(Received for publication, May 10, 1995; and in revised form, July 13, 1995)
From the
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 DNA-binding 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 hematopoietic 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, 10, 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 5`-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 5`-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 3` 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.
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).
The WT1, EGR-1, and Sp1 expression vectors consisted of the full-length coding region of each gene under the control of the cytomegalovirus immediate-early promoter(21) .
The noncoding strand primers were:
Quantitation of footprints was performed as described previously (22) with ImageQuant software version 4.15 (Molecular Dynamics). Percent protection values below 20% were considered too low and were not interpreted as footprints.
The oligonucleotides were synthesized with 5` overhangs and
end-labeled with [-
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 µg of poly(dI-dC),
0.5 ng of 10
cpm end-labeled DNA oligonucleotide probe, and
5-15 µg 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
Tris borate-EDTA, 5% polyacrylamide
gel. The samples were electrophoresed at 30 mA at 4 °C. For the
competition studies, a 100-fold molar excess of unlabeled competitor
oligonucleotide was added to the binding reaction.
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.
Figure 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 vitro-methylated 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 -628 and 89% at -622. Protection for the noncoding strand is 63% at positions -630 and -631, 72% at -627 to -623, and 87% at -621.
Figure 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 -454 and 66% for -448 and -447. Protection for the noncoding strand is 72% at -455 and 87% at -453 to -449.
Figure 3: Activity of the c-myb promoter in Molt-4 cells. A, results of transient transfection analysis of promoter deletion constructs. The luciferase activity is shown relative to the promoterless construct. The lines represent the standard deviation. B, results of transient transfection analysis with the -910 construct and the -910 construct with a mutated 5` WT1 site.
Figure 4: Activity of the c-myb promoter in DHL-9 cells. A, results of transient transfection analysis of promoter deletion constructs. The luciferase activity is shown relative to the promoterless construct. B, results of transient transfection analysis with the -910 construct and the -910 construct with a mutated 3` WT1 site.
Figure 5: EMSA of the EGR-1/WT1 sites with Molt-4 and DHL-9 nuclear extracts. A, EMSA of the two WT1 sites in the c-myb 5`-flanking region. Lane 1 is the labeled 5` WT1 site with Molt-4 nuclear extract, lane 2 is the labeled 3` WT1 site with DHL-9 nuclear extract, lane 3 is the same as lane 1 except for the addition of a 100-fold molar excess of unlabeled 3` WT1 site, and lane 4 is the same as lane 2 except for the addition of a 100-fold molar excess of unlabeled 5` WT1 site. B, EMSA of the mutated WT1 sites. Lane 1 is the labeled 5` WT1 site with a 100-fold molar excess of the mutated 5` WT1 site with Molt-4 nuclear extract, lane 2 is the labeled 3` WT1 site with a 100-fold molar excess of the mutated 3` WT1 site with DHL-9 nuclear extract, lane 3 is the labeled mutated 5` WT1 site with Molt-4 nuclear extract, and lane 4 is the labeled mutated 3` WT1 site with DHL-9 nuclear extract.
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.
Figure 6: EMSA Western analysis. A, EMSA with the labeled 5` WT1 site and Molt-4 nuclear extract (lane 1), the labeled 3` WT1 site with DHL-9 nuclear extract (lane 2) and with Molt-4 nuclear extract (lane 3). B, Western analysis of the proteins in the EMSA complexes. The lanes are labeled as in A. An antibody against WT1 was used at a dilution of 1:1000. C, Western analysis of the proteins in the EMSA complexes shown in A. An antibody against EGR-1 was used at a dilution of 1:1000. D, Western analysis of the proteins in the EMSA complexes shown in A. An antibody against Sp1 was used at a dilution of 1:1000.
Figure 7: Cotransfection of WT1, EGR-1, and Sp1 with the c-myb promoter constructs. A, transfections in Molt-4 cells were performed with either the WT1 expression vector (wt) or the empty expression vector (no wt) and the indicated c-myb promoter construct at a ratio of 1:1. B, transfections in DHL-9 cells with the same conditions as in A. C, transfections in Molt-4 cells were performed with either the empty expression vector (cont), the EGR-1 expression vector (Egr-1), or the Sp1 expression vector (Sp1) and the indicated c-myb promoter construct at a ratio of 1:1. D, transfections in DHL-9 cells with the same conditions as in C.
The c-myb gene is expressed primarily in immature hematopoietic cells. We have shown previously that two Myb binding sites in the 5`-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 5` EGR-1/WT1 site was not functional in the DHL-9 B cell line. A region of negative regulatory activity was mapped farther 3` 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 5` EGR-1/WT1 site was protected in Molt-4 cells and that the 3` 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 5` and 3` 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
platelet-derived 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.