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J Biol Chem, Vol. 274, Issue 8, 4939-4946, February 19, 1999
From the Divisions of The ETS domain transcription factor PU.1 is
necessary for the development of monocytes and regulates, in
particular, the expression of the monocyte-specific macrophage
colony-stimulating factor (M-CSF) receptor, which is critical for
monocytic cell survival, proliferation, and differentiation. The bZIP
transcription factor c-Jun, which is part of the AP-1 transcription
factor complex, is also important for monocytic differentiation, but
the monocyte-specific M-CSF receptor promoter has no AP-1 consensus
binding sites. We asked the question of whether c-Jun could promote the
induction of the M-CSF receptor by collaborating with PU.1. We
demonstrate that c-Jun enhances the ability of PU.1 to transactivate
the M-CSF receptor promoter as well as a minimal thymidine kinase
promoter containing only PU.1 DNA binding sites. c-Jun does not
directly bind to the M-CSF receptor promoter but associates via its
basic domain with the ETS domain of PU.1. Consistent with our
observation that AP-1 binding does not contribute to c-Jun coactivation
is the observation that the activation of PU.1 by c-Jun is blocked by
overexpression of c-Fos. Phosphorylation of c-Jun by c-Jun NH2-terminal kinase on Ser-63 and -73 does not alter
the ability of c-Jun to enhance PU.1 transactivation. Activated Ras
enhances the transcriptional activity of PU.1 by up-regulating c-Jun
expression without changing the phosphorylation pattern of PU.1. The
activation of PU.1 by Ras is blocked by a mutant c-Jun protein lacking
the basic domain. The expression of this mutant form of c-Jun also completely blocks
12-O-tetradecanoylphorbol-13-acetate-induced M-CSF receptor
promoter activity during monocytic differentiation. We propose
therefore that c-Jun acts as a c-Jun NH2-terminal
kinase-independent coactivator of PU.1, resulting in M-CSF receptor
expression and development of the monocytic lineage.
The ETS domain transcription factor PU.1 is preferentially
expressed in myeloid and B cells (1, 2) and plays a pivotal role in
their development (3, 4). Indeed, mice deficient in PU.1 display a
complete block in development of monocytes, macrophages, and B cells
(5, 6). During hematopoietic development, PU.1 mRNA is expressed at
low levels in murine embryonic stem cells and human CD34+ stem cells
and is specifically up-regulated upon myeloid differentiation, and
down-regulated upon erythroid differentiation (7, 8). PU.1 regulates
the expression of almost all characterized myeloid genes, including
growth factor receptors, and in particular directs the
monocyte-specific expression of the macrophage colony-stimulating
factor (M-CSF)1 receptor (9,
10). Thus, PU.1-deficient hematopoietic cells display minimal
expression of granulocyte colony-stimulating factor and
granulocyte-macrophage colony-stimulating factor receptors and no
detectable M-CSF receptors (11, 12).
The M-CSF receptor is critical for monocytic cell survival,
proliferation, and differentiation (3, 13). M-CSF is known to augment
monocyte survival and, therefore, to allow macrophage differentiation
(14). The responsiveness of hematopoietic progenitor cells to M-CSF is
regulated at the level of M-CSF receptor expression (15). Although the
important role of the M-CSF receptor for the development of monocytes
has been clearly demonstrated, little is known about the signaling
molecules or protein-protein interactions that modulate the effect of
PU.1 to regulate the M-CSF receptor promoter activity (3, 16).
c-Jun belongs to the bZIP group of DNA binding proteins and is a
component of AP-1 transcription factor complexes (17). c-Jun forms
homodimers or can heterodimerize with other Jun family members or with
other bZIP proteins including members of the Fos and ATF/cAMP response
element-binding protein (CREB) families (18, 19). AP-1 has been shown
to be involved in many cellular processes including proliferation,
differentiation, apoptosis, and stress responses (18). In particular,
there is evidence that c-Jun plays a role in monocytic differentiation.
c-Jun mRNA is up-regulated upon monocyte differentiation of
bipotential myeloid cell lines (20-22), while stable transfection of
c-Jun into myeloid cell lines results in partial differentiation (23,
24).
Although c-Jun and PU.1 are both pivotal for monocytic development, it
is still unclear whether c-Jun is involved in the regulation of the
M-CSF receptor, which is critical for monocyte survival, proliferation,
and differentiation. It has been shown that during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced monocytic
differentiation of U937 cells, c-Jun and M-CSF receptor mRNA
expression increases (25). However, the monocyte-specific M-CSF
receptor promoter (9, 26, 27) contains no AP-1 consensus binding sites.
As c-Jun and the regulation of the M-CSF receptor by PU.1 play
important roles in monocytic differentiation, we hypothesized that
c-Jun might be involved in the regulation of the M-CSF receptor, not by
binding to AP-1 sites, but possibly via a novel mechanism. Therefore,
we asked the question of whether c-Jun modulates the ability of PU.1 to
transactivate the human monocyte specific M-CSF receptor promoter.
Cell Lines and Cell Culture--
Monkey kidney CV-1 cells (ATCC
CCL-70; American Type Culture Collection, Rockville, MD) were
maintained in Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) supplemented with 10% calf serum (HyClone, Logan, UT). Murine
embryonal carcinoma F9 cells (ATCC CRL-1720; American Type Culture
Collection) and human kidney 293T cells (kindly provided by John
Blenis, Harvard Medical School, Boston, MA) were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum (HyClone). U937 cells (ATCC CRL 1593; American Type Culture
Collection) were maintained in RPMI 1640 medium (Life Technologies)
supplemented with 10% fetal bovine serum, and differentiated with
2 × 10 Reporter Constructs and Expression Plasmids--
The human
monocyte-specific M-CSF receptor promoter ranging from bp
The PU.1 mutants pcDNA1-PU.1/
Human pS3H-c-Jun containing wild-type c-Jun (34), pS3H-c-Jun-S63A/S73A
containing serine to alanine mutations in amino acid residues 63 and 73 of the human c-Jun cDNA (34), and murine pSV40-c-Fos (35) were
kindly provided by Jianmin Tian and Michael Karin (University of
California, San Diego). Murine pSV-SPORT1-c-Jun, pUC18-c-Jun/ Transient Transfections Using LipofectAMINE Plus or
Electroporation and Reporter Assays for Firefly and Renilla
Luciferase--
CV-1 cells, F9 cells, or 293T cells were transfected
using LipofectAMINE Plus (Life Technologies) as described by the
manufacturer. U937 cells were transiently transfected by
electroporation as described previously (9). Firefly luciferase
activities from the constructs pM-CSFR, pXP2, and pTK with PU.1 sites
and pTK with mutated PU.1 sites and Renilla luciferase
activity from the internal control plasmid pRL-null were determined
24 h after the initiation of the transfection protocols using the
Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase
activities were normalized to the Renilla luciferase values
of pRL-null. Results are given as means ± S.E. of at least six
independent experiments. The following DNA concentrations of the
reporter constructs and expression plasmids were used for LipofectAMINE Plus transfections: 0.3 µg of the human monocyte-specific M-CSF receptor promoter in pXP2, pXP2, the TK promoter with PU.1 sites, and
the TK promoter with mutated PU.1 sites; 0.05 µg of the internal control plasmid pRL-null; 0.5 µg of pEBG-PU.1; 0.2 µg of the other expression plasmids for PU.1 and PU.1 mutants; 0.25 µg of Ras(L61), Ras(N17), and MEKK1; 0.1 µg of c-Jun, c-Jun mutants, and c-Fos; and
the same concentrations of the empty expression vectors as controls,
respectively. For electroporation, 10 µg of the firefly luciferase
reporter constructs, 5 µg of expression plasmids, and 1 µg of the
internal control plasmid were used. pRL-null was chosen as internal
control plasmid, because it was not transactivated by Ras (30) or by
PU.1, c-Jun, c-Fos, or MEKK1 in CV-1, F9, 293T, or U937 cells (data not shown).
Electrophoretic Mobility Shift Assay--
Electrophoretic
mobility shift assays were performed as described previously (9, 26,
27, 29). As a positive control for c-Jun binding, a double-stranded
AP-1 probe from the collagenase promoter (5'-AAT TCG CTT GAT GAC TCA
GCC GGA A-3') was labeled with Klenow polymerase and
[ Protein Interaction Assay--
Protein interaction assays were
performed as described previously (27, 42). c-Jun and c-Fos were
in vitro transcribed and translated using the TNT
Reticulocyte Lysate System (Promega) and labeled with
[35S]methionine (NEN Life Science Products). 1 µl of
labeled in vitro translated c-Jun or c-Fos was mixed with 1 µg of bacterially expressed GST-PU.1 or equivalent amounts of GST or
glutathione-agarose beads (Sigma) for 1 h at 4 °C in NETN
buffer (20 mM Tris (pH 8.0), 200 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40). GST-PU.1 was recovered using glutathione-agarose beads, washed seven times with NETN buffer,
and separated by 10% SDS-polyacrylamide gel electrophoresis. Prior to
autoradiography, the gel was stained with Coomassie Brilliant Blue
(Bio-Rad) to verify that the protein concentrations of GST-PU.1 and GST
were the same in all lanes.
In Vivo Labeling, Phosphoamino Acid Analysis, and Phosphopeptide
Mapping--
To detect changes in the phosphorylation pattern of PU.1
upon stimulation with activated Ras in vivo, 0.5 µg of
pEBG-PU.1 either with 0.25 µg of activated Ras(L61) or with inactive
Ras(N17) was transfected into 293T cells using LipofectAMINE Plus (Life Technologies). 3 h after transfection, cells were starved in
serum-free Dulbecco's modified Eagle's medium. After 18 h,
cells were placed into serum-free and phosphate-free Dulbecco's
modified Eagle's medium (Life Technologies) for 30 min before they
were metabolically labeled with [32P]orthophosphate
(2.5 mCi/ml). After 4 h, cells were lysed with radioimmunoprecipitation assay buffer containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 5 mM EDTA, and 50 mM Tris (pH 8.0) and
supplemented with aprotinin, phenylmethylsulfonyl fluoride, pepstatin
A, leupeptin, antipain, and chymostatin as protease inhibitors (Sigma)
and sodium pyrophosphate, sodium fluoride, and sodium vanadate as
phosphatase inhibitors (Sigma). In parallel plates, 0.3 µg of the
M-CSF receptor promoter was co-transfected in 293T cells, and
luciferase activities were determined to ensure that Ras enhances the
transactivation function of PU.1 in the particular experiment used in
in vivo labeling and subsequent phosphoamino acid analysis
and phosphopeptide mapping.
GST-PU.1 was isolated from the radioimmunoprecipitation assay lysates
using glutathione-agarose beads (Sigma), washed four times with
radioimmunoprecipitation assay buffer, separated on 10%
SDS-polyacrylamide gels, and transferred to Immobilon-P membrane (Millipore Corp., Bedford, MA) for phosphoamino acid analysis or
nitrocellulose (Bio-Rad) for phosphopeptide mapping. After transfer,
the 69-kDa GST-PU.1 protein bands were excised. For phosphoamino acid
analysis, the samples were boiled at 100 °C for 1 h with 6 N HCl (Pierce), and the presence of serine, threonine, or
tyrosine phosphorylation was determined as described (43). To determine
the phosphorylated protein residues of PU.1, GST-PU.1 protein bands
were digested with 1-chloro-3-tosylamido-7-amino-2-heptanone-treated chymotrypsin (Worthington) and endoproteinase Glu-C (V8 protease) (Boehringer Mannheim) and processed for phosphopeptide mapping as
described previously (43).
Western Blot--
24 h after the start of transfection, cells
were lysed with radioimmunoprecipitation assay buffer. Equal amounts of
total protein were separated on 10% SDS-polyacrylamide gels and
transferred to Immobilon-P membrane (Millipore). Membranes were
incubated with anti-c-Jun antibody (catalog no. SC-45; Santa Cruz
Biotechnology, Inc., Santa Cruz, CA), anti-PU.1 antibody (catalog no.
SC-352; Santa Cruz Biotechnology), or anti- c-Jun Enhances the Ability of PU.1 to Transactivate the M-CSF
Receptor Promoter and a Minimal TK Promoter Containing PU.1 DNA Binding
Sites Only--
Since c-Jun and the regulation of the M-CSF receptor
by PU.1 are both important for monocytic development, we asked the
question of whether c-Jun enhances the ability of PU.1 to transactivate the M-CSF receptor promoter. CV-1 cells, which contain c-Jun (Fig. 4B), were transfected with a plasmid containing the human
monocyte-specific M-CSF receptor promoter (9, 26) cloned upstream of
the luciferase reporter gene along with expression plasmids for PU.1
and c-Jun, and reporter gene expression was determined 24 h
post-transfection. Transfection of a c-Jun expression construct
significantly enhanced the ability of PU.1 to transactivate the M-CSF
receptor promoter (Fig. 1A).
Moreover, in c-Jun-deficient F9 cells, PU.1 weakly transactivated the
M-CSF receptor promoter (2 fold), while co-expression of c-Jun with
PU.1 led to robust transactivation (33-fold) (Fig. 1B). The
cooperation of c-Jun with PU.1 is therefore important for M-CSF
receptor promoter activity.
We next asked the following questions: (a) whether the
binding of PU.1 to DNA was necessary for its activation by c-Jun and (b) whether a PU.1 binding site alone was sufficient for the
c-Jun-enhanced PU.1 activation. We observed enhanced PU.1
transactivation mediated by c-Jun using a reporter construct containing
four PU.1 binding sites cloned upstream of a minimal TK promoter (Fig.
1C). In control experiments, no effect of c-Jun on PU.1
activity was observed when the PU.1 binding sites were mutated (Fig.
1C). These data indicate that PU.1 binding to DNA is
necessary for its activation by c-Jun and that PU.1 binding sites are
sufficient to mediate this effect.
c-Jun Does Not Directly Bind to the M-CSF Receptor Promoter but
Associates with the ETS Domain of PU.1; the Activation of PU.1 by c-Jun
Is Blocked by Overexpression of c-Fos--
To elucidate the mechanism
by which c-Jun augments the transcriptional activity of PU.1, we
performed experiments to determine whether the activation of PU.1 by
c-Jun required the binding of c-Jun·AP-1 complexes to the M-CSF
receptor promoter. Since there are no AP-1 consensus sites in the human
monocyte-specific M-CSF receptor promoter from bp
It has previously been reported that c-Jun can physically and
functionally interact with ETS-1, which like PU.1 is an ETS family
transcription factor (44). In these same studies, it was reported that
c-Jun and PU.1 could physically interact, although the functional
consequences of this interaction were not examined. We were able to
confirm that c-Jun did indeed specifically interact with PU.1 in
vitro (Fig. 2B). Because c-Jun can bind to PU.1 but does not bind to DNA itself (Fig. 2A), we therefore conclude
that c-Jun acts as a coactivator (45) of PU.1.
Since c-Jun can form a heterodimer with c-Fos in AP-1 transcription
factor complexes (17-19), we asked the question of whether c-Fos
expression could modulate the synergy between c-Jun and PU.1 in F9
cells. Co-transfection of c-Fos did not enhance the synergy between
c-Jun and PU.1 but instead completely blocked it (Fig. 2C).
c-Fos did not bind to PU.1 (Fig. 2B) and therefore might
compete with PU.1 for the binding partner c-Jun. These results are
consistent with a model in which c-Jun mediates its effects through
direct interactions with PU.1 and not by independent DNA binding to an
AP-1 site.
Phosphorylation of c-Jun by c-Jun NH2-terminal Kinase
(JNK) on Ser-63 and -73 Does Not Alter the Ability of c-Jun to Enhance
PU.1 Transactivation--
PU.1 (Fig.
3A) and c-Jun (Fig.
3B) are composed of a number of discrete domains. We used
deletion mutants to determine which domains of PU.1 and c-Jun are
critical for the activation of PU.1 by c-Jun in the context of the
M-CSF receptor promoter. The transactivation domain of PU.1 (amino
acids 1-118) was necessary for the activation by c-Jun (Fig.
3A). The transactivation domain of c-Jun (amino acids
1-87); the basic domain of c-Jun, which can physically interact with
PU.1 (44) and mediates DNA binding to AP-1 sites; and the leucine
zipper domain, which is responsible for homodimerization and
heterodimerization with c-Fos, were all necessary for the activation of
PU.1 by c-Jun (Fig. 3B). The transcriptional activity of
c-Jun is increased following phosphorylation on Ser-63 and -73 by JNK
(34, 46). To determine whether phosphorylation of Ser-63 and -73 was
important for the function of c-Jun as a transcriptional coactivator of
PU.1, we used c-Jun constructs in which these sites were mutated to Ala
residues. Surprisingly, we found no difference in the ability of wild
type c-Jun and S63A/S73A c-Jun to enhance PU.1 transactivation of the
M-CSF receptor promoter in F9 cells (Fig. 3B). Furthermore,
co-transfection with an activated allele of Ras, which has been
demonstrated to enhance the transcriptional activity of c-Jun via
phosphorylation by JNK (34, 46), did not enhance the coactivator
function of wild type or S63A/S73A c-Jun (Fig. 3C). These
data indicate that the coactivator function of c-Jun to enhance the
transcriptional activity of PU.1 is independent of JNK
phosphorylation.
Activated Ras Enhances the Transcriptional Activity of PU.1 by
Up-regulating c-Jun Expression--
Although Ras signal transduction
has been demonstrated to play an important role in myeloid
differentiation (47-50), it has not been shown whether Ras increases
the activity of the monocyte-specific M-CSF receptor promoter. However,
Ras is known to induce the expression of c-Jun (51). Therefore, we
asked the question of whether Ras could augment the transcriptional
activity of PU.1 and if this was mediated by enhancing c-Jun
expression. In fact, activated Ras(L61) enhanced the ability of PU.1 to
transactivate the M-CSF receptor promoter in CV-1 cells to a similar
degree as c-Jun (Fig. 4A).
Furthermore, a dominant negative c-Jun mutant lacking the basic domain,
which is required for physical interaction with PU.1 in
vitro (44), blocked the activation of PU.1 by Ras (Fig. 4A). In the same experiment, Ras did not change the protein
expression of transfected PU.1 (Fig. 4B) but up-regulated
endogenous c-Jun protein expression 4-fold (Fig. 4B). Since
the transfection efficiency in the CV-1 cells was 40% (Fig.
4B), we estimate that Ras up-regulated c-Jun expression
10-fold in transfected cells.
The c-Jun promoter contains important TPA response elements that
preferentially bind heterodimers of c-Jun and ATF-2, both of which are
activated upon phosphorylation by JNK (17-19). We therefore tested the
effect of another JNK activator, the MAP kinase kinase kinase MEKK1
(40), on the ability of PU.1 to transactivate the M-CSF receptor
promoter. In fact, MEKK1 enhanced the PU.1 transactivation function to
a similar level as Ras (Fig. 4A).
Ras is known to modulate the activity of the ETS domain transcription
factors ETS-1 and ETS-2 by phosphorylation (52). However, co-expression
of Ras did not alter the phosphorylation pattern of PU.1 in
vivo (Fig. 4C), and furthermore, Ras enhanced the
transcriptional activity of known phosphorylation site mutants of PU.1
(Ser-41, -45, and -148) (53, 54) similar to wild type PU.1 (data not shown). In conclusion, these data suggest a model in which Ras enhances
the transcriptional activity of PU.1 by increasing the expression of
its coactivator c-Jun.
The Expression of the Mutant Form of c-Jun That Lacks the Basic
Domain Completely Blocks TPA-induced M-CSF Receptor Promoter Activity
during Monocytic Differentiation--
We next asked the question of
whether the coactivator function of c-Jun could play a biological role
during monocytic differentiation in vivo. We first showed
that Ras enhanced the ability of PU.1 to transactivate the M-CSF
receptor promoter in myeloid U937 cells (Fig.
5A). U937 cells can be
differentiated to monocytic cells upon treatment with TPA, and during
this process c-Jun and M-CSF receptor mRNA expression increases
(25). TPA increased reporter gene expression from our M-CSF receptor
promoter construct (Fig. 5A) and also increased the
expression of endogenous M-CSF receptor (Fig. 5B). This
effect on the human monocytic M-CSF receptor promoter was blocked by
co-expression with a dominant negative c-Jun mutant lacking the basic
domain (Fig. 5A). These results indicate that c-Jun function
is required for the increase in M-CSF receptor promoter activity
observed in myeloid cells differentiated toward the monocytic lineage
with TPA.
The transcription factor c-Jun (20-24) and the regulation of the
M-CSF receptor by the transcription factor PU.1 (11, 12, 55) both play
important roles in the development of the monocytic lineage. Therefore,
we asked the question of whether c-Jun could promote the induction of
the M-CSF receptor by PU.1. Here we demonstrate that c-Jun enhances the
ability of PU.1 to transactivate the human monocyte-specific M-CSF
receptor promoter (Fig. 1, A and B).
These data explain how c-Jun, PU.1, and the M-CSF receptor might
collaborate as major factors in the process of monocytic differentiation. Moreover, c-Jun and PU.1 are already known to cooperate in the induction of other monocytic genes containing PU.1 and
AP-1 DNA binding sites, such as the macrosialin gene (56) and
macrophage scavenger receptor gene (57). The c-Jun enhancement of the
ability of PU.1 to transactivate the M-CSF receptor promoter is novel,
because the human monocyte-specific M-CSF receptor promoter contains no
AP-1 consensus binding sites. In contrast, the macrosialin promoter and
macrophage scavenger receptor promoter contain PU.1 and AP-1 binding
sites, which are critical for their monocyte-specific expression (56,
57). In fact, our data suggest a novel mechanism by which c-Jun can induce gene expression. c-Jun does not bind to the M-CSF receptor promoter (Fig. 2A) and, moreover, enhances the ability of
PU.1 to transactivate a minimal promoter driven by PU.1 sites alone (Fig. 1C). Furthermore, c-Jun physically binds to PU.1 (Fig.
2B). Since c-Jun binds to PU.1 and functionally activates
PU.1 without binding to the M-CSF receptor promoter DNA, we conclude
that c-Jun acts as a coactivator (45) of PU.1. This is the first report demonstrating a coactivator function for c-Jun.
Usually, c-Jun forms heterodimers with c-Fos in AP-1 transcription
factor complexes (17-19). However, c-Fos does not cooperate with c-Jun
in its coactivator function. In contrast, c-Fos completely blocks the
coactivation of PU.1 by c-Jun (Fig. 2C). Since c-Fos does
not physically bind to PU.1 (Fig. 2B), it might compete with PU.1 for the binding partner c-Jun. Since c-Fos blocks coactivation of
PU.1 by c-Jun, the requirement of the leucine zipper domain of c-Jun
for the activation of PU.1 (Fig. 3B) suggests that a c-Jun
homodimer or heterodimer with a non-c-Fos partner mediates activation
of the M-CSF receptor promoter. These results are consistent with a
model in which c-Jun mediates its effects through direct interactions
with PU.1 and not by independent DNA binding to an AP-1 site (Fig.
5C).
Usually, the transcriptional activity of c-Jun is increased following
phosphorylation on Ser-63 and -73 by JNK (34, 46). Furthermore,
the general coactivator CBP/p300 stimulates
c-Jun-dependent transcription, and the c-Jun residues
Ser-63 and -73 are required for CBP/p300 stimulation in vivo
and CBP/p300 binding in vitro (58). Surprisingly, we found
no difference in the ability of wild type c-Jun and the S63A/S73A c-Jun
mutant to enhance PU.1 transactivation of the M-CSF receptor promoter
(Fig. 3B). Furthermore, co-transfection with an activated
allele of Ras, which has been demonstrated to enhance the
transcriptional activity of c-Jun via phosphorylation by JNK (34, 46),
did not enhance the coactivator function of wild type or S63A/S73A
c-Jun (Fig. 3C). Our data indicate that the coactivator
function of c-Jun to enhance the transcriptional activity of PU.1 is
independent of JNK phosphorylation.
In accordance with its pivotal role in B cell development (5, 6), PU.1
binds to the B cell-specific immunoglobulin kappa (Ig How is the coactivator function of c-Jun regulated? Since the
coactivator function of c-Jun cannot be regulated via phosphorylation by JNK, the regulation of c-Jun expression might be crucial for the
capacity of c-Jun to coactivate PU.1 and induce monocytic differentiation. The Ras signal transduction pathway, for example, induces the expression of c-Jun (51). The Ras family of proteins are
GTP-dependent molecular switches that are essential for
cell growth and differentiation (61, 62). Ras exerts its effect on cell
growth mainly via ETS (63) and AP-1 (64) transcription factors. For
example, cells with a null mutation in the c-jun gene lack
many characteristics of Ras transformation (64), and dominant negative
mutants of ETS-1, ETS-2, or PU.1 with just the DNA binding domain
inhibit Ras activation of transcription and revert Ras-transformed
cells (63). In particular, Ras has been demonstrated to play an
important role in myeloid differentiation. Macrophage differentiation
and M-CSF-dependent survival are altered in transgenic mice
that express dominant suppressors of Ras signaling (47), while a number
of hematopoietic cell lines undergo spontaneous monocytic
differentiation in response to expression of activated Ras (48, 49). In
addition, M-CSF, granulocyte-macrophage colony-stimulating factor, or
interleukin-3-induced monocytopoiesis of CD34+ cells is inhibited by
N-Ras antisense oligonucleotides (50).
Although Ras signaling plays an important role in monocytic
differentiation, Ras has not previously been shown to increase activity
of the monocyte-specific M-CSF receptor promoter. Our data suggest that
Ras activates the M-CSF receptor promoter via c-Jun and PU.1, because
in c-Jun-deficient F9 cells there is no effect of Ras on PU.1 (Fig.
3C), whereas Ras enhances the ability of PU.1 to
transactivate the M-CSF receptor promoter in c-Jun-containing CV-1
cells (Fig. 4A). Moreover, a c-Jun mutant lacking the basic domain blocks the Ras enhancement of PU.1 transactivation of the M-CSF
receptor promoter (Fig. 4A). Ras signaling has been reported to induce the expression of c-Jun (51), and here we demonstrate that
Ras induces c-Jun expression in CV-1 cells during activation of PU.1
(Fig. 4B). Thus, these data suggest that Ras enhances the
transcriptional activity of PU.1 by up-regulating the expression of its
coactivator c-Jun. In accordance with this model, another inducer of
c-Jun expression, MEKK1 (40), enhanced the PU.1 transactivation function to a similar level as Ras or c-Jun (Fig. 4A).
Ras modulates the activity of ETS domain transcription factors such as
ETS-1 or ETS-2 by phosphorylation (52). Moreover, the ETS domain factor
PU.1, in particular, can be phosphorylated by casein kinase (53, 65) or
JNK in vitro (66), and the activity of PU.1 is known to be
regulated by phosphorylation (53, 54, 65). Phosphorylation of PU.1 at
Ser-148 is necessary for interaction with NF-EM5 (53) or stimulation by
lipopolysaccharide (65), and a PU.1 mutant at Ser-41 and -45 decreases
the M-CSF- or granulocyte-macrophage colony-stimulating
factor-dependent proliferation of bone marrow macrophages
(54). However, co-expression of activated Ras does not alter the
phosphoamino acid (data not shown) or phosphopeptide pattern (Fig.
4C) of PU.1 in vivo. Furthermore, Ras enhances
the transcriptional activity of known phosphorylation site mutants of
PU.1 (Ser-41, -45, and -148) (53, 54) similar to wild type PU.1 (data
not shown). Our data are in accordance with a report that the ability
of PU.1 to rescue macrophage development in PU.1 Finally, we determined whether Ras and c-Jun could regulate the M-CSF
receptor promoter during differentiation of myeloid cells. In fact, Ras
enhanced the ability of PU.1 to transactivate the M-CSF receptor
promoter in myeloid U937 cells (Fig. 5A). It has been shown
that U937 cells can be differentiated with TPA to monocytic cells, and
during this process c-Jun and M-CSF receptor mRNA expression
increases (25). TPA increased reporter gene expression from our M-CSF
receptor promoter construct (Fig. 5A) and also increased the
expression of endogenous M-CSF receptor (Fig. 5B). This
effect on the M-CSF receptor promoter is blocked by co-expression with
a dominant negative c-Jun mutant lacking the basic domain, which
interacts with PU.1 (Fig. 5A). These results indicate that
c-Jun function is required for the increase in M-CSF receptor promoter
activity observed in myeloid cells differentiated toward the monocytic
lineage with TPA.
In summary, our data indicate that c-Jun is a JNK-independent
coactivator of PU.1 (Fig. 5C). These results suggest a model in which growth factors or other signals (3, 68) activate the Ras
pathway, which in turn increases c-Jun expression in monocytic progenitors. Increased c-Jun expression activates PU.1, resulting in
increased M-CSF receptor up-regulation and survival, proliferation, and
differentiation of the monocytic lineage.
We thank Laura Smith for excellent
suggestions and helpful discussions; Richard Maki, Michael Klemsz, and
Marian Koshland for PU.1 expression vectors; Bruce Mayer for the vector
pEBG; Joseph Sodroski for pcDNA3/EGFP; Jianmin Tian, Michael
Karin, Elisabetta Mueller, and Bruce Spiegelman for c-Jun and c-Fos
expression vectors; Larry Feig for Ras expression vectors; and John
Blenis for 293T cells.
*
This work was supported by National Institutes of Health
Grants CA41456 and P01 CA72009 (to D. G. T.) and Medical Research Council of Canada Grant MT-9734 (to T. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Recipient of a fellowship from the Deutsche Forschungsgemeinschaft.
§§
An investigator of the Howard Hughes Medical Institute.
¶¶
To whom correspondence should be addressed: Harvard
Institutes of Medicine, Room 954, 77 Ave. Louis Pasteur, Boston, MA
02115. Tel.: 617-667-5561; Fax: 617-667-3299; E-mail:
dtenen{at}bidmc.harvard.edu.
||
Senior Scientist of the Fonds de la recherche en
sante du Quebec.
The abbreviations used are:
M-CSF, macrophage
colony-stimulating factor;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
bp, base pair(s);
GST, glutathione S-transferase;
TK, thymidine kinase.
c-Jun Is a JNK-independent Coactivator of the PU.1 Transcription
Factor*
§,
,,
,¶¶
Hematology/Oncology and
Signal Transduction, Beth Israel Deaconess Medical Center and
Harvard Medical School, Boston, Massachusetts 02115, the
¶ Department of Biochemistry and Molecular Biology, University of
Massachusetts Medical School, Worcester, Massachusetts 01605, and the
** Clinical Research Institute of Montreal, Montreal, Quebec
H2W1R7, Canada
ABSTRACT
Top
Abstract
Introduction
References
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
7 M TPA (Sigma) (stock solution:
1 × 103 M in Me2SO) or
vehicle only.
88 to +71
with respect to the major monocytic transcription start site (9, 26)
was subcloned in the firefly luciferase vector pXP2 (28). pTK with PU.1
sites is a dimer of both PU.1 sites from the granulocyte
colony-stimulating factor receptor promoter from bp +28 to +54 (29)
subcloned into pTK81luc, a pXP2-based luciferase construct with a TATA
box only as a minimal promoter (28). pTK with mutated PU.1 sites is a
dimer of both mutated PU.1 sites from the granulocyte
colony-stimulating factor receptor promoter from bp +28 to +54
(primers: 5'-TCG AGT GGT TTC ACA AAC TTT TGT TGA CGA GAG-3' and 5'-TCG
ACT CTC GTC AAC AAA AGT TTG TGA AAC CAC-3') subcloned into pTK81luc and
was constructed as described for pTK with PU.1 sites (29). As an
internal control plasmid for co-transfection assays, the pRL-null
construct driving a Renilla luciferase gene (Promega,
Madison, WI) was used (30).
1-133, pcDNA1-PU.1/1-100,
and pcDNA1-PU.1/1-70 (31) were kindly provided by Marian
Koshland (University of California, Berkeley, CA). The PU.1
deletion mutants pECE-PU.1/119-160 and pECE-PU.1/8-32 and PU.1
serine to alanine mutants pECE-PU.1-S41A/S45A and pECE-PU.1-S148A (32)
were a gift from Richard Maki (the Burnham Institute, La Jolla, CA and Neurocrine Biosciences, San Diego, CA) and Michael Klemsz (Indiana University Medical Center, Indianapolis, IN). pEBG, a mammalian GST
expression vector using the strong constitutive EF1
promoter, was
kindly provided by Bruce Mayer (Harvard Medical School, Boston, MA). In
order to subclone PU.1 into pEBG, the PvuI/EcoRI
fragment of murine pECE-PU.1 (2), a gift from Michael Klemsz and
Richard Maki, was first subcloned into the
SmaI/EcoRI-cut vector pBluescript KS+/
(Stratagene, La Jolla, CA). Then the NaeI/NotI
fragment of pBS-PU.1 was subcloned into the
PmlI/NotI fragment of pEBG. The bacterial GST
expression vector pGEX-2TK-PU.1 has been described previously (33).
1-87,
pUC18-c-Jun/6-199, pSV-SPORT1-c-Jun/LZ lacking amino acids
281-313, and pSV-SPORT1-c-Jun/BD lacking amino acids 251-276 were
described previously (36). pSP65-c-Jun and pSP65-c-Fos (37) were a gift
from Elisabetta Mueller and Bruce Spiegelman (Dana Farber Cancer
Institute, Boston, MA). Human activated pMT3-Ha-Ras(L61) (38, 39) and
inactive pMT3-Ha-Ras(N17) (38, 39) were kindly provided by Larry Feig
(Tufts University, Boston, MA). pcDNA3-Flag-MEKK1 was constructed
by subcloning Flag-MEKK1 (40) into the EcoRI and
EcoRV sites of pcDNA3 (Invitrogen, Carlsbad, CA).
pcDNA3/EGFP (enhanced green fluorescence protein) was kindly
provided by Joseph Sodroski (Dana Farber Cancer Center Institute,
Boston, MA).
-32P]dCTP (NEN Life Science Products) and incubated
with 0.1 µg/µl of double-stranded poly(dI-dC) (Sigma) with 1 µl
of in vitro translated c-Jun or c-Fos. In some experiments,
a 100-fold molar excess of the AP-1 probe was added as specific
unlabeled competitor. Similarly, a double-stranded M-CSF receptor
promoter oligonucleotide extending from position bp 88 to +71 with
respect to the major transcription start site (9, 26) was
Klenow-labeled with [-32P]dCTP and incubated with 0.1 µg/µl double-stranded poly(dI-dC) with 1 µl of in
vitro translated PU.1, c-Jun, or c-Fos. In some experiments, a
100-fold molar excess of specific unlabeled competitor was added: an
oligonucleotide with the PU.1 DNA binding site in the CD11b promoter
(41), 3'-AGC CTA CTT CTC CTT TTC TGC CCT TCT TTG-5', to compete for
PU.1, and the AP-1 probe described above to compete for c-Jun.
-tubulin antibody as an
internal control (catalog no. 1111876; Boehringer Mannheim) for 60 min and then with Protein A-horseradish peroxidase conjugate (Amersham, Buckinghamshire, United Kingdom) for 45 min. For U937 cells, an anti-M-CSF receptor antibody (catalog no. SC-692, Santa Cruz
Biotechnology) was used. Signals were detected with ECL Western
blotting detection reagents (Amersham). In parallel plates, the M-CSF
receptor promoter construct was co-transfected, and luciferase
activities were determined to ensure that Ras enhances the
transactivation function of PU.1 in the particular experiment used for
Western blot analysis of c-Jun expression or PU.1 expression and that
the transfection efficacy was the same (less than 10% difference
between plates) in the particular experiment. Differences in protein
expression were quantitated by ImageQuant software (Molecular Dynamics).
RESULTS
View larger version (19K):
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Fig. 1.
Effect of c-Jun on the ability of PU.1 to
transactivate the M-CSF receptor promoter and a minimal TK promoter
containing only PU.1 DNA binding sites. A, CV-1 cells
were transfected with the human monocyte-specific M-CSF receptor
promoter or the promoterless vector pXP2 and with the expression
plasmids pECE-PU.1 and pSV-SPORT1-c-Jun. Luciferase activities were
determined 24 h after transient transfection with LipofectAMINE
Plus and normalized to the activities of the internal control plasmid
pRL-null. B, c-Jun-deficient F9 cells were transfected as
described for Fig. 1A. C, F9 cells were
transfected with a minimal TK promoter with PU.1 sites or a minimal TK
promoter with mutated PU.1 sites and with the expression plasmids for
PU.1 and c-Jun.
88 to +71 with
respect to the major monocytic transcription start site (9, 26) (Fig.
1, A and B) or in the TK promoter containing PU.1
sites (Fig. 1 C), our data suggested that c-Jun augmentation of PU.1
transactivation was not mediated by DNA binding of c-Jun. In order to
formally exclude DNA binding by c-Jun, we performed an electrophoretic mobility shift assay using a bp 88 to +71 M-CSF receptor promoter fragment or a bp 62 to 29 oligonucleotide containing the PU.1 binding site of the M-CSF receptor promoter (9). In vitro
translated c-Jun or a mixture of c-Jun and c-Fos specifically bound to
a double-stranded AP-1 oligonucleotide probe from the collagenase promoter (Fig. 2A), while no
specific binding was observed using the double-stranded bp 88 to +71
M-CSF receptor promoter (Fig. 2A) or bp 62 to 29
oligonucleotide containing the PU.1 binding site of the M-CSF receptor
promoter (data not shown). In control experiments, in vitro
translated PU.1 was shown to bind strongly and specifically to the same
M-CSF receptor promoter fragments (Fig. 2A). These results
indicate that binding of c-Jun to the M-CSF receptor promoter DNA is
not required to mediate its activating effect on PU.1.
View larger version (25K):
[in a new window]
Fig. 2.
Physical interactions among c-Jun, PU.1, and
the M-CSF receptor promoter and effect of c-Fos on the coactivation of
PU.1 by c-Jun. A, in lanes 1-6,
an AP-1 probe from the collagenase promoter was used as a positive
control for an electrophoretic mobility shift assay, and in
lanes 7-14, an M-CSF receptor promoter
oligonucleotide extending from position bp 88 to +71 with respect to
the major transcription start site was used. Probes were incubated with
no added protein, 1 µl of in vitro translated c-Jun, 1 µl of c-Fos, or both. In lanes 3, 6,
11, and 14, a 100-fold molar excess of
self-unlabeled competitor (AP-1 probe) was added to the electrophoretic
mobility shift assay. In lanes 8 and
9, 1 µl of in vitro translated PU.1 was added,
and in lane 9, a 100-fold excess of
oligonucleotide from the PU.1 binding site in the CD11b promoter was
added. B, for the protein interaction assay,
[35S]Met-labeled in vitro translated c-Jun
(top) or c-Fos (bottom) were incubated with 1 µg of bacterially expressed GST-PU.1 (lane 3)
or equivalent amounts of GST protein plus glutathione-agarose beads
(lane 2). GST-PU.1 was recovered using
glutathione-agarose beads and separated by SDS-polyacrylamide gel
electrophoresis prior to autoradiography.
[35S]Met-labeled in vitro translated c-Jun or
c-Fos were run directly in the first lane.
C, F9 cells were transfected with the human
monocyte-specific M-CSF receptor promoter or the promoterless vector
pXP2 and with the expression plasmids pECE-PU.1, pSV-SPORT1-c-Jun, and
pSV40-c-Fos. Luciferase activities were determined 24 h after
transient transfection with LipofectAMINE Plus.
View larger version (19K):
[in a new window]
Fig. 3.
Domains of PU.1 and c-Jun relevant for
coactivation of PU.1 by c-Jun. A, activation of the
M-CSF receptor promoter by PU.1 or different PU.1 deletion mutants
either with or without c-Jun in CV-1 cells as described in Fig.
1A. Also shown is a schematic representation of the
transcription factor PU.1 with transactivation domain (amino acids
1-118), PEST domain (amino acids 118-160), and the ETS DNA binding
domain (amino acids 161-255). B, activation of the M-CSF
receptor promoter by c-Jun or different c-Jun deletion or point mutants
either with or without PU.1 in F9 cells as described in the legend to
Fig. 1B. Also shown is a schematic representation of c-Jun
mutants, depicting the transactivation domain (amino acids 1-251),
basic domain (BD, amino acids 251-281), and the leucine
zipper (LZ, amino acids 281-313). C, activation
of the M-CSF receptor promoter by PU.1, c-Jun, c-Jun mutated in the JNK
phosphorylation sites (Ser-63 and -73), and activated pMT3-Ras(L61) in
F9 cells as described in the legend to Fig. 1B.
View larger version (36K):
[in a new window]
Fig. 4.
Effect of Ras on the transactivation
capacity of PU.1, on c-Jun expression, and PU.1 phosphorylation
pattern. A, activation of the M-CSF receptor promoter
by PU.1, Ras(L61), a c-Jun mutant lacking the basic domain, wild type
c-Jun, and MEKK1 in CV-1 cells as described in Fig. 1A.
B, CV-1 cells were transfected with the M-CSF receptor
promoter, PU.1, and/or activated Ras(L61), and Western blotting was
performed. To determine transfection efficiency, CV-1 cells were
co-transfected with pcDNA3/EGFP, and the percentage of transfected
cells was determined by fluorescence microscopy. C,
pEBG-PU.1 was transfected into 293T cells using LipofectAMINE
Plus either with inactive Ras(N17) (left) or activated
Ras(L61) (middle), and phosphopeptide mapping of PU.1 was
performed. The right part shows a mixture of the
left and middle samples.
View larger version (20K):
[in a new window]
Fig. 5.
Role of c-Jun in TPA-induced M-CSF receptor
expression and monocytic differentiation. A, shown is
luciferase activity directed from the human monocyte-specific M-CSF
receptor promoter or promoterless vector pXP2 following the addition of
no plasmid, Ras(L61), PU.1, c-Jun basic domain deletion mutant, TPA, or
combinations as indicated. B, Western blot analysis for
M-CSF receptor expression of the experiment shown in Fig.
5A. C, model of c-Jun as a JNK-independent
coactivator of PU.1.
DISCUSSION
) 3' enhancer
and can control transcriptional activity (59). The immunoglobulin 3'
enhancer is activated by PU.1, c-Jun, PIP, and c-Fos and contains
respective DNA binding sites for these factors (60). In this context,
mutants of PU.1 that lack the transcriptional activation domain are as
efficient at stimulating enhancer activity as the wild-type PU.1
protein (60). In contrast, the transactivation domain of PU.1 (amino
acids 1-118) is necessary for the basal transactivation of the M-CSF
receptor promoter by PU.1 (Fig. 3A), and c-Jun cannot exert
its coactivator function on PU.1 mutants that lack the transactivation
domain (Fig. 3A). These data suggest that the function of
different PU.1 domains might vary depending on the cell type (monocyte
versus B cell) and on the cooperating proteins (c-Jun as
part of the AP-1 transcription factor complex versus c-Jun
as a JNK independent coactivator).
/ ES cells is not
impaired by these same phosphorylation mutants (67). In conclusion,
these results suggest a model in which Ras enhances the transcriptional
activity of PU.1 by increasing the expression of its coactivator c-Jun
without modifying the activity of PU.1 by phosphorylation.
ACKNOWLEDGEMENTS
FOOTNOTES
A Leukemia Society of America Scholar.
ABBREVIATIONS
REFERENCES
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Abstract
Introduction
References
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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G. Behre, S. M. Singh, H. Liu, L. T. Bortolin, M. Christopeit, H. S. Radomska, J. Rangatia, W. Hiddemann, A. D. Friedman, and D. G. Tenen Ras Signaling Enhances the Activity of C/EBPalpha to Induce Granulocytic Differentiation by Phosphorylation of Serine 248 J. Biol. Chem., July 12, 2002; 277(29): 26293 - 26299. [Abstract] [Full Text] [PDF]
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V. A. Reddy, A. Iwama, G. Iotzova, M. Schulz, A. Elsasser, R. K. Vangala, D. G. Tenen, W. Hiddemann, and G. Behre Granulocyte inducer C/EBPalpha inactivates the myeloid master regulator PU.1: possible role in lineage commitment decisions Blood, June 28, 2002; 100(2): 483 - 490. [Abstract] [Full Text] [PDF]
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A. Iwama, M. Osawa, R. Hirasawa, N. Uchiyama, S. Kaneko, M. Onodera, K. Shibuya, A. Shibuya, C. Vinson, D. G. Tenen, et al. Reciprocal Roles for CCAAT/Enhancer Binding Protein (C/EBP) and PU.1 Transcription Factors in Langerhans Cell Commitment J. Exp. Med., February 25, 2002; 195(5): 547 - 558. [Abstract] [Full Text] [PDF]
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A. Alfranca, M. D. Gutierrez, A. Vara, J. Aragones, F. Vidal, and M. O. Landazuri c-Jun and Hypoxia-Inducible Factor 1 Functionally Cooperate in Hypoxia-Induced Gene Transcription Mol. Cell. Biol., January 1, 2002; 22(1): 12 - 22. [Abstract] [Full Text] [PDF]
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K. A. Gauss, P. L. Bunger, and M. T. Quinn AP-1 is essential for p67phox promoter activity J. Leukoc. Biol., January 1, 2002; 71(1): 163 - 172. [Abstract] [Full Text] [PDF]
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M. K. Anderson, X. Sun, A. L. Miracle, G. W. Litman, and E. V. Rothenberg Evolution of hematopoiesis: Three members of the PU.1 transcription factor family in a cartilaginous fish, Raja eglanteria PNAS, January 5, 2001; (2001) 21478998. [Abstract] [Full Text]
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I.-H. Oh, A. Lau, and C. J. Eaves During ontogeny primitive (CD34+CD38-) hematopoietic cells show altered expression of a subset of genes associated with early cytokine and differentiation responses of their adult counterparts Blood, December 15, 2000; 96(13): 4160 - 4168. [Abstract] [Full Text] [PDF]
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P. Zhang, X. Zhang, A. Iwama, C. Yu, K. A. Smith, B. U. Mueller, S. Narravula, B. E. Torbett, S. H. Orkin, and D. G. Tenen PU.1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding Blood, October 15, 2000; 96(8): 2641 - 2648. [Abstract] [Full Text] [PDF]
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I. Matsumura, A. Kawasaki, H. Tanaka, J. Sonoyama, S. Ezoe, N. Minegishi, K. Nakajima, M. Yamamoto, and Y. Kanakura Biologic significance of GATA-1 activities in Ras-mediated megakaryocytic differentiation of hematopoietic cell lines Blood, October 1, 2000; 96(7): 2440 - 2450. [Abstract] [Full Text] [PDF]
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 M. Takeda, H. Kato, A. Takamiya, A. Yoshida, and H. Kiyama Injury-Specific Expression of Activating Transcription Factor-3 in Retinal Ganglion Cells and Its Colocalized Expression with Phosphorylated c-Jun Invest. Ophthalmol. Vis. Sci., August 1, 2000; 41(9): 2412 - 2421. [Abstract] [Full Text]
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M. P. Coghlan, M. M. Chou, and C. L. Carpenter Atypical Protein Kinases Clambda and -zeta Associate with the GTP-Binding Protein Cdc42 and Mediate Stress Fiber Loss Mol. Cell. Biol., April 15, 2000; 20(8): 2880 - 2889. [Abstract] [Full Text]
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C. Nerlov, E. Querfurth, H. Kulessa, and T. Graf GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1-dependent transcription Blood, April 15, 2000; 95(8): 2543 - 2551. [Abstract] [Full Text] [PDF]
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S. Maitra and M. Atchison BSAP Can Repress Enhancer Activity by Targeting PU.1 Function Mol. Cell. Biol., March 15, 2000; 20(6): 1911 - 1922. [Abstract] [Full Text]
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N. Wara-aswapati, Z. Yang, W. R. Waterman, Y. Koyama, S. Tetradis, B. K. Choy, A. C. Webb, and P. E. Auron Cytomegalovirus IE2 Protein Stimulates Interleukin 1beta Gene Transcription via Tethering to Spi-1/PU.1 Mol. Cell. Biol., October 1, 1999; 19(10): 6803 - 6814. [Abstract] [Full Text] [PDF]
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P. Zhang, G. Behre, J. Pan, A. Iwama, N. Wara-aswapati, H. S. Radomska, P. E. Auron, D. G. Tenen, and Z. Sun Negative cross-talk between hematopoietic regulators: GATA proteins repress PU.1 PNAS, July 20, 1999; 96(15): 8705 - 8710. [Abstract] [Full Text] [PDF]
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X. Wang, E. Scott, C. L. Sawyers, and A. D. Friedman C/EBPalpha Bypasses Granulocyte Colony-Stimulating Factor Signals to Rapidly Induce PU.1 Gene Expression, Stimulate Granulocytic Differentiation, and Limit Proliferation in 32D cl3 Myeloblasts Blood, July 15, 1999; 94(2): 560 - 571. [Abstract] [Full Text] [PDF]
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Z. Yang, N. Wara-aswapati, C. Chen, J. Tsukada, and P. E. Auron NF-IL6 (C/EBPbeta ) Vigorously Activates il1b Gene Expression via a Spi-1 (PU.1) Protein-Protein Tether J. Biol. Chem., July 7, 2000; 275(28): 21272 - 21277. [Abstract] [Full Text] [PDF]
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A. Verger, E. Buisine, S. Carrere, R. Wintjens, A. Flourens, J. Coll, D. Stehelin, and M. Duterque-Coquillaud Identification of Amino Acid Residues in the ETS Transcription Factor Erg That Mediate Erg-Jun/Fos-DNA Ternary Complex Formation J. Biol. Chem., May 11, 2001; 276(20): 17181 - 17189. [Abstract] [Full Text] [PDF]
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M. K. Anderson, X. Sun, A. L. Miracle, G. W. Litman, and E. V. Rothenberg Evolution of hematopoiesis: Three members of the PU.1 transcription factor family in a cartilaginous fish, Raja eglanteria PNAS, January 16, 2001; 98(2): 553 - 558. [Abstract] [Full Text] [PDF]
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