THE JOURNAL OF B I O ~ I C A L CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Transcriptional Regulation of the Human Wilms’ Tumor Gene (WT1) CELL TYPE-SPECIFIC ENHANCER AND PROMISCUOUS PROMOTER*

(Received for publication, November 5, 1993)

Gail C. Fraizer, Ying-Ji Wu, Stephen M. Hewitt, Tapati Maity, Carl C. T. Ton& Vicki H 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- sues, mainly the developing kidney, genitourinary sys- tem, and mesothelium, and in immature hematopoietic cells. To develop an understanding of the role of WT1 in development and tumorigenesis, we have identified transcriptional regulatory elements that function in transient reporter gene constructs transfected into kid- ney and hematopoietic cell lines. We found three tran- scription start sites of the WT1 gene and have identified an essential promoter region by deletion analysis. The WT1 promoter is a member of the GC-rich, TATA-less, and CCAAT-less class of polymerase I1 promoters. Whereas the WT1 promoter is similar to other tumor suppressor gene promoters, the WT1 expression pattern (unlike Rb and p53) is tissue-restricted. The WT1 GC- rich promoter is promiscuous, functioning in all cell lines tested, independent of WT1 expression. This find- ing suggests that the promoter is not tissue-specific, but that tissue-specific expression of WT1 is modulated by additional regulatory elements. Indeed, we have identi- fied a transcriptional enhancer located 3’ of the WT1 gene >50 kilobases downstream from the promoter. This orientation-independent enhancer increases the basal transcription rate of the WT1 promoter in the human erythroleukemia cell line K562, but not in any of the other cell lines tested.

Wilms’ tumor (nephroblastoma) is one of the most common solid tumors of childhood, accounting for -6% of all childhood malignancies (Young and Miller, 1975). The disease occurs in both heritable and sporadic forms and has been postulated to require two allelic mutations (Knudson and Strong, 19721, one on each chromosomal homolog. Wilms’ tumors have been asso- ciated with both germline and somatic chromosomal deletions at l lp13 (Riccardi et al., 1978; Kaneko et al., 1981). The Wilms’ tumor gene, WT1, was isolated by positional cloning (Call et al., 1990; Gessler et al., 1990). Intragenic deletions of the WT1 gene in Wilms’ tumor patients (Haber et al., 1990; Huff et al., 1991; Pelletier et al., 1991a; Little et al., 1992) and high levels of expression of WT1 in the developing fetal kidney (Pritchard-

CA 46720, CA 34936, and CA 16672. The costs of publication of this * This work was supported by National Institutes of Health Grants

article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Center for Cancer Research, Massachusetts Insti- tute of Technology, Cambridge, MA 02139

5 Present address: Dept. of Experimental Pediatrics, Box 88, Univer- sity of Texas M. D. Anderson Cancer Center, Houston, TX 77030.

f To whom correspondence should be addressed: Dept. of Biochemis- try and Molecular Biology, Box 117, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792- 2690; Fax: 713-790-0329.

Jones et al., 1990) support the identification of WT1 as a gene that is important for Wilms’ tumorigenesis. At the same time, another putative Wilms’ tumor gene transcript, Wit-1 (Huang et al., 19901, was described as originating from within 600 bp’ of the start site of WT1, but transcribed in the opposite direc- tion. The Wit-1 gene was described as being expressed at one- tenth the level of the WT1 gene, and no protein product has been described for the Wit-1 gene.

The WT1 gene has two alternative splice sites resulting in four RNA transcripts that differ in abundance, but are ex- pressed in constant ratios (Haber et al., 1991). The WT1 gene encodes a transcription factor containing four zinc fingers in the carboxyl terminus with a proline-rich amino terminus. The zinc finger region of the WT1 protein binds DNA in vitro (Raus- cher et al., 1990; Bickmore et al., 19921, and the proline-rich region functions as a transcriptional repressor (Madden et al., 1991). WT1 has been demonstrated to repress the early growth response-1 (Madden et al., 1991), platelet-derived growth factor A-chain (Gashler et al., 19921, insulin-like growth factor I1 (Drummond et al., 1992), and insulin-like growth factor 1R (Werner et al., 1993) promoters in reporter gene constructs. Naturally occurring mutations in the zinc finger region of the WT1 gene have been detected in some Wilms’ tumors (Haber et al., 1990; Huff et al., 1991; Pelletier et al., 1991a; Little et al., 1992).

Histologically, Wilms’ tumor has a classic triphasic appear- ance with elements that resemble the embryonic kidney: an undifferentiated component (blastemic stem cells), an epithe- lial component (glomeruli and tubules), and a fibroblastic stro- mal component (Bennington and Beckwith, 1975). The tumor is believed to be derived from embryonic metanephric tissues per- sisting beyond fetal development (Kidd, 1984), perhaps failing to respond to normal differentiation signals (Beckwith et al., 1990). The Wilms’ tumor gene, WT1, has been proposed to play a role in kidney and genitourinary development based, in part, on its high level of expression during organogenesis (Pritchard- Jones et al., 1990; Eccles et al., 1992). Similarly, fetal spleen (Call et al., 1990) and immature leukemia cells (Miwa et al., 1992), presumably representing undifferentiated hematopoi- etic cells, also express WT1.

Our goal was to examine the regulation of WT1 expression in kidney and hematopoietic cells in order to understand its role in the development of these tissues. Our approach was to iden- tify regulatory elements that initiate and modulate WT1 re- porter gene expression in kidney and hematopoietic cell lines. The initiation site of WT1 transcription was mapped to a 650-bp genomic DNA fragment located 5‘ of the longest WT1 cDNA, LK15 (Gessler et al., 1990). We demonstrate that the promoter activity in this 650-bp fragment functions in all cell

The abbreviations used are: bp, base pairk); kb, kilobase(s); PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase.

8892

Regulation of WTl 8893 PEA3 PAX2 Spl CTCF

1 AAGCTTGACT GAGTTCTTTC TGCGCTTTCC TGAAGTTCCC GCCCTCTTGG

PAX8 APZ CTCFx2 TCF-1 51 AGCCTACCTG CCCCTCCCTC CAAACCACTC TTTTAGATTA A C A A C C m

CF1

AP2 101 ECTACTCCC ACCGCATTCG ACCCTGCCCG GACTCACTGC TTACCTGAAC

PAX8

FIG. 1. Sequence of the WTl proxi- mal promoter region. Potential tran- scription factor-binding sites are under- lined, with the consensus-binding sites listed in Table I. The transcription start

double-underlined. The major start site is sites are marked with bent arrows and are

the 104-bp minimal promoter. The 3'-ends also marked as +l. The stippled region is

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 itali- cized bases.

WT1 151 GGACTCTCCA GTGAGACGAG GCTCCCACAC TGGCGAAGGC CAAGAAGGGG

F-ACT1

H4TF1 +I r> r> AP4

201 AGGTGGGGGG AGGGTTGTGCCACCGGCC AGCTGAGAGC GCGTGTTGGG

253bp del 274bp del r> 1 CTCF

2 5 1 T T G ~ A G G A ' GGGTGTCTCd'.'GAGAGGGACG 'CTCCCPCGGA CCCGCCCTCA 1 CTCF SP1 CTCF

. .. . . primer 5 'c E2A AP4 WT1 .CTCF GCF

301 CC-Wc' AP2

. . :.-GAGGGCGCCCI GGaGGAGCA GCGCGCGCTG CC'TGGCC-

357bp del 1

351.:..;:-= . . TGAGTGAATG GAGCGGCCGA GCCTCCTGGC TCCTCCTCTT .:, : .. ..... ..... . .. . . . ..

AP2 GCFx4 CTCF NF-IL6 TCF-1 CTCF 401 CCCCGCGCCG CCGGCCCCTC TTTATTTGAG CTTTGGGAAG CTGAGGGCAG

E2A AP4 NF-IL6 I LKl5 end

WT1 TEF-2 AP2 451 CCAGGCAGCT GGGGTAAGGA GTTCAAGGCA GCGCCCACAC CCGGGGGCTC

WT 1 Spl WTlx2 CTCFx2 501 TCCGCAACCC GACCGCCTGT CCGCTCCCCC ACTTCCCGCC CTCCCTCCCA

primer 5 'b

PuFx3 WT1 551 CCTACTCATT CACCCACCCA CCCACCCAGA GCCGGGACGG CAGCCCAGGC

AP2

GCF AP2 GCF 601 GCCCGGGCCC CGCCGTCTCC TCGCCGCGAT CCTGGACTTC CTCTTGCTGC

PEA3 Ets-1 TCF-2a

NF-IL6 651 AG

lines tested. A 104-bp minimal promoter region has been iden- tified and is capable of supporting transcriptional initiation. Sequence analysis of this promoter region reveals that the WT1 promoter is GC-rich and contains multiple potential Spl- and WT1-binding sites (Wang et al., 1992; Pavletich and Pabo, 1991). The WT1 promoter is similar in structure to other tumor suppressor gene promoters, e.g. Rb and p53, which are also TATA- and CCAAT-less, but the expression pattern of WT1 is restricted to a few tissues and thus differs from these other tumor suppressor genes.

As the WT1 promoter functions in all cell lines tested, the tissue-specific expression of this gene must rely upon addi- tional regulatory elements. We have identified an orientation- independent transcriptional enhancer located 3' of the WT1 gene that is specific for K562 cells, a cell line derived from a chronic myelogenous leukemia in blast crisis that expresses WT1 mRNA. Using deletion analysis, we have identified the enhancer region that accounts for a majority of the enhancer activity. In constructs containing the WT1 promoter, the 3'- enhancer functions in a cell type-specific manner, increasing transcription from the WTl promoter in K562 cells, but not in any other cell line tested.

MATERIALS AND METHODS Cell Culture-The cell lines 293 (derived from adenovirus type

5-transformed human embryonic kidney cells (ATCC CRL1573)) and HeLa (a human cervical carcinoma cell line (ATCC CCL2)) were grown in Eagle's minimum essential medium supplemented with 10% fetal calf serum. Hep3B cells, from a human hepatocellular carcinoma cell line (ATCC HB8064), were maintained in a mixture of Eagle's minimum

essential medium and Waymouth medium (3:l) and 10% fetal calf se- rum. G401 cells, derived from a human rhabdoid tumor of the kidney (Garvin et al., 1993) (ATCC CRL1441), were maintained in a mixture of Dulbecco'slEagle's minimum essential medium and Ham's F-12 medium (1:l) with 10% fetal calf serum. K562 cells, from a human chronic myelogenous leukemia in blast crisis cell line (ATCC CCL243), were maintained in RPMI 1640 medium containing 10% fetal calf serum. Monolayers were seeded, and the suspension culture (K562) was split 48-72 h prior to transfection. Cells were transfected while in the log phase of growth, i.e. monolayers were <75% confluent, and the suspen- sion culture was at <5 x lo5 cellsfml.

RNA Preparation and Primer Extension Analysis-Total RNA was prepared from human fetal kidney tissues using guanidium isothiocya- nate (Chirgwin et al., 1979) and from cultured cells using RNAzol B (Tel-Test, Friendswood, TX) according to the manufacturer's recommen- dations. Fifty pg of RNA was hybridized a t 56 "C overnight in an aque- ous hybridization buffer (Kingston, 1990) to Ly-32PlATP end-labeled primers, which were then extended with avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim). The antisense primer 5'b (Fig. 1) was derived from sequence analysis of the 5'-end of the LK15 cDNA, +72 to +94 bp (Gessler et al., 1990). The primer 5'c was derived from our sequence analysis of the 5'-flanking region and is located 200 bp 5' of primer 5'b (Fig. 1). After cDNA synthesis, the DNARNA hybrid was digested with RNase A (Boehringer Mannheim), and the primer-extended products were analyzed on a 5% denaturing polyacrylamide gel. End-labeled DNA size markers and a dideoxy chain termination sequence ladder were loaded next to the extension products in order to map the transcription initiation sites precisely.

Cosmid Isolation-A pWE15 cosmid library (Evans and Wahl, 1987) prepared from human placental DNA was screened a t a density of 5-6 x lo3 colony forming unitdl50-mm plate. For isolation of fragments 5' of the WT1 gene, the colonies were first hybridized with the radiola- beled probe A24 (Compton et al., 1990). Colonies were replicated and processed according to Maniatis et al. (1982). A fragment from a result-

8894 Regulation of WT1

cB5-2 2-142 t I I I

M B NB B B B M E E W E E E E E E E E E E M , I II I , I 1 I I 1 1 1 I I, I I I , I , ,

I I I I I I I I I I I Genomic -30 -20 -10 0 10 20 30 40 50 60 70 lb

u u Proline Rlch

Regions P- 4 Zn Fingers Alternatively SPllCed

FIG. 2. Genomic organization of the WT1 gene. The limits of cosmids cB5-2 and 2 - 1 4 are defined by MboI (M) sites with internal NotI ( N ) and BamHI ( B ) sites shown for cB5-2 and internal EcoRI (E) sites shown for 2-1-C. A restriction map of the region between the two cosmids is adapted from Tadakoro et al. (1992). Map distances are marked from the NotI site 5' of the WT1 gene. The WTl proximal promoter and 3"enhancer are marked as boxes drawn below the line, and the 5'- and 3"untranslated regions are marked as hatched boxes. The positions of exons 1-10 are indicated. Coding regions are depicted with dotted lines extending from each exon; alternatively spliced exons are shown as devated boxes. The proline-rich transcriptional activation redon and the zinc finger DNA-binding region are marked (adapted from Huff and Saunders (1993) and Tadakoro et aZ. (1992)). enh350, 3'-enhancer region; aa, amino acids.

ant clone was then used to rescreen the library for adjacent clones. One of these, cB5-2, contained the 5'-end of the WT1 LK15 cDNA. To isolate genomic fragments from the 3'-portion of the WT1 gene, the pWE15 library was screened with the 848-bp Sau3AI fragment from the 3'- portion of WT33, another WT1 cDNA clone. The resultant clone, 2-1-C, contained the 3'-portion of the WTl locus.

Mapping and Sequencing the 5'-Flanking Region-The cosmid cB5-2, which contains the 5'-end of the WT1 LK15 cDNA (Gessler et al., 1990) and 32 kb of 5"flanking sequence, was mapped initially by hybridizing radiolabeled T3 and T7 primers to BarnHI or HindIII partially digested and NotI completely digested DNA (Fig. 2). Restriction fragments con- taining the 5"flanking region were identified by double digests with several restriction enzymes and hybridization to primer 5'b. The 3.1-kb XhoI fragment containing the 5'-end of the cDNA was subcloned into the sequencing vector Bluescript (Stratagene, La Jolla, CAI, and this fragment was, in turn, subcloned following HindIII digestion. The Hin- dIII clones were further subcloned by partial Tag1 or complete PstI digestion to generate two adjacent clones containing 0.53 and 0.65 kb, respectively, of 5'-flanking DNA. The 5"flanking region clones were sequenced by the dideoxy chain termination method (Sanger et al., 1977) using Sequenase (U. S. Biochemical Corp.) following the manu- facturer's recommendations. The sequence data were analyzed for po- tential regulatory elements using the Genetics Computer Group se- quence analysis software package, GCG (Genetics Computer Group, 1991).

Construction of WTl Promoter Plasmids-Gel-purified fragments were prepared from the 5"flanking clones described above and were subcloned into a pCAT reporter series of constructs (Promega). Both the distal 0.53-kb TaqI-Hind111 fragment and the proximal 0.65-kb HindIII- PstI fragment were subcloned into pCAT-Enhancer (Promega), contain- ing the SV40 enhancer, and pCAT-Basic (Promega), lacking the SV40 enhancer. To assess the bidirectionality of these promoter regions, both the distal promoter (0.53 kb) and the proximal promoter (0.65 kb) were subcloned into pCAT-Enhancer in both orientations. The distal pro- moter region was also cloned into the pCAT-Enhancer construct con- taining the proximal promoter region to create a 1.2-kb pCE proximal- distal promoter construct. 3"Deletions of the pCE proximal promoter were created by exonuclease IIVSl nuclease digestion of the PstI- and SmaI-double-digested pCE proximal promoter. These deleted frag- ments were religated and sequenced to determine the end points of their deletions. The minimal promoter region identified from the dele- tion constructs was subcloned into both pCAT-Enhancer and pCAT- Basic following PCR amplification of a 148-bp fragment containing an internal XbaI site at the vector junction and an artificial PstI site in the primer. Following PstI and XbaI digestion, the 104-bp fragment was subcloned into both the pCAT-Enhancer and pCAT-Basic vectors.

Mapping and Sequencing the 3"Flanking Region of WTl-The pla- cental cosmid library was rescreened with a radiolabeled 848-bp Sau3AI fragment derived from the 3'-end of the WT33 cDNAprobe (Call et al., 1990). The cosmid 2-1-C (Fig. 2), which contains the 3'-end of the WT1 cDNAand 12 kb of 3'-flanking sequence, was obtained. The cosmid 2-14 was restriction-mapped as described above using partial HindIII, EcoRI, or PstI digests and complete NotI digests. PstI fragments iden- tified in the cosmid map of 2-1-C were cloned directly into the PstI site

of the vector pCAT-Promoter (Promega) containing the SV40 promoter. This set of clones was transfected into K562 cells to assay for enhancer activity. One clone contained activity and was subjected to further anal- ysis.

Identification of the clone containing enhancer activity was accom- plished by hybridization to a radiolabeled PCR-amplified probe contain- ing exon 10 (zinc finger 4) of the WT1 gene. The PCR primer WTZN4 (5'-CTCGGGCCTTGATAGTTG) and the reverse 3"primer WT1762R (5"CCTGGGACACTGAACGGTC) were used to prepare the probe, and a 3.4-kb PstI fragment was detected. This PstI fragment was digested with AccI, generating a 1.85-kb AccI-PstI fragment (clone A4) that in- cludes 200 bp of the 3"untranslated region of exon 10. This AccI-PstI clone, A4, was sequenced as described above using both vector and internal primers and was analyzed for potential regulatory elements using the Genetics Computer Group package.

Construction of WT1 Enhancer Plasmids-The 1.85-kb AccI-PstI fragment clone A4 containing the 3"enhancer was used to generate the enhancer deletion constructs. Exonuclease IIUS1 nuclease deletion con- structs were created following XbaI and AccI digestion. The 5'-protrud- ing end of the XbaI site was protected from exonuclease 111 digestion by filling in the 3"recessed end with a-phosphorothioate dNTP. These deleted constructs were religated and sequenced to determine the end points of their deletions. On the basis of the functional analysis, a 414-bp fragment containing the minimal enhancer region was amplified by PCR. It has an internal BamHI site at the vector junction and an artificial BglII site in the primer. After digestion with BamHI and BglII, the 348-bp minimal enhancer fragment was inserted into the reporter vector PCB proximal promoter.

Dansfections and CAT Assays-Promoter constructs were trans- fected into 293, Hep3B, and G401 cells using the calcium phosphate transfection method (Graham and van der Eb, 1973) with 20 pg of CsCl gradient-purified plasmid DNA. The DNA was removed, and the me- dium wacchanged after 4 h for HepBB, HeLa, and G401 cells and after 18 h for 293 cells. K562 cells were transfected by electroporation using a modification of the protocol of Chu et al. (1987) adapted for leukemia cells (Oka et al., 1991). Four million cells were electroporated with the Gene Pulser (Bio-Rad) a t 200 V and 960 microfarads in 200 1.11 of serum- free medium containing 10 pg of plasmid DNAand were then kept on ice for 10 min and plated in T-25 flasks. As an internal control for trans- fection efficiency, K562 cells were also cotransfected with 5 pg of the expression vector pSV2p-Gal (Promega). Cells were harvested 48 h after transfection, and cytoplasmic extracts were prepared by three cycles of freeze-thawing (Gorman et al., 1982). The P-galactosidase ac- tivity in K562 cells was determined by standard methods (Maniatis et al., 1982). The protein content of cell extracts was determined by the assay of Bradford (19761, and extracts containing 25-50 pg of protein (for K562 cells) or 50-100 pg of protein (for 293, G401, HeLa, and Hep3B cells) were assayed for CAT activity (Gorman et al., 1982). After thin-layer chromatography, the acetylated [14Clchloramphenicol was quantitated by measuring the radioactivity with a Betascope 603 blot analyzer (Betagen Corp., Waltham, MA). The percent of acetylated chloramphenicol was normalized by the amount of P-galactosidase ac- tivity. Relative activities are averaged from at least three different experiments.

Regulation of WTl 8895

- A. B. - MW K562 HFK MW K562 -

369- -

FIG. 3. Primer extension analysis.A, 50 pg of total RNAfrom K562 cells (second lane) and human fetal kidney (HFK) tissue (third lane) was hybridized to the end-labeled primer 5'b (+72 to +94 bp of the cDNA LK15) and extended with reverse transcriptase. The primer-extended products were analyzed on a 5% denaturing polyacrylamide gel. The predominant extension products (350, 340, and 320 nucleotides) are denoted with arrows, and the 123-bp ladder (first lane) is marked (MW). B, K562 RNA was hybridized to primer 5'c (located 200 bp 5' of primer 5'b) and extended and analyzed as described above. The pre- dominant E O - , 140-, and 113-nucleotide products are marked (arrows), as is the 123-bp ladder (first lane; M W ) .

Enhancer constructs were tested for CAT activity in 293 and K562 cells as described above. For HeLa, G401, and Hep3B cells, 5-8 x lo6 cells were electroporated with 10-20 pg of DNA a t 230-250 V and 960 microfarads in 1 ml of HEPES-buffered saline containing 25 IIIM NaCl. Cells were plated and harvested a t 48 h. The protein content of extracts was determined as described above. Twenty to fifty pg of protein was assayed, and the CAT activity was quantitated and normalized by p-ga- lactosidase activity as described above. Relative activities are averaged from at least three different experiments.

RESULTS

Dunscription Initiation Sites within the GC-rich WTl Promoter-Primer extension analysis (Fig. 3 A ) of RNA ob- tained from cultured K562 cells and human fetal kidney tissue revealed several transcription start sites. Total RNAs from K562 cells (second lane) and human fetal kidney tissue (third lane) were hybridized to end-labeled primer 5'b (Fig. 11, and the primer was extended with reverse transcriptase. The pre- dominant extension products were 348, 339, and 312 nucleo- tides long. In addition to these extension products, smaller products were also observed (Fig. 3A ). These smaller products may reflect multiple start sites often observed in GC-rich pro- moters or artifacts resulting from reverse transcriptase termi- nation in the GC-rich region. In addition, a large extension product (750 nucleotides) was generated from tissue RNA samples (data not shown) and is believed to be an artifact due to residual DNA in the guanidium isothiocyanate-prepared tis- sue RNA sample. This possibility is supported by the finding that cDNA generated from cultured K562 and 293 kidney cell RNAs prepared by RNAzol lacked the larger extension product. Furthermore, the 5'-terminus of the 750-nucleotide primer ex- tension product mapped to a region that lacked promoter ac- tivity in all cell lines tested (Fig. 4). The positions of the tran- scription start sites were confirmed by use of a second primer, 5'c (Fig. l ) , whose sequence is located 200 bp 5' of primer 5'b. This primer generated three predominant extension products (Fig. 3 B ) that map to positions identical to those generated with primer 5'b, the region containing promoter activity. Two of three transcription start sites for the murine WT1 gene (Pelle- tier et al., 1991b) map to the corresponding region in the mu- rine 5'-flanking region; these two murine start sites are located 90 and 117 bp 5' of the major human WT1 start site. A com- parison of start sites in different tissues and cells revealed identical transcription start sites in Wilms' tumor tissue, adja- cent normal kidney tissue, acute myelogenous leukemia cells,

N B X H H T T H T P X I I I I I I Ilr I I

0 1 2 1 3 : 4 kb I i I I I

- d l 5 CDNA

Promoter 293 K562 I

Prox6 Dist WTl 11.3 18.0 k-5 Proximal WT1 66.0 45.0 - Proximal Witl 2.6 2.1 - Distal WTl 0.8 0.4 I=> Distal Witl 2.7 2.0 c-4 Activity Relative to pCAT Enhancer

"

FIG. 4. Activity of the W T 1 promoter constructs in K562 and 293 cells. The restriction map of the promoter region includes the Not1 (N), BarnHI ( B ) , and XhoI (X) sites 5' of the WT1 gene and the HindIII ( H ) , PstI (P), and Tu91 (7') sites within the 3.1-kb XhoI fragment ana- lyzed. The location of LK15 cDNA (Gessler et al., 1990) is marked, as is the major transcription start site (arrow). Promoter activity is ex- pressed relative to the vector pCAT-Enhancer. Transfections were car- ried out in 293 kidney and K562 leukemia cells, both of which express WT1 mRNA. The promoter constructs were tested for bidirectionality with the reverse orientation referred to as Wit-1 promoters, i.e. the direction of transcription is toward the Wit-1 gene (Huang et al., 1990).

and T cell leukemia cells (data not shown). Mapping and Sequencing the 5"Flanking Region of WTl-To

identify clones containing the 5'-flanking region of the WT1 gene, the 5'b primer-extended cDNA probe was hybridized to BamHI-, XhoI-, and BamHI-XhoI-digested cB5-2 cosmid DNA (Fig. 2). A 3.1-kb XhoI fragment containing the 5'-end of the WT1 cDNA was cloned into the sequencing vector Bluescript, and portions of the clone were sequenced using primer 5'b and M13 and reverse M13 primers, confirming the orientation of the 3.1-kb XhoI insert. The HindIII subclones described above were also sequenced using M13 and reverse M13 primers. Ini- tially, a 1.18-kb portion of 5'-flanking sequence was analyzed for potential regulatory elements. Our sequence analysis agrees with that presented by Hofman et al. (1993). The distal promoter (Fig. 4) contains many potential regulatory elements, but it lacks promoter activity. In contrast, the proximal pro- moter is GC-rich (67%) and lacks both TATA and CCAAT boxes. Comparison of the 5'-flanking sequence with the similar region of the murine WT1 gene (Pelletier et al., 1991b) reveals sub- stantial homology, >80% over the proximal promoter region. The homology extends from 10 bp 3' of the HindIII site of the human proximal promoter region, which is equivalent to the -90-bp position of the mouse promoter, and extends through the cDNA (Pelletier et al., 1991b). Within this homologous re- gion, there exist many conserved elements including some of the binding sites for the regulatory elements Spl, A P 2 , AP4, PAX2, and WT1 (Fig. 1).

Functional Analysis of the WTl Promoter-To localize the promoter within the Bl-flanking region of the WT1 gene, the 1.2-kb region containing the 5'-end of the cDNA was divided into two parts, the distal promoter and the proximal promoter. The primer extension analysis (Fig. 3) mapped the start of transcription to the proximal promoter. When the distal and proximal promoter regions were cloned into pCAT-Enhancer (containing the SV40 enhancer) and analyzed for functional activity in transient transfection assays, only the pCE proximal promoter had detectable promoter activity in K562 and 293 cells (Fig. 4). Activity is depicted relative to the vector pCAT- Enhancer, which lacks a promoter. When coupled with the SV40 enhancer, the proximal promoter region is >80 times more active than the distal promoter region in K562 and 293 cells. Addition of the distal promoter region to the proximal

8896 Regulation of WTl 70

I FIG. 5. Tissue specificity of the WT1

proximal and distal promoter re- gions. Left, proximal and distal W T 1 pro- moter regions were cloned into pCAT-En- hancer vectors containing the SV40 enhancer and were tested for CAT activity in 293, K562, HepBB, and G401 cells. Ac- W tivity is depicted relative to the vector !z pCAT-Enhancer, which lacks a promoter. Right, shown is a CAT assay of calcium phosphate-transfected 293 kidney cells c

(see text). Cells were transfected with the following DNA first lane, pCAT-Control, containing the SV40 promoter and en- hancer; second lane, pCAT-Enhancer, con- taining only the SV40 enhancer; third 10 lane, the proximal WT1 promoter and SV40 enhancer.

z 50

B c

0 30 a .- c - al U

. . . . .

. . . . .

. . . . .

. . . . .

-

. . . . . . . . . . . . . . . . . . . . . .o 293. . . .

. . . . . . . . . . . . . . . . . . . .

I I” I

n ~ 4 o 1 Q CL

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . @ e a I

pCAT Control ..... pCAT Enhancer ‘ I

Proximal WTl pro Distal WT1 pro Proximal WT1

promoter region construct resulted in some decrease in overall activity in deletion 274 to 16% of the CAT activity of the full- promoter activity (pCE proximal-distal uersus pCE proximal promoter). The distal promoter region was proposed to serve as a bidirectional promoter for the WT1 and Wit-1 transcripts (Huang et al., 1990). We tested constructs containing this re- gion 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 pro- moter for either orientation in 293 or K562 cells (Figs. 4 and 5). The proximal promoter has strong promoter activity in the WT1 orientation, but lacks bidirectionality, having no signifi- cant 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 ex- press moderate levels of WT1 mRNA as determined by North- ern blot analysis) and K562 erythroleukemia cells (which ex- press 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 that fail to express WT1 by Northern blot analysis. The proximal promoter region is promiscuous, expressing CAT activity in all cell lines tested with the reporter gene 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 the nonexpressing 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 WT1- binding sites suggest an autoregulatory system for WT1. Four Spl-binding sites (CCCGCCC) are present within the promoter and likely play an important role in initiation since there is no 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 is deleted. While there is essentially no promoter activity in the 3”deletion constructs in 293 cells, some activity remains in the K562 cells. This observation sug- gests that sequences upstream of the start site may contribute to promoter activity in K562 cells. However, the reduction of

length promoter indicates that the minimal promoter region is necessary for maximal promoter activity. The increase in pro- moter activity in K562 cells seen in deletion 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 transcrip- tion start sites.

To determine if the region demonstrated to be 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 se- quences, into the pCAT-Basic vector. This 104-bp minimal pro- moter construct is 79% 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 minimal promoter was assayed in K562 cells and found to contain a majority (53%) of the activity of the full-length proxi- mal promoter (Fig. 6). Like the full-length promoter, the mini- mal promoter is promiscuous, activating transcription in HeLa cells, which do not to express WT1, at 41% of the level of the full-length promoter.

Subcloning and Mapping the 3’-Flanking Region of WTl-To identify the 3’-flanking region of WT1, a PCR-amplified zinc finger 4 probe from exon 10 (the 3”terminal exon) was hybrid- ized to subclones derived from the cosmid 2-1-C (Fig. 2). The 3.4-kb PstI fragment containing exon 10 was restriction- mapped and used to generate the AccI-PstI clone A4. This clone, containing 1.6 kb of 3”flanking region, was sequenced to con- firm the orientation of the 1.85-kb insert.

Functional Analysis of the WTl 3”Enhancer Region- Functional analysis of the 3’-half of the WT1 gene contained within the cosmid 2-1-C and extending from 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 the developing spleen (Call et al., 1990). The only fragment containing en- hancer 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). Dele- tions of clone A4 created by exonuclease digestion were tested for enhancer activity in K562 cells, and activity is shown rela- tive to clone A4 (Fig. 7). The deletion construct d7.1 shows a >90% decrease in enhancer activity, suggesting the presence of an enhancer element in the 327-bp region between 711 and 1038 bp.

Regulation of WTl 8897

TABLE I Dunscription factor consensus-binding sites within the

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 from Faisst and Meyer (19921, except where noted.

AP1 TGASTMA HiNF-A ATTTNNNNATTT AP2 CCCMNSSS NF-IL6 TKNNGNAAK AP4 CAGCTGa PAX2 GTTCC CF1 ANATGG PAX8 TGCCCc CTCF CCCTC PEA3 AGGAAFt E2A RCAGNTG PUF GGGTGGG Ets-1 SMGGAWGY SPl GGGCGGd F-ACT1 TGGCGA TBP TATAAA GATA WGATAR TCF-1 MAMAG GCF SCGSSSC TCF-2a SAGGAAGY H4TFl GGGGGAGGG TEF-2 GGGTGTGG

WT1 GNGNGGGNGe

a Dang et al . , 1992. Dressler and Douglass, 1992. Zannini et al., 1992. Wang et al . , 1992.

e Pavletich and Pabo, 1991.

FIG. 6. Deletion analysis of the WT1 proximal promoter. A, the proximal promoter (652 bp) was cloned into pCAT- Enhancer, deleted with exonuclease, and assayed for the effect of deletion on tran- scription. 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 orienta- tion of the insert relative to the CAT re- porter gene are noted. Activities of the de- letion constructs are expressed relative to the full-length promoter of 652 bp. The positions of PAX2 (2), PAX8 (8), S p l (SI, and WT1 (W) DNA-binding motifs are marked. B, the 104-bp minimal promoter region was PCR-amplified and cloned into pCAT-Basic. Promoter activity was as- sayed and is expressed relative to the full- length promoter cloned into pCAT-Basic.

Relative Clone Activity "

A4

4d2

3d2

d7.1

d7.2

17d9

1 .oo

0.99

1.30

0.08

0.10

0.08

A. Relative CAT Activity

" 293 K562

1.00 1.00

0.48 0.69

0.03 0.16

0.07 0.28

8. Relative CAT ACtiVlty

HeLa K562

1.00 1.00 "

0.41 0.53

H LK15 cDNA

.I P

2s 8 w s w s w wswww

exon 10 enh350 1850 bp I I

1400 bp I

1038 bp I

711 bp

514 bp

192 bp

(enh2OO) -

2 = Pax2 8 = Pax 8 s = Sp1

FIG. 7. Deletion constructs of the 3'- enhancer. Deletion constructs are shown with sizes in base pairs. The 1.85-kb (A4) enhancer fragment was cloned into pCAT- Promoter, deleted by exonuclease, and as- sayed for the effect of the deletion on tran- scription from the SV40 promoter. Exon 10 of WT1 is marked, as is the 3'-en- hancer region (enh350) defined by dele- tion of clone 3d2. Deletion constructs were tested in K562 cells, and activity is ex- pressed relative to the full-length en- hancer construct A4.

Analysis of the Tissue Specificity of the WTI Enhancer-To G401 and HeLa. In K562 cells, the 1.04-kb enhancer fragment determine if the W T 1 enhancer is cell type-specific and re- in clone 3d2 increased transcription from the SV40 promoter by stricted in function to cells that express WT1, the enhancer 9.2-fold above the levels of the vector pCAT-Promoter without clone 3d2 was tested for CAT activity in the WT1-expressing enhancer. However, in all other cell lines tested, it failed to cell lines 293 and K562 and in the nonexpressing cell lines increase transcription above basal levels. In 293 and HeLa

8898 Regulation of WTl

Relative Actlvltv

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 containing the minimal enhancer. En- hancer activity is expressed relative to the WT1 proximal promoter (pro) con-

which the WT1 enhancer fragments were struct (PCB proximal promoter) into

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 HeLa 293

1.0 1.0 1.0 I WTl pro I CAT I “-

I WTI pro I CAT I I SV40Enh 0.0 17.0 2.4

10.4 0.5 0.2

L WTI pro I CAT I 362(-) 1 13.3 0.6 0.3 .

cells, the activity of the enhancer clone 3d2 coupled to the SV40 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 Posi- tion-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 the SV40 promoter-driven reporter constructs, yet the enhancer fragments tested all had similar activities, in- creasing 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 tested for its ability to modulate WT1 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 pro- moter. 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 (Gessler et 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 orientation- independent manner, but it confers tissue specificity to the WT1 promoter. In K562 cells, the WT1 enhancer increases ba- sal WT1 promoter transcription to greater levels compared with the SV40 enhancer (8.8-fold). However, the 3d2 enhancer fragment fails to increase the basal transcription levels 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 promoter- enhancer construct in 293 and HeLa cells is 10-30 times lower than the activity of the WT1 promoter-SV40 enhancer con- struct.

We confirmed that the minimal 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 analy- sis, enhances transcription from the WT1 promoter as effi- ciently as the larger 3d2 enhancer fragment. The sequence of the e350 fragment was determined and analyzed for potential

transcription factor-binding sites (Fig. 9). The minimal en- hancer region contains many transcription factor-binding sites that have been associated with enhancer 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-binding sites, and isolated a mini- mal promoter region capable of initiating transcription. This promoter functions in all cell types analyzed. A major 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 tissue specificity 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. Additional characteriza- tion 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 kid- ney (293) and erythroleukemia (K562) cell lines that express WT1. The promoter functions well in all cell lines tested, in- cluding 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 (posi- tion +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 site of the human WT1 gene maps to within 90 bp of one of the mouse start sites (Pelletier et al., 1991b).

Deletion analysis of the WT1 promoter has identified an 83-bp region that is essential for full promoter activity. Se- quence analysis of this minimal promoter region has identified four potential binding sites (Spl,AP4, GCF, and WT1) that may play an essential role 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 WTl 8899

TCF- 1 1 CTTTCTGTTC TGGTGTATGG TTTTTGAAGG TGAAATAAGG

CTCF GATA 51 GGTGCAGCCC CTCTCAGCTC CATTATCTTG GGGCTTGCAT

FIG. 9. Sequence of the WT1 3’-en- 101 TTTTATTTCT TCATTTAAAA TGCGTCTCAA ACAGATGGAA hancer. Potential transcription factor- binding sites are underlined, with consen- sus-binding sites listed in Table 11. The 5’-end of the 250-bp enhancer fragment is

HiNF-A NF-IL6 TCF-1 E2A CF1

TCF- 1 TCF-1 151 GGAGACTGTT TTACATTGAA GTGCAGCTCA AAGTTTGGGC

marked.

AGATTAATTT

enh250 1

GCATTCCGGG

GCCTAGCTAT

TCF-1 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

TABLE I1 Correlation of WTI mRNA expression with promoter and

enhancer activity

(tissue type) Cell line wT1 mRNA Promoter Enhancer

activity activity

293 (kidney) + + - G401 (kidney) - + - K562 (erytholeukemic) ++ + + Hep3B (liver) - + - HeLa (cervical) - + -

moter. A 104-bp minimal promoter construct containing the 83-bp essential region has been demonstrated to promote tran- scription in K562 and HeLa cells at 53 and 41%, respectively, of the level of the full-length promoter. While the start site for this minimal promoter has not been defined, it is likely to initiate near the Spl site within the 30-bp core that contains multiple transcription factor-binding sites (Fig. 1). Although the mini- mal promoter region was not examined, the 650-bp proximal promoter region has been shown to be Spl-responsive by co- transfection with an Spl expression vector in COS-7 African green monkey kidney cells (Hofman et al., 1993).

The WT1 promoter is similar in structure to other tumor suppressor gene promoters, e.g. Rb and p53, lacking CCAAT and TATA boxes, but differs from Rb and p53 because of its pattern of expression: limited to the kidney, genitourinary sys- tem, spleen, mesothelium, and hematopoietic tissue. Thus, the WT1 promoter is a member of a subclass of polymerase I1 promoters that lack typical TATA and CCAAT motifs, contain GC boxes, but are not housekeeping genes, e.g. genes encoding transforming growth factor-p (Jakobovits et al., 19881, c-Ha-ras (Ishii et al., 1985), the epidermal growth factor receptor (Pas- tan, 1985), and the nerve growth factor receptor (Sehgal et al., 1988). The specificity of selection of transcription start sites in this class of promoters is not clear, but the presence of GC boxes suggests a role for Spl, GCF, AP2, and other factors that bind GC boxes. Analyses of GC boxes in the dihydrofolate reductase promoter reveal that interactions between at least two of four GC boxes are required for transcription initiation and that these interactions affect start site utilization (Blake et al., 1990). As the WT1 minimal promoter 30-bp core region con- tains two overlapping GC-rich elements (GCF and WT1) lo- cated only 10 bp from an Spl element, it seems likely that these are essential elements for the initiation of transcription. The transcription start sites mapped by primer extension are lo- cated within 34 bp of the minimal promoter region, but second- ary structure problems, due to the high G + C content, may

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 revealed no element that enhances transcription of the WT1 promoter (Fig. 4). The combined construct of the distal and proximal promoter regions fails to significantly differ from the transcriptional activity of the proximal promoter alone (Fig. 4), and we do not believe that any WT1 regulatory elements exist in this region. We have examined the distal half of this 1.2-kb promoter region alone in the construct pCE distal promoter and found no evidence of significant promoter activity in either orientation (WT1 or Wit-1) in any cell line tested. Similarly, we found no significant evidence of bidirectional promoter activity in the proximal promoter region. Huang et al. (1990) note that the Wit-1 gene is expressed at a 10-fold lower level than WT1, initiating bidirectional transcription within a 600-bp fragment; however, we did not detect any promoter activity in the distal or proximal promoter regions to substantiate this. The finding of seven potential WT1-binding sites within the 650-bp promoter region of WT1 lends support to the possibility of autoregulation of WT1 expression. Of note are the WT1-binding sites on both sides of the transcription initiation site. Drummond et al. (1992) demonstrated the necessity of WT1-binding sites within the 5”untranslated region for WT1 repression of the insulin- like growth factor I1 promoter.

We have identified a cell line-specific enhancer in the 3’- flanking region of the gene. This enhancer can increase tran- scription from the SV40 promoter as well as the WT1 proximal promoter in transient transfection assays in K562 cells. Unlike the non-cell type-specific promoter, the 3”enhancer is hemato- poiesis-specific, functioning only in K562 cells. This may be due to the two hematopoiesis-specific GATA elements located within the 348-bp region. The 3”flanking region of the WT1 gene may have functional similarity to the 3’-flanking region of the hematopoiesis-specific p- and y-globin genes and the T cell receptor a- and &chain genes (Wall et al., 1988; KO et al., 1991; Ho et al., 1991). It is possible that GATA-binding proteins may facilitate transcription at the WT1 GC-rich promoter in some hematopoietic cells.

Regulation of the developmental and tissue-specific expres- sion of WT1 remains unclear. Clearly, the regulatory elements of the WT1 gene described here are not sufficient to confer kidney-specific regulation on the WT1 gene. Preliminary work in our laboratory has identified a tissue-specific transcriptional silencer.2 Defining additional regulatory elements that control

* S. M. Hewitt, unpublished data.

8900 Regulation of WTl tissue-specific developmental expression of WT1 will be essen- tial for in vivo studies of W l ’ s role in normal kidney develop- ment 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 muta- tions described in the Rb promoter (Bookstein et al., 1990; Sakai et al., 1991) will be found in the WT1 regulatory elements and possibly play a role in the pathogenesis of Wilms’ tumors and genitourinary abnormalities.

Acknowledgments-We acknowledge the assistance of Fernando Vil- lalba in computer analysis and our summer students Masayoshi Takashima, Derek Su, and Brian Le. We thank Dr. Brigitte Royer- Pokora for communicating unpublished results, Dr. Susan Fitzpatrick for a critical analysis of the manuscript, and Ruby Desiderio for the preparation of the manuscript.

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