©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
GATA-1 Transactivates the WT1 Hematopoietic Specific Enhancer (*)

(Received for publication, December 14, 1994; and in revised form, January 17, 1995)

Ying-ji Wu (§) Gail C. Fraizer Grady F. Saunders (¶)

From the Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Wilms' tumor gene, WT1, is believed to play a role in hematopoiesis as it is expressed in the spleen and in immature leukemias in addition to the developing genitourinary system. WT1 is down-regulated in differentiated leukemia cells both in vivo and in vitro and is up-regulated in fetal spleen and immature leukemia cells. The modulation of WT1 expression was examined in many cell types, and a hematopoietic-specific enhancer element has been identified. Here we describe the transcriptional response of this enhancer to hematopoietic-specific transcription factors. We found co-expression of WT1 and GATA-1 mRNA in K562 cells and in mouse spleen, suggesting potential interactions between these two transcription factors. We find that the activity of the 3` WT1 enhancer is positively correlated with the expression of GATA-1. Gel shift competition experiments and transactivation studies revealed that this functional activity is mediated via binding at a GATA-binding site in the WT1 enhancer. The transactivation of the WT1 enhancer by GATA-1 implies that GATA-1 plays a role in the regulation of WT1 during hematopoiesis.


INTRODUCTION

Wilms' tumor or nephroblastoma is one of the most common solid tumors in children and accounts for about 6% of all childhood malignancies (Knudson and Strong, 1972; Young and Miller, 1975). Histologically, classic triphasic Wilms' tumor consists of three cell types quite similar to those in the developing kidney (Kidd, 1984): poorly differentiated blastemal cells that are considered stem cells for nephrogenesis, epithelial cells, and a stromal component. A Wilms' tumor gene, WT1, has been isolated (Call et al., 1990; Gessler et al., 1990) by positional cloning techniques and shown to be involved in the etiology of at least some Wilms' tumor cases (Huff et al., 1991; Haber et al., 1990).

WT1 is expressed in normal fetal spleen, bone marrow, and immature leukemic cells (Call et al., 1990; Huang et al., 1990; Pritchard-Jones et al., 1994; Miwa et al., 1992; Miyagi et al., 1993), in addition to expression in fetal kidney cells (Pritchard-Jones et al., 1990) and cells of the genitourinary system (Pelletier et al., 1991, Kreidberg et al., 1993). Thus, WT1 is considered to have pleiotropic effects that relate to mesenchymal cell differentiation, and its expression is down-regulated during differentiation of pluripotent leukemic cell lines. In HL60 promyelocytic cells induced to differentiate to granulocytes or monocytes by treatment with Me(2)SO or phorbol 12-myristate 13-acetate, respectively, the WT1 mRNA levels decrease significantly (Sekiya et al., 1994). Similarly, in K562 cells, induction of erythroid or megakaryocytic differentiation by sodium butyrate or phorbol 12-myristate 13-acetate, respectively, resulted in a rapid drop in WT1 mRNA levels (Phelan et al., 1994). Thus, repression of WT1 may be required for differentiation of some hematopoietic lineages.

We have previously identified a tissue-specific regulatory region involved in the expression of the WT1 gene (Fraizer et al., 1994) and have now focused on identifying the trans-acting factors responsible for tissue-specific WT1 expression. As the WT1 promoter functions in all cell lines tested (Fraizer et al., 1994; Hofmann et al., 1993), the tissue-specific expression of this gene must rely upon additional regulatory elements. Previously we 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. Full enhancer activity is contained within a 250-bp (^1)minimal enhancer region. In constructs containing the WT1 promoter, the 3` enhancer functions in a cell type-specific manner, increasing transcription from the WT1 promoter in K562 cells. In this report we examine in detail the requirement of specific transcriptional regulatory factors for enhancer function. Our results indicate that GATA-1, a hematopoietic transcription factor, plays a major role in the regulation of WT1 expression in hematopoietic cells.


EXPERIMENTAL PROCEDURES

Cell Culture

Cell lines 293, derived from adenovirus 5-transformed human embryonic kidney cells (American Type Culture Collection, ATCC, CRL1573, Rockville, MD); HeLa, a human cervical carcinoma cell line (ATCC, CCL2); and Hep3B, a human hepatocellular carcinoma cell line (ATCC, HB8064) were grown in Eagle's minimal essential media supplemented with 10% fetal calf serum. K562 cells, a human cell line with erythroleukemia characteristics derived from a chronic myelogenous leukemia in blast crisis (ATCC, CCL243); HEL cells, a human erythroleukemia cell line (ATCC, T1B180); and CEM cells, a human acute lymphoblastic T cell leukemia (T-ALL) cell line (ATCC, CCL119), were maintained in RPMI 1640 medium containing 10% fetal calf serum. Monolayers were seeded, and the suspension cultures were 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 suspension cultures were at <5 times 10^5 cells/ml.

Construction of WT1 Enhancer Plasmids

The 1.85-kb AccI-PstI fragment derived from WT1 cosmid 2-1-c contains the 3` end of exon 10 and the 3`-flanking region of the gene (Fig. 1). The enhancer fragment 3d2 was derived from the AccI-PstI fragment during deletion analysis (Fraizer et al., 1994). The deletion clone 3d2 was used as a template for PCR amplification of the 351-bp enhancer fragment (e351) as described elsewhere (Fraizer et al., 1994).


Figure 1: Restriction map of the 3` enhancer. Top diagram, the cosmid 2-1-c, which contains the 3` end of the WT1 gene (exons 6-10) and 12 kb of 3`-flanking sequence was mapped with restriction enzymes: E = EcoRI, H = HindIII and N = NotI. Middle diagram, the 1.85-kb AccI (A)-PstI (P) fragment containing the 3` end of exon 10 (3`-untranslated region is shaded) was cloned into pCAT PRO and further restriction mapped with Bg = BglII and Pv = PvuII. Lower diagram, the deletion clone 3d2 containing 1.038 kb of enhancer sequence was then further restriction mapped using BstXI (Bs) and SphI (S). The enhancer clones e351 and e258 (striped) were constructed by PCR amplification as described in the text.



The same cloning strategy was used to generate the minimal enhancer construct e258 except that the PCR template was a derivative of clone 3d2, which lacked the 5`-most 93 bp of the enhancer fragment. After digestion of 3d2 with SphI, the 949-bp enhancer fragment was gel-purified and cloned into the SphI site of the pCAT promoter construct. The 5` PCR primer (5`-GCATTCTAGTTGTGGTTTGTC) was derived from the pCAT promoter vector and flanks the multiple cloning site. The 3` primer was derived from the enhancer sequence (5`-CTAGATCTTGCCATGCTTGTCCTGAG) and includes a 6-bp mismatch that creates a BglII restriction site. The PCR product was digested with BamHI and BglII, and the gel-purified 258-bp BamHI-BglII fragment was cloned in both orientations into the BamHI site of the 652-bp fragment containing the WT1 promoter. Additionally, the 258-bp minimal enhancer fragment was cloned in both orientations into the BamHI site of the WT1 104-bp minimal promoter (Fraizer et al., 1994). After the SphI digestion of 3d2 and removal of the 258-bp fragment, the 93-bp enhancer fragment remaining was ligated with the SphI site in the vector and contains only the 5` end of 3d2.

Transfections and CAT Assays

K562, 293, HEL, and CEM cells were transfected by electroporation as described previously (Fraizer et al., 1994) except that 2 times 10^7 CEM and HEL cells were electroporated at 230 V and then kept at room temperature for 10 min. HeLa cells were transfected as described previously except that cells were electroporated at 200 V in 200 µl of serum-free medium containing 10 µg of plasmid DNA. As an internal control for transfection efficiency, cells were also co-transfected with 5 µg of the expression vector pSV2beta-Gal (Promega Corp., Madison, WI). Cells were harvested, cytoplasmic extracts were prepared, and beta-galactosidase activity was determined as described (Fraizer et al., 1994). Equivalent amounts of beta-galactosidase activity were assayed for CAT activity (Gorman et al., 1982). The protein content of cell extracts was also determined by the Bradford assay (Bio-Rad), and extracts containing 25-50 µg of protein (for K562 cells) or 50-100 µg of protein (for other cells) were assayed for CAT activity (Gorman et al., 1982). After thin-layer chromatography, the acetylated [^14C]chloramphenicol was quantitated by measuring the radioactivity with a Betascope 603 blot analyzer (Betagen Corp., Waltham, MA). Relative activities are averaged from at least three different experiments.

Co-transfections were performed in 293 and HeLa cells as described above except that increasing amounts of the mouse GATA-1 cDNA expression construct (Tsai et al., 1990) were added to each transfection of 5 µg of reporter and 5 µg of beta-galactosidase control DNA. DNA concentrations were held constant by the addition of the expression vector DNA pXM (Tsai et al., 1990). The reporter construct (WT1 minpro.e258) contains no potential GATA-binding sites in the WT1 minimal promoter, but does contain a potential GATA-binding site in the WT1 minimal enhancer.

Reverse Transcriptase PCR

Total cell RNA was extracted from HEL, Hep3B, HeLa, 293, CEM, K562, and HL60, derived from a patient with acute promyelocytic leukemia (ATCC, CCL240), cells by the method of Chomczynski and Sacchi(1987) using STAT-60 (Tel Test, Friendswood, TX) according to manufacturer's recommendations. Total cell RNA was extracted from mouse spleen cells using guanidinium isothiocyanate (Chirgwin et al., 1979). The first strand cDNA synthesis was prepared using superscriptase II (Life Technologies, Inc.) according to the manufacturer's recommendations, except that 50 µM random hexamer primer and 20 units of an RNase inhibitor, RNasin (Pharmacia Biotech Inc.), were added, and the reaction was incubated for 2 h at 42 °C. PCR amplification was carried out using the following primer pairs: 5` GATA-1 (5`-ATTGTCAGTAAACGGGCAGG-3`) (exon 5) and 3` GATA-1 (5`-CACCACCATAAAGCCACCAGC-3`) (exon 6), 5` beta-actin (5`-GTGGGGCGCCCCAGGCACCA-3`) and 3` beta-actin (5`-GTCCTTAATGTCACGCACGATTTC-3`), and 5 ` WT1 (exon 7) (5`-GGCATCTGAGACCAGTGAGAA-3`) and 3 ` WT1 (exon 10) (5`-GAGAGTCAGACTTGAAAGCAGT-3`). Conditions of amplification were: 3 min denaturation at 95 °C followed by 35 cycles of 94 °C denaturation for 1 min, 58 °C annealing for 2 min, and 72 °C extension for 2 min, followed by a final 5-min extension at 72 °C. To compensate for the overabundance of actin mRNA, we used 1/10th the concentration of beta-actin primers as WT1 and GATA-1 primers (0.2 µM). PCR products were analyzed on a 1.5% SeaPlaque (FMC Corp., Bioproducts Division, Rockland, ME) agarose gel. The size marker used was the 123-bp DNA ladder (Life Technologies, Inc.). The triplex PCR amplification resulted in three PCR products of expected size: 540-bp beta-actin, 480-bp WT1, and 266-bp GATA-1.

Nuclear Extract Preparations and Gel Mobility Shift Assays

Crude nuclear ``mini-extracts'' were prepared from exponentially growing K562, 293, HeLa, and HepG2 (ATCC MB8065) (hepatocellular carcinoma) cells according to the method of Dignam et al.(1983) as modified by Lee et al.(1988). Gel mobility shift assays were performed essentially as described by Singh et al.(1986). The 258-bp minimal enhancer was gel-purified and end-labeled at the EcoRI site with Klenow fragment using [alpha-P]dATP (Amersham Corp.). One nanogram of the 3` end-labeled 258-bp fragment (10,000 cpm) was incubated with 0.5-5 µg of the crude nuclear protein extracts and 1 µg of poly(dI-dC) in a buffer of 20 mM HEPES, pH 7.6, 10% glycerol, 1 mM MgCl(2), 40 mM KCl, 0.1 mM EGTA, and 0.5 mM dithiothreitol. Nuclear extracts were replaced with 0.1% bovine serum albumin in the control. Following a 15-min incubation at room temperature, the reaction was analyzed by electrophoresis at 300 volts on a 5% polyacrylamide gel in 0.5 times TBE (0.045 M Tris borate, 0.001 M EDTA), dried, and visualized by autoradiography using XAR film (Eastman Kodak Co.) film. For competition assays, unlabeled double-stranded oligonucleotides containing the reverse complement of the GATA-binding site (5`-TTTTATCTTT-3`) found within e250 were preincubated for 15 min at room temperature with the extracts described above and then the end-labeled 258-bp fragment was added to the binding reaction and incubated for an additional 15 min and analyzed as described above.


RESULTS

Interaction of the 3` Minimal Enhancer with the WT1 Promoter

Since the WT1 promoter functions in all cell lines tested, the tissue-specific expression of this gene must rely upon additional regulatory elements. We initially identified an orientation-independent transcriptional enhancer located 3` of the WT1 gene that was specific for K562 cells, a cell line derived from a chronic myelogenous leukemia patient in blast crisis that has erythroleukemic characteristics and that expresses WT1 mRNA (Fraizer et al., 1994). The 3` enhancer region, e351, was cloned in both orientations into the 3` end of pCB-WT1pro, a CAT construct containing the 652-bp full-length 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 pCB-WT1pro (Fig. 2A). The enhancer region e351, located 1768 bp 3` of the stop codon of the WT1 gene (Gessler et al., 1992), increased transcription of WT1 promoter-CAT constructs in K562 cells 13-fold in either orientation. By further deleting the 5`-most 93 bp from e351, we created a 258-bp minimal enhancer element that contains the full enhancer activity, increasing transcription of the WT1 promoter-CAT constructs in K562 cells 13-fold in either orientation. This enhancer region located between +800 and +1050 bp 3` of the WT1 gene increases transcription from the WT1 promoter in an orientation-independent manner as efficiently as the larger e351 and the 1-kb enhancer fragment 3d2 described previously (Fraizer et al., 1994). The 258-bp minimal enhancer also functions in HEL cells, an erythroleukemic cell line, activating the WT1 promoter-CAT constructs 3-fold (data not shown). Thus, the 258-bp minimal enhancer is active in two different erythroleukemic cell lines.


Figure 2: Activity of the WT1 enhancer with the WT1 promoter. This diagram illustrates the interaction of the hematopoietic enhancer with the WT1 promoter. A, the activity of the enhancer region e351 was compared with the activity of the minimal enhancer e258, and both enhancers were cloned into the 652-bp full-length WT1 promoter and tested in both orientations. The enhancer activity is expressed relative to the WT1 full-length promoter construct into which the WT1 enhancer fragments were cloned. B, the activity of the 104-bp minimal enhancer fragment e258 was tested in both orientations, and activity is expressed relative to the 104-bp WT1 minimal promoter construct into which the WT1 enhancer fragment was cloned. C, sequence analysis of the WT1 minimal enhancer shows potential transcription factor binding sites. Note that GATA and NF-E2 are hematopoietic-specific transcription factor-binding sites and the E-box (E2A-binding site) could potentially bind an additional hematopoietic-specific helix-loop-helix transcription factor.



The enhancer fragment e258 was cloned into the WT1 minimal promoter construct in both orientations. The enhancer activates the minimal promoter CAT constructs 12-13-fold in K562 cells (Fig. 2B). Although the minimal promoter has only 50% of the activity of the larger, full-length promoter (Fraizer et al., 1994), e258 is able to fully activate the minimal promoter, that is, the enhancer does not depend upon target sequences contained within the larger promoter region for maximal enhancer activity.

The WT1 e258 enhancer confers tissue specificity to both the full-length and minimal WT1 promoter. In K562 and HEL cells the WT1 enhancer increases the WT1 promoter transcription levels even greater than the SV40 enhancer; however, in 293 cells the enhancer fragment has no activity, failing to increase the transcription levels of either WT1 promoter construct (Fig. 2, A and B). The larger e351 enhancer fragment also has no activity in 293 cells (Fig. 2A) and CEM cells (data not shown), although both cell lines express WT1 mRNA. This lack of enhancer activity in cell lines that express endogenous WT1 indicates the absence of transcription factors necessary for enhancer activity. The enhancer also failed to increase basal transcription in HeLa cells (Fig. 2B) which fail to express WT1 mRNA (Fig. 3).


Figure 3: GATA-1 expression in K562 cells. Reverse transcriptase PCR of total cell RNA from HEL (EL), K562 (K), U937 (U), HeLa (He), 293 (2), Hep3B (HB), HL60 (HL), CEM (C), and murine spleen (S) was performed as described in the text. The negative control (N) was the PCR product of the first strand cDNA synthesis done in the absence of any RNA. The 123-bp DNA ladder (MW) (Life Technologies, Inc.) was used as a size marker. The triplex PCR amplification resulted in three PCR products as labeled: 540-bp beta-actin, 481-bp WT1, and 266-bp GATA-1.



Because the e258 enhancer element maintains the strict tissue specificity of the larger e351 enhancer fragment, we analyzed the sequence of the e258 fragment for potential transcription factor- binding sites (Fig. 2C). The enhancer region contains many binding sites for transcription factors that have been associated with enhancer activity, but the hematopoietic-specific GATA and NF-E2 binding sites are most likely to play roles in hematopoietic gene expression. The E-box may also serve as a binding site for the Tal-1 (stem cell leukemia) transcription factor percent in some hematopoietic cells (Begley et al., 1989). Although a second GATA motif is located within the 93-bp fragment 5` of e258, inclusion of this region does not increase enhancer activity. When the 5`-most 93-bp fragment of the e351 enhancer was tested independently, no enhancer activity was found. Thus, we have identified the minimal enhancer and demonstrated that the enhancer functions in a cell type-specific manner, increasing transcription from the WT1 promoter in K562 and HEL cells but not in any other line tested.

GATA-1 and WT1 Co-Expression in K562 Cells

Both WT1 and GATA-1 are expressed in K562, HEL, and mouse spleen cells (Fig. 3). Although WT1 is expressed in 293, CEM, and HL60 cells, no GATA-1 expression was detected in these cells. HeLa and Hep3B cells failed to express either WT1 or GATA-1, although beta-actin expression was detected. Although WT1 is expressed in all the hematopoietic lines tested (Fig. 3), GATA-1 is only expressed in those cell lines in which the WT1 enhancer functions (e.g. K562 and HEL cells), and GATA-1 is not expressed in CEM cells, a leukemic line in which the WT1 enhancer fails to function. Thus, the 3` enhancer functions in a limited set of hematopoietic cell lines, apparently only those that express GATA-1.

Nuclear Protein Binding Analysis of the 3` Enhancer

The role of the GATA site in the minimal 3` enhancer was examined by both functional and protein-DNA binding assays. Evidence for a GATA-specific complex was obtained by gel retardation assays (Fig. 4). A unique complex (not observed with 293 extracts) is seen for the 258-bp fragment bound to K562 extracts (Fig. 4), and this complex is eliminated by competition with unlabeled GATA oligonucleotides. A higher molecular weight non-GATA-specific complex is observed with both the K562 and 293 extracts. This complex is not observed in the non-WT1-expressing cell lines: HeLa and HepG2. This higher molecular weight complex is not eliminated in competition experiments by unlabeled GATA or GAGA oligonucleotides.


Figure 4: Electrophoretic mobility shift assay of the minimal enhancer. A, nuclear extracts from K562, 293, HeLa, and HepG2 cells were bound to radiolabeled e258 fragment. The hematopoietic-specific complex is marked by an arrow. B, nuclear extracts from K562 and not 293 cells form a hematopoietic-specific complex with the e258 fragment, and that complex is specifically eliminated by competition with 100 times molar excess of unlabeled GATA oligonucleotides.



GATA-1 Transactivation of the WT1 3` Enhancer

The 3` enhancer fails to activate transcription in the 293 kidney cell line (lacking endogenous GATA-1 protein), but addition of the GATA-1 protein by co-transfection confers enhancer activity. In co-transfection assays using 5 µg of the WT1 reporter construct containing the e258 WT1 enhancer (with one GATA site) and the minimal WT1 promoter (lacking GATA binding sites) adding increasing amounts of a mouse GATA-1 cDNA (Tsai et al., 1990), expression construct resulted in increasing enhancer activity (Fig. 5). Ten micrograms of the mGATA-1 expression construct gave a 13-fold increase in WT1 promoter activity. Similar results were obtained when the co-transfection was carried out in HeLa cells. Thus, 3` enhancer activity correlates with increasing amounts of GATA-1 transcription factor. This positive correlation was also observed between endogenous GATA-1 expression in cultured cells and the ability of the 3` enhancer to transcriptionally activate the WT1 promoter in these cells as depicted in Table 1. This correlation explains the inability of the 3` enhancer to function in CEM cells, a T-ALL cell line that fails to express GATA-1. Interestingly, the 3` enhancer is not active in all cells that express WT1 mRNA. This suggests that additional unknown cis-acting elements regulate WT1 expression in these cells and indicates a very complex regulation of WT1 tissue-specific expression.


Figure 5: GATA-1 transactivates the WT1 enhancer. This figure shows the dose-dependent activation of the WT1 enhancer by GATA-1 co-transfection. Five micrograms of the reporter construct (WT1 minpro.e258) containing the 104 bp WT1 minimal promoter and the 258-bp minimal enhancer (e258) was co-transfected as described in the text. , WT1 minpro.e258.






DISCUSSION

This paper describes the isolation of an 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. Unlike the ubiquitously active promoter, the 3` enhancer is hematopoietic-specific, functioning only in K562 and HEL cells. This is probably the result of the hematopoietic-specific GATA element located within the 258-bp enhancer. It is possible that GATA-binding proteins may facilitate transcription at the WT1 GC-rich promoter in some hematopoietic cells, explaining the unique tissue specificity of the WT1 gene.

In addition to kidney and genitourinary expression, WT1 is expressed in normal fetal spleen, bone marrow, and immature leukemic cells (Call et al., 1990; Pritchard-Jones et al., 1994; Huang et al., 1990; Miwa et al., 1992; Miyagi et al., 1993). The hematopoietic-specific regulation of WT1 expression may require at least three cis-acting regulatory elements found within the promoters of many hematopoietic-specific genes: GATA (Evans et al., 1988), CACCC (Mantovani et al., 1988), and NF-E2 (Andrews et al., 1993); two of these three elements are also found within the WT1 enhancer region. Additionally, two potential TAL-1 (stem cell leukemia) (Begley et al., 1989) transcription factor-binding sites are found within the 258-bp enhancer region. As TAL-1 and GATA-1 are both expressed in acute myeloid leukemias particularly of the erythroid and megakaryocytic lineages (Orkin, 1992; Shimamoto et al., 1994), they may both interact with the WT1 enhancer. GATA-binding sites have been identified in both the GATA-1 and TAL-1 gene promoters (Tsai et al., 1991; Aplan et al., 1990), so activation of the WT1 enhancer may occur only after increased GATA-1 production has up-regulated its own expression and that of TAL-1.

Because we observed expression of WT1 and GATA-1 RNA in K562 and HEL cells and in mouse spleen, we evaluated potential interactions between GATA-1 transcription factors and the WT1 enhancer. The GATA family of transcription factors originally was classified as erythroid-specific zinc finger transcription factors, but GATA family members have also been shown to play critical roles in other hematopoietic lineages (Orkin, 1992). GATA-1 expression decreases during myelo/monocytic differentiation (Sposi et al., 1992) and is generally absent in the M3, M4, and M5 classes of acute myelogenous leukemia (Shimamoto et al., 1994). This pattern of expression in leukemia cells resembles that of WT1 (Miwa et al., 1992; Miyagi et al., 1993). Additionally, both GATA-1 and WT1 are down regulated during differentiation of HL60 cells to neutrophils (Sekiya et al., 1994; Orkin, 1992). However, the GATA-1 expression pattern only partially overlaps WT1 expression. GATA-1 is not sufficient to maintain WT1 expression, as WT1 is down-regulated during the differentiation of K562 cells (Phelan, 1994), whereas GATA-1 expression increases during erythroid differentiation (Orkin, 1992). Both WT1 and GATA-1 are likely to be expressed in an early pluripotent stem cell.

We have characterized the interactions of the hematopoietic- specific enhancer with the hematopoietic transcription factor GATA-1 by electrophoretic mobility shift assays and transactivation assays. We found that enhancer activity is correlated with the expression of GATA-1, implying that GATA-1 is necessary for the activation of the WT1 promoter by the 3` enhancer. Because GATA-1 is essential for erythroid development (Pevny et al., 1991) and expressed in only a subset of mature hematopoietic cells, primarily megakaryocytic and mast cells, we predict that the WT1 enhancer will activate transcription above basal levels in only a subset of hematopoietic cells. To date we have examined enhancer function in two erythroleukemia cell lines, HEL and K562 cells, and we plan to examine additional GATA-expressing leukemic cell lines.

In the present work we have identified both cis- and trans-acting factors that regulate expression of WT1 in the hematopoietic system. We have shown the dramatic effect GATA-1 has on the transcription of the Wilms' tumor gene WT1 and provided a framework for understanding the role of WT1 in hematopoiesis. As WT1 itself is a transcription factor expressed in at least two hematopoietic lineages, WT1 may be a hematopoietic target gene for the GATA-binding proteins and therefore part of the cascade of transcription factors involved in hematopoiesis. Elucidating the role of tumor suppressor genes such as WT1 in normal developmental processes will aid in understanding their roles in the development of cancer.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA 34936 and Core Grant CA 16672. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biochemistry, Zhongshan University, Guangzhou 510275, Peoples Republic of China.

To whom all correspondence should be addressed.

(^1)
The abbreviations used are: bp, base pair(s); kb, kilobase(s); ALL, acute lymphocytic leukemia; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase.


ACKNOWLEDGEMENTS

We thank Stephen Hewitt, Xiaohong Zhang, and Dr. Pimpawan Patmasiriwat for helpful discussions. We thank Drs. Michael Perry, Christine Stellrecht, and Stuart Orkin for review of this manuscript. We are grateful to Dr. Stuart Orkin for the GATA-1 expression clone and Dr. Hiro Miwa for tissue RNA samples. We appreciate the technical assistance of Lisa Mulcahey and Houyen Shen and Ruby Desiderio's help with the preparation of this manuscript.


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