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INTRODUCTION |
RB1 is a general tumor
suppressor that was identified by virtue of a predisposition to
childhood retinoblastoma when defects are present in its gene (1, 2).
The tumor suppressing property of this 110-kDa nuclear phosphoprotein
is associated with its central (A/B) and C-terminal (C) "pocket"
domains that were defined through their ability to bind cellular and
viral proteins that affect the cell division cycle (reviewed in Ref.
3). These pocket domains are also present in p107 and p130, structural
homologues of RB that exhibit similar protein binding and functional
characteristics pertaining to the cell division cycle (4-7). Together,
these homologues constitute the RB family of proteins and are linked by
substantial direct evidence to regulatory roles in cell growth, differentiation, and development (reviewed in Ref. 8). As a consequence
of the similar pocket domains, these three homologues exhibit a
noteworthy overlap of interacting proteins and in vivo functionality (reviewed in Ref. 9), although they also bind differentially to other proteins (10, 11) and display distinct molecular and cellular characteristics. One common characteristic is
the ability to restrain cell growth by inhibiting the pocket domain-binding E2F family of transcription factors, these factors being
crucial for the expression of genes that are vital for the S phase of
the cell division cycle. This repression of E2F activity and cell
growth requires the participation of yet another pocket domain binding
protein, hBRM (or its close homologue, BRG1) (12, 13).
hBRM and BRG1 are 200-kDa nuclear factors present in the large,
multisubunit human SWI/SNF complexes (14, 15) that contain eight or
more proteins depending on the origin and state of the cell (16-18).
These complexes function in reorganizing chromatin structure and thus
influence the activity of sequence-specific activator proteins by
affecting their access to regulatory sites (16). In the repression of
the S phase inducing transcription factor E2F1, RB physically contacts
both E2F1 and hBRM at the same time and thus targets the
hBRM-containing SWI/SNF complex to E2F1 (12). In other cases, the
SWI/SNF complex is targeted to specific promoters through interactions
with DNA-binding components of the transcription machinery. A case in
point is GR, a hormone-activated transcription factor that recruits the
SWI/SNF complex through direct binding to the hBRM/BRG1 and stimulates
nucleosome disruption at the glucocorticoidy response element (GRE)
(19, 20). Although the remodeling of the chromatin does not influence
the ability of GR to access and occupy the GRE, it is essential for
post-binding events involving the basic transcription machinery (20)
because the TFIID component cannot access the relevant DNA sites until after treatment with glucocorticoid hormone (21). Hence, an abundance
of hBRM can potentiate the transcriptional activity of the GR (14).
Interestingly, this potentiation is further enhanced by the interaction
of hBRM with RB (22).
The GR is a member of the nuclear hormone receptor
superfamily and consists of an N-terminal domain with a
transcription activation function, a central DNA binding and
dimerization domain, and a C-terminal ligand binding domain with an
inducible transcription activation function (reviewed in Ref. 23). It
binds as a homodimer to the canonical GRE, an imperfect palindrome, and
accelerates transcription initiation at the promoter of the target
genes. However, it functions as a monomer at composite GREs that are simultaneously occupied by other enhancer binding factors or at tethering elements where it is tethered to the DNA by an interacting protein. At these complex sites, GR may function to variously promote
or repress gene expression (24), contingent on the context of the
interacting proteins and DNA. The physiological outcome of these
diverse molecular interactions is the regulation, by GR, of critical
metabolic and developmental processes essential for survival (25, 26).
At the cellular level, activation of the GR by glucocorticoids
generally invokes antiproliferative effects, epitomized by the
apoptosis of immature thymocytes (27) and various leukemic cell lines
(28). It is because of these effects that glucocorticoids are employed
as immunosuppressive, anti-inflammatory, or cytostatic agents in the
treatment of rheumatoid arthritis, collagen diseases, lymphatic
leukemias, and lymphomas.
Developing on our earlier work, which showed that RB potentiates
GR-mediated transcription by interacting with hBRM (22), we queried
whether GR-associated apoptosis was similarly influenced by RB and hBRM
and whether RB could be functionally replaced by its pocket domain
homologues. We show here that both GR-mediated transcription and
apoptosis are dependent on RB and hBRM but not p107 or p130. This is
likely to result from the divergent N-terminal domain of RB that is
unique in promoting the interaction of GR with hBRM and hence the GR
targeted chromatin remodeling activity of the SWI/SNF complex.
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EXPERIMENTAL PROCEDURES |
Yeast Two-hybrid Interaction Analysis--
Vectors and strains
for the two-hybrid system (provided by Dr. S. J. Elledge) were
employed as previously described (29). The hBRM hybrid was constructed
by inserting a flush-ended ClaI fragment of the hBRM
cDNA into the NcoI- and SmaI-digested vector pACTII with flush ends. This created an in frame fusion of the coding
sequence for the Gal4 activation domain (GAL4A) with that for
amino acids Asp78-Ile1389 of hBRM. Fusion of
the RB pocket domain (Asn301-Lys928) with the
Gal4 DNA-binding domain (GAL4D) was achieved by inserting an RB
cDNA fragment with a flush EcoRI end and a cohesive
BamHI end into the pAS2 vector with a flush NcoI
end and a cohesive BamHI end. The p107 pocket domain fusion
(Arg337-His1068) was created by inserting a
flush-ended BsiI/KpnI cDNA fragment into the
SmaI-digested vector pAS2, and the p130 pocket domain fusion
(Leu322-His1139) was created by inserting a
flush-ended XbaI fragment into pAS2 with flush
NcoI ends. Double transfectants of Saccharomyces
cerevisiae strain Y190 verified for hybrid expression
through immunoblotting were analyzed for
-galactosidase activity as
a measure of GAL1-lacZ expression resulting from the
interaction between the GAL4A-hBRM hybrid and the GAL4D fusions.
Plasmids--
Expression vectors and cDNA for expressing
mutant cyclin D1, RB, HA-RB*(C706F), p107, p130, GR, hBRM, and E2F-1,
and the reporter construct DHFRpro-CAT have been previously described
(2, 4-7, 14, 22, 30-32). The GR mutant (R479D/D481R) was created with the TransformerTM site-directed mutagenesis kit
(CLONTECH). The GRE-CAT reporter was created by
placing a 180-base pair synthetic DNA fragment carrying an
appropriately positioned single GRE (20, 33) upstream of the coding
sequence for CAT. The RB
(1) expression plasmid was created by
replacing the RB coding sequence upstream of the unique
EcoRI restriction site with the codon for methionine. The coding sequences for the chimeric proteins p107(1-384)RB(373-928), RB(1-372)p107(385-1058), and RB(1-372)p130(417-1139) were
created via polymerase chain reaction fragment assembly.
hBRM-HA-H6 was created with the mutagenesis kit to have the
sequence YPYDVPDYAHHHHHH following the native C terminus of hBRM. All
constructions were verified by DNA sequence analysis.
Cell Culture and Transfections--
All cells were grown as
monolayers in Dulbecco's modified Eagle's medium with 10% fetal
bovine serum under 5% CO2. Transfections were performed
with 2 × 106 cells on 10-cm dishes, a total of 25 µg of DNA, and the LipofactAMINE reagent (Life Technologies, Inc.)
for 5 h in serum-free medium. Transfection efficiencies were
monitored and normalized across experiments by including 0.5 µg of
the constitutive RSV-luciferase reporter and analyzing for
luciferase activity with the Promega kit. 48 h after transfection,
stable transfectants were selected with 400 µg/ml gentamicin sulfate
and/or 100 µg/ml hygromycin B (Life Technologies, Inc.), and well
isolated colonies were recloned twice through limiting dilutions before
expanding the cultures. Where appropriate, GR was induced 24 h
after transfection by treating cells with 10 nM
dexamethasone for 24 h (transactivation assays) or 48 h
(apoptosis assays).
Cell and Nuclear Extracts, Immunoblotting, and
Immunoprecipitation--
Cells or isolated nuclei (34) were first
lysed in extraction buffer (22) and clarified. For immunoprecipitation,
extracts, with 100 µg of protein were precleared in extraction buffer
with 5% (w/v) protein A-Sepharose beads for 30 min and then incubated with 5 µg of the appropriate antibodies for 2 h at 4 °C
followed by 1 h with 5% protein A-Sepharose beads. The beads were
then washed extensively with extraction buffer before analysis.
For immunoblotting, equal amounts of protein were fractionated in each
track by SDS-polyacrylamide gel electrophoresis (35) and electroblotted
onto nitrocellulose membranes. The membranes were blocked with
phosphate-buffered saline, 0.1% Tween 20, 10% horse serum and
incubated with antibodies for 1 h at 4 °C. After extensive
washing with phosphate-buffered saline, 0.1% Tween-20, the retained
antibodies were detected with Enhanced Chemiluminescene reagents
(Amersham Pharmacia Biotech). Antibodies specific for hBRM (H290) have
been described before (22), and those specific for GR (P-20), RB
(C-15), p107 (C-18), p130 (C-20), actin (I-19), and HA (F-7) were from
Santa Cruz Biotechnology.
CAT and Caspase-3 Activity Assays--
CAT activity was
determined using both the chromatographic and the phase extraction
assays as previously described (34). For caspase-3 activity, monolayers
of cells on 15-cm dishes were harvested, processed and analyzed with
the Fluorometric CaspACETM Assay System (Promega) according to the
manufacturer's instructions. For both activities, results from a
minimum of three independent experiments, each performed in triplicate,
were normalized to transfection efficiencies where necessary and
reported as mean values with sample S.D. error bars.
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RESULTS |
Establishment of the Glucocorticoid-responsive Test System--
We
first established a glucocorticoid-responsive system that would enable
us to selectively introduce the factors of interest. Several subclones
of the cervical carcinoma cell line C33A were analyzed by Northern
blotting and immunoblotting and were found not to express GR,
full-length nuclear RB, or hBRM nor to exhibit glucocorticoid
receptor-dependent gene expression as measured with the CAT
cDNA placed downstream of a single GRE in the presence of the GR
inducer, dexamethasone. These cells were then transfected with a
neomycin-selectable mammalian expression vector carrying the GRE-CAT
fragment and a wild type or a dimerization defective mutant GR cDNA
downstream of the CMV promoter. Stable transfectants were screened for
low level expression of the receptor by immunoblotting and for the
contiguity of the integrated GRE-CAT element by polymerase chain
reaction analysis. The two clonal transfectants reported in this
study, C33A/GR and C33A/GR*, exhibited approximately equivalent levels
of wild type and mutant GR, respectively, but were still RB
/hBRM
. The mutant GR* carries two amino
acid substitutions (R479D/D481R) in the dimerization loop within the
DNA-binding domain, which leaves it with little or no transactivation
potential but capable of glucocorticoid-induced transcriptional
repression of specific genes (36). Accordingly, C33A/GR, but not
C33A/GR*, was able to support expression of GRE-CAT when induced with
10 nM dexamethasone (Fig.
1A). A similar system was also
established with subclones of the osteosarcoma cell line SAOS-2 that
were GR
/RB
but hBRM+. However,
in addition to the GR expression vector, these cells were also
transfected with a hygromycin-selectable mammalian expression vector
carrying a mutant cyclin D1 cDNA. The expression of mutant cyclin
D1 overcomes the growth arresting effects of RB overexpression in
SAOS-2 cells without other obvious effects (32). The stable double
transfectants reported here, SAOS-2 [cycD1mut/GR] and SAOS-2 [cycD1mut/GR*], were confirmed to express low levels of GR or GR*,
respectively, with no detectable expression of hBRM or RB. The validity
of using caspase-3 activity as a measure of apoptosis in these cell
lines was established by inducing cell death with etoposide,
staurosporin, or paxlitaxel and relating caspase-3 activity to the
fragmentation of chromatin as determined by DNA laddering assays and
the appearance of subdiploid DNA content as determined by flow
cytometry. With all three inducers, changes in caspase-3 activity
paralleled flow cytometry and DNA laddering data, had the added benefit
of high precision and reproducibility.

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Fig. 1.
Effect of RB and hBRM on GR-induced
apoptosis. A, C33A/GR, which expresses wild type GR,
but not C33A/GR*, which expresses the dimerization and transactivation
defective mutant GR*, exhibits glucocorticoid-responsive
transcriptional activation. B-E, glucocorticoid-induced
apoptosis requires RB and hBRM but not GR-mediated transcription.
48 h after dexamethasone treatment of cells transfected with the
indicated proteins, apoptosis was assessed by measuring caspase-3
activity in the extracts of C33A/GR, C33A/GR*, SAOS-2 (cycD1mut/GR),
and SAOS-2 (cycD1mut/GR).
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GR-dependent Apoptosis Requires RB and
hBRM--
Because GR-mediated transcription is enhanced by both RB and
hBRM (14, 22) and GR has been shown to induce apoptosis (28), we
examined whether GR-induced apoptosis was similarly influenced. To
determine this, cells were transfected with expression vectors for wild
type or mutant RB and/or hBRM, allowed to recover for 24 h, and
then treated with 10 nM dexamethasone for 48 h. Using C33A/GR cells, treatment with dexamethasone yielded no detectable apoptosis when RB or hBRM were present alone (Fig. 1B,
lanes 1-3). However, when RB and hBRM were both present,
significant apoptosis was detected as evidenced by the almost 10-fold
increase in caspase-3 activity (lane 4). When the
interaction between RB and hBRM was abrogated by introducing hBRM with
a defective LXCXE motif (lane 6) or RB with a pocket domain
disabling mutation (lane 7), then apoptosis was not
detected. The apoptosis detected here was clearly mediated by GR
because it was not observed in the absence of the GR inducer
dexamethasone (compare lanes 4 and 5) or with
cells of the GR-negative parental line C33A (data not shown). These results suggest that GR-mediated apoptosis requires the presence of
both RB and hBRM and is dependent on their interaction through the
pocket domain of RB and the LXCXE motif of hBRM.
Interestingly, the transfectant line C33A/GR*, which expresses the GR
(R479D/D481R) double mutant incapable of dimerization or
transactivation, also yielded results similar to C33A/GR with respect
to apoptosis. Specifically, dexamethasone treatment of C33A/GR* induced
apoptosis but only when both functional RB and hBRM were also present
(Fig. 1C). Hence, it can be inferred from these results that
GR-mediated apoptosis is clearly independent of GR-induced
transcription. These conclusions were also borne out by results
obtained with the osteosarcoma clones SAOS-2 (cycD1mut/GR) and SAOS-2
(cycD1mut/GR*) (Fig. 1, D and E). The only
difference that these cells yielded was that exogenous hBRM was not
necessary for GR-mediated apoptosis presumably because endogenous
levels were sufficient. Taken together, these results show that
GR-mediated apoptosis is dependent on the presence and mutual
interaction of RB and hBRM but does not require GR-mediated transcription.
p107 and p130 Interact with hBRM but Do Not Affect
GR-dependent Apoptosis and Transcription--
Because hBRM
binds to the pocket domains of RB that are conserved in the RB
homologues, p107 and p130, we investigated whether hBRM could also
interact with them. This was tested within the nuclear environment,
where hBRM and the RB family members normally reside and function,
using the yeast two-hybrid system (29). The bait for interaction was
formed by the fusion of GAL4D with the pocket domains of RB, p107 and
p130. These were coexpressed in S. cerevisiae strain Y190
with the prey hybrid, formed by fusing GAL4A to an almost full-length
hBRM (D78-I1389) that carries the RB-binding LXCXE motif but lacks the
bromo domain. Interactions between the hBRM and pocket domain fusions
were assessed by indirect measurement of GAL1-lacZ
expression. The results in Table I
demonstrate that, similar to RB, pocket domain fusions of p107 and p130
are able to interact with hBRM and that these interactions exhibit an
avidity that is of the same order as the hBRM-RB pocket domain interaction. Our results here are slightly different from a similar study reported earlier where no interaction was detected between p130
and hBRM (37), but it should be noted that only the C-terminal portion
of hBRM was used for interaction analysis in that study. The data in
Table I also show that this interaction is independent of the
N-terminal regions of the RB family members as these are absent in the
hybrids. This interaction was also assessed through coimmunoprecipitation of endogenous hBRM and RB family members from
nuclear extracts of HeLa, a human cervical carcinoma line. When
approximately a third of the hBRM was immunoprecipitated (Fig.
2A, first row),
about 15% of endogenous nuclear RB was also retrieved (second
row). In contrast, the transiently overexpressed HA-RB*(C706F)
mutant with a defective pocket domain was not detected in the
immunoprecipitates (row 3), verifying the specificity of the
assay. When the hBRM immunoprecipitates were analyzed for p107 and
p130, about 10% of each was consistently detected (rows 4 and 5).
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Table I
Interaction of hBRM with members of the RB family
Double-transfected yeast cells in the two-hybrid system were analyzed
for -galactosidase activity as a measure of GAL1-lacZ
expression resulting from the interaction between the GAL4A-hBRM hybrid
and the GAL4D fusions. The values are expressed relative to the
GAL4D/GAL4A double transfectant.
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Fig. 2.
All members of the RB family interact with
hBRM, but only RB supports GR-mediated transcription and
apoptosis. A, hBRM was immunoprecipitated from nuclear
extracts of untransfected (rows 1, 2,
4, and 5) or HA-RB*(C706F) overexpressing
(row 3) HeLa cells and the coretrieval of associated
proteins assessed by immunoblot analysis. B-D, SAOS-2
(cycD1mut/GR) cells transiently expressing the indicated factors were
treated with dexamethasone and extracts analyzed for GR-mediated
transcription by measuring the CAT activity resulting from the
expression of the chromosomally integrated GR-responsive reporter
construct GRE-CAT, the functional viability of the transfected RB
family proteins through their repression of E2F activity as reflected
by the expression of the E2F-responsive construct
DHFRpro-CAT, and the extent of glucocorticoid-induced
apoptosis as measured by caspase-3 activity.
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Because hBRM can also interact with p107 and p130, we next tested
whether these interactions were productive in affecting GR-dependent transcription and apoptosis. SAOS-2
(cycD1mut/GR) cells were transfected with expression vectors for RB,
p107, or p130, allowed to recover, and then treated with 10 nM dexamethasone to induce the GR. When the extracts of
cells treated for 24 h were analyzed for GR-mediated expression of
GRE-CAT, it was clear that unlike RB (Fig. 2B, lane
2), neither p107 nor p130 affected this activity in any way
(lanes 3 and 4). The abundant expression of all
three members of the RB family was assessed in parallel experiments by
cotransfecting with a vector for E2F-1 expression and the
E2F-responsive reporter DHFRpro-CAT. The heterologous expression of RB, p107, or p130 reduced the E2F-dependent
expression of DHFRpro-CAT (Fig. 2C),
indicating the overabundance of the respective proteins with functional
pocket domains capable of binding and inhibiting the transcription
factor E2F. When cells were treated with dexamethasone for 48 h
and analyzed for apoptosis by measuring caspase-3 activity in the cell
extracts, a similar profile of effects was observed. Again, unlike RB
(Fig. 2D, lane 2), neither p107 nor p130 (lanes 3 and 4) yielded any detectable apoptosis in the dexamethasone treated
GR+ cells. Thus, although p107 and p130 are capable of
interacting with hBRM, unlike RB, they are incapable of affecting
GR-mediated transcription or apoptosis.
Requirement for the N-terminal Domain of RB--
Although RB,
p107, and p130 exhibit extensive homology over the C-terminal
two-thirds where the pocket domains are located, RB differs
significantly from p107 and p130 at the N-terminal regions (4-7).
Because RB is the only member of this family that affects
GR-dependent transcription and apoptosis, we tested whether the divergent N-terminal domain of RB could account for this
distinguishing ability. Through a convenient deletion of the RB
expression vector, we were able to express the mutant form
RB
(1-300) (Fig. 3A) that lacks the first 300 amino acids. This removes 80% of the divergent N-terminal region of RB. When RB
(1-300) was expressed in SAOS-2 (cycD1mut/GR) cells, it was totally incapable of potentiating dexamethasone-induced expression of the GRE-CAT reporter (Fig. 3C, lane 3). The presence of abundant protein
with a functional pocket domain was confirmed by comparative
immunoblotting (Fig. 3B, lane 3) and by the
suppression of E2F-dependent DHFRpro-CAT expression in cells expressing RB
(1-300) (Fig. 3D,
lane 4). When these cells were analyzed for GR-inducible
apoptosis, it was clear that RB
(1-300) could not support this
activity either (Fig. 3E, lane 3). This clearly
established that the N-terminal domain of RB is necessary for its
effects on GR-dependent apoptosis and transcription but
apparently dispensable for its regulation of E2F activity. To map the
exact region within the 372-residue N-terminal domain responsible for
the effects of RB on GR activities, we performed incremental deletions
nested at either end of this region (Fig. 3A). After
transfection of the relevant expression vectors into SAOS-2
(cycD1mut/GR) cells, these deletion mutants of RB were confirmed to be
transiently expressed in abundance (Fig. 3B) and localized
to the nucleus by immunofluorescent analysis (data not shown). In
dexamethasone-treated cells, deletion of the first 20 N-terminal amino
acid residues did not diminish the potentiation of GRE-CAT by RB but
instead yielded a slight, but consistent, enhancement of the
potentiation (Fig. 3C, lane 4). However, deletion
of the N-terminal 40 residues or the 20 residues preceding the pocket
domain totally abrogated the transcription potentiating capability of
RB (lanes 5-7). All the deletion mutants, however, retained
the ability to suppress E2F activity (Fig. 3D), indicating
that the pocket domains of these RB species are functionally viable.
The profile of activities of the RB deletion mutants on GR-dependent transcription was mirrored by their ability to
support GR-induced apoptosis but with one difference. In this assay,
all the deletion mutants tested failed to support GR-induced apoptosis, including RB
(1-20), which exhibited an enhanced potentiation of
GR-dependent transcription (Fig. 3E). Similar
results were obtained with C33A/GR when these mutants were coexpressed
with hBRM (data not shown). Thus, these results clearly show that an intact N-terminal domain of RB is essential for GR-induced apoptosis or
for the potentiation of GR-dependent transcription.
Furthermore, the differential effects of RB
(1-20) indicate once
again that GR-induced apoptosis can be dissociated from GR-mediated
transcription.

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Fig. 3.
The N-terminal domain of RB is necessary for
GR/GR*-induced apoptosis and GR-mediated transcription.
A, schematic representation of the RB mutants created by
deleting various parts of the N-terminal domain. B, the
expression of the RB mutants in transiently transfected SAOS-2
(cycD1mut/GR) cells was assessed through immunoblot analysis of nuclear
extracts with antibodies specific for the C pocket of RB. C,
the effect of the RB mutants on GR-mediated transcription was
determined by measuring the expression of the GR-responsive reporter
GRE-CAT. D, the viability of the pocket domains of the RB
mutants was demonstrated by their ability to inhibit E2F activity as
measured by the expression of DHFRpro-CAT. E,
glucocorticoid-induced apoptosis in the presence of the RB mutants was
determined by measuring caspase-3 activity.
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The N-terminal Domain of RB Functions with the Pocket Domains of
p107 and p130--
Because the N-terminal domain of RB is necessary
for GR-induced apoptosis and GR-dependent transcription, we
tested whether the divergent N-terminal domains of p107 and p130
alone account for their inability to affect the GR activities. Through
in vitro mutagenesis, the N-terminal domains of p107 and
p130 were replaced with the corresponding 372 residues from the
N-terminal domain of RB to form RB(1-372)p107(385-1058) and
RB(1-372)p130(417-1139), respectively (Fig.
4A). Similarly, the N-terminal
region of RB was replaced with the corresponding sequence of p107 to
form p107(1-384)RB(373-928). These chimeric molecules were then
expressed in SAOS-2 (cycD1mut/GR) and verified for abundant nuclear
expression and the possession of viable pocket domains by testing for
their capability to suppress E2F activity (data not shown). When cells
were treated with dexamethasone, the chimera p107(1-384)RB(373-928)
exhibited no potentiation of GR-mediated expression of GRE-CAT (Fig.
4B, lane 3). This indicated that the
corresponding domain of a close homologue cannot functionally replace
the N-terminal domain of RB. In contrast, the chimera formed by the
N-terminal domain of RB and heterologous pocket domains,
RB(1-372)p107(385-1058) and RB(1-372)p130(417-1139), achieved up to
5-fold potentiation of GR-dependent transcription (lanes 5 and 7). Similar results were obtained
for GR-induced apoptosis in that p107(1-384)RB(373-928) failed to
induce any apoptosis (Fig. 4C) whereas
RB(1-372)p107(385-1058) and RB(1-372)p130(417-1139) clearly
promoted cell death (lanes 5 and 7). These
effects of RB(1-372)p107(385-1058), RB(1-372)p130417-1139), and
p107(1-384)RB(373-928) were also observed with C33A/GR cells
transiently expressing hBRM (data not shown). However, in SAOS-2
(cycD1mut/GR*) and hBRM supplemented C33A/GR* cells that express mutant
GR*, apoptosis but not transactivation was induced by the
expression of RB(1-372)p107(385-1058) and RB(1-372)p130417-1139) but not by p107(1-384)RB(373-928) (data not shown). This established that the N-terminal domain of RB possesses the ability to affect GR-dependent apoptosis and transcription and also that this
domain can confer p107 and p130 with the same capabilities when fused to their functional pocket domains.

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Fig. 4.
The N-terminal domain of RB renders the
pocket domains of p107 and p130 capable of supporting
GR-induced apoptosis and transcription. A, schematic
representation of the chimeric molecules created by shuffling the
N-terminal domains of RB, p107, and p130. B, total extracts
of cells transiently expressing the wild type or chimeric molecules
were analyzed for the expression of the cotransfected GRE-CAT as a
reflection of GR-mediated transcription. C, the extent of
apoptosis after 48 h of dexamethasone treatment of the transfected
cells was gauged by the caspase-3 activity.
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The N-terminal Domain of RB Enhances GR-hBRM Binding--
We next
investigated the molecular basis for the effect of the N-terminal
domain of RB on GR-dependent apoptosis and transcription. Using the yeast two-hybrid assay, we found that the N-terminal domain
of RB did not directly interact with GR in the presence or absence of
dexamethasone (data not shown) in agreement with a previous in
vitro analysis (38). The screening of Gal4 activation domain
fusion cDNA libraries derived from several human tissues, with a
bait comprising the N-terminal domain of RB fused to the Gal4
DNA-binding domain, also failed to identify any proteins that might
interact with the N-terminal domain. Nevertheless, the possibility
remained that the N-terminal domain of RB interacted with a factor(s)
with too low an avidity to be detected by the screen.
We reasoned that if the N-terminal domain of RB did indeed interact
with a factor(s) in affecting GR-dependent apoptosis and transcription, then the presence of excess amounts of this domain should interfere with the normal function of RB. We introduced this by
expressing the mutant RB*(C706F), which carries a nonfunctional pocket
domain but an intact N-terminal domain. This would serve to provide
excess N-terminal domain because RB* fails to achieve pocket
domain-dependent interactions (39). Using SAOS-2
(cycD1mut/GR) cells, the potentiation of GR-mediated
transcription was established with nonsaturating amounts of
RB (Fig. 5A, compare
lanes 2 and 3), before increasing amounts of RB*
were coexpressed. As expected, RB* itself failed to potentiate
GR-mediated transcription (lane 4). As shown, the presence
of increasing and excess amounts of RB* (lanes 5-7) also
failed to affect the potentiation achieved by RB (lane 3).
Similarly, the coexpression of the N-terminal domain alone with a
nuclear localization signal and an epitope tag in excess amounts also
failed to affect the potentiation of GR-mediated transcription by RB
(data not shown). This further suggests that the N-terminal domain of
RB is unlikely to interact with other factors because excess amounts of
this domain do not interfere with the effects of RB. This was also
borne out by similar experiments focusing on GR-induced apoptosis (Fig.
5B) where overexpression of RB* failed to affect the
RB-dependent cell death (compare lanes 5-7 with
lane 3). Similar results were obtained with C33A-H41 cells,
derived from a clonal transfectant of C33A that stably expresses low
levels of wild type GR (data not shown) and hBRM tagged at the C
terminus with a hemagglutinin (HA) epitope and a hexahistidine motif
(hBRM-HA-H6) (Fig. 5C, lanes 2 and
3). Taken together, this suggests that the N-terminal domain
of RB does not, by itself, interact with other factors that are
involved in the hBRM-dependent effects of RB on GR
activities.

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Fig. 5.
The N-terminal domain of RB does not
independently recruit or exclude factors but enhances the interaction
of GR and GR* with hBRM. A and B,
GR-mediated transcription (A) and GR-induced apoptosis
(B) in dexamethasone treated SAOS-2 (cycD1mut/GR) cells
expressing suboptimal levels of RB and increasing amounts of the pocket
domain mutant RB*. C, cloned C33A transfectants H41 and H3,
expressing GR and GR*, respectively, were confirmed to express low
levels of hBRM-HA-H6 through comparative immunoblotting of
total cell extracts from the indicated cell lines with hBRM specific
antibodies. Actin-specific antibodies indicate that comparable total
protein was analyzed. D, the effect of RB and RB (1-300)
on the incorporation of GR into the hBRM complex. C33A-H41 and C33A-H3
cells were transfected as indicated and treated with dexamethasone.
Proteins complexes immunoprecipitated with HA epitope specific
antibodies from total cell extracts were then analyzed by immunoblot
analysis with antibodies against hBRM, RB, and GR.
|
|
Because the N-terminal domain of RB functions in an
hBRM-dependent manner, we questioned whether it affected
the interaction of GR and hBRM. C33A-H41 cells were transfected with
expression vectors for RB and RB
(1-300), allowed to recover, and
exposed to dexamethasone for 24 h. Nuclear extracts prepared from
these cells were then immunoprecipitated with HA-specific antibodies to
isolate the complex containing hBRM-HA-H6. The precipitate was then analyzed for hBRM-HA-H6, RB, and GR. By
immunoblotting with antibodies specific for the C-terminal portion of
RB (Fig. 5D, second panel), both full-length RB
(lane 3) and RB
(1) (lane 4) were
detected in the hBRM-HA-H6 complex in approximately similar amounts, indicating that both species interacted equally well with
hBRM. With GR specific antibodies (Fig. 5D, third
panel), the amount of GR detected in the precipitate from
untreated cells (lane 1) was increased by 11-fold in
dexamethasone-treated cells (lane 2), indicative of the
expected ligand-dependent interaction between the receptor
and hBRM. However, when RB was transiently expressed, the amount of GR
detected was increased by another 5-fold (lane 3). This was
not observed with the expression of RB
(1-300) (lane 4).
This analysis was repeated with the cell line C33A-H3, which expresses
low levels of both hBRM-HA-H6 (Fig. 5C,
lane 3) and the transcription-defective, but repression- and apoptosis-capable double mutant GR*. When hBRM-HA-H6
immunoprecipitates were analyzed for the presence of GR* (Fig.
5D, fourth panel), it was clear that GR* also
exhibited a ligand-dependent interaction with hBRM (compare
lanes 1 and 2). Interestingly, the presence of RB
(lane 3), but not RB
(1) (lane 4), also
increased the amount of coimmunoprecipitated GR* by almost 3-fold.
These results demonstrate clearly that the presence of wild type RB
significantly increases the stable incorporation of GR or GR* into the
hBRM complex. In addition, the failure of the mutant RB
(1-300) to
enhance ligand-dependent receptor incorporation indicates
that the N-terminal domain of RB is essential for this activity,
although the pocket domain alone is sufficient for stable binding to
hBRM. Thus, RB is likely to achieve its potentiation of GR-mediated
transcription and the facilitation of GR and GR*-dependent
apoptosis by enhancing the incorporation of the nuclear hormone
receptor into the hBRM complex. This capability, which is intimately
linked to the divergent N-terminal domain of RB, functionally
distinguishes it from the other RB family members whose N-terminal
domains are more divergent.
 |
DISCUSSION |
Previous work has shown that the pocket domain of RB binds to the
transcription coactivator hBRM and that this interaction markedly
potentiates GR-mediated transcription (22). In the work reported here,
we investigated whether the other significant activity of GR, namely
glucocorticoid-induced apoptosis, is also similarly affected by RB and
hBRM and whether p107 and p130 are likewise potent modulators of GR
activities. To address these questions in a meaningful way, a
glucocorticoid-responsive cell culture system was established by
transfection to stably express wild type or mutant GR in initially
glucocorticoid refractory RB
cells. This rendered the
cells responsive to the GR inducer, dexamethasone, in terms of
GR-dependent apoptosis and transcription. The latter was
assessed through a chromosomally integrated glucocorticoid-responsive reporter system that likely existed in phased nucleosomes because the
nucleosome disrupting factor, hBRM (16-18), was necessary for its
expression. This mimics the response of endogenous
glucocorticoid-responsive genes within chromatin to the transcriptional
activity of GR. A further validation of our system was provided by the
observation that cells expressing GR*, incapable of transactivation,
were susceptible to GR-induced apoptosis. This is in accordance with other reports indicating that glucocorticoid-induced apoptosis is
independent of GR-mediated transcription (40, 41).
The introduction of RB and/or hBRM into our
RB
/hBRM
glucocorticoid-sensitive system
showed that the interaction between RB and hBRM is necessary for
glucocorticoid-induced apoptosis, regardless of whether this is
mediated by the wild type GR or the transactivation-deficient GR*.
However, neither p107 nor p130 affect the transactivation or apoptotic
activities of GR, although both factors interact with hBRM via their
pocket domains. Through deletion mutagenesis and domain swapping
experiments, it became clear that the N-terminal domain of RB is unique
among the RB family members in being able to affect GR-mediated
transcription and apoptosis. The clearest proof of this comes from the
chimeric molecules, formed by the N-terminal domain of RB and the
heterologous pocket domains of p107 and p130, which are competent in
affecting GR activities. In line with an earlier report (38), we did
not detect any direct interaction between RB and GR that might explain
our observations. However, because hBRM is necessary for the effects of
RB on the transactivation mediated by GR and the apoptosis induced by
both GR and GR*, we focused on the previously documented GR-hBRM
interaction (19). Indeed, we found that RB significantly enhances the
amount of GR, or GR*, that stably associates with hBRM in the nucleus. Moreover, the N-terminal domain of RB is essential for this effect. Hence, the promotion and/or stabilization of the GR-hBRM interaction is
likely to be the mechanism by which RB achieves its
hBRM-dependent effects on GR-mediated transcription and apoptosis.
In the case of GR-mediated transcription, the effect of RB is readily
explained and is consistent with the current understanding of GR
activity. Liganded GR can seek out and bind to its cognate response
elements even if the latter are assembled into phased arrays of
nucleosomes, but this binding does not lead to nucleosomal disruption
(42) and the subsequent initiation of transcription. However, this
occupation of DNA by the receptor attracts the nucleosome disrupting
activity of the hBRM-containing SWI/SNF complex (19, 20), through
ligand-dependent direct interaction between GR and hBRM
(19). This targeted and reversible (43) nucleosomal disruption would
enable other coactivators and the basic transcription machinery to
access the relevant region of DNA template, resulting in transcription
of the glucocorticoid-responsive gene. Hence, the ability of RB to
enhance the interaction of liganded GR with hBRM would in turn promote
and sustain the disrupted form within the context of a reversible
chromatin remodeling reaction and, consequently, increase GR-mediated
transcription. It is noteworthy that this model implicates the
remodeling of the chromatin as the rate-limiting step in
glucocorticoid-induced gene expression.
Rationalizing the effects of RB and hBRM on GR-induced apoptosis is
somewhat less straightforward. The ability of both the wild type GR and
the mutant GR* to effect apoptosis is consistent with earlier
demonstrations that transactivation by the receptor is not necessary,
and instead implicates the repressive functions of the GR (40, 41).
This repression of gene expression by the GR is not well understood, in
part because of the fact that most genes negatively regulated by GR do
not exhibit the classical GRE within their cis regulatory
elements. As such, various modes of action have been proposed for the
repression of gene expression by GR (24). These invoke negative GRE,
which GR binds to achieve direct transrepression as in the case of the
POMC gene (44), composite elements that GR binds together
with other transcription factors as in the case of the proliferin gene
(45), and tethering elements where GR itself does not bind to DNA but
is recruited and secured there through interactions with a bound
factor. The last is exemplified by the transcription factor
AP-1-dependent collagenase gene that is repressed by
liganded GR (46, 47). Significantly, AP-1 activity is associated with
cell proliferation and survival (48), and its repression by GR has been
implicated in GR-dependent apoptosis (40). Hence, it is
altogether plausible that RB and hBRM affect GR-dependent
apoptosis by promoting the transrepressive activity of GR. It is
already known that hBRM can function as a corepressor in partnership
with RB, as evidenced by their role in inhibiting E2F1 activity (12).
It is thus conceivable that GR, and GR*, achieve transcriptional
repression by recruiting hBRM as a corepressor to the regulatory site
of the target gene. The presence of RB, which serves to enhance GR-hBRM
interactions, would lead to a correspondingly stronger repression and
that would in turn manifest as enhanced apoptosis.
The molecular mechanism by which RB enhances the incorporation of GR
into the hBRM-containing complex is not clear, but our data indicate a
crucial requirement for the N-terminal domain of RB in addition to the
pocket domain. We did not detect any factors that might bind
exclusively to the N-terminal domain in explanation of its role, nor
could we relate the potency of the domain to any structural element or
motif. Instead, it is clear that the total structural information of
the domain determines its efficacy because the entire N-terminal domain
is necessary. It may participate in some very specific protein-protein
interactions because it functions only when attached to a functional
pocket domain that presumably brings it into the appropriate
environment through interactions with pocket domain binding proteins.
This environment might possibly involve the other members of the
chromatin remodeling and hBRM-containing SWI/SNF complex with which RB
would be closely associated when bound to hBRM. This interaction might possibly induce or stabilize a conformational state conducive to the
function of the SWI/SNF complex. Alternatively, RB may function as a
bridge to tether other RB-binding factors to the hBRM complex,
analogous to its ability to bind E2F1 and hBRM at the same time (12).
Yet another possibility arises from the fact that the N-terminal domain
of RB participates in its oligomerization (49), and it may thus
function as a multimeric bridge to tether pocket domain binding factors
to the hBRM complex.
In contrast to RB, neither p107 nor p130 exhibited the capacity to
affect GR-mediated transcription or apoptosis. This deficiency arises
from their more divergent N-terminal domains (4-7). This is aptly
demonstrated by the ability of the N-terminal domain of RB to confer
the pocket domains of p107 and p130 with the ability to affect
GR-dependent transcription and apoptosis. Presumably, the
N-terminal domains of p107 and p130 are involved in yet unascertained molecular events that would set these factors apart from RB with respect to cellular or gross physiological function. Investigations to
date have often highlighted the similarities in the molecular properties of the RB family members, with differences being restricted to subtle specifications in interaction properties or cellular effects.
This report provides a basis for functional distinction between RB and
its homologues p107 and p130 based on their differential modulation of
GR activities and defines the structural basis for this distinction.