|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2637-2644
Regulation of the Megakaryocytic Glycoprotein IX Promoter by the
Oncogenic Ets Transcription Factor Fli-1
By
L. Scot Bastian,
Boguslaw A. Kwiatkowski,
John Breininger,
Susan Danner, and
Gerald Roth
From the Hematology Section, Medical and Research Services, VA
Puget Sound Health Care System and the Divisions of Hematology
and Oncology, Department of Medicine, University of Washington,
Seattle, WA.
 |
ABSTRACT |
Glycoprotein (GP) IX is a subunit of the von Willebrand receptor,
GPIb-V-IX, which mediates adhesion of platelets to the subendothelium of damaged blood vessels. Previous characterization of the GPIX promoter identified a functional Ets site that, when disrupted, reduced
promoter activity. However, the Ets protein(s) that regulated GPIX
promoter expression was unknown. In this study, transient cotransfection of several GPIX promoter/reporter constructs into 293T
kidney fibroblasts with a Fli-1 expression vector shows that the
oncogenic protein Fli-1 can transactivate the GPIX promoter when an
intact GPIX Ets site is present. In addition, Fli-1 binding of the GPIX
Ets site was identified in antibody supershift experiments in nuclear
extracts derived from hematopoietic human erythroleukemia cells.
Comparative studies showed that Fli-1 was also able to transactivate
the GPIb and, to a lesser extent, the GPIIb promoter. Immunoblot
analysis identified Fli-1 protein in lysates derived from platelets. In
addition, expression of Fli-1 was identified immunohistochemically in
megakaryocytes derived from CD34+ cells treated with the
megakaryocyte differentiation and proliferation factor, thrombopoietin.
These results suggest that Fli-1 is likely to regulate lineage-specific
genes during megakaryocytopoiesis.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
HEMATOPOIETIC DEVELOPMENT is regulated by
a complex interplay of external and internal cell signaling that leads
to a decision process whereby primitive stem cells differentiate into
specific lineages.1 An integral part of this process is the
expression of lineage-restricted transcription factors that interact
with the complex regulatory machinery that controls differential gene
expression.2 Numerous laboratories have characterized hematopoietic transcription factors, which generally have been identified either on the basis of their binding to cis-acting promoter
elements or on the basis of their dysregulated expression, often as
fusion proteins, in leukemic cells.3
Megakaryocytes are the hematopoietic precursors of platelets, which
play an essential role in thrombosis and hemostasis.4,5 Several megakaryocyte-specific promoters, including platelet
factor-4,6 glycoprotein (GP) IIb,7-10
GPIb ,11 thrombopoietin receptor,12 and
GPIX13 have been characterized. These promoters are often short, generally less than 600 base pairs, lack TATA boxes, and usually
contain cis-acting regulatory elements regulated by the Sp1,14 GATA,15,16 and Ets17,18
families of transcription factors.
The Ets factors comprise a family of transcription factors that
regulate expression of several prominent hematopoietic genes. The
prototypical Ets protein, Ets-1, was originally named because of its
expression as part of an oncogenic protein by the
 - wenty- ix virus.19,20 Subsequent studies identified a number of Ets
transcription factors that share an 82 amino acid motif, termed the Ets
domain, that is required for binding to DNA sequences containing a
consensus core of 5'-GGAA/T-3' residues.21,22
Numerous hematopoietic genes have been described that are regulated by
Ets factors and several of these factors, including Tel, Erg, Ets-1,
Ets-2, and Fli-1 are involved with leukemia18 suggesting a
critical role in growth regulation of hematopoietic cells.
The Friend's leukemia integration-1 (Fli-1) protein, also known as
ErgB, is an Ets family member originally identified for its
overexpression in erythroleukemia in mice infected with Friend's leukemia virus.23 Fli-1 was independently discovered in
Ewing's sarcoma and peripheral neuroectodermal tumors with the Fli-1
carboxyterminus, including the DNA binding domain, linked to the
aminoterminus of the Ewing's sarcoma gene.24 Human Fli-1
is 97% homologous to the mouse gene.25 The mouse Fli-1
gene encodes two isoforms of 452 and 419 amino acids with molecular
weights of 51 and 48 kD respectively, which use alternate initiation
codons with the same reading frame.26 Both Fli-1 isoforms
have been identified in vivo27 and generated in
vitro.26 Fli-1 protein, like other Ets factors, has a
helix-turn-helix domain (amino acids 121-196), believed to be involved
with protein-protein interaction and an Ets domain (amino acids
277-360) that mediates DNA binding.28
The normal cellular target genes regulated by Fli-1 are unknown, but
recent experiments suggest that Fli-1 might play a prominent role in
the regulation of megakaryocytic genes. For example, Athanasiou et
al29 showed that the human erythro-megakaryocytic cell line K562, which normally lacks Fli-1, shows an increase in expression of
megakaryocytic features when transfected with a Fli-1 expression vector. Furthermore, promoters for the thrombopoietin
receptor,12 von Willebrand factor,30 and
GPIIb29 genes have been shown to be transactivated by
Fli-1.
GPIX is a subunit of the platelet von Willebrand factor receptor,
GPIb-IX-V, which is expressed in megakaryocytes.31
Deficiency in GPIX is associated with the rare human disorder,
Bernard-Soulier Syndrome.32 GPIX is expressed in and was
originally cloned from, the erythro-megakaryocytic human
erythroleukemia (HEL) cell line.33,34 Functional analysis
of the GPIX promoter identified an Ets site located between -35 and -49 relative to the GPIX transcriptional start site that, when disrupted,
reduced promoter activity as assessed by transient transfection into
HEL cells.13 The GPIX Ets site also bound to HEL cell
nuclear proteins in DNAse protection and gel mobility retardation
experiments. However, the Ets factor(s) that regulate the GPIX promoter
were unknown.13 Recent experiments35 showed
that the Ets factor, Tel, could abrogate Fli-1 transactivation when
reconstituted in the fibroblast-like 293T human embryonic kidney cell
line. Experiments described herein extend these observations comparing
Fli-1 transactivation of different GPIX constructs and showing Fli-1
binding in nuclear extracts derived from HEL cells. Additional
experiments compare Fli-1 transactivation of the GPIX, GPIb, and
GPIIb megakaryocyte promoters. Finally, Fli-1 protein was identified in
immunoblots of protein extracts derived from platelets and
immunohistochemically in human megakaryocytes. In summary, these data
provide evidence suggesting that Fli-1 is a likely candidate for
regulation of genes in megakaryocytes.
| |
MATERIALS AND METHODS |
Plasmid constructs.
Construction of plasmids GPIX5'-686Luc, GPIX5'-203Luc,
GPIX5'-69Luc, and pXP2 has been described.13,36
Plasmid GPIX5'-37Luc was constructed using polymerase chain
reaction (PCR) amplification of a GPIX promoter luciferase template
using a 5' sense primer that contained an Acc65 I restriction
site adaptor that bound at -37, relative to the GPIX promoter, in
conjunction with an antisense primer that bound in the luciferase gene.
The resulting fragment was restriction digested with Acc65 I and Bgl II
and inserted into the pXP2 luciferase expression plasmid that had been
predigested with the same enzymes. A similar strategy was used in the
construction of GPIb5'-567Luc, GPIIb5'-596Luc, and GPIIb5'-75Luc, inserting promoter sequences extending to 567, 596, and 75 base pairs, upstream of their respective transcriptional start sites into pXP2. GPIX5'-203MutLuc was constructed using PCR
amplification of a fragment of the GPIX promoter extending from -203 to
-45 relative to the GPIX transcriptional start site, followed by
restriction digestion and insertion into the HindIII and Acc 65 I sites of GPIX5'-37Luc. The resulting plasmid has GPIX promoter
sequence identical to GPIX5'-203Luc except that the 7 base region
that contains the Ets site between -37 and -45, ie,
5'-A C-3' was replaced with
5'-A C-3'. To screen for possible PCR
errors the fidelity of the GPIX constructs was confirmed by sequencing.
CMVFli-1 was constructed by insertion of a XhoI-NotI fragment
containing the human Fli-1 gene into pCRTM3 (Invitrogen,
Carlsbad, CA) predigested with the same enzymes.
Cell culture and transient transfections.
Human kidney 293T fibroblasts37 and K562
cells38 were cultured in Dulbecco's Modified Eagle Medium
(DMEM) supplemented with 0.584g/L glutamine, 4.5g/L glucose, 100 U/mL
penicillin, 0.1 mg/mL streptomycin, 25 mmol/L HEPES and 10%
heat-inactivated fetal bovine serum. The cells were cultured at 37°
in 5% CO2 and passaged three times per week. HEL
cells39 were cultured as described.13 Transient
transfections on cell lines were performed using the SUPERFECT TM
(Qiagen, Inc, Valencia, CA) according to the
manufacturer's instructions. Luciferase assays were performed as
previously described.13 Each assay was performed in
duplicate and adjusted for variations in transfection efficiency by
normalizing for activity generated by a cotransfected CMV gal
internal control plasmid.
Generation and purification of peripheral mobilized hematopoietic
CD34+ stem cells was performed on a service basis through
the laboratory of Scott Rowley at the Fred Hutchinson Cancer Research
Center. A male volunteer was injected with 5µg/kg/d with granulocyte
colony-stimulating factor followed by apheresis and purification of the
peripheral mobilized stem cells on an anti-CD34 antibody purification
column (CellPro, Borhell, WA). The CD34+
cells were cultured for 7 days in DMEM with Nutridoma (GIBCO-BRL, Gaithersburg, MD), 100 units/mL penicillin, 0.1 mg/mL streptomycin, and 10 ng/mL of thrombopoietin.
Gel mobility retardation assays.
Gel mobility retardation assays were performed as
described.40 Briefly, oligonucleotides that include the
GPIX Ets site, 5'-ATTTTCATCACTTCCTTCCGC-3' and its
complement were end-labeled with polynucleotide kinase and
( -32) adenosine triphosphate (ATP), followed by
annealing and purification on a nondenaturing polyacrylamide gel. To
test for factor binding a mixture was made of 30 µg of HEL nuclear
extracts, prepared as described,40 in 10 mmol/L Tris (pH
7.5), 50 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L dithiothreitol, 1 mmol/L EDTA, 12.5% glycerol, 0.1% Triton X-100 with
2 µg poly (dI-dC) as a nonspecific competitor. The 15 µL reactions
were preincubated for 20 minutess on ice before addition of probe
(30,000 disintegrations/minute ~.24 pmoles) and further incubation at
room temperature for 30 minutes. In samples containing antibodies, 5 µg of each antibody was added to the binding mixture followed by
incubation for an additional 30 minutes before loading on the gel.
Anti-Fli-1 (ab 1) is a purified mouse immunoglobulin G (IgG) antihuman
Fli-1 Ets domain monoclonal antibody (Pharmingen, San Diego, CA,
catalog # 15491A); anti-Fli-1 (ab 2) is a rabbit polyclonal IgG directed against the carboxyterminal 19 amino acids of
human Fli-1 (Santa Cruz Biotechnology, Santa Cruz, CA, catalog # sc-356) and the nonspecific control antibody is Mouse IgG
anti-IgG. After incubation the samples were separated on a 5%
polyacrylamide gel containing 50 mmol/L Tris, 380 mmol/L glycine, and 2 mmol/L EDTA running buffer. The gels were fixed in 10% methanol, 10% acetic acid, 5% glycerol, dried, and exposed to Kodak (Rochester, NY) X-OMAT AR film at  80°C with an intensifying screen.
Immunoblotting.
Immunoblotting was performed as described.41 Briefly, cell
lysates derived from 5 × 105 cells or 1.6 × 107 purified platelets42 were resolved on 7.5%
sodium dodecyl sulfate polyacrylamide gels followed by transfer to
polyvinylidene difluoride membranes (Bio-Rad cat # 162-184; Bio-Rad,
Hercules, CA). Primary antibodies ab 1 and ab 2 are
described above. The anti-Fli1 antibody used in Fig 4 has been
described.35 Secondary antibodies used were either the
monoclonal antirabbit -chain-specific IgG (Sigma catalog # A-2556;
Sigma, St Louis, MO) or the antirabbit Fab-specific IgG
(Sigma catalog # A-2179). The bands were visualized using Western Blue
stabilized substrate for alkaline phosphatase (Promega Catalog # S384B;
Promega, Madison, WI).
Immunofluorescence.
Megakaryocytes derived from thrombopoietin-treated CD34+
cells and 293T cells that were either mock transfected or transfected with CMVFli-1 expression vector were fixed in 4% paraformaldehyde in
phosphate buffered saline (PBS) (pH 7.2) for 10 minutes followed by
washing in PBS containing 1% bovine serum albumin. The cells were
incubated with rabbit anti-fli-1 antibody (Ab 2 described above) and
anti-GPIIb mouse monoclonal antibody (Pharmingen) for 30 minutes
followed by incubation with Goat antirabbit IgG linked to Cy3
fluorescent dye and Goat antimouse IgG linked to Cy2 fluorescent dye
(Jackson ImmunoResearch, West Grove, PA). The samples were incubated
for 45 minutes at room temperature in a humidified chamber in the dark
followed by washing in PBS and mounting in the DNA stain bisbenzimide
trihydrochloride (Sigma) (similar to Hoechst No. 33258, Kansas City, MO) dissolved in 15% polyvinyl alcohol, 10% glycerol,
0.01% Sodium azide, in 50 mmol/L Tris pH 9.0. Immunofluorescence was
analyzed on a Microcomputer Imaging Device using the M2 software package (Imaging Research, Ontario, Canada).
| |
RESULTS |
Transactivation of the GPIX promoter by Fli-1 in 293T fibroblasts.
To test whether Fli-1 could regulate GPIX promoter activity, constructs
were generated containing either intact, disrupted, or deleted GPIX Ets
sites linked to a luciferase reporter gene. The promoter constructs
were transiently cotransfected into fibroblast-like 293T cells in the
presence of either a Fli-1-expression vector, CMVFli-1, or nonspecific
plasmid, and the cells assayed for the presence of luciferase activity.
The left side of Fig 1A diagrams the five
luciferase constructs used in this experiment. GPIX5'-203Luc, GPIX5'-69Luc, and GPIX5'-37Luc contain promoter sequences
extending to 203, 69, and 37 base pairs upstream of the GPIX
transcriptional start site, respectively. GPIX5'-203Luc and
GPIX5'-69Luc contain intact GPIX Ets sites. GPIX5'-37Luc
contains GPIX promoter sequence that terminates just downstream of the
GPIX Ets consensus sequence. GPIX5'-203MutLuc is identical to
GPIX5'-203Luc, except the Ets site has been replaced with
irrelevant sequence. Plasmid pXP2 is the promoterless parental
luciferase construct from which the GPIX promoter constructs were
derived. The right side of Fig 1A shows activity generated by the
reporter vectors. Only constructs containing an intact Ets site,
GPIX5'-203 and GPIX5'-69, showed a significant increase,
approximately four- to fivefold, in luciferase activity in the presence
of CMVFli-1. In contrast, GPIX5'-203MutLuc, GPIX-37Luc, and pXP2,
which lack intact GPIX Ets sites, showed no significant increase in
activity in the presence of CMVFli-1. This indicates that the Fli-1
protein transactivates the GPIX promoter and that this activity is
mediated by the Ets site. Comparison of luciferase activities generated
in the presence of nonspecific plasmid, ie, the gray bars, identifies a
small but measurable Ets activity by both of the constructs containing
intact Ets sites, GPIX5'-203Luc and GP5'-69Luc, that is
absent in GPIX5'-203MutLuc, GPIX5'-37Luc, and pXP2, which
lack Ets sites. This indicates that 293T cells contain weak endogenous
Ets transactivation activity that is likely mediated by endogenous 293T
Ets factor(s) that operate through the GPIX Ets sequence. The identity
of this (these) factor(s) is unknown.
View larger version (15K):
[in this window]
[in a new window]
| Fig 1.
Fli-1 transactivation of the GPIX promoter in 293T kidney
fibroblasts. (A) Left: Diagrams of the luciferase reporter constructs
used in Fli-1 transactivation assays. The open boxes identify intact
GPIX Ets sites. The crossed out box identifies the region of the GPIX
promoter containing the Ets site that is replaced with irrelevant
sequence (See Materials and Methods). Plasmid pXP2 is a promoterless
construct that encodes the luciferase gene. (A) Right luciferase
activity, indicated as light units, generated in transiently
transfected 293T kidney fibroblasts. Each sample was transfected with
1.5 µg of luciferase reporter construct and 4.5 µg of the Fli-1
expression vector, CMVFli-1, or nonspecific plasmid, normalized using a
CMVgal control plasmid. Error bars represent deviations between
duplicate samples. Each plasmid was tested in at least seven
independent experiments. (B) shows an immunoblot analysis of lysates
derived from 293T kidney fibroblasts transfected with either pPCR3, the
empty expression vector lacking Fli-1, or CMVFli-1. Arrows identify the
two isoforms of Fli-1 and numbers indicate the migration pattern of
molecular weight markers.
|
|
To test for authentic Fli-1 expression lysates derived 293T cells that
were transfected with CMVFli-1 or plasmid pCR3, which is identical to
CMVFli-1 except that it lacks a Fli-1 complementary DNA, were analyzed
using immunoblot analysis using a Fli-1-specific antibody. Figure 1B
shows that the CMVFli-1 expression construct expressed two detectable
bands migrating with molecular weights of approximately 48 and 51 kD.
The two Fli-1 translation products are from different translational
start codons using the same reading frame, as has been previously
reported.27,26 The data indicate that the CMVFli-1 vector
encodes Fli-1 that is accurately translated into both isoforms of the
immunoreactive Fli-1 protein.
Identification of Fli-1 binding to the GPIX Ets site in the
erythro-megakaryocytic HEL cell line.
To test whether the GPIX Ets element can be regulated by Fli-1 in
hematopoietic cells, gel retardation supershift experiments were
performed using labeled, double-stranded oligonucleotide analogs of the
GPIX Ets site, nuclear extracts from HEL cells, and anti-Fli-1
antibodies. Figure 2A identifies three
DNA-protein complexes designated S1, S2, and S3. Previously published
experiments identified two of the DNA-protein complexes, S2 and
S3.13 Use of better quality nuclear extracts and improved
binding assay and gel conditions (see Materials and Methods) allowed
the identification of a third DNA-protein complex, S1, which migrates
more slowly than the other two complexes. Lanes b and c show patterns
of DNA-protein complex formation generated after incubation with a
monoclonal antibody directed against the Ets domain of Fli-1 (Ab-1) or
a polyclonal antibody directed against the Fli-1 carboxyterminus (Ab-2). Incubation with a nonspecific antibody shows a pattern of
DNA-protein complexes that is similar to the absence of antibody (compare Fig 2A Lanes a and d). Comparison of lanes b and c with a and
d indicate that in addition to formation of new retarded complexes,
designated SS1 and SS2, there is a diminution of intensity in the S1
band.
View larger version (44K):
[in this window]
[in a new window]
| Fig 2.
Fli-1 in the hematopoietic HEL cell line binds to GPIX
Ets sites. (A) Autoradiogram of a gel supershift experiment. Labeled
double-stranded oligonucleotide corresponding to bases -54 to -34 within the GPIX promoter was mixed with nuclear extracts derived from
HEL cells followed by addition of the indicated antibody. Shifted
complexes are indicated as S1, S2, and S3. Supershifted complexes,
observed only in lanes b and c, are indicated as SS1 and SS2. (B and C)
Immunoblots of separated extracts from HEL cells (left lanes), K562
cells (center), and 293T cells (right) that were transiently
transfected with the CMVFli-1 expression construct. (B) was probed with
the anti-Fli-1 (ab 1) antibody used in (A) lane b. (C) was probed with
the same anti--Fli-1 (ab 2) antibody that was used in (A) lane c.
Arrows identify bands of reactive protein that correspond to the sizes
of the two isoforms of Fli-1. Numbers on right indicate molecular
weight markers.
|
|
To test the specificity of the anti-Fli-1 antibodies, and to test for
the presence of authentic Fli-1 in HEL cells, immunoblots were
performed on membranes containing proteins derived from HEL, K562, and
293T cells transfected with CMVFli-1 expression vector. The K562 cell
line is a human erythro-megakaryocytic cell line that is known to not
express detectable Fli-1.29 Figure 2B and C shows that both
antibodies bound two prominent bands of approximately 48 and 51 kD,
that comigrated at the same rate as Fli-1 protein expressed in 293T
cells. There was no evident authentic Fli-1 expressed in K562 cells.
These data indicate that HEL cells contain Fli-1 protein of the
appropriate size and that the supershift complexes shown in Fig 2A are
very likely to contain Fli-1 protein.
Fli-1 transactivation of megakaryocyte promoters GPIb
and GPIIb.
To test whether Fli-1 could transactivate other megakaryocyte
promoters, 293T cells were transiently cotransfected with the CMVFli-1
expression vector and luciferase expression vectors containing sequences derived from the GPIX, GPIb, and GPIIb promoters. The left
side of Fig 3 diagrams the known Ets sites
in each promoter construct, indicated as boxes. GPIIb has two Ets sites
located at -515 and -40 and the GPIb promoter contains one Ets site
located at -144 relative to their respective transcription start sites. Both the GPIX GPIIb5'-596Luc and GPIIb5'-75Luc promoter
constructs show an increase in promoter activity in the presence of
Fli-1 of approximately four- to sixfold, whereas the GPIIb promoter constructs GPIIb5'-75Luc and GPIIb-596Luc appear to be less
responsive to Fli-1 showing an induction of approximately twofold.
View larger version (12K):
[in this window]
[in a new window]
| Fig 3.
Fli-1 transactivation of GPIX, GPIb, and GPIIb
promoter constructs. The left side of the figure diagrams the
luciferase reporter constructs used in Fli-1 transactivation assays.
The  identifies Ets sites. The exact locations of the Ets boxes are
identified in the text. The transfection was performed as described in
Materials and Methods and the legend to Fig 1. Error bars represent
deviations between duplicate samples. Each plasmid was tested in at
least three independent experiments, all with essentially with the same
results.
|
|
Identification of Fli-1 in platelets.
To investigate whether Fli-1 is a likely candidate for regulation of
genes expressed in megakaryocytes immunoblot analysis was performed on
lysates derived from human platelets.
Figure 4 shows an immunoblot comparing
Fli-1 protein in lysates derived from purified platelets with Fli-1
protein expressed in 293T cells transfected as described for Fig 1.
Platelets contain protein that comigrates with Fli-1 expressed in
transiently transfected 293T cells. Platelets do not have nuclei, and
therefore lack transcription. Furthermore, platelets have few ribosomes
and likely have very low levels of translational activity. This
suggests that the Fli-1 contained in platelets is protein that was
expressed in the megakaryocyte. Although platelets are a common source
for platelet integrins and other surface proteins,42 they
are not usually used as source for gene regulatory proteins. The
presence of Fli-1 in platelets may be a reflection of the abundance
and/or stability of the protein.
View larger version (27K):
[in this window]
[in a new window]
| Fig 4.
Fli-1 protein is contained in platelets. Immunoblot
analysis using an anti-Fli-1 antibody was performed on lysates derived
from platelets that were separated in parallel with molecular weight
markers (shown on right) and lysate derived from 293T cells transiently
transfected with the CMVFli-1 expression construct. Arrows identify the
two isoforms of Fli-1.
|
|
Identification of Fli-1 in megakaryocytes.
We next used indirect immunofluorescence to test whether Fli-1 is
expressed in megakaryocytes. Figure 5 (A
and B) shows 293T cells that were transfected with a nonspecific
plasmid (A) or with the CMVFli-1 expression construct (B). The cyan
color shows Hoechst staining in all cells, based on DNA content, and
the magenta color identifies cells positive for expression of Fli-1.
Only the population of 293T cells that that was transfected with the CMVFli-1 expression vector contained Fli-1-positive cells. (Compare 5A
to 5B.) This established the specificity of our indirect
immunofluorescence staining procedure. To test whether Fli-1 is
expressed in megakaryocytes, indirect immunofluorescence was performed
on human megakaryocytes derived from CD34+ cells treated
for 7 days with thrombopoietin. Figure 5C and 5D shows primary
megakaryocytes derived from peripheral mobilized CD34+
cells treated with the megakaryocyte differentiation and proliferation agent, thrombopoietin. Figure 5C shows double staining with both the
blue Hoechst DNA stain and red stain that identifies antibody directed
against Fli-1. Colocalized DNA and Fli-1 signals appear as magenta.
Figure 5D shows the same field of cells showing double staining with
antibodies directed against the megakaryocyte differentiation marker
GPIIb, green color, and Fli-1, which appears orange in 5D. The larger
cells show multilobed nuclei that are typical of mature, polyploid
megakaryocytes. The GPIIb antigen localizes to the cytoplasm and the
Fli-1 antigen is expressed predominantly in the nucleus. However, there
is detectable Fli-1 in the megakaryocyte cytoplasm (More easily
visualized in 5C). This experiment shows that Fli-1 is expressed in
megakaryocytes, consistent with a role for Fli-1 regulation of
megakaryocyte genes.
View larger version (69K):
[in this window]
[in a new window]
| Fig 5.
Fli-1 protein is expressed in human megakaryocytes. A and
B show immunohistochemical analysis for Fli-1 expression in 293T cells
that were transiently transfected with either nonspecific plasmid (A)
or the Fli-1 expression construct CMVFli-1 (B). Hoechst DNA staining is
cyan and Fli-1 staining is red. Regions that colocalize for DNA and
Fli-1 staining appear as magenta. Panels C and D show
CD34+ cells that were treated with 10 ng/mL of
thrombopoietin for 7 days before triple staining. Panel C shows Hoechst
DNA stain (blue) and Fli-1 (red). Areas of colocalization appear as
magenta. Panel D displays the same cells as panel C showing GPIIb
antigen (green) and Fli-1 (orange). In panel D Fli-1 appears as orange.
Original magnification of panels A and B was 40× and panels C and D
was 200×.
|
|
| |
DISCUSSION |
Because of their relative rarity, fragility, and apoptotic
developmental fate,43 gene expression in megakaryocytes is
difficult to study compared with other hematopoietic lineages. Although several megakaryocyte promoters have been characterized, most analyses
of these promoters have used hematopoietic cell lines to identify the
factors and sequences that regulate megakaryocyte expression. In fact,
a megakaryocytic-specific transcription factor has not yet been
described. Thus, it is difficult to confirm precisely which factors
govern megakaryocyte gene expression. Fortunately, comparisons of
activities of transcriptional elements in hematopoietic cell lines and
the few promoter regulatory studies in primary megakaryocytes have
confirmed that sites that are active in hematopoietic cell lines are
active in primary megakaryocytes.44 The recent cloning of
the megakaryocyte inducer thrombopoietin45-48 has made it
feasible to generate enough megakaryocytes to do biochemical-based experiments and analyze megakaryocyte developmental
regulators49 giving rise to the possibility that
megakaryocyte-specific factors, if such exist, may be identified.
Members of the Ets transcription factor family have been identified in
numerous different tissues, but seem particularly important to
hematopoietic development. There is little information available regarding the expression of Ets factors in megakaryocytes, although Pu.1 has been identified immunohistochemically.50 However,
erythro-megakaryocytic cell lines are known to express several Ets
factors, including Ets-1, Pu.1, Fli-1, Ets-2, Elf-1, Erm, and
SAP-1.12,51 Several reports have described transactivation
of megakaryocytic promoters using reconstitution assays in
nonhematopoietic cells. For example, Ets-1 is known to transactivate
the GPIIb promoter in HeLa cells that are transiently cotransfected
with GPIIb promoter constructs.52 It has also been shown
that the Ets factor Pu.1 can transactivate the promoter for the
megakaryocyte-specific platelet basic protein.53 The
thrombopoietin receptor promoter can be activated by both Ets-1 and
Fli-1,12 and the promoter for von Willebrand factor could
be transactivated by both Ets-1 and Erg,30 indicating that
promoters may be regulated by multiple Ets factors. A recent report
described transactivation of the GPIIb promoter by the Ets factor Pu.1
that was dependent on thrombopoietin stimulation.54 These
observations suggest that megakaryocyte promoters may be regulated by
multiple Ets factors.
The normal target(s) of Fli-1 is uncertain. Fli-1 is expressed in a
variety of hematopoietic cell lines and several tissues, including
spleen and thymus, and at low levels in embryonic endothelium, heart,
lung, and skeletal muscle.55 Fli-1 expression has also been
described in cells in embryonic mouse liver that have a characteristic megakaryocytic appearance but the identity of the cells was not definitively established.55 Transgenic studies showed that
overexpression of a Fli-1 transgene under control of the
H-2Kk promoter caused a lethal autoimmune renal disease,
but surprisingly, not an increase in cancer.27 Recently,
Melet et al55 described a partial disruption of the
amino-terminal 76 amino acids of the Fli-1, leaving intact activation
and Ets domains, which led to a reduction in thymic cellularity and an
extension in latency of onset of erythroleukemia mediated by Friend
Leukemia Virus infection.
The presence of the S2 and S3 complexes shown in Fig 2 indicates that
the GPIX Ets site binds, and may be regulated by, other Ets proteins.
Whether the GPIX promoter can be regulated by Ets factors other than
Fli-1 is currently under investigation. Further experiments are in
progress to test whether Ets factors may functionally interact. These
experiments may be particularly informative in light of the
differential transactivation by Fli-1 of the promoters tested in Fig 3.
Figure 4 shows the presence of Fli-1 protein in lysates derived from
purified platelets. It seems unlikely that Fli-1 is translated de novo
in platelets. It seems more probable that the presence of Fli-1 in
platelets represents residual protein found in the megakaryocyte.
Similar assays testing for the presence of Ets-1 were unsuccessful
(data not shown). Thus the presence of Fli-1 may reflect the abundance
and/or stability of the protein. It is also possible that Fli-1 serves
some unknown function in platelet physiology. Figure 5 shows that Fli-1
is expressed in human megakaryocytes and the presence of detectable
cytoplasmic Fli-1 may account for the presence of trace Fli-1 in platelets.
In summary, this study provides evidence that the oncogenic Ets factor
Fli-1 can regulate several megakaryocyte genes, including GPIX,
GPIb, and GPIIb. Furthermore, Fli-1 is present in the hematopoietic HEL cell line and it binds to the GPIX Ets site. In addition, Fli-1
protein was identified both in platelets and in megakaryocytes. In
toto, these data suggest that Fli-1 may be an important gene regulator
in megakaryocyte gene expression.
| |
ACKNOWLEDGMENT |
We thank Anna Zielinska-Kwiatkowska and Shawn Mohamed for technical
assistance, Kin Ritchie for critical reading of the manuscript, and
Sally Swedine for assistance with preparation of the figures.
| |
FOOTNOTES |
Submitted May 27, 1998; accepted December 2, 1998.
Supported by a National Research Service Award F32 HL09265 and
R01-DK49855-01 (L.S.B.). G.R. is supported by a Merit Review Grant from
the Veterans Administration and NIH grant HL39947.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Gerald Roth, MD, VA Puget Sound Health Care
System (M/S 111), 1660 S. Columbian Way, Seattle, WA 98108.
| |
REFERENCES |
1.
Graham GJ, Wright EG:
Haemopoietic stem cells: Their heterogeneity and regulation.
Int J Exp Pathol
78:197, 1997[Medline]
[Order article via Infotrieve]
2.
Shivdasani RA, Orkin SH:
The transcriptional control of hematopoiesis.
Blood
87:4025, 1996[Free Full Text]
3.
Tenen DG, Hromas R, Licht JD, Zhang DE:
Transcription factors, normal myeloid development, and leukemia.
Blood
90:489, 1997[Free Full Text]
4.
Packham MA:
Role of platelets in thrombosis and hemostasis.
Can J Physiol Pharmacol
72:278, 1994[Medline]
[Order article via Infotrieve]
5.
Roth GJ:
Platelets and blood vessels: The adhesion event.
Immunol Today
13:100, 1992[Medline]
[Order article via Infotrieve]
6.
Ravid K, Doi T, Beeler DL, Kuter DJ, Rosenberg RD:
Transcriptional regulation of the rat platelet factor 4 gene: interaction between an enhancer/silencer domain and the GATA site.
Mol Cell Biol
11:6116, 1991[Abstract/Free Full Text]
7.
Block KL, Poncz M:
Platelet glycoprotein IIb gene expression as a model of megakaryocyte-specific expression.
Stem Cells
13:135, 1995[Medline]
[Order article via Infotrieve]
8.
Block KL, Shou YP, Poncz M:
An Ets/Sp1 interaction in the 5'-flanking region of the megakaryocyte-specific alpha IIb gene appears to stabilize Sp1 binding and is essential for expression of this TATA-less gene.
Blood
88:2071, 1996[Abstract/Free Full Text]
9.
Prandini MH, Martin F, Thevenon D, Uzan G:
The tissue-specific transcriptional regulation of the megakaryocytic glycoprotein IIb gene is controlled by interactions between a repressor and positive cis-acting elements.
Blood
88:2062, 1996[Abstract/Free Full Text]
10.
Fong AM, Santoro SA:
Transcriptional regulation of alpha IIb integrin gene expression during megakaryocytic differentiation of K562 cells. Role of a silencer element.
J Biol Chem
269:18441, 1994[Abstract/Free Full Text]
11.
Hashimoto Y, Ware J:
Identification of essential GATA and Ets binding motifs within the promoter of the platelet glycoprotein Ib alpha gene.
J Biol Chem
270:24532, 1995[Abstract/Free Full Text]
12.
Deveaux S, Filipe A, Lemarchandel V, Ghysdael J, Rom'eo PH, Mignotte V:
Analysis of the thrombopoietin receptor (MPL) promoter implicates GATA and Ets proteins in the coregulation of megakaryocyte-specific genes.
Blood
87:4678, 1996[Abstract/Free Full Text]
13.
Bastian LS, Yagi M, Chan C, Roth GJ:
Analysis of the megakaryocyte glycoprotein IX promoter identifies positive and negative regulatory domains and functional GATA and Ets sites.
J Biol Chem
271:18554, 1996[Abstract/Free Full Text]
14.
Hagen G, Dennig J, Preiss A, Beato M, Suske G:
Functional analyses of the transcription factor Sp4 reveal properties distinct from Sp1 and Sp3.
J Biol Chem
270:24989, 1995[Abstract/Free Full Text]
15.
Orkin SH:
GATA-binding transcription factors in hematopoietic cells.
Blood
80:575, 1992[Free Full Text]
16.
Weiss MJ, Orkin SH:
GATA transcription factors: Key regulators of hematopoiesis.
Exp Hematol
23:99, 1995[Medline]
[Order article via Infotrieve]
17.
Papas TS, Bhat NK, Spyropoulos DD, Mjaatvedt AE, Vournakis J, Seth A, Watson DK:
Functional relationships among ETS gene family members.
Leukemia
3:557, 1997 (11 suppl)
18.
Bassuk AG, Leiden JM:
The role of Ets transcription factors in the development and function of the mammalian immune system.
Adv Immunol
64:65, 1997[Medline]
[Order article via Infotrieve]
19.
Nunn MF, Seeburg PH, Moscovici C, Duesberg PH:
Tripartite structure of the avian erythroblastosis virus E26 transforming gene.
Nature
306:391, 1983[Medline]
[Order article via Infotrieve]
20.
Leprince D, Gegonne A, Coll J, de T-C, Schneeberger A, Lagrou C, Stehelin D:
A putative second cell-derived oncogene of the avian leukaemia retrovirus E26.
Nature
306:395, 1983[Medline]
[Order article via Infotrieve]
21.
Macleod K, Leprince D, Stehelin D:
The ets gene family.
Trends Biochem Sci
17:251, 1992[Medline]
[Order article via Infotrieve]
22.
Seth A, Ascione R, Fisher RJ, Mavrothalassitis GJ, Bhat NK, Papas TS:
The ets gene family.
Cell Growth Differ
3:327, 1992[Medline]
[Order article via Infotrieve]
23.
Ben-David Y, Giddens EB, Letwin K, Bernstein A:
Erythroleukemia induction by Friend murine leukemia virus: Insertional activation of a new member of the ets gene family, Fli-1, closely linked to c-ets-1.
Genes Dev
5:908, 1991[Abstract/Free Full Text]
24.
Delattre O, Zucman J, Plougastel B, Desmaze C, Melot T, Peter M, Kovar H, Joubert I, de J-P, Rouleau G, Alain A, Thomas G:
Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours.
Nature
359:162, 1992[Medline]
[Order article via Infotrieve]
25.
Prasad DD, Rao VN, Reddy ES:
Structure and expression of human Fli-1 gene.
Cancer Res
52:5833, 1992[Abstract/Free Full Text]
26.
Zhang L, Lemarchandel V, Romeo PH, Ben D-Y, Greer P, Bernstein A:
The Fli-1 proto-oncogene, involved in erythroleukemia and Ewing's sarcoma, encodes a transcriptional activator with DNA-binding specificities distinct from other Ets family members.
Oncogene
8:1621, 1993[Medline]
[Order article via Infotrieve]
27.
Zhang L, Eddy A, Teng YT, Fritzler M, Kluppel M, Melet F, Bernstein A:
An immunological renal disease in transgenic mice that overexpress Fli-1, a member of the ets family of transcription factor genes.
Mol Cell Biol
15:6961, 1995[Abstract]
28.
Seth A, Papas TS:
The c-ets-1 proto-oncogene has oncogenic activity and is positively autoregulated.
Oncogene
5:1761, 1990[Medline]
[Order article via Infotrieve]
29.
Athanasiou M, Clausen PA, Mavrothalassitis GJ, Zhang XK, Watson DK, Blair DG:
Increased expression of the ETS-related transcription factor FLI-1/ERGB correlates with and can induce the megakaryocytic phenotype.
Cell Growth Differ
7:1525, 1996[Abstract]
30.
Schwachtgen JL, Janel N, Barek L, DuterqueCoquillaud M, Ghysdael J, Meyer D, KerbiriouNabias D:
Ets transcription factors bind and transactivate the core promoter of the von Willebrand factor gene.
Oncogene
15:3091, 1997[Medline]
[Order article via Infotrieve]
31.
Lopez JA:
The platelet glycoprotein Ib-IX complex.
Blood Coagul Fibrinolysis
5:97, 1994[Medline]
[Order article via Infotrieve]
32.
Clemetson KJ, McGregor JL, James E, Dechavanne M, Luscher EF:
Characterization of the platelet membrane glycoprotein abnormalities in Bernard-Soulier syndrome and comparison with normal by surface-labeling techniques and high-resolution two-dimensional gel electrophoresis.
J Clin Invest
70:304, 1982
33.
Lopez JA, Chung DW, Fujikawa K, Hagen FS, Davie EW, Roth GJ:
The alpha and beta chains of human platelet glycoprotein Ib are both transmembrane proteins containing a leucine-rich amino acid sequence.
Proc Nat Acad Sci USA
85:2135, 1988[Abstract/Free Full Text]
34.
Calverley DC, Yagi M, Stray SM, Roth GJ:
Human platelet glycoprotein V: Its role in enhancing expression of the glycoprotein Ib receptor.
Blood
86:1361, 1995[Abstract/Free Full Text]
35.
Kwiatkowski BA, Bastian LS, Bauer J, T. R, Tsai S, Zielinska-Kwiatkowska AG, Hickstein DD:
The ets family member Tel binds the Fli-1 oncoprotein and inhibits its transcriptional activity.
J Biol Chem
273:17525, 1998[Abstract/Free Full Text]
36.
Nordeen SK:
Luciferase reporter gene vectors for analysis of promoters and enhancers.
Biotechniques
6:454, 1988[Medline]
[Order article via Infotrieve]
37.
DuBridge RB, Tang P, Hsia HC, Leong PM, Miller JH, Calos MP:
Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system.
Mol Cell Biol
7:379, 1987[Abstract/Free Full Text]
38.
Gewirtz AM, Burger D, Rado TA, Benz EJ Jr, Hoffman R:
Constitutive expression of platelet glycoproteins by the human leukemia cell line K562.
Blood
60:785, 1982[Abstract/Free Full Text]
39.
Tabilio A, Rosa JP, Testa U, Kieffer N, Nurden AT, Del C-MC, Breton G-J, Vainchenker W:
Expression of platelet membrane glycoproteins and alpha-granule proteins by a human erythroleukemia cell line (HEL).
EMBO J
3:453, 1984[Medline]
[Order article via Infotrieve]
40.
Ausabel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K:
Current Protocols in Molecular Biology. New York, NY, Wiley-Interscience, 1991.
41.
Sambrook J, Fritsch EF, Maniatis T:
Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY, Cold Spring Harbor Press, 1989.
42.
Canfield VA, Ozols J, Nugent D, Roth GJ:
Isolation and characterization of the alpha and beta chains of human platelet glycoprotein Ib.
Biochem Biophys Res Commun
147:526, 1987[Medline]
[Order article via Infotrieve]
43.
Zauli G, Vitale M, Falcieri E, Gibellini D, Bassini A, Celeghini C, Columbaro M, Capitani S:
In vitro senescence and apoptotic cell death of human megakaryocytes.
Blood
90:2234, 1997[Abstract/Free Full Text]
44.
Block KL, Ravid K, Phung QH, Poncz M:
Characterization of regulatory elements in the 5'-flanking region of the rat GPIIb gene by studies in a primary rat marrow culture system.
Blood
84:3385, 1994[Abstract/Free Full Text]
45.
Lok S, Kaushansky K, Holly RD, Kuijper JL, Lofton D-CE, Oort PJ, Grant FJ, Heipel MD, Burkhead SK, Kramer JM, Bell LA, Sprecher CA, Blumberg H, Johnson R, Prunkard D, Ching AFT, Marhewes SL, Bailey MC, Forstrom JW, Buddle MM, Osborn SG, Evans SJ, Sheppard PO, Presnell SR, O'Hara PJ, Hagen FS, Roth GJ, Foster DC:
Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo.
Nature
369:565, 1994[Medline]
[Order article via Infotrieve]
46.
Kaushansky K, Lok S, Holly RD, Broudy VC, Lin N, Bailey MC, Forstrom JW, Buddle MM, Oort PJ, Hagen FS, Roth GJ, Papayannopoulou T, Foster DC:
Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin.
Nature
369:568, 1994[Medline]
[Order article via Infotrieve]
47.
Wendling F, Maraskovsky E, Debili N, Florindo C, Teepe M, Titeux M, Methia N, Breton G-J, Cosman D, Vainchenker W:
cMpl ligand is a humoral regulator of megakaryocytopoiesis.
Nature
369:571, 1994[Medline]
[Order article via Infotrieve]
48.
de Sauvage FJ, Hass PE, Spencer SD, Malloy BE, Gurney AL, Spencer SA, Darbonne WC, Henzel WJ, Wong SC, Kuang WJ, Oles KJ, Hultgren B, Solberg Jr LA, Goeddel DV, Eaton DL:
Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand.
Nature
369:533, 1994[Medline]
[Order article via Infotrieve]
49.
Lecine P, Blank V, Shivdasani R:
Characterization of the hematopoietic transcription factor NF-E2 in primary murine megakaryocytes.
J Biol Chem
273:7572, 1998[Abstract/Free Full Text]
50.
Hromas R, Orazi A, Neiman RS, Maki R, Van B-C, Moore J, Klemsz M:
Hematopoietic lineage- and stage-restricted expression of the ETS oncogene family member PU.1.
Blood
82:2998, 1993[Abstract/Free Full Text]
51.
Monte D, Baert JL, Defossez PA, de L-Y, Stehelin D:
Molecular cloning and characterization of human ERM, a new member of the Ets family closely related to mouse PEA3 and ER81 transcription factors.
Oncogene
9:1397, 1994[Medline]
[Order article via Infotrieve]
52.
Lemarchandel V, Ghysdael J, Mignotte V, Rahuel C, Roméo PH:
GATA and Ets cis-acting sequences mediate megakaryocyte-specific expression.
Mol Cell Biol
13:668, 1993[Abstract/Free Full Text]
53.
Zhang CY, Gadue P, Scott E, Atchison M, Poncz M:
Activation of the megakaryocyte-specific gene platelet basic protein (PBP) by the Ets family factor PU.1.
J Biol Chem
272:26236, 1997[Abstract/Free Full Text]
54.
Doubeikovski A, Uzan G, Doubeikovski Z, Prandini MH, Porteu F, Gisselbrecht S, Dusanter F-I:
Thrombopoietin-induced expression of the glycoprotein IIb gene involves the transcription factor PU.1/Spi-1 in UT7-Mpl cells.
J Biol Chem
272:24300, 1997[Abstract/Free Full Text]
55.
Melet F, Motro B, Rossi DJ, Zhang LQ, Bernstein A:
Generation of a novel fli-1 protein by gene targeting leads to a defect in thymus development and a delay in friend virus-induced erythroleukemia.
Mol Cell Biol
16:2708, 1996[Abstract]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
F. Bouilloux, G. Juban, N. Cohet, D. Buet, B. Guyot, W. Vainchenker, F. Louache, and F. Morle
EKLF restricts megakaryocytic differentiation at the benefit of erythrocytic differentiation
Blood,
August 1, 2008;
112(3):
576 - 584.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Pang, H.-H. Xue, G. Szalai, X. Wang, Y. Wang, D. K. Watson, W. J. Leonard, G. A. Blobel, and M. Poncz
Maturation stage-specific regulation of megakaryopoiesis by pointed-domain Ets proteins
Blood,
October 1, 2006;
108(7):
2198 - 2206.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Rainis, T. Toki, J. E. Pimanda, E. Rosenthal, K. Machol, S. Strehl, B. Gottgens, E. Ito, and S. Izraeli
The Proto-Oncogene ERG in Megakaryoblastic Leukemias
Cancer Res.,
September 1, 2005;
65(17):
7596 - 7602.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Hu, A. S. Philips, J. C. Kwok, M. Eisbacher, and B. H. Chong
Identification of Nuclear Import and Export Signals within Fli-1: Roles of the Nuclear Import Signals in Fli-1-Dependent Activation of Megakaryocyte-Specific Promoters
Mol. Cell. Biol.,
April 15, 2005;
25(8):
3087 - 3108.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. K. Zhang and D. K. Watson
The FLI-1 Transcription Factor Is a Short-Lived Phosphoprotein in T Cells
J. Biochem.,
March 1, 2005;
137(3):
297 - 302.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Jackers, G. Szalai, O. Moussa, and D. K. Watson
Ets-dependent Regulation of Target Gene Expression during Megakaryopoiesis
J. Biol. Chem.,
December 10, 2004;
279(50):
52183 - 52190.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Okada, R. Nagai, T. Sato, E. Matsuura, T. Minami, I. Morita, and T. Doi
Homeodomain proteins MEIS1 and PBXs regulate the lineage-specific transcription of the platelet factor 4 gene
Blood,
June 15, 2003;
101(12):
4748 - 4756.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. E. Elagib, F. K. Racke, M. Mogass, R. Khetawat, L. L. Delehanty, and A. N. Goldfarb
RUNX1 and GATA-1 coexpression and cooperation in megakaryocytic differentiation
Blood,
June 1, 2003;
101(11):
4333 - 4341.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Eisbacher, M. L. Holmes, A. Newton, P. J. Hogg, L. M. Khachigian, M. Crossley, and B. H. Chong
Protein-Protein Interaction between Fli-1 and GATA-1 Mediates Synergistic Expression of Megakaryocyte-Specific Genes through Cooperative DNA Binding
Mol. Cell. Biol.,
May 15, 2003;
23(10):
3427 - 3441.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Starck, N. Cohet, C. Gonnet, S. Sarrazin, Z. Doubeikovskaia, A. Doubeikovski, A. Verger, M. Duterque-Coquillaud, and F. Morle
Functional Cross-Antagonism between Transcription Factors FLI-1 and EKLF
Mol. Cell. Biol.,
February 15, 2003;
23(4):
1390 - 1402.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. L. Holmes, N. Bartle, M. Eisbacher, and B. H. Chong
Cloning and Analysis of the Thrombopoietin-induced Megakaryocyte-specific Glycoprotein VI Promoter and Its Regulation by GATA-1, Fli-1, and Sp1
J. Biol. Chem.,
December 6, 2002;
277(50):
48333 - 48341.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Furihata and T. J. Kunicki
Characterization of Human Glycoprotein VI Gene 5' Regulatory and Promoter Regions
Arterioscler Thromb Vasc Biol,
October 1, 2002;
22(10):
1733 - 1739.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. R. Hodge, W. Xiao, P. A. Clausen, G. Heidecker, M. Szyf, and W. L. Farrar
Interleukin-6 Regulation of the Human DNA Methyltransferase (HDNMT) Gene in Human Erythroleukemia Cells
J. Biol. Chem.,
October 19, 2001;
276(43):
39508 - 39511.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Eisbacher, L. M. Khachigian, T. H. Khin, M. L. Holmes, and B. H. Chong
Inducible Expression of the Megakarocyte-specific Gene Glycoprotein IX Is Mediated through an Ets Binding Site and Involves Upstream Activation of Extracellular Signal-regulated Kinase
Cell Growth Differ.,
August 1, 2001;
12(8):
435 - 445.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. D. Spyropoulos, P. N. Pharr, K. R. Lavenburg, P. Jackers, T. S. Papas, M. Ogawa, and D. K. Watson
Hemorrhage, Impaired Hematopoiesis, and Lethality in Mouse Embryos Carrying a Targeted Disruption of the Fli1 Transcription Factor
Mol. Cell. Biol.,
August 1, 2000;
20(15):
5643 - 5652.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
E. de Alava, A. Panizo, C. R. Antonescu, A. G. Huvos, F. J. Pardo-Mindan, F. G. Barr, and M. Ladanyi
Association of EWS-FLI1 Type 1 Fusion with Lower Proliferative Rate in Ewing’s Sarcoma
Am. J. Pathol.,
March 1, 2000;
156(3):
849 - 855.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION