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Mol Cell Biol, March 1998, p. 1312-1321, Vol. 18, No. 3
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Bone-Specific Expression of the Alpha Chain of the
Nascent Polypeptide-Associated Complex, a Coactivator Potentiating
c-Jun-Mediated Transcription
Alain
Moreau,
Wagner V.
Yotov,
Francis H.
Glorieux, and
René
St-Arnaud*
Genetics Unit, Shriners Hospital, and
Departments of Surgery and Human Genetics, McGill University,
Montréal, Québec H3G 1A6, Canada
Received 10 June 1997/Returned for modification 15 August
1997/Accepted 24 November 1997
 |
ABSTRACT |
The alpha chain of the nascent polypeptide-associated complex
(
-NAC) coactivator was shown to potentiate the activity of the
homodimeric c-Jun activator, while transcription mediated by the
c-Fos/c-Jun heterodimer was unaffected. The use of deletion mutants in
pull-down assays revealed that -NAC interacted with amino acids 1 to
89 of the c-Jun protein and that the coactivator could interact with
both the unphosphorylated and the serine 73-phosphorylated form of
c-Jun. N-terminal-deleted c-Jun protein failed to interact with -NAC
in mammalian two-hybrid assays, while mutant c-Jun proteins lacking the
leucine zipper or the basic domain retained interaction with -NAC in
vivo. Kinetics studies with purified c-Jun homodimer and recombinant
-NAC proteins allowed determination of the mechanism of coactivation
by -NAC: the coactivator stabilized the AP-1 complex formed by the
c-Jun homodimer on its DNA recognition sequence through an eightfold
reduction in the dissociation constant (kd) of
the complex. This effect of -NAC was specific, because -NAC could
not stabilize the interactions of JunB or Sp1 with their cognate
binding sites. Interestingly, the expression of -NAC was first
detected at 14.5 to 15 days postconception, concomitantly with the
onset of ossification during embryogenesis. The -NAC protein was
specifically expressed in differentiated osteoblasts at the centers of
ossification. Thus, the -NAC gene product exhibits the properties of
a developmentally regulated, bone-specific transcriptional coactivator.
| |
INTRODUCTION |
Tissue-specific gene expression is
regulated by a combination of sequence-specific DNA-binding
transcriptional activators, general or basal transcription initiation
factors, and associated cofactors. The sequence-specific transcription
factors can be divided into several classes or families based on either
the activation domain they possess or the protein motif they use for
DNA binding. In addition to these site-specific proteins, accurate
transcription initiation by RNA polymerase II requires at least six
general transcription initiation factors: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (reviewed in references 16 and
29).
While the general initiation factors are sufficient for basal-level
transcription, enhancement of transcription by transcriptional activator proteins bound to enhancer elements requires the presence of
additional mediator proteins, known as transcriptional coactivators (12, 16, 19, 38). Coactivators are defined functionally by
their ability to selectively potentiate the stimulatory activity of
specific subsets of enhancer binding transcriptional activators. Among
the best-characterized coactivators are the TAFs (TATA box-binding protein [TBP]-associated factors) (12), which are subunits
of TFIID. Purification and molecular cloning of several TAFs have confirmed that they provide protein interfaces to link the
sequence-specific factors to the basal transcriptional machinery, and
some exhibit specific enzymatic functions essential for activated gene
transcription (11, 27, 28). Moreover, coactivators distinct
from those associated with TBP have also been identified and cloned
(reviewed in reference 19).
We have recently demonstrated that the alpha chain of the nascent
polypeptide-associated complex (-NAC) protein, previously thought to
be involved in some aspects of translational control (41),
could translocate to the nucleus, where it was shown to function as a
transcriptional coactivator in conjunction with the chimeric activator
GAL4/VP-16 in vivo (43a). Specific interactions between
-NAC and TBP were detected. Examination of the expression pattern of
-NAC in adult tissues allowed us to identify and characterize a
muscle-specific isoform of -NAC, skNAC, also involved in the regulation of gene transcription (43). In this report, we
have examined the expression pattern of -NAC during embryogenesis and observed that it was selectively expressed in developing bone.
In an attempt to identify endogenous transcriptional activators that
could interact with -NAC to regulate gene transcription in
differentiating bone cells, we examined the possibility that AP-1
proteins, known modulators of bone development in vivo (17), might represent natural targets for -NAC. The AP-1 proteins are formed through the heterodimerization of Fos family members and Jun
family members through a structural motif called the leucine zipper
(30). The heterodimer can then bind DNA at a consensus site
termed the AP-1 site and act as a transcription factor to modulate the
expression of AP-1-responsive genes (30). All of the Jun
family members can also homodimerize to exert the same function
(30).
Interestingly, -NAC was shown to potentiate the activity of the
homodimeric c-Jun activator, while transcription mediated by the
c-Fos/c-Jun heterodimer was unaffected. We have delineated the domain
of the c-Jun protein that interacts with -NAC and measured the
influence of -NAC on kinetic parameters of c-Jun binding to AP-1
sites. These observations have allowed us to propose a model for the
-NAC-mediated coactivation of c-Jun-dependent transcription. The
observed restricted expression pattern of -NAC during embryogenesis
and the identification of its interaction with c-Jun suggest new
perspectives in the study of the regulation of gene transcription
during osteoblastic differentiation.
| |
MATERIALS AND METHODS |
Northern blot assay.
The commercially available mouse embryo
Northern blot (Clontech Laboratories, Palo Alto, Calif.) was probed
with the full-length, PCR-labeled -NAC cDNA. The membrane was
subsequently stripped and rehybridized with a probe directed against
the S28 ribosomal protein mRNA to monitor for variations in the quality
and quantity of the loaded mRNA.
Immunolocalization of -NAC.
Embryos were dissected,
embedded in Tissue-Tek O.C.T. compound (Miles, Inc., Elkhart, Ind.),
frozen, and sectioned at 6 µm. Immunohistochemistry was performed
according to standard protocols (40) with the anti-NAC
antibody (43) and a secondary goat anti-rabbit antibody
conjugated to fluorescein isothiocyanate (TAGO Immunologicals,
Burlingame, Calif.). The antibody dilutions were 1:1,000 and 1:50 for
the primary and secondary antibodies, respectively. Counterstaining of
the embryos was with toluidine blue.
Transient transfection assays.
All vectors were constructed
by standard molecular biology procedures, and full details and
sequences are available on request. C3H10T1/2 embryonic fibroblasts
(39) were cotransfected with 0.1 µg of a reporter vector
(AP-1-tk-luc) in which a canonical AP-1 binding site
(5'-TGACTCA-3') was subcloned upstream of the minimal herpes
simplex virus thymidine kinase promoter driving the luciferase reporter
gene together with 0.7 µg of cytomegalovirus (CMV)-driven expression
vectors for c-fos (8), c-jun, or
-NAC, alone or in combination. Variations in transfection efficiency were monitored with 0.1 µg of the expression plasmid pSV6tkCAT (7).
For mammalian two-hybrid assays, ROS 17/2.8 osteoblastic cells
(24) were cotransfected with combinations of simian virus 40-driven expression vectors for the GAL4 DNA binding domain (GAL4-DBD) (0.1 µg) (32) or the GAL4--NAC fusion protein (0.1 µg) and the jun and fos expression vectors (in
increasing amounts from 0.1 to 0.4 µg). The reporter gene used was
5Gal4-E1b-CAT (0.1 µg) (22), and transfection efficiency
was monitored with the luciferase vector pGL2control (Promega Corp.,
Madison, Wis.). Similar levels of expression were achieved for each
recombinant protein (data not shown).
All transfections were performed with 5 µl of the Lipofectamine
reagent (Gibco BRL, Gaithersburg, Md.) according to the instructions
of
the manufacturer, and cells were harvested 24 h posttransfection.
Chloramphenicol acetyltransferase (CAT) and luciferase activities
were
assayed as previously described (
34).
GST pull-down assays.
Pull-down assays were performed
essentially as described by Folkers and van der Saag (13)
with Promega's TNT in vitro transcription and translation kit in the
presence of [35S]methionine. In vitro-transcribed and
-translated plasmids included pSP65-c-fos (8), Jac7
(c-jun cDNA cloned into pGEM-7 [31]), pT7-GAL4/VP-16 (in which the GAL4/VP-16 fusion cDNA was subcloned into
the GPP-73 vector [37]), and the control plasmid
T7-luciferase supplied with the TNT kit (Promega). For pull-down assays
with deleted c-Jun fusion protein, glutathione-Sepharose beads loaded with glutathione S-transferase (GST)-jun(1-89) were
purchased from New England Biolabs (Mississauga, Ontario, Canada) and
phosphorylated with serum-induced whole-cell extracts from P19
embryonal carcinoma cells (25) according to the protocol
supplied by the manufacturer. Binding and washes were performed as
recommended with the buffers supplied. The anti-GST antibody was from
Pharmacia (Baie d'Urfé, Quebec, Canada).
Affinity chromatography.
Crude nuclear extracts from
serum-stimulated MC3T3-E1 cells (36) were prepared as
described by Dignam et al. (10). The nuclear extracts (2 mg
of total protein) were precleared on glutathione-Sepharose 4B beads
(Pharmacia) and then passed on a glutathione-Sepharose 4B column
previously loaded with GST--NAC (300-µl final bed volume). Bound
proteins were eluted with a step gradient of NaCl, precipitated with
trichloroacetic acid, and analyzed by Western blot assay (15). The antibodies directed against the serine
73-phosphorylated form of c-Jun were obtained from New England Biolabs.
On and off rate measurements.
Electrophoretic mobility shift
assays were performed as described previously (34), with the
exception that binding reactions were carried out at 4°C in a total
volume of 180 µl. Commercially available purified c-Jun and Sp1
protein (estimated at 7 × 10
8 M, final
concentration) (Promega Corporation), in vitro-translated JunB protein,
and purified recombinant GAL4/VP-16 and -NAC protein (3 × 107 M, final concentration) were used in the binding
assays. Twenty-microliter aliquots were removed at intervals and loaded
on the gels. Binding reactions and calculations were as described by
Chodosh et al. (5). The probes used were end-labeled
oligonucleotides corresponding to the AP-1 site from the hMT-IIA
promoter (21), the canonical GAL4 binding site
(22), or the consensus Sp1 DNA recognition sequence
(3a) (each estimated at around 2 × 107
M, final concentration).
| |
RESULTS |
-NAC is specifically expressed in bone during development.
We used both Northern blot assays and immunohistochemistry to address
the tissue specificity and developmental regulation of the expression
of -NAC. Figure 1 shows that the
-NAC mRNA could not be detected in poly(A)+ RNA isolated
from 7- and 11-day-postconception (p.c.) whole mouse embryos. However,
the -NAC transcript was readily detected in mouse embryos at 15 and
17 days p.c. (Fig. 1). Expression of the -NAC mRNA was widespread in
the adult mouse (43).
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FIG. 1.
Expression pattern of -NAC mRNA during mouse
embryogenesis. Whole mouse embryo mRNA was probed with the full-length
-NAC cDNA in a Northern blot assay. The membrane was subsequently
stripped and rehybridized with a probe directed against the S28
ribosomal protein.
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We next studied the pattern of expression of the
-NAC protein during
mouse development. Confirming the results obtained with
Northern blot
assays (Fig.
1), we could not detect the
-NAC protein
prior to 14 days p.c. (Fig.
2B and data not shown).
The onset
of mineralization is first detected at 14 to 14.5 days p.c.
(
20);
the ribs, for example, show evidence of endochondral
ossification
(Fig.
2C). Ossification of the limbs also begins at the
same time,
and the primary ossification centers of long bones can be
observed
along the periosteum in the midshaft region (Fig.
2E)
(
20).
The
-NAC protein was specifically detected in the
nucleus of
differentiated osteoblasts in the primary ossification
centers
of ribs and long bones at 14.5 days p.c. (Fig.
2D and F). No
signal
was detected in other tissues, as evidenced by the absence of
staining in the tissue flanking the ribs in Fig.
2C and D, and
control
staining with preimmune sera gave no signal (not shown).
This specific
pattern of expression was maintained until birth;
postnatally,
expression of the
-NAC protein was detectable in
other tissues
(
43).
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FIG. 2.
The -NAC protein is specifically expressed in
mineralizing bone during development. Cryosections of embryos at
various developmental ages were probed with the anti--NAC antibody
and fluorescein isothiocyanate-conjugated secondary antibody. (A, C,
and E) Phase-contrast photomicrographs of the toluidine blue-stained
sections. (B, D, and F) Indirect fluorescence photomicrographs. (A and
B) Sagittal section from a 12-day-p.c. embryo. No detectable signal
could be observed in any field examined. (C and D) Parasagittal section
from a 14.5-day-p.c. embryo showing ossification of the ribs and
expression of -NAC in differentiated osteoblasts. (E and F)
Longitudinal section of the ulna from a 14.5-day-p.c. embryo. The
ossification center is in the midshaft region and corresponds to the
area of expression of -NAC. Par, panniculus carnosus (cutaneous
muscle of the trunk); int, intercostal muscles; mu, muscle; per,
periosteum; ul, ulna.
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The AP-1 c-Jun homodimer may be a physiological target for
-NAC.
The tissue-restricted pattern of expression observed for
-NAC during development prompted us to search for transcriptional activators implicated in the control of gene expression during osteoblastic differentiation that could represent natural targets for
the -NAC transcriptional coactivation function. Considering the
large body of experimental evidence demonstrating that the AP-1
transcription factor plays a key role in the regulation of bone tissue
metabolism (17), we tested for a possible interaction between AP-1 family members and -NAC. The coactivation function of
-NAC was tested with both the c-Fos/c-Jun heterodimer and the c-Jun
homodimer.
As shown in Fig.
3A,
-NAC and c-Fos, alone or in combination, had no effect on the
expression of a luciferase reporter gene
under the control of a single
AP-1 binding site. The AP-1 Fos/Jun
heterodimer stimulated the
expression of the reporter gene by
about 10-fold; however,
-NAC had
no effect on the expression
levels driven by the heterodimer. The c-Jun
homodimer also stimulated
the transcription of the luciferase reporter;
mean fold induction
levels were measured as 6.2 ± 1.4 (mean ± standard error). Interestingly,
-NAC potentiated the
c-Jun-mediated transcriptional induction
by a further ninefold, to
53.1 ± 18.0. Similar levels of expression
were achieved for each
recombinant protein (data not shown).
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FIG. 3.
-NAC interacts with c-Jun to potentiate
c-Jun-mediated transcription. (A) Transient transfection assays with
expression vectors for c-fos, c-jun, and -NAC,
alone or in combination. The expression level detected in cells
transfected with the reporter alone (AP-1-tk-luc) was arbitrarily
ascribed a value of 1. Results are expressed as mean fold
induction ± standard error of four independent transfections. (B)
Pull-down protein-protein interaction assays.
[35S]methionine-labeled in vitro-translated proteins,
identified above each panel, were incubated with the fusion GST--NAC
protein or the free GST moiety. Input represents 1/10 of the total
input of in vitro-translated protein. M, molecular mass (kilodaltons)
markers.
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|
We next tested for specific protein-protein interactions between
-NAC and AP-1 family members by using the protein pull-down
assay.
As demonstrated in Fig.
3B, lane 5,
-NAC interacted with
the c-Jun
protein. The interaction was specific, because the GST
moiety of the
fusion GST-NAC protein did not bind c-Jun (lane
6). No interactions
were observed between
-NAC and the monomeric
c-Fos protein (lanes 1 to 3). The luciferase and GAL4/VP-16 proteins
served as negative and
positive controls for the pull-down assay,
respectively (lanes 7 to
12).
-NAC interacts with the N terminus of c-Jun.
A deletion
mutant of c-Jun fused to GST [GST-jun(1-89)] was utilized in
pull-down protein interaction assays to map the domain of c-Jun
interacting with -NAC. The GST-jun(1-89) fusion protein phosphorylated on the serine residue at position 73 by the specific action of the JNK kinase (9) was also tested in these
assays. As shown in Fig. 4, lane 5, -NAC interacted with GST-jun(1-89), demonstrating that the N
terminus of the c-Jun protein is involved in the binding of c-Jun to
-NAC. Two forms of the -NAC protein were detected by the
anti--NAC antibody in these interaction assays (lanes 5 and 6);
treatment of nuclear extracts from bone cells with protein phosphatases
prior to immunoblotting with the anti-NAC antibody eliminated the
protein form showing higher electrophoretic mobility (data not shown),
suggesting that it represents a phosphorylated form of -NAC.
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FIG. 4.
-NAC interacts with the N terminus of c-Jun.
Pull-down protein interaction assays were performed with the
phosphorylated or unphosphorylated GST-jun(1-89) fusion protein. Crude
cell extracts from P19 embryonal carcinoma cells were incubated with
glutathione-Sepharose beads loaded with the GST fusion proteins or the
negative control GST moiety. Following binding, centrifugation, and
washes, the bound proteins were analyzed by immunoblotting with
anti--NAC (lanes 4 to 6), anti-GST (lanes 7 to 9), or
anti-phospho-Ser 73 (lanes 10 to 12) antibodies. Background staining
was assessed with preimmune sera (lanes 1 to 3). M, molecular mass
(kilodaltons) markers.
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|
The c-Jun protein must be phosphorylated at positions 63 and 73 to
become transcriptionally active (
9). Interestingly,
-NAC
was shown to interact with both the phosphorylated and
nonphosphorylated
forms of c-Jun (Fig.
4, lanes 5 and 6). The observed
interactions
were specific because the cellular
-NAC proteins were
not pulled
down by the GST moiety of the GST-jun(1-89) fusion protein
(lane
4). The amounts of fusion protein loaded in the pull-down assays
were monitored with an anti-GST antibody (lanes 7 to 9), while
the
specificity of the phosphorylation of GST-jun(1-89) by the
JNK kinase
(
9) was assessed with an antiphosphoserine 73 antibody
(lanes 10 to 12). Preimmune sera did not detect any significant
cross-reactivity of the secondary antibodies used in the
immunodetection
(lanes 1 to 3).
The strength of the interaction between
-NAC and phospho-c-Jun was
next assessed by performing affinity chromatography of
crude nuclear
extracts from serum-stimulated osteoblasts on a
glutathione-Sepharose
column loaded with recombinant GST-
-NAC
fusion protein, followed by
immunoblotting of the step gradient
eluate with the antiphosphoserine
73 antibody. As illustrated
in Fig.
5,
upper panel, the native phosphorylated c-Jun protein
was retained on
the GST-
-NAC affinity column, and eluted with
a peak in the 0.5 M
salt fraction (lane 9), confirming the strength
of the interaction
between
-NAC and phospho-c-Jun. The phosphorylated
c-Jun did not
bind to a control column loaded with the GST moiety
alone (Fig.
5,
lower panel).
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FIG. 5.
Binding between -NAC and the phosphorylated form of
c-Jun is a strong interaction. (A) Affinity chromatography on a
GST--NAC column. The recombinant GST--NAC protein was immobilized
on a glutathione-Sepharose column. A nuclear extract from
serum-stimulated MC3T3-E1 osteoblastic cells was passed through the
column, allowing specific interactions between -NAC and nuclear
proteins to occur. Bound proteins were eluted with a step gradient of
salt and detected by Western blotting with a monoclonal antibody
directed against the phosphorylated form of c-Jun. Note that bound
phospho-c-Jun elutes at 0.4 to 0.5 M salt. (B) The same nuclear extract
was passed through a control GST column. Molecular mass (kilodaltons)
markers (M) are indicated to the left. In, input protein; F.T.,
flowthrough; lanes 1 and 2, Coomassie blue-stained molecular size
markers and input material, respectively.
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-NAC and c-Jun interact in vivo.
We performed mammalian
two-hybrid assays (Fig. 6A) in order to
confirm that c-Jun interacts with -NAC in vivo and to test the
specificity of this interaction. The full-length -NAC cDNA was
cloned in frame with the yeast GAL4-DBD and transfected in ROS 17/2.8
osteoblastic cells (24), together with a GAL4-dependent reporter construct and expression vectors for AP-1 family members. A
GAL4-DBD expression vector was used as a control in these experiments. As illustrated in Fig. 6B, c-Jun and c-Fos did not interact with the
GAL4-DBD. Moreover, the GAL4--NAC fusion protein did not significantly influence the expression of the reporter gene, confirming our previous observations that -NAC does not possess an intrinsic activation domain (43, 43a).
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FIG. 6.
-NAC binds the c-Jun N terminus in vivo. (A)
Schematic representation of the mammalian two-hybrid assay. (B) ROS
17/2.8 osteoblastic cells were transfected with the expression vectors
for the various proteins mentioned. The expression level detected in
cells transfected with the reporter and the GAL4 NAC vector was
arbitrarily ascribed a value of 1. The c-Jun and c-Fos proteins did not
interact with the GAL4-DBD. The fusion protein GAL4 NAC, which is
transcriptionally inert, did not bind c-Fos; however, the interaction
of c-Jun with -NAC tethers the c-Jun activation domain to the
GAL4-dependent promoter and results in the expression of the reporter
gene. (C) The mammalian two-hybrid assay was performed with c-Jun
deletion mutants. The expression level detected in cells transfected
with the reporter and the GAL4 NAC vector was arbitrarily ascribed a
value of 1. F.L., full-length c-Jun protein; 1-191, a truncated form
of c-Jun in which the C-terminal domain was deleted;  281-313,
leucine zipper deletion mutant; 251-276, basic domain deletion
mutant; 1-87, N-terminal deletion mutant.
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The expression of the reporter gene was stimulated when an expression
vector for c-Jun was coexpressed with the GAL4-
-NAC
fusion protein
(Fig.
6B). This result confirms the interaction
of the c-Jun protein
with
-NAC in vivo, which tethers the c-Jun
activation domain
(
1) to the GAL4-dependent promoter and allows
transcription
of the reporter gene. As previously observed in
vitro (Fig.
3B), the
c-Fos protein did not bind the GAL4-
-NAC
fusion and did not activate
the expression of the reporter construct
(Fig.
6B).
We also tested c-Jun deletion mutants in the mammalian two-hybrid assay
(Fig.
6C). As observed previously, the full-length
c-Jun protein
interacted with the GAL4-
-NAC fusion (Fig.
6C,
F.L.). Several c-Jun
mutant proteins also bound GAL4-
-NAC in
this assay, leading to the
activation of the reporter gene in
the range of two- to threefold (Fig.
6C): a truncated c-Jun comprising
only the N-terminal region (amino
acids 1 to 191 [1-191]), a leucine
zipper deletion mutant
(
281-313), and a basic domain deletion
mutant (
251-276). A
mutation deleting the first 87 amino acids
of the protein prevented the
interaction of c-Jun with GAL4-
-NAC,
resulting in expression levels
of the reporter gene that did not
exceed 1.5-fold over those of the
control (Fig.
6C,
1-87). These
in vivo results corroborate the data
obtained with the in vitro
pull-down protein interaction assays where
the domain of the c-Jun
protein interacting with the
-NAC
coactivator was identified
within the N-terminal amino acids region of
residues 1 to 89 (Fig.
4).
-NAC stabilizes the binding of the c-Jun homodimer to the AP-1
site.
In order to gain further understanding of the mechanism of
-NAC-mediated potentiation of c-Jun-activated transcription, we measured kinetic parameters of the c-Jun interaction with its cognate
AP-1 binding site in the presence or absence of -NAC by using the
electrophoretic gel mobility shift assay. -NAC did not influence the
on rate of association of the c-Jun homodimer with the AP-1 recognition
sequence (Fig. 7A). However, -NAC
significantly affected the off rate of c-Jun from its DNA binding site
(Fig. 7B), thus dramatically stabilizing the AP-1 complex formed by the
c-Jun homodimer. In three independent experiments, -NAC caused, on
average, an eightfold reduction in the dissociation constant (kd) of the complex (Table
1).
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FIG. 7.
-NAC stabilizes the binding of c-Jun to the AP-1
site. Binding reactions and electrophoretic mobility shift assays were
performed with purified c-Jun and -NAC proteins. (A) On rate
measurements. Samples were removed at intervals and analyzed for DNA
binding. Bound probe was measured by cutting out the bands on the gel
and counting in a gamma counter; calculations were done as described by
Chodosh et al. (5). Note that the slope of the binding
curve, which represents the on rate, is not affected by -NAC. A,
free c-Jun; AP, c-Jun-DNA complex; PT, total DNA. (B) Off
rate measurements. The bound complexes were formed as described, and a
50-fold excess of unlabeled AP-1 or GAL4 oligonucleotides was
subsequently added to the binding reaction mixture. Samples were
removed at intervals and processed as described above. The slope allows
calculation of kd, which is reduced in the
presence of -NAC, thus stabilizing the c-Jun AP-1 complex and the
GAL4/VP-16 complex. (C) Off rate measurement of the JunB AP-1 complex:
in vitro-translated JunB protein was used as described for c-Jun in
panel B. Note that -NAC does not stabilize the JunB AP-1 complex.
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We tested the specificity of the stabilizing effect of
-NAC on DNA
binding. While
-NAC stabilized the binding of GAL4/VP-16
to its
cognate site (Fig.
7B, right panel), it had no effect on
the stability
of binding of JunB (Fig.
7C) and Sp1 (data not shown)
to their
respective DNA recognition sequences. These data suggest
that
-NAC
only stabilizes the binding of factors with which it
interacts to
potentiate the transcriptional activation function.
These experiments demonstrate a direct interaction between
-NAC and
the c-Jun homodimer, resulting in increased transcriptional
activation
from an AP-1-responsive promoter. Combined with the
restricted
expression pattern observed for
-NAC during development,
these data
suggest that
-NAC may be implicated in the regulation
of gene
transcription during osteoblastic differentiation.
| |
DISCUSSION |
We have shown that the c-Jun AP-1 homodimer is a natural target
for the coactivation function of -NAC. Moreover, our data revealed
that -NAC represents one of the rare examples of a tissue-specific, developmentally regulated transcriptional coactivator. These results provide novel perspectives for our understanding of the specificity of
AP-1-dependent gene transcription and the regulation of
osteoblast-specific gene expression.
In a search for proteins interacting with c-Jun and implicated in
c-Jun-mediated transcriptional activation, Franklin et al. (14) have shown that the C terminus of the c-Jun protein
associates directly with TBP and TFIIB in vitro. While these
interactions may contribute to the molecular mechanisms regulating
c-Jun-driven transcription, they were measured as relatively weak
interactions (c-Jun dissociated from TBP in 0.2 M salt)
(14). The high affinity of -NAC for c-Jun (Fig. 5) and
TBP (43a) is thought to stabilize the interaction of the
c-Jun homodimer with the basal transcriptional machinery. Moreover,
-NAC stabilized the AP-1 complex formed by the c-Jun homodimer
through an eightfold reduction in the kd of the
complex (Table 1). We thus propose the following model for the
potentiation of c-Jun-mediated transcription by -NAC: the rate of
transcription from a c-Jun-dependent promoter is increased in the
presence of the -NAC coactivator through the stabilization of the
c-Jun dimer on its binding site and through the recruitment of the
basal transcriptional machinery by -NAC, which stabilizes the
interaction between c-Jun and TBP (Fig.
8).
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FIG. 8.
Model of the -NAC potentiation of c-Jun-activated
transcription. (Upper panel) The c-Jun homodimer binds the AP-1 site
and interacts weakly with TBP. (Lower panel) The -NAC coactivator
interacts with the N-terminal domain of c-Jun. This interaction leads
to a stabilization of the interaction of the c-Jun homodimer with the
AP-1 site through a reduction in the dissociation rate
(kd). Moreover, since -NAC interacts strongly
with both c-Jun and TBP, it strengthens the contacts between c-Jun and
the basal transcriptional machinery, resulting in enhanced
transcription from the c-Jun dimer (represented in the form of the
larger arrow than that in the upper panel). For simplicity, only the
phosphorylated form of c-Jun is depicted in the model; however, in
vitro protein interaction assays demonstrated that -NAC can also
interact with the unphosphorylated form of c-Jun.
|
|
Coactivator proteins that potentiate the transcriptional response to
the c-Jun homodimer have been reported previously. The interaction of
c-Jun with CBP (CREB binding protein) has been described previously
(2, 3). More recently, the JAB-1 coactivator which
selectively potentiates the activity of c-Jun and JunD, but not JunB or
v-Jun, was identified with the yeast two-hybrid protein interaction
assay (6). It is interesting to note the similarities
between the mechanisms of action of JAB-1 and -NAC, despite the
complete absence of sequence homology between them. Both proteins
interact with the N-terminal portion of c-Jun and can bind the
phosphorylated as well as the unphosphorylated forms of the
transcription factor (6). Moreover, the two coactivators appear to act by stabilizing the interaction of the c-Jun homodimer to
its cognate DNA binding site (6). However, the target
contacts of JAB-1 with the basal transcriptional machinery have not
been identified, and its tissue distribution has not been described. It
also remains unclear whether JAB-1 can interact with Fos family members
or affect the transcriptional response to the Fos/Jun AP-1 heterodimer.
Therefore, several distinct coactivators may contribute to the tissue
specificity of target gene activation by AP-1 proteins. Additional
specificity may also be provided by posttranslational modifications;
CBP, for example, interacts only with the phosphorylated form of c-Jun
(2).
There is a growing body of experimental evidence showing that AP-1 is
essential for proper bone development (reviewed in reference 17). While most of these studies have focused on the
c-fos component of the AP-1 family, it is well known that
the Fos protein needs a dimerizing partner in order to bind DNA and
activate transcription (1, 30). We have previously shown
that c-jun is expressed in osteoblasts (4);
indeed, expression of c-jun in bone cells has been reported
both during early embryogenesis (42) and at all stages of
osteoblastic differentiation (26). Thus, it is probable that
the c-Jun activator plays a physiological role in the control of gene
expression in bone cells.
Genetic and biochemical evidence has recently demonstrated that
transcriptional coactivators such as the TAFIIs mediate
transcriptional activation during development (33).
Moreover, coactivators have been shown to be involved in
tissue-specific gene expression. For example, the coactivation function
of the small subunit of TFIIA (TFIIA-S) has been shown to be required
for the Ras-mediated determination of photoreceptor cells during
Drosophila development (44). Several groups have
also identified a B-cell-specific coactivator (18, 23, 35).
These studies have led to the cloning and characterization of
OBF-1/Bob1, a coactivator interacting with some members of the Oct
family of octamer motif-binding proteins (18, 35). This
coactivator appears responsible for restricting the activity of the
immunoglobulin promoter to B cells (18, 35), thus converting
ubiquitously expressed transcription factors to cell-type-specific
activators. Since coactivators interact with one or several distinct
transcriptional activators, it is plausible that the developmental
regulation of the expression of a particular coactivator could lead to
the coordinate induction of a number of genes in a given tissue. We
propose that the restricted expression of -NAC to bone cells during
development serves such a function. The identification of additional
endogenous transcription factors interacting with -NAC, together
with the identification of -NAC-responsive genes in bone, should
further our understanding of the molecular mechanisms regulating
osteoblastic differentiation and function.
| |
ACKNOWLEDGMENTS |
A.M. and W.V.Y. participated equally in this study and should
both be considered first authors.
We thank the following investigators for their generous gift of
plasmids or reagents: Michael Green (5Gal4-E1b-CAT), Mark Ptashne
(pSGVP), Robert Tjian (pSV6tkCAT), Daniel Nathans (Jac7), Frank J. Rauscher III (CMV-c-fos and pSP65-c-fos), and Michael Karin and Trang
Hoang (c-Jun deletion mutants). We thank Edwin Wan for oligonucleotide
synthesis and Jane Wishart and Mark Lepik for preparing the figures.
This work was supported by a grant from the Shriners of North America.
W.V.Y. was a Shriners' Fellow, and R.S.-A. is a Chercheur-Boursier from the Fonds de la Recherche en Santé du Québec.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Genetics Unit,
Shriners Hospital, 1529 Cedar Ave., Montréal, Québec H3G
1A6, Canada. Phone: (514) 842-5964. Fax: (514) 842-5581. E-mail:
rst-arnaud{at}shriners.mcgill.ca.
Present address: Laboratoire de génétique
moléculaire, Institut de Recherches Cliniques de Montréal,
Montréal, Québec H2W 1R7, Canada.
Present address: Centre de cancérologie Charles Bruneau,
Hôpital Ste-Justine, Montréal, Québec H3T 1C5,
Canada.
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Abstract
Introduction
