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HEMATOPOIESIS
From the Department of Molecular and Developmental
Biology, Institute of Medical Science, Core Research for Evolutional
Science and Technology, Tokyo, Japan.
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 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, 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 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.
Chemicals, media, and cytokines
Plasmid construction
STAT5B- SR 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 µg/mL streptomycin. Stable transformants of BA/F3 cells expressing hGM-CSFR were grown in the same type of medium but supplemented with 500 µg/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 µg/mL streptomycin. 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 µg protein was
incubated in 12 µL 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 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 µg/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 µL 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 µg/mL DNase I, and 50 µg/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.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
Induction of DNA binding activity of STAT5 through the hGM-CSF
receptor in BA/F3 cells does not require 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
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 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 origin-dependent 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 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 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 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
dominant-negative 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
transcription-stimulating activity of these mutants (
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 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 (GAL4B781, 774, 769, and 748). Two different doses (0.5 µg, 2.0 µg) 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 µg of any one of
the mutants can induce replication of pPyG5OICAT. When we transfected 2 µg 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-STAT5B-6XSA, 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 µg) of GAL4-STAT5B-6XSA stimulated Py replication
to the same extent as did the wild-type GAL4-STAT5B, whereas a larger
amount (2 µg) 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 µg) 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 µg), 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.
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 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 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 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 We reported that activation of STAT5 is not essential or sufficient for
proliferation promotion of BA/F3 cells. 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 We previously found that
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.
Submitted June 5, 2000; accepted October 2, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Sumiko Watanabe, Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; e-mail: sumiko{at}ims.u-tokyo.ac.jp.
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Abstract
Introduction
locus by STAT5 has
been reported.