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HEMATOPOIESIS
Initiation of polyoma virus origin-dependent DNA replication through STAT5
activation by human granulocyte-macrophage colony-stimulating factor
Sumiko Watanabe, Rong Zeng, Yutaka Aoki, Tohru Itoh, and Ken-ichi Arai
Several lines of evidence indicate that
transcriptional activation is coupled with
DNA replication initiation, but the nature
of initiation of DNA replication in mammalian cells is unclear. Polyoma virus replicon is an excellent system to analyze the
initiation of DNA replication in murine
cells because its replication requires an
enhancer, and all components of replication machinery, except for DNA helicase
large T antigen, are supplied by host
cells. This system was used to examine
the role of signal transducer and activator
of transcription (STAT5) in replication initiation of polyoma replicon in the mouse
lymphoid cell line BA/F3. The plasmid
with tandem repeats of consensus STAT5
binding sites followed by polyoma replication origin was replicated by stimulation
with human granulocyte-macrophage
colony-stimulating factor (hGM-CSF) in
the presence of polyoma large T antigen
in BA/F3 cells. Mutation analysis of the
hGM-CSF receptor ␤ subunit revealed
that only the box1 region is essential, and
the C-terminal tyrosine residues are dispensable for the activity. Addition of the
tyrosine kinase inhibitor genistein suppressed this replication without affecting
transcriptional activation of STAT5. Because deletion analysis of STAT5 indicates the importance of the C-terminal
transcriptional activation domain of STAT5
for the initiation of replication, the role of
this region in the activation of replication
was examined with a GAL4–STAT5 fusion
protein. GAL4–STAT5 activated replication of the plasmid containing tandem
repeats of GAL4 binding sites and polyoma replication origin in BA/F3 cells. Mutation analysis of GAL4–STAT5 indicated
that multiple serine residues coordinately
have a role in activating replication. This
is the first direct evidence indicating
the potential involvement of STAT5 in
replication. (Blood. 2001;97:1266-1273)
© 2001 by The American Society of Hematology
Introduction
Roles of Janus kinase (JAK) and signal transducer and activator of
transcription (STAT) in cytokine signal transduction were first
identified in interferon (IFN) signaling pathways, and it was
revealed that all members of the cytokine receptor superfamily
activate JAK and STAT.1,2 To date, 7 members of the STAT family
with similar structural features, STAT1 to STAT6, have been
identified.3 A DNA binding domain is located in the amino-terminal
half, and linker and Src homology 2 (SH2) domains followed by
the transactivation domain are in the carboxyl-terminal half.2 A
conserved Y residue (single-letter amino acid code) is located in the
C-terminal region, and phosphorylation of this residue plays an
essential role in the dimerization and nuclear translocation of
STAT. S residues in the more C-terminal region of the Y residues
are phosphorylated by extracellular signal regulated kinase (Erk),
p38 mitogen activated protein kinase (p38 MAPK), or Jun N
terminal kinase (JNK), which is implicated in the transcriptional
activity of STAT1 and STAT3.4 Mechanisms related to the contribution of phosphorylated S residues in transcriptional activation are
not well understood. As observed initially in the IFN system,
accumulating evidence suggests that STAT proteins are involved in
the activation of cytokine-specific genes,5-8 and knockout studies of
various STATs strongly support this evidence.9-15
Receptors of granulocyte-macrophage colony-stimulating factor (GM-CSFR) consist of 2 subunits, ␣ and ␤, both of which are
members of the cytokine receptor superfamily.16,17 The ␣ subunit is
specific for each cytokine, and the ␤ subunit (␤c) is shared by
interleukin 3 (IL-3), GM-CSF, and IL-5 receptors.17 GM-CSF
induces tyrosine phosphorylation of ␤c and various cellular
proteins and activates early-response genes and cell proliferation in
hematopoietic cells and in fibroblasts.18 The ␤c contains conserved
box1 and box2 regions and 8 Y residues located in the cytoplasmic
region.19 We and others have analyzed biologic activities of various
mutants of ␤c20-23 and found that multiple distinct signaling
pathways that use different receptor domains or Y residues are
activated by hGM-CSFR.24-26 Apparently only the box1 region is
essential for cell proliferation, whereas signaling cascades such as
MAPK and phosphatidylinositol 3-kinase (PI3K), which are transduced through C-terminus Y residues, are dispensable. Because
box1 is assumed to bind JAK2, it is likely that activation of JAK2 is
sufficient for promotion of proliferation, and this notion was
supported by the experiments using chimeric JAK2 protein, which
can induce BA/F3 cell proliferation without the activation of
MAPK or PI3K cascades.27
GM-CSF activates JAK2 and STAT5 in various hematopoietic
cells.28,29 STAT5A and STAT5B genes encode proteins that are
approximately 95% identical in amino acid sequence.30 Mutation
analyses of hGM-CSFR showed that STAT5 activation is not
required for GM-CSF–dependent antiapoptosis or for proliferation.
Although STAT5 is activated by various cytokines such as erythropoietin (EPO), IL-3/IL-5/GM-CSF, prolactin, growth hormone, and
From the Department of Molecular and Developmental Biology, Institute of
Medical Science, Core Research for Evolutional Science and Technology,
Tokyo, Japan.
Minato-ku, Tokyo 108-8639, Japan; e-mail: [email protected].
Submitted June 5, 2000; accepted October 2, 2000.
Reprints: Sumiko Watanabe, Department of Molecular and Developmental
Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai,
1266
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
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BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
thrombopoietin (TPO), STAT5A and/or STAT5B knockout mice
showed an essential and a redundant role for physiologic responses
associated with growth hormone and prolactin.31 Because other
STATs may play pivotal roles for cell differentiation and function,
these results suggest that STAT5A and STAT5B are obligate
mediators of mammopoietic and lactogenic signaling rather than
cell proliferation.32
Because cytokines are strong proliferation-promoting factors
for various hematopoietic cells, attempts were made to clarify the
role of STATs in cell proliferation. Mutation analyses of the
receptor domains of IL-4, GM-CSF, and EPO receptors showed a
lack of correlation between cell growth and STAT6 (by IL-4) and
STAT5 (by GM-CSF and EPO).33,34 In contrast, dominant-negative
STAT5 partially suppressed IL-3–induced proliferation.35 Retardation of colony formation by IL-3, GM-CSF, or IL-5 of bone marrow
cells derived from STAT5A/B-deficient mice has been reported.31
Activation of hematopoietic cell proliferation through gp130 was
shown to depend on the activation of both STAT3 and SHP-2.36 The
requirement of STAT3 in Src-induced cell transformation is also
indicated,37 which means that STAT3 is probably involved in cell
proliferation.
We analyzed the direct role of STAT5 in the initiation of DNA
replication in BA/F3 cells using the polyoma (Py) replicon as a
model system. This system is widely used to analyze DNA
replication of mammalian cells because Py DNA replication makes
use of host DNA replication machinery, except for large T antigen
(LTag), a viral-encoded DNA helicase. In addition, it is an excellent
system to analyze the roles of transcription factors in DNA
replication because the Py origin of replication contains an
enhancer, which is an essential module in addition to the core
sequence of the origin.38 The enhancer sequence can be replaced
with multiple copies of a binding site for a single transcription
factor.39 Using this system, we examined the ability of STAT5 to
activate Py DNA replication. We found that STAT5 can activate
DNA replication in response to GM-CSF stimulation and that this
activation relies on the C-terminal transactivation domain of STAT.
Materials and methods
ACTIVATION OF POLYOMA VIRUS REPLICATION BY STAT5
1267
lated by T4 polynucleotide kinase, ligated with the Takara ligation kit
version II (Takara Biomedicals, Osaka, Japan), and digested with BamHI
and BglII to digest the head to head-ligated product. Four-tandem oligomer
was isolated by gel electrophoresis and inserted into the BglII site of
pGL3-promoter or pPyOICAT. pPyOICAT contains 4 tandem repeats of the
mutant STAT5 binding site30 and was constructed using oligonucleotides
5⬘-GATCTAGATTTATTTTAATTCAAATCG-3⬘, 5⬘-GATCCGATTTGAATTAAAATAAATCTA-5⬘. pPyG5OICAT,43 which contains 5 tandem repeats
of the GAL4 binding site fused with the polyoma replication origin, was
kindly provided by Dr Y. Ito (Kyoto University, Kyoto, Japan).
STAT5B-⌬68330 was kindly provided by Dr A. Miyajima (Tokyo
University, Tokyo, Japan). Truncation mutants STAT5B-⌬781 and STAT5B⌬721 were constructed by deletion of downstream BamHI and NarI sites,
respectively. Either the BamHI or NarI site was blunt-ended and ligated to
the blunted NotI site of pME18S vector, which contains SR␣ promoter.44
STAT5B-Y699F was constructed by introduction of a mutation in Y699 to F
(TTC) by polymerase chain reaction (PCR) mutagenesis. The AgeI-NotI
region of STAT5B was replaced with the AgeI-NotI digested 2-round PCR
product, which contains a point mutation within Y699. Primers used for
PCR were 5⬘-GGCATCACCATTGCTTGGAAG (sense) and 5⬘-TGGCTTCACGAATCCGTCAGCTGCTT (antisense) and 5⬘-CTGACGGATTCGTGAAGCCACAGATCA (sense) and antisense primer of pME18S vector for
the first round. For the second round of PCR, we used 5⬘-CTGACGGATTCGTGAAGCCACAGATCA (sense) and antisense primer of pME18S
vector. Construction of GAL4–STAT5A, GAL4–STAT5B, and their mutants will be described elsewhere (Itoh, Watanabe et al, manuscript in
preparation).
SR␣-PyLTag, which contains the SR␣ promoter 44 and Py large T
antigen, was constructed by insertion of the coding region of Py LTag
(blunted BamHI and SalI sites) into the pKU2 vector (blunted SpeI site and
XhoI site), which contains the SR␣ promoter 44 for mammalian cell
expression.
Cell lines and culture methods
An mIL-3–dependent pro–B-cell line, BA/F3 was maintained in RPMI
1640 medium containing 5% FCS, 1 ng/mL mIL-3, 100 U/mL penicillin,
and 100 ␮g/mL streptomycin. Stable transformants of BA/F3 cells expressing hGM-CSFR were grown in the same type of medium but supplemented
with 500 ␮g/mL G418. Expression levels of ␣ and ␤ subunits of these cell
lines were examined using fluorescence-activated cell sorter analysis.22
Cells with almost equivalent levels of the subunits were used.
COS7 cells were maintained in DMEM containing 10% FCS, 100
U/mL penicillin, and 100 ␮g/mL streptomycin.
Chemicals, media, and cytokines
Fetal calf serum (FCS) was from Sera-Tech Zellbiologische Produkte (St.
Salvatol, Germany). RPMI 1640 and Dulbecco modified Eagle medium
(DMEM) were from Nikken Biomedical Laboratories (Kyoto, Japan).
Recombinant human (h) GM-CSF and G418 were kindly provided by
Schering-Plough (Madison, NJ). Mouse (m) IL-3 produced by silkworm,
Bombyx mori, was purified as described.40 Plasmids encoding wild-type
STAT5A and B were gifts from Dr A. Miyajima (Tokyo University,
Tokyo, Japan).
Plasmid construction
Four tandem repeats of the proximal STAT5 binding site of ␤-casein
promoter8 were used as an enhancer of plasmids to analyze replication
(4XSTOICAT) and luciferase (4XST-Luc). pPyOICAT contains Py fragment (nt 5267 to nt 152), which includes the replication origin core
sequence (nt 5267 to nt 56) and early gene promoter but lacks the enhancer
region, and chloramphenicol acetyltransferase (CAT) coding sequence.41,42
pGL3-promoter (Promega, Madison, WI) has the luciferase coding region
and SV40 minimal promoter, but lacks the enhancer. The oligonucleotide
containing the proximal STAT5 binding site of ␤-casein promoter, 5⬘GATCTAGATTTCTAGGAATTCAAATCG-3⬘, 5⬘-GATCCGATTTGAATTCCTAGAAATCTA-3⬘, is designed to place the BglII site at the 5⬘ end and
the BamHI site at the 3⬘ end. Both strands were annealed and phosphory-
Gel shift analysis
The proximal STAT5 binding site of the ␤-casein promoter (5⬘AGATTTCTAGGAATTCAATCC-3⬘) served as a probe. The nuclear
extract was prepared as described,26 and 5 ␮g protein was incubated in 12
␮L binding buffer (10 mM Tris-HCl, pH 8.0, 100 mM KCl, 5 mM MgCl2, 1
mM dithiothreitol, 10% glycerol, 0.1 mg/mL poly dI-dC, 0.5 mg/mL bovine
serum albumin) for 30 minutes at room temperature. Samples were
subjected to electrophoresis through 5% polyacrylamide gel in 0.25 ⫻ TBE
buffer (22.5 mM Tris-borate, 0.5 mM ethylenediamine-N, N, N⬘, N⬘tetraacetic acid [EDTA]) and visualized using a Fuji Image analyzer (model
BAS-2000, Tokyo, Japan).
Transient transfection, Py replication assay, and
luciferase assay
DNA replication of the transfected plasmid was assayed by DpnI analysis,25,41 and transcription activity was monitored according to luciferase
activity. Plasmids were introduced into semiconfluent BA/F3 cells (2 ⫻ 106
cells per sample) by electroporation, as described.26 Cells resuspended in
factor-depleted media were incubated for 5 hours and then stimulated with 5
ng/mL hGM-CSF. After 24 hours of incubation, the cells were harvested
and used for either replication assay or luciferase assay. For replication
assay, low-molecular-weight DNA was isolated by the Hirt extraction
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1268
WATANABE et al
method, as described.25,45 Ten microliters of DNA solution was digested
with HindIII (4XSTOICAT) or BamHI (pPyG5OICAT), which linearizes
template plasmid, and DpnI. Because DpnI digests only methylated or
hemimethylated recognition sites of DNA, newly synthesized DNA is
resistant to DpnI digestion. DNA was separated by electrophoresis and
transferred to Hybond-N⫹ (Amersham Pharmacia Biotech Limited, Buckinghamshire, England) by alkaline blotting.46 DNA blots were hybridized
with denatured HindIII-digested template plasmid labeled with 32P by a
random priming kit (United States Biochemical, Cleveland, OH) with the
use of QuikHyb rapid hybridization solution (Stratagene, La Jolla, CA).
Blots were visualized and quantified using a Fuji Image Analyzer (model BAS-2000).
For the luciferase assay, proteins were extracted by freezing and
thawing of the cells18 and the assay was done, as described, using a
luciferase assay substrate (Promega) and a luminometer (model LB9501;
Berthold Lumat, Tokyo, Japan). Transfection efficiency was normalized by
the alkaline phosphatase activity of the cotransfected CMV–alkaline
phosphatase plasmid.
Isolation of chromatin fraction of BA/F3 cells
Chromatin fractions were isolated as described.47 Briefly, cells (2 ⫻ 107 per
sample) treated with or without genistein (20 ␮g/mL) were incubated for 30
minutes and then stimulated with hGM-CSF (10 ng/mL) for 15 minutes.
The harvested cells were suspended in 1 mL cytoskeleton buffer (100 mM
NaCl, 300 mM sucrose, 10 mM 1,4-peperazinebis (ethanesulfonic acid)
(PIPES), pH 6.8, 3 mM MgCl2, 1 mM ethylenebis (oxyethylenenitrilo)
tetraacetic acid (EGTA), 0.5% Triton X-100, and 1.2 mM phenylmethylsulfonyl fluoride [PMSF]) and incubated for 10 minutes on ice. The
suspensions were centrifuged and the supernatants were stocked as soluble
fractions. Precipitates were resuspended in 500 ␮L digestion buffer (50 mM
NaCl, 400 mM sucrose, 1 mM PIPES, pH 6.8, 3 mM MgCl2, 1 mM EGTA,
0.5% Triton X-100, 1.2 mM PMSF, 100 ␮g/mL DNase I, and 50 ␮g/mL
RNase A) and incubated for 20 minutes at room temperature. Next,
ammonium sulfate (final concentration 250 mM) was added; the supernatants were retained as the chromatin fraction and precipitates were referred
to as nuclear matrix fractions.
BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
examined the role of ␤c cytoplasmic Y residues in induction of
STAT5 DNA binding activity, using gel shift analysis and the
proximal STAT5 binding site of ␤-casein promoter8 as a probe.
Although the extent of binding activity was much less than that
seen with the wild-type receptor, DNA binding activity was clearly
induced by the addition of GM-CSF through the Fall mutant, and
any one of the Y-series mutants also enhanced DNA binding of
STAT5 by GM-CSF stimulation (Figure 1A). The extent of binding
strength through Y-series mutants correlates with that observed
with tyrosine phosphorylation of STAT5 through these mutants.22
We analyzed the transcriptional activation of STAT5 through these
mutant receptors using 4XST-Luc, which contains 4 tandem
repeats of the proximal STAT5 binding site of the ␤-casein
promoter, followed by the luciferase coding region. As expected,
Fall can activate luciferase activity of 4XST-Luc, and adding back
of any Y residues enhanced the activity (data not shown). These
results suggest that DNA binding activity and luciferase activity
through mutant ␤c are correlated. We also examined the possible
requirement of box1 and box2 motifs, which are conserved among
cytokine receptors, using internal deletion mutant of ␤c, which
Transfection to COS7 cells and preparation of cytoplasmic
and nuclear fractions
Transfection of plasmids to COS7 cells was done by electroporation, and
nuclear fractions were isolated as described.26 Briefly, cells were electroshocked and cultured in DMEM (10% FCS) for 48 hours. Cells were
stimulated with hGM-CSF (10 ng/mL) for 30 minutes and harvested. The
cells were incubated in buffer (10 mM N-2-Hydroxyethylpiperazine-N⬘ethanesulphonic acid (HEPES), pH 7.9, 10 mM KCl, 2 mM MgCl2, 1 mM
dithiothreitol, 0.1 mM EDTA, and 0.1 mM PMSF) for 15 minutes on ice,
and NP40 was added at a final concentration of 1%. Cells were mixed
vigorously for 15 seconds and centrifuged. The supernatant was stored as
the cytosol fraction. Nuclear proteins were extracted as described.26
Results
Induction of DNA binding activity of STAT5 through the
hGM-CSF receptor in BA/F3 cells does not require
␤c tyrosine residues
Human GM-CSF activates STAT5 in BA/F3 cells, and we earlier
reported the detailed region requirement of hGM-CSFR ␤c for
STAT5 tyrosine phosphorylation by receptor mutation analysis.22
Y-series mutants of ␤c contain only a single intact Y residue, with
the remaining Ys mutated to F. Fall mutant has substitutions of all 8
Y residues together. Using these mutants, we found that the level of
STAT5 tyrosine phosphorylation was dramatically decreased with
lack of all of the ␤c Ys (Fall), but was increased when any one (or
2, in the case of Y12) Y was added back.22 In the present work, we
Figure 1. Activation of STAT5 DNA binding and STAT-dependent Py origin
replication in BA/F3 cells through a series of mutants of hGM-CSFR. (A) BA/F3
cells expressing wild-type ␣ subunit and various ␤ mutants were depleted of mIL-3 for
5 hours and restimulated with hGM-CSF (10 ng/mL) for 15 minutes. Gel shift assay
was done using an oligonucleotide corresponding to the STAT binding site. Arrow
indicates specific STAT binding complex to the probe. (B) Plasmid containing 4
tandem repeats of STAT5 binding sites or mutant STAT5 binding sites fused to Py
virus replication origin were transfected to Ba/F-wild cells and left for 5 hours in
depletion media. Cells were restimulated with hGM-CSF (10 ng/mL) and harvested
after 24 hours of incubation, followed by replication assay. Transfected plasmids were
extracted using Hirt solution and digested with DpnI, which selectively degrades an
unreplicated plasmid, and then the plasmids were separated through an agarose gel
and analyzed by Southern blotting. (C) Plasmid containing wild-type STAT5 binding
site followed by Py virus replication origin was transfected to BA/F3 cells expressing
various hGM-CSFR mutants, and replication induced by hGM-CSF was analyzed as
described above. Arrow indicates DpnI-resistant replicated bands.
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BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
ACTIVATION OF POLYOMA VIRUS REPLICATION BY STAT5
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lacks either box1 (⌬box1) or box2 (⌬box2).48 As shown in Figure
1A, ⌬box1 did not activate DNA binding activity of STAT5, but
⌬box2 did transduce signals for DNA binding of STAT5 in
BA/F3 cells.
Activation of STAT leads to initiation of polyoma replicon
DNA replication
Because transcription factors are apparently involved in regulating
DNA replication in eukaryotic cells, we evaluated whether STAT5
would activate the initiation of DNA replication with the use of
polyoma replicon. Plasmids containing 4 tandem repeats of STAT
binding sites followed by polyoma replication origin (4XSTOICAT) were transfected to BA/F3 GM-CSFR cells and incubated
with or without hGM-CSF (10 ng/mL). After 24 hours of culture,
DpnI assay was done as described in “Materials and methods.” As
shown in Figure 1B, replication of the 4XSTOICAT was induced
by stimulation of hGM-CSF. When we used plasmids containing 4
tandem repeats of mutant STAT5 binding sites followed by
polyoma replication origin, no replication was induced with the
addition of hGM-CSF to BA/F-wild cells. Because the mutant site
cannot bind to STAT530 (our unpublished results), the essential role
of STAT5 and its binding site for initiation of polyoma origindependent replication was suggested. We then analyzed activities
of STAT-dependent DNA replication with various hGM-CSF
receptor mutants in BA/F3 cells. A stable line of BA/F3 cells
expressing hGM-CSF ␣ subunit was transfected with various
mutants of hGM-CSFR and 4XSTOICAT, and DpnI assay was
done. ⌬box2, but not ⌬box1, induced replication, indicating that
box1 is essential but box2 is dispensable for replication initiation
(lanes 3, 4). Any one of the Y-series mutants induced DNA
replication of 4XSTOICAT. When we examined levels of activation of DNA replication, no correlation was observed with that of
DNA binding activity. However, because Fall induced DNA replication,
Y residues of ␤c may not be essential, and the requirement of the ␤c
region for Py DNA replication seems the same as that for the STAT5
DNA binding activity induced by hGM-CSF.
Genistein inhibits Py replication, but not STAT5
chromatin localization
We earlier found that adding the tyrosine kinase inhibitor genistein
suppressed proliferation promotion by hGM-CSF, but not cell
survival or MAPK cascade activation.24,25 Therefore, genistein may
be a specific inhibitor of signaling pathways for cell proliferation.
Here, we tested the effects of genistein on replication and transcription
activation by STAT5. Both 4XST-Luc and 4XSTOICAT were transfected to BA/F-wild cells, and then the cells were stimulated with
hGM-CSF in the presence of the indicated doses of genistein. After
24 hours of culture, the cells were harvested and divided into 2
samples for luciferase and replication analyses. The addition of
genistein completely suppressed DNA replication of 4XSTOICAT
(Figure 2A). In contrast, the addition of genistein induced the
luciferase activity of 4XST-Luc to a great extent in response to
hGM-CSF (Figure 2B).
To clarify the target of genistein, we examined the subcellular
localization of STAT5 of the fractionated cells. Cells were stimulated with hGM-CSF in the presence or absence of genistein, and
soluble chromatin fractions were separated. Both fractions were
subjected to polyacrylamide gel electrophoresis, and Western
blotting was done using anti-STAT5 (Transduction Laboratories,
Lexington, KY) or antiphosphotyrosine (4G10; Upstate Biotechnology, Lake Placid, NY) antibodies. As shown in Figure 2C (lower
Figure 2. Characterization of Py origin-dependent replication by activation of
STAT5. Plasmids containing 4 tandem repeats of STAT5 binding sites fused to Py
virus replication origin (A) or luciferase (B) were transfected to BA/F-wild cells. Cells
were restimulated with hGM-CSF (10 ng/mL) and harvested after 24 hours of
incubation. The tyrosine kinase inhibitor genistein (10 or 20 ␮g/mL) was added 30
minutes before restimulation. Either DpnI (A) or luciferase assay (B) was done as
described in “Materials and methods.” Arrow in (A) indicates the replicated plasmid.
(C) Subcellular translocation of STAT5 by hGM-CSF in the presence or absence of
hGM-CSF. Cells were stimulated with hGM-CSF (10 ng/mL) in the presence or
absence of genistein (20 ␮g/mL) and separated into soluble and chromatin fractions.
Recovery of STAT5 in these fractions was analyzed by Western blotting with
anti-STAT5 or phosphotyrosine antibodies.
panel), in response to hGM-CSF stimulation, recovery of STAT5B
in chromatin fractions was dramatically increased and these
fractions were tyrosine phosphorylated (Figure 2C, upper panel),
which means that tyrosine-phosphorylated STAT5 was moved to
the chromatin fraction after the stimulation. In contrast, slight
decreases in the soluble fractions were observed. When cells were
treated with genistein, no change in STAT5B recovery of chromatin, soluble fractions was observed, and the presence of genistein
did not affect the tyrosine phosphorylation of STAT5. The findings
suggest that genistein suppressed replication through specific inhibition
of the replication machinery. Further experiments of immunoprecipitation of STAT5 followed by antiphosphotyrosine antibody Western
blotting and gel shift analysis indicated that tyrosine phosphorylation
and DNA binding activity of STAT5 were not suppressed by genistein
(data not shown); hence, the target of genistein seems to be events after
chromatin opening by STAT5.
C terminus of STAT5 is required for DNA replication initiation
STAT family proteins have common structural features, including a
C-terminal transcriptional activation domain, tyrosine residue, and
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WATANABE et al
Figure 3. Requirement of a region of STAT5 for Py origin-dependent replication.
(A) Mutants with deletion or Y-residue point mutation are shown schematically. The
mutant STAT5 was transfected with 4XSTOICAT (B) or 4XST-Luc (C) into BA/F-wild
cells, and the cells were depleted of mIL-3 for 5 hours. hGM-CSF (10 ng/mL) was
added and culture was continued for another 24 hours. Cells were harvested, and
DpnI (B) or luciferase (C) assay was done as described in “Materials and methods.”
Arrow in (B) indicates the replicated plasmid.
SH2 region.49 To determine the STAT5B region required for
replication initiation, we constructed STAT5B mutants and analyzed their activity in BA/F3 cells. Wild-type as well as mutant
STAT5B, shown schematically in Figure 3A, were transfected
together with 4XSTOICAT and SR␣-LTag. Cells were cultured in
the presence or absence of hGM-CSF for 24 hours, and DpnI assay
was done. Without hGM-CSF stimulation, no replicated band was
observed (Figure 3B, lanes 1-6). Because endogenous STAT5
exists, a replicated band was observed in the vector control sample
(lane 12), but cotransfection of STAT5B dramatically enhanced the
intensity of the band (lane 7). When we transfected mutant
STAT5B instead of wild-type STAT5B, deletion up to amino acid
781 did not affect replication activity, but further deletion up to 721
resulted in loss of the activity (lanes 8, 9). Further deletion up to
683 confirmed this result (lane 10). The C-terminal Y residue of
BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
STAT5B is phosphorylated upon cytokine stimulation and is thought to
be essential for dimerization through the SH2 region of STAT. When we
mutated STAT5B Y699 to F (Y699F), DNA replication was abrogated
(lane 11), and complete loss of the replicated band suggests that this
mutant acts in a dominant-negative fashion to endogenous STAT5B. We
also analyzed the transcriptional activation potential of these STAT5B
mutants using the 4XST-Luc plasmid (Figure 3C). As shown in Figure
3C, mutant ⌬781 can induce transcription of 4XST-Luc, but further
deletion up to 721 resulted in loss of the activity. Mutation of Y699 also
resulted in a complete loss of luciferase activation. In both transcription
and replication, ⌬683, which lacks Y residue, showed weaker dominantnegative effects than those observed with Y699F. We speculate that this
is caused by differences in expression levels of ⌬683 and Y699F, which
can be deduced from expression levels of these mutants in COS7 cells.
These results indicate that Y699 and the C-terminal transactivation
domain of STAT5B are essential for both transcription and replication
activation by hGM-CSF in BA/F3 cells.
To analyze the mechanism of lack of replication and transcriptionstimulating activity of these mutants (⌬683, ⌬721, and Y699F), we
examined tyrosine phosphorylation and nuclear translocation of these
mutants with a transient expression system in COS7 cells. Mutants of
STAT5 were transfected to COS7 cells with hGM-CSFR ␣ and ␤c
plasmids and cultured for 2 days. Cells were stimulated for 30 minutes
with hGM-CSF (10 ng/mL), and total cell lysates of nuclear and
cytoplasmic fractions were analyzed by Western blotting with antiphosphotyrosine and anti-STAT5 antibodies. As shown in Figure 4 (upper
panel), wild type, ⌬781, and ⌬721 were tyrosine phosphorylated in
response to hGM-CSF (open arrowhead). Because no tyrosine phosphorylation was observed with ⌬683 or Y699F, the results suggest that Y699
is a major phosphorylation site of STAT5B. When we examined nuclear
translocation by blotting with ␣ STAT5 antibody, wild type as well as
⌬781 and ⌬721, but not ⌬683 or Y699F, were translocated to the
nucleus. A weak Y699F band was observed, but this band is assumed to
be caused by an incomplete purity of fractionation because a trace
amount of SHP-2, which is assumed to localize exclusively in the
cytoplasm, was also observed in the nuclear fraction. Exclusive localization of Y699F and ⌬683 in the cytoplasm was also supported by
immunostaining with anti-STAT5 antibody (data not shown). These
results suggest that failure of activation of DNA replication by ⌬683 and
Y699F was caused by a lack of nuclear translocation, whereas requirement of the transcriptional activation domain for DNA replication was
suggested in the case of ⌬721.
C-terminus transcriptional activation domain of STAT5 fused
with GAL4 DNA binding domain can activate Py replication
through the GAL4 binding sequence
We next analyzed the role of the STAT5 transcriptional activation
domain using the yeast GAL4 fusion protein system. GAL4 protein
and various hybrid proteins composed of the GAL4 DNA binding
Figure 4. Characterization of mutant STAT5B. Wild-type as well as mutant STAT5B
with hGM-CSFR ␣ and ␤c subunits were transfected to COS7 cells. After 2 days of
culture, the cells were stimulated with hGM-CSF (10 ng/mL) for 30 minutes and
harvested. Cells were separated into soluble and nuclear fractions, and phosphorylation and translocation of STAT5 were analyzed by Western blotting.
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BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
Figure 5. Activation of Py replicon by GAL4–STAT fusion protein. (A) Schematic
diagram of GAL4–STAT fusion proteins used. (B) GAL4–STAT5A or GAL4–STAT5B
was transfected with Py LTag and pPyG5OICAT, which contains a 5-tandem repeat of
GAL4 binding site and Py replication origin, into BA/F3 cells. After 24 hours of culture,
replication was analyzed by DpnI assay. The replicated DpnI-resistant plasmid is
indicated by the arrow. (C) The indicated doses of GAL4–STAT5 or its mutants were
transfected with SR␣-LTag and pPyG5OICAT into BA/F-wild cells. After 24 hours of
stimulation, DpnI assay was done.
domain and the activating domain of other transcription factors
were shown to transactivate replication of the plasmid containing
the Py replicon and GAL4 binding sequence in the upstream of
replication origin.50 It has been reported that fusion proteins in
which the DNA binding domain of the yeast GAL4 transcription
factor is linked to the transactivation domain of STAT5A can lead
to transactivation of a luciferase reporter construct with a promoter
that contains 3 binding sites for the GAL4 protein.51 We constructed similar plasmids, GAL4–STAT5A and GAL4–STAT5B;
the mouse STAT5 C-terminal transcriptional activation domains
(amino acids 709-793 of STAT5A and amino acids 714-786 of
STAT5B) were fused with the C terminus of GAL4 DNA binding
element (Figure 5A). These fusion proteins do not contain Y
residues, which were shown to be a major phosphorylation site. As
a replicon, the 5-tandem repeat of GAL4 binding domain fused
with the polyoma replication origin (pPyG5OICAT) was used.43
Either GAL4–STAT5A or GAL4–STAT5B was transfected with Py
LTag into BA/F-wild cells. After 24 hours of culture with or
without hGM-CSF, DpnI assay was done. As shown in Figure 5B
(right panel), cotransfection of either GAL4–STAT5A or GAL4–
STAT5B induced DNA replication by the addition of hGM-CSF to
BA/F-wild cells. Replication occurs in a GM-CSF–dependent
manner because no band was observed with the nonstimulated
samples (left panel). To analyze the role of the transcriptional
activation domain of STAT5B, we tested the induction of replication by C-terminal deletion mutants of GAL4–STAT5B
(GAL4B⌬781, 774, 769, and 748). Two different doses (0.5 ␮g, 2.0
␮g) of GAL4–STAT5B and its mutants were transfected, and the
ability to induce replication of pPyG5OICAT was analyzed. As
shown in Figure 5C, transfection of 0.5 ␮g of any one of the
ACTIVATION OF POLYOMA VIRUS REPLICATION BY STAT5
1271
mutants can induce replication of pPyG5OICAT. When we transfected 2 ␮g GAL4–STAT5 or its mutants, deletion up to 769 did not
affect replication induction ability, but further deletion up to 748
resulted in loss of replication. These results indicate that the
transcriptional activation domain between amino acids 748 and 769
is essential for replication activation.
Six S residues exist in the C-terminal transcriptional activation
domain of STAT5B, and the role of these residues in transactivation
was not clarified. To determine the requirement of S residues of the
STAT5B transcriptional activation domain, we constructed a mutant carrying all the S residues substituted with A (GAL4–STAT5B6XSA, Figure 5A). Because the effects of deletion of the transactivation domain were observed only when we transfected a high
amount of GAL4–STAT5B mutants, we transfected 3 different
amounts of GAL4–STAT5B or GAL4–STAT5B-6XSA with
pPyG5OICAT and Py LTag into BA/F-wild cells. As shown in
Figure 6A, a small or middle amount (0.1, 0.5 ␮g) of GAL4–
STAT5B-6XSA stimulated Py replication to the same extent as did
the wild-type GAL4–STAT5B, whereas a larger amount (2 ␮g) of
transfected GAL4–STAT5B-6XSA did not induce Py replication.
We then looked at the role of each S residue by constructing
GAL4–STAT5B mutants with one S mutated with A and other Ss
left intact (Figure 5A). The mutants (2 ␮g) were then transfected to
BA/F-wild cells. As shown in Figure 6B, none of the mutants
induced Py replication. When we transfected these mutants with a
low dose (0.5 ␮g), all the mutants induced Py replication, as was
seen with the wild type (data not shown). These results suggest that
these S residues coordinately play a role in replication initiation.
Discussion
We obtained evidence that the activation of STAT5 can lead to initiation
of Py DNA replication and that the transcriptional activation domain is
required for this activity. This is the first direct evidence indicating the
potential involvement of STAT5 in replication.
All transcription factors with replication-enhancing activity
appear to require an activation domain, in addition to the DNA
binding domain. Deletion or mutation analysis of STAT showed
that the region required for transcription and that for replication
activation are not separable. The activation domain for replication
Figure 6. Role of S residues of STAT5 for initiation of Py replication. (A) Various
amounts of GAL4–STAT5B-wild or GAL4–STAT5B-6XSA were transfected to BA/Fwild cells together with SR␣-LTag and pPyG5OICAT. (B) Two micrograms of
GAL4–STAT5B or GAL4–STAT5B S-residue mutants were transfected with SR␣LTag and pPyG5OICAT. Cells were cultured for 24 hours in the presence of hGM-CSF
(10 ng/mL). Replication was analyzed by DpnI assay. The arrows indicate replicated plasmid.
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1272
BLOOD, 1 MARCH 2001 䡠 VOLUME 97, NUMBER 5
WATANABE et al
overlaps that for transcription in many cases, such as GAL4, VP16,
and c-Jun.39,50,52 However, in the case of p53 and c-Rel, no identical
requirement of the region for Py DNA replication and transcription
was found.43,53 Our results obtained with the mutant STAT5 ⌬721
indicate that the transcriptional activation domain is essential and
sufficient for the activation of Py replication. Because deletion of
the transactivation domain does not affect STAT5 nuclear translocation or DNA binding, we speculate that the transactivation domain
may play a role to recruit molecules to the replication machinery.
Initiation of Py DNA replication occurs when the chromatin
structure around the origin opens for the binding of LTag. LTag forms a
double hexamer in a manner dependent on adenosine triphosphate and
induces a structural change at the origin. Unwinding of double-stranded
DNA then begins in the presence of replication protein A (RP-A), and
DNA polymerase ␣-primases initiate DNA synthesis on the unwound
DNA covered with RP-A. The activation domain of VP16, GAL4, E2,
and p53 binds to a single-stranded DNA binding protein, RP-A, which is
essential for the initiation of Py DNA replication,54-56 and c-Jun interacts
with LTag.57 To examine the potential involvement of STAT5 in the
recruitment of RP-A, we overexpressed VP-16 in the STAT-Py system.
Because no interference was observed (data not shown), VP-16 and
STAT5 may not use the same mechanism. Similarly, the target protein of
polymavirus enhancer binding protein 2␣B1 is probably different from
that of RP-A or other VP16 binding proteins.47
Mutation of S residues of the STAT5 transactivation domain
suggested the role of S residues for replication activation. Because
mutation of any one of the S residues resulted in loss of replication
activation, we could not define a specific role of each S residue.
Among the members of the STAT family, the role of S is reported
only for STAT1 and STAT3 for their transcriptional activation.58 In
contrast, although phosphorylation of S730 of STAT5B by prolactin stimulation occurs, this phosphorylation is not essential for
DNA binding or transcriptional activation.59
Core binding protein and p300 can interact with STAT5, and
histone acetyltransferase activity may participate to maintain a
transcriptionally active chromatin structure.60 Induction of germline transcription in the T-cell receptor ␥ locus by STAT5 has been
reported.61 Therefore, it is feasible that STAT5 plays roles in both
the activation of chromatin accessibility and recruitment of replication machinery.
We reported that activation of STAT5 is not essential or
sufficient for proliferation promotion of BA/F3 cells. ␤c fused with
JAK2 can promote proliferation without activation of STAT527; on
the other hand, GyrB fused with JAK2 cannot sustain the survival
or proliferation of BA/F3 cells, although it can lead to activation of
STAT5.62 There are several possible explanations as to why STAT5
cannot induce proliferation even though it can lead to DNA
replication. STAT5 is not sufficient to promote other cell-cycle
machinery such as the cyclin/CDK complex. As another possibility,
it should be considered that STAT5 can initiate polyoma replicondependent replication but not cellular DNA replication. The former
possibility is of interest because it was reported that coactivation of
the ras/MAPK pathway, in addition to the constitutively active
STAT5, promotes proliferation of BA/F3 cells, which means that an
additional signal is required to promote cell proliferation.63 Acute
myelogenous leukemia 1, c-Rel, c-Jun, and c-Fos can initiate
polyoma replicon DNA replication, which means that STAT5 is not
the only factor related to activation of replication. GM-CSF can
lead to replication of Py replicon, whose enhancer does not contain
STAT binding site.25 This notion is supported by our previous
results that activation of STAT5 is not essential to promote
proliferation in BA/F3 cells. It also suggests that other transcription
factors can activate initiation of DNA replication.
Because STAT is assumed to play a role in cytokine-specific
functions, it probably is not like a major role related to the general
cell proliferation. Our results are important because it seems highly
likely that STAT functions as a part of the DNA replication
machinery. Selection of transcription factors for initiation of DNA
replication may depend on cell types and other factors.
Our previous results indicated that no specific Y residue of ␤c was
required for tyrosine phosphorylation of STAT5 in BA/F3 cells,22 and
the same requirement was observed for the initiation of replication.
Therefore, STAT5 is probably activated independently of receptor Y
residues. This implication is inconsistent in that other STATs are
considered to recognize and bind to a specific sequence surrounding
certain Y residues of cytokine receptors. In addition, we reported that the
GyrB–JAK2 fusion protein can activate STAT5 without involving
GM-CSF receptor activation.62 Taking these data together, we speculate
that STAT5 is directly phosphorylated by JAK2.
We previously found that ␤-casein luciferase is not activated by
Fall, although Fall can induce tyrosine phosphorylation of STAT5.22
Other modifications such as serine phosphorylation may be required for transcriptional activation of STAT5, and this modification depends on Y residues. When we examined transcriptional
activation of tandem repeats of STAT5 binding sites derived from
the ␤-casein promoter, even Fall activated luciferase activity;
hence the Y residue is not required for the transcriptional activation
of STAT5. Because ␤-casein promoter contains various putative
transcription factor binding sites,8 a combination of activation of
multiple transcription factors may be required for full activation of
␤-casein promoter, and receptor Y residues may play a role in
activating other transcription factors.
Acknowledgments
We thank Yukitaka Izawa for excellent technical support, Drs
Yoshiaki Ito and Kosei Ito for providing materials and for helpful
discussion, and Mariko Ohara for comments. T.I. is a recipient of a
research fellowship from the Japan Society for the Promotion of
Science for Young Scientists.
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2001 97: 1266-1273
doi:10.1182/blood.V97.5.1266
Initiation of polyoma virus origin-dependent DNA replication through
STAT5 activation by human granulocyte-macrophage colony-stimulating
factor
Sumiko Watanabe, Rong Zeng, Yutaka Aoki, Tohru Itoh and Ken-ichi Arai
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