Repression of the c-myb gene by WT1 protein in T and B cell lines.

The c-myb gene is primarily expressed in immature hematopoietic cells, and it is overexpressed in many leukemias. We have investigated the role of negative regulatory sites in the c-myb promoter in the Molt-4 T cell line and in the DHL-9 B cell line. A potential binding site for either the EGR-1 or WT1 protein was identified by in vivo footprinting in the 5′-flanking region of c-myb in a region of negative regulatory activity in T cells. We showed by electrophoretic mobility shift assay and electrophoretic mobility shift assay Western that WT1, EGR-1, and Sp1 bound to this site. A mutation of this site which prevented protein binding increased the activity of the c-myb promoter by 2.5-fold. In the DHL-9 B cell line, this site was nonfunctional; however, we found a potential EGR-1/WT1 site located more 3′ in a region of negative regulatory activity. We showed that WT1, EGR-1, and Sp1 bound to this site, and that mutation of this site increased the activity of the c-myb promoter by 3.2-fold. Cotransfection of a WT1 expression vector repressed the activity of the c-myb promoter in both cell lines, and this repression was relieved when the EGR-1/WT1 sites were removed. Cotransfection of either an EGR-1 or Sp1 expression vector had no significant effect on the activity of the c-myb promoter. We conclude that WT1 is a negative regulator of c-myb expression in both T and B cell lines.

The c-myb gene is primarily expressed in immature hematopoietic cells, and it is overexpressed in many leukemias. We have investigated the role of negative regulatory sites in the c-myb promoter in the Molt-4 T cell line and in the DHL-9 B cell line. A potential binding site for either the EGR-1 or WT1 protein was identified by in vivo footprinting in the 5-flanking region of c-myb in a region of negative regulatory activity in T cells. We showed by electrophoretic mobility shift assay and electrophoretic mobility shift assay Western that WT1, EGR-1, and Sp1 bound to this site. A mutation of this site which prevented protein binding increased the activity of the c-myb promoter by 2.5-fold. In the DHL-9 B cell line, this site was nonfunctional; however, we found a potential EGR-1/WT1 site located more 3 in a region of negative regulatory activity. We showed that WT1, EGR-1, and Sp1 bound to this site, and that mutation of this site increased the activity of the c-myb promoter by 3.2-fold. Cotransfection of a WT1 expression vector repressed the activity of the c-myb promoter in both cell lines, and this repression was relieved when the EGR-1/ WT1 sites were removed. Cotransfection of either an EGR-1 or Sp1 expression vector had no significant effect on the activity of the c-myb promoter. We conclude that WT1 is a negative regulator of c-myb expression in both T and B cell lines.
The c-myb protooncogene is the cellular homologue of the avian myeloblastosis virus and avian leukemia virus (E26) transforming genes (1,2). Myb is a sequence-specific DNAbinding protein with the ability to transactivate promoters with the specific consensus sequence PyAAC(G/T)G (3,4). Reduction of c-myb expression results in a block to hematopoietic precursor cell proliferation (5), and homozygous c-myb mutant mice demonstrate greatly impaired fetal hepatic hematopoiesis (6). The importance of the c-myb gene product in leukemic cell proliferation is demonstrated by the inhibition of cellular proliferation by c-myb antisense oligonucleotides (7). Leukemic cells were shown to be more sensitive to this inhibitory effect than normal hematopoietic cells (8).
The central role that c-Myb plays in the regulation of hema-topoietic cell development has fueled research into the regulation of its expression. The regulation of c-myb expression appears to be complex and occurs at several levels. An important mechanism for regulation of mouse c-myb expression is a block to transcription elongation within the first intron of the c-myb locus, recognized as a pause site (9 -11). A correlation between protein binding to the intron 1 pause site and c-myb mRNA levels has been demonstrated using DNA mobility shift assays (12). It has been shown that in vitro translated c-Myb can bind to Myb binding sequences found in the c-myb 5Ј-flanking region and that in cotransfection studies c-Myb is involved in positive autoregulation of the c-myb gene in hamster fibroblasts (13). Recent studies conducted in mouse T cell lines suggest that murine c-myb expression is dependent on a GC-rich sequence of the 5Ј-flanking region and that the c-myb promoter is functional in diverse T cell lines (14). We have shown that two Myb binding sites function as negative regulators of c-myb expression in T cell lines (15). Further studies of the regulation of expression of c-myb have shown that c-Jun and JunD are positive regulators of the c-myb promoter in hamster fibroblasts. A second promoter in the 3Ј end of intron 1 has been identified recently (16).
The putative Wilms' tumor suppressor gene (wt1) encodes a zinc finger DNA-binding protein that functions as a transcriptional repressor (17,18). The WT1 protein binds to the target sequence GCGGGGGCG which is also recognized by the zinc finger transcription factors EGR-1, EGR-2, and EGR-3. The wt1 gene is mainly expressed in the developing kidney, testis, ovary, and spleen (19).
In this report we have characterized a site identified by in vivo footprinting in T cells. We show that WT1 binds to this site in vitro and, by cotransfection experiments, that WT1 negatively regulates c-myb expression. In the DHL-9 B cell line, a similar sequence located farther downstream was protected in vivo. We found that WT1 bound to this site in vitro, and, in cotransfection experiments, it negatively regulated c-myb expression in B cells through this site.

MATERIALS AND METHODS
Cell Lines-Molt-4 and DHL-9 cell lines were cultured in RPMI medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 0.5 ϫ penicillin/streptomycin.
Construction of Reporter Plasmids-The HindIII-BamHI fragment of the luciferase reporter vector was derived from pSV232AL-A⌬5Ј (obtained from D. Helinski, UCSD). The fragment was cloned into pUC18 and subsequently recut as a HindIII-KpnI fragment and ligated into the multiple cloning site of Bluescript II KS ϩ . The 2.3-kilobase EcoRI-EcoRI 5Ј-flanking region of human c-myb (a kind gift from R. Dalla-Favera, Columbia) was ligated into the EcoRI site 5Ј of the luciferase gene in Bluescript. To remove a confounding ATG site, the HindIII-NcoI fragment was removed, HindIII linkers were added, and the plasmid was religated. A deletion of the 5Ј-flanking region was made using a unique BamHI restriction site located at Ϫ910 from the ATG codon. Further deletions were made using Bal31 endonuclease. Deletions were made at 30-s intervals from BamHI-linearized plasmid. The deleted end was then treated with Klenow polymerase, cut with HindIII, and sep-* This work was supported in part by a grant from the Department of Veterans Affairs. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  arated by gel electrophoresis. A vector was prepared by removing the HindIII/NotI (NotI site treated with Klenow polymerase) fragment of the luciferase/Bluescript construct, and the fragment and luciferase vector were ligated. Other deletions at specific locations were made by the polymerase chain reaction (PCR) 1 and confirmed by sequence analysis.
Mutagenesis of the WT1 binding sites was achieved using a technique previously described by Higuchi (20). Mutants were screened by restriction enzyme analysis and subsequently sequenced using the fmol Sequencing Kit (Promega) or the Sequenase Kit (U. S. Biochemical Corp.). Compressions were resolved with dITP or deaza-dGTP. The oligonucleotide sequences used for PCR primers are (mutated bases are in boldface).
5Ј WT site: CCGCTTGCGCACCTTCGACAGTG 3Ј WT site: GCCGTCGCGCACCTTGCCACCCC The WT1, EGR-1, and Sp1 expression vectors consisted of the fulllength coding region of each gene under the control of the cytomegalovirus immediate-early promoter (21).
In Vivo Dimethylsulfate Treatment and DNA Isolation-DNA isolation after dimethylsulfate treatment was performed as described previously (22)(23)(24). Cleavage with piperidine was performed according to the Maxam-Gilbert procedure (25).
Ligation-mediated PCR-Chemically modified and cleaved DNA was then subjected to amplification by ligation-mediated PCR essentially as described by Mueller and Wold (26), Pfeifer et al. (27), and Garrity and Wold (28). Sequenase was used for first strand synthesis, and Taq DNA polymerase was used for PCR. Conditions used for amplification were 95°C for 2 min, 61°C for 2 min, and 76°C for 3 min. After 20 to 22 cycles of PCR, samples were hybridized with end-labeled primers (primer 3 of each primer set) and amplified by one more cycle of PCR. The reaction mixtures were resolved in a 6% polyacrylamide denaturing gel. Footprinting on each strand was repeated at least four times with genomic DNA samples prepared from at least three separate batches of dimethylsulfate-treated cells. The primers used for PCR were synthesized in an Applied Biosystems 380B DNA synthesizer and purified on Applied Biosystems oligonucleotide purification cartridges. The common linkers used were GCGGTGACCCGGGAGATCTGAATTC and GAATTCAGATC. The primers for the coding strand were: The oligonucleotides were synthesized with 5Ј overhangs and endlabeled with [␣-32 P]dCTP and Klenow. Binding conditions were as follows: 10 mM HEPES, pH 7.9, 5 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 2 g of poly(dI-dC), 0.5 ng of 10 4 cpm end-labeled DNA oligonucleotide probe, and 5-15 g of protein from crude nuclear extract. The binding reaction was conducted at room temperature for 15 min, and the samples were then loaded onto a 0.5 ϫ Tris borate-EDTA, 5% polyacrylamide gel. The samples were electrophoresed at 30 mA at 4°C. For the competition studies, a 100fold molar excess of unlabeled competitor oligonucleotide was added to the binding reaction.
Gel Shift Western-EMSA was carried out as described above. The complexes were transferred to stacked nitrocellulose and DEAE-membranes as described (29). The DEAE-membrane was dried briefly, and the radiolabeled complexes were detected by autoradiography. For protein detection, the nitrocellulose membrane was processed as recommended by Amersham for enhanced chemiluminescence. The rabbit polyclonal antibodies against WT1, EGR-1, and Sp1 (from Santa Cruz Biotechnology) were used at a dilution of 1:1000.
Transfections and Luciferase Assays-Transfections were performed on cells in log phase. Cells were washed and resuspended in unsupplemented RPMI medium to a final concentration of 3 ϫ 10 7 cells/ml and incubated for 10 min at room temperature after the addition of DNA plus DEAE-dextran (30). Electroporations were carried out with the Bio-Rad Gene Pulser at 290 mV, 960 microfarads for Molt-4 cells, and 350 mV, 960 microfarads for DHL-9 cells. The cells were then incubated again for 10 min at room temperature. Transfected cells were cultured in 10 ml of supplemented RPMI for 48 h.
Cell lysis and luciferase assays were conducted according to the protocol and with reagents supplied with Promega's Luciferase Assay System. Luciferase measurements were performed on an LKB 1251 luminometer. The Rous sarcoma virus long terminal repeat-␤-galactosidase plasmid was used to control for variations in transfection efficiency. Each transfection was repeated at least six times with at least three different DNA preparations. The average value with the standard deviation is plotted.

Identification of in Vivo Footprints over EGR-1/WT1
Sites-In vivo footprinting by ligation-mediated PCR was performed on the c-myb 5Ј-flanking region in Molt-4 T cells and in DHL-9 cells. A protected region extended from Ϫ630 to Ϫ621 in Molt-4 cells (all numbers are relative to the translation start site) (Fig. 1). This sequence is identical with the EGR-1/WT1 consensus binding site. In the B cell line, DHL-9, this region did not show any protection, but a similar sequence extending from Ϫ455 to Ϫ446 was protected (Fig. 2). This site differs by one base from the EGR-1/WT1 consensus binding sequence. We also found that the Ϫ455 to Ϫ446 sequence was not protected in Molt-4 cells.

The Region of the c-myb Promoter Containing the EGR-1/ WT1 Site Functions as a Negative Regulator in Molt-4 T Cells-To determine whether the regions identified by in vivo
footprinting correlated with functional activity of the c-myb promoter, a number of deletion constructs of the 5Ј-flanking region of c-myb were made and linked to the luciferase gene. The activities of several of these constructs in Molt-4 cells are shown in Fig. 3A. A region of negative regulatory activity was observed between Ϫ632 and Ϫ594. The EGR-1/WT1 binding site is located in this region. To determine whether this site was responsible for the negative activity of this region, we made a construct with a mutated EGR-1/WT1 site. As shown in Fig. 3B, the activity of the promoter construct with the mutated EGR-1/WT1 site increased by approximately 2.5-fold. The increase in activity with mutation of the EGR-1/WT1 site accounted for essentially all of the negative regulatory activity in this region.

The 3Ј EGR-1/WT1 Site in the c-myb Promoter Functions as a Negative Regulator in DHL-9 B Cells-The region between
Ϫ632 and Ϫ594 had no functional activity in DHL-9 cells. A negative regulatory region located between Ϫ455 and Ϫ444 was identified (see Fig. 4A). The site which was protected in DHL-9 cells in vivo is located in this region. To determine whether this was a functional site, the sequence was mutated. As shown in Fig. 4B, the activity of the promoter construct with the mutated site increased by approximately 3.2-fold. Mutation of the EGR-1/WT1 site accounted for all of the negative regulatory activity in the region from Ϫ455 to Ϫ444 in DHL-9 cells.
Both Sequences Bind the WT1 Protein-Because WT1 has been shown to act as a transcriptional repressor, we investi-gated whether it was responsible for the negative activity associated with the EGR-1/WT1 sites in the c-myb 5Ј-flanking region. A previous study demonstrated that the myeloid cell lines, K562 and HL60, expressed WT1 protein (31,32). We have confirmed these results and have shown that both Molt-4 and DHL-9 cells express WT1 protein as well. 2 To determine whether WT1 bound to the two sites, EMSAs were performed. Three complexes of altered mobility were formed with each site, and the mobility of each complex appeared to be the same with both sites (Fig. 5A). Cross-competition studies demonstrated that the 5Ј site competed against the 3Ј site and vice versa (Fig. 5A). We confirmed that the mutated sites did not compete against the complexes formed with the wild-type sequences and that the mutated sites did not yield EMSA complexes similar to those with the wild-type sequences (Fig. 5B).
We used EMSA Western to determine whether the WT1 protein was present in one of the complexes (29). As seen in Fig.  6, A and B, the fastest migrating complex reacted with a WT1 antibody. These results suggested that the WT1 protein was found in the fastest migrating EMSA complex. To identify the proteins in the other two EMSA complexes, Westerns were performed with antibodies against EGR-1 (Fig. 6C) and Sp1 (Fig. 6D). The Sp1 protein was found in the slowest migrating EMSA complex, and the EGR-1 protein was identified in the complex of intermediate mobility.

FIG. 1. In vivo footprint analysis by ligation-mediated PCR of a region of the c-myb 5-flanking region (5 EGR-1/WT1 site) in Molt-4 and DHL-9 cells.
The region illustrated is labeled with nucleotide numbers relative to the ATG codon. Lanes (from left to right for both gels): Vv, in vivo-methylated DNA from Molt-4 cells; Vt, in vitromethylated DNA from Molt-4 cells; Vv, in vivo-methylated DNA from DHL-9 cells; Vt, in vitro-methylated DNA from DHL-9 cells. The protected guanines are indicated by *. Protection of guanine for the coding strand is 84% at position Ϫ628 and 89% at Ϫ622. Protection for the noncoding strand is 63% at positions Ϫ630 and Ϫ631, 72% at Ϫ627 to Ϫ623, and 87% at Ϫ621.

FIG. 2. In vivo footprint analysis by ligation-mediated PCR of the 3 EGR-1/WT1 site in DHL-9 and Molt-4 cells.
The lanes are labeled as in Fig. 1. Protection of guanine for the coding strand is 52% for position Ϫ454 and 66% for Ϫ448 and Ϫ447. Protection for the noncoding strand is 72% at Ϫ455 and 87% at Ϫ453 to Ϫ449. expression vector at a ratio of 1:1. As shown in Fig. 7A, cotransfection of the WT1 expression vector in Molt 4 cells repressed the c-myb promoter until the WT1 site was removed in the Ϫ594 construct. Similarly, in DHL-9 cells, cotransfection of the WT1 expression vector repressed the c-myb promoter until the functional WT1 site was removed in the Ϫ444 construct. These results demonstrate that increased levels of WT1 protein repress the activity of the c-myb promoter. As shown in Fig. 7C, cotransfection of either an EGR-1 or Sp1 expression vector had no significant effect on the c-myb promoter activity in Molt-4 cells. Similar results were obtained in the DHL-9 cell line (Fig. 7D).

DISCUSSION
The c-myb gene is expressed primarily in immature hematopoietic cells. We have shown previously that two Myb binding sites in the 5Ј-flanking region are negative regulators of c-myb expression in T cells (15). We have now characterized another negative regulatory region of the c-myb promoter and have shown by cotransfection experiments that WT1 is a negative regulator of c-myb expression in T cells. Mutation of this site increased c-myb expression by 2.5-fold. Mutation of the two Myb binding sites increased expression by 1.85-fold (15); together, the deletion of the Myb and WT1 sites brings the c-myb promoter activity to its maximal value. These results suggest that these sites are major negative regulatory sites for the c-myb promoter in T cells.
We have shown by EMSA and Western analysis that the WT1 protein binds to this site in the c-myb promoter. Three bands of altered mobility are observed with both WT1 site oligonucleotides. We have demonstrated that WT1 protein is found in the fastest migrating band. The EGR-1 protein is located in the intermediate complex, and Sp1 is found in the slowest migrating EMSA complex.
The 5Ј EGR-1/WT1 site was not functional in the DHL-9 B cell line. A region of negative regulatory activity was mapped farther 3Ј in the promoter. A potential binding site for EGR-1/ WT1, which differed from the consensus sequence by one base, was located in this region. We demonstrated that WT1, EGR-1, and Sp1 bound to this site by EMSA and by Western analysis. This site is identical with the B2 site in the insulin-like growth factor II gene which binds purified WT1 protein (33). Increased expression of WT1 in both DHL-9 and Molt-4 cells led to repression of the c-myb promoter while cotransfection of EGR-1 or Sp1 expression vectors had little effect.
In vivo footprinting by ligation-mediated PCR demonstrated that the 5Ј EGR-1/WT1 site was protected in Molt-4 cells and that the 3Ј EGR-1/WT1 site was protected in DHL-9 cells. We had initially thought that the EGR-1 protein might be responsible for the in vivo footprints because it is a transcriptional activator and the c-myb gene is expressed at moderately high levels in both cell lines. Our transient transfection experiments demonstrated that both the 5Ј and 3Ј EGR-1/WT1 sites were negative regulatory elements in the Molt-4 and DHL-9 cell lines, respectively. Although we cannot be certain that the in vivo footprints represent binding of WT1 and not EGR-1 or Sp1 to these sites, the fact that the sites are negative regulatory elements makes it more likely that WT1 is responsible for this activity. In addition, we demonstrated that cotransfection of a WT1 expression vector with the c-myb promoter-luciferase constructs caused repression of luciferase activity while EGR-1 and Sp1 expression vectors had no significant effect.
It has been shown previously that WT1 represses a plateletderived growth factor gene (34), the colony-stimulating factor 1 gene (35), the transforming growth factor ␤1 gene (36), the insulin-like growth factor I receptor (37), and the insulin-like growth factor II gene (33,38) at the transcriptional level. It is interesting to note that WT1 has been shown to regulate negatively genes that encode positive regulators of cell growth. Both WT1 and c-Myb are expressed in immature hematopoietic cells. We have now demonstrated by cotransfection experiments that the c-myb gene, which also encodes a protein involved in cell proliferation, is negatively regulated by the WT1 protein in T and B cell lines.
In summary, we have characterized several negative regulatory factors involved in the control of c-myb expression in both B and T cell lines. In T cells, both Myb and WT1 are negative regulators, while in B cells WT1 is a negative regulator of c-myb expression. We have preliminary evidence that the positive regulators of c-myb expression differ in T versus B cell lines as well.