(Received for publication, July 30, 1996, and in revised form, October 25, 1996)
From the Center for Molecular Biology in Medicine, Palo Alto Veterans Affairs Medical Center and the Department of Medicine, Stanford University School of Medicine, Stanford, California 94305
The c-myb gene is primarily expressed
in hematopoietic cells, and it is overexpressed in many leukemias. The
regulation of its expression is of critical importance in hematopoietic
cells. We identified the major positive regulatory sites in the
5-flanking sequence of the human c-myb gene, and we found
that the positive regulators differed in cells of different lineages.
In the Molt-4 T-cell line, two Ets-like binding sites were required for
the expression of c-myb. The 5
site played a minor role in
the regulation of c-myb expression, and we demonstrated
that a protein of 67 kDa bound to this site. Antibodies against Ets
proteins showed no cross-reactivity with this protein. We showed that
Ets-1 bound to the 3
-regulatory site in the c-myb promoter
by electrophoretic mobility shift assay and antibody studies. Both of
these Ets-like binding sites were nonfunctional in the DHL-9 B-cell
line and the K562 myeloid cell line. We identified a novel
transcription factor of 50.5 kDa that was required for expression of
c-myb in these cell lines.
The c-myb protooncogene is the cellular homologue of the avian myeloblastosis virus and avian leukemia virus (E26) transforming genes (1, 2). The c-myb gene product is a 75-83-kDa phosphoprotein that is predominantly expressed in hematopoietic cells and in leukemic cells. c-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). It is involved in the regulation of a number of cellular genes associated with proliferation and cell growth as well as genes that are expressed in a lineage-specific manner (4-8). Reduction of c-myb expression results in a block to hematopoietic precursor cell proliferation (9), and homozygous c-myb mutant mice demonstrate greatly impaired fetal hepatic hematopoiesis (10). 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 (11). Leukemic cells were shown to be more sensitive to this inhibitory effect than normal hematopoietic cells (12).
c-Myb plays a central role in the regulation of hematopoietic cell development, and the control of its expression is critically important. The regulation of c-myb expression is not well understood; it appears to be complex and occurs at several levels. An important mechanism for regulation of murine c-myb expression is a block to transcription elongation within the first intron of the c-myb locus, recognized as a pause site (13-15). A correlation between protein binding to the intron 1 pause site and c-myb mRNA levels has been demonstrated using DNA mobility shift assays (16).
In cotransfection studies c-Myb is involved in positive autoregulation
of the c-myb gene in hamster fibroblasts (17). We have shown
that two c-Myb binding sites function as negative regulators of
c-myb expression in T-cell lines (18). The Wilms' tumor
gene product, WT1, is also a negative regulator of c-myb
expression in both T- and B-cell lines (19). 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 (20), and there are regulatory elements in the first
intron as well (21, 22).
The Ets family of transcription factors is comprised of proteins that
bind to the core sequence GGA (for reviews, see Janknecht and Nordheim
(23) and Wasylyk et al. (24)). The Ets-1 protein, which is
predominantly expressed in B and T lymphocytes, has been found to act
as a transcriptional activator of several T-cell-specific genes,
including the T-cell receptors and
and CD4 (25-29). Mutation
of the two guanines in the core binding area results in a loss of 95%
of the promoter activity of the T-cell receptor
gene (25). Studies
of lymphocytes that lack Ets-1 have shown a significant increase in
apoptosis and a subsequent decrease in the number of T-cells (30,
31).
In this report we have characterized the major positive regulatory
sites in the human c-myb 5-flanking sequence. In the T-cell line, Molt-4, we find that two Ets-like binding sites are required for
c-myb expression. These Ets binding sites are nonfunctional in both a B- and myeloid cell line, and we find that a novel
transcription factor is required for c-myb expression in
these cell lines.
Molt-4, DHL-9, Jurkat, Nalm-6, and K562 cell lines were cultured in RPMI medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 50 units/ml penicillin and 50 µg/ml streptomycin. For activation of Jurkat cells, 20 ng of phorbol myristate acetate/ml and 1 µM ionomycin were added for 8 h prior to nuclear extract preparation.
Construction of Reporter PlasmidsThe construction of the
human c-myb promoter luciferase construct has been described
previously (18, 19). A deletion of the 5-flanking region was made
using a unique BamHI-restriction site located at
910 from
the ATG codon. Further deletions were made using Bal-31 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 separated 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 and confirmed by sequence analysis. All
numbers are relative to the translation start site.
Mutagenesis of the T-cell and the B- and myeloid cell binding sites was
achieved using a technique previously described by Higuchi (32). The
oligonucleotide sequences used were (mutated base pairs are in
boldface type): 5 T-cell site,
GAAGAAGAATTAAAAAAAA; 3
T-cell site,
CTCATTCAAAGGCGCCGTCGCGCCCC; and B-myeloid site,
GGCCGCGGCAGGGCGCGTACACACTGCA. Mutants were screened by restriction enzyme analysis and subsequently sequenced using the Fmol sequencing kit (Promega Biotech Inc.,) or the Sequenase kit (U. S. Biochemical Corp.). Compressions were resolved with dITP or
deaza-dGTP.
Transfections were performed on cells in log phase. Cells were washed and resuspended in unsupplemented RPMI medium to a final concentration of 3 × 107 cells/ml for Molt-4 and DHL-9 and 8 × 106 cells/ml for K562. The cells were incubated for 10 min at room temperature after the addition of 15 µg of DNA plus 25 µg/ml DEAE-dextran for Molt-4 and DHL-9 cells or 20 µg of DNA for K562 cells. Electroporations were carried out with the Bio-Rad Gene Pulser at 290 mV, 960 µF for Molt-4 cells; 350 mV, 960 µF for DHL-9 cells; and 350 mV, 500 µF for K562 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 (Molt-4) or 24 h (DHL-9 and K562).
Cell lysis and luciferase assays were conducted according to the
protocol and with the 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 four
times with at least three different DNA preparations. The average value
with the standard deviation was plotted.
The oligonucleotide sequences
used are shown in Table I. The oligonucleotides were
synthesized with 5 overhangs and end-labeled with
[
-32P]dCTP and Klenow. Binding conditions for the 5
T-cell site were as follows: 10 mM HEPES, pH 7.9, 1.25 mM sodium phosphate, 25 mM KCl, 0.175 mM EDTA, 0.075 mM EGTA, 1 mM
dithiothreitol, 5 mM MgCl2, 10% glycerol, 2 µg of poly(dI-dC), 0.5 ng of 104 cpm end-labeled DNA
oligonucleotide probe, and 8 µg of protein from Molt-4 nuclear
extract. Binding conditions for the 3
T-cell site were as follows: 10 mM HEPES, pH 7.9, 10 mM Tris, pH 7.5, 25 mM KCl, 1.25 mM dithiothreitol, 1.1 mM EDTA, 10% glycerol, 1.5 µg of poly(dI-dC), 0.5 ng of
104 cpm end-labeled DNA oligonucleotide probe, and 8 µg
of protein from Molt-4 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, 100-fold molar excess of unlabeled competitor oligonucleotide was added to the binding reaction. For the supershifts, 0.5 µg of an
antibody specific for Ets-1 (Barbara Graves, University of Utah) (33),
Ets-1/Ets-2 (C-275, Santa Cruz Biotechnology), or preimmune serum was
added to the binding reaction above, and incubation was for 60 min at
4 °C. As a positive control, 10 ng of an N-terminal deletion mutant
N331 (Barbara Graves, University of Utah) was used in EMSA with the
conditions above with the addition of 4 µg of bovine serum
albumin.
|
Binding conditions for the B-myeloid cell site were as follows: 12 mM HEPES, pH 7.9, 5 mM NaCl, 50 mM KCl, 0.6 mM dithiothreitol, 0.4 µg poly(dI-dC), 12% glycerol, and 5-10 µg of DHL-9 or K562 nuclear extract. Incubation was for 25 min at room temperature. Electrophoresis was as described above.
UV Cross-linking and SDS-Polyacrylamide Gel ElectrophoresisEMSA was performed as described above. The wet gel was exposed to film to locate the complexes. UV cross-linking was performed essentially as described previously (34) on a short wave UV light box at 4 °C for 45-60 min. The regions of the gel containing the complexes were cut out, and the complexes were eluted at room temperature overnight in 50 mM Tris-HCl, pH 7.9, 0.1% SDS, 0.1 mM EDTA, 5 mM dithiothreitol, 150 mM NaCl, 0.1 mg/ml bovine immunoglobulin. The eluted protein was precipitated with 4 volumes of acetone, washed with ethanol, and air-dried. After resuspension in Laemmli loading buffer, SDS-polyacrylamide gel electrophoresis was performed, and the labeled proteins were visualized by autoradiography.
In Vitro Methylation Interference5 end-labeled
oligonucleotide (labeled with T4 kinase) was methylated with 0.5%
dimethylsulfate for 30-55 s at room temperature. This probe was used
in EMSA as described above. The wet gel was exposed to locate the
complexes, and both the bound and free probe were excised and
transferred to DEAE membranes. The DNA was eluted and cleaved with
piperidine, and equal counts of bound and free samples were resolved in
a 15% acrylamide sequencing gel.
Transient transfections with a
number of deletion constructs of the c-myb promoter were
performed in Molt-4 cells. As shown in Fig.
1A, there are two major regions of positive
regulatory activity. These are located between 784 and
758
(8.5-fold change in activity) and
474 and
455 (36-fold change in
activity). Comparison of the sequence in each of these regions revealed
potential Ets binding sites with the core sequence GGA.
Since c-myb is expressed in all hematopoietic cells, we
wished to determine whether the same promoter regions were involved in
positive regulation in B- and myeloid cell lines. We found that the two
active regions in T-cells showed no activity in either DHL-9 B-cells
(Fig. 1B) or K562 myeloid cells (Fig. 1C). The
major positive regulatory region in each of these cells lines was
located between 292 and
271. This region mediated a 12-fold change
in activity in DHL-9 cells and a 5-fold change in K562 cells.
Comparison of the sequence in this region with both of the positive
regulatory regions identified in T-cells revealed no similarities, and
no known transcription factor binding sites were identified in this region.
EMSA was performed with an oligonucleotide that encompassed
the 5 region of positive regulatory activity in Molt-4 cells. As shown
in Fig. 2A, lane 1, two specific
complexes were formed. Competition with a 100-fold molar excess of
unlabeled cold oligonucleotide prevented formation of the two complexes
(Fig. 2A, lane 2) while competition with an
irrelevant oligonucleotide (Fig. 2A, lane 5) had
no effect. To determine whether the two guanine residues at
779 and
778 were required for binding, we used an oligonucleotide with the
two guanine residues replaced by thymines. This oligonucleotide did not
form the same two complexes as the wild-type oligonucleotide did (Fig.
2A, lane 6), and it did not compete against the
two complexes formed by the wild-type sequence (Fig. 2A,
lane 4). Because the more 3
region of positive regulatory
activity contained a similar sequence, we used an oligonucleotide of
this region as a competitor against the 5
site. As shown in Fig.
2A, lane 3, it did not compete against the two
complexes.
In activated T-cells we had identified two proteins that bound to this
region (35). CMAT was present only in activated Jurkat cells, and a
60-kDa protein was constitutively expressed. We wished to determine
whether the EMSA complexes formed in Molt-4 cells were similar to the
ones we had observed in Jurkat cells. As shown in Fig. 2B,
the mobility of the EMSA complexes formed with Molt-4 nuclear extract
differed from that of the complexes formed with either unactivated or
activated Jurkat nuclear extracts (Fig. 2B, lanes
1-3 and 4-6). This was true with both the 5 site
probe or the CMAT site probe. (The CMAT site probe is 7 base pairs
longer than the 5
site probe.)
To determine the molecular mass of the protein that bound to the 5
site, EMSA followed by UV cross-linking was performed. Both the upper
and lower EMSA complexes yielded a single protein that migrated at 79 kDa (Fig. 3, lanes 1 and 2). When
corrected for the bound oligonucleotide, the molecular mass was 67 kDa. The corrected molecular mass of the CMAT protein was 90 kDa (Fig. 3,
lane 3) and that of the constitutively expressed protein was 60 kDa (Fig. 3, lane 4). Because the binding site contains a
potential Ets sequence, we performed a Western blot on the UV
cross-linked protein with an antibody that cross-reacts with many Ets
proteins. The cross-linked proteins were not reactive with this
antibody.2 In addition, no supershifted
complex was seen when this antibody was used in EMSA with either the 5
or CMAT site.
Methylation interference was performed to determine which guanine
residues contacted the protein that bound to the 5 site. On the coding
strand, methylation of the two guanines in the GGA sequence (
779 and
778) interfered with binding (Fig. 4). Methylation of
the other two guanine residues had no effect on binding of the protein.
There are no guanines in this region of the noncoding strand.
To confirm the importance of this site, we mutated the guanines at
779 and
778 in the c-myb promoter-luciferase construct. The activity of this construct was 9.5-fold lower than the construct with the wild-type site (Fig. 1A, Mut 5
).
EMSA was performed with an oligonucleotide of the positive
regulatory sequence located more 3 in the c-myb promoter.
With nuclear extracts from Molt-4 cells, two complexes were visible (Fig. 5A, lane 4). Further studies
demonstrated that only the slower migrating complex was a specific one
(marked with an arrow in Fig. 5). Competition with a
100-fold molar excess of unlabeled oligonucleotide prevented formation
of only the slower migrating complex (Fig. 5A, lane
5). Competition with an irrelevant oligonucleotide or the 5
c-myb site did not prevent formation of either complex (Fig.
5A, lanes 9 and 7, respectively). EMSA
with an oligonucleotide in which the two guanines at
469 and
468
were changed to thymines revealed only the nonspecific
complex.2 The mutated oligonucleotide did not compete
against the slower migrating complex (Fig. 5A, lane
8). We used an Ets-1 consensus sequence as a competitor and found
that it prevented formation of the slower migrating complex (Fig.
5A, lane 6). When the Ets-1 sequence was labeled
and used in EMSA with Molt-4 nuclear extract, two complexes were
visible. The faster migrating complex co-migrated with the specific
complex formed with the c-myb 3
site (Fig. 5A,
lanes 1 and 4). In addition, the c-myb
3
site competed against the labeled Et-1 consensus site in EMSA (Fig.
5A, lane 3).
An antibody that is specific for Ets-1 was used in EMSA experiments
with the 3 c-myb site. As shown in Fig. 5B,
lane 2, a supershifted complex was formed and the original
complex disappeared when this antibody was added. This antibody also
supershifted the complexes formed with the consensus Et-1
oligonucleotide (Fig. 5, lane 5), and a complex formed with
a truncated Ets-1 protein (Fig. 5B, lane 8).
UV cross-linking followed by SDS denaturing gel analysis of the
specific complex formed with the 3 site and the two complexes formed
with the Ets-1 consensus site revealed a single protein of the same
molecular mass in each case. After correction for the bound
oligonucleotide, the molecular mass was 55 kDa, which is the size
reported for Ets-1.2 Methylation interference demonstrated
that the two guanines at
470 and
469 in the coding strand were
required for binding of this protein (Fig. 6). The
guanines at
474 and
472 in the noncoding strand were also required
for protein binding (Fig. 6).
We changed the two guanines at 470 and
469 to thymines in the
c-myb-promoter luciferase construct. The activity of the
promoter was decreased by 18-fold compared to the wild-type
523
promoter (Fig. 1A, Mut 3
523) and
by 28-fold compared to the wild-type
474 promoter.
EMSA
analysis with an oligonucleotide that encompassed the positive
regulatory sequence from the c-myb promoter in DHL-9 cells revealed a single specific complex (Fig. 7A,
lanes 1 and 4). A 100-fold molar excess of
unlabeled oligonucleotide prevented formation of this complex (Fig.
7A, lane 2). We changed the guanines at 280 and
278 to adenines and found that this sequence no longer competed
against the wild-type sequence in EMSA (Fig. 7A, lane 3). The specific complex was not seen in EMSA with the labeled mutant sequence (Fig. 7A, lane 5).
EMSA with K562 nuclear extract revealed a single specific complex with the same mobility as the one formed with DHL-9 nuclear extract (Fig. 7, B, lanes 1 and 4, and C, lanes 1 and 2). The mutant sequence did not compete against this complex (Fig. 7B, lane 3) or form a similar complex when labeled (Fig. 7B, lane 5). EMSA with nuclear extracts from two T-cell lines and another B-cell line revealed that a complex of identical mobility was formed with the wild-type oligonucleotide (Fig. 7C).
EMSA followed by UV cross-linking and denaturing SDS gel
electrophoresis was performed to determine the molecular mass of this
protein. A single protein of 63.5 kDa was observed (see Fig. 8 for results with DHL-9 cells). When corrected for the
bound oligonucleotide, the molecular mass of this protein was 50.5 kDa.
Methylation interference was performed to determine the guanine
residues that contact the protein (Fig. 9). On the
coding strand, methylation of guanines 282,
280, and
278
interfered with binding, and on the noncoding strand, methylation of
guanines
279 and
277 interfered with binding.
We changed the guanines at 280 and
278 to adenines in the
c-myb promoter-luciferase construct and found that the
promoter activity decreased by 14-fold in DHL-9 cells (Fig.
1B, Mut) and in K562 cells by 5.7-fold compared
to the wild-type
455 construct (Fig. 1C, Mut
455) and by 6-fold compared to the wild-type
292 construct (Fig. 1C, Mut
292).
We have identified the major positive regulatory regions of the c-myb promoter in T-, B-, and myeloid cell lines. Although c-myb is expressed in all hematopoietic lineages, the regulation in T-cells is different from that in B- and myeloid cells. We have previously shown that c-Myb negatively regulates the c-myb promoter only in T-cells (18). We have also shown that WT1 is a negative regulator of c-myb expression in both T- and B-cell lines, but that a different WT1 site is functional in each cell lineage (19).
There are two major positive regulators of c-myb expression
in T-cells. One is a 67-kDa protein that appears to be a novel transcription factor. Although the recognition sequence contains a
potential Ets binding site, we have not been able to demonstrate that
this protein is a member of the Ets family. Our studies have shown that
the 67-kDa protein does not comigrate with either CMAT or the 60-kDa
protein found in Jurkat cells on UV cross-linking SDS gel analysis.
Both the 67-kDa protein and CMAT contact the two guanines at 779 and
778 and the CMAT protein also contacts guanine
782, while the
constitutively expressed protein does not contact any of these guanine
residues (35). In Jurkat cells, the 5
region appears to be important
only for the increase in expression seen with activation and there is
very little constitutive activity associated with this region (35). The
3
region is the major positive regulatory region in Jurkat
cells.3
The two c-Myb binding sites between 815 and
793 are responsible for
the negative regulatory activity seen in Fig. 1A between constructs
910 and
784 (18). The WT1 site between
630 and
621
is responsible for the negative regulatory activity seen in Fig.
1A between the
758 and
594 constructs (19). There appears to be another region of negative regulatory activity between
594 and
523; we have not characterized this region further. It is
possible that another transcription factor binds near the Ets site in
the region from
523 to
474. We noted a small decrease in activity
when this region was deleted, and mutation of the Ets site did not
decrease the promoter activity to the base-line level when this region
was present (Fig. 1A, Mut 3
523).
Although we demonstrated that Ets-1 bound to the 3-regulatory site
in vitro, it is possible that a different Ets family member is a positive regulator of the c-myb promoter through this
site. From the results of chimeric mice with Ets-1-deficient
lymphocytes, it has been proposed that Ets-1 serves to keep T-cells is
a resting, G0 cell cycle stage (30, 31). It would thus seem
less likely that Ets-1 would positively regulate the expression of a
gene such as c-myb since the c-Myb protein is a positive
regulator of cell proliferation. However, Ets-1-deficient T-cells were
also very susceptible to cell death (30, 31). While the c-Myb protein is involved in the positive regulation of cell proliferation, it also
plays a role in the induction of programmed cell death if conditions
are not right for proliferation (36). It is interesting to note that
Ets-1 was thought to regulate the T-cell receptor gene
by binding
to a specific site in the enhancer (25). In Ets-1-deficient T-cells,
the T-cell receptor
gene is expressed (30, 31), suggesting that
Ets-1 is not required for this expression or that another Ets protein
can replace Ets-1. It is possible that there are several Ets proteins
in T-cells that play overlapping roles in the regulation of a set of
genes expressed in T-cells.
The 5- and 3
-positive regulatory sites identified in Molt-4 T-cells
are not functional in either the DHL-9 B-cell line or the K562 myeloid
cell line. Instead a transcription factor binds to a sequence from
282 to
277. Based on the molecular mass and the DNA recognition
sequence, we believe that this is a novel transcription factor. It is
expressed in different hematopoietic lineages, but this site in the
c-myb promoter is nonfunctional in T-cell lines. In the
DHL-9 B-cell line, the WT1 site between
455 and
447 serves as a
negative regulatory element (19). Neither of the WT1 sites is
functional in the myeloid cell line, K562.
We did not find any evidence for a functional role for either the E2F
site at 259 or the AP-1 site at
370 in the human c-myb promoter. The activity of the E2F site was described in a glioblastoma cell line (37), and the activity of the AP-1-like site was described in
fibroblasts (38). It is interesting to note that the two binding sites
for the zinc finger protein MZF1 were not functional in Molt-4, DHL-9,
or K562 cells. These sites appear to be negative regulators of
c-myb expression and are required during hematopoietic cell
development from embryonic stem cells (39). It is possible that these
sites are no longer functional in more differentiated hematopoietic
cells.
In summary, we have identified the major positive regulatory sites in
the human c-myb 5-flanking sequence. In Molt-4 T-cells, two
Ets-like sites are required, although we have no evidence that an Ets
protein binds to the 5
site. In both DHL-9 B-cells and K562 myeloid
cells, the Ets-like sites are nonfunctional. A novel transcription
factor is involved in the regulation of c-myb expression in
these cell lines.