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Identi®cation of a tumor-speci®c methylation site in the Wilms tumor suppressor

Elena V Kleymenova, Xiaoqin Yuan, Michael E LaBate and Cheryl L Walker

Department of Carcinogenesis, The University of Texas MD Anderson Center, Science Park-Research Division, Smithville, Texas 78957, USA

Malignant is one of the very few extra- tion has been shown for several tumor suppressor renal in which the Wilms tumor suppressor , such as retinoblastoma gene in retinoblastomas gene (wt1) is expressed. We examined wt1 for alterations (Ohtani-Fujita et al., 1993), von Hippel-Lindau gene in in rat , a well characterized animal model renal cell carcinomas (Herman et al., 1995) and in for the human disease. Southern analysis revealed a epithelial (Merlo et al., 1995). In tumor cells de 3.5 kb EcoRI wt1 fragment readily detectable in novo DNA methylation is proposed to occur as a result majority of mesothelioma cell lines and primary of increased activity of DNA methyltransferase (el- mesotheliomas but not in normal rat tissues. Cloning Deiry et al., 1991), an enzyme thought to be and sequencing of this fragment revealed that the responsible for maintenace and de novo DNA presence of this EcoRI fragment resulted from an in- methylation in . However, the molecular ability of this enzyme to cut at a EcoRI site in intron 1 determinants of DNA methylation at speci®c CpG of wt1. This site contains potential motifs for cytosine sites in tumor cells are unknown at present. methylation and treatment of mesothelioma cells with 5- The short arm of human 11, where the azadeoxycytosine restored the normal EcoRI digestion Wilms (WT1) resides is known pattern of wt1 in these cells indicating that cleavage was to contain methylated regions in both normal tissues inhibited by methylation at this site. Southern analysis and tumors (de Bustros et al., 1988), and recently it using HpaII/MspI digestion revealed no di€erences in was demonstrated that transcription of the H19 and methylation between mesothelioma cell lines and normal insulin-like II (IGFII) genes, which reside at other CpG sites in wt1 5' region. Renal on , is regulated by methylation (Li et cell carcinoma lines which did not express wt1 were also al., 1993). WT1, which encodes a zinc-®nger DNA- methylated at this EcoRI site. Our identi®cation of a site binding with properties of a transcription frequently methylated in malignant cells, independent of factor and a putative splicing regulator (Drummond , provides a new model system to study et al., 1992; Larsson et al., 1995), was identi®ed by its determinants of site-speci®c methylation in tumors. location in overlapping 11p13 deletions occurring in Wilms' tumors (Call et al., 1990; Gessler et al., 1990). Keywords: EcoRI restriction enzyme; rat model; Wilms' tumor is an embryonic nephroblastoma which mesothelioma; renal cell carcinoma; leiomyoma/leio- occurs in both familial and spontaneous forms myosarcoma (Francke et al., 1979; Fearon et al., 1984). Loss of heterozygosity of the WT1 gene is a common ®nding in Wilms' tumors in addition to other alterations, including gross and small deletions, insertions, and Introduction point (Cowell et al., 1991; Kikuchi et al., 1992; Little et al., 1992). These data are consistent with DNA methylation has been proposed to be involved in Knudson and Strong's model of tumor suppressor gene adverse genetic alterations associated with neoplastic function for the WT1 gene in Wilms' tumors (Knudson transformation (Baylin et al., 1991; Rainier and and Strong, 1972). However, WT1 expression is Feinberg, 1994; Jones, 1996). The abnormal DNA observed in half of Wilms' tumors (although the methylation observed in tumors includes both loss and biological meaning of this expression is unknown) gain of methyl-groups at the C5 position of cytosine in and the low frequency detected in sporadic CpG dinucleotides, which are targets for DNA tumors expressing WT1 suggests that genetic altera- methylation in mammalian cells. In the human tions other than inactivating mutations in WT1 may be , CpG sites are normally heavily methylated involved in tumorigenesis. in adult tissues; however, there are speci®c CpG-rich In mice, knocking out wt1 has a striking e€ect on regions that are unmethylated in these tissues. These development of the mesothelium (Kreidberg et al., CpG-rich regions are commonly found in the 5' 1993), and malignant mesothelioma (MM) is one of a regulatory domains of genes and are believed to very few extrarenal neoplasms that express WT1 de®ne an `open' chromatin conformation and active (Miyagi et al., 1993; Park et al., 1993; Walker et al., state for these genes when unmethylated and a 1994). Development of MM in humans is closely condensed chromatin structure which is `inactive' associated with environmental or occupational expo- when methylated. Gene silencing via DNA methyla- sure to asbestos or other carcinogenic mineral ®bers. This correlation was clearly established in the 1960s Correspondence: EV Kleymenova (Wagner et al., 1960), but asbestos continues to be Received 1 May 1997; revised 22 September 1997; accepted 22 intensively mined and used, presenting signi®cant September 1997 health risks for occupationally exposed workers. The wt1 site-specific methylation EV Kleymenova et al 714 incidence of MMs, which are rare tumors in the CpG site in intron 1 of the wt1 gene was detected in general population, continues to increase and in the the majority of MM cell lines and primary mesothe- United States about 2000 ± 3000 cases per year are liomas analysed. In addition, methylation of this site expected until the end of the century (Antman et al., was detected in rat renal cell carcinoma cell lines, 1993). Mesothelial cells, which like epithelial cells of which do not express wt1, suggesting that transformed the renal nephron originate from the mesoderm, are cells become programmed to methylate this site and believed to be the progenitor cells for these tumors. that this programming is driven by determinants other Like epithelial cells of the renal nephron, mesothelial than gene expression. cells undergo a mesenchymal-epithelial transition in which the WT1 gene product has been shown to participate (Hastie, 1994). The long latency period of Results MM (20 ± 40 years) suggests that multiple genetic alterations are necessary for the genesis of this Mutations within the WT1 coding region, which are disease, and recent studies revealed that the p16 and frequently detected in tumors, are believed to alter the neuro®bromatosis 2 tumor suppressor genes are ability of the WT1 protein to bind DNA and so may frequently damaged in human MMs (Cheng et al., modify its putative tumor suppressor function. wt1 1994; Sekido et al., 1995; Bianchi et al., 1995; Xiao et exons 7 ± 10, encoding the DNA-binding domain, in al., 1995). No mutations were found in WT1 coding which most of these mutations occur, were examined region in one report which examined 32 asbestos- for mutations in rat MM cell lines II45, II14, ME1T, associated human mesotheliomas (Park et al., 1993); NRM1, NRM2, NRM3, and NRM4 by direct however, a detailed study of this gene in MM has not sequencing of reverse transcription (RT) ± polymerase yet been conducted. chain reaction (PCR) products ampli®ed using primers The rat is an important experimental model for shown in Figure 1a. In addition, RT ± PCR fragments, studying mechanisms of mesothelioma development, which span exons 1 ± 7 were sequenced in the rat MM and for testing the carcinogenic potential of mineral cell lines II45, II14 and NRM3. At least two ®bers. Expression of the wt1 gene in rat mesotheliomas independent RT ± PCR products for each cell line has been reported previously (Walker et al., 1994), but were sequenced in both directions. No mutations were no other information about the status of this gene in detected in any of the regions analysed (data not these tumors is available. We report here that shown). consistent with human data, no mutations were found To examine the wt1 gene for other structural in wt1 exons 7 ± 10 encoding the DNA-binding domain alterations, we performed Southern analysis of of wt1 in rat MM cell lines. However, methylation of a genomic DNA digested with one of the restriction

wt 1

Figure 1 Schematic representation of the rat wt1 gene (a) and the clone wk 1.4 (b). The shaded boxes represent wt1 exons (not drawn to scale). PrE - the intronic EcoRI site, protected from cleavage in MM. Potential CpG sites methylated in MM and ERC cell lines, are boxed. The horizontal arrows indicate the location and direction of primers used for PCR and sequencing. The vertical arrows indicate binding sites for transcriptional factors Ap2 (A), Sp1 (S), and (G) and for histone H4 (H) wt1 site-specific methylation EV Kleymenova et al 715 present at an approximately single copy level in the transformed mesothelial cells. Normal liver DNA from II14 II45 III2 T08 T10 Liver six other rat strains exhibited the same pattern as 8-10 kb normal Fisher rat tissues, with only a faint 3.5 kb band observable after prolonged exposure (Figure 3). In- complete digestion did not account for the novel EcoRI 5 kb band, as digestion with 6 ± 10 units of enzyme per mgof 3.5 kb DNA gave the same pattern by Southern analysis as the standard digestion with 3 units/mg, which was sucient to digest completely control Lambda phage DNA (data not shown). Using probes speci®c for rat 1.7 kb wt1 exons 1 ± 7, 4 ± 7, or 1 ± 3, the 3.5 kb band was shown to contain wt1 sequence homologous to exons 1 ± 3, and subsequent studies focused on this region. To determine the nature of this novel band, the 3.5 kb EcoRI fragment from MM cell line II45 was cloned into the EcoRI site of Lambda ZAP II. The Lambda ZAP II library was screened with a probe to

Stomach Spleen Lung Diaphragm Brain Testis Mesothelium wt1 exons 1 ± 3, and clone wk 1.4 was chosen for pBluescript excision. Sequencing of wk 1.4 revealed 8-10 kb that the clone contained only the 5' end of exon 2 of the rat wt1 gene. As expected, the 3.5 kb insert in wk 1.4 contained EcoRI sites at both ends, but it also had 5 kb an internal EcoRI site 300 bp from the 3' end of this clone (100 bp from the 5' end of the exon 2, Figure 3.5 kb 1b), presumably in intron 1 of the rat wt1 gene. To Figure 2 Southern analysis of the wt1 gene in rat MM cell lines con®rm that the sequence adjoining exon 2 containing and normal rat tissues. MM lines which have the 3.5 kb fragment this EcoRI site in clone wk 1.4 was present in the were II45, III2, T08, T10 and ME1, ME1T, NRM1, NRM2, normal rat wt1 gene, we directly sequenced a PCR NRM3, NRM4 (data not shown). The DNA was digested with product ampli®ed from normal rat liver DNA using EcoRI and hybridized to a PCR probe for wt1 exons 1 ± 7. Slight primers 4581 and r618 (Figure 1a). This PCR product di€erences in apparent band size are an artifact resulting from slight di€erences in electrophoretic mobility in di€erent experi- spanned the rat wt1 gene from the 3' end of exon 1 to ments the 5' end of exon 3 and was con®rmed by DNA sequencing to contain the EcoRI site 100 bp upstream of exon 2 observed in clone wk 1.4 isolated from MM cell line II45. The presence of this intronic EcoRI site 300 bp upstream of the EcoRI site in exon 2 predicted that a 300 bp fragment containing exon 2 should be released by EcoRI digestion of the normal rat wt1 gene.

II14 II45 Long Evans ACI Lewis Brown Norway Nobel Copenhagen Southern analysis revealed that a 300 bp EcoRI fragment that hybridized to exon 2 of the wt1 gene 8-10 kb was readily detectable only in normal rat tissues and the MM cell line II14 which did not have the extra 5 kb 3.5 kb fragment (Figure 4). The inverse correlation observed between the presence of the 3.5 kb and 3.5 kb 300 bp EcoRI fragments suggested that the EcoRI site 100 bp upstream to exon 2 was present in the ®rst intron in both normal tissues and MMs, but protected from EcoRI cleavage in tumors giving rise to the 1.7 kb additional 3.5 kb fragment. Direct sequencing of intron 1 of the wt1 gene from normal liver and mesothelioma cell line II45, which Figure 3 Southern analysis of the wt1 gene from various rat strains. DNA from six di€erent strains of laboratory rats was had the extra 3.5 kb EcoRI fragment, revealed no digested with EcoRI and hybridized to a probe for wt1 exon 1 ± 7 di€erences in the sequence of the protected EcoRI (prE) site, which was present in both DNAs. However, the site was ¯anked 5' by a C and 3' by a G (Figure enzymes, EcoRI, BglII, PstI, PvuI, BamHI, HindIII, 1a), which created potential sites for cytosine methyla- RsaI and HhaI, and hybridized with a 32P-labeled RT ± tion that could make the prE site refractory to PCR product spanning exons 1 ± 7 of rat wt1 gene. Of enzymatic digestion in MMs. the 11 MM cell lines and ®ve asbestos-induced MM To demonstrate that the mesothelioma-speci®c examined, 10 cell lines and four primary tumors had an EcoRI digestion pattern was due to cytosine methyla- additional 3.5 kb EcoRI fragment (Figure 2) that was tion of the prE site, cell line II45 was treated with 5- absent or only apparent as a very faint band in the azadeoxycytosine (5-aza-dC) to inhibit cytosine methy- DNA from normal rat tissues after prolonged (4 lation. DNA was isolated from cells grown for 72 h in weeks) exposure (Figure 2). This 3.5 kb fragment was medium with 2 mM 5-aza-dC, digested with EcoRI and wt1 site-specific methylation EV Kleymenova et al 716 Liver II45 III2 II14 II45 II45 treated with 5-aza-dC

5.0 kb

5.0 kb 3.5 kb

3.5 kb

0.3 kb

0.3 kb Figure 4 Detection of 0.3 kb EcoRI fragment in normal rat tissues and MM II14 which did not have the 3.5 kb wt1 fragment. The DNA was digested with EcoRI and hybridized with wt1 exon 2 probe Figure 5 Southern analysis of demethylated DNA. DNA obtained from MM cell line II45 treated with 5-aza-dC and control DNA from II45 were digested with EcoRI and hybridized hybridized with a wt1 exon 2 probe. After DNA to a PCR probe spanning wt1 exons 1 ± 3 hypomethylation by 5-aza-dC treatment, cell line II45 exhibited the same digestion pattern as normal rat tissues, with a substantial decrease in the 3.5 kb EcoRI Table 1 Levels of DNA methylation in normal and tumor cells fragment and the appearance of the 300 bp fragment Mean group incorporation (Figure 5), indicating that methylation of the prE site (c.p.m./ng DNA+s.e.m.) was responsible for the lack of enzymatic digestion at Sample Assay I Assay II this site in MM DNA. MM II45 5830+453 4280+471 To determine if methylation of the prE site was a MM II14 5148+296 3480+228 MM II45 treated with 9540+628 6390+385 re¯ection of a global increase in DNA methylation in 5-aza-dC mesothelioma cells we estimated the levels of DNA Mesothelium 890+82 490+86 methylation using the in vitro methyl-accepting assay in Liver not determined 750+91 DNA from normal rat tissues, cell line II45 (which Lung not determined 440+68 contained the tumor-speci®c 3.5 kb EcoRI fragment resulting from methylation within prE site) and cell line II14 (which like normal rat tissues, did not exhibit the 3.5 kb EcoRI fragment). As summarized in Table 1, no studied yielded the same pattern as digestion of DNA di€erences were found in methyl-group incorporation from normal mesothelium with regard to both into DNA from II14 or II45, indicating that DNA methylated and unmethylated CpG sites present in methylation levels were roughly equal in both cell lines. this region of the wt1 gene. Figure 6 shows that wt1, Methyl-group incorporation into DNA from normal intron 1 unmethylated (1.6 and 0.6 kb bands present in tissues was signi®cantly lower than incorporation into both HpaII and MspI lanes) and methylated (0.8 kb tumor DNA, indicating that DNA from the MM cell band visible only in HpaII lanes) sites recognized by lines was less methylated than DNA from normal cells. these isoschizomers were the same in normal mesothe- Therefore, the methylation of the prE site in MM was lium and MM II45. However, in the 5' region of wt1, not simply a re¯ection of a global increase in DNA methylation at the prE site did not follow this general methylation in tumor cells. methylation pattern as normal cells, while methylated To assay methylation of other CpG sites in the 5' at the same sites in wt1 intron 1 as II45 (Figure 6), did region of the wt1 gene Southern analysis of DNA from not display methylation at the prE site seen in MM cell lines II14, II45, III2, TO8, ME3 and normal mesotheliomas and MM cell lines. mesothelium was performed using HpaII/MspI diges- To determine whether methylation of the prE site tion and probes for wt1 exons 1 ± 3 and intron 1. was speci®c for MM or occurred in other tumors as HpaII/MspI digestion of DNA from all MM cell lines well, we analysed by Southern analysis the wt1 EcoRI wt1 site-specific methylation EV Kleymenova et al 717 digestion patterns of rat renal cell carcinomas, including primary tumors and ERC cell lines, and ELT cell lines derived from rat leiomyomas/leiomyo- sarcomas. The expression pattern of the wt1 gene in 5A ELT ERC 18 II 45 ERC and ELT cell lines was determined by Northern analysis with a wt1 probe for exons 1 ± 7. In contrast to 8-10 kb rat mesotheliomas (Walker et al., 1994), neither ERC (Figure 7) nor ELT cell lines (data not shown) expressed the wt1 gene. However, while the normal EcoRI digestion pattern was observed in primary renal 5.0 kb cell carcinomas and ELT cell lines (Figure 8), ®ve of seven ERC cell lines exhibited methylation at the prE 3.5 kb site as evidenced by 3.5 kb EcoRI band (Figure 8), indicating that transformed cells other than mesothelial cells also speci®cally methylated this site, even in the absence of wt1 gene expression. 1.7 kb

Discussion Figure 8 Detection of the 3.5 kb EcoRI fragment in ELT and Although mutations in the wt1 gene were not observed ERC cell lines. DNA from these lines was digested with EcoRI and hybridized with a PCR probe spanning wt1 exons 1 ± 7. ERC in rat mesothelioma cell lines, a CpG site in intron 1 of lines which exhibited the 3.5 kb EcoRI fragment were ERC 18 the wt1 gene was found to be methylated in almost all and ERC 15, ERC 21, ERC 24, and ERC 37 (data not shown) rat MM, including primary tumors and cell lines, and in most rat RCC cell lines studied. Methylation of this prE site prevented normal cleavage of the wt1 gene by EcoRI and so produced an altered EcoRI digestion pattern of this gene in rat MM and ERC cell lines. Treatment of MM cell line II45, which had the altered EcoRI digestion pattern, with the well established II45 Mesothelium inhibitor of DNA methylation 5-aza-dC restored prE site cleavage, indicating that the altered digestion H M H M pattern was caused by methylation of one or both cytosine residues at this site. Sensitivity of EcoRI to cytosine methylation is well known for prokaryotes 1.6 kb and has been reported for synthetic oligomers (Brennan et al., 1986), but not for eukaryotic genomic DNA. The ability of methyl cytosine to alter EcoRI cleavage of genomic DNA in mammals is not generally appreciated, and could cause misinterpretation of 0.8 kb Southern analysis data if not considered. 0.6 kb Methylation of the prE site in two di€erent types of rat tumor cells suggests that a common tumor-speci®c Figure 6 Methylation of the CpG sites in 5' region of the wt1 mechanism of DNA methylation may regulate gene in rat MM cell lines. DNA was digested with HpaII (H) or MspI (M) and hybridized to clone wk 1.4 that spans 200 bp of methylation of this site. Prolonged exposure of wt1 exon 2 and 3.3 kb of wt1 intron 1 Southern blots with DNA from normal cells revealed a very low level of methylation at this site in normal DNA suggesting that a quantitative rather than qualitative change in methylation occurs at this site ERC in tumor DNA. A previous study in our laboratory nK 15 17 19 21 (Walker et al., 1994) demonstrated wt1 expression in normal rat tissues and MM cell line II14, in which the prE site was unmethylated, and in MM cell lines II45, III2, ME1T, and NRM2, in which the prE site was wt1 3.0 kb methylated, indicating that transcription of the wt1 gene in rat mesotheliomas is not the primary determinant for methylation at this site. Southern analysis of wt1 5' region revealed presence both methylated and unmethylated CpG sites in wt1 intron 1 in MM indicating that methylation of prE site was actin not due to hypermethylation of this region. Moreover, methylation of this site does not correlate with methylation of other CpG sites within 5' region of wt1 gene o€ering unique opportunities for studying Figure 7 Northern analysis of wt1 expression in ERC cell lines. determinants of tumor-speci®c methylation. Signi®cant Poly(A)+ RNA from normal rat neonatal kidney (nK) and four di€erent ERC cell lines was hybridized to a PCR probe spanning increases in methylation of the prE site observable in wt1 exons 1 ± 7 and reprobed with actin both transformed cells that expressed (mesothelioma) wt1 site-specific methylation EV Kleymenova et al 718 and did not express (renal cell carcinoma) wt1 suggests patterns via de novo DNA methylation events at that regardless of wt1 expression tumor cells can speci®c sites. Future studies using this model system speci®cally recognize this site for DNA methylation. may yield new insights into the mechanisms of de novo Why the prE site is methylated in tumor cells with a methylation in normal and neoplastic cells. high frequency is not clear at present. Our data are consistent with the hypothesis that there are `hot spots' Materials and methods that are preferentially recognized by DNA methyl- transferase (Smith et al., 1991). We identi®ed several Cell lines and tumors consensus sequences for DNA-binding , including transcriptional factors GCN4, SP1 and The cell lines II14, II45 and III2 were derived from chrysotile-induced rat mesotheliomas (Craighead et al., AP2, and histone H4 in intron 1 of the rat wt1 gene, 1987). Cell lines T10 and T08 were derived from the H4 binding site being nearest to the prE site crocidolite-induced rat mesotheliomas (Palekar et al., (Figure 1a). Histone H4 is one of the four core histones 1988). Cell lines ME1 and ME1T were derived from a that pack DNA into nucleosomes, and its absence or spontaneous rat MM (Funaki et al., 1991). Cell lines modi®cation by deacetylation, for example, results in NRM1, NRM2, NRM3, and NRM4 are rat pleural local chromatin perturbations (Wol€e, 1994). Future mesothelial cells spontaneously transformed in vitro studies of chromatin structure near prE site in MM (Bermudez et al., 1990). Cell lines ERC 15, ERC 17, and ERC cell lines may shed some light on the cause ERC 18, ERC 19, ERC 21, ERC 24, and ERC 37 were for its tumor-speci®c methylation. The lack of derived from renal cell carcinomas (Freed et al., 1990) and methylation at the prE site in ELT cell lines, in cell lines ELT 3, ELT 4, ELT 5A, ELT 5B, and ELT 6 were derived from leiomyomas/leiomyosarcomas (Howe et contrast to MM and ERC cell lines in which this site al., 1995); both of which develop spontaneously in Eker was frequently methylated, suggests that tissue-speci®c rats. All cell lines were maintained as described by authors. events may also be involved in site-speci®c DNA Five chrysotile-induced rat mesotheliomas obtained by methylation in tumor cells. Methylation of the prE site installation of Ural-mined milled chrysotile (a single dose in the wt1 gene was detected in a majority of primary of 60 mg per rat) into Wistar rat peritoneal cavities and mesotheliomas, indicating that methylation at this site nine spontaneous renal cell carcinomas from Eker rats is not a cell culture artifact. The extent of tumor- were also used. speci®c methylation at the prE site in primary tumors was variable (data not shown), suggesting either tumor PCR heterogeneity exists or re¯ecting the presence of normal Genomic DNA was obtained by phenol-chloroform cells, such as stromal cells, that do not methylate prE extraction from cell lines in the log phase of growth and site, in the tumor tissue. quick-frozen normal tissues and tumors pulverized under Methylation of the WT1 gene in normal or tumor liquid nitrogen. The DNA was con®rmed by electrophor- cells has not been well studied, although human esis to be of high molecular weight and was quanti®ed by chromosome 11 where the WT gene is located is spectrophotometry. Total RNA was isolated as described known to be imprinted and contains genes such as H19 (Chirgwin et al., 1979) from cell lines and tissues under and IGFII whose expression is regulated by DNA conditions mentioned above. Poly(A)+ RNA was obtained methylation (Li et al., 1993). Methylation of 11p13 by oligodeoxythymidylate cellulose anity chromatogra- region in Wilms' tumors was studied by Royer-Pokora phy. Reverse transcription and PCR of one half of the and Schneider (1992). In that study, CpG methylation reverse transcribed product or 0.5 ± 1 mg of DNA were performed under conditions described previously (Kley- in the wt1 5' region was found in two of 29 Wilms' menova et al., 1997). The LA PCR from TaKaRa (Pan tumors, whereas normal kidney and blood were Vera Corp., Madison, WI) was used according to unmethylated; however, no correlation was observed manufacturer's instructions to amplify a 6 kb fragment of between methylation and WT1 expression. In addition, the wt1 gene from exon 1 to exon 3. The primers used to several methylated CpG sites 100 kb distal to the WT1 amplify various exons of the rat wt1 gene were based on gene and 100 ± 150 kb proximal to WT1 were the rat wt1 sequence published previously (Sharma et al., demethylated in most tumors analysed. Interestingly, 1992), and are shown in Figure 1a. Primers used were: exon Wilms' tumors which were hypomethylated proximal 1 ± 10 probe - 4581+4887; exon 1 ± 7 probe - 4581+4585; to WT1 expressed high levels of WT1, although a exon 4 ± 7 probe - 4883+4585; exon 1 ± 3 probe - direct link between WT1 methylation and transcription 4581+r618; exon 2 probe - T7B + T7. Primers P1, T3A, T3B, T3C, T7A, T7C, T7 and T3 were used for sequencing. in Wilms' tumors remains to be established. In our Primers T3 and T7 were purchased from Promega Corp., study, the 5-aza-dC treatment that restored normal Madison, WI; sequence of other primers is available upon EcoRI cleavage did not change the expression of the request. The PCR products were electrophoresed on a 1% wt1 gene in MM cell lines T08 and T10 (data not LMP agarose gel (Gibco BRL, Gaithersburg, MD), and shown), which were methylated at the prE site and bands of the expected sizes were gel-puri®ed with the which had been previously shown to express low levels Wizard PCR Preps System (Promega Corp., Madison, WI), of wt1 (Walker et al., 1994). and used for sequencing or as probes for hybridization. The ability of DNA methylation to regulate gene expression points to an important role for de novo Southern analysis methylation in and di€erentiation. How- Fifteen micrograms of genomic DNA was digested with ever, how speci®c CpG sites, substrates for DNA restriction enzymes under conditions suggested by manu- methyltransferase, are recognized in mammalian cells, facturer, electrophoresed in 0.8% agarose gel, and especially tumor cells, remains largely unknown. Our denatured with 0.5 M NaOH and 1.5 M NaCl. After identi®cation of a CpG site frequently methylated in neutralization in 1.5 M NaCl and 1 M Tris-HCl, the DNA tumors provides a useful model system for studying was transferred to nitrocellulose membranes in 206SSC. how malignant cells establish DNA methylation The Prime-it Random Primer Labeling kit (Stratagene, La wt1 site-specific methylation EV Kleymenova et al 719 Jolla, CA) was used to label probes for hybridization. incorporation of methyl-groups into DNA. Two hundred Hybridization was performed at 428C in 50% formamide, nanograms of DNA was treated with 4 units of M.Sss I 56SSC, and 0.25% SDS for 48 h. Blots were washed in CpG Methylase (New England Biolabs, Beverly, MA) in 0.16SSC and 0.05% SDS, dried, and exposed to X-ray the presence of 5 mM [3H]S-adenosylmethionine (SAM) and ®lm with an intensifying screen at 7708C. an equal amount of nonradioactive SAM for 3 h at 378C. Methyl-group incorporation was determined by measuring 3H c.p.m./ng DNA. All samples were performed in DNA cloning duplicate. Reactions without either DNA or enzyme Forty-®ve micrograms of DNA from MM cell line II45 was added served as negative controls. digested with EcoRI and electrophoresed in a 1% LMP agarose gel. Using a 1 kb DNA Ladder (Gibco BRL, 5-aza-dC treatment Gaithersburg, MD) stained with ethidium bromide as a reference, 3 ± 4 kb DNA fragments were gel-puri®ed with MM cell lines II45, T08, and T10 were plated in the Wizard PCR Preps System. A genomic library appropriate medium in 150 mm dishes at low density containing these inserts cloned into the EcoRI site of the (15% con¯uence) and allowed to proliferate for 24 h. The vector Lambda ZAP II (Stratagene, La Jolla, CA) was medium was then removed and replaced with medium screened by Lark Sequencing Technologies Inc. (Houston, containing 2 mM 5-aza-dC (Sigma Chemical Co., St. Louis, TX). By using a PCR product spanning wt1 exons 1 ± 3 as a MO) prepared immediately before use. The cells were probe, positive clones were isolated, and after two rounds grown for 96 h (75 ± 85% con¯uence) in the presence of 5- of screening, one clone was chosen for pBluescript excision. aza-dC, and DNA was obtained by Qiagen kit (Qiagen The pBluescript phagemid containing the cloned insert was Inc., Chatsworth, CA). excised to generate the phagemid clone wk 1.4.

DNA sequencing Puri®ed PCR products and clone wk 1.4 were directly Acknowledgements sequenced with the fmol DNA Sequencing System kit We are very grateful to Drs J Craighead, D Con and A (Promega Corp., Madison, WI) according to manufac- Knudson for the cell lines; Drs J-P Issa and S Baylin for turer's instructions. Sequences were analysed with the assistance with the in vitro methyl-accepting assay; and Wisconsin Sequence Analysis Package (Genetic Computer Drs G Fraiser, D Swa€ord and K Burroughs for their Group, Madison, WI). critical reviews of the manuscript. We also thank K Sowell and D Trono for technical assistance and M Gardiner for assistance in preparation of the manuscript. Methyl-accepting assay The editorial assistance of the MD Anderson Department To determine levels of DNA methylation, in vitro methyl- of Scienti®c Publications is appreciated. This work was accepting assay (Wu et al., 1993) was performed. In this supported in part by NIH grants ES06658 and CA63613 assay DNA methylation levels are inversely related to the to CLW.

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