Binding of ETS Family Members Is Important for the Function of the c

Vol. 9, 523-534,
July
Cell Growth & Differentiation
1998
523
Binding of ETS Family Members
Is Important
for the Function
of the c-sis/Platelet-derived
Growth Factor-B
TATA
Neighboring
Sequence
in I 2-O-Tetradecanoylphorbol-1
3acetate-treated
K562 Cells1
Gregory
William
C. Kujoth,2
Donald
F. Robinson,
and
E. Fahl3
McArdle Laboratory
for Cancer Research,
Madison,
53706
Wisconsin
University
of Wisconsin,
Abstract
The c-sis/platelet-derived
growth factor (PDGF)-B
TATA neighboring
sequence
(TNS) is a promoter
element that is required for the full induction of this
gene in K562 erythroleukemia
cells undergoing
12-0tetradecanoylphorbol-13-acetate-mediated
megakaryoblastic
differentiation.
Nuclear factors from
K562 cells can bind to the c-s1sIPDGF-B
TNS,
generating
four complexes
in electrophoretic
mobility
shift
assays.
One of these complexes
was previously
shown to contain Sp family members.
In this work, we
provide evidence
implicating
two of the remaining
complexes
as belonging
to the ETS family of
transcription
factors.
This includes the identification
of
a novel constitutive
TNS-binding
complex containing
the ETS family
member
ELK-I. The binding of both
ETS-like
complexes
was disrupted
by mutations
in a
central CCGGAA core within the TNS and, for one of
the complexes,
could be promoted
by bringing the
sequences
flanking
the core closer to a consensus
ETS binding site. The molecular
weights of these TNSbinding factors were estimated
by UV cross-linking
analysis and found to be consistent
with those of
several ETS family transcription
factors,
including
ELKI. A consensus
ELK-I binding site could compete for
the binding of both putative ETS-like factors, and the
novel complex could be disrupted
by the addition of an
antibody
raised
against ELK-I
Transient
transfection
analysis using mutant TNS promoter-reporter
constructs
demonstrated
a strong
correlation
between
the binding of the ETS-like factors and the
transcriptional
activity
of the TNS. In contrast,
.
mutations
that prevented
the binding of Sp family
transcription
factors
had no effect on promoter
activity.
Thus, ETS family members,
such as ELK-I
are
not only capable of binding to the TNS but seem to be
necessary
for the function of this element in
differentiating
K562 cells.
,
Introduction
The ETS family of transcription
family of 30 or more members
,
as sponges, fruit flies, and mammals
(1 2). All family members share a conserved
-85-amino
acid DNA-binding
domain,
termed
the ETS domain,
that directs binding to recognition
sites
containing
GGA
cores;
sequences
flanking
this
trinucleotide
core contribute
to the binding
specificity
of
individual family members (reviewed in Refs. 3-5). ETS proteins
are important
mediators
of growth-regulating
and
dif-
ETS proteins
are effectors
of growth factor and phorbol ester signaling
pathways involving RAS/MAPK4-dependent
signaling events
(6-1 0). Not surprisingly,
some
ETS members
have trans-
ferentiation
forming
signaling
activity
pathways.
In particular,
(1 1-1 3), and this
is also
true of the E26 avian
retrovirus that has transduced
its v-ets oncogene
from the
cellular c-ets-1 gene (1 4-1 7). The ETS genes PU. 1/Spi-i and
Fll-1 are activated
by proviral insertion in erythroid
tumors
induced by spleen focus-forming
retrovirus
and Friend murime
leukemia
virus,
respectively
(1 8-20).
Chromosomal
translocations
involving members of the ERG/Fll-i
of ETS genes are frequently
seen in various forms
tumors
(21-27).
These
translocations
can
subgroup
of Ewing’s
produce
fusion
proteins that bind to ETS recognition
sites and display increased
transcriptional-activating
abilities
relative to their
wild-type
ETS counterparts
(28-30).
PDGF is the major mitogen in human serum for cells of
mesenchymal
origin and is composed
of two subunits,
PDGF-A and PDGF-B. These subunits
can form homo- or
heterodimers
and bind to cell surface PDGF receptors
to
elicit their biological
effects, such as chemotaxis
and mitogenesis (31-33). The PDGF-B gene is the cellular homologue
of the
Received
1/29/98;
revised 5/14/98;
accepted
5/20/98.
The costs of publication
of this article
were defrayed
in part by the
payment
of page charges.
This article must therefore
be hereby marked
advertisement
in accordance
with 1 8 U.S.C. Section
1 734 solely to mdi-
factors
is a large and growing
found in organisms
as diverse
(34-36),
v-sis
oncogene
from
the
simian
and both v-sis and c-sis/PDGF-B
of transforming
cells
in experimental
sarcoma
retrovirus
genes are capable
systems
(37-40).
Al-
cate this fact.
1 Supported
by American
Cancer
Society
Grant BE-268
and National
Cancer Institute
Grant CAO71 75.
2 Present
address:
Department
of Medical
Genetics,
University
of Wisconsin, Madison,
WI 53706.
3 To whom
requests
for reprints should be addressed,
at McArdle
Laboratory for Cancer
Research,
University
of Wisconsin,
1400 University
Avenue, Madison,
WI 53706. Phone: (608) 262-1275;
Fax: (608)262-2824;
E-mail: [email protected].
4 The abbreviations
used are: MAPK, mitogen-activated
protein kinase;
Ab, antibody; BrdUrd, 5’-bromodeoxyuridine;
CBP, cAMP-responsive
ek
ement
binding
protein-binding
protein;
EGR-1 , early growth
response
gene 1 ; EMSA, electrophoretic
mobility
shift assay; PDGF, platelet-derived growth
factor; SPE, sis proximal
element;
TNS, TATA neighboring
sequence;
WA, 12-O-tetradecanoylphorbol-i
3-acetate.
524
ETS Factors
Bind
TNS
the PDGF-B
though the expression
of c-sis/PDGF-B
is normally restricted
to a limited number
of cell types, PDGF-B
expression
is
commonplace
in many human tumor cells (41). The mechanisms
by which
c-sis/PDGF-B
gene expression
can be
turned on in tumor cells are therefore
of interest. Although
the expression
of c-sis/PDGF-B
can be regulated at multiple
levels, transcriptional
regulation
has been found to be a
major means of controlling
PDGF-B production
in many cell
types (42).
One model system that has been used for studying c-sis/
PDGF-B transcriptional
regulation
is that of K562 erythroleukemia cells undergoing
TPA-mediated
megakaryoblastic
differentiation
(43). The
steady-state
expression
level
of c-sis/
novel TNS-binding
complex is also observed and revealed to
transcription
factor ELK-i
In addition,
the previously
reported
band 2 complex
is implicated
as
belonging
to the ETS family, although
its specific identity is
not yet known. Importantly,
the binding of ELK-i and of the
band 2 complex
to mutant TNSs is shown to strongly correlate with the transcriptional
activity of these sequences
when placed into promoter-reporter
constructs.
Thus, we
propose that ETS family transcription
factors
are the critical
TNS-binding
components
necessary
for full induction
of the
c-sis/PDGF-B
promoter
in differentiating
K562 cells.
the ETS family
contain
.
Results
PDGF-B mRNA is induced in differentiating
K562 cells by at
least 200-fold,
and much of this up-regulation
takes place at
the transcriptional
level (44, 45). Studies of the c-sis/PDGF-B
promoter
in TPA-treated
K562 cells have indicated
that two
Identification
of an ETS-like
CCGGAA
Core within the
TNS. The TNS identified in our previous study (47) is at least
1 0 bp in length and is bound by several unidentified
nuclear
regulatory
the TNS that are necessary
for nuclear factor binding, we
constructed
high-resolution
TNS mutants.
These i-3-bp
substitution
mutants (Mi-M7)
are shown in Fig. iA. Doublestranded
DNAS containing
these mutations
were tested for
nuclear factor binding by EMSA, using new preparations
of
elements,
the
SPE and
the TNS,
control
the
pro-
moter’s response to TPA (46, 47). The SPE is also critical for
the basal expression
of c-sisIPDGF-B
in a number of cell
types (48-51),
as well as for c-sis/PDGF-B
expression
induced by thrombin
(52) or vascular injury in endothelial
cells
(53) and by human T-cell leukemia virus I Tax in Jurkat T cells
(54). The SPE contains
a CCACCC
core to which multiple
cellular transcription
factors can bind. This includes EGR-1
(53-55)
as well
as members
of the
Sp family,
such
as Spi
and Sp3 (49, 53-57).
Simultaneous
binding
of Spi
and
EGR-1 to the SPE does not seem to occur; rather, it seems
that Spi occupies
the SPE in unstimulated
cells and is
replaced
by EGR-1 on induction
or injury (53). The SPE is a
lower-affinity
binding site than a canonical
EGR-1 recognition sequence.5 Therefore,
it is possible that a threshold level
of EGR-1 protein must be reached before Spi is efficiently
displaced
off the SPE. In any event, interplay between Spi
and EGR-1 on overlapping
binding sites is an increasingly
common
mechanism
for regulating
inducible
gene expression (53, 58-60).
The second regulatory element, the TNS, consists of 10 bp
located immediately
downstream
of the c-sis/PDGF-BTATA
box and is necessary
for the full induction
of the c-sis/
PDGF-B
promoter
in differentiating
K562 cells (47). The TNS
acts synergistically
with the SPE to mediate
induction
in
TPA-treated
K562 cells, although the mechanism
underlying
this cooperation
is not yet known. Three specific complexes
representing
nuclear factor binding to the TNS have been
demonstrated
using
the EMSA
(47). One of these
complexes
is comprised
of Sp family members,
predominantly
Spi and
Sp3 (47). The remaining
complexes
are as yet unidentified.
Furthermore,
the relative importance
of these TNS-binding
complexes
with respect
to TNS function
is unresolved.
Therefore,
we have decided to pursue further characterization of TNS-binding
components.
In this work, high-resolution substitution
mutations
of the TNS have been analyzed
for their effects on EMSA band formation
and transcriptional
activity.
UV cross-linking
analysis has been used to estimate
the molecular
weights of selected TNS-binding
proteins.
A
5
D. F. Robinson
and W. E. FahI, unpublished
observation.
factors
in EMSAs.
nuclear
extract.
To determine
In these
the
core
experiments,
sequences
within
complexes
corre-
sponding to previously
reported bands 1-3 were again seen
on the wild-type
TNS probe (Fig. 1 B, Lane 1). Band 2 appeared as a doublet containing
a thin upper band (band 2a)
and a thicker lower band (band 2b). However,
both bands
cosegregated
in all subsequent
experiments
and
are there-
fore referred to as band 2 for the sake of simplicity.
An
additional
complex was noted that had not been observed
in
earlier experiments,
and this was designated
as band 4 (Fig.
1B, Lane 1). Band 4 appeared
in at least three independent
preparations
of nuclear extracts
(data not shown) and was
specific for the TNS because it could be competed
by wildtype TNS (Fig. iB, Lane 2) and Ji6 competitors,
but not by
the Ji 7 sequence
(Fig. 2, Lanes 1-3). Consistent
with our
earlier work, no differences
in any of these EMSA complexes
were observed
between TPA-treated
and untreated
nuclear
extracts. Additional
bands present below band 4 were cornpeted similarly and poorly by all of the competitors
and were
deemed nonspecific.
Using the TNS substitution
mutants Mi-M7
as cornpetitors in the EMSA revealed a striking result. Mutants M4 and
M5 failed to compete for band 2 and band 4 (Fig. 1B, Lanes
6 and
All of the remaining mutants competed
well for both
of these complexes,
although
M3 did so slightly less efficiently than did the wild-type
TNS (Fig. 1B, Lane 5). Interestingly, M4 and M5 alter a GGM
sequence
that is present
in the TNS. Members
of the ETS family of transcription
factors bind to sites containing
GGAA core sequences;
hence,
this result suggested
that band 2 and band 4 might contain
ETS family members.
Consistent
with this possibility,
a CC
7).
dinucleotide
is present
immediately
upstream
of the
GGM
core in many ETS binding sites (see Table 1), and the TNS
contains this feature.
Band 1 was previously
shown to contain members
of the
Sp family of transcription
factors (47). Consistent
with this
observation,
mutants
competition
for band
M2,
M4,
and
1 (Fig. iB, Lanes
M6
showed
impaired
4, 6, and 8). The Sp
Cell Growth
probe)
Competitor
& Differentiation
525
(TNS
A
TNS(WT)
ccc
Ml
Probe
..i1-i:
M2
w
‘
.0
(rJ
c
N-
TT
M3
AA
M4
TT
cc
MS
Band1/Sp-#{248}.*-.
M6
Band
Band
AC
M7
M8
T
.
2..._.
!I:
3--
Band4
T
J16
J17
!i
AACGATCGAT
AACGATGCAT
B
Cl-i
Competitor:
-
--
Band
l/Sp-...
....,.
Band
I
Band
3
Band
4
-
Fig. 2.
ns
EMSA
analysis
using
-
The
3
2
4
5
6
7
8
9
10
Thisprobe
Fig. 1. EMSA analysis
using mutant TNS competitor
sequences.
A, sequences
of the wild-type
TNS probe corresponding
to the -34 to -8
region of the c-sis/PDGF-B
promoter
and sequences
of the TNS mutants,
Mi-M8.
The TNS is shaded, and dashes indicate wild-type
sequence.
J16
and J17 contain
linker-scanning
mutations
of the TATA box and INS,
respectively.
B, EMSA using nuclear extracts
from untreated
K562 cells
and a radiolabeled
wild-type
INS probe as described
in “Materials
and
Methods.”
Unlabeled
competitor
sequences
used at a 100-fold
molar
excess are as indicated
in A. Bands 1-4 discussed
in the text are mdicated by arrows. ns, nonspecific
complexes.
binding
site
within
these
on
impaired
1B,
3), despite
M4, and
has a high G/C
M6 mutations
all change
the TNS, the detrimental
band
Somewhat
markedly
Lane
(5’-GGGGCGGGGC)
the M2,
G or C residues
mutations
1 complex
surprisingly,
in its ability
formation
the
Ml
to compete
its addition
effects
were
mutant
was
for band
of a string
9), whereas
1B, Lane
more
7).
9
for band
M5,
which
efficiently
1 (Fig.
not
also
1 (Fig.
of cytosines
2, Lane
2). The
M3
for band 1 to roughly
the same
TNS competitor
(Fig. 1B, Lanes 5
makes
than
did
the TNS
the
more
wild-type
probes.
untreated
INS
more
competed
10
EMSA
K562
11
12
analysis
cells
and
competitors
difficult
well
for band
complexes
petitors.
slightly
The
from
difference
M4,
and
for band
was
radiola-
M5 were
migration
that of
18, Lanes 5
1 B, Lane 8).
to at least
substitution
the
with
artifactual
mutants
even
4 complexes
GAA ETS-like
the M6 probe
more
did
2, Lanes
(indicating
bands
The
2 and
were
marked
not
6-8).
partial
form
also
on the
Mutants
than
4 was
Ml , M2,
4 at
did the wild-type
bound
similarly
when
M4,
alter
and
levels
M7 mutant
similar
M7
EMSA
probes
and
M5
the CCG-
bases
bound
probes
to those
but
in
2 and
of
site
imrneband 2
INS
probe
(Fig. 2,
by both M6 and INS
seen
M3,
paired
band
M3,
of purine-pyrirnidine
core), and this probe
G/C-rich,
and
as probes
The
M3-M5
TNS
M5,
used
results.
corn-
complete
of novel
core of the TNS. Intriguingly,
the sequence
more closely
matches
an ETS consensus
(through
its arrangement
diately
after the GGAA
efficiently
9). Band
partially
of these residual
complexes
differed
band
3. It is unknown
whether
this
was
complexes.
The INS
(Fig.
3
and
3 (Fig. 1B, Lanes 3, 4, 6, and 7), although
were seen in the presence
of these corn-
in mobility
probes
M3
M7 did so
(Fig.
(Fig.
petition
of band 3 by these mutants)
or represented
competition
of band 3 and the subsequent
formation
EMSA,
band
The
3, although
type INS probe (Fig. 2, Lanes 4, 5, and
Formation
of the band 1 complex
was
(Fig.
on
to assess.
able
Ml , M2,
probes.
to compete
and M7 mutants
competed
extent as did the wild-type
INS
from
mutant
were
Mutants
band
of
of the
compete
residual
quence
competed
8
INS
poorly
more
Lane
and
7
less efficiently
than did the wild-type
and 9), whereas
M6 competed
only
upstream
of the GGCCGG
region of the TNS. However, this
result
is consistent
with the observation
that the J16 Sefails
6
mutant
extracts
formation
M7 mutants
1
unexpected.
5
4
using
nuclear
effects
complex
Because
3
beled wild-type
(TNS) or mutant
TNS probe sequences
(M1-M8),
as
described
in Fig. 1A. Unlabeled
competitor
sequences
in Lanes 2 and 3
were used at a 1 00-fold
molar excess and are as indicated
in Fig. 1A.
Bands
1-4 discussed
in the text are indicated
by arrows.
The open
arrowhead
designates
a complex
formed on the M8 probe that exhibits
different
competition
and Ab reactivity
patterns
than the band 2 complex
formed on the wild-type
INS probe.
I.q!
,,-.
content.
2
..
performed
consensus
“-
all generated
with
10).
observed
was
the Ml , M2, M4, and M6 probes
the
using
dramatically
were
used
wild-
the
irn(Fig.
526
ETS Factors Bind the PDGF-B
Table
1
Literature-based
INS
comparison
of huma n ETS fam ily tra nscription
M
ETS family
factors
DNA
K562L
binding
characteristicsc
member
(xicP)
-WA
+IPA
EIS-1
48-54
-
+
ETS-1 VlI
39-42
56
+
+
ETS-2
ELK-i
SAP-la
SAP-lb
SAP-2
58-62
58
52
Selected
ERG-2
ERG-3
Other ERG
FLI-i/ERG-B
+
+
52-57
59
38/49
51-55
ELF-i
MEF
GGAT
+
+
+
+
ACCGGAAGTG/A
NACCGGANtG/aT/cA/C/G
+
-
+
+
G/A/cG/AC/A/gC/AGGAAJtG/a/cCiT/ac
C/G C/aGGMG/aIc
W
W
G/MCCGGAAG/aT/cAIG
AAJtC/aCCGTA/c/g
A’gG/C/ANG/AAIIGGAA/rGWrNMNAJtT/a
+
-
+
-
+
+
T/AC/ACNCGGMGTAd
+
W
+
-
-
90-100 (68)
-100 (70)
NERF-la
NERF-ib
NERF-2a
NERF-2b
E1A-FIPEA3
ER81/ETV-i
ERM
Spi-1/PU.i
Spi-B
GGAA
A’gCC/aGGAA/rG/aT/CN
-45 (mh)
41
ERG-i
site
+
+
56
57
63e
60
62
70 (58)
35
46
60
E4TF1-60/GABP-a
TELJETV6
+
+
W
G/aG/cC/aGGAWFG/aT/cN
+
+
+
M.AA/tG/cA/C/GG/cTA/GG/C
+
-
-
AAAJtG/cA/cG/cTAiTN
+
-
G/AC/gC/aGGAA/tG/aT/cN
+
+
57-60
+
50-52
PE-i
+
ERF
a
b
C
75
+
+
binding
is indicated
by W. Note that in many binding sites, the GGAAJI
core is immediately
followed
d Sequences
derived from a comparison
of known binding
sites rather than the site selection
assay.
.
The calculated
molecular
weight
in those
cases
in which
the protein
W
+
Molecular
weight was determined
by PAGE as reported
in the literature.
The size of SAP-2 is for mouse homologue
Net/ERP.
Expression
in K562 cells at either the mRNA or protein level as reported
in the literature.
Weak expression
is indicated
by W.
The preferred
binding site was determined
by site selection
assays and the ability to bind GGAAIT cores (underlined)
as reported
migrates
2). Band 1 complex
formation
occurred
more efficiently
on
the M5 probe than it did on the wild-type
INS probe (Fig. 2,
Lane 8). This band i binding pattern is identical to the band
i competition
pattern that was observed for the mutant INS
competitors
(Fig. iB).
Band 3 complexes
were present on all of the mutant TNS
probes, although slight differences
in mobility could again be
seen for several of the mutant probes. This would suggest
that band 3 is not dependent
on sequences
within the INS
for binding. This result is inconsistent
with the inability of the
Ji 7 sequence
to compete
for band 3 (Fig. 2, Lane 3). Currently, the binding activity of band 3 is not fully understood.
However, this complex is unlikely to be the functionally
irnportant TNS-binding
component,
as will be discussed
below
(see Fig. 6).
A subset of ETS family members
are able to recognize
a
GGAT core sequence
in addition to the GGAA core recognized by all ETS family members.
We were therefore
interested in determining
whether bands 2 and 4 were capable of
binding to a version of the INS with a GGAT core (M8; Fig.
iA). When used as a competitor
in the EMSA, M8 competed
for both bands 2 and 4 somewhat
less efficiently than did the
wild-type
INS (Fig. iB, Lane 10). A more definitive
result
emerged when the M8 mutant was used as an EMSA probe.
Neither band 2 nor band 4 formed on the M8 probe (Fig. 2,
significantly
more
slowly
by a purine
than would
and then
in the literature.
Weak
a pyrimidine.
be expected
from
its cDNA
sequence.
Lane 1 1). Note that a complex
of slightly different
mobility
than band 2 did appear on the M8 probe. This M8 probespecific complex
could be competed
only by M8 DNA and
not by any of the other mutants or by the wild-type INS (data
not shown). Moreover,
formation
of the M8 probe-specific
complex could be disrupted
by the addition of an Ab against
ETS family
member
FLI-i
(data
not shown),
a behavior
not
shared by the wild-type
INS band 2 complex (Fig. 5A, Lane
6). Thus, competition
and Ab analysis revealed that this M8
probe complex was distinct from the band 2 complex formed
on the wild-type
INS probe. Therefore, the band 2 and band
4 INS complexes
both contain factors that display some
preference
for GGAA over GGAT core sequences.
Sizes of Band 2 and Band 4 Proteins Are Consistent
with ETS Family Members.
The binding patterns of bands
2 and 4 toward the INS substitution
mutant probes and
competitors
suggested
that these two complexes
contained
members of the EIS family of transcription
factors. To provide additional
support for this hypothesis,
we performed
UV
cross-linking
analysis on these complexes.
This method allows one to determine
an approximate
molecular
weight of
proteins that are in close proximity to DNA. We could therefore compare the resulting molecular weight values for band
2 and band 4 with the sizes of known EIS family members.
For the UV cross-linking
analysis, we opted to use a BrdUrd-
Cell Growth
M6
.
Band 2 slices
probe
Competitor:
-
J17
-
527
4 slices
-
M6
J17
6
7
Cl-i
,N.
Competitor/Ab:
Band
M6
-
& Differentiation
.
\C
-
200 kD
-
97.4 kD
-
.
.
#{149}.
68kD-
43 kD
-
29 kD
-
1.
III
18.4 kD.:
.
14.3 kD1
2
3
MW
4
probe
M6 UVXL
1
2
3
5
4
6
8
7
9
10
11
Fig. 3. EMSA complexes
formed
on the M6 probe behave similarly
to
those formed on the wild-type
INS probe. EMSA analysis was performed
using nuclear extracts
from untreated
K562 cells and radiolabeled
wildtype INS or M6 mutant probes. Unlabeled competitor
sequences were
used at a 1 00-fold
molar excess.
The sequence
of the consensus
Spi
competitor
is 5’-ATTCGATCGGGGCGGGGCGAGC.
The Lane 6 binding
reaction
was preincubated
with 2 g of an a-ELK-i
antiserum
(Santa
Cruz) before addition
of the radiolabeled
probe. ELK-i
SS, a complex
supershifted
by the a-ELK-i
Ab. A novel M6 band formed only on the M6
probe is also indicated.
5
Fig. 4. UV cross-linking
analysis
of selected
TNS-bmnding
components.
EMSA analysis
was performed
using nuclear
extract
from IPA-treated
K562 cells and a radiolabeled
BrdUrd-substituted
M6 probe (M6UVXL)
either in the presence
or absence
of the indicated
unlabeled
competitors.
Gel slices containing
band 2 and band 4 complexes
were excised,
exposed to UV light, and denatured
in SDS-containing
sample
buffer as
indicated
in “Materials
and Methods.”
Cross-linked
protein-DNA
com-
plexes were separated
labeled
protein
(ELK-i
weight
The sizes and positions
markers
6). This Ab
on the wild-type
formation
the
band
M6 probes
2 and
band
also affected
probe
(see
4 complexes
on both
INS
to be identical
by all of the criteria
M6 probe
for two reasons:
(a) the M6 probe
sequence
displayed
a more abundant
band 2 in the EMSA
(Fig. 2, Lane 9); and (b) BrdUrd substitution
increases
the
This indicated
that
the UV cross-linking
the M6 probe
analysis.
was
efficiency
cross-links
slices
with
which
are formed.
In a control
EMSA
UV-induced
experiment,
to determine
the
whether
covalent
M6 probe
protein-DNA
was
it displayed
tested
the
in the
same
band
2
and band 4 binding patterns as the wild-type INS probe. As
expected,
the M6 probe bound bands 2-4 as well as or
better than the wild-type
INS probe but bound band 1 very
poorly (Fig. 3, Lanes 1 and 2). Additionally,
a novel band
(designated
as the M6 band) was observed
only on the M6
probe (Fig. 3, Lane 2) and may represent the binding of an
additional
ETS-like
factor.
Of
note,
bands
2 and
4 were
competed
by the M6, J16, INS,
and M2 sequences
(Fig. 3,
Lanes 3, 5, 8, and 9) but not by the J17, Spi, or M5 sequences
(Fig.
by the
Ji6,
cause
they
and
M2
binding.
for both
of the
band
wild-type
competition
pattern
2 and
INS
was
2 and 4 on the wild-type
family
sequences
was
of band
incomplete,
lack the M6 mutation
that increases
The
M8 competitor
showed
a reduced
compete
that
4, 7, and 10). Competition
3, Lanes
INS,
member
ELK-i
band
4 complexes
competitor
identical
INS
supershifted
to that
observed
Finally,
band
band
ability
relative
(Fig. 3, Lane
probe.
2
be-
EMSA
IPA-treated
K562
tuted
M6 probe.
tion and boiled
cross-linked
was
2 and
experiment
cells
and
performed
using
nuclear
in a SDS-containing
were
tein
markers
and
for
a
from
BrdUrd-substi-
buffer,
the
gel
from
extract
exposed
to UV irradia-
and the extracted
by SDS-PAGE.
The
cross-linked
proteins
with 14C-labeled
pro-
separated
corrected
by isolating
4 complexes
were
and
tested.
for use in
band
a radiolabeled
The gel slices
proteins
appropriate
apparent
molecular
weights
of the
were calculated
through comparison
size
of
the
cross-linked
single-stranded
M6 probe DNA (M
9,900).
A major
protein
band of Mr
57,000
(corrected
size) was detected
in band 2
complexes
formed in the absence of competitor
(Fig. 4, Lane
1). Ihis
protein
band was greatly
diminished
when
a M6
(homologous)
EMSA
competitor
binding
to
corresponding
for bands
band
preparative
Ji 7 competitor
an Ab to ETS
analysis
containing
2
to
1 1). This
4 on the M6 probe
UV cross-linking
“C-
band 4
Fig. 5A).
seemed
substituted
of
(MW) are given.
SS; Fig. 3, Lane
complex
Thus,
by SDS-PAGE.
molecular
cross-linked
65,000,
with
Lanes 4 and
species
in size
the
5).
6 and
7).
included
in the
but was negligibly
was used
were
instead
observed
and
not to the Ji7
molecular
when
a
2 and 3). Two
for the band
to approximately
The calculated
preparatory
affected
(Fig. 4, Lanes
latter species
being
Again, these complexes
to the M6 competitor
Lanes
was
reaction
4 complex,
M, 58,000
and
Mr
predominant
(Fig. 4,
were sensitive
only
competitor
weights
(Fig.
4,
of all three
528
ETS Factors
Bind the PDGF-B
INS
A
,
C-i
-
-
F-
,.;‘
CL.
F-
LU
Antibodies:
,i
r1
Cl-i
(t
LU
-
LU
LU
CL,
0
B
B
B
-
BandliSp-.-
-
U
.<
<
LU
LU
c
Cl-i
B
-
Cli
B
Lr-i
Competitors:
B
-
4/ELK-i
-
U,
-
-,
:‘
__-
Band 2
Band3-#{248}
4/ELK-i
Band
;z
--
8
TNS
-
Band1/Sp-..-
-
Band
Ln1
c
-
-
B
9
10
.
0
I
11
234
TNS probe
probe
-
probe
TNS
Fig. 5. A, ETS family member
ELK-i binds to the INS. EMSA analysis was performed
using nuclear extracts from untreated
K562 cells and a radiolabeled
wild-type
INS probe. Binding reactions
were preincubated
with 2 j.g of antisera to the indicated
ETS family members
before addition
of the radiolabeled
probe. Lanes 8-i i are assembled
from nonadjacent
lanes of the same gel and are from a different
experiment
than Lanes 1-7. B, a consensus
ELK-i
binding
site competes
for band 2 and band 4. EMSA analysis
was performed
using nuclear extracts
from IPA-treated
K562 cells and a radiolabeled
wild-type
INS probe. Unlabeled
competitors
were used at a 1 00-fold
molar excess. The sequence
of the consensus
ELK-i
binding site (ELK- 1 CS) is
5’-AAAGAAGGCAAACCGGAAGTACCATCG-3’.
The INS and MS competitor
sequences
are given in Fig. iA.
of these
sizes
protein
species
of many
Identification
Cells. Results
from
the
are
ETS family
of ELK-I
from
transcription
band
4 INS-binding
mine
whether
bers
within
against
strongly
we
could
these
We
identify
specific
ETS
binding
reactions
type INS probe.
Binding
scriptional
TNS
in K562
and
complexes
observed
function
of the INS,
ETS
to those
assayed
CR5-Luc
background.
wild-type
INS
implicated
of the band
next
individual
complexes.
reported
mutagenesis
as components
complexes.
the
1).
to the
substitution
analysis
factors
with
(Table
Binding
the INS
UV cross-linking
family
consistent
members
wanted
to deterETS family
mem-
To do so, we added
family
transcription
2 and
factors
antisera
to
EMSA
before
the addition
of radiolabeled
Strikingly,
the addition
of an a-ELK-i
wildAb
of ETS-like Factors Correlates
with INS TranActivity.
To determine
which ofthe INS-binding
in the
selected
in the
EMSA
were important
for the
INS mutations
corresponding
EMSA
This
sequence
were
reporter
introduced
construct
in its synthetic
into
a
(Fig.
6A)
promoter
and is fully inducible
by IPA (47). When compared
similar reporter
construct
lacking the INS (CR4-Luc;
CR5-Luc
displayed
K562 cells (Fig. 6B).
INS
of CR5-Luc
the
contains
with a
Fig. 6A),
3.3-fold
greater
activity
in TPA-treated
Introduction
of the M2 mutation
into the
(CR5m2-Luc;
Fig. 6A) had a negligible
effect
disrupted
formation
of the band 4 complex
(Fig. 5A, Lane 5).
None
of the other antibodies
assayed
had any such disruptive effect on either band 2 or band 4. Consistent
with the
on promoter
the binding
effect
2 and
critical
for INS function
in differentiating
K562 cells. Placement of the M5 or MB mutations
into the TNS of the reporter
family
construct
(CR5m5-Luc
completely
abrogated
of the a-ELK-i
sus ELK-i
band
members
ELK-i
multiple
binding
4 in the
site
EMSA
share
binding
ETS
Ab on the band
competed
(Fig.
similar
site
factors.
5B,
effectively
Lane
consensus
used
4 complex,
in Fig.
5B
for band
3). Many
sites
Therefore,
a consen-
(lable
should
competition
ETS
i),
be
so
bound
of band
the
by
2 in
Therefore,
activity
(Fig. 6B). In the EMSA,
M2 maintained
of bands
2-4 but failed
to generate
band
1.
the binding
because
bands
1 and 3 in the EMSA
family
2 and
band
transcription
includes
factor
ELK-i
a yet-to-be-identified
.
Ihe
band 2 complex
ETS family
member.
very
complexes
and
M8
reduced
6B).
probe
4 complexes.
as being
critical
1) to the INS
CR5mB-Luc;
promoter
to the level
sequences
were
but were
This
Fig.
6A)
unable
implicates
for INS-mediated
is not
nearly
transcriptional
and CR5m8-Luc
were
addition
to band 4 was not surprising.
These results
clearly
demonstrate
that the band 4 complex
contains
the ETS
likely
M5
cells
and
(band
TNS-specific
CR5m5-Luc
TPA-treated
of Sp factors
activity,
activities
in
of CR4-Luc
both
(Fig.
bound
to generate
these
by
band
ETS-like
transcription
Cell Growth
& Differentiation
529
A
I
SPE
-64
I
ITATA
-45
-31
AdML
mr
-jTdT
-25
-15
+6
+1
I
J
SPE
-64
CR5-Luc
1TATAI4I-ITdT1nrI
-45
-31
25
1[%.1
+6
EMSA
Band
Fig. 6. Binding
of ETS family members
correlates with the transcriptional
activity of the mutant
INS sequences.
A, schematic
representation
of
chimeric
promoters
containing
mutated
c-sis/
PDGF-B
TNSs. The -64 to +6 promoter
regions
of the CR4-Luc
and CRS-Luc
chimeric
reporter
constructs
are diagrammed.
CRS-Luc
differs from
CR4-Luc
only in containing
a wild-type
c-sis/
PDGF-B TNS. Mutations
of the INS corresponding to those used in the EMSA analyses
in Figs. 1
and 2 were introduced
into the CR5-Luc
background.
TATA,
consensus
TATAAA
sequence;
TNS, c-sis/PDGF-B
INS; AdML,
INS from the
Adenovirus
major late promoter;
TdT lnr, initiator
element
from the murine terminal
deoxynucleotidyl transferase
gene. B, relative activities
of mutant INS promoter-reporter
constructs
in transiently
transfected
K562
cells.
The indicated
reporter
constructs
were transfected
into K562
cells and subsequently
treated
with IPA or solvent, as described
in “Materials
and Methods.”
Luciferase
activity
was normalized
to f3-galactosidase activity and calculated
relative to CR4-Luc,
which was defined as i .0. Mean values of three to
four replicates
are shown.
Error
bars,
SE of the
mean.
CR5-Luc
CR5m2-Luc
I
Probe
Band
2
Binding
Band
3
Band
GGCCGGAACA
+
+
+
+
TT
-
+
+
+
CR5m5-Luc
++
CC--
CR5m6-Luc
AC
CR5m8-Luc
T--
-
+
-
-
++
+
+
+
-
+
4
B
T
-TPA
5
4
I
C)
.<
I
3
2
1
ill
0
73
-
c
indicates
sufficient
that
for
bound well
with respect
TNS
band
The
1 nor band
M6
probe
(CR5m6-Luc;
comparable
to that
Fig. 6A) displayed
of the wild-type
CR5-Luc
promoter
in IPA-treated
cells
ence between
CR5-Luc
and CR5m6-Luc
significant
dent’s
at the
t test.
strongly
mutant
P
0.05
=
Therefore,
correlated
TNSs.
This
members,
3 binding
sequence
is
was
including
the c-sis/PDGF-B
level
only
when
the
binding
(Fig.
was
ELK-i
,
is important
in differentiating
that
of bands
for the
2 and
function
TNS-binding
presented
factors
here
more
that
were
extensively
first
4
of
cells.
characterizes
observed
in our
the
nuclear
extract
INS.
factors
the
earlier
and
complex
labile
B
INS-binding
TPA-treated
in earlier
nature
complex
untreated
is identified
K562
experiments
of its protein
cells.
The
presumably
component
during
preparation.
core
nuclear
Iwo
mutational
sequence
factors
complexes,
TNS variants
with
in these
analysis
critical
and the transcriptional
band
2 and
mutations
two
of the TNS has idenfor both the binding
of
band
in this GGAA
complexes
show
activity
of the
4, are unable
core.
to bind
Furthermore,
a preference
for bind-
GGAA core sequence
over a INS
containing
a
GGAT core. These binding patterns suggest that both coming to the
plexes
Discussion
The work
reflects
J1
B
B
a novel
from
of this
selected
Stu-
activity
of the
of ETS family
K562
Ui
7
‘:
High-resolution
tified a GGAA
not statistically
using
n
:n
7
2
In addition,
absence
INS-containing
6B); the differ-
analyzed
with the transcriptional
argues
that the binding
TNS
activity
report.
n
cii
7::
r
in extracts
by band 4 in the EMSA and was an “up” mutant
to band 2 binding.
A reporter
construct
contain-
ing this mutation
was
neither
activity.
n
cii
c
C-
and
n
73
.N
contain
members
factors.
UV cross-linking
57,000
and
Mr
of the
ETS
analysis
58,000/65,000
family
gives
for
teins,
respectively.
Such
sizes
are
ported
molecular
weights
of
many
of transcription
estimated
band
2 and
consistent
ETS
family
sizes
band
with
of Mr
4 pro-
the
members.
re-
5:,
ETS Factors
Bind the PDGF-B
TNS
Indeed, a consensus
binding site for ETS family members
competes
efficiently
for both the band 2 and band 4 cornplexes, and antiserum
against EIS factor ELK-i disrupts
band 4 complex formation.
ELK-i is a member of the ternary complex factor subfamily
of ETS transcription
factors
(6i). It forms a ternary
complex
with a homodimer
of serum response factor, p67SRF, bound
to the serum response
element
of the c-fos promoter
to
regulate
serum-inducible
(or IPA-inducible)
expression
of
that gene (62-65).
Ihis ternary complex
is present before
and after induction
(66, 67) but undergoes
mitogen/TPAinduced phosphorylation
(68-70). Phosphorylation
of ELK-i
can lead to increased ternary complex formation under some
circumstances
(68, 71-73) and is required for the transcriptional activity of the ternary complex (65, 70, 72, 74). ELK-i
has an apparent M, of 58,000-62,000
by PAGE (62, 75) and
is capable
of autonomous
DNA
binding,
displaying
a prefer-
ence for GGAA core sequences
over GGAI cores (76, 77).
ELK-i expression
in K562 cells has been reported previously
(78). Band 4 clearly fits this profile.
The protein doublet observed for band 4 in the UV crosslinking
analysis
could
have
several
explanations,
including
differences
in the posttranslational
modifications
of the two
protein species. For example, phosphorylation
of proteins is
capable
of altering their migration
in polyacrylamide
gels.
ELK-i is known to be phosphorylated
in its COOH terminus
in response to mitogen stimulation or to environmental
stresses
by members
of the extracellular
signal-regulated
kinase and
stress-activated
protein kinase/c-jun-NH2-terminal-kinase
subfamilies
of MAPKS,
respectively
(65, 68-70,
72-74, 79, 80).
Additionally,
the use of phorbol
esters or other intracellular
activators
of the MAPK pathway
(e.g., activated forms of ,as,
raf, mos, and src) also results in phosphorylation
of the ELK-i
COOH-terminal
domain (69, 74, 81, 82). Phosphorylation
of this
“C box” domain increases the ability of ELK-i to activate transcnption, whether bound to DNA autonomously
or in a ternary
complex
(65, 69, 72, 74, 8i-83).
In K562
cells,
WA
treatment
increases generalized
MAPK activity
(84). Therefore,
it is reasonable to expect that ELK-i will be phosphorylated
in WAtreated K562 cells, and that this could be important for franscnptional activation ofthe c-sis/PDGF-B
promoter by ELK-i in
these cells. The phosphorylation
status of the INS EMSA cornplexes or the involvement
of phosphorylation
in the WA induction ofthe c-sis/PDGF-B
gene has not been directly addressed.
It is interesting to note, however, that c-fos mRNA is constitutively expressed
in mature human megakaryocytes,
providing
indirect support for the activation of ELK-i during megakaryocytic development
(85).
Band 2 remains unidentified,
although
it is very likely another EIS family transcription
factor. It is perhaps a bit surprising
that another member of the ternary complex
factor
subfamily,
SAP-i
was not identified
as binding to the INS
(Fig. 5A, Lane 10). SAP-i is expressed
in K562 cells (78) and
binds to a similar but more relaxed set of EIS binding
sites
compared
with ELK-i (77). The Ab used in this study recognizes only the SAP-i a isoform; thus, the use of additional
antisera against the SAP-i b isoform might be of interest.
Obtaining
additional
antisera to other EIS family members
would probably be the fastest way to specifically
identify the
,
band 2 protein component.
However, it is also quite possible
that band 2 represents
an as-yet-uncloned
EIS transcription
factor.
The importance
of the individual
INS-binding
complexes
for the transcriptional
activity
of the INS has also been
addressed
by this work. Promoter-reporter
constructs
with
mutations
that prevent the binding of bands 2 and 4 do not
show INS-mediated
transcriptional
induction
by IPA, despite retaining
the binding of the band 3 complex.
Conversely, mutant INS promoter-reporter
constructs
that maintam band 2 and band 4 binding retain induction,
despite
losing the binding of the band i/Sp factor complex.
These
results provide strong evidence that the ELK-i and ETS-like
band 2 factors are important
for the function
of the INS.
However, we are unable to determine whether one of the two
EIS-like
factors is a more critical INS-binding
component,
because none of the current INS mutants distinguish
between the binding of the band 2 and band 4 complexes.
Identification
of ETS-like factors binding to the INS may
provide insights into the mechanism
of action of the INS.
The transcriptional
activation
domains
of ELK-i and other
EIS factors
have been delineated
(69, 74, 89, 90). This
information
will facilitate future studies of potential
interactions between
EIS factors and components
of the general
transcriptional
machinery.
The location of an ETS binding site adjacent to the IAIA
box is not unique to the c-sis/PDGF-B
gene. For example,
the granulocyte
macrophage
colony-stimulating
factor,
HER-
2/neu, and interleukin
13 promoters
all contain ETS binding
sites located 2-i 0 bp upstream
of their IAIA
sequences
(86-88).
At least one ETS family member,
PU.i/Spi-i
is
capable of binding directly to the IAIA-binding
protein (91).
ELK-i associates
with CBP, and this physical interaction
is
dependent
on the COOH-temiinal
transactivation
domain of
ELK-i
(92). CBP is a transcriptional
coactivator
that can
associate
with the TAIA-binding
protein
in vitm and in vivo
(93, 94), with IFIIB in vitm (95), and with RNA polymerase
II
holoenzyme
complexes
in vivo (96, 97). Interestingly,
phosphorylation
of ELK-i
is not necessary
for binding to CBP but
is required for functional
cooperation
with CBP, whose transcriptional
activity itself is also enhanced
by extracellular
signal-regulated
kinase i -mediated
phosphorylation
(92).
The contribution
of EIS-like
factors to the regulation
of
c-sis/PDGF-B
expression
in K562
cells
undergoing
,
megakaryoblastic
differentiation
fits
nicely
with
the
larger
role of ETS factors in megakaryocytic
gene expression.
EIS
binding sites are frequently found in the regulatory
regions of
megakaryocyte-specific
c-mpl
genes.
Some
examples
(98), gpllb (99, 100), gplba (ioi),
include
the
gplX (i02), -thro-
moglobulin
(i03), platelet
factor 4 (102, i04), and P-selectin
genes (i 05). Among these genes, those ETS binding sites
that have been tested thus far have been found to be functionally important (98-ioi).
Forced expression
of EIS factor
ELI-i is sufficient to induce megakaryoblastic
differentiation
of K562 cells (i 06), underlying the importance
of ETS factors
for development
along this hematopoietic
lineage. As another gene expressed
in megakaryocytes,
it is perhaps not
surprising
involves
that the transcriptional
ETS family members.
regulation
What
of c-sis/PDGF-B
is particularly
intriguing
Cell Growth & Differentiation
is that
several
ETS-like
factors
are
involved
in human
can-
cers. For example,
chromosomal
translocations
in human
Ewing’s sarcoma result in fusion proteins consisting
of one of
several ETS factors (ELI-i ERG-i
ETVi
E’lAF, or FEy) and
the Ewing’s sarcoma gene product that display greater transcriptional
activation
than do the corresponding
wild-type
ETS factors
(2i-30).
Perhaps
wild-type
or altered ETS family
transcription
factors can contribute
to the aberrant expression of c-sis/PDGF-B
that is observed
in a variety of nec-
, , ,
plastic
and
Materials
nonneoplastic
and
disease
states.
Methods
cell cultue.
Human K562 erythroleukemia
American
Type Cuiture
Collection
(CCL243;
tamed in RPMI 1640 supplemented
with 10%
g/ml
gentamicin
sulfate (complete
cells were obtained
from
Rockville,
MD) and mainfetal bovine serum and 25
RPMI) a 37#{176}C/5%
CO2 incubator.
EMSAS.
Nuclear
extracts
from WA-treated
or untreated
K562 cells
were prepared
according
to the method
of Dignam et a!. (i07). Cycling
cultures
of K562 cells were treated
with 2 ng/ml WA for 2-i2 h before
harvest. The protein content of the nuclear extracts was determined
by the
Coomassie
protein assay (Pierce). Probe and competitor
sequences
used
in the EMSAS are given in the figures or figure legends.
Commercially
synthesized
oligonucleotides
(Genosys)
were purified by PAGE. Complementary single-stranded
oligonucleotides
were mixed in annealing
buffer
(iO mi Tris-HCI,
50 mM NaCI, iO m MgCl2, and i mi Dli),
heated
briefly, and cooled to room temperature
for al h. Alternatively,
a common
complementary
partial-length
oligonucleotide
was hybridized
to the various full-length
oligonucleotides
and extended
using Klenow fragment
in
the presence
ofailfourdeoxynucleotidetriphosphates.
PAGE was used to
isolate full-length
double-stranded
probes and competitors.
Probes were
end-labeled
using 50 Ci of [y-32PJATP
(Amersham;
3000 Ci/mmol)
and
14 polynucleotide
kinase (New England Biolabs); unincorporated
radiola-
bel was removed
through
Sephadex
G-50 chromatography
(Pharmacia
Nick columns).
Nuclear extract (15 g) was premixed
with unlabeled
competitors
(used at a 100-fold
molar excess over radiolabeled
probe)
and 2-3 p.9 of poly(deoxyadenylate-deoxythymidylic
acid) in binding
buffer [20 mM HEPES (pH 7.9), 40 m KCI, 2 mri MgCl2, 0.5 mpi DTT, 0.i
mM EGIA,
and 4% Flcoll (M 400,000)]
for 10 mm at room temperature.
Radiolabeled
probe (-175 fmol) was added to this mixture and incubated
for an additional
iO mm before the binding reaction
was loaded onto a
native 5% polyacrylamide
gel. The gel was electrophoresed
at 4#{176}C
for 90
mm at235 V, dried undervacuum,
and exposed to Phosphorimager
plates
(Molecular
Dynamics)
or autoradiogram
film. Commercially
available
antibodies
against
ETS family members
(Santa Cruz) were used in some
EMSA experiments;
2 g of Ab were added to the binding reaction
and
incubated
at room temperature
for iO-20
mm before the addition
of
radiolabeled
probe.
uv cross-uning
Analysis.
UV cross-linking
analysis was performed
using
BrdUrd-substituted
a
M6
probe
[5’-CCTTTATAAAGGCCGcorrespond
to positions sub-
GMCGCTGAAAGGG
(underlined
bases
stituted with BrdUrd in the bottom
strand)]. The probe was prepared
by
hybridizing
a short bottom-strand
oligonucleotide
(5’-CCCTTTCAG)
with
the full-length
(50 mi
top-strand
Tris-HCI,
oligonucleotide
(shown
10 msi MgCl2, 1 mri DII,
above)
in Klenow
and 50 g/ml
buffer
BSA) and
extending
for 60-90 mm at 25-37#{176}Cusing Klenow fragment
in the presence of 50 .LM each of 5’-bromo-dUTP,
dAIP, and dGTP; 5 p dCTP; and
iOO Ci of [a-32PJdCTP (Amersham;
3000 CVmmol).
Unincorporated
radiolabel
was removed
through
Sephadex
G-50 chromatography
(Pharmacia Nick columns),
and PAGE was used to isolate full-length
doublestranded
M6.UVXL
Approximately
used in standard
from WA-treated
molar excess. The
probe.
i .7 X i0 cpm (-58 fmol) of the M6.UVXL
probe were
EMSA binding
reactions
with 20 p.g of nuclear extract
K562 cells. Competitors
were used at a iOO-200-fold
binding reactions
were incubated
at room temperature
for iS mm before and after addition of the M6.UVXL probe and run on a
0.75-mm,
5% native polyacrylamide
gel for 2 h at 235 V at 4#{176}C,
slightly
longer than usual for a standard
EMSA. The gel was exposed
wet to a
Phosphorimager
screen for 1 h, and this image was used as a template for
cutting out gel slices containing
the bands of interest. The gel slices were
exposed
to UV light (i 20 millijoules
at 254 nm) for 22 mm at a distance of
approximately
i cm. Next, 2x SDS sample buffer [i25 m Iris (pH 6.8),
4% SDS, 20% glycerol,
iO% p-mercaptoethanol,
and 0.002% bromphe-
nol blue] was added to the gel slices, which were then boiled for iO mm
and loaded onto a 1.5-mm, 10% SDS protein polyacrylamide
gel along
with 14C-labeled,
high molecular
weight protein markers
(Life Technologies, Inc.). Electrophoresis
was carried out at 4”C for i8 h at iO-30
mA.
The gel was dried and exposed to a Phosphorimager
screen
for 52 h.
Migration of the molecular weight markers was plotted versus the log of
the molecular
weight to generate
a standard
curve, which was then used
to calculate the molecular weight of the proteins present in the EMSA gel
slices. A value of Mr 9900, corresponding
to the size of 30 nucleotides
of
single-stranded
DNA, was subtracted
from the calculated
molecular
weight values to correct for the presence
of the M6.UVXL
probe.
Plasmid
enstructIons.
CR4-Luc
and CR5-Luc
have been described
previously
(47). CR5m2-Luc,
CR5m5-Luc, CR5m6-Luc, and CR5m8-Luc
were constructed
by PCR. The SPE (-64) to +6 region of the promoter
in
CR5-Luc
was amplified
using primers that contained
Hindlll sites at their
ends and the desired base changes within the INS. The PCR products
were cloned
into the Hindlll site of the promoterless
Iuciferase vector
pSVOA-1A5’,
and the resulting constructs
were confirmed
by sequencing.
TransIent
Transfection
Assays. Cycling cuitures of K562 cells were
counted,
harvested
by centrifugation,
and resuspended
in complete
RPMI
to a concentration
of i .25 x i O cells/mI.
For each transfection,
5 x 106
cells (0.4 mO were mixed on ice with 5 p.g of c-sis/POGF-B
promoterreporter
plasmid
and 4 L9 of 3-galactosidase
expression
vector.
The
ceIVDNA mixtures
were pulsed at 300 V/960 F capacitance
(giving time
constants
in the range of 25-35 ms) using a Bio-Rad
gene pulser and
immediately resuspended in complete RPMI. Approximately
24 h after
electroporation,
the cell culture volumes were split equally into two dishes
and treated with either WA (Sigma Chemical
Co.; final concentration,
2
ng/ml) or solvent (ethano.
Cultures were harvested approximately 48 h
after electroporation
by centrifugation,
and cell pellets were lysed in i 50
d of lysis buffer [i % Triton X-iOO, 25 mu glycylglycmne
(pH 7.8), 15 m,i
MgSO4, 4 mM EGTA, and 1 m Dli].
Cell lysates were stored at -80#{176}C
or used immediately
in the reporter
assays. The luciferase
assays were
performed
as described
previously
(i08) using 70 d of the lysate. The
3-gaIactosidase
reporter assays using chlorophenolred-p-o-galactopyr(i09) were performed
by adding 50 d of the lysate
to 150 el of working buffer [150 mi sodium phosphate (pH 7.3), 15 mpi
anoside as a
MgSO4,
substrate
12 mM chlorophenclred---galactopyranoside,
and 12 mi
DIll,
incubating
at 37#{176}C
for iO-90 mm, and reading optical densities at 574 nm.
Luciferase
activity
for variations
was normalized
in transfection
to f3-galactosidase
efficiency
activity
to correct
and set relative to the reference
construct
as indicated
in the figure legend. At least three independent
transfections
were performed with each reporter
plasmid,
and the mean
values
for relative activity are reported,
with error bars representing the SE
of the mean.
Acknowledgments
We thank
Loo for technical assistance
in the construction
series of promoter-reporter
constructs.
Deborah
CR5mX-Luc
of the
References
i . Degnan, B. M., Degnan, S. M., Naganuma, T., and Morse, D. E. The ets
multigene
family is conserved
throughout
the Metazoa.
Nucleic
Acids
Res., 21: 3479-3484,
i993.
2. Laudet, V., Niel, C., Duterque-Coquillaud,
helm, D. Evolution
of the ets gene family.
mun., 190: 8-i4, 1993.
3. Wasylyk,
factors.
B., Hahn,
M., Leprince,
D., and SteBiochem.
Biophys.
Res. Com-
S. L, and Giovane, A. The Eta family of transcription
21 1: 7-18, i993.
Eur. J. Biochem.,
4. Crepieux,
P., CoIl, J., and Stehelin, D. The Ets family of proteins:
modulators
of gene expression
in quest for transcriptional
partners.
Rev. Oncog.,
5: 615-638,
1994.
5. Janknecht,
Biochim.
R., and Nordheim,
Biophys.
Acta,
A. Gene regulation
1 155: 346-356,
6. Ireisman,
R. Temary
complex
scriptional
activators.
Curr. Opin.
weak
Crit.
by Ets proteins.
1993.
factors:
growth
factor regulated
Genet. Dev., 4: 96-i 01 , 1994.
tran-
531
532
Bind the PDGF-B
ETS Factors
INS
ETS-family
7. Galang, C. K., Der, C. J., and Hauser, C. A. Oncogenic
Ras can induce
transcriptional
activation
through
a variety of promoter
elements,
including tandem
c-Ets-2
binding sites. Oncogene,
9: 2913-2921
, 1994.
8. O’Neill, E. M., Rebay, I., Ijian, R., and Rubin, G. M. The activities of two
Ets-related
transcription
factors required
for Drosophila eye development
are modulated
by the Ras/MAPK
pathway.
Cell,
9. Wasylyk, C., Maira, S. M., Sobieszczuk,
of Ras transformed
3665-3673,
cells
78: i37-i47,
1994.
P., and Wasylyk,
by Ets transdominant
B. Reversion
mutants.
Oncogene,
9:
1994.
10. O’Hagan,
R. C., Tozer, R. G., Symons,
M., McCormick,
sell, J. A. The activity of the Ets transcription
factor PEA3
two distinct
MAPK cascades.
Oncogene,
i3: 1323-1333,
F., and Hasis regulated
by
1996.
11 . Seth, A., Watson, D. K., Blair, D. G., and Papas, T. S. c-ets-2
oncogene
has mitogenic
and oncogenic
USA, 86: 7833-7837, 1989.
12. Seth, A., and Papas,
activity
and is positively
activity.
Proc.
proto-
NatI. Acad.
Scm.
T. S. The c-ets-i
proto-oncogene
has oncogenic
autoregulated.
Oncogene,
5: 1761-1767,
1990.
13. Hart, A. H., Comck,
C. M., Tymms,
M. J., Hertzog,
P. J., and Kola, I.
Human ERG is a proto-oncogene
with mitogenic
and transforming
activity. Oncogene, iO: 1423-1430,
i995.
14. Leprince, D., Gegonne, A., CoIl, J., De Taisne, C., Schneeberger A.,
Legrou, C., and Stehelin, D. A putative second cell-derived oncogene of
the avian leukemia retrovirus E26. Nature (Lond.), 306: 395-397,
1983.
15. Nunn, M., Seeburg,
P. H., Moscovici,
tite structure
of the avian erythroblastosis
Nature (Lond.), 306: 391-395,
1983.
C., and Duesberg,
P. H. Triparvirus E26 transforming
gene.
1 6. Nunn, M., and Hunter, I. The ets sequence
is required
of erythroblastosis
in chickens
by avian retrovirus
E26.
398-402,
i989.
17. Metz, I., and Graf, I. v-myb
chicken
cells and ccoperate
both
ation phenotypes.
Genes
distinct
18. Moreau-Gachelin,
tative
oncogene
in
(Lond.), 33i: 277-280,
F., Tavitian, A., and Tambourin,
virally
induced
1988.
19. Moreau-Gachelin,
vitian,
and v-eta transform
in trans and in cis to induce
Dev., 5: 369-380,
1991 .
for induction
J. Virol., 63:
murine
erythroid
P. Spi-i
F., Ray, D., Mattei, M-G., Tambourin,
K., and Bernstein,
E., Letwen,
is a pu-
Dev., 5: 908-918,
.
Delattre,
B., Desmaze,
chromosome
165, 1992.
translocation
22.
J., Delattre,
Zucman,
in human
0.,
tumours.
Desmaze,
C., Melot,
I., Peter,
A., and
caused
by
359:
162-
B., Joubert,
I.,
Cloning and characterization
of the Ewing’s sarcoma
and peripheral
neuroepithelioma
t(1 1 22) translocation
breakpoints.
Genes Chromosomes
Cancer,
5: 271-277,
1992.
23. Zucman,
J., Melot, T., Desmaze,
C., Ghysdael,
J., Plougastel,
B.,
Peter, M., Zucker, J. M., Triche, T. J., Sheer, D., Turc-Carel,
C., Ambros,
P., Combaret,
V., Lenoir, G., Aurias, A., Thomas,
G., and Delattre,
0.
Combinatorial
generation
of variable fusion proteins
in the Ewing family of
tumours.
EMBO J., i2: 4481-4487,
1993.
25. Jeon,
I-S., Davis,
Lessnick,
S. L, Lopez-Terrada,
I. A second Ewing’s sarcoma
to another ETS-family
D., Liu, X. F., Triche,
translocation,
t(2i 22),
transcription
factor. ERG. Nat.
1994.
J. N., Braun,
B. S., Sublett,
J. E., Roussel,
M. F.,
Denny, C. I., and Shapiro, D. N. A variant Ewing’s sarcoma translocation
(7;22) fuses the EWS
1234, 1995.
26.
Kaneko,
V., Sasaki,
gene to the ETS gene
E7V1.
Oncogene,
S. L, Braun, B. S., Klemsz, M., Lewis, B. C.,
B., Hromas,
A., and Denny,
C. I. The Ewing’s
fusion gene encodes
a more potent transcriptional
and is a more powerful transforming
7393-7398,
29. Ohno,
gene than FLI-i
sarcoma
activator
Mol. Cell. Biol., 13:
.
1993.
I.,
Rao, V. N., and Reddy,
is a transcriptional
activator.
E. S. P. EWS/Fli-i
Cancer Res., 53: 5859-5863,
chimeric
1993.
protein
30. Bailly, R-A., Bosselut, A., Zucman, J., Cormier, F., Delattre, 0., Roussel, M. Thomas, G., and Ghysdael, J. DNA-binding and transcriptional
activation
properties
of the EWS-FLI-i
fusion protein resulting
from the
t(i 1 ;22) translocation
1994.
in Ewing
31 . Ross, R., Raines,
platelet-derived
growth
E. W.,
factor.
sarcoma.
Mol. Cell.
and Bowen-Pope,
Cell, 46: 155-169,
Biol.,
14: 3230-3241,
D. F. The
biology
of
1986.
32. Heldin,
C-H. Structural
and functional
studies
growth factor. EMBO J., 11: 4251-4259,
1992.
on
platelet-derived
33. Claesson-Welsh,
L
action. In: B. Westermark
Growth Factor, Cytokines
and
mechanism
34. Devare, S.
S. A. Nucleotide
PDGF
receptors:
structure
of
and C. Sorg (eds.), Biology of Platelet-derived
5, pp. 31-43.
Basel,
Switzerland:
Karger,
i993.
G., Reddy, E. P., Law, D. J., Robbins, K. C., and
sequence
of the simian
sarcoma
Aaronson,
genome:
demon-
virus
stration that its acquired cellular sequences encode the transforming
product p28#{176}’#{176}.
Proc. NatI. Acad. Sci. USA, 80: 73i-735,
1983.
R. F., Hunkapiller,
gene
M. W., Hood, L E., Devare, S. G., Rob-
K. C., Aaronson,
S. A., and Antoniades,
H. N. Simian sarcoma
virus
onc gene, v-sis, is derived from the gene (or genes) encoding
a plateletderived growth factor. Science
(Washington),
221: 275-277, i983.
37. Clarke,
G., Aurias,
C., Plougastel,
L
M. D., Scrace,
G. I., Whittle,
N., Stroobant,
P., Johnsson,
transforming
35-39,
i983.
Melot, I., Peter, M., de Jong, P., Rouleau, G., Aurias, A., and Thomas, G.
24. Sorensen,
P. H.,
I. J., and Denny, C.
fuses the 84’S gene
Genet., 6: 146-151,
Lunsford,
EWS/FLI-i
localization
Oncogene,
A. Erythroleu-
domain
Nature (Lond.),
1996.
28. May, W. A., Lessnick,
10: i229-
V., Yoshida,
K., Handa, M., Toyoda, Y., Nishihira,
H., Tanaka,
V., Ishida, S., Higashino,
F., and Fujinaga,
K. Fusion of an
protein
p28#{176}’#{176}
of simian
M. F., Westin,
sarcoma
E, Schmidt,
A., lgarashi,
H., Chiu,
l-M.,
S. R., Robbins, K. C., and Aaronson,
sis/PDGF-2
coding
89-97,
1984.
sequence
virus.
Nature
D., Josephs,
Wong-Staal,
F., Gallo, R. C., and Reitz,
NIH3T3 cells by a human c-sis cDNAclone.
1984.
38. Gazit,
J., Plougastel,
M., Kovar, H., Joubert, I., de Jong, P., Rouleau,
Thomas, G. Gene fusion with an ETS DNA-binding
trans-
Chromosomes
P., and Ta-
1991.
0., Zucman,
Genes
27. Peter, M., Couturier,
J., Pacquement,
H., Michon,
J., Thomas,
G.,
Magdelenat,
H., and Delattre,
0. A new member
of the ETS family fused
to EWS in Ewing tumors.
Oncogene,
14: 1 159-i 164, 1997.
36. Waterfleld,
by Friend murine leukemia virus: insertional activation of
a new member of the ets gene family FLI- 1 , closely linked to c-etsi . Genes
21
15: ii5-i2i,
chromosome
of infancy.
A., Wasteson, A., Westermark, B., Heldin, C-H., Huang, J. S., and Deuel,
I. F. Platelet-derived
growth factor is structurally related to the putative
oncogene
Spi-i : murine chromosomal
and transcription activation in murine acute erythroleukemias.
4: 1449-1456,
1989.
V., Gidden,
Cancer,
sarcoma
bins,
Nature
A. The putative
20. Ben-David,
kemia induction
in an undifferentiated
35. Dcolittle,
differenti-
erythroleukemias.
gene, E1AF, to EWS by t(i7;22)(qi2;qi2)
location
(Lond.),
304:
S. F., Ratner, L,
M. S., Jr. Transformation
of
Nature(Lond.),
308: 464-467,
Srinivasan,
A., Yaniv,
A., Tronick,
S. A. Expression ofthe normal human
Induces
cellular
transformation.
Cell,
39:
39. Stevens,
C. W., Brondyk,
W. H., Burgess,
J. A., Manoharan,
I. H.,
Hane, B. G., and Fahl, W. E. Partially
transformed,
anchorage-independent human diploid
fibroblasts
result from overexpression
of the c-sis
oncogene: mitogenic activity of an apparent monomeric
growth factor 2 species. Mol. Cell. Biol., 8: 2089-2096,
platelet-derived
1988.
40. Pech, M., Gazit,
A., Amstein, P., and Aaronson, S. A. Generation of
in vivo by a retrovirus that expresses the normal B chain of
platelet-derived growth factor and mimics the alternative splice pattern of
the v-s/s oncogene.
Proc. Natl. Acad. Sci. USA, 86: 2693-2697,
1989.
fibrosarcomas
41 . Westermark,
Westermark
Cytokines
B. PDGF and its receptors
in human
and C. Borg (eds.), Biology of Platelet-derived
5, pp. 146-162.
Basel, Switzeriand:
Karger,
42. Betsholtz,
C. The PDGF
genes
and their regulation.
tumor cells. In: B.
Growth Factor,
1993.
In: B. Westermark
and Borg, C. (eds.), Biology of Platelet-derived
Growth Factor, Cytokines
5, pp. 1 1-30. Basel, Switzeriand: Karger, 1993.
43. Alitalo, R. Induced
differentiation
of K562 leukemia
studies
of gene expression
in early megakaryoblasts.
501-514,
cells: a model
Leuk. Res.,
for
14:
1990.
44. Colamonici,
0. R., Irepel,
J. B., Vidal,
Phorbol ester induces c-sis gene transcription
Cell. Biol., 6: 1847-1850,
1986.
C. A., and Neckers,
L M.
in stem cell line K562. Mol.
Cell Growth & Differentiation
45. Pech, M., Durga Rao, C., Robblns, K. C., and Aaronson,
tional
identification
platelet-derived
of regulatory
elements
within
the promoter
S. A. Funcregion of
growth factor 2. Mol. Cell. Biol., 9: 396-405,
i989.
46. Jin, H-M., Brady, M. L, and FahI, W. E. Identification and characterization of an essential,
activating regulatory element of the human SIS/
PDGF-B promoter in human megakaryocytes.
Proc. NatI. Aced. Sci. USA,
90: 7563-7567,
1993.
47. Kujoth, G. C., and FahI, W. E. c-sis/PDGF-B
promoter
requirements
for induction
during the i2-O-tetradecanoylphorbol-i3-acetate-mediated
megakaryoblastic
differentiation
of K562 human
erythroleukemia
cells.
Cell
Growth Differ.,
48. Jin,
8: 963-977,
H. M., Robinson,
1997.
V., and Fahl, W. E. SIS/PDGF-B
of regulatory
elements
necessary
of the SIS/PDGF-B
gene in U2-OS osteosarcoma
269: 28648-28654,
1994.
isolation and characterization
for basal
expression
cells. J. Biol. Chem.,
49.
Khachigian,
L M., Fries, J. W. U., Benz, M. W., Bonthron,
D. I., and
I. Novelc/s-acting
elements
in the human platelet-derived
growth
factor B-chain
core promoter
that mediate
gene expression
in cultured
vascular
endothelial
cells. J. Biol. Chem., 269: 22647-22656.
1994.
Collins,
50. Dirks,
Bloemers,
A. P. H., Jansen,
H. J., van Gerven,
B., Onnekink,
C., and
H. P. J. In vivo footprinting
and functionalanalysis
ofthe human
c-sis/PDGF-B
gene promoter
transcriptional
activators.
64. Graham,
R., and Gilman,
response
at the c-fos serum
192, 1991.
65.
Hill, C. S., Marais,
R. Functional
complex.
Cell,
provides evidence for two binding sites for
Nucleic Acids Res., 23: 1 1 i9-i i26, 1995.
68.
Gille,
H., Shan’ocks,
scription
factor
tion at c-fos
promoter.
Identification
phorylation
in the promoter
of the
52. Scarpati,
E. M., and DiCorieto,
P. E. Identification
response
element
in the human platelet-derived
growth
(c-s/s) promoter. J. Biol. Chem., 271: 3025-3032,
i996.
53. Khachigian,
induced
Science
PDGF
B-
theme
status
A. A,, Pingoud,
V., and Nordheim.
A. c-fos tranand repression
correlate
temporally
with the phosof TCF. EMBO J., 12: 2377-2387,
1993.
of a thrombin
factor B-chain
Chem.,
266: 8576-8582,
72. Gille,
in vascular
injury.
1991.
H., Kortenjann,
M., Thomae,
0.,
951-962,
of MAP kinase signal transduction
J. Biol.
sponse
element.
i996.
C., Slaughter,
C.,
1995.
gration
i4584-i4590,
Mcomaw,
Cobb, M. H., and Shaw, P. E. ERK phosphorylation
potentiates Elk-i
mediated ternary complex formation and transactivation.
EMBO J., 14:
73. Whitmarsh,
271:
forma-
R. The SRF accessory protein
transcriptional
activation do-
54. Trejo, S. R., FahI, W. E., and Ratner, L c-sis/PDGF-B
promoter
transactivation
by the Tax protein of human T-cell leukemia virus type 1.
Chem.,
of tran-
7i . Malik, R. K., Roe, M. W., and Blackshear, P. J. Epidermal growth
factor and other mitogens induce binding of a protein complex to the c-fos
serum response
element in human astrocytoma
and other cells. J. Biol.
L M., Lindner, V., Williams, A. J., and Collins, T. Egr-1-
endothelial
gene expression:
a common
(Washington),
271: 1427-1431 , 1996.
P. E. Phosphorylation
69. Marais, R., Wynne, J., and Treisman,
Elk-i contains a growth factor-regulated
main. Cell, 73: 381-393,
1993.
A., Hipskind,
cell lines. Biochim. Biophys. Acta,
A. D., and Shaw,
by MAP kinase stimulates ternary complex
Nature (Lond.), 358: 4i4-4i7,
1992.
p62TcF
activation
sequences
S., Wynne, J., Dalton, S., and Treisman,
factor-responsive
transcription
factor
1993.
67. Konig, H. Cell-type
specific
multiprotemn
complex
formation
over the
c-fos serum response
element in vivo: ternary complex
formation
is not
required
for the induction
of c-fos. Nucleic
Acids Res., 19: 3607-36i
1,
1991.
scriptional
mesothelioma
R., John,
73: 395-406,
70. Zinck,
of regulatory
targets for signals acting
(Washington),
251: 189-
analysis of a growth
51 . Prins, J. B., Langerak, A. W., Dirts, R. P. H., Vanderlindenvanbeurden,
C. A. J., Delaat, P. A. J. M., Bloemers, H. P. J., and Versnel, M. A.
chain gene in malignant
1317: 223-232, i996.
M. Distinct protein
element.
Science
66. Herrera, A. E., Shaw, P. E., and Nordheim,
A. Occupation
of the c-fos
serum response
element in vWo by a multi-protein complex is unaltered by
growth factor induction.
Nature (Lond.), 340: 68-70,
1989.
D. F., Liang,
promoter
63. SchrOter,
H., Mueller, C. G. F., Meese, K., and Nordhemm, A. Synergism in ternary
complex
formation
between the dimeric glycoprotein
75RF
polypeptide
2TcF
and the c-fos serum
response
element.
EMBO J., 9: 1i23-ii3O,
1990.
A. J., Shore,
Science
P., Sharrocks,
(Washington),
A. D., and Davis,
pathways
269: 403-407,
A. J. Inte-
at the serum
1995.
55. Trejo, S. R., Fahl, W. E., and Ratner, L The Tax protein of human
I-cell leukemia virus type 1 mediates the transactivation
of the c-s/s/
74. Janknecht,
platelet-derived
growth
factor-B
zinc finger transcription
factors
272: 27411-27421,
1997.
75. Rao, V. N., Huebner,
K., lsobe, M., ar-Rushdi,
A., Croce, C. M., and
Reddy. E. S. P. elk, tissue-specific
eta-related
genes on chromosomes
X
and 14 neartranslocation
breakpoints. Science (Washington), 244: 66-70,
1989.
promoter
Spi and
through
interactions
with the
NGFI-NEgr-i.
J. Biol. Chem.,
56. Liang, V., Robinson,
D. F., Denig, J., Suske, G., and FahI, W. E.
Transcriptional
regulation
of the SIS/PDGF-B
gene in human osteosarcoma cells by the Sp family of transcription
factors.
J. Biol. Chem., 271:
11792-11797,
1996.
57. Liang, V., Robinson,
D. F., Kujoth, G. C., and FahI, W. E. Functional
analysis of the SIS proximal
element and its activating
factors:
regulated
transcription
of the c-SIS/PDGF-B
gene in human erythroleukemia
cells.
Oncogene,
13: 863-871,
1996.
58. Khachigian,
L M., Williams, A. J., and Collins, I. Interplay of Spi and
Egr-i in the proximal
platelet-derived
cultured
vascular
endothelial
cells.
1995.
growth factor
J. Biol. Chem.,
A-chain
promoter
270: 27679-27686,
59. McCoy,
C., Smith, D. E., and Comwell,
M. M. 12-O-Tetradecanoylphorbol-13-acetate
activation
of the MDR1
promoter
is mediated
EGR1. Mol. Cell. Biol., 15: 6100-6108,
1995.
in
by MAP
Kinases.
EMBO
J., 12: 5097-5104,
77. Shore, P., and Sharrocks, A. D. The ETS-domain transcription
Elk-i and SAP-i exhibit differential
DNA binding
specificities.
Acids Res.,
23: 4698-4706,
Lin, J-X., and Leonard, W. J. The immediate-early
gene product
Egr-1
regulates the human interleukin-2
receptor
frchain
promoter
through
noncanonical
Egr and Spi binding sites. Mol. Cell. Biol., 17: 3714-3722,
1997.
61 . Hipskind,
R. A., Rao, V. N., Mueller,
C. G. F., Reddy, E. S. P., and
Nordheim,
A. Eta-related
protein Elk-i is homologous
to the c-fos regulatory factor p62T.
Nature (Lond.), 354: 531-534,
1991.
of a ternary
with serum
1989.
factors
Nucleic
1995.
78. Magnaghi-Jaulin,
L, Masutani,
H., Lipmnski, M., and Hare-Bellan,
A.
Analysis of SRF, SAP-i and ELK-i transcripts and proteins in human cell
lines. FEBS Left., 391: 247-251 , 1996.
79. Gille, H., Strahl, T., and Shaw, P. E. Activation
by
A. Activation
1993.
76. Rao, V. N., and Reddy, E. S. P. A divergent ets-related protein, Elk-i,
recognizes
similar c-ets-i proto-oncogene
target sequences and acts as
a transcriptional
activator. Oncogene, 7: 65-70, 1992.
factor
1995.
60.
62. Shaw, P. E., Schroter,
H., and Nordheim,
A. The ability
complex
to form over the serum response
element correlates
inducibility
of the human c-fos promoter.
Cell, 56: 563-572,
of TCF Elk-i
R., Ernst, W. H., Pingoud, V., and Nordheim,
re-
Elk-i
by stress-activated
protein
80. Cavigelli,
M., Dolfi, F., Claret,
expression
through JNK-mediated
14: 5957-5964,
i995.
kinases.
of ternary complex
Curr. Blol., 5: i i9i-i
200,
F. X., and Karmn, M. Induction
of c-fos
TCF/Elk-i
phosphorylation.
EMBO J.,
. Nebreda,
A. R., Hill, C. S., Gornez, N., Cohen, P., and Hunt, T. The
protein kinase mos activates MAP kinase kinase in vitro and stimulates the
MAP kinase pathway
in mammalian
somatic cells in vivo. FEBS Left., 333:
8i
183-187,
1993.
82. Price, M. A., Rogers, A. E., and Treisman,
R. Comparative
analysis of
the ternary complex
factors Elk-i , SAP-ia
and SAP-2 (ERP/NET). EMBO
J., 14: 2589-2601,
1995.
83. Kortenjann,
M., Thomae, 0., and Shaw,
dependent
c-fos expression
and transformation
P. E. Inhibition
of v-rafby a kinase-defective
533
534
9_S Factors Bind the PDGF-B INS
mutant of the mitogen-activated
4815-4824,
1994.
84. Kang,
protein
kinase
Erk2.
14:
Mol. Cell. BioI.,
C. D., Lee, B. K., Kim, K. W., Kim, C. M., Kim, S. H., and Chung,
of PMA-induced differentiation of K562 cells.
Biophys.
Res. Commun., 221: 95-100, 1996.
B. S. Signaling mechanism
Biochem.
85.
Panterne,
B., Hatzfeld,
J., Blanchard,
J-M., Levesque,
J-P., Berthier,
A. c-fos mRNA constitutive
expression
megakaryocytes.
Oncogene,
7: 234i-2344,
1992.
Kee, B. L, Arias, J., and Montminy, M. R. Adaptor-mediated
ment of RNA polymerase II to a signal-dependent
activator.
Chem., 271: 2373-2375,
1996.
96.
97. Nakajima,
T., Uchida,
C., Anderson,
S. F., Parvin,
J. D., and
Montminy,
M. Analysis
of a cAMP-responsive
activator
reveals a twocomponent
mechanism
for transcriptional
induction
via signal-dependent
R., Ginsbourg, M., and Hatzfeld,
factors.
by mature
98.
human
86. Scott, G. K., Daniel, J. C., Xiong, X., Maki, R. A., Kabat, D., and Benz,
C. C. Binding of an ETS-related
protein within the DNase I hypersensitive
site of the HER2/neu
promoter
in human
breast cancer
cells. J. Biol.
Chem., 269: 19848-19858,
1994.
87. Wang, C. V., Bassuk, A. G., Boise, L H., Thompson,
C. B., Bravo, R.,
and Leiden, J. M. Activation
of the granulocyte-macrophage
colonystimulating factor promoter in I cells requires ccoperative binding of Elf-i
and AP-i transcription factors. Mol. Cell. Biol., 14: i 153-i 159, i994.
88. Buras, J. A., Reenstra, W. R., and Fenton, M. J. NF-j3A, a factor
required
for maximal
interleukin-i
3 gene expression is identical to the eta
family
member
PU.1 . Evidence
for structural
alteration
following
LPS
activation.
Mol. Immunol.,
32: 54i-554,
1995.
89. Bhattacharya,
tional activation
8: 3459-3464,
G., Lee, L, Reddy, E. S. P., and Rao, V. N. Transcripof elk-i , Aelk-i and SAP-i proteins.
Oncogene,
domains
i993.
90. Janknecht,
R., Zinck, R., Ernst, W. H., and Nordheim, A. Functional
dissection
of the transcription
factor Elk-i . Oncogene,
9: 1 273-i 278,
1994.
9i
Hagemeler,
C., Bannister,
A. J., Cook, A., and Kouzarides,
I. The
activation
domain
of transcription
factor PU.1 binds the retinoblastoma
(RB) protein and thetranscription
factorlFllD
in vitro: RB shows sequence
similarity
to WIlD and TFIIB. Proc. NatI. Aced. Sci. USA, 90: 1580-1584,
i993.
92.
.
Janknecht,
tional coactivation
Commun.,
228:
R., and Nordheim,
A. MAP
by Elk-i and its cofactor
831-837, 1996.
kinase-dependent
transcrip-
CBP. Biochem. Biophys. Res.
recruitJ. Biol.
Genes Dev., 11: 738-747,
1997.
Deveaux,
S., Filipe, A., Lemarchandel,
V., Ghysdael,
J., Romeo, P-H.,
and Mignotte,
V. Analysis
of the thrombopoietmn receptor (MPL) promoter
implicates
GAlA and Ets proteins
in the coregulation
of megakaryocytespecific
genes. Blood, 87: 4678-4685,
1996.
99. Prandini,
Characterization
glycoprotemn
M-H., Uzan, G., Martin, F., Thevenon,
D., and Marguerie,
of a specific erythromegakaryocytic
enhancer
within
lb promoter.
J. Biol. Chem., 267: 10370-10374,
1992.
100. Lemarchandel,
Romeo,
P-H. GAlA
cyte-specific
V.,
Ghysdael,
J.,
Mignotte,
V.,
Rahuel,
C.,
G.
the
and
and Eta c/s-acting
expression.
sequences mediate megakaryoMol. Cell. Biol., 13: 668-676,
1993.
101 . Hashimoto, Y., and Ware, J. Identification of essential GAlA and Ets
binding motifs within the promoter
of the platelet glycoprotein
ba gene.
J. Biol. Chem., 270: 24532-24539,
1995.
102.
Hickey,
M. J., and Roth,
G. J. Characterization
ofthe
gene encoding
human platelet glycoprotein IX. J. Biol. Chem., 268: 3438-3443,
1993.
103. Majumdar, S., Gonder, D., Koutsis, B., and Ponz, M. Characterization of the human -thromboglobulin
platelet factor 4. J. Biol. Chem.,
266:
gene. Comparison
with the gene for
5785-5789,
1991.
104. Eisman, R., Surrey, S., Ramachandran, B., Schwartz, E., and Poncz,
M. Structural
and functional
comparison
of the genes for human platelet
factor
105.
4 and PF4,,.
Blood,
Pan, J., and McEver,
76: 336-344,
1990.
R. P. Characterization
of the promoter
for the
human P-selectin gene. J. Biol. Chem., 268: 22600-22608,
1993.
106. Athanasiou,
M., Clausen,
P. A., Mavrothalassitis,
G. J., Zhang, X-K.,
Watson, D. K., and Blair, 0. G. Increased
expression
of the ETS-related
transcription
megakaryocytic
factor
FLI-1/ERGB
correlates with and can
phenotype.
Cell Growth Differ., 7: 152-134,
induce the
1996.
93. Swope, D. L, Mueller, C. L, and Chrivia, J. C. CREB-binding
protein
activates
transcription
through
multiple
domains.
J. Biol. Chem.,
271:
28138-28145,
1996.
107. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. Accurate transcription initiation by RNA polymerase
II in a soluble extract from isolated
94. Dallas, P. B., Yacluk,
P., and Moran, E. Characterization
of monoclonal antibodies
raised against p300: both p300 and CBP are present in
intracellular
TBP complexes.
J. Virol., 71: i 726-i 73i , 1997.
108. Brasier, A. A., Tate, J. E., and Habener,
J. F. Optimized
use of the
firefly luciferase assay as a reporter gene in mammalian
cell lines. Biotechniques,
7: 1 116-1 122, 1989.
95.
109. Eustice, D. C., Feldman, P. A., Colberg-Poley,
A. M., Buckery,
R. M.,
and Neubauer, R. H. A sensitive method for the detection of 3-galactosidase in transfected
mammalian cells. Biotechniques,
11: 739-742,
Kwok,
R. P., Lundblad,
J. R., Chrivia,
J. C., Richards,
J. P., Bachinger,
H. P., Brennan, R. G., Roberts, S. G., Green, M. R., and Goodman,
Nuclear
protein
CBP
is a coactivator
1994.
Nature (Lond.), 370: 223-226,
for the transcription
factor
R. H.
CREB.
mammalian
1991.
nuclei.
Nucleic
Acids
Res.,
5: 1475-1489,
1983.