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NEOPLASIA
Myeloblastin is an Myb target gene: mechanisms of regulation in myeloid
leukemia cells growth-arrested by retinoic acid
Pierre G. Lutz, Anne Houzel-Charavel, Christel Moog-Lutz, and Yvon E. Cayre
A pivotal role has been assigned to Myb
in the control of myeloid cell growth.
Although Myb is a target of retinoic
acid, little is known about the mechanisms by which it may contribute to
induced growth arrest in leukemia cells.
Indeed, few Myb target genes are known
to be linked to proliferation. Myeloblastin is involved in the control of proliferation in myeloid leukemia cells. It is
expressed early during hematopoiesis
and is a granulocyte colony-stimulating
factor–responsive gene. Myeloblastin
can confer factor-independent growth
to hematopoietic cells, an early step in
leukemia transformation. The myeloblastin promoter contains PU.1, C/EBP,
and Myb binding sites, each of which
are critical for constitutive expression
in myeloid cells. Inhibition of myeloblastin expression in leukemia cells
growth-arrested by retinoic acid is demonstrated to depend on Myb downregulation. Myb is shown to induce myeloblastin expression and abolish its
down-regulation by retinoic acid. Alto-
gether, the data offer a clue as to how a
myeloid-specific transcriptional machinery can be accessible to regulation by
retinoic acid and point to myeloblastin
as a novel target of Myb. This link
between Myb and myeloblastin suggests a previously nonidentified Myb
pathway through which growth arrest is
induced by retinoic acid in myeloid leukemia cells. (Blood. 2001;97:2449-2456)
© 2001 by The American Society of Hematology
Introduction
Retinoic acid (RA) plays a major role in inducing growth arrest and
differentiation of myeloid leukemia cells both in vitro and in
vivo.1-5 However, little is known about those myeloid genes that
respond to RA and are involved in growth control. Myeloblastin
(MBN)6 is a serine protease with a broad spectrum of proteolytic
activity. Inside myeloblastic cells, a main enzymatic target appears
to be the Sp1 transcription factor, which is physiologically
truncated by MBN and is known to contribute to growth regulation.7 An involvement of MBN in the control of leukemia cell
growth was initially suggested by the fact that its expression was
serum-dependent and down-regulated by growth-inhibiting inducers such as all-trans retinoic acid (ATRA).6 Furthermore, downregulation of MBN expression by antisense oligodeoxynucleotides
inhibited proliferation of promyelocytic-like leukemia cells.6 MBN
is overexpressed in myeloid leukemia cells,8 and several reports
suggest that T-cell responses to MBN peptides can be used for
adaptative T-cell therapy in acute and chronic myeloid leukemias.9,10 MBN is a granulocyte colony-stimulating factor (G-CSF)
target gene and can confer factor-independent growth when
ectopically overexpressed in an early hematopoietic cell line.11
This has strongly reinforced the view that MBN is a key protease
involved in the control of proliferation in normal hematopoietic
progenitors and in contributing to early steps in their transformation.
The Myb family of transcription factors is strongly implicated
in the regulation of cell growth and differentiation. In the murine
system, inappropriate expression of c-Myb clearly contributes to
leukemia transformation.12 The c-Myb promotes proliferation and
blocks differentiation of hematopoietic cells in several experimen-
tal models.13,14 Targeting of c-Myb with antisense oligodeoxynucleotides inhibited hematopoiesis as well as proliferation of myeloid
leukemia cells.15,16 Mice homozygous for the inactivated c-Myb
gene had impaired definitive hematopoiesis with a drastic decrease
in the number of progenitors likely to reflect a proliferation
abnormality.17 Expression of c-Myb declines in RA-treated myelomonocytic leukemia cells, suggesting that the retinoic acid
receptor (RAR) acts in part by down-regulating c-Myb expression.
This may account for the observation that transcriptional activation
of a Myb-responsive gene can be inhibited by RA.18 Furthermore,
introduction of an exogenous RAR␣ into v-Myb–transformed
monoblasts permitted RA-dependent differentiation, indicating that
transformation by v-Myb is recessive to RAR␣.18 Conversely, Myb
functions as a potent inhibitor of RA-induced biologic responses.19
Although little is known about the mechanisms involved in these
processes, a physical interaction exists between c-Myb and RAR.19
However, despite a pivotal role assigned to Myb in the control of
cell growth, few Myb-target genes are known to be linked to
proliferation, and its relevance to ATRA-induced growth arrest in
leukemic cells remains unresolved.
Because MBN is associated with growth control in myeloid
leukemia cells, we have analyzed its transcriptional regulation by
RA. For this, we have cloned the human and murine MBN
promoters, which both harbor binding sites for the PU.1, C/EBP,
and Myb transcription factors. Our data show that these factors are
all critical for constitutive MBN expression and establish MBN as a
novel target of Myb, mediating its regulation by ATRA in leukemia
cells. This, together with the fact that MBN is involved in growth
From Unité INSERM U417, Hôpital Saint Antoine, Paris, France; and
Department of Microbiology/Immunology, Kimmel Cancer Institute, Thomas
Jefferson University, Philadelphia, PA.
Reprints: Yvon E. Cayre, Unité INSERM U417, Hôpital Saint Antoine, 184 Rue
du Faubourg Saint Antoine, 75012 Paris, France; e-mail: cayre@st-antoine.
inserm.fr.
Submitted August 4, 2000; accepted December 21, 2000.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by INSERM and grants from the Association Pour la Recherche sur
le Cancer, the Lady Tata Memorial Trust, and the Ligue Nationale Contre le
Cancer.
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
© 2001 by The American Society of Hematology
2449
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2450
LUTZ et al
control and can provide factor-independent growth to hematopoietic cells, suggests that its association with Myb may play a role in
mediating ATRA-induced growth arrest in myeloid leukemia cells.
Materials and methods
Leukemia cell lines culture conditions
Myeloblastic PLB-98520 and promyelocytic NB421 cells were cultured in
RPMI 1640 medium with 10% fetal bovine serum and 2 mM L-glutamine
(Gibco, Life Technologies, Paisley, UK). COS-7 cells were grown on 9-cm
Petri dishes in Dulbecco modified Eagle medium containing 5% fetal
bovine serum. Cells were grown in a humidified atmosphere of 5% CO2.
Cell viability was assessed by standard trypan blue dye exclusion assay.
Exponentially growing PLB-985 or NB4 cells (1 ⫻ 106/mL) were used to
start suspension cultures. PLB-985 and NB4 cells (2 ⫻ 105/mL) were
seeded 16 hours prior ATRA treatment. ATRA (Sigma-Aldrich, St Louis,
MO) dissolved in ethanol was used at a final concentration of 10⫺6 M.
Differentiation was assessed by the percentage of cells with cell-associated
nitroblue tetrazolium (Sigma) and cell morphology under light microscopy
on May-Grünwald-Giemsa–stained cytospin slides.
Northern blot analysis
Total RNA extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany)
was electrophoresed and transferred onto Hybond-N nylon filters (Amersham Pharmacia Biotech, Uppsala, Sweden). Filters were prehybridized for
2 hours at 42°C in 50% formamide, 5 ⫻ SSC, 0.5% sodium dodecyl sulfate
(SDS), 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 50 mM sodium pyrophosphate (pH 6.5), 1% glycine, and 500 ␮g/mL single-stranded DNA.
Hybridization was conducted for 15 hours at 42°C in 50% formamide, 5 ⫻
SSC, 0.5% SDS, 0.04% polyvinylpyrrolidone, 0.04% Ficoll, 20 mM
sodium pyrophosphate (pH 6.5), 10% dextran sulfate, and 100 ␮g/mL
single-stranded DNA. Filters were washed 30 minutes in 2 ⫻ SSC and
0.1% SDS at room temperature, followed by 60 minutes in 0.1 ⫻ SSC and
0.1% SDS at 60°C. The MBN probe was the full-length complementary
DNA (cDNA). The c-Myb probe was a polymerase chain reaction (PCR)
product obtained using 5⬘-AATTAAATACGGTCCCCTGAAGATG-3⬘ sense
and 5⬘-CAGGTACTGCTACAAGGCTGCAAGG-3⬘ antisense primers. The
glyceraldehydes-3-phosphate dehydrogenase (GAPDH) probe was a PCR
product obtained using 5⬘-ATCACCATCTTCCAGGA-3⬘ sense and 5⬘CCTGCTTCACCACCTTCTTG-3⬘ antisense primers. Probes were labeled
with [␣-32P]deoxycytidine triphosphate 110 TBq/mM (3000 Ci/mM) using
a random primer labeling kit (Amersham Pharmacia Biotech).
Recombinant plasmids
Expression vectors for C/EBP␣ (MSV-C/EBP␣), C/EBP␤ (MSV-C/EBP␤),
and C/EBP␦ (MSV-C/EBP␦); for c-Myb (CMV-c-Myb)22; for PU.1 (pECEPU.1)23; and for E1A 13S and E1A 12S Ntdl814 24 were obtained from A.
Friedman, B. Lüscher, R. Maki, and P. Whyte, respectively.
A genomic library of SV129 mouse embryonic stem cell DNA was a gift
from P. Chambon. A total of 1 ⫻106 recombinants were screened using a 5⬘
probe (85-base pair [bp] NotI-PvuII fragment) of the mouse MBN cDNA
kindly provided by L. Hellman.25 BamHI digestion of a positive clone and
hybridization with the 5⬘-TCTGGAAGCTACCCATCC-3⬘ oligonucleotide
yielded a 1.1-kilobase fragment that was subcloned and submitted to
automated sequencing (Eurogentec, Seraing, Belgium). A 658-bp human
MBN promoter region was subcloned into the pBLCAT6 vector,26 generating the pMBN-658 reporter plasmid. For constructing the pMBN-194,
pMBN-137, and pMBN-91 deletion reporter plasmids, 194-bp, 137-bp, and
91-bp fragments, respectively, were generated using PCR and cloned into
BamHI/XhoI sites of the pBLCAT6 vector. The 5⬘ primers were 5⬘ATGGATCCGACTTGGGTGGGTGA-3⬘ from positions ⫺194 to ⫺180
(194-bp fragment), 5⬘-ATGGATCCAAAGCCCCCACCT-3⬘ from positions ⫺137 to ⫺124 (137-bp fragment), and 5⬘-ATGGATCCAAGGCAAAAGGAGGAAGT-3⬘ from positions ⫺91 to ⫺72 (91-bp frag-
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
ment); the 3⬘ primer was 5⬘-ATCTCGAGGATATCGAATTCCTGCAG-3⬘.
Constructions of pMBN-658PU.1mut, pMBN-658C/EBPmut, and pMBN658Mybmut constructs were obtained by mutagenesis of the PU.1, c/EBP,
and Myb binding sites using the QuickChange site-directed mutagenesis kit
(Stratagene, La Jolla, CA). The oligonucleotide sequences used were
(mutated base pairs are in bold): PU.1, 5⬘-CAAGGCAAAAGGATTAAGTGGGGACCCAG-3⬘; C/EBP, 5⬘-CCAGCCTGGGCGTAGTGGGACTCAACGGCC-3⬘; c-Myb, 5⬘-GGGCAACTCATGGGCCTCTGGC-3⬘. Promoter constructs were verified by DNA sequencing.
In vivo expression assays
COS-7 cells were transfected using calcium phosphate coprecipitation27
of DNA vectors (adjusted to 14 ␮g per 9-cm Petri dish with pBluescript
carrier DNA). Medium was changed after 16 hours. Cells were
harvested after an additional 20-hour culture. Exponentially growing
PLB-985 cells were washed twice in serum-free medium, once in
Optimem medium, resuspended in the same medium at 15 ⫻ 106 cells in
0.5 mL, and electroporated (Gene Pulser, Biorad, Hercules, CA) at 300
V, 960 ␮F with 20 ␮g of (1) pBabe Puro28 alone or together with
CMV–c-Myb (cells were then cultured for 48 hours prior to selection
with 1 ␮g/mL puromycin [Sigma]); (2) reporter plasmid. Cells transfected with reporter plasmid were cultured in 10 mL of medium for 40
hours. Plasmid (5 ␮g) containing the luciferase cDNA driven by the
cytomegalovirus promoter was an internal control for transfection
efficiency. All transfections were performed at least in triplicate with
independent template preparations. Assays to measure luciferase29 and
chloramphenicol acetylase30 were conducted as described.
Electrophoretic mobility shift assays
For whole-cell extracts, COS-7 cells were harvested in ice-cold phosphatebuffered saline, pelleted, washed twice in the same buffer, and resuspended
in extraction buffer (0.4 M KCl, 20 mM Tris-HCl [pH 7.9], 20% glycerol, 5
mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, and 2.5 ng each
of leupeptin, pepstatin, aprotinin, antipain, and chymostatin [Sigma] per
milliliter). After 2 freeze-thaw cycles with liquid nitrogen, the resulting cell
lysate was cleared by centrifugation 15 minutes (4°C) at 10 000g and used
for electrophoretic mobility shift assays (EMSAs). PLB-985 nuclear
extracts were prepared essentially as described31; 2.5 ng each of leupeptin,
pepstatin, aprotinin, antipain, and chymostatin (Sigma) per milliliter were
added in both lysis and extraction buffers. EMSAs were carried out using
PU.1 (human MBN promoter position ⫺84 to ⫺62), 5⬘-AGCTTAGGCAAAAGGAGGAAGTGGGGACG-3; mut PU.1, 5⬘-AGCTTAGGCAAAAGGATTAAGTGGGGACG-3⬘; C/EBP (human MBN promoter
position ⫺51 to ⫺36), 5⬘-AGCTTGGGCATTGGGCAACTCG-3⬘; mut
C/EBP, 5⬘-AGCTTGGGCGTAGTGGGACTCG-3⬘; Myb (human MBN
promoter position ⫺42 to ⫺22), 5⬘-ATGGATCCGCAACTCAACGGCCTCTGGCAT-3⬘; and mut Myb, 5⬘-ATGGATCCGCAACTCATGGGCCTCTGGCAT-3⬘ oligonucleotides. Approximatively 0.25 ng (15 000 cpm)
of a 32P-5⬘ end-labeled synthetic double-stranded oligonucleotide was
incubated with 5 ␮g COS-7 cell lysate or PLB-985 nuclear extracts in the
presence of poly(dIdC) as a nonspecific competitor in 10 mM Tris-HCl (pH
7.9), 50 mM KCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 0.05%
Nonidet P-40, and 0.3 mg/mL bovine serum albumin. After 15 minutes at
room temperature, DNA-protein complexes were separated on a 5%
polyacrylamide gel (acrylamide:bisacrylamide ratio, 37.5:1) in TBE (25
mM Tris base, 25 mM boric acid, 0.5 mM EDTA) at 4°C. For competition
experiments, extracts were preincubated with poly(dIdC) and unlabeled
competitor oligonucleotides for 15 minutes before addition of the probe and
further incubation. For antibody supershift assays, affinity-purified polyclonal anti-PU.1, anti-C/EBP␣, anti-C/EBP␤, anti-C/EBP␦, or anti–c-Myb
antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) (1 ␮L) were added
to the mixture and incubated for an extra 30 minutes before loading into
the gel.
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BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
Results
PU.1, C/EBP, and Myb binding sites are all critical
for constitutive MBN promoter activity
To assess critical MBN regulatory sequences, we have cloned
human and mouse MBN promoter regions (GenBank accession
numbers M96628 and AJ007030, respectively). Both promoter
sequences had a TATA box and potential binding sites for Myb,
C/EBP, and PU.1 (Figure 1A). These sites exhibited consensus
sequences in both the mouse and human promoters (Figure 1B). In
addition, the human sequence harbored a CG element (Figure 1A).
These elements were in agreement with putative binding sites
indicated by Sturrock et al,32 except for C/EBP, which was not
conserved between human and mouse and fit poorly with the
C/EBP consensus sequence (data not shown). Mutation of this site
did not modify the MBN promoter activity in transfected myeloid
cells (data not shown). In contrast, we have identified another
putative C/EBP binding site that differs from the C/EBP consensus
sequence only by a G-to-C transition at the fifth position. A similar
Figure 1. The human MBN promoter harbors conserved PU.1, C/EBP, and Myb
binding sites: importance for MBN expression and down-regulation in ATRAinduced PLB-985 cells. (A) Sequence homology between human and murine MBN
promoters. Both strands were sequenced. The numbering is relative to the human
transcription initiation site. The TATA box is underlined; the CG element as well as the
PU.1, C/EBP, and Myb binding sites are boxed. (B) Comparison of the human and
murine putative PU.1, C/EBP, and Myb binding sites with their consensus binding
sites. The C/EBP site is in the reverse orientation. (C) ATRA affects MBN promoter
activity. The pMBN 5⬘ deletion promoter mutants were fused to the CAT gene (i).
PLB-985 cells were transfected with 20 ␮g each of the indicated MBN 5⬘ deletion
mutants and cultured with or without ATRA (10⫺6 M). CAT activity was measured after
normalization to the luciferase internal control (ii). The graph on the left shows
untreated (⫺ATRA, u) and ATRA-treated (⫹ATRA, f) cells. CAT activities presented
for each pMBN construct were calculated relative to the pMBN-658. On the right are
calculated ⫺ATRA/⫹ATRA ratios as well as standard errors for each construct.
MYELOBLASTIN IS A TARGET OF MYB
2451
C/EBP site is present in the granulocyte-macrophage colonystimulating factor receptor ␣ promoter, harbors the same G-to-C
transition, and binds C/EBPs.33 The Myb site is identical to a
functional Myb binding site present in the neutrophil elastase
promoter, which binds c-Myb with much lower affinity than, for
example, the mim-1 Myb site.34
To investigate the mechanisms involved in MBN downregulation in ATRA-treated growth-arrested leukemia cells, deletion constructs of the human MBN promoter (Figure 1Ci) cloned
into the pBLCAT6 reporter plasmid were transfected into myeloblastic PLB-985 cells. Deletion from position ⫺658 to ⫺91 had
minimal effect on MBN promoter constitutive activity, indicating
that the remaining proximal promoter region retained the major
elements (Figure 1Cii). The activity of the pMBN-658 reporter
plasmid was reduced by 7-fold in ATRA-induced cells (Figure 1C).
The ⫺91 deletion construct was still responsive to ATRA, indicating that this part of the promoter harbored responsive elements
involved in the inhibition of MBN expression in myeloid leukemia
cells growth-arrested by ATRA (Figure 1Cii).
EMSAs were first conducted using total extracts from COS-7
cells transfected with vectors expressing recombinant PU.1,
C/EBP␣, C/EBP␤, C/EBP␦, and c-Myb. Total extracts from cells
expressing the PU.1, C/EBP␣, C/EBP␤, C/EBP␦, or c-Myb
recombinants specifically bound to their respective PU.1, C/EBP,
and c-Myb probes (Figure 2A) as confirmed by competition
experiments. Addition of anti–c-Myb antibodies specifically supershifted the c-Myb complex (Figure 2A). Although, similarly to
other investigators in the field, we were unable to assess c-Myb
DNA binding in myeloid cell nuclear extracts, likely because of
high levels of protease activity, our results indicate that c-Myb
binds specifically to its site in the MBN promoter. The PU.1 and
C/EBP binding sites in the MBN promoter were able to bind their
respective nuclear factors present in proliferating myeloid cells as
confirmed by EMSA with nuclear extracts from untransfected
PLB-985 cells. Indeed, PU.1- and C/EBP-specific complexes were
detected with the PU.1- and C/EBP-specific probe (Figure 2B),
respectively. Addition of the anti-PU.1 antibody specifically supershifted this complex (Figure 2B). Similarly, C/EBP complexes
were specifically supershifted with C/EBP␣, C/EBP␤, and C/EBP␦
antibodies (Figure 2B). Altogether, our results demonstrate that the
PU.1, C/EBP, and Myb binding sites in the MBN promoter were
able to bind their respective factors.
Deletion fragments of the MBN promoter were not adequate for
studying the respective importance of the C/EBPs and Myb
elements because removal of the PU.1 site resulted in a drastic
diminution of the MBN promoter activity (data not shown). We
therefore introduced point mutations into these sites. Mutation of
PU.1, C/EBP, or Myb sites resulted in a drastic decrease of the
promoter activity, indicating that all 3 sites were critical for the
constitutive activity of the MBN promoter (Figure 2C) and
suggesting that their action could be combinatorial. Indeed, it has
been shown that c-Myb requires direct and specific interaction with
the cyclic-AMP response element-binding protein (CREB)-binding
protein (CBP) for its transactivation function.35,36 Similar observations were made with PU.137 and C/EBP␤,38 which also have
physical and functional interaction with CBP/p300. We therefore
investigated whether CBP/p300 could be required for full MBN
expression and therefore be a potential candidate to support a
combinatorial transcriptional effect of the 3 transcriptional factors
on the MBN promoter. As previously described to demonstrate
c-Myb35 and C/EBP38 interactions with CBP/p300, we used the
E1A 13S protein as a competitor that specifically interacts with
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2452
LUTZ et al
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
Figure 2. Functional PU.1, C/EBP, and c-Myb binding sites are
present in the human MBN promoter. (A) Recombinant human
PU.1, C/EBPs, and c-Myb specifically bind functional sites in the
MBN promoter. Five micrograms of whole-cell extract from COS-7
cells either untransfected (lanes 1, 5, 15) or transfected with PU.1
(lanes 2-4), C/EBP␣ (lanes 6-8), C/EBP␤ (lanes 9-11), C/EBP␦
(lanes 12-14), or c-Myb (lanes 16-18) were preincubated with 2 ␮g
poly(dIdC) and a 400-fold molar excess of the wild-type (wt) or
mutated (mut) competitors as indicated, before addition of the
specific probe (PU.1, lanes 1-4; C/EBP, lanes 5-14; and c-Myb,
lanes 15-18). Binding reaction mixtures were assayed by EMSA.
The asterisks mark unspecific complexes. Supershift experiments
were performed with 1 ␮L c-Myb antibody (Ab) and unrelated
control Ab (lanes 19-22). Arrowhead indicates the specific supershifted complex. (B) Myeloid nuclear factors bind to the PU.1 and
C/EBP sites. Nuclear extracts (1 ␮g) from exponentially growing
PLB-985 cells were subjected to EMSA (lanes 1-7 and 11-19).
DNA binding assays were performed with the PU.1 (lanes 1-10) or
the C/EBP (lanes 11-24) probe in the presence of 5 ␮g and 0.2 ␮g
poly(dIdC), respectively. Competition assays were carried out with
a 40- and 200-fold molar excess of the wt or mut oligonucleotides.
Arrows indicate the specific PU.1 or C/EBP complexes. Supershift
experiments were performed with 1 ␮L anti-PU.1 (PU.1 Ab) (lanes
7-9), anti-C/EBP␣, anti-C/EBP␤, anti-C/EBP␦ (C/EBP␣, C/EBP␤,
C/EBP␦ Abs) (lanes 17-19) and unrelated (control Ab) Abs (lanes
6, 8, 16, 20). Arrowheads indicate specific supershifted complexes. (C) PU.1, C/EBP, and Myb binding sites are all critical for
MBN promoter activity in PLB-985 cells. Wt or indicated point
mutation constructs (PU.1mut, C/EBPmut, and Mybmut) were
transfected into cells. Transfection efficiencies were normalized
by cotransfection of a CMV-luciferase vector. CAT activities
relative to the wt (⫺658) are presented for PU.1mut, C/EBPmut,
and Mybmut constructs. Error bars indicate SD from at least 3
different experiments. (D) CBP/p300 or related molecules are
involved in MBN expression. PLB-985 cells were transfected with
20 ␮g pMBN-658 alone (⫺) or together with 2 and 10 ␮g of
expression vector encoding E1A (13S) or the E1A 12S NTdl814
mutant. The average of 3 experiments are shown as relative CAT
activity with SD as indicated. (E) Both c-Myb and C/EBP␦ can
cooperate to activate the MBN promoter. Five micrograms of
pMBN-194 was transfected into COS-7 cells alone (⫺) or together
with 1 ␮g of the CMV, CMV-Myb, MSV, MSV-C/EBP␣, MSV-C/
EBP␤, MSV-C/EBP␦, pECE, or pECE-PU.1 expression vectors or
the indicated combinations of these vectors. CAT activities were
assayed after 40 hours. The mean activations and standard errors
observed in 3 determinations are shown.
CBP/p300 and blocks their activities. Transfection of an E1A 13S
expression vector in PLB-985 cells reduced MBN promoter
activity by 2.5-fold (Figure 2D). In contrast, no inhibition was
detected using an E1A deletion mutant unable to interact with
CBP/p300 (Figure 2D). Although E1A 13S can potentially interact
with a variety of different target molecules, these results suggest
that, in the context of the MBN promoter, CBP/p300 or related
molecules are required for full MBN expression. To assess a
potential cooperativity between c-Myb, PU.1, and C/EBP, the
pMBN-194 reporter plasmid was transfected into COS-7 cells
alone or together with the CMV, CMV-Myb, MSV, MSV-C/EBP␣,
MSV-C/EBP␤, MSV-C/EBP␦, pECE, or pECE-PU.1 expression
vectors or combinations of these vectors. Compared with the empty
vectors, c-Myb–, C/EBP␦-, and C/EBP␣-containing vectors stimulated CAT expression by 20-, 6-, and 2-fold, respectively (Figure
2E), while C/EBP␤ and PU.1 alone did not activate the MBN
promoter in COS-7 cells. Cooperative activation was observed
when c-Myb was cotransfected with C/EBP␦ (Figure 2E). Cooperative DNA binding by c-Myb and C/EBP could not be demonstrated by EMSA analysis (data not shown). Altogether, these
results suggest that (1) combinatorial action of Myb, PU.1, and
C/EBP supports MBN promoter activity and (2) restricted positive
cooperativity exists between C/EBP␦ and Myb. Furthermore,
together with Figure 2C, these results indicate that the binding of
each factor to its specific site was necessary for constitutive activity
of the MBN promoter but not sufficient individually for its full
transactivation.
Inhibition of MBN expression in growth-arrested ATRA-treated
leukemia cells is mediated by Myb down-regulation
ATRA-induced growth inhibition in differentiating PLB-985 and
NB4 leukemia cells (Figure 3Ai,ii, respectively) was accompanied
by progressive down-regulation of the MBN messenger RNA
(mRNA), which was barely detected at days 4 and 2, respectively
(Figure 3Aiii). During these processes, the time at which MBN
mRNA expression was inhibited closely paralleled that of c-Myb
(Figure 3Aiii). We therefore overexpressed c-Myb in PLB-985
cells to test whether its down-regulation was required for ATRA to
down-regulate the MBN promoter. In contrast to the expected
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BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
MYELOBLASTIN IS A TARGET OF MYB
2453
Figure 3. Transcriptional down-regulation of MBN by ATRA requires Myb down-regulation. (A) ATRA-induced down-regulation of MBN mRNA in PLB-985 and NB4 cells
correlates with growth arrest and down-regulation of c-Myb. Growth (i) and morphologic (ii) assessments of PLB-985 (left) and NB4 (right) at 2, 4, and 6 days of treatment with
10⫺6 M ATRA. (iii) Regulation of MBN and c-Myb mRNAs in PLB-985 cells untreated or treated for 0.5, 1, 2, 4, and 6 days with 10⫺6 M ATRA. For RNA blots, 3 ␮g total RNA was
loaded in each lane. The lower part is a control hybridization of the same blots with GAPDH probe for assessment of RNA quantities. Panels Ai-iii are for PLB-985 and NB4 cells
on left and right column, respectively. (B) Overexpression of c-Myb in PLB-985 cells abolished transcriptional down-regulation of MBN by ATRA. (i) Twenty micrograms of the
pMBN-658 (⫺658) or pMBN-658Mybmut (⫺658 Mybmut) constructs were individually transfected (⫺) or cotransfected with 1, 2, or 5 ␮g CMV-Myb (c-Myb) into PLB-985 cells
and cultured with (f) or without (u) ATRA (10⫺6 M) for 40 hours. Transfection efficiencies were normalized by cotransfection of a CMV-luciferase vector. CAT activities were
calculated relative to the wt (⫺658). The means and standard errors of 3 determinations are shown. (i) Autoradiogram of MBN mRNA expression in PLB-985 cells transfected
with 5 ␮g of either the empty (⫺) or CMV-Myb (c-Myb) vector. The lower part is an autoradiogram of GAPDH mRNA for assessment of RNA quantities. Fold increase is
indicated. (C) Overexpression of C/EBP and PU.1 in PLB-985 cells did not affect transcriptional down-regulation of MBN by ATRA. (i,ii) Induced PLB-985 growth arrest
correlates with increased PU.1- and C/EBP-binding activity. Nuclear extracts from PLB-985 cells untreated or treated with 10⫺6 M ATRA for the indicated days were assayed for
PU.1 binding and C/EBP binding by EMSA performed with the PU.1 and C/EBP probes in the presence of 5 ␮g and 0.2 ␮g poly(dIdC), respectively (left panels). Competition
experiments were carried out with a 400-fold molar excess of the indicated wt or mut competitor, and supershift experiments were performed with 1 ␮L PU.1 Ab; C/EBP␣,
C/EBP␤, C/EBP␦ Abs; and unrelated control Abs (right). Arrows indicate the specific PU.1 or C/EBP complexes. Arrowheads indicate specific supershifted complexes. (iii)
Twenty micrograms of the pMBN-658 (⫺658) was transfected (⫺) or cotransfected with 5 ␮g of the expression vectors for C/EBP␣ (MSV-C/EBP␣), C/EBP␤ (MSV-C/EBP␤),
C/EBP␦ (MSV-C/EBP␦), and PU.1 (pECE-PU.1) into PLB-985 cells and cultured with (f) or without (u) ATRA (10⫺6 M) for 40 hours. Transfection efficiencies were normalized
by cotransfection of a CMV-luciferase vector. CAT activities calculated relative to the wt (⫺658) as a reference are presented. This is representative of 3 independently
performed experiments.
ATRA-induced inhibition of MBN promoter activity in PLB-985
cells, overexpression of c-Myb (5 ␮g plasmid) resulted in an 8-fold
induction of the MBN promoter activity (Figure 3Bi), which could
no longer be decreased following treatment of the cells with ATRA
(Figure 3Bi). To verify that potential ATRA-induced effects on
MBN expression were not blunted by saturated levels of MBN as a
result of c-Myb overexpression in PLB-985 cells, we used lower
levels of engineered c-Myb expression (2 ␮g), which, following
ATRA treatment, resulted in an MBN promoter activity similar to
that found in untreated cells transfected with the empty plasmid
(Figure 3Bi). This suggested that transfection of 2 ␮g c-Myb
plasmid allowed for a quasi “normal” levels of c-Myb after ATRA
treatment. Under these conditions, resistance to ATRA was still
detectable. Indeed, while ATRA treatment resulted in a 10.6-fold
decrease of the MBN promoter in control cells, a 2.9-fold and
2.4-fold decrease was observed with the same treatment in cells
transfected with 1 and 2 ␮g c-Myb plasmid, respectively (Figure
3Bi). Resistance to ATRA was therefore likely due to exogenous
c-Myb expression, which resulted in increased MBN promoter
activity. In both untreated and ATRA-treated cells, induction of the
MBN promoter activity by c-Myb overexpression and resistance to
ATRA were dependent upon the integrity of the Myb binding site
(Figure 3Bi). This strongly suggests that the binding of Myb to the
MBN promoter is required for its regulation by ATRA. Notably,
overexpression of c-Myb resulted in a 3.6-fold induction of the
endogenous MBN mRNA (Figure 3Bii).
We then analyzed whether changes in PU.1 and C/EBP binding
to their sites could be responsible for ATRA-induced MBN
down-regulation. For this, EMSAs were conducted out with
PLB-985 cell nuclear extracts using the PU.1 or the C/EBP probe
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
2454
LUTZ et al
(Figure 3Ci,ii, respectively). ATRA-induced growth arrest and
differentiation of PLB-985 cells were accompanied by a progressive increased binding to both the PU.1 (Figure 3Ci, left) and
C/EBP (Figure 3Cii, left) probes. Competition assays attested the
specificity of the resulting DNA-protein complexes (Figure 3Ci,ii,
right). The PU.1-retarded complex was specifically supershifted by
PU.1 antibodies (Figure 3Ci, right). Incubation with anti-C/EBP␤
and anti-C/EBP␦ antibodies resulted in supershift of the C/EBP
complexes and to a lesser extent of C/EBP␦ complexes, respectively; no supershift was observed with anti-C/EBP␣ antibodies
(Figure 3Cii, right). Overexpression of PU.1 in PLB-985 cells
resulted in a slight decrease (about 2-fold) of the MBN promoter
activity (Figure 3Ciii). In contrast to our observations with c-Myb
and PU.1, overexpression of C/EBP␣, C/EBP␤, and C/EBP␦ had
no effect on MBN transcriptional expression in ATRA-treated
PLB-985 cells (Figure 3Ciii). This indicated that, despite increased
binding to their specific sites in ATRA-treated PLB-985 cells,
C/EBPs had no inhibitory effect on the ATRA response. These
results indicate that MBN is a Myb target gene and that its
down-regulation in growth-arrested ATRA-treated leukemia cells
is dependent upon Myb down-regulation. Altogether, our results
establish that (1) down-regulation of c-Myb mRNA correlated with
that of MBN; (2) ectopic expression of c-Myb induced MBN
promoter activity and enabled it to resist down-regulation by
ATRA; (3) these 2 effects required the integrity of the specific Myb
binding site; (4) the inhibitory effect of ATRA on the MBN
promoter is not dependent upon C/EBPs; and (5) PU.1 may act as a
transrepressor of MBN promoter activity via transcriptional repression of the c-Myb promoter.
Discussion
MBN is a myeloid growth-related gene.6,11 Although PU.1, C/EBP,
and Myb transcription factors were all critical for constitutive
expression of MBN, its down-regulation in leukemia cells growtharrested by ATRA was dependent upon Myb. This and the fact that
overexpression of c-Myb in leukemia cells inhibited downregulation of MBN by ATRA point to MBN as a novel target of
Myb in the context of proliferation.
The mechanism by which myeloid-specific genes can be
expressed in immature myeloid cells has been exemplified by the
study of neutrophil elastase, which is regulated as a result of a
combinatorial activation by at least 3 factors, c-Myb, PU.1, and
C/EBP, none of which by itself is myeloid-specific.34 PU.1 and
C/EBP are involved in myeloid-specific gene expression.23,39 The
fact that introduction of mutations in the PU.1 and C/EBP sites of
the MBN promoter resulted in a drastic decrease in its constitutive
transcriptional activity is consistent with the observation that MBN
mRNA was markedly reduced in vivo in fetal liver of PU.1 and
C/EBP␣ knockout mice.40 Furthermore, Zhang et al41 demonstrated
that loss of both the interleukin-6 and G-CSF receptors contributed
to the absolute block in granulocyte maturation observed in
C/EBP␣-deficient hematopoietic cells and suggested that additional C/EBP␣ target genes are also important for the block in
granulocyte differentiation observed in vivo in C/EBP␣-deficient
mice. Our data show that c-Myb can positively regulate MBN
expression in myeloid leukemia cells. This is consistent with the
fact that c-Myb is involved in the control of proliferation of
definitive hematopoietic stem cells or multipotential progenitors as
well as proliferation of myeloid leukemia cells.15-17 Expression of
c-Myb decreases as normal hematopoietic stem or progenitor cells
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
go into growth arrest and differentiation.42 It is also known that
MBN mRNA is barely expressed in differentiated myeloid cells.6
Therefore, the fact that in leukemia cells inhibition of both MBN
and c-Myb mRNA expression is obtained through treatment by
ATRA draws a parallel with their down-regulation during normal
hematopoiesis. This is consistent with the view that, in leukemia
cells, ATRA may mimic a process occurring during normal
hematopoiesis. To this extent, it should be noted that, although
G-CSF can induce MBN expression,11 a potential ATRA-induced
increase in G-CSF receptor expression43 in PLB-985 cells did not
affect the capacity of ATRA to induce MBN down-regulation as
well as growth arrest and differentiation. Unlike c-Myb, B-Myb is
ubiquitously expressed; A-Myb is expressed in a subpopulation of
normal activated B lymphocytes and is not detected significantly in
other hematopoietic cells. Although they have different expression
patterns, a common feature of the Myb proteins is their expression
in proliferating cells.44 Because anti–B-Myb antibodies suitable for
EMSA are not available, our data do not indicate whether B-Myb
can bind to the MBN Myb site. However, this is likely because
Myb proteins display a high degree of homology within their
DNA-binding domains recognizing a PyAACG/TG consensus
sequence. The c-Myb and B-Myb can activate transcription of the
same reporter genes,45,46 suggesting that they can regulate similar
set(s) of growth control genes.47 Furthermore, expression of
B-Myb closely parallels that of c-Myb in myeloid cells, particularly
in ATRA-induced HL-60 cells.48
Studies of few genes regulated by Myb have indicated that it
frequently acts in cooperation with other transcription factors.
Indeed, our finding that functional PU.1, C/EBP, and Myb binding
sites exist in the MBN promoter is reminiscent of previous
observations with the neutrophil elastase34 and myeloperoxidase
promoters. Our data are in agreement with previous work32 that
established the importance of PU.1 for MBN promoter activity.
However, our results contrast with previous indications32 that
C/EBP and c-Myb were unlikely to play a significant role in the
expression of MBN. Furthermore, it is of interest that PU.1
appeared to be involved in the regulation of MBN by phorbol
myristate acetate (PMA), which decreased PU.1 binding to its
site.32 Although there was no evidence that overexpression or
change in phosphorylation status of PU.1 would affect PMAinduced MBN transcriptional inhibition, these results are consistent
with our observation that the PU.1 site is critical to MBN
constitutive activity. Our results indicate that Myb specifically
regulates MBN transcriptional inhibition by ATRA. We also found
that, in contrast to PMA,32 ATRA up-regulates PU.1 binding to its
site though overexpression of PU.1 could not abolish the negative
effect of ATRA on MBN promoter activity. Altogether, previous
work conducted with PMA32 and our present data suggest that
PMA and ATRA down-regulate MBN promoter activity by affecting specific bindings to 2 different sites that are each critical to their
constitutive transcriptional expression. Notably, overexpression of
PU.1 resulted in a slight decrease in the MBN promoter activity. In
PLB-985 cells, we have observed an increased PU.1 binding to its
site upon ATRA treatment. In these cells, overexpression of PU.1
appeared to transrepress MBN promoter activity. Therefore, a
transrepressor role for PU.1 may contribute to the down-regulation
of MBN expression via inhibition of c-Myb transcription during
myeloid differentiation.49
Cooperation between myeloid transcription factors for maximal
promoter transactivation has been demonstrated.34,50,51 Recruitment of CBP, which has a strong histone acetyltransferase activity,
by c-Myb and NF-M potentiated their transcriptional activities by
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
BLOOD, 15 APRIL 2001 䡠 VOLUME 97, NUMBER 8
MYELOBLASTIN IS A TARGET OF MYB
bridging these 2 proteins.35 Our data show that C/EBP␦ and Myb
cooperatively activated the MBN promoter, reinforcing the view
that cooperation between these factors is relevant to the activation
of early myeloid genes. We have shown that CBP/p300 is likely to
be required for full MBN expression. In view of the fact that the
Myb, C/EBP, and PU.1 sites were all critical for MBN promoter
activity, there is a possibility that CBP/p300 may support a
combinatorial transcriptional effect of the 3 factors. Another
possibility would be that, after recruitment to the MBN promoter
by C/EBP and c-Myb, CBP/p300 may render the chromatin more
accessible to the transcriptional machinery, allowing MBN transactivation by C/EBP and c-Myb.
It is assumed that leukemic cells descend from a small pool of
progenitors with high proliferative activity.52 This suggests that
genes that are involved in controlling limited factor-dependent
growth in normal hematopoietic progenitors might be continuously
expressed, providing factor-independent growth to preleukemic
cells. Such genes might be inhibited de novo when leukemia cells
2455
are forced into growth arrest by inducers such as ATRA. Our data
provide a way by which ATRA can access a myeloid transcriptional
machinery in which Myb is instrumental. Myb is known for its
pivotal role in controlling cell growth and may therefore provide a
way for ATRA to control proliferation in myeloid leukemia cells.
Few Myb target genes are known to be linked to proliferation, and
little is known about Myb involvement in growth inhibition
induced by ATRA in leukemia cells. The fact that MBN, which can
confer factor-independent growth to hematopoietic cells,11 is a
target of Myb suggests a mechanism by which growth arrest may
be induced by ATRA in myeloid leukemia cells.
Acknowledgments
We thank Drs A. Friedman, B. Lüscher, R. Maki, and P. Whyte for
generous gifts of plasmids.
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2001 97: 2449-2456
doi:10.1182/blood.V97.8.2449
Myeloblastin is an Myb target gene: mechanisms of regulation in myeloid
leukemia cells growth-arrested by retinoic acid
Pierre G. Lutz, Anne Houzel-Charavel, Christel Moog-Lutz and Yvon E. Cayre
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