Centre Oncologia Molecular, Institut de Recerca Oncologica, Hospitalet, Barcelona 08907, Spain
* Author for correspondence (e-mail: abigas{at}iro.es )
Accepted 13 December 2001
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Summary |
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Key words: NFB, N-CoR, Notch, Transcriptional regulation
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Introduction |
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SMRT and N-CoR are highly homologous proteins, coded by two different genes
with different splicing isoforms (Wong and
Privalsky, 1998; Park et al.,
1999
). Although both proteins share many common structural and
functional features, inactivation of the N-CoR gene by homologous
recombination has shown that their functions are not physiologically redundant
(Jepsen et al., 2000
).
In recent years, coactivators and corepresors of nuclear receptors, and
other transcription factors, have been identified as fundamental components in
the regulation of eukaryotic gene expression (reviewed in
Xu et al., 1999;
Pazin and Kadonaga, 1997
).
Although transcriptional coactivator complexes possess intrinsic histone
acetyl-transferase (HAT) activity, nuclear receptor corepressors achieve their
function by recruiting histone deacetylases (HDACs). There is a positive
correlation between core histone acetylation and gene transcriptional
activity. It is generally accepted that acetylation causes local changes in
chromatin structure, thus facilitating the assembly of the transcriptional
machinery. Many transcription factors such as RBPJ
(Kao et al., 1998
),
NF
B, AP-1, SRF (Lee et al.,
2000
), MyoD (Bailey et al.,
1999
) and Pbx (Saleh et al.,
2000
) have been shown to regulate gene transcription by recruiting
acetylase/deacetylase activities.
The NFB/Rel family of transcription factors participates in the
regulation of disparate cellular processes such as proliferation,
differentiation, immune response and transcription of viral promoters
(reviewed in Baldwin, 1996
;
Ghosh et al., 1998
). The Rel
family includes p65 (RelA), p105/p50, p100/p52, RelB, c-Rel and the viral
oncoprotein v-Rel. These proteins associate as homo or heterodimers to form
transcriptional regulatory complexes known as nuclear factor kappa B
(NF
B). Transcriptional activity of NF
B is specified by the
composition of the dimers and depends on their subcellular localization and
their association with the inhibitory protein I
B (reviewed in
Karin, 1999
).
There is emerging evidence supporting a functional interplay between Notch
and NFB signaling pathways. For example, Notch can interact with
p50-NF
B, thus modulating NF
B-dependent gene transcription
(Guan et al., 1996
).
Conversely, NF
B induces the expression of the Notch ligand Jagged1,
triggering Notch activation in adjacent cells
(Bash et al., 1999
).
Interaction between both pathways is specially intriguing since NF
B can
either promote or inhibit differentiation in various cell types
(Kim et al., 2001
;
Feng and Porter, 1999
;
Kaliman et al., 1999
;
Kaisho et al., 2001
) (L.E. and
A.B, unpublished), whereas Notch activity is primarily involved in blocking
this process (Fortini et al.,
1993
; Sternberg,
1988
; Milner et al.,
1996
). We have now studied the putative effect of p65-NF
B
on modulating the Notch/RBPJ
transcriptional activity. We describe here
how the overexpression of p65-NF
B or a mutant that lacks the
transcriptional activation (TA) domain facilitates Notch-IC-mediated
transcription of the Hes1 promoter by inducing cytoplasmic retention of the
nuclear corepressor N-CoR. This effect applies to other promoters repressed by
N-CoR, such as those containing SRF or AP-1 sites. Further physiological
studies are needed to understand the contribution of this new mechanism of
gene regulation in the control of the different cellular events.
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Materials and Methods |
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Antibodies
Anti-flag (clone M2) was purchased from Sigma and used at a dilution of
1:1,000 for western blot and 1:750 for immunofluorescence analysis;
anti-p65-NFB (sc-109, Santa Cruz Biotechnology) was used at 1:400.
9E10, anti-myc-tag, was used at 1:1,000 for western blot and 1:200 for
immunofluorescence. mN1A antibody (Huppert
et al., 2000
) was used 1:200 for western blot. Secondary
antibodies conjugated with horseradish peroxidase (HRP) were purchased from
DAKO and used 1:2,000 for western blot. Fluorescein-conjugated goat anti-mouse
or Cy3-conjugated goat anti-rabbit (Amersham) secondary antibodies were
diluted 1:200 and 1:1,000 respectively.
Cell culture and transfections
NIH-3T3 and 293T cells were cultured in Dulbecco's modified Eagle medium
and 10% FBS (fetal bovine serum). Cells were plated at subconfluence and
transfected by calcium phosphate. Medium was changed after 12 hours, and cells
were processed 24 hours later for luciferase assays, immunofluorescence or
western blots. Leptomycin B (LMB) was purchased from SIGMA and used at 10-40
ng/ml. Trichostatine A (TSA) was purchased from Calbiochem and used at 600 nM
for 12 hours.
Western blot
293T cells were transfected with the different DNA plasmids by calcium
phosphate treatment, and 48 hours later were lysed during 30 minutes at
4°C in a buffer containing 20% glycerol, 20 mM Hepes pH 7.6, 350 mM NaCl,
50 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.25% NP-40, 5 mM Na Fluoride, 1
mM EGTA, 0.25 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.4 mM
Na-ortovanadate and 1 µg/ml Bestatin. Protein extracts were electrophoresed
in 6% polyacrylamide gels and transferred to PVDF membranes overnight.
Membranes were blocked with 5% non-fat dried milk in TBS and incubated with
the appropriate antibody in TBS and 0.5% tween20 (TBS-T) with 5% non-fat dried
milk for 90 minutes. Membranes were washed and incubated with a secondary
HRP-conjugated antibody for 1 hour. After extensive washing, immunoreactive
proteins were detected by using the Enhanced Chemiluminiscent Detection System
(ECL, Amersham Pharmacia Biotech) as specified by the manufacturer.
Northern blot analysis
Total RNA was extracted from cells using the Chomczynski and Sacchi method
(Chomczynski and Sacchi,
1987). RNAs were size-fractionated by electrophoresis, transferred
onto Hybond-N+ nylon membranes (Amersham) and then hybridized with a
radiolabeled Hes1 probe. Radioactivities of each signal were measured by a
PhosphorImager using Quantity One software (Bio-rad). Densitometric analysis
was performed by using Phoretics software.
Luciferase assays
NIH-3T3 were plated on 12-well plates and transfected with the indicated
expression vectors or the empty vector as a control. In the different
experiments we used 1 µg of Hes1-luc, 2xB-luc,
3xAP-1-luc or 2xSRE-luc as reporter plasmids and 0.5 µg
RSV-ß-gal as internal control. pCS2 vector was added when necessary to
keep the amount of DNA constant. A luciferase assay (Luciferase Assay System,
Promega) was performed 48 hours after transfection, following the
manufacturer's instructions. Luciferase values were normalized for
ß-galactosidase activity. At least three independent experiments were
performed in duplicates.
Immunofluorescence
NIH-3T3 or 293T cells were seeded on slides at 20% confluence and
transfected with 10 µg N-CoR, 4 µg p65-NFB or 4 µg N1-IC.
After 48 hours, cells were fixed in 3% paraformaldehyde in PBS for 25 minutes
at 4°C, washed in PBS, permeabilized in 0.1% Triton X-100 in PBS, 5%
non-fat dry milk for 25 minutes at 4°C. After washing, cells were
incubated with the indicated primary antibody for 90 minutes at 4°C and
extensively washed in PBS 1% non-fat dry milk. After 90 minutes of incubation
with the appropriate secondary antibody, slides were extensively washed and
mounted with Vectashield plus DAPI (Vector). Cells were visualized in an
Olympus BX-60 microscope with the appropriate filters. Representative cells
were photographed and slides were digitalized with Adobe Photoshop.
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Results |
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|
We next tested whether the previously reported interaction between Notch-IC
and p50-NFB (Guan et al.,
1996
) was responsible for the above-described effect. We
hypothesized that addition of p65-NF
B would liberate Notch-IC from a
putative p50 inhibition, resulting in an increase in Notch activity.
Cotransfection of p50-NF
B did not affect the Notch activation of the
Hes1 promoter nor the observed p65-mediated upregulation
(Fig. 1D), although it
increased NF
B-dependent transcription (data not shown). Altogether,
these results demonstrate that p65-NF
B can synergize with Notch-IC to
activate Hes1 transcription. Moreover, this effect is not due to a competition
of both transcription factors for binding to the p50-NF
B subunit,
indicating that other mechanisms may be involved.
Activation of NFB-dependent transcription is not required for
Notch and p65-NF
B synergism
Since NFB participates in the transcriptional activation of several
genes including the Notch ligand Jagged1
(Bash et al., 1999
), we
reasoned that the observed effect on the Hes1 promoter could be mediated by
p65-NF
B transcriptional activation of any Notch pathway gene. To check
this hypothesis, we repeated the p65/Notch-IC cotransfection experiments in
the presence of the constitutive repressor of NF
B,
I
B
32-36 (DiDonato et al.,
1996
). As expected, expression of I
B
32-36 inhibited
p65-NF
B-mediated transactivation of the 2x
B-luc reporter
in a dose-dependent manner (Fig.
2A) by sequestering NF
B in the cytoplasm even in the
presence of Notch-IC (Fig. 2C).
In these conditions, inhibition of NF
B transcriptional activity did not
abrogate the p65-mediated upregulation of the Hes1 promoter
(Fig. 2B). These results
indicate that the synergistic effect of p65-NF
B and Notch on the Hes1
promoter requires neither colocalization of Notch and p65-NF
B in the
nucleus nor NF
B transcriptional activity. To further demonstrate this
observation, we coexpressed N1-IC with a deletion mutant of p65-NF
B
(p65
TA), which lacks the transactivation domain (AA 451-551)
(Harhaj and Sun, 1999
). In the
presence of N1-IC, expression of p65
TA resulted in the increased
dose-dependent activation of the Hes1 promoter
(Fig. 2D). Moreover, the effect
of p65
TA was even stronger (up to four-fold) than the maximum effect
observed with the p65 wild type (p65 wt).
|
Hes1 promoter activity is repressed by N-CoR, and this effect is
reversed by ectopic expression of p65-NFB
We have demonstrated that neither transcriptional activation by NFB
nor nuclear localization of p65 subunit are required for upregulating the
Notch-dependent Hes1 promoter activation. Thus, we examined whether
p65-NF
B was facilitating Notch activity by displacing a Hes1 repressor
molecule.
As previously described for the SMRT corepressor
(Kao et al., 1998), we found
that N-CoR was able to repress Hes1 promoter activity in a dose-dependent
manner (Fig. 3A). To
investigate whether p65-NF
B was able to modulate N-CoR-mediated
repression, we cotransfected N-CoR and increasing amounts of p65 wt or
p65
TA along with the Hes1 promoter. Our results demonstrate that
ectopic expression of p65wt (data not shown) or p65
TA were able to
reverse the inhibitory effect of N-CoR in a dose-dependent manner
(Fig. 3B). This suggests that
the previously observed effect of p65-NF
B on the Hes1 promoter may be
mediated through its interaction with N-CoR.
|
We next asked whether overexpression of p65-NFB was also modifying
the expression of the endogenous Hes1 gene. We observed a moderate
increase (1.5-fold by densitometric analysis) in the Hes1 mRNA from 293T cells
transfected with Notch-IC and N-CoR in the presence of p65 wt
(Fig. 3C) or p65
TA (data
not shown).
p65TA expression triggers cytoplasmic translocation of
N-CoR
We then examined the subcellular localization and protein levels of N-CoR
in the presence or absence of p65TA. In the absence of p65
TA,
N-CoR was localized in the nucleus, displaying a speckled distribution (upper
panels), as previously reported (Horlein
et al., 1995
). However, in 293T cells cotransfected with N-CoR and
p65
TA, we observed colocalization of both proteins in the cytoplasm,
N-CoR being absent from the nucleus (Fig.
4A, lower panels). Identical results were obtained in NIH-3T3
cells (data not shown).
|
We next quantified N-CoR protein levels in 293T cells cotransfected with
N1-IC and N-CoR in the presence or absence of p65TA
(Fig. 4B). N-CoR was detected
at comparable levels in both conditions, suggesting that protein stability was
not affected.
p65-NFB nuclear export is required for N-CoR cytoplasmic
translocation and Hes1 upregulation
There is increasing evidence of continuous nuclear-cytoplasmic shuttling of
p65-NFB within the cell (Carlotti
et al., 2000
). p65-NF
B subcellular localization is
regulated by its NES domain and by its interaction with the p50-NF
B
subunit and I
B (Harhaj and Sun,
1999
; Huang et al.,
2000
). It has previously been reported that incubation of cells
with the CRM-1-mediated nuclear export inhibitor LMB
(Nishi et al., 1994
) results
in nuclear accumulation of p65-NF
B
(Huang et al., 2000
). We next
investigated whether inhibition of p65 nuclear export by LMB was blocking the
cytoplasmic translocation of N-CoR. 293T cells were cotransfected with
p65
TA and N-CoR and incubated in media alone or in the presence of 10
ng/ml of LMB. Fig. 5A shows
that when CRM1-dependent nuclear export was inhibited, both p65
TA and
N-CoR remained in the nucleus.
|
To investigate whether p65 nuclear retention but not LMB incubation per se
is responsible for inhibiting N-CoR cytoplasmic translocation, we used a p65
mutant (p65NES) lacking the NES domain, which localizes exclusively in
the nucleus (Harhaj and Sun,
1999
). When coexpressed with p65
NES, N-CoR shows an
exclusively nuclear localization (Fig.
5B). Consistent with this observation, increasing levels of
p65
NES with N1-IC did not result in upregulation of Hes1 promoter
(Fig. 5C). These results
strongly suggest that p65 cytoplasmic translocation is required for N-CoR
cytoplasmic retention and that this mechanism is responsible for p65-mediated
Hes1 upregulation. Moreover, we have observed that incubation with the HDAC
inhibitor trichostatine A (TSA) results in a more nuclear localization of
p65-NF
B, as reported by Chen et al.
(Chen et al., 2001
).
Nevertheless, the colocalization of N-CoR with p65-NF
B is not modified
in these conditions, suggesting that deacetylase activity is not necessary for
interactions between both proteins (data not shown).
p65-NFB increases the activity of other promoters repressed by
N-CoR
Repression of genes dependent on NFB, Activator Protein-1 (AP-1) or
Serum Response Factor (SRF) has been associated with the interaction of these
transcription factors with SMRT (Lee et
al., 2000
). We next speculated that p65-induced N-CoR cytoplasmic
translocation should also be affecting the expression of genes other than
Hes1. We transfected N-CoR with or without p65
TA into NIH-3T3 cells and
determined the transcriptional activity of SRF- and AP-1-dependent luciferase
reporters. When incubated in 20% FBS, expression of p65
TA upregulates
SRE reporter activity in a dose-dependent manner, up to a maximum of a
three-fold increase (Fig. 6B).
When the AP-1-dependent promoter was assayed in the presence of p65
TA,
luciferase activity was five-fold higher compared with the maximum activity of
this reporter in the presence of c-fos.
(Fig. 6C). Moreover, and
similar to its effect on the Hes1 reporter, we show that N-CoR was able to
repress both promoters and that this repression was overridden by coexpression
of p65
TA (Fig. 6A,B,C).
These results clearly demonstrate that p65-NF
B can regulate gene
expression not only through binding to promoters containing
B-sites but
also by triggering cytoplasmic translocation of the nuclear corepressor
N-CoR.
|
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Discussion |
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First, we show that p65-NFB cooperates with Notch in activating the
Hes1 promoter. Second, we show that this effect is independent of
transcriptional activity and colocalization of p65-NF
B and Notch-IC.
Next, we demonstrate that ectopic expression of p65-NF
B can modify
subcellular localization of the nuclear corepressor N-CoR. Finally, we
demonstrate that this new mechanism affects not only Notch-dependent gene
expression but also the expression of other genes (such as SRF- and
AP-1-responsive genes) that are targets for N-CoR repression.
Notch and NFB signaling pathways
Several interactions between Notch and NFB pathways have already
been reported; however, the physiological significance of their interplay is
not well understood. For instance, a Notch-IC and p50-NF
B direct
interaction leads to the inhibition of NF
B transcriptional activity,
supporting the idea that Notch activation promotes downregulation of
NF
B-dependent genes (Guan et al.,
1996
). Conversely, it has been shown that Notch3 homologue is able
to upregulate the expression of NF
B-dependent genes, probably by
promoting degradation of the NF
B inhibitor I
B
(Bellavia et al., 2000
). On the
other hand, NF
B participates in the transcriptional regulation of the
Notch ligand Jagged1 gene (Bash et al.,
1999
), thus modifying the balance between receptors and ligands
that is critical in Notch pathway regulation
(Heitzler and Simpson,
1991
).
Notch and NFB can play different roles in regulating cell
proliferation (Baonza and Garcia-Bellido,
2000
; Carlesso et al.,
1999
; Kontgen et al.,
1995
), cell differentiation
(Kaisho et al., 2001
;
Kaliman et al., 1999
;
Guttridge et al., 2000
;
Feng and Porter, 1999
;
Egan et al., 1998
;
Milner and Bigas, 1999
) and
apoptosis (reviewed in Barkett and Gilmore,
1999
; Jehn et al.,
1999
; Shelly et al.,
1999
; Ohishi et al.,
2000
). However, it is tempting to speculate that they should exert
antagonistic or synergistic effects depending on the cellular context. In
myogenic systems, NF
B can both promote or inhibit differentiation
depending on the external stimuli. For example, it mediates
TNF-
-induced inhibition of differentiation in C2C12 cells
(Guttridge et al., 1999
), and
it is required for the IGFII-induced differentiation of L6E9 myoblasts
(Kaliman et al., 1999
). In
contrast, Notch activation is mainly associated with maintaining the
undifferentiated phenotype (Kopan et al.,
1994
) by upregulating Hes1
(Kuroda et al., 1999
),
inhibiting MEF2C (Wilson-Rawls et al.,
1999
) or by combined mechanisms.
In T cell development, constitutive activation of Notch
(Robey et al., 1996) and
suppression of NF
B activity by expression of
I
B
32-36 (Boothby
et al., 1997
) results in opposite phenotypes for the acquisition
of the CD8+CD4- T cell fate. In fact, in a previous
step, both Notch and NF
B activities are necessary for the survival of
double-positive CD4+CD8+ cells when the appropriate TCR
signal is received (Deftos et al.,
2000
; Hettmann and Leiden,
2000
). Synergistic mechanisms of crosstalk between NF
B and
Notch pathways (Oswald et al.,
1998
; Bash et al.,
1999
; Bellavia et al.,
2000
) (this paper) may be controlling thymocyte-positive selection
by regulating the expression of anti-apoptotic genes. Consistent with this,
both Notch and NF
B can activate bcl-2 expression in lymphoid cells
(Zong et al., 1999
;
Deftos et al., 2000
).
On the other hand, alterations in the Notch or NFB pathways have
been associated with oncogenic processes. Several cancer cells show
constitutive nuclear NF
B activity, including those from lymphoid,
breast, ovarian, lung, thyroid and melanoma origin (reviewed in
Rayet and Gelinas, 1999
).
Moreover, there is emerging evidence that alteration of the Notch pathway
affects many tumorigenic transformation and tumor progression processes
(Capobianco et al., 1997
;
Leethanakul et al., 2000
;
Gallahan and Callahan, 1997
).
The NF
B constitutive activity found in Notch3-IC-transformed T cells is
the first evidence that suggests a possible synergistic effect of both
pathways in tumorigenesis (Bellavia et al.,
2000
). Taken together, these results indicate that coordination
between Notch and NF
B pathways may be crucial in controlling cellular
events.
Transcriptional activation mediated by p65-NFB
Different combinations of Rel/NFB subunits bind to specific target
genes, directly regulating their transcriptional activity. Although
NF
B-mediated transcriptional regulation has been extensively studied
(reviewed in Siebenlist et al.,
1994
), NF
B-mediated effects on promoters lacking
b-sites or possible interactions of single NF
B subunits with
other transcriptional complexes need further clarification.
For example, it has been reported that p65-NFB can upregulate
SRE-containing promoters independently of classical NF
B activity
(Franzoso et al., 1996
). In
addition, the transforming ability of v-rel, a mutated version of the avian
c-Rel that lacks the transactivation domain (reviewed in
Luque and Gelinas, 1997
),
correlates with high levels of c-jun and c-fos proteins and increased AP-1
transcriptional activity (Kralova et al.,
1998
). Consistent with this, we have shown that overexpression of
p65-NF
B regulates not only the transcriptional activity of Hes1 but
also the activity of promoters containing SRE and AP-1 elements that lack
NF
B sites. Thus, our results can provide an explanation of the
previously published observations, reinforcing the idea that p65-NF
B
exerts a positive effect over a broad spectrum of transcription factors, even
in the absence of its transcriptional activation (TA) domain. In this sense,
our results suggest that the p65
TA mutant may be more effective at
overriding N-CoR transcriptional repression than the full-length protein,
perhaps reflecting the presence of some regulatory elements in the C-terminal
half of the protein.
Although more physiological studies should be done, these observations may
reflect a new crosstalk between NFB and other signaling pathways, which
may be responsible for regulating transcription of specific sets of genes.
A role for p65-NFB in regulating chromatin modifying
activities
Assembly of multimeric protein complexes at their appropriate target DNA
sequences results in transcriptional activation or repression, thus generating
specific gene expression patterns (reviewed by
Merika and Thanos, 2001).
Recruitment of coactivators or corepresors by transcription factors and
hormone receptors is crucial to determine the nature and activity of these
complexes. Coactivators and corepressors exert their function by increasing or
decreasing histone acetylation and thus modifying chromatin structure. N-CoR
and SMRT nuclear corepressors are highly homologous, although N-CoR contains a
unique N-terminal region that might be involved in unreported specific
functions. Although preliminary studies associated both proteins with
repression of hormone nuclear receptors (reviewed by
Xu et al., 1999
), SMRT can
also repress c-fos-, NF
B-, SRF-
(Lee et al., 2000
) and
Notch/RBPJ
-dependent genes (Kao et
al., 1998
). Here we show that most of these promoters are also
repressed by N-CoR, and we demonstrate that this repression can be modulated
by p65-NF
B.
Physical association of SMRT and HDACs with p65-NFB has been
reported to repress NF
B-regulated genes
(Lee et al., 2000
;
Ashburner et al., 2001
;
Chen et al., 2001
). Our
results suggest that p65-NF
B may interact with N-CoR, inducing its
translocation to the cytoplasm and acting as a rapid and potent tool for
regulating gene expression. Although experiments with TSA suggest that this
mechanism does not depend on deacetylase activity, the role of HDACs or other
proteins known to interact with p65 or N-CoR is currently under investigation.
It is also worth considering that phosphorylation or acetylation events
affecting N-CoR or p65-NF
B may regulate their interaction. In this
sense, it has been reported that SMRT can translocate to the cytoplasm in
response to phosphorylation events (Hong
and Privalsky, 2000
; Jang et
al., 2001
).
Coactivators and corepressors bind to nuclear receptors competing for
similar consensus regions (Nagy et al.,
1999). Interestingly, p65-NF
B can synergize with IRF1 and
ATF2/c-jun to activate transcription of the IFNß promoter by recruiting
transcriptional coactivators through a leucine-rich domain (AA 430-458)
similar to that present in nuclear receptors
(Merika et al., 1998
). In this
system, the effect of p65-NF
B is dependent on the binding to DNA of all
three transcription factors. By contrast, the mechanism that we describe here
does not require the presence of a
B-binding site in the target
promoter. Since the NES is included in the p65-NF
B leucinerich domain,
it is tempting to speculate that there is a potential physical interaction
between p65-NF
B and N-CoR involving this domain.
Taken together, our results indicate that p65-NFB can exert an
important role in coordinating gene transcription of specific sets of genes by
a new mechanism that involves cytoplasmic translocation of N-CoR. We speculate
that this new p65-NF
B function may be crucial in the integration of
multiple signal transduction pathways.
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Acknowledgments |
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References |
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