Enhancement of B-MYB Transcriptional
Activity by ZPR9, a Novel Zinc Finger Protein*
Hyun-A
Seong
,
Kyong-Tai
Kim§, and
Hyunjung
Ha
From the
Department of Biochemistry, School of Life
Sciences, Research Center for Bioresource and Health, Chungbuk National
University, Cheongju 361-763 and the § Department of Life
Science, Pohang University of Science and Technology,
Pohang 790-784, Republic of Korea
Received for publication, July 25, 2002, and in revised form, January 6, 2003
 |
ABSTRACT |
By using the yeast two-hybrid system, the zinc
finger protein ZPR9 was identified as one of the B-MYB
interacting proteins that associates with the carboxyl-terminal
conserved region of B-MYB. ZPR9 was found to form in
vivo complexes with B-MYB, as demonstrated by in vivo
binding assay and coimmunoprecipitation experiments of the endogenously
and exogenously expressed proteins. Deletion analysis revealed that
this binding was mediated by all three functional domains, an
amino-terminal DNA-binding domain, a transactivation domain, and a
carboxyl-terminal conserved region of B-MYB. We show that
the interaction of ZPR9 with B-MYB is functional because cotransfection
of ZPR9 significantly up-regulates B-MYB transcriptional activity in a dose-dependent manner. In
addition, coexpression of ZPR9 with B-MYB
caused the accumulation of B-MYB, as well as ZPR9, in the nucleus.
Furthermore, constitutive expression of ZPR9 in human
neuroblastoma cells induces apoptosis in the presence of retinoic acid.
These results strongly suggest that ZPR9 plays an important role in
modulation of the transactivation by B-MYB and cellular
growth of neuroblastoma cells.
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INTRODUCTION |
B-MYB is a member of the MYB family of
transcription factors, which is ubiquitously expressed and is involved
in controlling cell proliferation and differentiation (1-5). B-MYB is
phylogenetically the most divergent among the three MYB
proteins, A-MYB, B-MYB, and c-MYB (6). Recent reports showed that the
CDK2-cyclin A complex could induce phosphorylation of B-MYB and
potentiate the B-MYB transactivating function and that this
activation was also induced by truncation of the carboxyl terminus of
B-MYB, suggesting that post-translational modifications are
required for relieving the constitutive repression of B-MYB
(7-10). In addition, a recent study (11) indicated that the
B-MYB transactivation correlates with the binding of some
cofactors to the carboxyl-terminal conserved region, suggested as a
protein binding domain and a putative phosphorylation site.
The MYB proteins are composed of three functional domains for
transactivation, an amino-terminal DNA-binding domain, a central acidic
region (transactivation domain), and a carboxyl-terminal negative
regulatory domain containing the leucine zipper motif (12). Recently,
all these domains have been reported to be involved in interactions
with several cellular proteins. The DNA-binding domain of
c-MYB was found to bind with several proteins such as p100
coactivator (13, 14), c-Maf transcription factor (15), Cyp-40
peptidylprolyl isomerase (16), HSF3 (17), nucleolin (18), and retinoic
acid receptor (19). In addition, recent reports have shown that the
DNA-binding domain of A-MYB and B-MYB interacts
with several nuclear proteins (18, 20) and poly(ADP-ribose) polymerase
(PARP),1 which is associated
with chromatin (21), respectively. On the other hand, the
cAMP-response element-binding protein has been demonstrated to
interact directly with the transactivation domain of both
c-MYB and A-MYB and potentiate their
transcriptional activity (22, 23). The leucine zipper motif of the
carboxyl-terminal domain was also found to associate with several
proteins, including p26/28 (24), p67, and p160 (25, 26), and ATBF1
transcription factor (27), but these interactions except for ATBF1 have
not been implicated in the regulation of MYB function so
far. From these results, it is tempting to speculate that additional
proteins may be involved in the regulation of transactivation by
B-MYB, probably by association with the carboxyl-terminal
conserved region that shows significant homology with other members of
the MYB gene family such as A-MYB and
c-MYB.
ZPR9, a zinc finger protein, was originally identified as a novel
cellular partner for the MPK38 serine/threonine kinase that may be
involved in early T cell activation by concanavalin A (28) and
embryonic development (29, 30). ZPR9 is a 52-kDa protein containing
three zinc finger motifs and a physiological substrate of MPK38 kinase
in vivo (31).
Here we show that ZPR9 binds to B-MYB in vivo and that the
overexpression of ZPR9 induces apoptosis, instead of neural
differentiation, in the neuroblastoma cells treated with retinoic acid.
Binding of ZPR9 to B-MYB can stimulate the B-MYB
transcriptional activity. In addition, we provide evidence that all
three functional domains of B-MYB physically interact with
ZPR9 in vivo. We also demonstrate that the coexpression of
ZPR9 with B-MYB causes the accumulation of both
ZPR9 and B-MYB in the nucleus.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
The eukaryotic glutathione
S-transferase (GST) expression vector (pEBG) and pFLAG-CMV-2
vector with a FLAG epitope were obtained as described previously (32).
The anti-GST antibody was as described (32). The pT81luc 3xA reporter
plasmid (33), containing three copies of the "A box" Myb-binding
sites from the nim promoter, was a kind gift from Dr. Scott
A. Ness (the University of New Mexico, Albuquerque, NM). The expression
vector pCEV27 was kindly provided by Dr. D-Y. Shin (Danguk
University, Chonan, Korea). The anti-FLAG (M2) antibody,
all-trans-retinoic acid (RA), BisBenzimide (H 33258),
isopropyl-
-D-thiogalactopyranoside, dithiothreitol, aprotinin, and phenylmethylsulfonyl fluoride were purchased from Sigma.
Polyvinylidene difluoride membrane was obtained from Millipore Corp.
[
-32P]ATP was purchased from PerkinElmer Life
Sciences. The human B-MYB antibody (C-20) raised against the carboxyl
terminus was used for immunoprecipitation and Western analysis (Santa
Cruz Biotechnology). Oligonucleotides were synthesized from Bioneer Corp. (Cheongwon, Chungbuk, Korea).
Cell Culture--
The human neuroblastoma cell line SK-N-BE (2)C
and 293T cells, a derivative of human kidney embryonal fibroblast
containing SV40 T antigen, were cultured in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% FBS, 100 units/ml
penicillin-streptomycin, and 1 mM glutamine as described
(34). For cell differentiation experiments, SK-N-BE (2)C cells grown in
DMEM supplemented with 10% FBS were plated in 6-well flat-bottomed
microplates at a concentration of 4 × 105 cells per
well the day before retinoic acid (RA) treatment, and the medium was
replaced with fresh medium without FBS, containing 5 µM
all-trans-retinoic acid, every 3 days. The 293T cells were transfected by the calcium phosphate precipitation method as described previously (34).
Plasmid Constructions--
The pEBG-B-MYB, an
amino-terminally truncated version containing part of the acidic region
and a complete conserved region, and pEBG-WT B-MYB,
containing a full-length B-MYB cDNA, have been described
previously (32). The deletion constructs, pEBG-B-MYB R1 and
pEBG-B-MYB R2, were generated by PCR as described (32). To
generate two deletion constructs, pFLAG-DBD and pEBG-TA, we performed a
PCR using the full-length B-MYB cDNA as the template. The forward primers for DBD (5'-GCGAATTCATGTCTCGGCGG-3')
and TA (5'-GCAAGCTTGAGGACAAGGAC-3') contain an
EcoRI and a HindIII site (underlined). The
reverse primers for DBD (5'-GCGGATCCCTCGAGCTCCAG-3') and TA
(5'-GCGGATCCCAGGCGGTACTC-3') contain a BamHI
site (underlined). The amplified PCR products for deletion mutants were
cut with EcoRI plus BamHI and HindIII
plus BamHI and cloned into pBluescript KS (Stratagene) to
generate the KS-DBD and KS-TA constructs, respectively. The pFLAG-DBD
was generated after subcloning of an EcoRI/BamHI fragment of KS-DBD into the EcoRI/BamHI site of
pFLAG-CMV-2. The pEBG-TA was generated by subcloning of a
ClaI/NotI fragment from KS-TA into pEBG vector.
The identity of all PCR products was confirmed by nucleotide sequencing
analysis on both strands with the T7 SequencingTM kit
(Amersham Biosciences). The pEBG-CR, a carboxyl-terminally truncated
version containing a DNA-binding domain, a transactivation domain, and
a complete conserved region, was made by PCR amplification. The
amplified PCR product was cut with HindIII plus
BamHI and cloned into pBluescript KS to generate the KS-CR
construct. The pEBG-CR was created by subcloning of a
ClaI/NotI fragment from KS-CR into pEBG vector.
The pEBG-TA1, a carboxyl-terminally truncated version containing part
of the transactivation domain and a complete DNA-binding domain, was
constructed in several steps. We first cloned the
HindIII/EcoRI fragment of full-length
B-MYB into pBluescript KS and digested with ClaI
plus NotI and subcloned into pEBG, yielding pEBG-TA1. A
full-length ZPR9 cDNA obtained from a human normal keratinocyte cDNA library was cloned into pEBG and pFLAG-CMV-2 to
generate the pEBG-ZPR9 and pFLAG-ZPR9,
respectively (31). The pCEV27-ZPR9 for expression of human
ZPR9 was prepared by subcloning of a
BamHI/XhoI fragment from pBacPAK9-ZPR9
into pCEV27 vector. The pBacPAK9-ZPR9 was generated by
subcloning of an EcoRI/XbaI fragment from
pFLAG-ZPR9 into pBacPAK9 (Clontech). For
a confocal microscopy, the GFP-B-MYB was created by
subcloning of a KpnI/BamHI fragment from
KS-B-MYB into pRSGFP-C1 vector (Clontech).
Yeast Two-hybrid Specificity Test--
A fish plasmid, pJG4-5
harboring a carboxyl-terminal conserved region of B-MYB, was
transformed back into EGY48 cells along with either the bait plasmid,
pEG202 harboring ZPR9, or other several bait plasmids
available in our laboratory (32). For selection of proteins interacting
with the B-MYB, the plate assays of
-galactosidase expression were
carried out.
In Vivo Binding Assay--
293T cells grown in DMEM supplemented
with 10% FBS were plated in 6-well flat-bottomed microplates at a
concentration of 2 × 105 cells per well the day
before transfection. 1-5 µg of each plasmid DNA was transfected into
293T cells with a calcium phosphate precipitation method. Forty eight
hours after transfection, cells were washed three times with ice-cold
phosphate-buffered saline (PBS) and solubilized with 100 µl of lysis
buffer (20 mM Hepes (pH 7.9), 10 mM EDTA, 0.1 M KCl, and 0.3 M NaCl) containing 0.1% Nonidet P-40, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 mM
sodium fluoride, 2 µg/ml
1-antitrypsin, 2 mM sodium pyrophosphate, 25 mM sodium
-glycerophosphate, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride. Detergent-insoluble
materials were removed by centrifugation at 13,000 rpm for 15 min at
4 °C. Approximately 80 µl of the cleared lysates were mixed with
15 µl of glutathione-Sepharose beads (Amersham Biosciences) and
rotated for 2 h at 4 °C. Beads were washed three times with the
lysis buffer. The bound proteins were eluted by boiling in SDS sample
buffer, subjected to SDS-PAGE, and then transferred to polyvinylidene
difluoride membranes. The membranes were probed with an anti-FLAG (M2)
antibody and then developed using an ECL detection system (Amersham Biosciences).
Luciferase Reporter Assay--
293T cells were transfected
according to the calcium phosphate precipitation method. After 48 h, the cells were harvested, and luciferase activity was monitored with
a luciferase assay kit (Promega) following the manufacturer's
instructions. Light emission was determined with a Berthold luminometer
(Microlumat LB96P). The cell extracts containing equal amounts of B-MYB
and ZPR9, determined by Western blot analysis, were used for luciferase assay. The values were adjusted with respect to expression levels of a
cotransfected
-galactosidase reporter control, and experiments were
repeated at least three times.
Northern Blot Analysis--
Total cytoplasmic RNAs were prepared
from cells using RNAzolTM B (Biotex Laboratories) as
described (28). Approximately 30 µg/ml total RNA was electrophoresed
through 1.2% agarose-formaldehyde gel and transferred to GeneScreen
PlusTM nylon membrane (PerkinElmer Life Sciences). The
membranes were hybridized with a 32P-labeled
ZPR9 cDNA probe at 42 °C overnight in 50% formamide, 10% dextran sulfate, 7% SDS, 0.25 M NaHPO4,
0.25 M NaCl, 1 mM EDTA, and 100 µg/ml
denatured salmon sperm DNA. The membranes were washed to a final
stringency of 0.25× SSC and 0.2% SDS at 55-60 °C for 20-30 min.
Glyceraldehyde-3-phosphate dehydrogenase mRNA was used as an
internal control.
Apoptosis Analysis--
Cells undergoing apoptosis were
quantitated by staining with the fluorescein isothiocyanate-conjugated
annexin V and the fluorescent dye propidium iodide according to the
manufacturer's recommendations (Roche Molecular Biochemicals). The
RA-treated cells of 6-cm dishes were harvested and incubated for 10 min
at room temperature in annexin V- and propidium iodide-containing
buffer and then washed with PBS. 10,000 events were analyzed per sample
using a FACSCalibur-S system (BD Biosciences).
293T and SK-N-BE (2)C cells grown in sterile coverslips were
transfected with pEGFP, an expression vector encoding GFP, together
with expression vectors encoding the indicated proteins. In 293T cells,
after 24 h of transfection, the cells were treated with TNF-
(20 ng/ml) and cycloheximide (10 µg/ml) for 14 h. In SK-N-BE
(2)C cells, after 24 h, the medium was replaced with fresh medium
without FBS, containing 5 µM RA, and the cells were further incubated for 2-3 days. Stimulations were terminated by aspirating the culture medium and fixing cells with ice-cold 100% methanol for 5 min at room temperature. The cells were washed three
times with PBS and then stained with a BisBenzimide (H 33258) in PBS.
The coverslips were washed two times with PBS, then mounted on glass
slides, using Gelvatol, and visualized under a fluorescence microscope.
The percentage of apoptotic cells was calculated as the number of
GFP-positive cells with apoptotic nuclei divided by the total number of
GFP-positive cells.
Confocal Microscopic Analysis--
293T cells were grown on
sterile coverslips and transfected with GFP-B-MYB and/or
FLAG-tagged ZPR9 constructs by the calcium phosphate
precipitation method, placed on ice, and washed three times with
ice-cold PBS prior to fixation with 4% paraformaldehyde for 10 min at
room temperature. The mouse monoclonal anti-FLAG (M2) antibody was
applied for 2 h at 37 °C. The cells were then incubated with
Texas Red-conjugated anti-mouse secondary antibody (Amersham
Biosciences) at 37 °C for 1 h as described (31). The coverslips
were washed three times with PBS and then mounted on glass slides,
using Gelvatol. Confocal laser scanning microscopy observations were
done on a Bio-Rad MRC 1024 (15-milliwatt argon-krypton laser; mounted
on a Zeiss Axioskop (Jena, Germany) equipped with a ×63 (NA 140)
oil-immersion objective, using a 488- (GFP) and 568-nm (Texas Red)
bandpass filter).
 |
RESULTS |
B-MYB Physically Interacts with ZPR9 in Vivo--
We have
identified recently that B-MYB proteins associate in
vivo with each other (32). In an effort to identify further proteins that interact with B-MYB, we used a two-hybrid specificity test technique that was usually employed to verify the interaction specificity between bait and the cDNA-encoded proteins and to eliminate quickly the majority of false positives detected in the yeast
two-hybrid assay. Specific interacting proteins confer the
galactose-dependent Leu+/LacZ+
phenotype to yeast containing the related baits but not to yeast containing unrelated baits. To test this, the B-MYB library
plasmid was rescued from the galactose-dependent
Leu+/LacZ+ yeast and re-introduced into the
ZPR9 bait strain as well as the other strains containing
approximately 20 different baits available in our laboratory. From this
random screening, B-MYB cDNA was found to interact with
the total seven baits tested (results not shown), including
ZPR9 and B-MYB baits, suggesting that ZPR9, like
B-MYB (32), can interact with B-MYB physically in mammalian cells.
To determine whether B-MYB and ZPR9 interact in vivo, we
performed cotransfection experiments using GST- and FLAG-tagged
eukaryotic expression vectors. In these experiments, the
ZPR9 and wild-type B-MYB were coexpressed as a
GST fusion protein and a FLAG-tagged protein in 293T cells,
respectively. The interactions of FLAG-tagged B-MYB proteins to
the GST-ZPR9 fusion proteins were analyzed by immunoblotting with an
anti-FLAG antibody. As shown in Fig.
1A, the B-MYB was detected in
the coprecipitate only when coexpressed with the GST-ZPR9
but not with the control GST alone, demonstrating that B-MYB physically
interacts with ZPR9 in vivo. In order to verify further the
interaction of B-MYB with ZPR9 in vivo, we performed
coimmunoprecipitation experiments using 293T cells transiently transfected with the vector alone or FLAG-tagged ZPR9 (Fig.
1B). Endogenous B-MYB was immunoprecipitated from cell
lysates, and Western blot analysis shows that B-MYB was precipitated
(Fig. 1B, lower panel). The binding of ZPR9 was
subsequently analyzed using Western blotting with an anti-FLAG
antibody, and as shown in Fig. 1B (upper left
panel), ZPR9 was present in the B-MYB immunoprecipitate. In
conclusion, our results clearly demonstrate that B-MYB associates with
ZPR9 in vivo.

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Fig. 1.
Interaction of B-MYB with ZPR9 in mammalian
cells. A, GST alone (pEBG), as a control, and
pEBG-ZPR9 (GST-ZPR9) containing the full-length human
ZPR9 cDNA were cotransfected with pFLAG-B-MYB
containing the full-length human B-MYB cDNA (FLAG-B-MYB)
into 293T cells. Cells were extracted in a lysis buffer as described
previously (34). GST fusion proteins were purified on
glutathione-Sepharose beads (GST puri.) and analyzed on an
SDS-polyacrylamide gel, and the complex formation (upper
left, GST puri.) and the FLAG-tagged B-MYB
of the amount used for the in vivo binding assay
(upper right, Lysate) were determined by
anti-FLAG antibody immunoblot. The same blot was stripped and re-probed
with an anti-GST antibody (lower panel) to confirm
expression of the GST fusion protein (GST-ZPR9) and the GST
control (GST). B, 293T cells were transiently
transfected with the vector alone (CMV), as a control, or
FLAG-ZPR9 containing the full-length human ZPR9
cDNA, lysed, and immunoprecipitated with an anti-B-MYB antibody.
The immunoprecipitate of B-MYB was analyzed for the presence of ZPR9 by
Western blot (WB) using anti-FLAG antibody. Bands
representing coimmunoprecipitating ZPR9 by anti-B-MYB antibody are
indicated (upper left, IP: -B-MYB). The amount
of immunoprecipitated B-MYB was analyzed using anti-B-MYB antibody
(lower panel). Total cell lysates were analyzed for ZPR9
(upper right, Lysate).
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Three Functional Domains of B-MYB Are Involved in ZPR9
Binding--
Recently, together with other data (22, 23), it was
reported that poly(ADP-ribose) polymerase binds to the B-MYB
DNA-binding domain and enhances the transcriptional activity of
B-MYB (21). Therefore, we speculated that ZPR9 might
interact with the DNA-binding or transactivation domain of
B-MYB, in addition to the carboxyl-terminal conserved
region, and cause the modulation of B-MYB transactivation. To determine which regions of B-MYB were required for
binding of ZPR9 in vivo, we generated nine deletion
constructs fused to GST (Fig. 2,
A and B). The GST-WT B-MYB, GST-CR,
GST-B-MYB, GST-TA1, GST-B-MYB R1, GST-TA, and
GST-B-MYB R2 constructs were expressed in 293T cells (Fig.
2, C and D, middle left panels) and
used for the in vivo binding assay with ZPR9 and
Two9, a partial clone of ZPR9 comprising amino
acids 206-452 (31). The binding of the FLAG-tagged ZPR9 and
Two9 with all six constructs tested, except for
GST-B-MYB R2, was readily detectable (Fig. 2, C
and D, top left panels). These results suggest
that all three functional domains of B-MYB, a DNA-binding
domain, a transactivation domain, and the carboxyl-terminal conserved
region, are responsible for ZPR9 binding in vivo. To narrow
down further the binding motif, we generated a DBD deletion construct
(amino acids 1-206) and carried out a similar experiment. As a result,
the FLAG-tagged DBD was coprecipitated with GST-tagged ZPR9
(or Two9) but not with GST alone (Fig. 2, C and
D, top right panels). These findings, together
with the binding of ZPR9 to GST-TA, clearly indicate that both
DNA-binding and transactivation domains are required for ZPR9 binding.
However, the GST-B-MYB R2 was not coprecipitated with
FLAG-tagged ZPR9 or Two9, indicating that the
conserved region is only required for ZPR9 binding within the
carboxyl-terminal domain of B-MYB. Taken together, these
results suggest that each functional domain of B-MYB is
sufficient for its association with ZPR9.

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Fig. 2.
Domains of B-MYB involved in
ZPR9 binding. A, schematic representation of the
wild-type B-MYB (WT B-MYB) and its deletion
mutants, B-MYB (for partial B-MYB),
B-MYB R1, B-MYB R2, CR, TA1, DBD, and TA. The
structure of the wild-type human B-MYB is depicted with the
relative locations of its DNA binding domain, acidic region, and
conserved region indicated. Arrows represent the three
tandem repeats in the DNA-binding domain. B, schematic
representation of the ZPR9 and Two9, a partial
clone of ZPR9. The predicted domain structure of
ZPR9 includes three zinc fingers (ZF1, ZF2, and
ZF3). Amino acid number of domain boundaries is indicated.
C and D, mapping of the site on B-MYB
involved in the association with ZPR9 and Two9. 293T cells were
cotransfected with GST alone (pEBG) or the deletion mutants as
indicated, together with pFLAG-ZPR9 (FLAG-ZPR9) or
pFLAG-Two9 (FLAG-Two9). Transfected cells were extracted and
purified with glutathione-Sepharose beads (GST purification) and
immunoblotted with an anti-FLAG antibody as in Fig. 1. A complex
formation between B-MYB proteins and ZPR9 (or Two9) was
determined by Western analysis (WB) using anti-FLAG antibody
(top panels). The same blot was re-probed with an anti-GST
antibody to examine the expression of GST fusion proteins in the
coprecipitates (middle panels), and the expression level of
FLAG-tagged proteins in total cell lysates (Lysate) was
analyzed by Western analysis using anti-FLAG antibody (bottom
panels). GST alone (pEBG) and either pEBG-ZPR9
(GST-ZPR9) or pEBG-Two9 (GST-Two9)
were cotransfected with pFLAG-DBD (FLAG-DBD) into 293T
cells. Transfected cells were extracted and purified with
glutathione-Sepharose beads and immunoblotted with an anti-FLAG
antibody to analyze a complex formation (top right panels).
The expression level of FLAG-tagged and GST fusion proteins in total
cell lysates and the coprecipitates was analyzed by anti-FLAG
(bottom right panels) and anti-GST (middle right
panels) immunoblot assays. The asterisks indicate the
expressed GST fusion proteins.
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ZPR9 Enhances the Transcriptional Activity of B-MYB--
Because
ZPR9 is binding to the DNA-binding and transactivation domain of
B-MYB (Fig. 2), it is likely that the interaction may affect
the transactivation by B-MYB. To investigate the functional significance of binding of ZPR9 to B-MYB, we cotransfected the pT81luc
3xA reporter plasmid, containing three Myb-binding sites from the
chicken mim-1 gene (33), with mammalian expression vectors
encoding for ZPR9 and B-MYB. As shown in Fig.
3A, the addition of ZPR9 to
B-MYB led to a significant enhancement of B-MYB
transcriptional activity. To investigate further whether the expression
of ZPR9 protein levels could influence the B-MYB transactivation, a dose dependence experiment by increasing the ZPR9 expression plasmid was performed. As shown in Fig.
3B, the stimulatory effect of ZPR9 in the B-MYB
transactivation increased in a dose-dependent manner.
However, the transfection of ZPR9 alone, as a control, did
not influence a significant change in the basal transcription. These
findings strongly suggest that ZPR9 is a potential coactivator of
B-MYB.

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Fig. 3.
ZPR9 enhances B-MYB
transactivating activity by direct interaction.
A, 293T cells were transiently transfected with the pT81luc
3xA reporter (8 µg) together with empty pFLAG-CMV2 vector (10 µg)
or pFLAG-CMV plasmid expressing wild-type B-MYB (6 µg) and
pFLAG-ZPR9 (4 µg). Normalized luciferase expression from
triplicate samples is presented relative to the LacZ expressions, and
the standard deviations are less than 5%. Whole cell extracts from
cells transfected with the indicated expression plasmids were analyzed
by Western blotting (WB) using anti-FLAG antibody, and the
cell extracts containing approximately equivalent amounts of B-MYB and
ZPR9 were used for luciferase assay (lower panel).
B, ZPR9 synergizes with B-MYB in the transactivating
activity of B-MYB. 293T cells were transfected as described
above with increasing amounts of ZPR9 (4, 8, and 16 µg) or
B-MYB (2, 4, and 8 µg) as indicated in the presence or
absence of B-MYB (4 µg). Luciferase assays were performed
as described for A. The bottom panel
shows the expression of the increasing amounts of ZPR9 and B-MYB in the
cell extracts used for luciferase assay.
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ZPR9 Stimulates the Nuclear Localization of B-MYB--
Because, in
addition to the ZPR9 binding to the DNA-binding and transactivation
domain of B-MYB, as shown in Fig. 3, coexpression of
ZPR9 and B-MYB resulted in synergistic activation
of the B-MYB-responsive promoter, pT81luc 3xA reporter, it is likely
that ZPR9 could modify B-MYB movement. To address this point, 293T
cells were transfected with FLAG-tagged ZPR9 alone or
together with GFP-B-MYB. Cells expressing B-MYB
and ZPR9 exhibit both cytosolic and nuclear staining, but
the coexpression of ZPR9 with B-MYB resulted in
nuclear localization of the B-MYB protein, as well as the ZPR9,
with an average increase of 2.8- and 3.8-fold in the four experiments,
respectively, when the percentage of the nuclear localization of B-MYB
and ZPR9 was calculated as the number of GFP- (for B-MYB) and Texas
Red-positive (for ZPR9) cells with nuclear staining divided by the
total number of GFP- and Texas Red-positive cells (Fig.
4). These data show that ZPR9 is able to
cooperate with B-MYB for the transactivation by B-MYB.

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Fig. 4.
Subcellular localization of B-MYB and
ZPR9. A, effect of ZPR9 on B-MYB subcellular
localization. 293T cells were transfected with GFP-B-MYB
alone (B-MYB), as a control, or GFP-B-MYB was
coexpressed in cells together with FLAG-tagged ZPR9
(B-MYB/ZPR9). Forty eight hours after
transfection, cells were washed three times with ice-cold PBS and fixed
with 4% paraformaldehyde for 10 min at room temperature prior to
incubation with the anti-FLAG (M2) monoclonal antibody, which was
followed by an incubation with a Texas Red-conjugated anti-mouse
secondary antibody to label the ZPR9 construct. Slides were
mounted and analyzed by confocal microscopy. B, effect of
B-MYB on ZPR9 subcellular localization. 293T cells were transfected
with FLAG-tagged ZPR9 alone (ZPR9), as a control,
or GFP-B-MYB was coexpressed in cells together with
FLAG-tagged ZPR9 (ZPR9/B-MYB).
Cells were washed and fixed as described above. Cells were
immunostained with the anti-FLAG (M2) monoclonal antibody, followed by
Texas Red-conjugated anti-mouse secondary antibody, and analyzed by
confocal microscopy. GFP is in green, and areas of
colocalization appear as yellow (Merge).
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Constitutive Expression of ZPR9 Induces Apoptotic Neuroblastoma
Cell Death by Retinoic Acid--
To analyze the effect of
ZPR9 gene expression on the differentiable or
apoptotic potential of SK-N-BE (2)C, a human neuroblastoma cell line,
we constructed an expression vector pCEV27-ZPR9, where a
full-length human ZPR9 cDNA was placed under the control
of Moloney murine leukemia virus long terminal repeat promoter.
ZPR9-transfected SK-N-BE (2)C cells were selected in medium
containing G418 (800 µg/ml). Overexpression of the ZPR9
transcript in the selected transfectants was analyzed by Northern blot
analysis. As shown in Fig. 5A,
compared with parental SK-N-BE (2)C cells and pCEV27 vector
transfectants, as negative controls, the ZPR9 transcripts were identified at a high level in the selected ZPR9
transfected clones. In addition, similar results were obtained with all
selected ZPR9 transfectant clones (results not shown). The
growth rates under normal serum conditions were comparable in
ZPR9 transfectants, pCEV27 vector transfectants, and
parental SK-N-BE (2)C cells, suggesting that the ectopic expression of
ZPR9 did not affect the proliferative activity on
neuroblastoma cells (results not shown). RA treatment resulted in a
more rapid loss of viability in all ZPR9-expressing clones
compared with the parental SK-N-BE (2)C cells and the cell lines
transfected with the pCEV27 vector (Fig. 5B). To confirm if
the marked decrease in cellular viability of the RA-treated
ZPR9 transfectants is due to apoptosis, we performed dual
annexin V/propidium iodide staining as described under "Experimental Procedures" and obtained an experimental result similar to those in
Fig. 5B. A significant increase in the number of apoptotic cells was observed in the ZPR9 transfectants after RA
treatment, suggesting that the overexpression of ZPR9 may
induce apoptosis, instead of the neural differentiation, in the
presence of RA (Fig. 5C). To investigate further the
physiological roles of ZPR9 during apoptosis, 293T cells were
transiently transfected with GFP alone, GFP and ZPR9, and
GFP and B-MYB. In addition, cells were cotransfected with
ZPR9 and B-MYB, together with GFP. After inducing
apoptosis by TNF-
treatment, apoptotic cells were scored by a change
in nuclear morphology among GFP-positive cells. As shown in Fig. 5D, ~59% of 293T cells expressing ZPR9 were
apoptotic following TNF-
treatment. In contrast, ~16% of cells
transfected with B-MYB underwent TNF-
-induced apoptosis,
similar to the percentage (about 15%) of control apoptotic cells
expressing GFP alone. On the other hand, B-MYB coexpression
markedly inhibited the apoptotic stimulation induced by ZPR9 (about
35% inhibition).

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|
Fig. 5.
ZPR9 transfectants undergo apoptotic
cell death after RA treatment. A, SK-N-BE (2)C cells were
stably transfected with a ZPR9 expression plasmid
(pCEV27-ZPR9) or the parental plasmid (pCEV27) as a control.
The relative expression levels of ZPR9 mRNA in
G418-resistant clones were examined by Northern blot analysis. Total
cytoplasmic RNAs were extracted from ZPR9 transfectants
(ZPR9-18, -34, -37, and -38), pCEV27
transfectants (V11 and V16), and parental SK-N-BE
(2)C cells (SK), and the membrane was probed with a
32P-labeled ZPR9 cDNA insert. Exo-ZPR9
represents the exogenous ZPR9 mRNA, and endo-ZPR9
represents the endogenous ZPR9 mRNA. The
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as
an internal control. B, cells were seeded in 6-well plates
at 4 × 105 cells per well the day before RA
treatment. The cell number of viable cells at the indicated times
following RA treatment of cells (SK, V16, ZPR9-34, and ZPR9-37),
determined by trypan blue exclusion, was counted with a hemocytometer.
Each point indicates the means ± S.E. of two separate experiments
carried out in triplicate. C, cells were plated at the
density of 3-4 × 105 cells/cm2 and grown
the day before RA treatment in the presence of 10% serum. Serum
starvation was induced by changing the medium to 0% serum and then
cultured in the presence (shaded bars) or absence
(white bars) of RA for 2 days. Apoptotic cell death was
determined by flow cytometry for annexin V and propidium iodide.
Results shown are the average of duplicate samples and are
representative of two independent experiments. D, 293T cells
were transiently transfected with expression vectors encoding B-MYB (5 µg) and ZPR9 (5 µg) along with an expression vector encoding GFP (3 µg) as indicated. Transfected cells were incubated for 24 h and
treated with TNF- (20 ng/ml) and cycloheximide (10 µg/ml) for
14 h to induce apoptosis. E, SK-N-BE (2)C cells were
transiently transfected with an expression vector encoding GFP and the
indicated combinations of expression vectors encoding B-MYB and ZPR9.
After 24 h, the medium was changed with 0% serum, and then cells
were cultured in the presence of RA for 2 or 3 days to induce
apoptosis. D and E, GFP-positive cells were
analyzed for the presence of apoptotic nuclei with a fluorescence
microscope. The data shown are the mean ± S.D. of duplicate
assays and are representative of at least three independent
experiments.
|
|
To confirm further the involvement of ZPR9 in the enhancement of
RA-induced apoptosis, we carried out a similar transient transfection
experiment using SK-N-BE (2)C cells. As shown in Fig. 5E,
the results obtained in this experiment were very similar to those in
Fig. 5D. These findings suggest that the overexpression of
ZPR9 is sufficient to stimulate apoptosis induced by various stimuli and raise the possibility that ZPR9 may be a potential pro-apoptotic protein.
 |
DISCUSSION |
In this report, we demonstrate that ZPR9 interacts with B-MYB
in vivo and that each functional domain of B-MYB
is necessary and sufficient to mediate direct protein-protein
interactions with ZPR9. We found that ZPR9 enhanced the transactivating
activity of B-MYB by direct interaction. Furthermore, we
show that B-MYB moves to the nucleus following the coexpression of
ZPR9, implying that ZPR9 may behave as an activator of the bound
transcription factor, B-MYB.
The novel zinc finger protein, termed ZPR9 (zinc
finger-like protein 9), was originally
discovered as a protein partner for the MPK38 serine/threonine kinase
(31). Recently, evidence has emerged that several zinc finger proteins
such as ZPR1, tumor necrosis factor receptor-associated factor, CD40
receptor-associated factor, enigma, and LMP-associated protein act as
modulators for receptor signaling, and that the formation of
multiprotein complexes in many transcription factors results in an
increased diversity and specificity in the regulation of gene
expression (35-40). Zinc finger motifs of the
Cys2-His2 type have been found in
numerous transcription factors, including ZPR9. In this respect, the
self-association of ZPR9 containing zinc finger motifs and the
interaction of ZPR9 with the kinase catalytic domain of MPK38 provide
an interesting aspect to the regulation of this factor (31). In
addition, our recent study strongly suggests a possible role for
phosphorylation of ZPR9 proteins in their translocation to the nucleus
(31). Thus, these data open a new area of investigation on the
potential interaction of ZPR9 with other cellular proteins.
Several lines of evidence indicate that the DNA-binding domain of
MYB proteins has the potential of mediating contact with both
DNA and proteins. It has been shown that PARP binds to the DNA-binding
domain of B-MYB and enhances its transactivating activity and that the physical interaction between PARP and B-MYB is critical for the coactivating function (21), suggesting an important role for
the direct interaction in the regulation of the B-MYB transcriptional activity. Recently, we have shown that B-MYB interacts in vivo with each other via the carboxyl-terminal conserved
region (32). In addition, we have observed that the conserved region of
B-MYB binds to several cellular proteins as well as ZPR9 in the yeast two-hybrid tests and in vivo binding assays
(results not shown). This evidence led us to investigate whether ZPR9, a potential transcription factor containing zinc finger motifs, participates in the B-MYB-mediated transactivation. As shown
in Fig. 3, a significant increase was observed in the transactivating activity of B-MYB by direct binding of ZPR9, suggesting that
the in vivo association of B-MYB and ZPR9 plays a pivotal
role in the modulation of B-MYB transcriptional activity.
Based on this result, we imagine that rather than direct interaction
with the carboxyl-terminal conserved region, the conformational change mediated through the DNA-binding or transactivation domain of B-MYB by direct binding of ZPR9 likely plays a role that is
important in the regulation of B-MYB transcriptional
activity. For activation of B-MYB transcriptional activity,
our results, together with existing data (21-23), suggest a distinct
mechanism in which, in addition to the truncation of the carboxyl
terminus of B-MYB and the phosphorylation by cyclin A-CDK2
complex (7-10), cellular cofactors are bound to the B-MYB, for example
through the binding of PARP and ZPR9, resulting in the enhancement of
the B-MYB transcriptional activity (21, 31).
Recent studies (41) showed that all-trans-retinoic acid
reduces human neuroblastoma growth by inducing either differentiation or apoptosis. To determine whether RA-treated ZPR9 stable
transfectants can influence the differentiation or apoptosis in human
neuroblastoma cells, the morphological and apoptotic analysis of
ZPR9 transfectants was performed in addition to the
examination of the number of viable cells (Fig. 5 and results not
shown). The ZPR9 transfectants undergo apoptosis rather than
differentiation after RA treatment, suggesting that ZPR9 may be one of
the regulators controlling cellular growth arrest induced by RA in
neuroblastoma cells. To test whether the observed apoptotic cell death
after RA treatment in the ZPR9 stable transfectants is a
consequence of direct interaction of B-MYB with ZPR9, we performed two
separate transient transfection experiments using 293T and SK-N-BE (2)C
cells in the presence of TNF-
and RA, respectively (Fig. 5,
D and E). In these experiments, coexpression of
B-MYB significantly inhibited rather than stimulated TNF-
and RA-induced apoptosis enhanced by ZPR9. In contrast, the percentage
of apoptosis in cells transfected with B-MYB alone was
similar to the percentage of control apoptotic cells expressing GFP
alone in both 293T and SK-N-BE (2)C cells. Thus, it is tempting to
suggest that the apoptotic cell death in RA-treated ZPR9
stable transfectants may be derived from the overexpression of
ZPR9 itself, not through binding with B-MYB. On the other
hand, one may raise the argument that ZPR9, like other zinc finger
proteins, could be nuclear for its effect on target genes. Based on
this, one possible explanation for ZPR9-induced apoptosis is that a subcellular location of ZPR9 may contribute to its apoptotic function in the presence of retinoic acid because a markedly increased nuclear
accumulation of ZPR9, compared with the untreated control cells, was
observed when the cells transfected with ZPR9 were treated
with RA (results not shown). Additionally, it is not clear that the
repression of ZPR9-induced apoptosis by B-MYB is dependent on the
direct interaction of B-MYB with ZPR9 because B-MYB thought to mediate
the anti-apoptotic functions is also involved in interactions with
other cellular proteins that may compete with ZPR9 for binding (see
Refs. 21 and 32 and results not shown). The biochemical and molecular
mechanisms underlying the pro-apoptotic properties of ZPR9 are unknown
at present. However, it seems that the most likely mechanism by which
ZPR9 may accelerate apoptosis would be through the modulation of the
potential cellular targets for ZPR9. In this context, future studies
aimed at identifying cellular physiological targets for ZPR9 will be
necessary to elucidate the exact mechanism through which ZPR9 can
induce apoptotic cell death.
In addition, ZPR9 proteins, like PARP (21), are nuclear and enhance
B-MYB transactivation. In this regard, it will be of further
interest to determine the mechanistic interaction between B-MYB and
ZPR9 to gain more insight into the role of ZPR9 in the B-MYB
transactivation. Moreover, because B-MYB is thought to have a general
role in cell growth control, differentiation, and cancer, the
ZPR9-dependent modulation of this transcription factor may contribute to elucidate the mechanism by which B-MYB affects these processes as well as the mechanism of transcription activation by
B-MYB.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Scott A. Ness for providing the
pT81luc 3xA reporter plasmid. We also thank Taenam Kim, Se-Yeon Kim,
and Myeong-Suk Choi for technical assistance.
 |
FOOTNOTES |
*
This work was supported by Grants M10016000009-01A190000910,
M10106000053-01A200001000, and 00-J-BS-01-B-26 from the Ministry of
Science and Technology.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
School of Life Sciences, Chungbuk National University, Cheongju
361-763, Republic of Korea. Tel.: 82-43-261-3233; Fax: 82-43-267-2306;
E-mail: hyunha@cbucc.chungbuk.ac.kr.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M207478200
 |
ABBREVIATIONS |
The abbreviations used are:
PARP, poly(ADP-ribose) polymerase;
ZPR9, zinc finger-like protein 9;
RA, retinoic acid;
GST, glutathione S-transferase;
GFP, green
fluorescent protein;
FBS, fetal bovine serum;
PBS, phosphate-buffered
saline;
DMEM, Dulbecco's modified Eagle's medium;
TNF-
, tumor
necrosis factor-
;
DBD, DNA binding domain.
 |
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