From the Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo 2, 166 37 Prague 6, Czech Republic
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ABSTRACT |
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Using the yeast two-hybrid system, the
transcription factor ATBF1 was identified as v-Myb- and c-Myb-binding
protein. Deletion mutagenesis revealed amino acids 2484-2520 in human
ATBF1 and 279-300 in v-Myb as regions required for in
vitro binding of both proteins. Further experiments identified
leucines Leu325 and Leu332 of the Myb leucine
zipper motif as additional amino acid residues important for efficient
ATBF1-Myb interaction in vitro. In co-transfection experiments, the full-length ATBF1 was found to form in
vivo complexes with v-Myb and inhibit v-Myb transcriptional
activity. Both ATBF1 2484-2520 and Myb 279-300 regions were required
for the inhibitory effect. Finally, the chicken ATBF1 was identified,
showing high degree of amino acid sequence homology with human and
murine proteins. Our data reveal Myb proteins as the first ATBF1
partners detected so far and identify amino acids 279-300 in v-Myb as
a novel protein-protein interaction interface through which Myb
transcriptional activity can be regulated.
The v-myb oncogene carried by the chicken retrovirus
AMV1 is a truncated and
mutated version of its cellular progenitor c-myb proto-oncogene. The c-myb gene is expressed mainly in
hematopoietic cells, where it is involved in maintaining their immature
proliferative stages, while the viral v-myb transforms
myelomonocytic cells in vitro and causes acute monoblastic
leukemia in virus-infected chicks (1-5). Both c-myb and
v-myb have been useful in studying the development of
hematopoietic cells, and much work has been devoted to analyzing their
properties and regulation.
Myb proteins are transcription factors with a unique N-terminal
DNA-binding domain (DBD), a central transactivation domain (TA) and a
C-terminal negative regulatory domain containing the leucine zipper
structure (LZ) (6-13). The DBD and TA domains are crucial for
biological activities of Myb proteins (11, 12, 14). Recently, the LZ
motif has been reported to be required for proliferation of
v-Myb-transformed monoblasts and for in vivo leukemogenicity
of v-Myb (15). In addition to leucine zipper, other parts of v-Myb C
terminus were also found to be important for the biological activities
of the oncogene.2 All these
domains are engaged in interactions with other proteins. Several
interactions of Myb DBD and TA have been implicated in regulation of
Myb functions in growth and differentiation. The interaction of c-Myb
DBD with the EVES motifs of both p100 transcriptional coactivator and
c-Myb itself down-regulates c-Myb activity (16). Similarly, the direct
binding of c-Maf transcriptional activator to c-Myb DBD was found to
repress Myb transcriptional activity and to inhibit Myb- and
Ets1-dependent activation of CD13/APN transcription (17).
The Myb DBD was also found to interact with the retinoic acid receptor.
This interaction appears to inhibit retinoic acid-dependent
transactivation (18). On the other hand, the interaction of C/EBP In an attempt to detect and identify additional protein partners of
v-Myb C terminus that could potentially affect biological activities of
v-Myb, the v-Myb sequence downstream from transactivation domain was
used in this work as a bait in a yeast two-hybrid screen applied to a
human cDNA library. These experiments led to the isolation of ATBF1.
ATBF1 gene was first identified in human hepatoma cells. Its protein
product binds to and represses the activity of an AT-rich enhancer
element of the human In this work, it is shown that ATBF1 binds specifically to Myb proteins
both in vitro and in vivo. Binding of ATBF1 to
v-Myb can repress transcriptional activity of the oncoprotein in
chicken fibroblasts. In this interaction, v-Myb amino acids
279-300 are involved and represent a novel protein-protein
interaction interface through which Myb transcriptional activity can be
regulated. We also describe the detection of chicken ATBF1 and point to
the very high ATBF1 conservation between mammalian and avian species.
Cells and Viruses--
Sf9 insect cells grown in SF 900 II (Life Technologies, Inc.) medium were used to produce v-Myb and
c-Myb from recombinant baculovirus genomes (28). Chicken embryo
fibroblasts (CEF) were grown in Dulbecco's modified Eagle's medium
supplemented with 8% fetal calf serum, 2% chicken serum, and antibiotics.
Plasmids--
The bait molecule pB-137 was constructed by
inserting the v-myb SalI-XbaI fragment
(nucleotides 714-1163) in frame with GAL4 DNA-binding domain in pGBT9
(CLONTECH). In pB-85 bait, the shorter v-myb
fragment (nucleotides 714-971) was used. The numbering of v-myb starts
at the first coding nucleotide of the gag gene in AMV v-myb
(29).
To express various forms of ATBF1 fragment (encoded by the two-hybrid
system-selected RX-28 plasmid) as GST fusion proteins in
Escherichia coli, the following plasmids were prepared.
Plasmid GST-AT438 was constructed by inserting
EcoRI-HindIII fragment of ATBF1 from RX-28 into
EcoRI-Tth111I site of pGEX-3X (Amersham Pharmacia
Biotech) after converting HindIII and Tth111I
into blunt ends by Klenow fragment. To make GST-AT175, GST-AT438 was
used as a template in PCR reaction using the following primers:
5'-CGTGGGATCCACGGGAATTC-3' and 5'-CTGGCTCTTCAGGGAGTTTC-3'. The
amplified ATBF1 fragment was digested with BamHI and
inserted into pGEX-3X digested with BamHI and
SmaI; GST-AT139 and GST-AT81 were made by inserting
BamHI-SmaI and BamHI-StuI
fragments of GST-AT175 into BamHI-EcoRI site of pGEX-3X after converting EcoRI site into blunt end; in-frame
deletions of BamHI-StuI fragment in GST-AT139 and
GST-AT175 created GST-AT58 and GST-AT94, respectively. BamHI
and StuI ends were partially filled to recreate
BamHI site and maintain the reading frame.
To express various v-myb proteins in TnT (Promega), the wild type
v-myb EcoRI-XbaI fragment (nucleotides 360-1163)
and its in-frame deletions, namely
For transient expression in CEFs, the full-length wild type (wt) or
mutant AMV v-myb genes were inserted into pneo vector (30),
creating pneo-wt v-myb, pneo- Yeast Two-hybrid Cloning--
Yeast two-hybrid screening was
performed using the MATCHMAKER two-hybrid system
(CLONTECH). The yeast strain HF7c was transformed with transcriptionally inert pB-85 construct using lithium acetate method. Cells that grew on synthetic medium lacking tryptophan were
transformed with a HeLa cDNA library directionally cloned into
EcoRI-XhoI site of GAL4 activation domain vector
pGAD GH. Approximately 1 × 106 yeast transformants
were plated on 20 15-cm plates containing synthetic medium lacking
tryptophan, leucine, and histidine, and including 10 mM
3-aminotriazol. Plates were incubated at 30 °C for 5 days. Two
hundred growing individual Leu+, Trp+,
His+ colonies were picked and purified on the same
selection medium. Fifty well growing purified colonies were transferred
to the filter paper and assayed for In Vitro Binding Assays--
Logarithmically growing bacterial
cultures (10 ml) harboring different GST-ATBF1 constructs were induced
for 1 h with 1 mM isopropyl- Co-transfection Transactivation Assay and Preparation of Nuclear
Extracts--
Transient transfections were performed by a calcium
phosphate precipitation method (33). Four µg of v-Myb-expressing
plasmids, 2 µg of reporter plasmids, 5 µg of ATBF1 expression
plasmids, 1.5 µg of internal control RSV- Immunoprecipitation and Western Blotting--
Nuclear proteins
were extracted from 2 × 106 CEF transiently
expressing v-Myb and ATBF1-HA using salt and detergent buffer essentially as described (34). A mouse monoclonal anti-HA antibody was
used to immunoprecipitate ATBF1-HA, according to the published protocol
(35). The antigen-antibody complexes were collected on a protein
A-Sepharose beads (Amersham Pharmacia Biotech), washed, and analyzed on
10% SDS-PAGE. For Western blots, the fractionated proteins were
transferred onto HybondTM ECLTM nitrocellulose membrane (Amersham
Pharmacia Biotech) and v-Myb was detected using myb-specific polyclonal
antibody. The blots were developed using ECL reagents (Amersham
Pharmacia Biotech).
Reverse Transcriptase-PCR of Chicken ATBF1 Fragment--
Two
µg of total RNA prepared (36) from the whole chicken embryo was
reverse transcribed using Superscript II reverse transcriptase (Life
Technologies, Inc.), and cDNA was used to amplify a chicken ATBF1
fragment. Degenerated PCR primers were derived from homeodomain HD2
(5'-ACN JGN TTY ACN GAY TAY CAR-3') and zinc finger motif Z12 (5'-RCA
YTT YTT RCA XXX RTA RTT-3') that are well conserved in
Drosophila ZfH-2 and mammalian ATBF1. PCR was performed as follows: 5 min at 94 °C; 1 min at 94 °C, 2 min at 55 °C, 1 min at 72 °C for 35 cycles, and then 1 µl of the PCR reaction was used
for reamplification with HD3 (5'-GTN CAR GTN TGG TTY CAR AAY-3') and
Z12 primers. The final PCR product (138 base pairs) was cloned into
pCRTMII vector (Invitrogen) to yield pCR-chATBF1,
sequenced, and used as a probe for RNase protection assay.
RNase Protection Assays--
pCR-chATBF1 was linearized by
XhoI and transcribed by SP6 polymerase (Roche Molecular
Biochemicals) in the presence of [ v-Myb and ATBF1 C Termini Interact in the Yeast Two-hybrid
System--
In an attempt to isolate a protein(s) interacting with
v-Myb C terminus, the MATCHMAKER yeast two-hybrid system was used. In
this system, the bait molecule pB-137 containing v-myb
nucleotides 714-1163 fused in frame to GAL4 DBD and carrying the gene
for TRP1 bound to regulatory regions of HIS3 and
lacZ reporter genes in HF7c cells auxotrophic for Trp, Leu,
and His. The plasmids containing cDNA (HeLa) library fused to GAL4
TA and carrying the gene for LEU2 were introduced into bait-containing
cells and transformants were selected on Trp-, Leu-, and His-lacking
plates. Interaction of GAL4 TA/cDNA encoded protein with the bait
protein resulted in HIS3 transcription and growth of
transformants on selective medium. Growing colonies were subsequently
screened for the expression of the second lacZ reporter
gene. The pB-137 construct alone, however, activated both
HIS3 and lacZ genes in HF7c cells. Since v-Myb
leucine zipper region has previously been shown to activate transcription in yeast (37), the C-terminal part of LZ was deleted from
pB-137 yielding transcriptionally inactive pB-85 (v-myb
nucleotides 714-971 fused to GAL4 DBD). Upon introduction of GAL4
TA/cDNAs into pB-85 containing cells, 1 × 106
transformants were obtained, 50 of which reproducibly grew well in the
absence of histidine. Among them five colonies were also positive for
lacZ expression. cDNA-containing plasmids were isolated from them and sequenced. One clone (RX-28) contained 440 C-terminal codons (amino acids 2345-2783) of human ATBF1 (Ref. 26; Fig. 1A). Four other positive
clones carried inserts not homologous to any known sequence. To verify
the interaction, the RX-28 plasmid was introduced into HF7c cells
containing pB-85. In this case, all the transformed colonies exhibited
both HIS+ and LacZ+
phenotype. Finally, instead of HF7c also SFY526 yeast strain was
transfected with pB-85, pGBT9 (containing only GAL4 DBD), or pLAM
(encodes human lamin C/GAL 4 DBD). LacZ expression appeared only when pB-85 and RX-28 were cotransfected, suggesting that Myb and
ATBF1 fragments interact in yeast cells.
ATBF1 C Terminus Interacts with Full-length v-Myb and c-Myb in
Vitro--
To confirm the v-Myb-ATBF1 interaction observed in yeast
cells, the ATBF1 sequence from RX-28 was expressed in bacteria as GST
fusion protein from GST-AT 438 vector (Fig. 1A) and bound to
glutathione-agarose column. Ly-sates of Sf9 cells synthesizing either v-Myb or c-Myb were applied to the column, and retained proteins
were analyzed by the Western blot procedure. As shown in Fig.
1B, both v-Myb and c-Myb proteins bound efficiently to GST-AT 438 (lanes 3 and 6), but not to GST alone
(lanes 2 and 5). Smaller-sized proteins detected
in lane 1 represent degradation products of c-Myb. Protein
molecules larger than p48 v-Myb seen in lanes 4,
5, and 6 represent irreversible reaction products of v-Myb, often generated during prolonged manipulation with v-Myb preparations.
ATBF1 Amino Acids 2484-2520 Are Required for Myb Binding--
In
order to delineate the ATBF1 interaction domain, six deletion mutants
fused to GST were prepared. The GST-AT 175, GST-AT 139, GST-AT 81, GST-AT 94, and GST-AT 58 constructs (schematically represented in Fig.
1A) were expressed in bacteria (Fig. 1D) and used
for the binding assay with v-Myb as above. Fig. 1C shows that only the constructs GST-AT 438, GST-AT 175, and GST-AT 94 bind Myb
oncoprotein. Similar results were obtained with c-Myb (not shown).
Thus, the ATBF1 amino acids in GST-AT 94 represent the minimal
interaction domain where the C-terminal 37-amino acid region (amino
acids 2484-2520) referred to as ATBF1-MIS is necessary for binding the
full-length Myb proteins in vitro. This proline-rich region
covers the C-terminal part of the third ATBF1 PQ box.
Two Myb C-terminal Segments Are Involved in ATBF1 Binding--
In
order to define the Myb sequences engaged in ATBF1 binding, several
v-Myb deletion mutants were prepared. As shown in Fig. 2A, v-Myb devoid of
DNA-binding domain with short in-frame deletions covering the bait
fragment was used. The [35S]methionine-labeled Myb
proteins (Fig. 2B, lanes 1-6) were mixed with
glutathione-agarose-bound GST-AT 438. Labeled proteins retained on the
agarose were revealed by electrophoresis (Fig. 2B, lanes 7-12). Smaller sized forms of Myb proteins were probably
initiated at an internal ATG codon located 96 nucleotides downstream
from the first ATG. None of the Myb proteins bound to the control GST column (data not shown). It has been observed that deletion of Myb
amino acids 279-300 encoded by nucleotides 835-900 severely reduced
binding to ATBF1 (lane 9). Identical results were obtained with agarose-bound GST-AT 175 (not shown). Since all the Myb mutants contain the LZ region active in protein-protein interactions (15, 24,
25), the potential contribution of LZ to ATBF1 binding was assessed
using the L3,4A v-Myb mutant. This mutation was shown to reduce binding
of v-Myb LZ to other proteins (15, 24). In our experiments mutation of
Myb LZ, leucines 3 and 4 (Leu325 and Leu332) to
alanines resulted in a significant decrease of binding to GST-AT 438 (lane 11) as well as to GST-AT 175 (not shown). Thus, the
data indicate that there are two adjacent sequences in Myb, namely the
amino acids 279-300 present in the bait molecule and also leucines L3
and L4 of the zipper domain, required for efficient binding of ATBF1
in vitro.
Detection of Chicken ATBF1--
Previous experiments revealed the
interaction between chicken Myb and human ATBF1 proteins. To
substantiate further functional studies with these proteins, we
searched for the existence, evolutionary conservation, and expression
of ATBF1 homologue in chicken cells. Southern blot analysis of the
chicken chromosomal DNA detected a set of restriction fragments that
hybridized with the human ATBF1 probe at high stringency (Fig.
3A). Degenerated PCR primers, derived from ATBF1 sequence highly conserved in man and
Drosophila, were than used for amplification of an ATBF1
fragment from the chicken embryo cDNA. The isolated cDNA
fragment displayed very high homology with the human sequence.
Additional upstream and downstream sequences were obtained by 5'- and
3'-rapid amplification of cDNA ends and aligned with the human and
murine ATBF1 sequences. The chicken fragment shown in Fig.
3B displayed 97% identity of amino acid and 84%
conservation of nucleotide sequences (data not shown). Based on the
sequence comparison of other chicken ATBF1 cDNA fragments covering
approximately one third of mRNA, it can be assumed that the
evolutionary conservation of human and chicken ATBF1 proteins is
approximately 95%.3
ATBF1 Is Expressed in Many Chicken Tissues--
RNase protection
analysis was conducted to characterize the distribution of ATBF1
mRNA in various chicken tissues, cultured v-Myb transformed primary
monoblasts (AMV blasts), and CEF. As shown in Fig. 3C, ATBF1
transcripts were detected in all samples examined. The threshold levels
were found in CEF, while the highest expression was detected in spleen
and brain.
These results document the existence of chicken ATBF1 and substantiate
functional studies with human ATBF1 and chicken v-Myb. Moreover, they
show that chicken fibroblasts, which do not express endogenous Myb and
synthesize only background amounts of ATBF1 mRNA, are suitable for
Myb-ATBF1 co-expression experiments.
Full-length ATBF1 Forms in Vivo Complexes with v-Myb and Inhibits
v-Myb Transcriptional Activity--
To document the direct binding of
transiently expressed full-length ATBF1 and v-Myb proteins in CEF, a
co-immunoprecipitation experiment was performed using a hemagglutinin
epitope-tagged ATBF1. CEF nuclear proteins were immunoprecipitated with
monoclonal anti-HA epitope antibody, and immunocomplexes were resolved
by SDS-PAGE. Western blots were then developed with Myb-specific polyclonal antibody. As shown in Fig.
4A, v-Myb was detectable in
anti-HA immunoprecipitates (lane 1). In the control
experiment, where the identical procedure was applied to cells
transfected only with v-Myb, no signal was obtained (lane
2).
In order to assess whether ATBF1 can modulate transcriptional activity
of v-Myb, both cDNAs were expressed in CEF in the presence of
3xMRE-CAT, the mim-1 MRE-based reporter. In these
experiments, deletion mutants ATBF1- The aim of this work was to identify a protein(s) interacting with
v-Myb C terminus, as this part of the oncoprotein is involved in a
subset of v-Myb activities, namely in the in vitro growth of
transformed monoblasts as well as in the dynamics of the leukemic process in chicks (15).2 It was impossible, however, to use
the entire v-Myb C terminus as a bait in the two-hybrid system due to
the presence of the leucine zipper motif, which behaves as
transcription activating domain in yeast (37). Deletion of C-terminal
portion of leucine zipper (including L3, L4, and L5) provided a
transcriptionally inactive bait pB-85, which was then used to search
for proteins interacting with Myb sequences upstream from the leucine
L3. Screening of a HeLa cDNA library resulted in the isolation of
the 1.2-kilobase fragment encoding C terminus of the complex
homeodomain/zinc finger protein, ATBF1 (26), which bound efficiently
not only the bait fragment, but also full-length v-Myb and c-Myb
proteins in vitro. In co-transfection transactivation
assays, ATBF1 behaved as both the general and v-Myb specific repressor
of transcription. In ATBF1, a proline-rich sequence represented by
amino acids 2484-2520 was found to be crucial for interaction with Myb
proteins and was therefore designated as MIS. This sequence was
required for in vitro and in vivo interaction and
brought about the ATBF1 repressive effect on v-Myb transactivation. No
other functional data on the MIS-containing part of ATBF1 protein are
available to date.
On the other hand, two v-Myb sequences were required for an efficient
in vitro binding of ATBF1: the 22-amino acid region 279-300
(encoded by nucleotides 835-900) and leucines L3 and L4 of v-Myb LZ
(not present in the bait molecule). Only amino acids 279-300, however,
appeared to mediate the ATBF1 inhibitory effect on v-Myb
transactivation, since their deletion eliminated the Myb-specific
repression effect of ATBF1. No function has been assigned to this part
of v-Myb yet. Our data indicate, however, that it contributes to the
transcriptional activity of the oncoprotein, since its deletion reduced
v-Myb transactivation to one third (as shown in Fig. 4B,
d). In biological assays, the In addition to v-Myb specific inhibitory activity mediated by amino
acids 2484-2520 and 279-300 in ATBF1 and v-Myb, respectively, ATBF1
displayed a general inhibitory effect on expression of different reporters. This supports a view of ATBF1 as a transcriptional inhibitor
with a domain(s) of general effect and domains specific for particular
tissue-specific transcription factors.
Recently, a related member of the ZfH family, ZEB, the vertebrate
homologue of Drosophila ZfH-1, was shown to block
transcriptional activity of c-Myb (38). Since our results suggest that
c-Myb is likely to be affected by ATBF1 as well, the ZfH/ATBF1 family and Myb proteins might cooperate in regulation of development of
specific tissues. The very high evolutionary conservation of the
chicken ATBF1 and its high expression in spleen and brain tissues,
where also c-Myb is expressed, support such a view.
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ABSTRACT
INTRODUCTION
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DISCUSSION
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with v-Myb DBD positively affects Myb activity and results in
synergistic activation of the mim-1 promoter by both
proteins (19). Binding of Cyp-40 (20) and HSF3 (21) further document
the complex regulation of Myb DBD by protein-protein interaction. The
Myb TA was found to interact with CBP (CREB-binding protein). This
interaction results in activation of Myb transcriptional properties
(22, 23). Recently, several proteins, including p67 and p160 (24, 25)
and p26/28 (15), were also found to associate with Myb LZ. No
biological activity has been assigned to these interactions so far.
-fetoprotein gene (26). ATBF1, a vertebrate
homologue of Drosophila ZfH 2 protein, is a 306-kDa protein
containing 4 homeodomains, 17 zinc finger motifs, and a number of
segments potentially involved in transcriptional regulation. More
recently, its longer form, termed ATBF1A, was isolated (27).
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721-750,
721-834,
835-900, and
904-933, were constructed in pGEM-4Z.
835-900 v-myb,
and pneo-L3,4A v-myb (15), respectively. The
pneo-
v-myb vector containing no myb sequences was used as
a control. Cytomegalovirus expression vector pKW-2T was used to express
various ATBF1 constructs. To generate pKW-AT, an 8.6-kilobase
HindIII fragment of full-length ATBF1 from pCATBF1A (kindly
donated by T. Morinaga; Ref. 31) was used. In pKW-
MISAT, the
Myb-interacting sequence (MIS) spanning amino acids 2426-2516 was
deleted. In pKW-ATHA, three tandemly arranged hemagglutinin epitopes
(HA) were attached in frame to the 3' terminus of ATBF1. To measure
transcriptional activities, four CAT reporter plasmids were used;
3xMRE-CAT contained three wild type mim-1a Myb recognition
elements (15); the SN0.5-CAT contained SmaI-NotI
fragment of the chicken c-myb promoter (nucleotides
481 to
15 in Ref. 32) in front of TATA box derived from herpes simplex virus
thymidine kinase gene; the ATBF1 recognition element (AFE)-containing
reporters, namely AFE SN0.5-CAT and AFEm SN0.5-CAT, contained the wild
type or mutated AFE (31), respectively, upstream from SN0.5 sequence.
RSV-
Gal plasmid has been described (15). All constructs were checked
by restriction mapping or by sequencing.
-galactosidase. Five colonies
were positive. Plasmids containing human cDNAs were isolated, and
cDNA inserts were sequenced. ATBF1-containing plasmid (RX-28) was
co-transformed with the bait construct or control plasmids (see
"Results") into SFY526 cells to verify the specificity of
two-hybrid assay.
-D-thiogalactoside, sonicated briefly on ice
in 1 ml of 25 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.5% Nonidet P-40 (Fluka), and 50 µg/ml aprotinin (Sigma), and soluble fraction was incubated with 25 µl of glutathione-agarose beads (Amersham Pharmacia Biotech) for 10 min at 4 °C. Agarose beads were washed four times with the above
lysis buffer and then incubated in 200 µl of lysis buffer with insect
cell lysates containing v-Myb or c-Myb. Lysates were prepared by
sonication of 1 × 104 insect cells in lysis buffer,
and soluble fraction was used. Alternatively, 20,000 cpm of
35S-labeled in vitro synthesized (TnT) Myb
proteins were used. After rocking at 4 °C for 2 h, beads were
washed four times with lysis buffer and bound proteins were analyzed by
SDS-PAGE followed by autoradiography or Western blotting.
Gal plasmid, and
pBluescript carrier DNA up to 25 µg were co-transfected into
approximately 7 × 105 CEF/10-cm Petri dish. Empty
expression vectors pKW-2T or pneo-
v-myb were used as
controls. Forty-eight hours later, cells were harvested and nuclear or
whole cell extracts were prepared. CAT activity was determined in whole
cell extracts by CAT enzyme-linked immunosorbent assay (Roche Molecular
Biochemicals), normalized for
-galactosidase activity, and plotted
as -fold activation. The -fold activation represents CAT values
obtained with v-Myb expression vectors divided by CAT values obtained
with pneo-
v-myb vector. For co-immunoprecipitation experiments, nuclear extracts of transfected cells were prepared as
described (34). In these experiments, pKW-ATHA was used.
-32P]GTP to produce
radioactive 257-base pair fragment protecting 138 nucleotides of ATBF1
mRNA. As a referential probe, chicken actin was used. RNA probes
were purified by polyacrylamide gel electrophoresis and hybridized to
10 µg of total cellular RNA in 40 mM PIPES, pH 6.4, 80%
formamide, 0.4 M NaCl, and 1 mM EDTA at
65 °C overnight. After digestion with 5 µg/ml RNase A and 50 units/ml RNase T1 at 37 °C for 1 h, samples were analyzed in
6% polyacrylamide-urea gel and detected by autoradiography.
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ABSTRACT
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Fig. 1.
Binding of c-Myb and v-Myb to ATBF1 in
vitro. A, structure of GST-ATBF1 fusion
proteins used for the binding assay. The potential functional domains
of ATBF1 are depicted (top): rectangles HD1-HD4,
homeodomains; closed rectangles, zinc finger motifs;
PQ rectangles, segments rich in proline and glutamine.
Structures of six types of GST-ATBF1 fusion proteins with various
deletions are shown below. The results of binding assays are
indicated at right. B, binding of c-Myb and v-Myb to ATBF1.
Glutathione-agarose beads containing GST (lanes 2 and
5) or GST-AT 438 (lanes 3 and 6) were
mixed with protein lysate from insect cells expressing c-Myb
(lanes 2 and 3) or v-Myb (lanes 5 and
6). Bound proteins were analyzed on 10% SDS-PAGE, followed
by Western blotting. Lanes 1 and 4 show the input
of c-Myb and v-Myb used for binding assay. C, binding of
v-Myb to GST-ATBF1 fusion proteins. Agarose beads containing each of
the fusion protein or control GST were mixed with baculovirus expressed
v-Myb, as in B. D, analysis of GST-ATBF1 fusion proteins.
Protein lysates from 0.5 ml of bacterial culture expressing various
GST-ATBF1 fusion proteins or GST were mixed with agarose beads. Bound
proteins were analyzed on 10% SDS-PAGE, followed by silver
staining.
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Fig. 2.
Domains of Myb required for binding to
ATBF1. A, structure of Myb mutants used for binding
assay. Top, various domains of v-Myb are indicated:
DBD, DNA binding domain; TA, transactivation
domain; PP, multiple phosphorylation sites; LZ,
leucine heptad repeat (leucine zipper). Shaded box
represents a portion of Myb used in the bait construct. The structures
of Myb mutants are shown below. Blank spaces and
numbers at left represent deleted nucleotides.
Mutations introduced within the leucine zipper in L3,4A construct are
indicated by asterisks. The results of binding assays are
indicated at right. B, binding of Myb mutants to
ATBF1. Various forms of Myb shown above each lane were synthesized
in vitro. 35S-Labeled proteins were mixed with
agarose beads containing GST-AT 438 (lanes 7-12). Bound
proteins were resolved on 12% SDS-PAGE, followed by autoradiography.
Lanes 1-6 show the input of proteins used for binding
assay.
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Fig. 3.
Detection of chicken ATBF1.
A, Southern blot analysis of human and chicken DNA. Genomic
DNA prepared from chicken spleen and human HeLa cells were digested
with EcoRI (lanes 1 and 2) or
HindIII (lanes 3 and 4) and separated
on a 1% agarose gel, blotted onto a nylon membrane, and hybridized
with 32P-labeled ATBF1 cDNA probe spanning nucleotides
7260-7690. B, alignment of amino acid sequences of chicken
(ch), mouse (m), and human (h) ATBF1.
Diverse amino acids are printed in bold type. Homeodomain
HD2 and zinc finger motif Z12 are boxed. The sequence from
which the probe for RNase protection was derived is underlined.
C, expression of ATBF1 mRNA in adult chicken tissues and
chicken cells. RNase protection analyses were conducted using
32P-labeled 138-base pair RNA probe (underlined
in B) and 10 µg of total cellular RNA isolated from
indicated chicken tissues and cells. As a referential probe, the
chicken actin was used.
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Fig. 4.
ATBF1-Myb interaction in chicken embryo
fibroblasts. A, intracellular association of ATBF1 with
v-Myb. Nuclei were isolated from cells co-transfected with v-Myb and
ATBF1-HA expression plasmids (lane 1) or v-Myb expression
plasmid alone (lane 2). Detergent-extracted proteins were
immunoprecipitated with anti-HA antibody. Immunoprecipitates were
separated on 10% SDS-PAGE and analyzed by Western blotting using v-Myb
specific antibody. B, repression of
v-Myb-dependent transcription activation by ATBF1. Plasmids
expressing either no ATBF1 (pKW-2T), or ATBF1 deletion mutant
(ATBF1- MIS) or full-length ATBF1 (ATBF1) were cotransfected with
reporter and Myb expression plasmids as indicated above each
column diagram.
MIS,
835-900 v-Myb and point
mutant L3,4A v-Myb were also used to characterize the effect of protein
regions required for ATBF1-Myb binding in vitro. Both ATBF1
and ATBF1-
MIS displayed a general inhibitory effect, as they reduced
the basal activity of the reporter as well as the expression of
internal control plasmids having lacZ gene placed under
RSV-long terminal repeat or cytomegalovirus promoters. ATBF1 proteins
also inhibited v-Myb-dependent activation of 3xMRE-CAT
reporter. The full-length ATBF1 appeared to be a stronger inhibitor of
v-Myb than ATBF1-
MIS, but both CAT values were so close to the
background that it was hard to draw a reliable conclusion using them.
Therefore, SN0.5-CAT reporter containing a chicken c-myb
promoter fragment (SN0.5) was used. Although there is no consensus MRE
in the SN0.5, v-Myb binds to it in the electrophoretic mobility shift
assay and activates the downstream reporter gene (CAT) by an order of
magnitude stronger than multimerized mim-1 MRE
sequences.4 With this
reporter it was possible to demonstrate that full-length ATBF1 was a
more efficient inhibitor than ATBF1-
MIS, as it reduced v-Myb
activity to 65% of that observed in the presence of ATBF1-
MIS (Fig.
4B, a). Insertion of ATBF1 binding sequence (AFE)
upstream from the SN0.5 fragment (AFE SN0.5-CAT reporter) resulted in a further reduction of v-Myb activity by full-length ATBF1 to 25% of
that obtained with ATBF1-
MIS (Fig. 4B, b).
This effect appeared to be specifically dependent on ATBF1 binding
site, since the point mutation in AFE (AFEm), known to abrogate ATBF1
binding (see AFE[IV] element in Ref. 32), eliminated the increment of inhibition observed with AFE SN0.5-CAT (Fig. 4B-c). The
conclusion that ATBF1 inhibits v-Myb via direct interaction has been
further supported by experiments with
835-900 v-Myb, the mutant
lacking ATBF1 binding region found in pull-down experiments (see Fig. 2B, lane 9). This v-Myb mutant was inhibited to
the same extent with both the ATBF1 and ATBF1-
MIS, independently of
the presence of MIS (Fig. 4B, d). The L3,4A
mutant of the v-Myb leucine zipper was inhibited by ATBF1 and
ATBF1-
MIS similarly as the wt v-Myb, suggesting that Myb LZ may not
participate in down-regulation of Myb transcriptional activity (Fig.
4B, e). In conclusion, the data show that ATBF1
can exert transcriptional repression of v-Myb, which is dependent on
the presence of interacting amino acid sequences 279-300 (encoded by
nucleotides 835-900) and 2484-2520 (encoded by nucleotides 7581-7689
in Ref. 26) in v-Myb and ATBF1, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
835-900 v-Myb mutant (in
which amino acids 279-300 are missing) displayed reduced transformation and leukemia formation abilities compared with wild type
v-Myb.2 Thus, v-Myb amino acids 279-300 can be viewed as a
part of v-Myb transactivation domain that might activate transcription
machinery through interaction with its factor(s). Binding of ATBF1 to
Myb could prevent such interaction and result in down-regulation of v-Myb transcriptional activity similarly as deletion of this region. In
agreement with that, ATBF1 did not show specific repression of the
835-900 v-Myb mutant. On the contrary, leucines L3 and L4 of v-Myb
LZ do not mediate ATBF1 inhibition of v-Myb transactivation, since
mutation of leucines to alanines (that eliminated Myb-ATBF1 binding
in vitro) had no effect on ATBF1 inhibitory activity. This
mutation of leucines, which partially inactivates the growth promoting
and leukemogenic abilities of v-Myb (15), does not inhibit but rather
activates transcriptional activity of v-Myb (Ref. 15; Fig.
4B, e). Thus, v-Myb LZ leucines do not seem to take part in v-Myb interaction with the transcription machinery and
could be involved in a regulatory mechanism not directly connected with
transcription. If this is the case, ATBF1 could modulate also this Myb
activity. Alternatively, leucines of v-Myb LZ would only stabilize the
in vitro binding of ATBF1. Further work is necessary to
address this question.
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ACKNOWLEDGEMENT |
---|
We thank Dr. . Vl
ek for a part
of sequencing work and Dr.
. Taká
ová for
editorial assistance. We are indebted to Dr. P. Bart
n
k
for the help with preparation of graphic files.
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FOOTNOTES |
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* This work was supported by Commission of the European Communities Grant CIPA-CT93-0143), Academy of Sciences of Czech Republic Grant A5052805, Grant Agency of the Czech Republic Grants 204/97/1146 (to M. D.) and 204/96/0014 (to Z. K.), and Howard Hughes Medical Institute Grant 75195-540401 (to M. D.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF104331.
International Research Scholar of the Howard Hughes Medical
Institute. To whom correspondence should be addressed. Fax:
420-2-24310955; E-mail: mdvorak{at}img.cas.cz.
2
M. Dvoáková, unpublished observations.
3 P. Pajer, unpublished data.
4
M. Dvoák, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: AMV, avian myeloblastosis virus; DBD, DNA-binding domain; TA, transactivation domain; LZ, leucine zipper structure; ZfH, zinc finger homeodomain protein; CEF, chicken embryo fibroblasts; GST, glutathione S-transferase; HA, hemagglutinin; CAT, chloramphenicol acetyltransferase; MRE, Myb-responsive element; AFE, ATBF1 recognition element; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; MIS, ATBF1-Myb-interacting sequence; RSV, Rous sarcoma virus; wt, wild type; PIPES, 1,4-piperazinediethanesulfonic acid.
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