Differential Effects of Nuclear Receptor Corepressor (N-CoR) Expression Levels on Retinoic Acid Receptor-Mediated Repression Support the Existence of Dynamically Regulated Corepressor Complexes
Mats Söderström,
Annie Vo,
Thorsten Heinzel,
Robert M. Lavinsky,
Wen-Ming Yang,
Edward Seto,
Daniel A. Peterson,
Michael G. Rosenfeld and
Christopher K. Glass
Cellular and Molecular Medicine (M.S., A.V., C.K.G.) and, Howard
Hughes Medical Institute (T.H., R.M.L., M.G.R.), Graduate Program in
Biology (R.M.L.),University of California, San Diego, La Jolla,
California 92093-0651,
H. Lee Moffitt Cancer Center and
Research Institute (W.Y., E.S.), University of South
Florida,Tampa, Florida 33612,Salk Institute for Biology
(D.A.P.), La Jolla, California 92037
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ABSTRACT
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Thyroid hormone and retinoic acid receptors are
members of the nuclear receptor superfamily of ligand-dependent
transcription factors that stimulate the transcription of target genes
in the presence of activating ligands and repress transcription in
their absence. Transcriptional repression by the thyroid hormone and
retinoic acid receptors has been proposed to be mediated by the nuclear
receptor corepressor, N-CoR, or the related factor, SMRT (silencing
mediator of retinoic acid and thyroid hormone receptors). Recent
studies have suggested that transcriptional repression by N-CoR
involves a corepressor complex that also contains mSin3A/B and the
histone deacetylase, RPD3. In this manuscript, we demonstrate that
transcriptional repression by the retinoic acid receptor can be either
positively or negatively regulated by changes in the levels of N-CoR
expression, suggesting a relatively strict stoichiometric relationship
between N-CoR and other components of the corepressor complex.
Consistent with this interpretation, overexpression of several
functionally defined domains of N-CoR also relieve repression by
nuclear receptors. N-CoR is distributed throughout the nucleus in a
nonuniform pattern, and a subpopulation becomes concentrated into
several discrete dot structures when highly expressed. RPD3 is also
widely distributed throughout the nucleus in a nonuniform pattern.
Simultaneous imaging of RPD3 and N-CoR suggest that a subset of each of
these proteins colocalize, consistent with the existence of coactivator
complexes containing both proteins. In addition, a substantial fraction
of both N-CoR and mSin3 A/B appear to be independently distributed.
These observations suggest that interactions between RPD3 and
Sin3/N-CoR complexes may be dynamically regulated.
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INTRODUCTION
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Thyroid hormone and retinoic acid receptors are members of the
nuclear receptor superfamily of ligand-dependent transcription factors
that control diverse aspects of development and homeostasis by
regulating the expression of target genes (1, 2). In addition to
activation of gene expression in the presence of thyroid hormone
(T3) or retinoic acid, respectively, thyroid hormone and
retinoic acid receptors are representative of a subset of nuclear
receptors that repress transcription in the absence of regulatory
ligands (3, 4, 5, 6, 7, 8, 9, 10). This ligand-independent repression function has been
localized to the C-terminal ligand-binding domain (3, 4, 8, 11). Fusion
of the ligand-binding domains of the thyroid hormone or retinoic acid
receptor to the DNA-binding domain of GAL4 results in chimeric proteins
that are capable of repressing transcription from promoters containing
multimerized GAL4-binding sites (8). Addition of activating ligands
switch the ligand-binding domains of the thyroid hormone and retinoic
acid receptors from transcriptional repressors to activators. These
observations suggested that unliganded receptors interact with
corepressor complexes that are replaced by a set of coactivator
complexes that mediate transcriptional activation after the binding of
activating ligands.
Biochemical studies of cellular proteins that were capable of
interacting with the unliganded thyroid hormone and retinoic acid
receptors led to the identification of a 270-kDa protein, termed N-CoR
(nuclear receptor corepressor), that exhibited several properties
suggesting that it might serve a role as a corepressor (12, 13). N-CoR
interacted with the ligand-binding domains of both the thyroid hormone
and retinoic acid receptors and was released from DNA-bound receptors
by T3 and retinoic acid, respectively. Interaction of N-CoR
with the thyroid hormone and retinoic acid receptors required a region
within the N-terminal end of the ligand-binding domain, termed the CoR
box (12, 13). Mutations within this region of the thyroid hormone and
retinoic acid receptors abolished ligand-independent repression,
suggesting that interaction with N-CoR is required for repression
function. The isolation of cDNAs encoding N-CoR indicated that the
primary transcript can be alternatively spliced to generate several
distinct protein products (13, 14). Western blotting experiments
indicated that the major protein products migrate at approximately 270
kDa, consistent with the forms initially identified to interact with
the retinoic acid and thyroid hormone receptors (12, 13). Functional
analysis of N-CoR deletion mutants indicate that it contains two
distinct C-terminal domains required for interactions with nuclear
receptors (14, 15). Fusion of N-CoR to the DNA- binding domain of GAL4
resulted in a chimeric protein that strongly repressed the
transcription of a promoter containing GAL4-binding sites (13).
Analysis of subregions of N-CoR linked to the GAL4 DNA-binding domain
led to the identification of three distinct regions, termed repressor
domains I, II, and III, that possessed intrinsic repressor activity
(Fig. 1
). Finally, overexpression of the C-terminal
domain of N-CoR lacking repression domains I-III relieves repression by
the unliganded thyroid hormone receptor (16). Based on these
observations, N-CoR was proposed to function as a nuclear receptor
corepressor (12, 13).

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Figure 1. Schematic Diagram of N-CoR and N-CoR Deletion
Mutants Used in These Studies
Domains that transfer repression to the GAL4 DNA-binding domain are
indicated as RI, RII, and RIII. Regions that mediate interactions with
mSin3 are indicated as SID1 and SID2. Regions that mediate nuclear
receptor interactions are labeled NRI and NRII. The solid
box at the amino terminus of N-CoR deletion mutants indicates
the presence of an engineered nuclear localization signal. The
solid box at the carboxy terminus of N-CoR indicates an
epitope (FLAG) tag that was used to facilitate immunolocalization
experiments. Plus and minus signs at the right of the
figure indicate whether or not the corresponding proteins localized to
discrete dot structures.
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In addition to N-CoR, expression cloning studies led to the
identification of a related protein, termed SMRT (silencing mediator of
retinoic acid and thyroid hormone receptors) (17, 18). Like N-CoR, SMRT
interacts with the unliganded thyroid hormone and retinoic acid
receptors via conserved nuclear receptor interaction domains and
strongly represses transcription of promoters containing GAL4-binding
sites when linked to the DNA-binding domain of GAL4. Intriguingly, SMRT
appears to lack regions homologous to N-CoR repression domains I and
II, but contains an N-terminal region with significant sequence
similarity to N-CoR repression domain III.
Recent investigations into the mechanisms by which N-CoR might exert
repressive effects have suggested a link with members of the Sin3
family of transcriptional corepressors (34a). Sin3 was initially
identified by genetic screens for suppresser mutations of a Swi 5
defect in yeast (19, 20). Mammalian homologs of Sin3 were subsequently
discovered in the course of investigating mechanisms responsible for
transcriptional repression by bHLHZ proteins of the Mad family (21, 22). Members of the Mad family, which are believed to be involved in
the induction of terminal differentiation in a wide range of cell
types, have previously been demonstrated to act as transcriptional
repressors upon heterodimerization with Max (23, 24, 25, 26, 27, 28, 29). The Mad proteins
contain an N-terminal region that is required for transcriptional
repression. Utilizing this repression domain to identify interacting
proteins, mammalian homologs of Sin3 (mSin3A and mSin3B) were
identified (21, 22).
Genetic studies in yeast have linked transcriptional repression by Sin3
to the global transcriptional regulator RPD3 (30, 31, 32, 33). A human homolog
of RPD3, hdac1, was isolated by virtue of its interaction with
trapoxin, a histone deacetylase inhibitor (34). These observations led
to the demonstration that RPD3 and hdac1 both posses histone
deacetylase activity, suggesting a direct link between histone
deacetylation and transcriptional repression.
Based on the parallels between transcriptional repression by nuclear
receptors and Mad/Max heterodimers, studies have been performed to
determine whether there might be functional interactions between mSin3,
N-CoR, and RPD3. Consistent with this possibility, mSin3 and N-CoR have
recently been found to directly interact in vitro (34a). Two
Sin interaction domains within N-CoR were demonstrated to be capable of
mediating these interactions, an N-terminal SIN interaction domain (SID
1) extending from amino acids 254 to amino acid 312 in repression
domain I and a second domain (SID 2), extending from amino acids 1829
to 1940, in the C terminus of N-CoR (Fig. 1
and Ref. 34a). Evidence
that the interactions between N-CoR and mSin3A/B are relevant to the
repressive activities by nuclear receptors was provided by
microinjection experiments in which cells were injected with antibodies
directed against either N-CoR or the murine Sin3 homologs (34a). These
experiments provided evidence that mSin3A/B and N-CoR were each
required for transcriptional repression by nuclear receptors and by
Mad.
A potential role for RPD3 in repression events was suggested by
immunoprecipitation experiments, in which anti-Sin3A/B antibodies
coprecipitated RPD3 (R. Eisenman, personal communication). Furthermore,
microinjection of anti-RPD3 antibodies relieved repression by the
nuclear receptor ligand-binding domain and by Mad (34a). In concert,
these observations suggested the existence of a corepressor complex
containing N-CoR, mSin3A/B, and RPD3 that is required for
transcriptional repression by at least two distinct families of
transcription factors.
To further examine the possibility that N-CoR, Sin3A/B, and RPD3
function as components of a corepressor complex in cells, we have
performed a series of experiments examining the functional properties
of N-CoR deletion mutants and the subnuclear localization of N-CoR and
RPD3 using specific antibodies. Paradoxically, marked overexpression of
N-CoR relieves, rather than potentiates, the repressive effects of the
unliganded retinoic acid receptor. These observations suggest that
alterations in the stoichiometry of corepressor function result in
inhibition of the repressor complex. At low levels of expression, N-CoR
is widely distributed throughout the nucleus in a nonuniform pattern,
while at high levels of expression, an additional subpopulation of
N-CoR becomes localized to multiple discrete dot structures. This
discrete pattern of localization requires the presence of the N-CoR C
terminus. RPD3 is also widely distributed throughout the nucleus in a
nonuniform pattern. RPD3 and N-CoR appear to partially colocalize,
consistent with the possibility that they are involved in the formation
of a corepressor complex. RPD3 does not colocalize with N-CoR in the
dot structures, however, and RPD3 and N-CoR are independently
distributed throughout much of the nucleus. These observations suggest
that the interactions between N-CoR and RPD3 may be dynamically
regulated and are consistent with biochemical studies of histone
deacetylases in yeast that suggest functionally distinct complexes.
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RESULTS
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A schematic diagram of N-CoR and a series of N-CoR deletion
mutants used in these studies are illustrated in Fig. 1
. Domains
involved in transcriptional repression, nuclear receptor interaction,
and mSin3A/B interaction are indicated. In the case of deletion mutants
lacking the extreme amino terminus, a nuclear localization signal was
incorporated into the expression vector to ensure appropriate targeting
of N-CoR derivatives to the nucleus. In addition, to facilitate
immunolocalization of these various derivatives, an epitope (FLAG)-tag
was engineered into the carboxy terminus.
Functional analysis of N-CoR and N-CoR deletion mutants was initially
performed by assessing their effects on the transcriptional properties
of retinoid acid receptor (RAR)/retinoid X receptor (RXR) heterodimers
bound to the DR1 element present in the CRBPII promoter (Fig. 2
). On this element, the RAR has been shown to
constitutively repress transcription by RXR in CV1 cells (35, 36). We
have previously demonstrated that N-CoR remains bound to RAR on this
element, even in the presence of retinoic acid (12). As illustrated in
Fig. 2A
, several deletion mutants of N-CoR resulted in
ligand-independent derepression of the RAR/RXR heterodimer on the DR1
element, consistent with the possibility that they functioned as
dominant negative inhibitors of endogenous N-CoR function.
Surprisingly, however, overexpression of the full-length N-CoR protein,
rather than potentiating transcriptional repression, also resulted in
increased levels of transcription from the DR1 element (Fig. 2A
). This
effect was dependent on the presence of the DR1 elements because
overexpression of N-CoR or N-CoR 15862453 had no effect on the
thymidine kinase (TK) promoter containing GAL4-binding sites (Fig. 2B
).
One possible interpretation of this paradoxical effect of N-CoR might
be that at high levels of expression, N-CoR titrates out additional
components required for the function of a corepressor complex. To
assess this possiblility, transcriptional activity of the
DR1-containing promoter was evaluated over a wide range of N-CoR
expression by transfecting between 10 ng to 5 µg of N-CoR expression
plasmid. At low levels of N-CoR expression (10100 ng expression
plasmid per well), transcription from the DR1-containing promoter was
reduced, while at 15 µg of N-CoR expression plasmid, transcription
was significantly increased (Figs. 2C
and 3A
). Indeed, when 15 µg
of N-CoR expression plasmids were transfected, there was not only an
increase in basal expression from the DR1-containing promoter, but the
transcriptional response to the RAR-specific ligand, TTNPB, was also
significantly increased (Fig. 3A
). When evaluated on a
promoter containing two copies of the DR5 element present in the
RARß2 promoter, transfection of 15 µg of N-CoR expression plasmid
similarly resulted in a marked increase in promoter activity in the
absence of ligand, consistent with relief of ligand-independent
repression (Fig. 3B
).

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Figure 2. Effects of Overexpression of Full-Length N-CoR and
N-CoR Deletion Mutants on Transcriptional Activity of a DR1-Containing
Promoter
A, Effects of N-CoR and N-CoR deletion mutants on transcription from
the HSV-TK promoter containing three copies of the DR1 response element
from the CRBPII gene. The 3xDR1 TK luciferase reporter plasmid was
transiently transfected with 1 µg of expression vector for each of
the indicated N-CoR deletion mutants, as described in Materials
and Methods. Reporter gene activity was determined 24 h
later. Promoter activity in the presence of overexpressed N-CoR or
N-CoR deletion mutants is expressed relative to the activity of the
3x/DR1-TK-luciferase reporter gene alone. B, Effects of N-CoR and N-CoR
15862453 overexpression on transcription from the HSV-TK promoter
containing five copies of a binding site for the GAL4 DNA-binding
domain. Cells were transfected with 1 µg of the 5xGAL4-TK reporter
and 1 µg of the N-CoR or N-CoR 15862453 expression plasmids and
assayed for luciferase activity 24 h later. C, Influence of the
levels of N-CoR expression on transcriptional activity of the 3xDR1-TK
promoter. Cells were cotransfected with the indicated amounts of N-CoR
expression plasmid and 100 ng of a CBP expression plasmid to increase
basal levels of promoter activity.
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Figure 3. Effects of N-CoR Overexpression on Ligand-Dependent
Transcription from DR1- and DR5-Containing Promoters
Panel A, CV1 cells were transfected with 1 µg of the 3xDR1-TK
promoter and the indicated amounts of N-CoR expression plasmid. Cells
were treated with the RAR-specific ligand, TTNPB, as indicated and
harvested for luciferase activity 24 h later. B, CV-1 cells were
transfected as in panel A, except that the HSV-TK reporter gene
contained two copies of the DR5 response element from the
RARß2 promoter.
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These observations raised the question of whether N-CoR might be
expressed at different levels in specific cell types or during programs
of differentiation. To examine this question, levels of N-CoR
expression were evaluated in embryonic stem (ES) cells by Far-Western
and Western blot experiments (Fig. 4
). Undifferentiated
ES cells exhibited very high levels of N-CoR. Intriguingly, N-CoR
levels were markedly down-regulated during the in vitro
differentiation of ES cells into embryoid bodies after removal of
leukemia-inhibiting factor (LIF). These observations indicate that
N-CoR levels can be dynamically regulated in cells.

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Figure 4. Expression of N-CoR in Undifferentiated ES Cells
and Embryoid Bodies
D3 ES cells were maintained in LIF (control), or in media lacking LIF
for 3 and 20 days, resulting in the formation of embryoid bodies.
Whole-cell extracts were prepared and N-CoR was captured on a GST-RAR
affinity matrix. Proteins bound to RAR were resolved by SDS-PAGE,
transferred to a nitrocellulose membrane, and detected using either
[32P]GST-RAR (left) or a specific anti
N-CoR antisera (right).
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To evaluate the subcellular distribution of N-CoR, indirect
immunofluorescence microscopy was performed using cells transiently
expressing epitope-tagged N-CoR. As illustrated in Fig. 4A
, transient
expression of epitope-tagged N-CoR in CV1 cells revealed a finely
granular nuclear pattern of staining that was excluded from the
nucleolus. In many cells, N-CoR staining was also observed in a number
of discrete dot structures. The accumulation of N-CoR in these
structures appears to be a consequence of high levels of N-CoR
expression, as parallel experiments using antibodies directed against
the N- or C-terminal regions of the endogenous N-CoR in nontransfected
cells exhibited the identical granular staining pattern as exogenously
expressed N-CoR, but did not stain the discrete dot structures (data
not shown). Furthermore, increased levels of N-CoR expression, achieved
by transfection of increasing amounts of N-CoR expression plasmid, were
correlated with an increase in the number and size of these dot
structures (Fig. 5
, panels B-D). A similar pattern of
N-CoR staining was observed in HeLa cells and P19 cells (data not
shown), indicating that localization of N-CoR into these discrete
structures was not cell type-specific. Treatment of cells with retinoic
acid did not alter the staining pattern of N-CoR (data not shown).

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Figure 5. Subcellular Distribution of N-CoR in CV-1 Cells
CV-1 cells were plated on glass coverslips and transfected with
increasing amounts of a vector directing the expression of full-length,
epitope-tagged N-CoR (corresponding to 0.1 to 2.2 µg DNA/10
cm2 of plating surface). Twenty four hours later, the cells
were fixed and prepared for indirect immunofluorescence labeling using
the anti-epitope antibody as described in Materials and
Methods. Panels A through D are images of representative cells
that express progressively higher levels of N-CoR in dot structures.
The immunostaining patterns were exclusively nuclear, and the cell
bodies are not apparent. (Magnification of original images, 100x).
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We next examined the staining patterns of the series of N-CoR deletion
mutants evaluated functionally in Fig. 2
. As illustrated in Fig. 6B
, the N-terminal region of N-CoR (amino acids 11461)
containing repressor domains 1, 2, and 3 was localized to the nucleus
in a finely granular pattern, but failed to stain the dot-like
structures observed for full-length N-CoR. In contrast, a construct
containing C-terminal amino acids from 758 to 2453 exhibited both the
granular and dot-like patterns of staining (Fig. 5C
). Additional
amino-terminal deletion mutants beginning at amino acids 979 and
1586 also exhibited both granular and dot-like patterns of staining
(Fig. 6
, D and E). A further N-terminal deletion to amino acid 2218
resulted in a diffuse pattern of staining in both the cytoplasm and
nucleus. An internal region of N-CoR extending from 1586 to 2211 also
failed to exhibit a dot-like staining pattern. Most of the N-CoR
deletion mutants that accumulated in nuclear dot structures were also
observed to relieve repression. However, relief of repression was also
observed for some N-CoR derivatives that did not localize to nuclear
dot structures. For example, N-CoR 15862211 did not accumulate in dot
structures but was effective at relieving repression. This may reflect
the fact that N-CoR 45862211 retains one of the nuclear receptor
interaction domains (NRI) and would be expected to compete with
endogenous N-CoR for interaction with RAR.

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Figure 6. The C-Terminal Domain of N-CoR Is Required for
Localization to Discrete Nuclear Structures
CV-1 cells were plated on glass coverslips and transfected with vectors
directing the expression of the indicated epitope-tagged N-CoR deletion
mutants. Twenty four hours later, the cells were fixed and prepared for
indirect immunofluorescence labeling. Representative cells are shown.
Staining is exclusively nuclear in panels A-E. Some extranuclear
staining is also observed in panel F. (Magnification of original
images, 100x)
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We next evaluated the subcellular localization of RPD3. As illustrated
in Fig. 7A
, indirect immunofluoresence staining of RPD3
exhibited an exclusive nuclear staining pattern. RPD3 was excluded from
the nucleolus, but was otherwise widely distributed in a nonuniform
pattern. To determine whether RPD3 and N-CoR were colocalized, confocal
laser scanning microscopy was performed. At high image magnification,
RPD3 was localized to many small, diffuse clusters (Fig. 7A
). In
addition to the discrete dot-like structures, N-CoR was also localized
to diffuse clusters, although this pattern was somewhat less distinct
than that observed for RPD3 (Fig. 7C
). Examination of cells exhibiting
low, moderate, and high levels of transfected N-CoR expression revealed
similar patterns of staining, with the exception that the discrete dot
pattern was only observed at higher levels of N-CoR expression (data
not shown). Upon merging the images obtained for N-CoR and RPD3 within
the same optical section, some colocalization of the two antibodies was
observed, as evidenced by the yellow staining pattern (Fig. 7B
). A
significant fraction of the staining for the two proteins did not
colocalize, however, and several regions were observed that stained
brightly for RPD3, but not N-CoR, or brightly for N-CoR, but not RPD3.
Thus, although these observations are consistent with biochemical
experiments indicating that RPD3 and N-CoR interact, they suggest that
a substantial fraction of these proteins reside in separate
compartments within the nucleus.

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Figure 7. Colocalization of RPD3 and N-CoR in the Nucleus
CV-1 cells were plated on glass coverslips and transfected with
epitope-tagged N-CoR (A, B, C) or N-CoR1586-2453 (D, E, F). Cells were
harvested 24 h later and immunostained with anti-RPD3 antisera
(Cy3) and anti-epitope (FLAG) antibody (FITC). Images were collected
using a Bio-Rad (Hemel Hempstead, UK) confocal imaging system linked to
an Axiovert microscope (Carl Zeiss, Thornwood, NY) at an optical
magnification of 63x. The RPD3 signal alone is illustrated in panels A
and D. The N-CoR signal alone from the same optical section is
illustrated in panels C and F. The sections are merged in panels B and
E. Regions in which N-CoR and RPD3 colocalize appear
yellow.
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DISCUSSION
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Transcriptional repression has been demonstrated to be an
important strategy for the regulation of growth, development, and
homeostasis in several experimental systems (37, 38, 39, 40, 41, 42). In the cases of
the retinoic acid and thyroid hormone receptors, the specific
biological roles of their transcriptional repression functions are not
well understood. However, cErb-A, the oncogenic counterpart
of the thyroid hormone receptor, is thought to collaborate with other
oncogenes and promote uncontrolled cell growth by virtue of its
activity as a constitutive transcriptional repressor (5, 7, 43, 44). In
contrast, transcriptional repression by Mad is correlated with growth
arrest and the establishment of differentiated cell phenotypes
(23, 24, 25, 26, 27, 28, 29). Thus, the repressive functions of unliganded thyroid hormone
and retinoic acid receptors and members of the Mad family can be
considered to have opposing effects on programs of growth and
differentiation and are therefore likely to regulate distinct sets of
genes.
Investigation of the mechanisms responsible for transcriptional
repression by the retinoic acid and thyroid hormone receptors led to
the identification of N-CoR and the related factor SMRT, which were
subsequently demonstrated to mediate transcriptional
repression by v-Erb A and Rev-erb (12, 13, 15, 18). In contrast,
mSin3A/B were identified as putative corepressors of Mad (21, 22).
Protein-protein interaction assays and coimmunoprecipitation studies
have recently demonstrated that N-CoR and mSin3 A/B are components of a
corepressor complex that is required for the repression functions
of both Mad and unliganded RAR and thyroid hormone receptor (34a).
Intriguingly, Sin3 has been demonstrated previously to inhibit the
activity of the progesterone receptor in yeast, suggesting that it may
play a more general role in regulating nuclear receptor function (45).
Although direct interactions have not yet been established,
immunoprecipitation of mSin3 A/B or of N-CoR from cells
coprecipitates the histone deacetylase, RPD3. RPD3 appears to be
required for repression by unliganded nuclear receptors and Mad because
microinjection of anti-RPD3 antibodies into cells reverses their
repressive effects (34a).
The link between histone deacetylases and corepressor proteins that
directly interact with Mad and unliganded retinoic acid and thyroid
hormone receptors suggests an attractive mechanism by which these
factors repress the transcription of target genes. It has been well
established that nucleosomes play important roles in both positive and
negative regulation of transcription (reviewed in Refs. 39, 46, and
47). The dynamic effects of nucleosomes on transcription have been
suggested to be due, in part, to the acetylation of lysine residues
present in the amino-terminal ends of the core histones (46). Numerous
studies have demonstrated that nucleosomes containing hyperacetylated
histones are colocalized with actively transcribed regions of the
genome (48, 49, 50). Conversely, histone hypoacetylation is associated with
heterochromatic regions of DNA that are transcriptionally silent (51, 52). The mechanisms by which the state of histone acetylation
influences transcription have not been established, but acetylation of
the N-terminal tails of histones H3 and H4 appears to facilitate the
association of other transcription factors with nucleosomal DNA
(53).
The acetylated state of the core histones is thought to be
determined by the competing activities of histone acetylases and
deacetylases. In this regard it is intriguing to note that
transcriptional activation by liganded nuclear receptors has been
demonstrated to require cAMP response element binding protein (CBP)
and/or p300 (54, 55, 56, 57). CBP and p300 each posses intrinsic histone
acetylase activity (58, 59) and can associate with p/CAF, which also
exhibits histone acetylase activity (60). These observations suggest a
highly symmetric model in which transcriptional repression is mediated
by a complex containing N-CoR, mSin3, and RPD3, which effects local
histone deacetylation. Upon binding of activating ligands, this
deacetylase-containing complex dissociates from nuclear receptors
and is replaced by a coactivator complex containing CBP/p300 and p/CAF,
which acetylates histones and facilitates entry of core transcription
factors.
In the present study, we have evaluated the consequences of
overexpression of N-CoR and N-CoR deletion mutants and have examined
the intracellular distributions of N-CoR and RPD3 in the cell. At low
levels of expression, both N-CoR and RPD3 are distributed in the
nucleus in a nonuniform, finely granular pattern. Based on images
obtained using confocal laser scanning microscopy, a fraction of N-CoR
and RPD3 appear to be colocalized, consistent with biochemical evidence
for their presence in a corepressor complex. However, a significant
fraction of the two proteins also appears to be independently
distributed. These observations suggest heterogeneity in the
composition of corepressor complexes containing RPD3 and/or the
possibility that the formation of mSin3/N-CoR/RPD3 is dynamically
regulated. Biochemical evidence for heterogeneity in histone
deacetylase complexes has been obtained in yeast, in which two
deacetylase complexes have been identified, termed HDA and HDB, that
exhibit different sensitivities to deacetylase inhibitors (61).
Overexpression of several regions of N-CoR involved in nuclear receptor
or mSin3 interaction resulted in derepression of RAR/RXR heterodimers
bound to a DR1 element, consistent with the possibility that they serve
as dominant negative inhibitors of endogenous N-CoR activity.
Surprisingly, marked overexpression of N-CoR relieved
receptor-dependent repression. These observations raised the
possibility that active repression by N-CoR might require a specific
stoichiometry of corepressor components, and that overexpression of
N-CoR might cause redistribution of some of these components away from
the target promoter. Localization of N-CoR by indirect
immunofluorescence revealed that, when expressed at high levels, a
significant fraction of N-CoR was redistributed to discrete dot
structures within the nucleus that were not observed for endogenous
N-CoR. Localization of overexpressed N-CoR to these structures required
an extended region of the N-CoR C terminus, containing the nuclear
receptor and C-terminal mSin3 interaction domains. The redistribution
of N-CoR to these discrete structures is consistent with the
possibility that disruption of corepressor complexes accounts for the
relief of repression by unliganded RAR/RXR heterodimers observed after
N-CoR overexpression. RPD3 was not colocalized to these structures,
consistent with the lack of evidence for a direct interaction between
RPD3 and N-CoR or mSin3. Additional studies localizing nuclear
receptors, mSin3, and other putative components of the corepressor
complex should help resolve the possible function of these discrete
structures.
Studies of N-CoR expression in ES cells revealed very high levels in
undifferentiated cells and marked down-regulation during their
differentation into embryoid bodies. These results are consistent with
in situ hybridization studies that indicate very high levels
of N-CoR mRNA in embryonic tissues. These levels markedly decline
during later stages of development and reach much lower levels in adult
tissues (M. G. Rosenfeld, unpublished observation). Thus, N-CoR levels
appear to be dynamically regulated during development and
differentiation. Such changes in N-CoR expression are likely to have
significant effects on repression mediated by nuclear receptors and
Mad. Intriguingly, the RAR/RXR heterodimer does not constitutively
repress transcription from DR1-containing promoters in several ES cell
lines (62). These observations raise the possibility that high levels
of N-CoR in ES cells may result in derepression, rather than enhanced
repression, if other components of the corepressor complex are
limiting. It will be of considerable interest to determine whether the
marked variations in levels of N-CoR expression result in significant
alterations in patterns of transcriptional repression that might have
important consequences for normal programs of growth and
development.
 |
MATERIALS AND METHODS
|
---|
Construction of N-CoR Deletion Mutants
Two oligonucleotides corresponding to two copies of the FLAG
sequence, including a BamHI site (5') and a
NotI site (3') for cloning, were synthesized (sense:
5'-GCGGGATCCGACTACAAGGACGACGATGACAAGGACTACAAGGACGACGATGA-CAA-GTGAGCGGCCGCGGGCCCAGAG-3')
annealed, digested with BamHI and NotI, fused
in frame at the C terminus of N-CoR, and placed under the control of a
cytomegalovirus transcription unit. Two oligonucleotides corresponding
to a nuclear localization signal (NLS) (PKKKRK) and an ATG start site
(sense: 5'-AGCTTGACCACCATGGGTACCATGCCAAAGAAGAAGAGGAAGGTAC) were
annealed and fused at the HindIII-KpnI sites of
the multiple cloning site of pcDNA 3 (Invitrogen Carlsbad, CA) creating
an open reading frame polycloning site (pcDNA3-NLS). N-CoR 758-2453 was
constructed by fusing the XhoI-NotI fragment of
N-CoR with the pcDNA3-NLS vector digested in the same way. N-CoR
979-2453 was constructed by fusing the NsiI-NotI
fragment of N-CoR with the pcDNA3-NLS vector digested in the same way.
N-CoR 15862453 was constructed by fusing the BamHI
fragment (48717480) of N-CoR with the pcDNA3-NLS vector digested in
the same way and screened for orientation. The FLAG epitope was put in
place by replacing the HindIII-NotI fragment with
the corresponding fragment cut out from full-length N-CoR/FLAG. N-CoR
22182453 was constructed by digesting N-CoR with
HindIII-NotI, filled and fused with Rexp,
digested with BamHI, and filled and placed under the control
of a rous sarcoma virus transcription unit. A nuclear
localization sequence was engineered by synthesizing two
oligonucleotides encoding the NLS and fused in frame at the
NcoI site (6766). N-CoR 11461 was constructed by digesting
N-CoR with SacI and NotI and fused with pcDNA3. A
FLAG epitope was constructed by using oligonucleotides encoding to two
copies of the FLAG sequence and fused in frame at the C terminus. N-CoR
15861817 were constructed by digesting N-CoR 15862453 with
ApaI and religating the vector. N-CoR 15862211 were
constructed by digesting N-CoR with HindIII, filled, and
recut with BamHI. The fragment was ligated into pcDNA3-NLS
digested with BamHI and EcoRV. A FLAG epitope was
put in frame at the N terminus of N-CoR 15861817 and N-CoR 15862211
constructs by using oligonucleotides encoding two copies of the FLAG
sequence, fused in frame at the BamHI site directly after
the NLS signal.
Transient Transfection Assays
Transfections were performed in six-well plates by the calcium
phosphate method as described previously (34). If not otherwise
indicated, each well was transfected with 1 µg reporter plasmid and 1
µg N-CoR constructs. The precipitate was washed away with PBS after
12 h, and the media was replaced. The cells were harvested after
an additional 24 h and assayed for luciferase activity.
Indirect Immunofluorescence Analysis
Cells were grown on coverslips in 6-cm dishes and were
transiently transfected with N-CoR constructs as indicated. Cells were
washed after 12 h, after which the media were replaced, and the
cells were incubated for an additional 24 h. Cells were then fixed
in 2% paraformaldehyde in PBS for 45 min at room temperature, washed
in PBS, and permeabilized with 0.1% Triton X-100 for 10 min at room
temperature. Monoclonal FLAG antibody M2 (Kodak, New Haven, CT) (50
µg/ml in PBS, 1% BSA) was added for 1 h at room temperature.
For double-staining, cells were incubated with monoclonal FLAG antibody
M2 (50 µg/ml) plus rabbit-mRPD3 antisera (1:50 dilution in PBS, 1%
BSA) for 1 h, followed by five washes in PBS, 0.1% BSA.
Fluorochrome-conjugated secondary antibody (anti-mouse fluorescein
isothiocyante (FITC) affinipure F (ab')2 goat IgG diluted
1:150 was applied for 60 min followed by five washes. Double-stained
cells were incubated for 60 min with fluorochrome-conjugated secondary
antibody (anti-mouse FITC affinipure F (ab')2 goat IgG plus
biotinylated anti-rabbit goat antisera (diluted 1:150), washed five
times in PBS-0.1% BSA, and incubated with Cy3-conjugated streptavidin
(diluted 1:1000), followed by five washes in PBS-0.1% BSA.
Confocal Laser Scanning Microscopy
Fluorescent sections were imaged using a Bio-Rad MRC600 confocal
microscope (Hemel Hempstead, UK) equipped with a krypton/argon laser
and coupled to a Zeiss Axiovert 135 M microscope (Carl
Zeiss, Thornwood, NY) as described (63).
Analysis of N-CoR in ES Cells
D3 ES cells were maintained in an undifferentiated
state by culture in the presence of leukemia-inhibitory factor (LIF).
To induce embryoid body formation, ES cells were transferred to
LIF-deficient media and plated at 1 x 104 cells/ml.
Embryoid bodies formed over the next 512 days and exhibited
morphological evidence of hematopoietic development and the development
of contractile cells. Whole-cell extracts were prepared from
undifferentiated ES cells and embryoid bodies as described by Halachmi
et al. (64). N-CoR was purified on a
glutathione-S-transferase (GST)-RAR affinity matrix,
resolved by SDS-PAGE, and transferred to nitrocellulose membranes as
described by Kurokawa et al. (12). N-CoR was detected using
[32P]GST-RAR or anti N-CoR antisera as described
(12).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Fred H. Gage for access to and assistance with
confocal laser scanning microscopy. We also thank Tanya Schneiderman
for assistance with preparation of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Christopher K. Glass, Division of Cellular and Molecular Medicine, University of California, San Diego, Room 217A, Mail Code 0656, 9500 Gilman Drive, La Jolla, California 92093-0651.
M.S. is supported by the Swedish Natural Science Research Counsel
scholarship. M.G.R. is an investigator of the Howard Hughes Medical
Institute. C.K.G. is supported by NIH Grant 5 RO1CA-52599-07.
Received for publication February 21, 1997.
Accepted for publication March 24, 1997.
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