From the Unité INSERM U453, Centre Léon Bérard, 69373 Lyon Cedex 08, France
Received for publication, September 7, 2000, and in revised form, November 26, 2000
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ABSTRACT |
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We have reported previously the
physical interaction of B-cell translocation gene proteins (BTG)1 and
BTG2 with the mouse protein CAF1 (CCR4-associated factor 1) and
suggested that these proteins may participate, through their
association with CAF1, in transcription regulation. Here we describe
the in vitro and in vivo association of these
proteins with hPOP2, the human paralog of hCAF1. The physical and
functional relationships between the BTG proteins and their partners
hCAF1 and hPOP2 were investigated to find out how these interactions
affect cellular processes, and in particular transcription regulation.
We defined their interaction regions and examined their expression in
various human tissues. We also show functional data indicating their
involvement in estrogen receptor The pathways that inhibit cell proliferation allow normal cycling
cells to exit from the cell cycle in response to changes in
environmental conditions (e.g. nutrient deprivation,
growth-inhibiting factors, or high cell density). The
BTG1 family,
whose founding member is BTG1 (B-cell translocation gene 1)
(1, 2), is a family of functionally related genes involved in the
negative control of the cell cycle. In vertebrate, this family
comprises at least nine distinct members: BTG1,
BTG2/TIS21/PC3, BTG3/ANA, TOB,
TOB2, B9.10, PC3K, PC3B,
and B9.15. Two short conserved domains (Box A and Box B)
define the signature of this family (3). BTG family proteins have been
reported to be involved in some aspects of cell growth,
differentiation, and survival (4-9). For example BTG1, BTG2/PC3, TOB,
TOB2, and ANA were reported to display antiproliferative properties (2,
10-14). Furthermore, BTG2 expression is regulated by p53
and has been found to be involved in DNA damage-induced
G2/M cell cycle arrest (8). Rat BTG2, known as PC3 (for
pheochromocytoma cell-3) was recently shown to inhibit S-phase entry in
an Rb-dependent fashion, correlated with the inhibition of
cyclin D1 expression (15). Therefore the authors suggest that PC3 may
act as a transcriptional regulator of cyclin D1. These results support
our previous work, indicating that BTG1 and BTG2 may play a role in
transcription regulation. We have shown that these proteins associate
physically and functionally with the homeoprotein HOXB9 and enhance
HOXB9-mediated transcription (16). In addition, we have demonstrated
that both BTG1 and BTG2 interact with mCAF1 (17), whose yeast homolog
is a component of the CCR4·NOT transcriptional complex, which
can affect transcription either positively or negatively (18).
The association of BTG1 with hCAF1, the human homolog of mCAF1, has
been confirmed by a study (19) that showed that hCAF1 overexpression in
different cell lines leads to a proliferation block, demonstrating its
involvement in growth suppression. Furthermore, a search in GenBank
with the hCAF1 cDNA sequence revealed sequence identity
with a human cosmid (DDBJ/EMBL/GenBank accession number AB020860),
localized in the short arm of chromosome 8 at 8p21.3-p22, a region
frequently deleted in numerous human tumors (20-22). CAF1 was
subsequently shown to interact with two other members of the BTG
family, TOB and TOB2 (13). Recently, the paralog of the hCAF1 gene, hPOP2, has been identified (23)
(EMBL/GenBank accession number AF053318). The authors mapped this gene
on chromosome 5q31-q33 and suggested that hPOP2 might be the
tumor suppressor gene associated with the development of the
myelodysplastic (5q-) syndrome. The human POP2 protein was later
described (under the name of CALIF) as interacting with hNOT2 and
hNOT3, the human homologs of the yeast proteins involved in the
formation of the CCR4·NOT complex (24).
In other words, CAF1 and POP2 seem to be involved
in transcriptional regulation, and both are localized on chromosome
regions frequently deleted in human tumors. The structural and
functional characterization of these genes should help to establish
their role in transcription regulation and in tumorogenesis. In the present study, we demonstrate that POP2 protein, like CAF1, interacts with both BTG1 and BTG2, and we define their interaction regions. We
also examine the expression of CAF1 and POP2,
together with BTG1 and BTG2, in different human
tissues. Finally, we present functional results indicating the
involvement of these proteins in estrogen receptor Cloning of hPOP2 Open Reading Frame and Preparation of
Specific Probes--
The cDNA encompassing the entire
hPOP2 open reading frame, along with the 3'-nontranslated
regions (NT) of hCAF1 and hPOP2 mRNA, were
cloned by reverse transcriptase-PCR. 1 µg of total RNA from a human
lymphoblastoid cell line was reverse-transcribed using 100 ng of
oligo(dT) as primer, following the Superscript 2 (Life Technologies,
Inc.) protocol. One-tenth of this mixture was used as a template for
PCR. 50 pmol of specific oligonucleotides were added, and the reaction
was carried out following Promega's instructions, at an annealing
temperature of 50 °C. The expected cDNAs were recovered, cloned,
and sequenced. Sequences of the synthetic oligonucleotides used were as
follows:
hPOP2 3': 5'-TAAGCACCACTCTGGGATCA-3';
hPOP2 5': 5'-ACTTCTCAGGTTTCTTCAGG-3';
hCAF1 3'-NT 3': 5'-AGTTTATCAAAATATTCAATGATG-3';
hCAF1 3'-NT 5': 5'-TTGTATGGCCTTGGTTCTGG-3';
hPOP2 3'-NT 3': 5'-ATGGAGTAGAGAAGTGGGAG-3';
hPOP2 3'-NT 5': 5'-TGATCCCAGAGTGGTGCTTA-3'.
Bacterial Expression Constructs--
To generate the bacterial
expression vectors for BTG1, BTG2, and CAF1, their full-length coding
sequences were inserted into the pGEX-ET expression vector (Amersham
Pharmacia Biotech) in-frame with the glutatione
S-transferase (GST) coding sequence.
Mammalian Reporter Plasmid--
The pP1-CAT reporter plasmid
contains the P1 promoter (nucleotides Mammalian Expression Constructs--
All mammalian expression
constructs used are derivatives of the SV40 promoter-driven expression
vector pSG5 (Stratagene). The plasmid pSG5Flag was derived from pSG5 by
insertion between the EcoRI and BamHI sites of an
oligonucleotide containing the Flag peptide sequence (IBI Flag system;
Eastman Kodak Co.) and a polylinker. The full-length BTG1
cDNA and the fragments coding for the regions containing amino
acids 1-96, 1-117, 1-126, and 38-171, obtained by PCR, were cloned
in-frame with the Flag epitope to generate pSG5FlagBTG1,
pSG5FlagBTG1/1-96, pSG5FlagBTG1/1-117, pSG5FlagBTG1/1-126, and
pSG5FlagBTG1/38-171. pSG5FlagBTG1S159A and pSG5FlagBTG1
The cloned products were verified by DNA sequencing, and the correct
expression of all the proteins was checked.
Transfections--
The plasmids used for transfection were
prepared by the alkaline lysis method and purified by polyethylene
glycol/LiCl. HeLa cells were grown in Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal
calf serum and seeded at 2.5 × 105 cells/well in
six-well microtiter plates 8 h prior to transfection. The
transfected DNA included various amounts of reporter and expression vectors, as detailed in the figure legends, with 50 ng of the pCMV- CAT-ELISA and Luciferase Assay--
CAT-ELISA and luciferase
assay were performed using the CAT-ELISA kit (Roche Molecular
Biochemicals), and the Luc Kit (Promega) following the manufacturers'
instructions. The transfected cells were lysed in 0.150 ml of lysis
buffer. The supernatants were assayed for CAT and luciferase protein
production. All transfection data were normalized by Immunoblot Analysis--
For protein expression assays, 50 µg
of transfected HeLa cell lysate was subjected to electrophoresis on a
10% polyacrylamide-SDS gel. The separated proteins were transferred to
a polyvinylidene difluoride membrane (Millipore) by electroblotting.
Equal amounts of protein were loaded onto each lane, as measured by
Bradford assay and confirmed by Red Ponceau staining of the transferred membrane. ER Glutathione S-Transferase (GST) "Pull-down"
Experiments--
GST and GST fusion proteins were expressed in
Escherichia coli DH5 Far Western Analysis--
For in vitro
protein-protein interaction assays, 5 µg of GST, GST-CAF1, or
GST-FLRG purified proteins were subjected to 10% SDS-PAGE and
transferred to membrane. After denaturation in 6 M and
renaturation in 0.187 M guanidine-HCl in HB buffer
(25 mM Hepes, pH 7.2, 5 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol), the blots were saturated at 4 °C in buffer H (20 mM Hepes,
pH 7.7, 7.75 mM KCl, 0.1 mM EDTA, 25 mM MgCl2, 1 mM dithiothreitol,
0.05% Nonidet P-40, 1% milk), then incubated for 2 h at 4 °C
with 50 µl of [35S]methionine-labeled in
vitro translated ER Northern Blot Analysis--
The pre-made multi-tissue Northern
blots (CLONTECH) were hybridized to the indicated
32P-probes labeled by random priming (Rediprime kit,
Amersham Pharmacia Biotech).
The Two Members of the CAF/POP Family Are Widely Expressed in Human
Tissues: Relationship with BTG1 and BTG2--
hCAF1 and hPOP2 proteins
exhibit 76% sequence identity (see Fig.
1A), but their corresponding
cDNA are divergent in the 5'- and 3'-nontranslated regions. To
determine the tissue-specific patterns of CAF1 and
POP2 gene expression, Northern blots containing human RNA
from various tissues were performed using specific probes corresponding
to the 3'-nontranslated regions of each transcript.
Transcripts of hCAF1 and hPOP2 were observed in a
wide variety of tissues, with the highest levels for hCAF1,
in the skeletal muscle, heart, and pancreas and for hPOP2,
the heart and pancreas. A 2.4-kb mRNA species was detected with
both the CAF1 and POP2 probes (see Fig.
1B). Besides the 2.4-kb transcript, the hCAF1 probe also detected significant amount of a 4.3-kb transcript in the
skeletal muscle and a 1.35-kb transcript, which was the most abundant
in the testis and was not detected in the lung or brain (Fig.
1B). Both forms were absent in the stomach, small intestine,
and thymus. The nature and function of these variant transcripts are
still unknown. At the same time we monitored the expression of
BTG1 and BTG2 (see Fig. 1B):
BTG1 transcript was found, though at barely detectable
levels, in most tissues assayed other than the pancreas, heart, and
lung, unlike BTG2 expression, which was abundant in the
majority of the tissues analyzed, less so in the brain, and absent from
the liver and testis.
Interaction of hPOP2 with BTG1 and BTG2--
Given that hPOP2 and
hCAF1 exhibit 76% amino acid sequence identity, we next investigated
whether hPOP2 interacted with BTG1 and BTG2. We also tested the
possibility that hPOP2 could interact with CAF1.The hCAF1 and mCAF1
proteins have only one amino acid sequence difference (Fig.
1A), so we used CAF1 to indicate both human and mouse
proteins. The cDNA encompassing the entire hPOP2 open reading frame
was cloned by reverse transcriptase-PCR, as described under
"Experimental Procedures." The interaction assay was performed in
the mammalian two-hybrid system, as already described (17). As
shown in Fig. 2A both BTG1 and
BTG2 interacted with hPOP2 in mammalian cells, but hPOP2 did not
associate with CAF1. To verify that hPOP2 can interact directly with
BTG1 and BTG2, we performed in vitro association assays with
purified recombinant GST fusion proteins. GST-BTG1, GST-BTG2, and, as a
control, GST alone, were coupled to glutathione-Sepharose beads and
incubated with [35S]methionine-labeled hPOP2. As shown in
Fig. 2B, the specific retention of hPOP2 was observed with
the GST-BTG1 and GST-BTG2 beads, but not with the control GST beads.
The incubation of GST-BTG1 and GST-BTG2 with
[35S]methionine-labeled luciferase, used as a control,
failed to show any specific interaction (Fig. 2B). These
results point to a direct physical interaction of hPOP2 with both BTG1
and BTG2 and indicate that POP2 does not interact with CAF1 and that it does not homodimerize (data not shown).
Mapping of the Protein Domains Required for BTG-CAF1
Interaction--
Our previous studies indicated that BTG1 and BTG2
were able to interact with mCAF1 in yeast and in mammalian cells and
that Box B was necessary to this interaction (17). More recent
work has confirmed that BTG1 protein interacts with hCAF1 (19), but the
authors indicate that this interaction is mediated by the phosphorylation of BTG1 Ser-159. To define further the BTG1 regions involved in the interaction with CAF1, we used the mammalian two-hybrid system, given that proteins are more likely to be appropriately modified post-transcriptionally and that the results are therefore more
likely to represent biologically significant interactions. Several
deletion mutants of BTG1, described in Fig.
3A, were fused to the DNA
binding domain of the GAL4 protein and assayed for possible interaction
with CAF1 fused to the VP16 transactivation domain (VP16CAF1) in HeLa
cells (see Fig. 3B). As expected, no interaction was found
with the GALBTG1
Seeking then to delineate the regions of CAF1 which are important for
its association with BTG1 and BTG2, we made a series of deletion
constructs of CAF1 (Fig. 4A).
Using the mammalian two-hybrid assay (Fig. 4B), we found
that two regions, corresponding to residues 11-31 and 229-247, are
important for the interaction between CAF1 and both BTG2 (Fig.
4B) and BTG1 (data not shown). None of the hybrid proteins
activated expression of CAT reporter gene on its own. In fact, as
described in our previous studies (17), CAF1 and BTG proteins do
not seem to be capable of stimulating transcription when tethered to
multimerized DNA sites through a GAL4 binding domain in HeLa cells. We
confirmed all of these interactions by in vitro interaction
using a GST pull-down assay, showing that the interactions observed in
HeLa cells are direct (Fig. 4D). As these regions are
conserved between CAF1 and POP2, they are probably also involved in
BTG-POP2 interactions.
Modulation of ER
We performed similar transfection assays using a CAT
reporter gene driven by the P1 promoter (nucleotides
Taken together, the results of these transfection studies suggest that
BTG1 and BTG2 can function either as coactivator or corepressor of
ER Role of NR Motifs in Transcriptional Regulation by
BTG1--
To determine the relative importance of the two
LXXLL motifs, which are common to both BTG1 and BTG2, we
constructed a series of full-length BTG1 derivatives bearing individual
leucine to alanine substitutions as illustrated in Fig.
6A. These constructs were
expressed in HeLa cells and assayed for ER Mechanisms for BTG Regulation of ER
Mouse CAF1 was identified as interacting with the CCR4 complex, a
general transcription multisubunit complex that, in yeast, regulates
the expression of different genes involved in cell cycle regulation and
progression. Our previous data suggested that BTG proteins might
participate, through association with CAF1, in transcription
regulation. To explore further the functional significance of the
interaction between BTG proteins and CAF1, we tested the possibility
that the overexpression of CAF1 could affect
ER
Because BTG1, BTG2, and CAF1 acted as coactivators of ER
At this point we looked at the possibility of a direct CAF1-ER
We next investigated whether the LXXLL motifs might be
involved in the BTG1-CAF1 interaction. As Fig.
8 shows, the BTG1 LXXLL mutant
that can modulate ER In this study we compared structural and functional features of
CAF1 and POP2 gene products and their relations
with the BTG proteins. The POP2 coding region has a high
degree of homology with mouse and human CAF1, resulting in a protein
that has 76% amino acid identity with mouse and human CAF1. A
noticeable difference between POP2 and CAF1 is found in the C-terminal
region, where POP2 contains an extension that is not found in CAF1 (see
Fig. 1A). The present study shows that POP2, like CAF1, can
interact with BTG1 and BTG2 and that the interactions involve two BTG1 regions (amino acids 1-38 and 98-126). Because these two regions are
highly conserved between BTG1 and BTG2, it is probable that they are
necessary for interactions of CAF1 and POP2 with BTG2. Again, two CAF1
regions are involved in the interaction with BTG proteins. It is
possible that both regions are necessary for BTG-CAF interaction or
that one of them is important for the association, and the other is
essential for maintaining the appropriate structure of the proteins.
In an effort to understand how POP2 and CAF1 function in
vivo we examined expression of their mRNA in numerous human
tissues. Along the 2.4-kb transcript that was detected by both the
hCAF1 and hPOP2 probes, the hCAF1
probe also detected two variant transcripts whose structure and
function are still unknown. CAF1 and POP2 being partners of BTG
proteins, we monitored the expression of BTG1 and
BTG2 as well. The high degree of evolutionary conservation of CAF1 and POP2 among eukaryotes indicates that these proteins play an
important biological role. Because CAF1 and POP2 were found to be
expressed in every cell type tested, it is possible that functional
specificity and selectivity could be achieved by interactions with
different partners. The fact that hPOP2/CALIF but not hCAF1, is able to
interact with hNOT2 and hNOT3 (24) suggests that the two proteins are
functionally distinct and that they could participate in the formation
of different complexes in mammals. This observation is particularly
interesting because in yeast the CCR4·NOT complex appears to be
composed of at least two groups of proteins which are physically and
functionally distinct (34).
Different findings indicated the possibility that BTG1 and BTG2
play a role in transcription regulation: (a) both proteins interact with HOXB9 and modulate its transcription activity (16); (b) both of them interact with CAF1 (17) and POP2 (this
report), which are homologs of a yeast transcription factor;
(c) BTG2 acts as a transcriptional regulator of cyclin D1
(15); (d) BTG1 and BTG2 contain two copies of an
LXXLL motif (see Fig. 6A), which has been
identified as being essential for the interaction of a number of
coactivators with nuclear receptors (29); (e) they also
interact with the protein-arginine N-methyltransferase
(PRMT1) (35), and a relationship between the methylation of proteins and the transcription regulation of nuclear receptors has recently been
described (36, 37). We therefore investigated the possible involvement
of the BTG proteins in the transcriptional regulation of the estrogen
nuclear receptor ER BTG1 and BTG2 exhibit either corepressor or coactivator characteristics
depending on the promoter context. The major difference between the two
promoters used for the transcription assay was that the P1 promoter of
ER It is possible that BTG and CAF1 proteins, whose expression inhibits
cell proliferation, can function as corepressors for genes whose
expression activates cell proliferation and, vice versa, as
coactivators in the context of antiproliferative gene promoters. The
localization of hCAF1 and hPOP2 on chromosome
regions frequently deleted in human tumors suggests a tumor-suppressing role for these proteins. The mechanism by which BTG and CAF1 modulate ER (ER
)-mediated transcription
regulation. We found that BTG1 and BTG2, probably through their
interaction with CAF1 via a CCR4-like complex, can play both positive
or negative roles in regulating the ER
function. In addition, our
results indicate that two LXXLL motifs, referred to
as nuclear receptor boxes, present in both BTG1 and BTG2, are involved
in the regulation of ER
-mediated activation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(ER
)-mediated
transcription regulation.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
900+13) of the human
ER
gene fused to the CAT gene (25). The
pERE-Luc, which contains three copies of the ER
consensus elements
(ERE) upstream from the TATA box fused to the luciferase gene, was
provided by V. Laudet (ENSL, Lyon, France). The pG4-TK-CAT reporter
plasmid contains six GAL4 consensus elements upstream from the
thymidine kinase (TK) promoter region, fused to the CAT gene.
BoxB were
obtained from pSG5FlagBTG1 by directed mutagenesis (USE mutagenesis
kit, Amersham Pharmacia Biotech), respectively, replacing the AGC codon
(coding for serine 159) by a GCG (coding for alanine), and deleting
bases 292-351, corresponding to Box B. pSG5FlagBTG1ML2, M3L2, ML1L2,
and M3L1 were obtained from pSG5FlagBTG1 by the same technique,
replacing, at one or both LXXLL sites (bases 130-144 and
280-294), one or three CTN codons (coding for leucine) by a GCG
(coding for alanine). pSG5FlagCAF1 has been described (17); for
pSG5FlagPOP2, the full-length hPOP2 coding sequence,
amplified by reverse transcriptase-PCR as described above, has been
cloned in pSG5Flag vector. pSG5HEO has been described (26). All of the
GAL4 fusion plasmids were obtained by subcloning the different coding
regions from pSG5Flag vectors into the Gal4PolyII plasmid (27) in-frame
with the yeast GAL4 DNA binding domain coding sequence, as described
(17). The fragment corresponding to BTG1 amino acids 76-171, used to
generate pGALBTG1/76-171, was obtained by digesting the
BTG1 cDNA with BamHI. pVPBTG1, pVPBTG2, and
pVPCAF constructs were obtained by cloning the respective coding
regions in-frame with the VP16 transactivation domain coding sequence
into the pSG5FNV vector, as already described (17). The fragments
coding for the regions containing amino acids 1-267 and 1-226 of CAF1
were obtained by digesting the 3'-end of CAF1 cDNA with
the Bal31 exonuclease (Roche Molecular Biochemicals) and then used to
construct pVPCAF/1-267 and pVPCAF/1-226. The fragments coding for the
regions containing amino acids 1-52 and 53-285 of CAF1 were obtained
by digesting the CAF1 cDNA with EcoRI and
then used to construct pVPCAF/1-52 and pVPCAF/53-285.
pVPCAF
229-247 and pVPCAF
11-31 were obtained from pSG5FlagCAF by
directed mutagenesis (USE mutagenesis kit), deleting, respectively,
bases 724-780 (corresponding to amino acids 229-247) and 70-132
(corresponding to amino acids 11-31).
GAL plasmid as an internal control, in 5 µl of LipofectAMINE (Life Technologies, Inc.), and 1 ml of Opti-MEM (Life Technologies, Inc.). The amount of transfected SV40 promoter was kept constant by the
addition of pSG5 to the transfection mixture. After 24 h, the
cells were washed and treated, where necessary, with a medium
containing 10 nM 17
-estradiol for 24 h. The
transfected cells were washed and collected 48 h after transfection.
-galactosidase
assays, quantified by o-nitrophenly
-D-galactopyranoside assay using a standard
linear curve. Reporter activity was expressed as the ratio of fold
induction to the activity of the reporter vector alone or as pg of CAT
protein/ml of cell lysate. Each set of experiments was repeated at
least three times, and similar results were obtained in each case.
was detected with an anti-human monoclonal antibody (HC-20; Santa Cruz Biotechnology, Inc.). The membranes were then incubated with horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulins. The protein was visualized using the enhanced chemiluminescence kit (Amersham Pharmacia Biotech), following the
manufacturer's instructions.
, purified on glutathione-Sepharose
beads (Amersham Pharmacia Biotech), and quantified using the Bio-Rad
method. SDS-PAGE and Coomassie staining were used to confirm the
integrity of the full-length fusion proteins. For in vitro
protein-protein interaction assays, 10 µg of GST and GST fusion
proteins was incubated for 1 h at room temperature with 50 µl of
glutathione-Sepharose beads in the binding buffer (20 mM
Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1% milk, 10% glycerol) and packed in minicolumns. 15 µl of [35S]methionine-labeled proteins
synthesized in vitro (TNT T7 Quick Kit, Promega) were
suspended in the binding buffer and passed through GST-, GST-BTG1-,
GST-BTG2-, or GST-CAF1-glutathione-Sepharose minicolumns. After
washing, the retained proteins were eluted with 10 mM
glutathione in 50 mM Tris-HCl, pH 8, analyzed on a 12% SDS
polyacrylamide gel, and visualized by autoradiography.
, synthesized in a reticulocyte
lysate-coupled transcription/translation system (Promega) in the
presence or absence of 100 nM 17
-estradiol. After
washing in buffer H for 1 h at 4 °C, the filters were dried and autoradiographed.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of CAF1,
POP2, BTG1, and BTG2
in human tissues. Panel A, alignment
of the human CAF1 and POP2 proteins. Panel B,
Northern blot analysis of the distribution of CAF1,
POP2, BTG1, and BTG2 transcripts in
adult human tissues. The same Northern blots were successively stripped
and rehybridized with the indicated probes. Actin hybridization was
used as a control.
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Fig. 2.
hPOP2 interacting with BTG1 and BTG2 in
vitro and in vivo. Panel A,
hPOP2-BTG interaction in vivo using the mammalian two-hybrid
system. 200 ng of GAL4 and VP16 expression plasmids corresponding to
indicated fusion proteins were transiently cotransfected into HeLa
cells with 0.5 µg of a reporter gene containing six GAL4 binding
sites upstream from a minimal thymidine kinase promoter fused to the
CAT gene. Cells were transfected as described under
"Experimental Procedures." Total DNA was kept constant at 1 µg.
Reporter activity, which is expressed as pg of CAT protein/ml of
lysate, was normalized with -galactosidase activity. Bars
indicate standard deviations from the mean of at least three
independent transfections. Panel B, physical
interaction of hPOP2 with BTG1 and BTG2 in vitro.
35S-Labeled full-length in vitro translated
hPOP2 and luciferase were suspended in binding buffer and passed
through GST, GST-BTG1, and GST-BTG2-glutathione-Sepharose minicolumns.
The beads were washed and then eluted with 10 mM
glutathione. The eluted proteins and one-third of input radiolabeled
proteins were analyzed by 12% SDS-PAGE and visualized by
autoradiography. Molecular size markers are given in kDa.
BoxB chimera, lacking Box B (amino acids 98-117),
whereas GALBTG1S159A, in which the BTG1 Ser-159 is mutated into
Ala, did not prevent BTG1 interaction with CAF1. The results of this
assay indicate that the phosphorylation of serine 159, unlike Box B, is
not indispensable for BTG1-CAF1 interaction. In contrast to the results
obtained with yeast (17), the deletion mutant GALBTG1/1-117 was unable
to interact with CAF1, showing that Box B is necessary but not
sufficient for this interaction in HeLa cells. As the deletion mutant
GALBTG1/1-126 was still able to interact with CAF1, we conclude that
the interaction in question does not require the C terminus of BTG1 but
is strictly dependent on the short sequence flanking Box B (amino acids
118-126), which is highly conserved between BTG1 and BTG2. The
deletion mutant GALBTG1/38-171 did not interact with CAF1, showing
that the N terminus of BTG1 is required for the interaction. This
region (amino acids 1-38) is also involved in the interaction of both BTG1 and BTG2 with HOXB9 protein in yeast (16) and contains a short
motif (EIAAAV) that is conserved in all the members of the BTG family.
It is possible that this motif has some functional significance, at
least in protein-protein interactions. All of the results obtained with
the GALBTG1 chimeric mutants were confirmed in vitro, using
a GST pull-down method with purified GST-CAF1 and
35S-radiolabeled mutant BTG1 proteins (Fig. 3D).
We conclude that the interactions observed were direct and not modified
by the presence of the GAL4 domain in the fusion proteins. Analysis of the interactions of the VP16POP2 chimeric protein with the GALBTG1 fusion mutants gave similar results (data not shown). Taken together, these results demonstrate that the direct interaction of CAF1 and POP2
with BTG1 involve two regions of BTG1 (amino acids 1-38 and 98-126).
As these two regions are highly conserved between BTG1 and BTG2, it is
probable that they are also necessary for the interactions of CAF1 and
POP2 with BTG2.
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Fig. 3.
Mapping of BTG1 interaction(s) domain(s) with
CAF1. Panel A, schematic diagram of various
BTG1 mutant proteins expressed either as Gal4 or Flag fusion products.
Panel B, interaction assay of GAL4BTG1 mutant
fusion proteins with VP16CAF1 in the mammalian two-hybrid system.
Production of CAT protein is expressed as the ratio of fold induction
to the activity of the reporter vector alone. Bars indicate
standard deviations from the mean of at least three independent
transfections. Panel C, all GAL4BTG1 mutants are stably
expressed at levels comparable to the wild type protein, as assayed by
Western blotting of cellular extracts from transfected HeLa cells using
the anti-GAL4 antibody (RK5Cl; Santa Cruz Biotechnology, Inc.).
Panel D, GST pull-down assay showing interactions
between 35S-labeled in vitro translated FlagBTG1
mutants and GST-CAF1, as described for Fig. 2B. Molecular
size markers in panel D are given in
kDa.
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Fig. 4.
Regions of CAF1 which mediate the interaction
with BTG. Panel A, schematic representation
of various CAF1 mutant expressed as VP16 fusion proteins.
Panel B, interaction assay of VP16CAF1 mutant
fusion proteins with GAL4BTG2 in the mammalian two-hybrid system.
Production of CAT protein is expressed as described for Fig.
3B. Panel C, Western blot showing that CAF1 and
derivative mutants are expressed to similar levels in
transfected HeLa cells. The anti-Flag (M2) monoclonal antibody
(Stratagene) was used to detect the VP16Flag-fusion proteins.
Panel D, GST pull-down assay showing interactions
between 35S-labeled in vitro translated VP16CAF1
mutants and GST-BTG2, as described in Fig. 2B. Comparable
results were obtained with BTG1 constructs. Molecular size
markers are given in kDa.
-mediated Transcriptional Activation by BTG
Proteins--
The fact that BTG1 and BTG2 interact with CAF1 and POP2,
which are homologs of the yeast transcription factor yCAF1/POP2 and act
as cofactors for HOXB9-mediated transcription (16), supports the
hypothesis that these proteins play a role in transcription regulation.
In addition, BTG1 and BTG2 contain two copies of an LXXLL
motif known as the NR (nuclear receptor) box
(Fig. 6A), which was identified as being essential for the
interaction of a number of coactivators with nuclear receptors (29).
One motif (referred to below as L1) is located in the N-terminal part
of the two proteins; the other motif (referred to below as L2) is located within the middle part, at the beginning of Box B, one of the
two conserved domains that constitute the BTG family signature. These
observations incited us to study the possible role of the BTG proteins
in the transcriptional regulation of the nuclear receptors. We first
focused on ER
because both BTG and ER
are involved in the
regulation of cell proliferation: hormone binding to ER
induces
conformational changes leading to the recruitment of transcriptional
auxiliary factors, binding of ER
to EREs in gene promoters, and
regulation of transcriptional activity of genes involved in
proliferation, development, and differentiation (for review, see Refs.
30-33). HeLa cells, which lack endogenous ER
, were transfected with
a vector expressing ER
, pSG5HEO, and a luciferase reporter linked to
a multimer palindromic ERE sequence, pERE-Luc, along with either a
control plasmid or vectors expressing BTG1 and BTG2, in the presence of
17
-estradiol. The ER
expressed by pSG5HEO contained a
substitution of a valine for a glycine (point mutation in codon 400)
which made its binding to ERE strictly dependent on estradiol, at least
in vitro (26). Both BTG1 and BTG2 significantly enhanced the
ER
-mediated activation of the luciferase reporter gene, as shown in
Fig. 5A. No effect of BTG1 or
BTG2 on reporter gene activity was observed in the absence of ER
,
and the cotransfection of pSG5FlagFOLL, which encodes an unrelated
protein that was used as a control, showed no effect on reporter gene
activation (Fig. 5A).
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Fig. 5.
Promoter-selective coactivator or repressor
effect of BTG1 and BTG2 for ER . Panel A,
coactivator effect of BTG1 and BTG2 on ER
function. HeLa cells were
transiently transfected with 100 ng of pERE-Luc plasmid in the presence
of 40 ng of pSG5HEO and increasing amounts (white bar, 20 ng; gray bar, 100 ng; black bar, 200 ng) of BTG1-
and BTG2-expressing vectors or 200 ng of control plasmids. After
24 h, the cells were washed and treated for 24 h with a
medium containing 10 nM 17
-estradiol. The transfected
cells were washed and collected 48 h after transfection, then
assayed for luciferase and
-galactosidase activity. Normalized
values are expressed as in Fig. 3B. Panel
B, inhibition of ER
-mediated activation of transcription
on the P1 promoter by BTG1 and BTG2. 200 ng of expression plasmids,
corresponding to the indicated proteins, was transiently cotransfected
into HeLa cells with 0.5 µg of pP1-CAT reporter plasmid, as described
under "Experimental Procedures." Reporter activity, which is
expressed as pg of CAT protein/ml of lysate, was normalized by
-galactosidase activity. Bars indicate S.D. from the mean
of at least three independent transfections. The pERE-Luc and pP1-CAT
reporter vectors are represented at the bottom of the
figure.
900+13) of the human ER
gene (pP1-CAT), which is responsive to estradiol through three half-EREs dispersed in the promoter (25). HeLa cells were transfected with the pSG5HEO vector, expressing ER
, and pP1-CAT, with vectors expressing BTG1 and BTG2, in the presence of
17
-estradiol. In marked contrast to the previous results, both
proteins significantly inhibited ER
-mediated activation of the
CAT reporter as shown in Fig. 5B. The control
reporter construct pBLCAT3, which lacks the P1 promoter, was not
activated when cotransfected with the ER
-expressing vector (data not
shown), and no effect of BTG1 or BTG2 on reporter gene activity was
observed in the absence of ER
. Cotransfection of pSG5FlagFOLL used
as a control, showed no effect on reporter gene activation (Fig.
5B). Similar results were obtained in COS-7 and MCF7 cells
(data not shown).
(Fig. 5, A and B), depending on the
promoter context, which in turn suggest that the BTG proteins can act
as effectors of the ER
signaling pathway.
-dependent
transcriptional activation. The results of four independent experiments
(Fig. 6, B and C) demonstrated that the mutation
of LXXLL to LXXAL in both NR boxes (see Fig. 6B;
ML1L2) were fully responsive to ER
. In contrast, mutations that
converted the three hydrophobic leucine to alanine (M3L1 and M3L2) in
the L1 or in the L2 motif, prevented BTG1 for having an effect on ER
transcriptional activity, suggesting that both motifs participate in
the observed regulation. As expected the mutant BTG1/1-96, lacking Box
B and L2, had no effect on ER
-mediated transcriptional activation.
Thus, the activity of BTG1 and BTG2 on ER
appears to depend on the
presence of two functional NR motifs.
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Fig. 6.
Involvement of BTG1 NR box motifs in the
modulation of ER -mediated transcription. Panel
A, schematic representation of the LXXLL sites of
BTG1 and the corresponding mutants. L1 and L2
refer to the mutated site, and M and M3 refer to
the number of leucine(s) replaced by alanine(s). Panels
B and C, HeLa cells were transiently transfected
with the ER
and the NR BTG1 mutants expression vectors and either
the pERE-Luc (panel B) as indicated in Fig. 5A or
pP1-CAT (panel C), as indicated in Fig. 5B.
Data are presented as described for Fig. 5. Panel D,
expression of FlagBTG1 and the indicated mutants in lysates from
transfected HeLa cells analyzed by Western blotting with the anti-Flag
(M2) antibody.
-mediated
Activation--
To investigate whether the ER
-BTG functional
interaction takes place directly, we carried out GST pull-down
experiments. In vitro translated ER
did not appear to
interact directly with either GST-BTG1 or GST-BTG2 used as baits,
either in the presence or the absence of the ligand (data not shown).
But although the BTG proteins seem to not interact directly with ER
,
they possibly interact with other components of regulatory complexes
involved in ER
-dependent transactivation.
-dependent activation. As shown in Fig.
7, A and B, CAF1,
like its partner proteins BTG1 and BTG2, regulates ER
-mediated
transactivation and can function either as coactivator or corepressor
depending on the promoter context. To ensure that this effect on ER
transcriptional activity was not a result of a modification of ER
expression, we performed a Western blot analysis. As Fig. 7C
illustrates, ER
protein is expressed at a comparable level in both
the presence and absence of exogenous BTG1, BTG2, and CAF1.
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Fig. 7.
Effect of CAF1 on ER -dependent
transcription. Panels A and B, HeLa cells
were transiently transfected with the ER
, CAF1, or FOLL expression
vectors, and either the pERE-Luc (panel A) as described in
Fig. 5A or pP1-CAT (panel B), as indicated in
Fig. 5B. Data are presented as described in Fig. 5.
Panel C, expression of ER
in lysates from nontransfected
HeLa cells (HeLa), or from HeLa cells transfected with
pSG5HEO (200 ng), either alone (ER
) or in the presence of
BTG1-, BTG2-, or CAF1-expressing vectors (200 ng) (ER+BTG1,
ER+BTG2, ER+CAF1). The cell lysates were
subjected to Western blot analysis after SDS-PAGE and immunoblotted
with the anti-ER
antibody. Panel D, ER
interacts
directly with CAF1 in the presence of 17
-estradiol. Purified GST,
GST-CAF1, and the control protein GST-FLRG, shown as Coomassie
staining, were subjected to SDS-PAGE, transferred from the gel to
membrane, and probed with [35S]methionine-labeled ER
protein suspended in binding buffer in the presence or absence of 100 nM 17
-estradiol. Specific hybridization was observed
with GST-CAF1, but not with control GST or GST-FLRG, after incubation
with labeled ER
in the presence of 17
-estradiol. No interaction
was observed in the absence of hormone. Molecular size markers are
shown in kDa.
, we tested
for additive or synergistic effects on ER
-dependent activation when both proteins were expressed in the transient transfection assays. The simultaneous expression of CAF1 and BTG did
not shown a synergistic effect but produced an enhancement over the
activation observed with each protein alone, depending upon ER
, BTG,
and CAF1 expression levels and the cellular context (data not shown).
interaction performing a far Western blot analysis. Purified GST,
GST-CAF1, and GST-FLRG, an unrelated protein used as control, were
subjected to SDS-PAGE, transferred from the gel to membrane, and probed
with [35S]methionine-labeled ER
protein in the
presence or absence of 17
-estradiol. As shown in Fig. 7D,
specific hybridization was observed with GST-CAF1, but not with GST and
GST-FLRG after incubation with labeled ER
in the presence of
hormone. [35S]Methionine-labeled ER
protein in the
absence of hormone failed to show any interaction. This finding
suggests that BTG1 and BTG2 may exert their coactivator function on
ER
-mediated transcription by means of CAF1.
-dependent transcription (BTG1ML1L2) was still able to interact with CAF1. In contrast, the LXXLL
mutations that abolish the effect of BTG1 on ER
transcription
(BTG1M3L1 or BTG1M3L2), also strongly decrease the interaction of BTG1
with CAF1 (Fig. 8). These results were confirmed by mammalian
two-hybrid assay (data not shown). Furthermore, the interaction of BTG1
with CAF1 is consistent with its ability to mediate transcription
activation and inhibition.
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Fig. 8.
Dependence of the physical interaction of
BTG1 with CAF1 on the integrity of its LXXLL
sites. Shown is a GST pull-down assay with interactions between
35S-labeled in vitro translated BTG1 proteins
mutated in LXXLL boxes, as described in Fig. 6A,
and GST-CAF1, as described in Fig. 2B.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Our results indicate that BTG1 and BTG2 can
function as coactivators and corepressors of ER
(Fig. 5) and that
the LXXLL sequences are involved in the effect of BTG
proteins on ER
-dependent transcription (Fig. 6). This
result along with the fact that (a) the BTG proteins need the LXXLL motifs to modulate ER
-mediated transcription
and to interact with CAF1 and (b) that CAF1 also acted as a
modulator in this assay and can bind directly to ER
in
vitro strongly suggests that BTG proteins modulate ER
-mediated
transcription through their interaction with CAF1 via a
CCR4-like complex or complexes. As regards yeast, it is thought that a
complex of this type may affect transcription either positively or
negatively (18). This hypothesis is supported by our recent results
showing that in mammalian cells CAF1 and BTG1 bind together in a large
multiprotein complex.2
Alternatively, it may be that the LXXLL sites are important
for the correct conformation of BTG proteins and for their biological activity. In fact this motif normally takes on a helical conformation and facilitates protein-protein interaction. BTG proteins may function
as bridges between nuclear receptors associated with coactivators or
corepressors and component(s) of the transcription machinery. We have
also tested the ability of BTG1 and BTG2 to affect transactivation by
other members of the nuclear receptor family, and our results indicate
that BTG1 and BTG2 also modulate the transcriptional activity of the
thyroid hormone receptor
(data not shown).
is a natural promoter containing three half-ERE dispersed in the
promoter, whereas the ERE-Luc contains multimerized palindromic ERE. In
the case of the P1 promoter, ER
can bind as monomer, and its
regulation can be different from that of a dimeric conformation. In
accordance it has been demonstrated that SRC1, an ER
coactivator,
requires the presence of ER
dimer for binding (38). Although the
mechanistic basis of the inhibitor effect of BTG1 and BTG2 on
ER
-dependent transcription remains unclear, a number of
plausible hypotheses may be put forward: inhibition of DNA binding of
ER
, direct antagonism of cofactors functioning via competition, or
transrepression via recruitment of a putative repressor. BTG1 and BTG2
overexpression in vivo may sequester limiting factors
required for ER
transactivation, thus leading to an imbalance of
limiting constituents that generate the functional ER
activation
complex and leading to transcriptional squelching. Recent studies have
shown that PC3, the rat homolog of BTG2, inhibits the expression of
cyclin D1, which stimulates ER transactivation (28, 39). We can
speculate that BTG2 inhibits the activation of the ER
P1 promoter by
inhibiting cyclin D1 expression. The characterization of the components
of the multiprotein complex containing CAF1 and BTG should tell us
whether BTG1 and BTG2 modulate ER
-mediated transcription directly or
through the control of ER
activator expression.
-activated transcription remains to be elucidated. Further investigation is also required to understand how BTG-CAF1 interactions mediate transcription regulation in normal physiology and in neoplasia.
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ACKNOWLEDGEMENTS |
---|
We thank Isabelle Treilleux (Centre Léon Bérard, Lyon, France) for providing pP1-CAT plasmid and for helpful advice on the work, Helene Gaude for the help in mutant constructions, and Vincent Laudet (Ecole Normale Supérieure, Lyon France) for providing the pERE-Luc plasmid.
![]() |
FOOTNOTES |
---|
* This work was supported by the Ligue Nationale Contre le Cancer, Comité du Rhône, by the Association pour la Recherche sur le Cancer, and the Région Rhône-Alpes.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.
Holder of a Ministère de l'Education Nationale, de la
Recherche et de la Technologie (MENRT) fellowship.
§ To whom correspondence should be addressed: Unité INSERM U453, Centre Léon Bérard, 28 Rue Laënnec, 69373 Lyon Cedex 08, France. Tel.: 33-4-7878-2691; Fax: 33-4-7878-2720; E-mail: corbo@ lyon.fnclcc.fr.
Published, JBC Papers in Press, January 2, 2001, DOI 10.1074/jbc.M008201200
2 A.-P. Morel, D. Prévôt, T. Voeltzel, M.-C. Rostan, and L. Corbo, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
BTG, B-cell
translocation gene;
CAF, CCR4-associated factor;
PC3, pheochromocytoma
cell-3;
ER, estrogen receptor;
NT, nontranslated;
PCR, polymerase chain
reaction;
GST, glutathione S-transferase;
CAT, chloramphenicol acetyltransferase;
ERE, ER response element, Luc,
luciferase;
ELISA, enzyme-linked immunosorbent assay;
PAGE, polyacrylamide gel electrophoresis;
kb, kilobase;
NR, nuclear
receptor.
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