From the Laboratory of Medical Chemistry and Medical
Oncology, Pathology, University of Liege, Sart-Tilman, 4000 Liege,
Belgium and the
Laboratory of Immunoregulation, NIAID, National
Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Combinatorial interactions between distinct
transcription factors generate specificity in the controlled expression
of target genes. In this report, we demonstrated that the HOXB7
homeodomain-containing protein, which plays a key role in development
and differentiation, physically interacted in vitro with
I Multiple transcription factors establish combinatorial
interactions to achieve their in vivo specificity. These
protein-protein interactions modulate the activating or repressing
abilities of the complexes. The identification of all the partners
interacting with a transcription factor is thus essential for the
understanding of its biological functions.
Homeodomain-containing proteins are transcription factors that play a
crucial role in the development of many species, including humans
(1-4). They share a highly conserved 60-amino acid DNA-binding domain,
the homeodomain, and control the expression of many target genes, most
of which remain unknown (5). These proteins are encoded by 39 HOX genes, which are organized in four clusters (loci A, B,
C, and D) located on chromosomes 7, 17, 12, and 2, respectively (6).
Interestingly, their pattern of expression along the anteroposterior
axis of the developing embryo is closely related to their chromosomal
position on the cluster (7), defining a "spatial colinearity."
Although homologous recombination experiments have clearly demonstrated
their in vivo specificity, all the HOX gene
products bind to very similar sequences in vitro (8-10). Their specificity may thus be achieved not only through DNA-protein interactions but also through protein-protein interactions with other
transcription factors whose identities remain largely unknown. Among
these partners, the extradenticle/Pbx
homeodomain-containing proteins were the first to be identified as
co-factors for HOX proteins (11). Interaction with the PBX protein
requires the pentapeptide, a conserved domain located upstream of the
DNA-binding domain of most HOX gene products and required
for the interactions of HOX proteins with other peptides (11), as well
as the HOX cooperativity motif, a sequence C-terminal to the Pbx
homeodomain (12). Because AbdB-like HOX proteins do not harbor any
pentapeptide-like sequence, they cannot interact with Pbx proteins
(13), thus suggesting that other partners might be involved. Indeed, a
recent report has illustrated the existence of heterodimeric complexes between HOX and Meis1 proteins (13). Moreover, it is likely that other
proteins yet to be identified also interact with HOX proteins and
contribute to their biological function.
HOXB7 cDNA was initially isolated from an
SV40-transformed human fibroblast cDNA library (14). The HOXB7
protein is involved in a variety of developmental processes, including
hematopoietic differentiation and lymphoid development (15-17).
Because of its expression in lymphoid and nonlymphoid cells, the HOXB7
protein might be involved in the regulation of a common transcriptional event rather than in lineage-specific gene expression (18). However,
despite the demonstration of HOXB7 protein binding to DNA (19), little
is known about its transcriptional properties and interacting partners
in vivo. We first demonstrated that the HOXB7 protein as
well as a naturally occurring mutant harboring a truncated C-terminal
tail both transactivate from a HOX-binding consensus sequence in breast
cancer cells (20) and physically interact with the coactivator
CREB-binding protein.1
The NF- In this report, we demonstrated that I Cell Cultures--
The MDA-MB231 cell line was obtained from the
American Type Tissue Collection (Rockville, MD). The cells were
maintained in RPMI 1640 medium supplemented with 10% fetal bovine
serum (Life Technologies, Inc.) and antibiotics.
Plasmids--
Coding sequences for the HOXB7 protein and for a
naturally occurring protein lacking 2 amino acids in its C-terminal
sequence (B7*) (20) were subcloned by
PCR2 into the expression
vectors pcDNA3 (Invitrogen, San Diego, CA) and pMT2T.
The constructs were sequenced to confirm the integrity of the
amplified regions. pcDNA3 expression vectors coding for HOXB7
proteins deleted either in the N- or the C-terminal domain were
constructed by PCR amplification. The constructs B7-
The mammalian PMT2T expression vectors for p50, RelA, and
I
Both the pT109 and pTCBS reporter plasmids were provided by Dr.
Zappavigna (Laboratory of Gene Expression, Department of Biology and
Technology, Instituto Scientifico H. S. Raffaele, Milan, Italy). The
pTCBS plasmid contains an 8-fold multimerized form of a homeodomain consensus-binding sequence (CBS) cloned upstream of an HSV-TK promoter
and a luciferase reporter gene, whereas the pT109 construct does not
contain the CBS sequence and was thus used as a negative control. The
For GST interaction experiments, various functional domains of
I In Vitro Translation--
In vitro transcription and
translation were performed using the Wheat Germ TNT kit provided by
Promega (Madison, WI) with 1 µg of various DNA templates and
[35S]methionine, according to the protocol provided by
the manufacturer.
In Vitro Protein-Protein Interactions--
GST fusion proteins
were produced in the Escherichia coli BL21 bacterial strain.
Bacteria were grown in 500 ml of Luria broth to an
A600 nm of 0.6, induced with 0.5 mM
isopropyl-1-thio- Transient Transfections and Luciferase Assays--
Transfections
of MDA-MB231 cells were performed as described (41), using 1 µg of
reporter plasmid (either pTCBS or pT109) and various amounts of vectors
expressing HOXB7, RelA, p50, and/or I p50, RelA, and I
When p50 and RelA expression vectors were transfected with the pTCBS or
pT109 reporter plasmids, weak inductions of luciferase activity were
observed (Fig. 2, columns 5 and 6). Moreover, a very weak increase in luciferase activity was observed when the plasmid
encoding I
To further characterize the transcriptional properties of the HOXB7
protein, additional transient expression experiments were performed
using the I
To determine whether other domains were involved in the interaction,
additional constructs including a naturally occurring HOXB7 mutant that
lacks two amino acids at the C-terminal tail (B7*) (20) and HOXB7
products deleted in their C-terminal domain (B7- The N-terminal Domain of the HOXB7 Protein Is Required for the
Interaction with I The Ankyrin Repeats and the C-terminal Domain of I
To confirm these results in vivo, we performed transient
expression experiments using both pT109 and pTCBS constructs as
reporter plasmids and a variety of vectors generating distinct
I This report has demonstrated a physical interaction between the
HOXB7 homeodomain-containing protein and I All the homeodomain-containing proteins encoded by the 39 HOX genes share a highly conserved 60-amino acid DNA-binding
domain, the homeodomain, and bind to very similar sequences in
vitro (8-10). Their in vivo specificity may thus
involve protein-protein interactions with other transcription factors.
In this context, the homeodomain proteins derived from the
extradenticle/Pbx genes act as co-factors for
HOX gene products that contain a pentapeptide sequence (11), whereas the AbdB-like HOX proteins, which do not harbor a pentapeptide, interact with Meis1 (13). We have provided here evidence that the
NF- We have shown that the inhibitor I Interestingly, we demonstrated that both the I Several models, which are not mutually exclusive, can explain how
IB-
, an inhibitor of NF-
B activity. This interaction was
mediated by the I
B-
ankyrin repeats and C-terminal domain as well
as by the HOXB7 N-terminal domain. In transient transfection
experiments, I
B-
markedly increased HOXB7-dependent
transcription from a reporter plasmid containing a homeodomain
consensus-binding sequence. This report therefore showed a novel
function for I
B-
, namely a positive regulation of transcriptional
activation by homeodomain-containing proteins.
INTRODUCTION
Top
Abstract
Introduction
References
B proteins form a family of transcription factors that play a
central role in the cellular responses to stress, cytokines, and
pathogens (22-24). Indeed, these transcription factors are activated
in response to a variety of extracellular signals such as phorbol
esters, tumor necrosis factor-
, interleukin-1, lipopolysaccharide, UV irradiation, viral infection, and growth factors (24, 25) and
regulate a wide spectrum of immune and inflammatory responses (26). In
unstimulated cells, NF-
B activity is inhibited by another class of
proteins that includes I
B-
(27, 28), I
B-
(29), I
B-
(30), p105, and p100. These inhibitory proteins all share ankyrin
repeats, sequester the NF-
B complexes in the cytoplasm, and block
their binding to
B DNA sequences. Initially described as a
cytoplasmic protein (28), I
B-
has since been detected in the
nucleus of transfected Vero cells (31) as well as after serum
stimulation (32). The nuclear localization of I
B-
is mediated by
its second ankyrin repeat, which acts as a nuclear import sequence
(33). Once in the nucleus, I
B-
can remove NF-
B dimers from
their
B DNA sequences, thus inhibiting NF-
B activity (34). When
fused to the GAL-4 DNA-binding domain, I
B-
displays
transactivation abilities (35, 36), a property not possessed by the
naturally occurring I
B-
protein (32). These results raised the
possibility that I
B-
can interact with other transcription
factors and modulate their activity.
B-
is able to physically
interact with the HOXB7 homeodomain-containing protein and to
enhance HOXB7 transcriptional activity. We further identified HOXB7 and
I
B-
domains involved in this interaction. Our results thus
demonstrate a novel function of I
B-
.
EXPERIMENTAL PROCEDURES
N18,
N54,
N86, and
N129 generate HOXB7 proteins lacking 18, 54, 86, and 129 N-terminal amino acids, respectively. The constructs B7-
C12,
B7-
C34, B7-
C80, and B7-
C97 encode HOXB7 proteins deleted of
12, 34, 80, and 97 C-terminal amino acids, respectively.
B-
were previously described (37, 38). The I
B-
coding
sequence was also subcloned by PCR into the expression vector
pcDNA3. The PMT2T expression vectors for I
B-
N
and I
B-
C lacking the first 53 codons and the last 42 codons of
I
B-
, respectively (39), are schematically illustrated in Fig.
1A. The PMT2T expression vector for I
B-
N+C GST codes for a protein where the ankyrin repeats of I
B-
have
been replaced by the GST peptide (Fig. 1A) as described
(39).
B-ICAM-1 reporter plasmid construct has been previously described;
it harbors three NF-
B-like sites from the ICAM-1 promoter cloned
upstream of the herpes simplex virus thymidine kinase minimal promoter
and the luciferase gene (40).
B-
were subcloned by PCR into the
BamHI/EcoRI polylinker of the pGEX-2TK vector
(Amersham Pharmacia Biotech) to create GST fusion proteins. These
constructs include pGEX I
B-
C, pGEX I
B-
N, pGEX
ankyrins, pGEX NI
B, and pGEX CI
B and are
schematically illustrated in Fig.
1B. The sequence of primer 1 is 5'-TATAGGATCCATGTTCCAGGCGGCC-3'; primer 2, 5'-TATAGGATCCCTCGAGCCGCAGGAGGT-3'; primer 3, 5'-TATAGGATCCAACCTTCAGATGCTGCCAGAG-3'; primer 4, 5'-ATATGAATTCCTCGAGGCGGATCTCCT-3'; primer 5, 5'-ATATGAATTCTTCTAGTGTCAGCTGGCC-3'; and primer 6, 5'-ATATGAATTCTCATAACGTCAGACGCTGGCC-3'.
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Fig. 1.
Schematic illustration of the
I B-
expression
vectors (A) and the
GST-I
B-
constructs
(B). The ankyrin repeats are illustrated by
dark rectangles. The primers designed for PCR amplification
are numbered from 1 to 6 and represented by
arrows. Primers 4-6 are derived from the complementary
strand.
-D-galactopyranoside for 3 h and
harvested. Bacterial pellets were washed once with phosphate-buffered
saline, resuspended in 10 ml of NENT buffer (250 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8, Nonidet P-40 1.5%) and sonicated three times for 15 s at 4 °C. Insoluble materials were removed by centrifugation. GST fusion proteins were purified after
incubation of 1 ml of the supernatant with 10 µl of
glutathione-Sepharose beads for 1 h at 4 °C (Amersham Pharmacia
Biotech). The beads were then washed twice with 1 ml of NENTM buffer
(NENT + 0.5% milk) and once with 1 ml of TWB buffer (20 mM
Hepes, pH 7.9, 60 mM NaCl, 1 mM
dithiothreitol, 6 mM MgCl2, 8.2% glycerin, 0.1 mM EDTA). In each case, the expected fusion proteins were
visualized on a 12% polyacrylamide gel stained by Coomassie Blue.
Protein-protein interactions were performed by incubating an aliquot of
the GST-I
B
fusion protein bound to the glutathione-Sepharose
beads with 10 µl of in vitro translated protein in 200 µl of TWB buffer for 1 h at 4 °C. Beads were then washed six
times with 1 ml of NENTM buffer, resuspended into migrating buffer, and
loaded on an SDS-polyacrylamide gel before autoradiography.
B-
. Total amounts of
transfected DNA were kept constant throughout by adding appropriate
amounts of either pcDNA3 or pMT2T empty vectors. Cells
were harvested 48 h after transfection, and luciferase activities
were measured with the Luciferase Reporter Gene Assay kit (Boehringer
Mannheim), as recommended by the manufacturer. The luciferase
activities were normalized to the protein concentration of the extracts.
RESULTS
B-
Enhance Transactivation by the HOXB7
Protein--
To investigate whether the HOXB7 protein can interact
with transcription factors from other families, we transiently
transfected MDA-MB231 cells with a HOXB7 expression vector and a
variety of constructs coding for different members of the NF-
B/I
B
families. Both the pTCBS and pT109 constructs were used as reporter
plasmids: the pTCBS plasmid contains a luciferase reporter gene driven
by a multimerized HOX CBS that is recognized by most HOX proteins, whereas the pT109 vector does not harbor any HOX-binding sequence and
was used as a negative control (42). A 3.6-fold induction over basal
luciferase activity was measured when the HOXB7 expression vector was
transfected with pTCBS (Fig. 2,
column 3), as described previously (20, 41). This effect was
mediated by the binding of the HOXB7 protein to the CBS sequence,
because no significant effect was observed with the pT109 reporter
plasmid (Fig. 2, column 4).
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Fig. 2.
NF- B and
I
B-
increase
transactivation by HOXB7 through a HOX consensus DNA-binding
sequence. MDA-MB231 cells were transfected with 1 µg of HOXB7,
p50, RelA, and/or I
B-
expression vectors together with 1 µg of
reporter plasmid, as indicated in the figure. The pT109 does not
contain any HOX-binding sequence and was used as a negative control.
The figure shows the relative luciferase activity over the basal
activity observed with 1 µg of the pTCBS or pT109 reporter plasmid
alone. Each value represents the mean (± S.D.) of at least three
independent experiments after normalization to the protein
concentration of the extracts.
B-
was co-transfected with either the pTCBS or pT109
reporter constructs (Fig. 2, columns 7 and 8),
indicating that, as expected, I
B-
did not transactivate through
these promoters in MDA-MB231 cells. When the HOXB7 expression construct
was co-transfected with p50, RelA, and pTCBS, a 4.9-fold induction over
basal luciferase activity was observed (Fig. 2, column 9),
indicating that NF-
B members enhanced HOXB7 transcriptional
activity. To determine whether I
B-
could inhibit the
transactivation observed with HOXB7 and p50-RelA, we co-transfected an
I
B-
expression vector with the plasmids generating the HOXB7,
p50, and RelA proteins as well as with the pTCBS construct.
Surprisingly, a further increase in luciferase activity (7.2-fold
induction over basal luciferase activity) was measured (Fig. 2,
columns 11). Moreover, the luciferase activity was even more
elevated (13.7-fold induction over basal luciferase activity) when we
co-transfected only the HOXB7 and I
B-
expression vectors with the
pTCBS reporter plasmid (Fig. 2, column 13).
B-ICAM-1 reporter plasmid harboring three
B-like
binding sites upstream of a CAT gene. As expected, transfection of the
p50 and RelA expression vectors induced CAT activity (Fig. 3, column 2), and this effect
was inhibited by simultaneous expression of I
B-
(Fig. 3,
column 3). Transfection of increasing amounts of HOXB7
expression vector did not lead to any significant induction of CAT
activity (Fig. 3, columns 4-6). Moreover, when we
co-transfected HOXB7 with both p50 and RelA expression vectors, the CAT
activity was close to that measured in the absence of HOXB7 (Fig. 3,
columns 2 and 8) and was attenuated by the
inhibitor I
B-
(Fig. 3, columns 10-12). No significant
induction of CAT activity was measured when HOXB7 and I
B-
expressing vectors were co-transfected (Fig. 3, columns
14-16). These results suggest that the HOXB7 protein does not
significantly modulate the transcriptional abilities of NF-
B
members.
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Fig. 3.
HOXB7 does not modify
NF- B transcriptional activity. MDA-MB231
cells were transfected with 1 µg of p50, RelA, and/or I
B-
expression vectors together with various amounts of HOXB7 expression
vector (0.5, 1, or 2 µg) and 1 µg of the
B-ICAM-1 reporter
plasmid, as indicated in the figure. The figure shows the relative CAT
activity over the basal activity observed with 1 µg of the
B-ICAM-1 reporter plasmid alone. Each value represents the mean (± S.D) of at least three independent experiments after normalization to
the protein concentration of the extracts.
B-
Physically Interacts in Vitro with the N-terminal Domain
of HOXB7--
To determine whether I
B-
physically interacted
with the HOXB7 protein, purified GST-I
B-
fusion protein bound to
glutathione-Sepharose beads was incubated with in vitro
translated HOXB7. After precipitation of the beads, a positive signal
was detected (Fig. 4, lane 2), whereas HOXB7 did not interact with the GST protein (lane
3), thus demonstrating the existence of an in vitro
interaction between HOXB7 and I
B-
. To map the HOXB7 domain
involved in this process, we designed several constructs generating
HOXB7 gene products progressively deleted in their N-terminal domain
and designated as B7-
N18,
N54,
N86, and
N129. All these
HOXB7 proteins shared an intact homeodomain, whereas only the
N129
peptide was deleted of the pentapeptide. These products were then
in vitro translated and incubated with the GST-I
B-
fusion protein as described above. None of these proteins were able to
significantly interact with I
B-
(Fig. 4, lanes 5,
8, 11, and 14).
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Fig. 4.
In vitro protein-protein
interaction between HOXB7 and
I B-
requires the
HOXB7 N-terminal domain. The HOXB7 expression vectors are
schematically represented. B7-
N18,
N54,
N86, and B7-
N129
products are deleted in their N-terminal domain. The homeodomain is
illustrated by a large shaded rectangle, whereas the
pentapeptide is represented by a small shaded box upstream
of the homeodomain, and the acidic C-terminal tail is shown as a
cross-hatched box. The expected molecular mass of the
resulting proteins is mentioned on the right.
35S-Labeled in vitro translated wild-type and
deleted HOXB7 proteins were incubated with a GST-I
B-
fusion
protein attached to glutathione-Sepharose beads (lanes 2,
5, 8, 11, and 14),
precipitated and run on an SDS-polyacrylamide gel. Beads carrying the
GST protein alone were used as negative controls (lanes 3,
6, 9, 12, and 15). In
vitro translated proteins (10% of the amounts used in the
precipitation experiments) were run on lanes 1,
4, 7, 10, and 13.
C12, B7-
C34,
B7-
C80, and B7-
C97) were in vitro translated (Fig.
5, lanes 1, 4,
7, 10, 13, and 16) and
incubated with the GST-I
B-
fusion protein bound to
glutathione-Sepharose beads. All these proteins were still able to
interact with I
B-
despite the absence of a complete homeodomain
sequence for B7-
C80 and of the pentapeptide region for B7-
C97
(Fig. 5, lanes 5, 8, 11, 14, and 17). These results indicate that a
HOXB7/I
B-
physical interaction can occur independently of the
homeodomain sequence and depends exclusively on an intact HOXB7
N-terminal sequence.
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Fig. 5.
In vitro protein-protein
interaction between HOXB7 and
I B-
does not require
the HOXB7 C-terminal domain. The HOXB7 expression vector
generating proteins deleted in their C-terminal domain are
schematically represented (B7-
C12, B7-
C34, B7-
C80, and
B7-
C97). The B7* protein is a naturally occurring mutant deleted of
two C-terminal amino acids. The homeodomain is illustrated by a
large shaded rectangle, whereas the pentapeptide is
represented by a small shaded box upstream of the
homeodomain, and the acidic C-terminal tail is shown as a
cross-hatched box. The expected molecular mass of the
resulting proteins are mentioned on the right.
35S-Labeled in vitro translated wild-type and
deleted HOXB7 proteins were incubated with a GST-I
B-
fusion
protein attached to glutathione-Sepharose beads (lanes 2,
5, 8, 11, 14, and
17), precipitated, and run on an SDS-polyacrylamide gel.
Beads carrying the GST protein alone were used as negative controls
(lanes 3, 6, 9, 12,
15, and 18). In vitro translated
proteins (10% of the amounts used in the precipitation experiments)
were run on lanes 1, 4, 7,
10, 13, and 16.
B-
in Vivo--
We previously demonstrated
that both the N-terminal domain and the acidic C-terminal tail of the
HOXB7 protein mediated its transcriptional properties.1
Because the N-terminal domain of HOXB7 was required for the interaction with I
B-
in vitro, we transfected MDA-MB231 cells with
the B7
N129 expression vector and the pTCBS or pT109 reporter
plasmid. The B7
N129 product, alone or co-expressed with I
B-
,
did not induce any luciferase activity (Fig.
6). Moreover, the B7-
C12 protein, which lacks the acidic C-terminal domain but still interacts with I
B-
in vitro (Fig. 5), did not behave as a
transcriptional activator (Fig. 6). Interestingly, when both the
B7-
C12 and I
B-
expression vectors were transfected
simultaneously with the pTCBS reporter plasmid, an induction of the
luciferase activity similar to that measured with both HOXB7 wild-type
and I
B-
proteins was observed (Fig. 6). These results suggest
that the inhibitor I
B-
potentiates HOXB7 transactivating
activities through a physical interaction with the HOXB7 N-terminal
domain.
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Fig. 6.
The HOXB7 N-terminal domain is required for
transactivation by
HOXB7/I B-
.
MDA-MB231 cells were transfected with 1 µg of expression vectors
coding for wild-type or deleted HOXB7 and 1 µg of I
B-
expression vector together with 1 µg of reporter plasmid. The figure
shows the relative luciferase activity over the basal activity observed
with the pTCBS or the pT109 reporter plasmid alone. Each value
represents the mean (± S.D.) of at least three independent experiments
after normalization as described above.
B-
Are
Required for in Vitro and in Vivo Interaction with HOXB7--
To map
the I
B-
domain(s) involved in the physical interaction with
HOXB7, we inserted the ankyrin domain of I
B-
in the pGex-2TK
vector (Fig. 7A, lane
2) and produced the corresponding fusion protein. Incubation of
this protein with in vitro translated HOXB7 demonstrated a
physical interaction between the two proteins (Fig. 7A,
lane 2), whereas HOXB7 could not interact with the GST protein purified from E. coli (Fig. 7A,
lane 3). Because the signal was weaker than that observed
with the full-length GST-I
B-
fusion protein (Fig. 7A,
lane 1), it is likely that other functional domains were
also involved in this process. An I
B-
peptide deleted of the
C-terminal domain was fused to GST (Fig. 7B) and purified. As illustrated in Fig. 7B (lane 4), this fusion
protein bound to glutathione-Sepharose beads interacted with HOXB7,
although this interaction was weaker than that obtained with the
full-length I
B-
product (Fig. 7B, lane 1).
Moreover, a GST-I
B-
-C-terminal domain fusion protein was still
able to interact with HOXB7 (Fig. 7B, lane 5). To
confirm that both the ankyrin and C-terminal domains of I
B-
were
responsible for the interaction, a fusion protein that does not contain
the I
B-
-N-terminal domain was produced and incubated with HOXB7.
As illustrated in Fig. 7C (lane 6), a signal
similar to that obtained with the wild-type I
B-
protein was
observed, suggesting that the N-terminal domain of I
B-
is not
involved in the interaction with HOXB7. Indeed, incubation of the
GST-I
B-
-N-terminal domain fusion protein with HOXB7 did not
generate any significant signal (Fig. 7C, lane
7). These results indicate that both the I
B-
ankyrin and
C-terminal domains are required for physical in vitro
interaction with HOXB7.
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Fig. 7.
In vitro protein-protein
interaction between HOXB7 and
I B-
requires
I
B-
ankyrin repeats
and the C-terminal domain. The GST-I
B-
constructs are
schematically illustrated. 35S-Labeled in vitro
translated wild-type HOXB7 protein was incubated with full-length
(lane 1) or deleted (lanes 2-7) GST-I
B-
fusion proteins attached to glutathione-Sepharose beads, precipitated,
and run on an SDS-polyacrylamide gel. Beads carrying the GST protein
alone were used as negative controls (lane 3).
B-
peptides (Fig. 1A). Full-length I
B-
,
I
B-
C, I
B-
N, and
NI
BGSTCI
B proteins did not significantly
induce luciferase activity when co-transfected with any of the reporter
plasmids (Fig. 8, rows 5-12).
However, cotransfection of the plasmids generating the I
B-
N
and HOXB7 peptides led to a 13-fold induction of luciferase activity
(Fig. 8, row 17) that was dependent on the binding to the
CBS sequence (row 18) and comparable with that measured with the full-length I
B-
expression vector (row 19).
Cotransfection of the expression plasmids for I
B-
C and HOXB7
generated only a 4.3-fold induction over the basal activity (Fig. 8,
row 15). To confirm that the ankyrin domain was also
required, we cotransfected the pTCBS or the pT109 reporter plasmids
with the HOXB7 plasmid and the NI
BGSTCI
B
construct generating an I
B-
-related protein in which the ankyrin
domain had been replaced by the GST sequence. As illustrated in Fig. 8
(row 13), we observed only a 6.4-fold induction of the
luciferase activity. Our results clearly demonstrate that the ankyrin
repeats and the C-terminal domain of I
B-
are required for
in vitro and in vivo interaction with the HOXB7
homeodomain-containing protein.
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Fig. 8.
The
I B-
ankyrin and
C-terminal domains are required for transactivation by
HOXB7/I
B-
.
MDA-MB231 cells were transfected with expression vectors
coding for HOXB7 (1 µg) and for wild-type or deleted I
B-
(1 µg) as well as with the reporter plasmid (1 µg), as indicated in
the figure. The figure shows the relative luciferase activity over the
basal activity observed with 1 µg of the pTCBS or the pT109 reporter
plasmid alone. Each value represents the mean (± S.D) of at least
three independent experiments after normalization as described
above.
DISCUSSION
B-
, resulting in an
enhanced transactivation by this HOX gene product. Moreover, we identified the HOXB7 and I
B-
functional domains mediating this
interaction. These results provide new insights into the transcription
properties of the homeodomain-containing proteins and reveal a novel
function of the inhibitor I
B-
.
B proteins, including the p50-p65 heterodimer, can enhance the
transcription potential of the HOXB7 protein in transient expression
experiments. This effect is presumably mediated by physical
interactions between the p50-p65 complex and HOXB7. Preliminary in vitro experiments have indeed confirmed this hypothesis
(data not shown). It is tempting to speculate the existence of a
p50-p65-HOXB7 complex that could bind the CBS consensus sequence and
transactivate through the HOXB7 and p65 activation domains. This
complex, however, did not display a similar effect on a
B-binding
site under our experimental conditions.
B-
can enhance the HOXB7
transactivating effect. This is the first demonstration that I
B-
interacts with proteins from other families of transcription factors.
Previous reports had demonstrated that I
B-
can translocate to the
nucleus, using its second ankyrin repeat as a nuclear import sequence
(33) and subsequently remove the NF-
B complex from its binding site
(34). Moreover, I
B-
can repress the 9-cis-retinoic acid-induced
transcriptional activity of retinoid X receptor in lipopolysaccharide-treated cells (43). Thus, in both cases, I
B
proteins localized in the nucleus negatively regulate the transcriptional activity of their interacting partners. Surprisingly, our study demonstrates that I
B-
can also positively regulate the
transcriptional properties of a homeodomain-containing protein. A
similar phenomenon was described previously for Bcl3, another member of
the I
B protein family. Indeed, Bcl3 can transactivate through
B
sites when physically associated with p52 and p50 (37, 44), and it has
been demonstrated that the N- and C-terminal domains of Bcl3 are
transcriptional activation domains (37). Moreover, Bcl3, but not
I
B-
, can also act as a coactivator of the retinoid X receptor
(45). These results and the present report strongly suggest that
distinct I
B proteins can modulate, positively as well as negatively,
the transcriptional properties of their interacting partners, including
transcription factors that do not belong to the NF-
B family.
B-
ankyrin and
C-terminal domains mediate interaction with HOXB7. The same domains are
also required for the regulation of c-Rel by I
B-
in the nucleus
(46). Taken together, these results suggest a critical role for the
ankyrin repeats and C-terminal domains in the function of I
B-
in
the nucleus.
B-
enhances HOXB7 transcriptional activity (Fig.
9). The first model does not imply a
direct interaction between HOXB7 and I
B-
but rather an indirect
mechanism mediated by NF-
B. Indeed, we can postulate that NF-
B
activates the expression of HOX genes encoding repressors. Therefore,
NF-
B inhibition by I
B-
would lead to a decreased expression of
these HOX genes and to increased luciferase activity in transient
expression experiments (Fig. 9A). This first model is the
only one that does not require I
B-
nuclear localization. The
second model is based on a report demonstrating that ankyrin repeats
stabilize the DNA binding of other transcription factors (47). The
enhanced HOXB7 transcriptional activity would then be mediated by a
stronger HOXB7 DNA binding affinity for its target sequence in the
presence of I
B-
(Fig. 9B). This hypothesis is
supported by the observation that the physical interaction between
HOXB7 and I
B-
requires the ankyrin repeats. In the third model, a
HOXB7-I
B-
complex is bound to the CBS sequence through the HOXB7
homeodomain and transactivates through I
B-
. This hypothesis is
supported by previous studies demonstrating that I
B-
can
transactivate when fused to a GAL4 DNA-binding domain (35, 36).
Moreover, Bcl3, another member of the I
B-
family, also harbors
transactivating domains (37). The fourth model implies a
HOXB7-RelA-I
B-
complex activating the luciferase gene through
both the HOXB7 and RelA transactivation domains. The last two
hypotheses are supported by the induction of luciferase activity
observed with I
B-
and B7
C12 expression vectors, whereas the
B7
C12 protein is not able to transactivate by itself but can
physically interact with I
B-
. However, the last model cannot
account for the fact that the transcriptional activity was higher after
transfection of the HOXB7 and I
B-
expression vectors than in the
presence of the same vectors plus RelA (Fig. 2). Further experiments
are required to determine which of these models is correct.
Unfortunately, the available HOXB7 antibodies did not allow us to study
more precisely the HOXB7 multimeric complexes in transfected or
unmodified cells.
View larger version (19K):
[in a new window]
Fig. 9.
Models for HOXB7 transcriptional activation
by
NF- B/I
B-
proteins. A, the NF-
B heterodimer
transactivates the expression of a HOX gene that codes for a
transcription repressor. Upon transfection of I
B-
, the inhibitor
sequesters NF-
B in the cytoplasm, thus preventing the expression of
the HOX target gene. The luciferase gene is consequently activated.
This first model does not imply a direct interaction between HOXB7 and
I
B-
. B, I
B-
stabilized the HOXB7 binding to the
CBS sequence, thus allowing the induced expression of the luciferase
gene. C, upon transfection of I
B-
, a HOXB7-I
B-
complex is formed on the CBS sequence and activates the expression of
the luciferase gene through HOXB7 and I
B-
transactivation
domains. D, upon transfection of I
B-
, a
HOXB7-p65-I
B-
complex is formed on the CBS sequence and activates
the expression of the luciferase gene through HOXB7 and p65
transactivation domains.
The functional link between NF-B-I
B-
and homeodomain proteins
was unexpected because of the distinct physiological processes they
control. However, a first link between these two families during the
outgrowth of the vertebrate limb has recently been described (48, 49).
Indeed, NF-
B gene expression has been detected during limb
morphogenesis and the alteration of NF-
B activity causes an arrest
of the outgrowth (48, 49). Moreover, I
B-
is the human homologue
of cactus, a protein that plays a crucial role in the
dorsoventral patterning of the Drosophila embryo (21).
Because HOX genes are clearly required to establish the
anteroposterior axis of the developing embryo, it is tempting to
speculate that the interaction between I
B-
and HOX proteins might
determine the antero-posterior and dorsoventral polarities of the embryo.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. P. T. van der Saag for
providing the B-ICAM-1 reporter plasmid and are grateful to Dr.
Zappavigna for the pTCBS and pT109 plasmids.
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FOOTNOTES |
---|
* This work was supported by grants from the National Fund for Scientific Research, Télévie (Belgium) and the Centre Anti Cancéreux (University of Liège, Belgium).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.
§ Present address: Lab. of Immunoregulation, NIAID, NIH, Bethesda, MD 20892.
¶ Research Associates of the National Fund for Scientific Research (Belgium).
** To whom correspondence should be addressed: Medical Oncology, C.H.U. B35, University of Liege, Sart-Tilman, 4000 Liege, Belgium. Tel.: 32-4-366-24-82; Fax: 32-4-366-45-34; E-mail: vbours{at}ulg.ac.be.
1 A. Chariot, C. van Lint, M. Chapelier, J. Gielen, M.-P. Merville, and V. Bours, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; CBS, consensus-binding sequence; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase.
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REFERENCES |
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