 101 are necessary and sucient for the inhibition of the WT1 transcriptional activation domain. Figure 5c shows the sequence of the WT1 Nterminus from various species with totally conserved residues indicated. Across the entire region mouse and chick show 72% identity (52% for mouse and puer ®sh), compared to 83% for the suppression domain (67% for mouse and puer ®sh). Moreover, most substitutions are conservative indicating an important function for this domain. In order to determine if this region was also necessary and sucient to inhibit a heterologous activation domain we constructed fusions of WT1 residues 71 ± 101 and 99 ± 180 to the activation domain of SP1 (Figure 6a). As seen with the WT1 activation domain, residues 99 ± 180 did not signi®cantly inhibit the SP1 activation domain, but residues 71 ± 101 did (Figure 6b). GAL4-SP1 eciently activates transcription in vitro (e.g. Roberts et al., 1995). We next tested if the minimal suppression domain of WT1 was able to inhibit the SP1 transcriptional activation domain in vitro. GAL4, GAL4-R(71-101)-SP1 and GAL4- SP1 were produced and puri®ed from E. coli (Figure 6c)

6549


6550

Figure 4 (a) The WT1 suppression domain functions in a context-independent manner relative to the activation domain. A diagram of the constructs and an anti-GAL4 immunoblot of whole cell lysates (293 cells) is shown in conjunction with a graph of the CAT activities elicited by the indicated GAL4 expression constructs (1 mg) at the G5E4CAT reporter (0.5 mg). (b) The WT1 suppression domain functions on a heterologous transcriptional activation domain. The WT1 suppression domain was fused in frame to the SP1 activation domain (residues 143 ± 230; diagrammed at bottom left) and transfected into 293 cells with the reporter G5E4CAT (0.5 mg). Relative CAT activity produced by the various constructs (1 mg) is shown graphically. An anti-GAL4 immunoblot detecting the expressed fusion constructs is at bottom left. (c) The WT1 suppression domain can inhibit activation domains in trans. Left panel, G0TKCAT or G5TKCAT (0.5 mg) was transfected into 293 cells along with 2 mg of the GAL4 expression construct indicated. Relative CAT activity compared to empty expression vector alone is shown. Right panel, as above except that G0HIVCAT or G6HIVCAT were used as reporters

and tested in vitro using a HeLa cell nuclear extract (Figure 6d). GAL4 and GAL4-R(71-101)-SP1 had no eect on transcription, but GAL4-SP1 elicited transcriptional activation. Thus, the minimal WT1 transcriptional suppression domain is able to inhibit a heterologous transcriptional activation domain both in vivo and in vitro. Transcriptional activation domains exert their eects by interacting with both basal and coactivator components of the transcription apparatus (reviewed in Hampsey, 1998). We next tested if the WT1 suppression domain can aect the ability of the transcriptional activation domain to interact with a potential target component in the general transcription machinery. We used a GST pull-down assay to determine if the general transcription factor TFIIB can interact with the region of WT1 used in this study. Figure 7 shows that TFIIB interacts directly with the WT1 transcriptional activation domain (GST-WT1A), but not with GST-WT1R. Furthermore GST-WT1RA interacts with TFIIB with equivalent anity to GSTWT1A. Thus, the suppression domain does not directly

prevent the WT1 activation domain from interacting with a potential target component in the transcription complex. Discussion A major problem encountered with the study of intact WT1 in vivo is that the DNA binding speci®city of WT1 is similar to that of other zinc ®nger proteins. It is therefore possible that the eects observed are due to competitive DNA binding with endogenous transcription factors rather than direct transcriptional eects. Furthermore, as WT1 may play a role in RNA processing, it is dicult to draw de®nitive conclusions regarding transcription function with transfection data alone. Our studies suggest that WT1 contains a distinct transcriptional activation domain. However, this transcriptional activation domain is tightly regulated both in vivo and in vitro. Compared to the EVE repression domain, the region of WT1 used in this study (71 ± 250) did not signi®cantly repress transcrip-


6551

Figure 5 A minimal WT1 suppression domain. (a) GAL4-fusion constructs containing the indicated regions of the WT1 R-domain linked to the activation domain are shown. An anti-GAL4 immunoblot demonstrating the expression of each construct is below. (b) The GAL4-fusion constructs (1 mg) were transfected into 293 cells with 0.5 mg G5E4CAT and relative CAT activity measured and presented graphically. (c) Mouse WT1 residues 1 ± 140 (single letter code) are shown aligned with the corresponding region from the indicated organisms. Identical residues are dark shaded. The minimal suppression domain is indicated

tion of any of the basal level core promoters in any of the cell lines we tested. Rather, we observed inhibition of the WT1 activation domain by the region previously described as a repression domain. The core promoters that we used were devoid of any regulatory elements and were therefore subject to regulation by the GAL4derivatives alone. This is the ®rst study of the eect of WT1 on such promoters. On the other hand, we did observe signi®cant inhibition of the intrinsically active HSV TK and HIV LTR promoters. The TK promoter has been tested by others with similar GAL4 constructs and our results are in agreement with these previous data (Madden et al., 1993; Wang et al., 1995a). In addition, a GAL4-fusion of the R-domain has been shown to suppress the SV40 early promoter (Wang et al., 1995a). Moreover, in the latter study WT1 residues 85 ± 97 were required for the WT1 repression activity, which is within the region we found to mediate suppression of a transcriptional activation domain. However, they also found that residues 104 ± 124 were

required for suppression, which is in contrast to our minimal domain (residues 71 ± 101). We determined the minimal suppression domain in the context of an activation domain fusion, where as in the study of Wang et al. (1995a) they directly assessed the eect of the suppression domain in trans. It is possible that residues 102 ± 124 provided stabilizing ¯anking sequence for the unfused C-terminus of the suppression domain that was substituted by the activation domain in our experiments. Taken together with previous studies our data strongly suggests that the WT1 suppression domain inhibits the function of the activators that drive intrinsically active promoters. Such mechanisms of repression are distinct from that described for `active' repressors such as EVE that function by making non-productive contacts with the basal transcription machinery (Austin and Biggin, 1995; Um et al., 1995; Li and Manley, 1998). We have delineated the inhibition function of WT1 to a 30 amino acid region that was also able to inhibit


6552

Figure 6 The minimal WT1 suppression domain inhibits a heterologous activation domain in vivo and in vitro. (a) GAL4-WT1 Rdomain residues 71 ± 101 or 99 ± 180 were fused to the SP1 activation domain and transfected into 293 cells (1 mg) with the reporter G5E4CAT (0.5 mg). An anti-GAL4 immunoblot con®rming equivalent expression of the constructs is shown. (b) Graph showing relative CAT activity produced by the indicated constructs. (c) Coomassie-stained gel of the puri®ed GAL4 derivatives. (d) In vitro transcription assay showing the eect of 100 ng and 500 ng of the indicated recombinant GAL4 fusion proteins at the G5E4 promoter in a HeLa cell nuclear extract

Figure 7 The WT1 suppression domain does not aect the interaction of the activation domain with general transcription factor TFIIB. The indicated GST-fusion proteins (1 mg) immobilized on glutathione agarose were used in a binding assay with bacterial lysate containing recombinant human TFIIB. Speci®cally bound TFIIB was eluted in SDS ± PAGE buer, resolved by SDS ± PAGE and detected by immunoblotting with anti-TFIIB antibody. INPUT represents 5% of the bacterial lysate used in each assay. Molecular weight markers are shown at left

a heterologous activation domain in cis both in vivo and in vitro. How does the WT1 suppression domain inhibit the transactivation domain? One attractive possibility is that the suppression domain directly interacts with the activation domain, preventing its correct presentation to the general transcription machinery. Indeed, WT1 has previously been shown to self associate (Moett et al., 1995; Reddy et al., 1995b; Holmes et al., 1997). However, we do not believe this is the case for three reasons; Firstly, the region we ®nd necessary and sucient for suppression of the activation domain does not contain the WT1 sequence required for self association. Secondly, the WT1 suppression domain can function on a heterologous transcriptional activation domain. Finally, the

WT1 suppression domain does not prevent the transcriptional activation domain from interacting with a target factor in the preinitiation complex, TFIIB. We favour the idea that the WT1 suppression domain associates with a nuclear protein that mediates the inhibition of the transcriptional activation domain. A previous report provided evidence that a titrateable nuclear protein may interact with WT1 to mediate transcriptional repression (Wang et al., 1995a). The region mapped in that study was similar to the region we ®nd to inhibit a transcriptional activation domain. The most likely explanation for our data therefore is that a cosuppressor protein directly interacts with the suppression domain to mediate inhibition of the transcriptional activation domain. This may involve a direct interaction between the cosuppressor and components of the basal transcription machinery to counteract the productive eects of the transcriptional activation domain. Although we can not rule this out, we have shown that the suppression domain does not interact with TFIIB, but the activation domain does. Even so, if such a mechanism were involved it must speci®cally aect the activation of transcription and not the basal activator-independent function of the general transcription machinery. We observed suppression of the WT1 transcriptional activation domain in several dierent cell types, including NIH3T3 cells that do not express WT1. Thus, the cosuppressor does not show the cell-type restriction that is observed with WT1, suggesting that it has additional functions, perhaps in the regulation of other transcription factors. This would be similar to previously reported factors that can inhibit transcriptional activation domains. For example, MDM2 inhibits the p53 transcriptional activation domain, but is also able to coactivate E2F1 (Martin et al., 1995).


Several other transcriptional activator proteins have also been shown to contain regions that inhibit the activation domain, including E2F (e.g. Brehm et al., 1998), Fos (Brown et al., 1995), SP3 (Dennig et al., 1996), Oct 1 (Liu et al., 1998) and Pho4 (Jayaraman et al., 1994). Some inhibitory domains can act on a heterologous activation domain (e.g. SP3), whereas others are only able to inhibit related transcriptional activation domains (e.g. Fos). These observations underlie a complex and diverse array of mechanisms at play in the regulation of transcriptional activation domains. The transcriptional activation function of WT1 may be restricted to particular conditions regardless of its ability to bind to DNA elements present in the promoter regions of several genes. This would be consistent with a transcriptional role for WT1 at speci®c stages in development. Indeed, a recent study found that overexpression of WT1 alone did not have discernible eects on the transcript levels of several of the genes previously suggested to be under WT1 control (Thate et al., 1998). Moreover, some studies have failed to detect transcriptional eects by WT1 in transient transfection assays (Maheswaran et al., 1998b). These observations may be due to a requirement for a speci®c cellular and promoter context required for WT1 function. This may also help explain the diversity of eects attributed to WT1 under dierent experimental conditions. As mentioned previously, the zinc ®ngers of WT1 are an interface for interaction with several other transcription factors. Interactions with the WT1 zinc ®ngers may form the basis for the modulation of the suppression domain, perhaps involving a conformational change in WT1 that disturbs the interaction with the cosuppressor. These and other possibilities will require further studies to dissect the mechanism of transcriptional regulation by WT1.

Materials and methods Plasmids The reporter G5E4CAT has been described previously and contains nucleotides 738 to +27 of the Adenovirus E4 promoter ¯anked by 5 GAL4 DNA binding sites (5') and the Chloramphenicol acetyltransferase gene (3'; Lin and Green, 1991). The HSV tk CAT derivatives were a gift from A Bannister and T Kouzarides (Wellcome/CRC, Cambridge). The HIVLTR and G5MLCAT plasmids have been described previously (Southgate and Green, 1991; Hawkes and Roberts, 1999). G5IGFIICAT was constructed by replacing the E4 sequence in G5E4CAT with the IGFII P3 core promoter (750 to +30). A GAL4-fusion expression vector was constructed by amplifying the coding region of GAL4 (residues 1 ± 93) by PCR and cloning into pCDNA3 (Invitrogen) under a CMV promoter. Coding sequences of mouse WT1 as indicated were cloned into this vector as BamHI/EcoRI fragments in frame with GAL4 (1 ± 93). Deletions within the WT1 residues 71 ± 180- (R-domain) were constructed by amplifying the appropriate regions of WT1 by PCR producing BamHI (5') and NcoI (3') restrictions sites. These were then cloned in frame with the WT1 activation domain (an NcoI/EcoRI fragment encoding residues 180 ± 250). GAL4-AR was constructed by cloning the WT1 activation domain (residues 180 ± 250) as a BamHI fragment in frame with the R-domain (BamHI/NcoI

fragment). CMV-GAL4(1 ± 93) EVE was constructed by cloning of an XmaI fragment of EVE (encoding residues 89 ± 376) into pCDNA3-GAL4 (1 ± 93). Sequence encoding SP1 residues 143 ± 250 were ampli®ed by PCR from pRJRSP1 (Roberts et al., 1995) as an NcoI/EcoRI fragment. This was then cloned independently into pCDNA GAL4 or fused to the WT1 R-domain (or fragments). Plasmids for expression of the GAL4-fusion proteins in bacteria were constructed by cloning the indicated fragments into the vector pRJR, which contains 6-His tagged GAL4 (1 ± 93) under the control of a Tac promoter (Reece et al., 1993). All PCR products were sequenced to ensure that they were free of mutations. Transfection assays Human embryonic kidney 293 cells, NIH3T3 cells and human foreskin ®broblasts (HFFs) were cultured as monolayers in Dulbecco's Modi®ed Eagle Medium (DMEM) containing 10% Foetal calf serum, 5 mM Lglutamine, 100 mg/ml streptomycin and 100 u/ml penicillin. Transient transfection assays were performed as described previously (Hawkes and Roberts, 1999). CAT assays were performed with cleared whole cell extracts prepared from equal packed cell volumes. Assays were quantitated by phosphorimager and are presented relative to the activity of the reporter cotransfected with pCDNA3. For immunoblot, equal volumes of cells were added to SDS ± PAGE loading dye, the proteins resolved by electrophoresis and immunoblotting performed with anti-GAL4 antibody (raised against His-tagged GAL4 DNA binding domain by the Scottish antibody production unit). Detection was performed by chemiluminescence (ECL, Amersham). All transfections were performed at least three times and representative data are shown. In vitro transcription and DNA binding assays In vitro transcription assays were performed as described previously (Lin and Green, 1991). GAL4-fusion proteins were prepared as 6XHIS tagged fusion proteins according to the method of Reece et al. (1993). GAL4-RA, GAL4-A and GAL4-EVE were puri®ed from the insoluble fraction by denaturation/renaturation. GAL4-WT1 (71 ± 101)-SP1 and GAL4-SP1 were puri®ed from the soluble fraction. Electrophoretic mobility shift assays were performed as described previously using a 5% native polyacrylamide gel (Carey et al., 1990). HeLa cell nuclear extracts were obtained from Computer Cell Culture Centre (Mons, Belgium). Transcriptionally active nuclear extracts were prepared from human embryonic 293 cells as described previously (Lee et al., 1988). In vitro binding assays GST-fusion proteins were prepared as described previously (Lin and Green, 1991). Bacterial lysate containing TFIIB was prepared by IPTG-induction of 10 ml log phase BL21DE3 bacteria containing pET11a TFIIB for 3 h at 378C. After harvesting the bacteria they were resuspended in 1 ml buer D (20 mM HEPES pH 8, 20% [v/v] glycerol, 0.2 mM EDTA, 100 mM Kcl, 1 mM DTT, 0.2 mM PMSF), sonicated and insoluble debris removed by centrifugation in a microfuge at full speed for 10 min. Bacterial lysate (2 ml) was incubated with 25 ml glutathione agarose beads containing 1 mg GSTfusion protein in 0.6 ml binding buer (40 mM HEPES pH 8, 10% [v/v] glycerol, 150 mM Kcl, 5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 0.2 mM PMSF) for 2 h at 48C. The beads were washed four times with binding buer and bound proteins eluted with SDS ± PAGE loading dye. TFIIB was detected by immunoblotting with anti-TFIIB antibodies (Hawkes and Roberts, 1999).

6553


6554

Acknowledgements We would like to thank Rachel Davies and Nick Hastie for WT1 plasmids. Also, Andy Bannister and Tony Kouzarides for transfection reporters. We thank Tom OwenHughes, Neil Perkins, Joost Zomerdijk and members of the lab for comments on the manuscript. This work was

funded by the Wellcome Trust (047674/Z/96/Z), Cancer Research Campaign [CRC] (SP2410/0101) and Association for International Cancer Research (98 ± 39). SGE Roberts is a Research Career Development Fellow of the Wellcome Trust.

References Austin RJ and Biggin MD. (1995). Mol. Cell Biol., 15, 4683 ± 4693. Bardeesy N and Pelletier J. (1998). Nucleic Acids Res., 26, 1784 ± 1792. Bickmore WA, Oghene K, Little MH, Seawright A, van Heyningen V and Hastie ND. (1992). Science, 257, 235 ± 237. Brehm A, Miska EA, McCance DJ, Reid JL, Bannister AJ and Kouzarides T. (1998). Nature, 391, 597 ± 601. Brown HJ, Sutherland JA, Cook A, Bannister AJ and Kouzarides T. (1995). EMBO J., 14, 124 ± 131. Carey M, Lin Y-S, Green MR and Ptashne M. (1990). Nature, 345, 361 ± 364. Caricasole A, Duarte A, Larsson SH, Hastie ND, Little M, Holmes G, Todorov I and Ward A. (1996). Proc. Natl. Acad. Sci. USA, 93, 7562 ± 7566. Cook DM, Hinkes MT, Bern®eld M and Rauscher III FJ. (1996). Oncogene, 13, 1789 ± 1799. Davies RC, Calvio C, Bratt E, Larsson SH, Lamond AI and Hastie ND. (1998). Genes Dev., 12, 3217 ± 3225. Dennig J, Beato M and Suske G. (1996). EMBO J., 15, 5659 ± 5667. Drummond IA, Madden SL, Rohwer-Nutter P, Bell GI, Sukhatme VP and Rauscher III FJ. (1992). Science, 257, 674 ± 678. Englert C. (1998). TIBS, 23, 389 ± 393. Hampsey M. (1998). Microbiol. Mol. Biol. Rev., 62, 465 ± 503. Hastie ND. (1994). Annu. Rev. Genet., 28, 523 ± 558. Hawkes NA and Roberts SGE. (1999). J. Biol. Chem., 274, 14337 ± 14343. Holmes G, Boterashvili S, English M, Wainwright B, Licht J and Little M. (1997). B.B.R.C., 233, 723 ± 728. Jayaraman P-S, Hirst K and Goding C. (1994). EMBO J., 13, 2192 ± 2199. Johnstone RW, See RH, Sells SF, Wang J, Muthukkumar S, Englert C, Haber DA, Licht JD, Sugrue SP, Roberts T, Rangnekar VM and Shi Y. (1996). Mol. Cell Biol., 16, 6945 ± 6956. Johnstone RW, Wang J, Tommerup N, Vissing H, Roberts T and Shi Y. (1998). J. Biol. Chem., 273, 10880 ± 10887. Kennedy D, Ramsdale T, Mattick J and Little M. (1996). Nature Genetics, 12, 329 ± 332. Kent J, Coriat A-M, Sharpe PT, Hastie ND and van Heyningen V. (1995). Oncogene, 11, 1781 ± 1792. Kreidberg JA, Sariola H, Loring JM, Maeda M, Pelletier J, Housman D and Jaenisch R. (1993). Cell, 74, 679 ± 691. Larsson SH, Charlieu J-P, Miyagawa K, Engelkamp D, Rassoulzadegan M, Ross A, Cuzin F, van Heyningen V and Hastie ND. (1995). Cell, 81, 391 ± 401. Lee KAW, Binderief A and Green MR. (1988). Gene Anal. Tech., 5, 22 ± 31. Li C and Manley JL. (1998). Mol. Cell Biol., 18, 3771 ± 3781. Lin Y-S and Green MR. (1991). Cell, 64, 971 ± 981. Liu Y-Z, Lee I-K, Locke I, Dawson SY and Latchman DS. (1998). Nucleic Acids Res., 26, 2464 ± 2472.

Maheswaran S, Englert C, Bennett P, Heinrich G and Haber DA. (1995). Genes Dev., 9, 2143 ± 2156. Maheswaran S, Englert C, Lee SB, Ezzel RM, Settleman J and Haber DA. (1998a). Oncogene, 16, 2041 ± 2050. Maheswaran S, Englert C, Zheng G, Bong Lee S, Wong J, Harkin DP, Bean J, Ezzell R, Garvin AJ, McCluskey RT, DeCaprio JA and Haber DA. (1998b). Genes Dev., 12, 1108 ± 1120. Madden SL, Cook DM, Morris JF, Gashler A, Sukhatme VP and Rauscher III FJ. (1991). Science, 253, 1550 ± 1553. Madden SL, Cook DM and Rauscher III FJ. (1993). Oncogene, 8, 1713 ± 1720. Martin K, Trouche D, Hagemeier C, Sorensen TS, Lathangue NB and Kouzarides T. (1995). Nature, 375, 691 ± 694. Moet P, Bruening W, Nakagama H, Bardeesy N, Housman D, Housman DE and Pelletier J. (1995). Proc. Natl. Acad. Sci. USA, 92, 11105 ± 11109. Nachtigal MW, Hirokawa Y, Enyeart-vanHouten DL, Flanagan JN, Hammer GD and Ingraham HA. (1998). Cell, 93, 445 ± 454. Novina CD and Roy AL. (1996). TIG, 12, 351 ± 355. Pritchard-Jones K, Fleming S, Davidson D, Bickmore W, Porteous D, Gosden C, Bard J, Buckler A, Pelletier J, Housman D, van Heyningen V and Hastie ND. (1990). Nature, 346, 194 ± 197. Pritchard-Jones K and King-Underwood L. (1997). Leukemia Lymphoma, 27, 207 ± 220. Reddy JC, Hosono S and Licht JD. (1995a). J. Biol. Chem., 270, 29976 ± 29982. Reddy JC, Morris JC, Wang J, English MA, Haber DA, Shi Y and Licht JD. (1995b). J. Biol. Chem., 270, 10878 ± 10884. Reddy JC and Licht JD. (1996). Biochim. Biophys. Acta., 1287, 1 ± 28. Reece RJ, Rickles RJ and Ptashne M. (1993). Gene, 126, 105 ± 107. Renshaw J, King-Underwood L and Pritchard-Jones K. (1997). Genes Chromosomes Cancer, 19, 256 ± 266. Roberts SGE, Choy B, Walker SS, Lin Y-S and Green MR. (1995). Curr. Biol., 5, 508 ± 516. Southgate CD and Green MR. (1991). Genes Dev., 5, 2496 ± 2507. Thate C, Englert C and Gessler M. (1998). Oncogene, 17, 1287 ± 1294. Um M, Li C and Manley JL. (1995). Mol. Cell Biol., 15, 5007 ± 5016. Wang Z-Y, Qiu Q-Q and Deuel TF. (1993). J. Biol. Chem., 268, 9172 ± 9175. Wang Z-Y, Qiu Q-Q, Gurrieri M, Huang J and Deuel TF. (1995a). Oncogene, 10, 1243 ± 1247. Wang ZY, Qiu Q-Q, Huang J, Gurrieri M and Deuel TF. (1995b). Oncogene, 10, 415 ± 422.