Transcriptional regulation of the human Wilms' tumor gene (WT1). Cell ...

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tumor gene, WT1, was isolated by positional cloning (Call et al.,. 1990; Gessler et ... sity of Texas M. D. Anderson Cancer Center, Houston, TX 77030. f To whom ...
THEJOURNAL OF B I O ~ I C CHEMISTRY AL

Vol. 269, No. 12, Issue of March 25, pp. 8892-8900, 1994 Printed in U.S.A.

0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Transcriptional Regulation of the Human Wilms’ Tumor Gene(WT1) CELL TYPE-SPECIFIC ENHANCERAND PROMISCUOUS PROMOTER* (Received forpublication, November 5, 1993)

Gail C. Fraizer, Ying-Ji Wu, Stephen M. Hewitt, Tapati Maity, Carl C. T. Ton& VickiH a , and Grady F. Saundersn From the Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

The Wilms’ tumor gene, WT1, is expressed in few tis- Jones et al., 1990) support theidentification of WT1 as a gene sues, mainly the developing kidney, genitourinary sys- that is importantfor Wilms’ tumorigenesis. At the same time, tem, and mesothelium, andin immature hematopoietic another putative Wilms’ tumor gene transcript,Wit-1 (Huang cells. To develop an understanding of the role of WT1 in et al., 19901, was described as originatingfrom within 600 bp’ developmentandtumorigenesis,wehave identified of the start siteof WT1, but transcribed in theopposite directranscriptionalregulatoryelementsthatfunctionin tion. The Wit-1 gene was described as being expressed at onetransient reporter gene constructs transfected into kid- tenth the level of the WT1 gene, and no protein product has ney and hematopoietic cell lines. We found three tran- been described for the Wit-1 gene. scription startsites of the WT1 gene and have identified The WT1 gene has two alternative splice sites resulting in an essential promoter region by deletion analysis. The four RNA transcripts that differ in abundance, but are exWT1 promoter is a member of the GC-rich, TATA-less, pressed in constant ratios (Haber et al., 1991). The WT1 gene and CCAAT-less class of polymerase I1 promoters. Whereas the WT1 promoter is similar to other tumor encodes a transcription factor containing four zinc fingers in suppressor gene promoters, the WT1 expression pattern the carboxyl terminus witha proline-rich amino terminus. The (unlike Rb and p53) is tissue-restricted. The WT1 GC- zinc finger regionof the WT1 protein bindsDNA in vitro (Rausrich promoter is promiscuous, functioning in all cell cher et al., 1990; Bickmore et al., 19921, and the proline-rich lines tested, independent ofWT1 expression. This find- region functions asa transcriptional repressor (Madden etal., ing suggests that the promoter is not tissue-specific,but 1991). WT1 has been demonstrated to repress the early growth that tissue-specific expression of WT1 is modulated by response-1 (Madden etal., 1991), platelet-derivedgrowth factor additional regulatory elements. Indeed, we have identi- A-chain (Gashler et al., 19921, insulin-like growthfactor I1 fied a transcriptional enhancer located 3’of the WT1 (Drummond et al., 1992), and insulin-like growth factor 1R gene >50 kilobases downstream from the promoter. This(Werner et al., 1993) promoters in reporter gene constructs. orientation-independent enhancer increases the basal Naturally occurring mutations in thezinc finger region of the transcription rate of the WT1 promoter in the human WT1 gene havebeen detected in some Wilms’tumors (Haberet erythroleukemia cell line K562, but not in any of the al., 1990; Huff et al., 1991; Pelletier et al.,1991a; Little et al., other cell lines tested. 1992). Histologically, Wilms’ tumor has a classic triphasic appearance with elements that resemble the embryonic kidney: a n Wilms’ tumor (nephroblastoma) is one of the most common undifferentiated component (blastemic stem cells), an epithesolid tumors of childhood, accounting for -6% of all childhood lial component (glomeruliand tubules), anda fibroblastic stromalignancies (Young and Miller, 1975). The disease occurs in mal component (Bennington andBeckwith, 1975). The tumor is both heritable and sporadic forms and has been postulated to believed to be derived from embryonicmetanephric tissuesperrequire two allelic mutations (Knudson and Strong, 19721, one sisting beyond fetal development (Kidd, 1984), perhaps failing on each chromosomal homolog. Wilms’ tumors have beenasso- to respond to normal differentiation signals (Beckwith et al., ciated with both germline and somatic chromosomal deletions 1990). The Wilms’ tumor gene,WT1, has been proposed to play development based, in part, at llp13 (Riccardi et al., 1978; Kanekoet al., 1981). The Wilms’ a role in kidney and genitourinary tumor gene,WT1, was isolatedby positional cloning(Call et al., on its highlevel of expression during organogenesis (Pritchard1990; Gessler et al., 1990). Intragenic deletionsof the WT1 gene Jones et al., 1990; Eccles et al., 1992). Similarly, fetal spleen in Wilms’ tumor patients (Haberet al., 1990; Huff et al., 1991; (Call et al., 1990) and immature leukemia cells (Miwa et al., Pelletier et al., 1991a; Little et al., 1992) and high levels of 1992), presumably representing undifferentiated hematopoiexpression of WT1 in the developing fetal kidney (Pritchard- etic cells, also express WT1. Our goal was to examine the regulation of WT1 expression in * This work was supported by National Institutes of Health Grants kidney and hematopoietic cells in order to understand its role CA 46720, CA 34936, and CA 16672. The costs of publication of this in thedevelopment of these tissues. Our approach was toidenarticle were defrayed in part by the payment of page charges. This tify regulatory elements that initiate and modulate WT1 rearticle must therefore be hereby marked “advertisement” in accordance porter gene expression in kidney and hematopoietic cell lines. with 18 U.S.C. Section 1734 solely to indicate this fact. a of WT1 transcriptionwasmappedto $ Present address:Center for Cancer Research, Massachusetts Insti- Theinitiationsite 650-bp genomic DNA fragment located 5‘ of the longest WT1 tute of Technology, Cambridge, MA 02139 5 Present address: Dept. of Experimental Pediatrics,Box 88, Univer- cDNA, LK15 (Gessler et al., 1990). We demonstrate that the sity of Texas M. D. Anderson Cancer Center, Houston, TX 77030. promoter activity in this 650-bp fragment functions in allcell f To whom correspondence should be addressed: Dept.of Biochemistry and Molecular Biology, Box117, University of Texas M. D. Anderson The abbreviations used are: bp, base pairk); kb, kilobase(s); PCR, Cancer Center, 1515 Holcombe Blvd., Houston,TX 77030. Tel.:713-792polymerase chain reaction; CAT, chloramphenicol acetyltransferase. 2690; Fax: 713-790-0329.

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Regulation of WTl

8893

1

PEA3 PAX2 S p l CTCF AAGCTTGACT GAGTTCTTTC TGCGCTTTCC TGAAGTTCCC GCCCTCTTGG

51

CF1 PAX8 APZ CTCFx2 TCF-1 AGCCTACCTG CCCCTCCCTC CAAACCACTC TTTTAGATTA A C A A C C m

PAX8 AP2 101 ECTACTCCC ACCGCATTCG ACCCTGCCCG GACTCACTGC TTACCTGAAC

WT1 F-ACT1 151 GGACTCTCCA GTGAGACGAG GCTCCCACAC TGGCGAAGGC CAAGAAGGGG

+I H4TF1 r> r> AP4 201 AGGTGGGGGG A G G G T T G T G C C A C C G G C C AGCTGAGAGC GCGTGTTGGG FIG.1. Sequence of the WTl proximal promoter region. Potential transcription factor-binding sites are underlined,withtheconsensus-bindingsites listed in Table I. The transcription start sites are marked withbent arrows andare double-underlined. The major start site is also marked as +l. Thestippled region is the 104-bp minimal promoter. The 3'-ends of the deletion constructs (see Fig. 6) are marked with straight arrows, as is the 5'-end ofthe LK15 cDNA. Primers 5'c and 5'b are the antisense sequence of the italicized bases.

del 274bp del 253bp 1 CTCF SP1 CTCF CTCF 2 51 T T G ~ A. G ..G .A .'GGGTGTCTCd'.'GAGAGGGACG 'CTCCCPCGGA CCCGCCCTCA primer 5 'c AP2 E2A AP4 WT1 .CTCF GCF . :.-GAGGGCGCCCI . GGaGGAGCA GCGCGCGCTG CC'TGGCC301 CC-Wc'

r> 1

351.:...:,;::..-=..... ..... . ...

. . . ..

357bp del 1 . TGAGTGAATG GAGCGGCCGA GCCTCCTGGC TCCTCCTCTT

AP2 GCFx4 CTCF NF-IL6 TCF-1 CTCF 401 CCCCGCGCCG CCGGCCCCTC TTTATTTGAG CTTTGGGAAG CTGAGGGCAG LKl5 end WT1 TEF-2 AP2 I E2A AP4 NF-IL6 451 CCAGGCAGCT GGGGTAAGGA GTTCAAGGCA GCGCCCACAC CCGGGGGCTC

primer 5 'b Spl WTlx2 CTCFx2 WT 1 501 TCCGCAACCC GACCGCCTGT CCGCTCCCCC ACTTCCCGCC CTCCCTCCCA AP2 PuFx3 WT1 551 CCTACTCATT CACCCACCCA CCCACCCAGA GCCGGGACGG CAGCCCAGGC GCF AP2 GCF

PEA3 Ets-1 TCF-2a

601 GCCCGGGCCC CGCCGTCTCC TCGCCGCGAT CCTGGACTTC CTCTTGCTGC

NF-IL6 651 AG

lines tested.A 104-bp minimal promoter region has been iden- essential medium and Waymouth medium (3:l) and 10% fetalcalf serum. G401 cells, derived from a human rhabdoid tumor of the kidney tified and is capable of supporting transcriptional initiation. of Sequence analysis of this promoter region reveals that theWT1 (Garvin etal., 1993) (ATCC CRL1441), were maintained in a mixture Dulbecco'slEagle's minimum essential medium and Ham's F-12 medium promoter is GC-rich and contains multiple potential Spl- and (1:l) with 10% fetal calf serum. K562 cells, from a human chronic WT1-binding sites (Wang et al., 1992;Pavletich and Pabo, myelogenous leukemia in blast crisis cell line (ATCC CCL243), were 1991). The WT1 promoter is similar in structure to other tumor maintained in RPMI 1640 medium containing 10% fetal calf serum. suppressor gene promoters, e.g. Rb and p53, which are also Monolayers were seeded, and the suspension culture (K562) was split TATA- and CCAAT-less, but the expression pattern of WT1 is 48-72 h prior to transfection. Cells were transfected while in the log phase of growth, i.e. monolayers were 80 times identical transcription start sites inWilms' tumor tissue, adja- more active than the distal promoter region in K562 and 293 cent normal kidney tissue, acute myelogenous leukemia cells, cells. Addition of the distal promoter region to the proximal

Regulation of WTl

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70

FIG.5. Tissue specificity of the WT1 proximal and distal promoter regions. Left, proximal and distal W T 1 promoter regions were cloned into pCAT-Enhancer vectors containing the SV40 enhancer and were tested for CAT activity in 293, K562, HepBB, and G401 cells. Activity is depicted relative to the vector pCAT-Enhancer, which lacks a promoter. Right, shown is a CAT assay of calcium phosphate-transfected kidney cells 293 (see text). Cells were transfected with the following DNA first lane, pCAT-Control, containingthe SV40 promoterandenhancer; second lane, pCAT-Enhancer, containing only the SV40 enhancer; third lane,theproximal WT1 promoterand SV40 enhancer.

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Proximal WTl pro

promoter region construct resulted in some decrease in overall promoter activity (pCE proximal-distal uersus pCE proximal promoter). The distalpromoter region was proposed to serveas a bidirectional promoter for the WT1 and Wit-1 transcripts (Huang et al., 1990). We tested constructs containing this region in both orientations: pCE distal WT1 and pCE distal Wit-1 promoters and pCE proximal WT1 and pCE proximal Wit-1 promoters. No significant activity was found at the distal promoter for either orientation in293 or K562 cells (Figs. 4and 5). The proximal promoter has strong promoter activity in the WT1 orientation, but lacks bidirectionality, having no significant activity in the Wit-1 orientation in 293 and K562 cells. Analysis of WTl Promoter Tissue Specificity-To determine if the WT1 promoter is cell type-specific and restricted in function to cells that express WT1, the distal and proximal promoter regions were tested for CAT activity in several cell lines. The WT1-expressing cell lines included 293 kidney cells (which express moderate levels of WT1 mRNA as determined by Northern blot analysis) and K562 erythroleukemia cells (which express significant levels of WT1 mRNA). Cell lines that do not express WT1 that were examined included G401 cells (derived from a rhabdoid tumor of the kidney), Hep3B (a hepatocellular carcinoma cell line), and HeLa (a cervical carcinoma cell line), all cell lines thatfail to expressWT1 by Northern blot analysis. The proximal promoter region is promiscuous, expressing CAT activity inall cell lines testedwith the reportergene constructs (Fig. 5). The distal promoter construct lacks significant CAT activity in both the WT1-expressing cell lines 293 and K562 (Figs. 4 and 5)and in thenonexpressing cell line G401 (Fig. 5). Identification of the Minimal Promoter Region-Sequence analysis reveals several potential regulatory elements within the 650-bp proximal promoter (Fig. 1).Potential binding sites for several transcription factors, including Spl, WT1, PAX2, and PAX8, are present (Fig. 1 and Table I ) . The seven WT1binding sites suggest an autoregulatory system for WT1. Four Spl-binding sites(CCCGCCC)are presentwithin the promoter and likely play an importantrole in initiation since there isno CCAAT or TATA box in the promoter. The relative activity of deletion constructs created by exonuclease digestion of the proximal promoter was assayed in 293 and K562 cells and is shown in Fig. 6. Promoter activity in 293 and K562 cells is significantly reduced when the 83-bp segment containing the central WT1 and Spl sites deleted. is While there isessentially no promoter activity in the 3”deletion constructs in 293 cells, some activity remains in theK562 cells. This observation suggests thatsequences upstream of the start site may contribute to promoter activity in K562 cells. However, the reduction of

Distal WT1 pro

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a

‘ I

pCAT Control pCAT Enhancer Proximal WT1

activity in deletion 274 to 16% of the CAT activity of the fulllength promoter indicates that theminimal promoter region is necessary for maximal promoter activity. The increase in promoter activityin K562 cells seen indeletion 253 (28%)suggests that a negative element may be located between positions 274 and 253. Both the 253- and 274-bp promoter deletion fragments have significantly reduced promoter activity compared to that of the full-length promoter, although they retain the transcription start sites. To determine if the region demonstrated tobe necessary for significant promoter activity in the deletion constructs was sufficient to initiate transcription, we cloned a 104-bp fragment containing the 83-bp region, independent of the upstream sequences, into the pCAT-Basic vector. This 104-bp minimal promoter construct is79% GC-rich and contains seven overlapping potential transcription factor-binding sites within the core of 30 bp, including an SP1- and a WT1-binding site (Fig. 1).The minimalpromoter was assayed in K562 cells and found to contain a majority (53%)of the activity of the full-length proximal promoter (Fig. 6). Like the full-length promoter, the minimal promoter is promiscuous, activating transcriptionin HeLa cells, which do not to express WT1, at 41% of the level of the full-length promoter. Subcloning and Mappingthe 3’-FlankingRegion of WTl-To identify the 3’-flanking region of WT1, a PCR-amplified zinc finger 4 probe from exon 10 (the 3”terminalexon) was hybridized to subclones derived from the cosmid 2-1-C (Fig. 2). The 3.4-kb PstI fragment containing exon 10wasrestrictionmapped and used to generate the AccI-PstI clone A4. This clone, containing 1.6 kb of 3”flanking region, was sequenced to confirm the orientation of the 1.85-kb insert. Functional Analysis of the WTl 3”Enhancer RegionFunctional analysis of the 3’-half of the WT1 gene contained within the cosmid 2-1-Cand extendingfrom intron 5 to 12 kb 3’ of WT1 was carried out in K562 cells. The K562 cell line was chosen because it expresses abundant WT1 mRNA as do other immature leukemia cells (Miwa et al., 1992) and thedeveloping spleen (Call et al., 1990). The only fragment containing enhancer activity when assayed with the SV40 promoter was the 1.85-kb AccI-PstI cloneA4, containing 320 bp of exon 10 and 1.6 kb of 3’-flanking sequence (Fig. 7) (Gessler et al., 1992). Deletions of clone A4 created by exonuclease digestion were tested for enhancer activity in K562 cells, and activity is shown relative to clone A4 (Fig. 7). The deletion construct d7.1 shows a >90% decrease in enhanceractivity, suggesting the presence of a n enhancer element in the 327-bp region between 711 and 1038 bp.

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TABLEI Dunscription factor consensus-binding sites withinthe regulatory elements of WTl Potential transcription factor-binding sites found within the W T 1 promoter and 3"enhancer regions are listed. Consensus transcription fador-binding sites were obtained fromFaisst and Meyer (19921, except where noted.

PAX8

enh350

HiNF-A NF-IL6 PAX2

TGASTMA CCCMNSSS CAGCTGa ANATGG CCCTC RCAGNTG SMGGAWGY TGGCGA

AP1 AP2 AP4 CF1 CTCF E2A Ets-1 F-ACT1 GATA GCF H4TFl

ATTTNNNNATTT TKNNGNAAK GTTCC TGCCCc AGGAAFt GGGTGGG GGGCGGd TATAAA MAMAG SAGGAAGY GGGTGTGG GNGNGGGNGe

PEA3

PUF SPl TBP TCF-1 TCF-2a TEF-2 WT1

SCGSSSC GGGGGAGGG

Dang et al., 1992. Dressler and Douglass, 1992. Zannini et al., 1992. Wang et al., 1992. e Pavletich and Pabo, 1991. a

LK15 cDNA

A. FIG. 6.Deletion analysisof the WT1 proximalpromoter. A, the proximal promoter (652 bp) was cloned into pCATEnhancer, deleted with exonuclease, and assayed for theeffect of deletion on transcription. The start sites of transcription are marked with arrows, and the end of the LK15 cDNA is marked. The sizes of the deletion constructs and the orientation of the insert relative to the CAT reporter gene are noted. Activities of the deletion constructs are expressed relative to the full-length promoter of 652 bp. The S p l (SI, positions of PAX2 (2), PAX8 (8), andWT1 (W) DNA-bindingmotifs are marked. B , the 104-bp minimal promoter region wasPCR-amplified and cloned into pCAT-Basic. Promoteractivitywasassayed andis expressed relative to the fulllength promoter cloned into pCAT-Basic.

Relative Clone Activity 10 1.oo A4

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4d2

0.99

3d2

1.30

d7.1

0.08

d7.2

0.10

17d9

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Relative CAT Activity 293

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Relative CAT ACtiVlty

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0.53

exon

= Pax2 = Pax 8 s = Sp1 2 8

bp

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1400 bp

1038 bp

711 bp

514 bp

FIG.7. Deletion constructsof the 3'enhancer. Deletion constructs are shown with sizes in base pairs. The 1.85-kb (A4) enhancer fragment wascloned into pCATPromoter, deletedby exonuclease, andassayed for the effect of the deletion on transcription from the SV40 promoter. Exon 10 of WT1 is marked, as is the 3'-enhancer region (enh350) defined by deletion of clone 3d2. Deletion constructs were tested in K562 cells, and activity is expressedrelativetothefull-lengthenhancer constructA4.

192 bp (enh2OO)

Analysis of the Tissue Specificity of the WTI Enhancer-To determine if the W T 1 enhancer is cell type-specific and restricted in function to cells that express WT1, the enhancer clone 3d2 was tested for CAT activity in the WT1-expressing cell lines 293 and K562 and in the nonexpressing cell lines

G401 and HeLa. In K562 cells, the 1.04-kb enhancer fragment in clone 3d2 increased transcriptionfrom the SV40 promoter by 9.2-fold above the levels of the vector pCAT-Promoter without enhancer. However, in all other cell lines tested, it failed to increase transcription above basal levels. In 293 and HeLa

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FIG.8. Activity of the 3”enhancer with the W T 1 proximal promoter.The activity of the 3”enhancer region (clone 3d2) was tested in both orientations and compared with the activity of e350, the region identified by deletion analysis as containingtheminimalenhancer. Enhanceractivityisexpressedrelative to the WT1 proximalpromoter ( p r o ) construct (PCB proximal promoter) into which the WT1 enhancer fragments were cloned. The effect of the SV40 enhancer (SV40Enh) on W T 1 promoter activity is included for comparison. Activity was tested in K562, HeLa, and 293 cells.

K562

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WTl pro

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CAT

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WT pro I

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CAT

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WT pro I

cells, the activity of the enhancerclone 3d2 coupled to theSV40 promoter was 1.1- and O.5-fold, respectively, of the level of the SV40 promoter alone in the vector pCAT-Promoter. Thus, the 3”enhancer is tissue-specific, functioning only in K562 cells among the lines tested. The WT1 Enhancer Functions in a Tissue-specific and Position-independent Manner-The genomic position of the WT1 enhancer is -50 kb 3‘ of the WT1 promoter, but within the reporter constructs, it has been repositioned to a distance of only 2.7 kb 5’ of either the WT1 or SV40 promoter. Depending upon the size of the enhancer fragment tested, the minimal enhancer region has been positioned from 1.7 to 4.1 kb 3‘of the CAT gene in theSV40 promoter-driven reporter constructs, yet the enhancer fragments tested all had similar activities, increasing CAT activity 5.8-7.1-fold above the SV40 promoter basal transcription levels. Znteraction of the 3”Enhancer with the WTl Promoter-The 3”enhancer was inserted downstream of the CAT reporter gene and testedfor its ability to modulateWT1 promoter activity in a tissue-specific manner. The 3”enhancer region from clone 3d2 was cloned in both orientations into the PCB proximal promoter, a CAT construct containing the proximal WT1 promoter. This WT1 promoter-enhancer construct was tested for CAT activity in K562, 293, and HeLa cells, and CAT activity is shown relative to the PCB proximal promoter(Fig. 8). The enhancer region 3d2, located 1768 bp 3’ of the stop codon of the WT1 gene (Gessleret al., 19921, increased transcription of WT1 promoter-CAT constructs in K562 cells 10-13-fold in either orientation. Not only does the WT1 enhancer function in an orientationindependent manner, but it confers tissue specificity to the WT1 promoter. In K562 cells, the WT1 enhancer increases basal WT1 promoter transcription to greater levels compared with the SV40 enhancer (8.8-fold). However, the 3d2 enhancer fragment fails to increase the basal transcriptionlevels of the WT1 promoter in 293 or HeLa cells, i.e. the activity of the WT1 promoter-enhancer construct in 293 and HeLa cells is 0.2- and O.B-fold, respectively, of the level of the basal WT1 promoter construct (Fig. 8). Thus, the activity of the WT1 promoterenhancer construct in 293 and HeLa cells is 10-30 times lower than the activity of the WT1 promoter-SV40 enhancer construct. We confirmed that theminimal enhancer region is sufficient for the enhancement of transcription from the WT1 promoter by cloning the minimal enhancer region independent of the 3“flanking sequences. Fig. 8 shows that e350, containing the 327-bp minimal enhancer region identified by deletion analysis, enhances transcription from the WT1 promoter as efficiently as the larger 3d2 enhancer fragment. The sequence of the e350 fragment was determined andanalyzed for potential

I

CAT

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HeLa

293

1.0

1.0

1.0

0.0

17.0

2.4

10.4

0.5

0.2

13.3

0.6

0.3

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SV40Enh

.

362(-)

1

transcription factor-binding sites (Fig. 9).The minimal enhancer region contains many transcriptionfactor-binding sites that havebeen associated withenhancer activity, including two GATA-binding sites (Fig. 9 and Table I) likely to play a role in hematopoietic gene expression. DISCUSSION

We have identified multiple WT1 transcription start sites, cloned and characterized the WT1 promoter region, identified potential regulatory element-bindingsites, andisolated a minimal promoter region capable of initiating transcription. This promoter functionsin allcell types analyzed. Amajor finding of this work was the isolation of a 3”enhancer fragment that is capable of increasing basal transcription levels from both the SV40 and WT1 promoters in transient transfection assays in K562 cells. This enhancer fragment has been characterized with respect to its tissuespecificity and potential transcription factor-binding sites. Table I1 correlates the activities of the WT1 promoter and enhancer with the WT1 mRNA expression levels in five different cell lines. Clearly, additional regulatory elements play a role in the tissue-specific regulation of WT1 expression, and we plan to examine additional intron regions as well as to more extensively examine the 5’-flanking region for kidney-specific enhancer elements. Additionalcharacterization of transcription factors binding the 3”enhancer will also clarify the nature of its limited tissue specificity. The proximal promoter region, which is 5’ of WT1, includes 180 bp of the 5‘-untranslated region of LK15 and functions as a promoter in transient transfection assays in embryonic kidney (293) and erythroleukemia (K562) cell lines that express WT1. The promoter functions well in all cell lines tested, including Hep3B, HeLa, and G401, regardless of WT1 mRNA expression (Table 11). The unrestricted function of the WT1 promoter suggests that tissue-specific expression of WT1 is conferred by additional regulatory elements. WT1 transcription appears to initiate from multiple sites within the proximal promoter, with the major start site (position +1)located 251 bp upstream from the 5’ terminus of LK15 (Fig. 1).Mapping precisely the start site of the WT1 promoter has been hampered by the high GC content (67%). The major start siteof the human WT1 gene maps to within90 bp of one of the mouse start sites (Pelletieret al., 1991b). Deletion analysis of the WT1 promoter has identified an 83-bp region that is essential for full promoter activity. Sequence analysis of this minimal promoter region has identified four potential bindingsites (Spl,AP4,GCF, and WT1) that may play an essentialrole in WT1 transcription. Although the WT1 start sites do not correspond to this minimal promoter region (Fig. 6), this region contains transcription factor-binding sites that are sufficient to initiate transcription from the WT1 pro-

Regulation of W T l

FIG.9. Sequence of the WT1 3’-enhancer. Potential transcription factorbinding sites are underlined, with consensus-binding sites listed in Table 11. The 5’-end of the 250-bp enhancer fragment is marked.

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1

TCF- 1 CTTTCTGTTC TGGTGTATGG TTTTTGAAGG TGAAATAAGG AGATTAATTT

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enh250 1 CTCF GATA GGTGCAGCCC CTCTCAGCTC CATTATCTTG GGGCTTGCAT GCATTCCGGG

HiNF-A NF-IL6 TCF-1 E2A CF1 101 TTTTATTTCT TCATTTAAAA TGCGTCTCAA ACAGATGGAA GCCTAGCTAT TCF-1 TCF- 1 TCF-1 151 GGAGACTGTT TTACATTGAA GTGCAGCTCA AAGTTTGGGC AGCCTAAAAG CTCF NF-IL6 TCF-1 201 TCAGGTCCAG AGGCCCCTCT TATTTGCATC TGGCTCTTGC ATCACTGTTA AP1 TBP GATA TCF-1 251 ATTATAGCGA GTGTGGTGAC TCATTTATAT CAGCCGTTTT TATCTTTTCC PEA3 301 TGCCAGAAGA CAGCATTTCT CTGGAGAAGC TCAGGACAAG CATGGCAA

have prevented a more precise mapping of the predominant start sites. Analysis of the 1.2-kb region directly 5’ of the WT1 gene has wT1 mRNA Promoter Enhancer Cell line revealed no element that enhances transcription of the WT1 activity activity (tissue type) promoter (Fig. 4). The combined construct of the distal and + + 293 (kidney) proximal promoter regions fails to significantly differ from the + G401 (kidney) transcriptional activity of the proximal promoter alone (Fig.4), ++ + + K562 (erytholeukemic) and we do not believe that any WT1 regulatory elements exist + Hep3B (liver) in this region. We have examined the distal half of this 1.2-kb + HeLa (cervical) promoter region alone in the construct pCE distal promoter and found no evidence of significant promoter activity in either moter. A 104-bp minimal promoter construct containing the orientation (WT1 or Wit-1) in anycell line tested. Similarly, we 83-bp essential region has been demonstrated topromote tran- found no significant evidence of bidirectional promoter activity scription in K562 and HeLa cells at 53 and 41%, respectively, of in the proximal promoter region. Huang et al. (1990) note that the level of the full-length promoter. While the start site for this the Wit-1 gene is expressed at a 10-fold lower level than WT1, minimal promoter has not been defined, it is likely to initiate initiating bidirectional transcription withina 600-bp fragment; or near the Spl site within the 30-bp core that contains multiple however, we did not detect any promoter activityin the distal proximal promoter regions to substantiate this. The finding of transcription factor-binding sites (Fig. 1). Although the minimal promoter region was not examined, the 650-bp proximal seven potential WT1-binding sites within the650-bp promoter region of WT1 lends support to the possibility of autoregulation promoter region has been shown to be Spl-responsive by coof WT1 expression. Of note are the WT1-binding sites on both transfection with an Spl expression vector in COS-7 African sides of thetranscriptioninitiationsite. Drummond et al. green monkey kidney cells (Hofman et al., 1993). (1992) demonstrated the necessity of WT1-binding sites within The WT1 promoter is similar in structure to other tumor suppressor gene promoters, e.g. Rb and p53, lacking CCAAT the 5”untranslated region for WT1 repression of the insulinand TATA boxes, but differs from Rb and p53 because of its like growth factor I1 promoter. We have identified a cell line-specific enhancer in the 3’pattern of expression: limited to thekidney, genitourinary system, spleen, mesothelium, and hematopoietic tissue. Thus, the flanking region of the gene. This enhancer can increase tranWT1 promoter is a member of a subclass of polymerase I1 scription from the SV40 promoter as well as theWT1 proximal promoters that lack typical TATA and CCAAT motifs, contain promoter in transienttransfection assays inK562 cells. Unlike GC boxes, but are nothousekeeping genes, e.g. genes encoding the non-cell type-specific promoter, the 3”enhancer is hematotransforming growth factor-p(Jakobovitset al., 19881, c-Ha-ras poiesis-specific, functioning only in K562 cells. This may be due (Ishii et al., 1985), the epidermal growth factor receptor (Pas- to the two hematopoiesis-specific GATA elements located tan, 1985), and the nervegrowth factor receptor (Sehgalet al., within the 348-bp region. The 3”flanking region of the WT1 3’-flanking region of 1988). The specificity of selection of transcription start sites in gene may have functional similarity to the this class of promoters is notclear, but thepresence of GCboxes the hematopoiesis-specific p- and y-globin genes and the T cell suggests a role for Spl, GCF, AP2, and other factors that bind receptor a- and &chain genes (Wall et al., 1988; KOet al., 1991; GC boxes. Analyses of GC boxes in thedihydrofolate reductase Ho et al., 1991). It ispossible that GATA-bindingproteins may promoter reveal that interactions between at least two of four facilitate transcription at the WT1 GC-rich promoter in some hematopoietic cells. GC boxes are required for transcription initiation and that Regulation of the developmental and tissue-specific exprestheseinteractions affect start site utilization (Blake et al., 1990). As the WT1 minimal promoter 30-bp core region con- sion of WT1 remains unclear. Clearly, the regulatory elements tains two overlapping GC-rich elements (GCF and WT1) lo- of the WT1 gene described here are not sufficient to confer cated only 10 bpfrom a n S pelement, l it seems likely that these kidney-specific regulation on the WT1 gene. Preliminary work are essential elements for the initiation of transcription. The in our laboratory has identified a tissue-specific transcriptional transcription start sites mapped by primer extension are lo- silencer.2 Defining additional regulatory elements that control cated within34 bp of the minimalpromoter region, but secondary structure problems, due to the high G + C content, may * S. M. Hewitt, unpublished data. TABLE I1 Correlation of WTI mRNA expression with promoter and enhancer activity

8900

Regulation of WTl

tissue-specific developmental expression of WT1 will be essential for in vivo studies of W l ’ s role in normal kidney development and for determining the mechanism of pathogenesis for both Wilms’ tumors and genitourinary abnormalities. At the same time, it is likely that mutations analogous to the mutations described in the Rb promoter (Bookstein et al., 1990; Sakai et al., 1991)will befound in theWT1 regulatory elements and possibly play a role in the pathogenesis of Wilms’ tumors and genitourinary abnormalities. Acknowledgments-We acknowledge the assistanceof Fernando Villalba in computer analysisand our summerstudents Masayoshi Takashima, Derek Su, and Brian Le. We thank Dr. Brigitte RoyerPokora for communicating unpublished results, Dr. Susan Fitzpatrick for a critical analysis of the manuscript, and Ruby Desiderio for the preparation of the manuscript. REFERENCES Beckwith, J. B., Kiviat, N. B., and Bonadio, J . F. (1990)Pedintr: Pathol. 10, 1-36 Bennington, J., and Beckwith, J. B. (1975)TLmors of the Kidney, RenalPeluis, and Ureters, Armed Forces Institute of Pathology, Bethesda, MD Bickmore, W. A,, Oghene, K., Little, M. H., Seawright,A., van Heyningen, V., and Hastie, N. D. (1992) Science 267, 235-237 Blake, M. C., Jambou, R. C., Swick, A. G . , Kahn, J. W., and Azizkhan, J. C. (1990) Mol. Cell. Biol. 10, 66324641 Bookstein, R., Rio, P., Madreperla, S. A,, Hong, F., Allred, C., Grizzle, W. E., and Lee, W.-H. (1990)Proc. Natl. Acad. Sci. U. S. A . 87,7762-7766 Bradford, M. M. (1976)Anal. Biochem. 72, 248-254 Call, K. M., Glaser, T., Ito, C. Y., Buckler, A. J., Pelletier, J., Haber,D. A,, Rose, E. A,, Kral, A,, Yeger, H., Lewis, W. H., Jones, C., and Housman,D. E. (1990) Cell 60,509-520 Chirgwin, J. M., Przybyla,A. E., MacDonald,R. J., Cowan, N. J., Rutter,W. J., and Kirschner, M.W. (1979) Biochemistry 18, 5294-5299 Chu, G . , Hayakawa, H., and Berg, P. (1987) Nucleic Acids Res. 16, 1311-1326 Compton, D. A,,Weil, M. M., Bonetta, L., Huang, A,, Jones,C., Yeger, H., Williams, B. R. G . , Strong, L. C., and Saunders, G . F. (1990) Genomics 6, 309-315 Dang, C. V., Dolde, C., Gillison, M. L., and Kato, G . J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 599-602 Dressler, G . R., and Douglass, E. C. (1992) Proc. Natl. Acad. Sei. U. S. A. 89, 1179-1183 Drummond, I. A,, Madden, S. L., Rohwer-Nutter, P., Bell, G . I., Sukhatme, V. P., and Rauscher, F. J . , 111 (1992)Science 257, 674478 Eccles, M. R., Wallis, L. J., Fidler, A. E., Spurn, N. K., Goodfellow, P. J., andReeve, A. E. (1992) Cell Growth & Differ: 3,279-289 Evans, G . A., and Wahl, G. (1987) Methods Enzymol. 152,604-610 Faisst, S., and Meyer, S. (1992)Nucleic Acids Res. 20, 3-26 Garvin, A. J., Re, G . G . , Tarnowski, B. I., Hazen-Martin, D. J., and Sens, D. A. (1993)Am. J. Pathol. 142,375-380 Gashler, A. L., Bonthron, D. T., Madden, S. L., Rauscher, F. J., 111, Collins, T., and Sukhatme, V. P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10984-10988 Genetics Computer Group(1991) Program Manual for the GCG Package, Version 7, University of Wisconsin, Madison, WI Gessler, M., Poustka, A,, Cavenee, W., Neve, R. L., Orkin, S. H., and Bruns, G . A. P. (1990)Nature 343,774-778 Gessler, M., Konig, A,, and Bruns, G . A. P. (1992) Genomics 12, 807-813 Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982)Mol. Cell. Biol. 2, 10441051 Graham, E, and van derEb, A. (1973) Virology 62,45&457 Haber, D. A., Buckler, A. J., Glaser, T., Call, K. M., Pelletier, J., Sohn, R. L.,

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