The DNA-Binding and
2 Transactivation Domains of the Rat Glucocorticoid Receptor Constitute a Nuclear Matrix-Targeting Signal
Yuting Tang,
Robert H. Getzenberg,
Barbara N. Vietmeier,
Michael R. Stallcup,
Martin Eggert,
Rainer Renkawitz and
Donald B. DeFranco
Departments of Biological Sciences (Y.T., D.B.D.), Neuroscience
(D.B.D.), and Pharmacology (D.B.D.) University of Pittsburgh
Pittsburgh, Pennsylvania 15260
Departments of Pathology,
Medicine, Surgery, and Pharmacology and The University of
Pittsburgh Cancer Institute (R.H.G., B.N.V.) University of
Pittsburgh School of Medicine Pittsburgh, Pennsylvania 15213
Departments of Pathology and Biochemistry and Molecular
Biology (M.R.S.) University of Southern California Los
Angeles, California 90033
Genetisches Institut der
Justus-Liebig-Universität (M.E., R.R.) D35392, Giessen,
Germany
 |
ABSTRACT
|
---|
Using an ATP-depletion paradigm to augment
glucocorticoid receptor (GR) binding to the nuclear matrix, we have
identified a minimal segment of the receptor that constitutes a nuclear
matrix targeting signal (NMTS). While previous studies implicated a
role for the receptors DNA-binding domain in nuclear matrix
targeting, we show here that this domain of rat GR is necessary, but
not sufficient, for matrix targeting. A minimal NMTS can be generated
by linking the rat GR DNA-binding domain to either its
2
transactivation domain in its natural context, or a heterologous
transactivation domain derived from the Herpes simplex virus VP16
protein. The transactivation and nuclear matrix-targeting activities of
2 are separable, as transactivation mutants were identified that
either inhibited or had no apparent effect on matrix targeting of
2.
A functional interaction between the NMTS of rat GR and the RNA-binding
nuclear matrix protein hnRNP U was revealed in cotransfection
experiments in which hnRNP U overexpression was found to interfere with
the transactivation activity of GR derivatives that possess nuclear
matrix-binding capacity. We have therefore ascribed a novel function to
a steroid hormone transactivation domain that could be an important
component of the mechanism used by steroid hormone receptors to
regulate genes in their native configuration within the nucleus.
 |
INTRODUCTION
|
---|
Physiological effects of steroid hormones are mediated primarily
by the transcriptional regulatory functions of steroid hormone
receptors. These signal transduction proteins are members of a large
superfamily of nuclear receptors that possess highly conserved
zinc-finger DNA-binding domains (DBDs), carboxyl-terminal
ligand-binding domains (LBDs), and multiple transactivation domains (1, 2). Transcriptional regulation of hormonally responsive genes is
brought about either by the direct binding of receptors to specific
response elements linked to target gene promoters (3), or interactions
between receptors and specific transcription factors (4, 5, 6) or
co-activators (7, 8) in the absence of direct receptor-DNA binding.
Three distinct transactivation domains have been identified within
various members of the nuclear receptor superfamily. The
carboxyl-terminal AF-2 transactivation domain is highly conserved
within the nuclear receptor superfamily (9, 10, 11) and is recognized
by various transcriptional coactivators (7, 8, 12). Another
transactivation domain (i.e. AF-1) has been identified
within the amino-terminal region of some nuclear receptors (9, 13, 14).
In contrast to AF-2, the amino-terminal AF-1 transactivation domain in
retinoic acid and retinoid X receptors differs in sequence (15) and
properties from the amino-terminal
1/enh2 transactivation domain of
steroid receptors (9, 13, 14). Finally, a third transactivation domain,
2/AF-2a, has been localized within the amino-terminal portion of
steroid receptor LBDs (7, 9, 16). The mechanism of action of this
transactivation domain has not been elucidated.
Much of the in vivo characterization of steroid
receptor transactivation domains has used templates (i.e. in
transient transfection assays) that do not accurately reflect the
natural organization of steroid-responsive genes within the
nucleus. It has become increasingly apparent that a high degree of
organization exists within the nucleus that restricts distinct
biochemical processes to unique subnuclear compartments (17). The
nuclear matrix, an interconnected network of ribonuclear protein
filaments (18, 19), may play an important role in transcriptional
regulation by providing the framework for both cell type-specific
attachment of active genes (20, 21, 22) and high-affinity binding of
specific transcription factors (23, 24, 25, 26). Although steroid receptors
were the first transcription factors shown to be associated with the
nuclear matrix (27, 28), the physiological significance of this
interaction has not been definitively established.
We had previously used a reversible ATP-depletion paradigm to reveal a
dynamic interaction between glucocorticoid receptors (GRs) and the
nuclear matrix (29). Specifically, GR binding to the nuclear matrix was
found to be dramatically increased upon depletion of cellular ATP pools
(29). GRs rapidly release from the matrix upon restoration of ATP pools
(29). Based upon these results, we hypothesized that while GR has the
capacity to exchange between the nuclear matrix and soluble nuclear
compartments, at least one step in this subnuclear trafficking pathway,
i.e. nuclear matrix release, requires ATP (29).
In this manuscript we have set out to define the minimal segment of rat
GR that is required for its nuclear matrix targeting. Although the DBD
of GRs has been implicated to possess a nuclear matrix targeting signal
(NMTS) (29, 30), we show here that while necessary, the rat GR DBD is
not sufficient for nuclear matrix targeting. A minimal NMTS can be
generated by linking the rat GR DBD to either the
2 transactivation
domain in its natural context, or a heterologous transactivation
domain. Furthermore, we have identified at least one nuclear matrix
protein, hnRNP U, which through direct or indirect interactions with
the GR NMTS, could play a role in directing the receptor to the nuclear
matrix.
 |
RESULTS
|
---|
The Rat GR DBD Is Not Sufficient to Target Receptors to the Nuclear
Matrix
In previous studies, we have used ATP depletion to augment nuclear
matrix binding of GRs (29). The validity of this paradigm to examine
specific targeting of GR to the nuclear matrix was further verified by
the use of two-dimensional gel electrophoresis, which showed that the
composition of the nuclear matrix was not altered by ATP depletion (not
shown). Furthermore, our ATP depletion conditions did not generate a
denaturing environment within the nucleus as the enzymatic activity of
a nuclear targeted ß-gal chimera was indistinguishable in
metabolically active (+ATP) vs. ATP-depleted Chinese hamster
ovary (CHO) cells (not shown).
From the analysis of various GR deletion mutants, we previously showed
that either 407 amino-terminal or 239 carboxyl-terminal amino acids
were dispensable for receptors to associate with the nuclear matrix
(29). However, nuclear matrix binding of LBD-deleted receptors was
dramatically reduced upon deletion of a 38-amino acid segment of the
DBD (29). This result suggested that the rat GR DBD was necessary for
nuclear matrix binding, which was consistent with the results of
independent studies examining the binding of human GR to the nuclear
matrix (30). We therefore set out to reveal whether the rat GR DBD was
also sufficient for nuclear matrix binding using the identical
ATP-depletion paradigm.
In our initial experiments to examine nuclear matrix-binding properties
of the rat GR DBD, we used CHO cells stably transfected with a
DBD-ß-gal chimera. The DBD-ßgal protein localized predominantly
within the nucleus of both metabolically active (not shown) and
ATP-depleted cells (Fig. 1B
). Both
monoclonal anti-ß-gal and anti-GR antibodies were used to detect this
chimera and gave identical results (not shown). The binding of the GR
DBD-ß-gal protein to the nuclear matrix of ATP-depleted cells was
visualized in situ after a nuclear matrix preparation. As
shown in Fig. 1E
, the GR DBD-ß-gal protein was not found to be
associated to an appreciable extent with the nuclear matrix of
ATP-depleted cells. Costaining of extracted cells with the anti-NuMA
antibody (Fig. 1
, A and D) and 4,6-diamidino-2-phenylidole (DAPI) (Fig. 1
, C and F) confirmed the efficiency of the nuclear matrix preparation.
Thus, it appears that while the DBD is necessary for nuclear matrix
binding of rat GR, it is not sufficient.

View larger version (47K):
[in this window]
[in a new window]
|
Figure 1. rGR DBD-ß-gal Is Not Targeted to the Nuclear
Matrix of ATP-Depleted CHO Cells
CHO cells stably expressing a DBD-ß-gal chimera were depleted of ATP
and either directly fixed (AC) or subjected to a nuclear matrix
preparation before extraction (DF). DBD-ß-gal protein was detected
using a polyclonal anti-GR antibody (B and E), while the nuclear matrix
(A and D) and DNA (C and F) were visualized by anti-NuMA and DAPI
staining, respectively.
|
|
The
2 Transactivation Domain along with the DBD of Rat GR
Comprises a NMTS
Transient transfection assays were used to search for a NMTS
within the rat GR. The efficiency of our transient transfection
protocol was reproducibly high (i.e.
25% of transfected
cells staining positive for GR) enabling the use of differential
extractions to visualize nuclear matrix binding of transfected GRs.
Furthermore, extracted and nonextracted samples were always compared in
the same transfection experiment that used a single transfection
mixture for multiple plates of cells. In such transient transfections
we confirmed the inefficient association of the rat GR DBD-ß-gal
chimera with nuclear matrices prepared from both metabolically active
and ATP-depleted cells (data not shown).
We had previously established that in the rat GR, amino acids both
amino terminal and carboxyl terminal to the DBD contribute to nuclear
matrix binding (29). In this report we have focused exclusively on the
region carboxyl terminal to the rat GR DBD to delineate the minimal
amino acids that, together with the DBD, constitute a NMTS. Thus, rat
GR derivatives that were tested for nuclear matrix targeting have been
deleted of amino acids amino terminal of serine 407. Figure 2
depicts the structures of rat GR (rGR)
(and Gal4) DBD derivatives that were used in our studies. The first rGR
segment assessed for nuclear matrix binding possessed a
carboxyl-terminal extension of the rGR DBD to alanine 574. This
truncated receptor (i.e. including amino acids 407574)
possesses an intact DBD linked in its natural context to the
2
transactivation domain (31).

View larger version (17K):
[in this window]
[in a new window]
|
Figure 2. DNA Constructs Used in This Study
rGR (and Gal4) DBD constructs are depicted with amino acid end points
of domains indicated.
|
|
As shown in Fig. 3a
, the DBD of rGR
(i.e. amino acids 407545), although capable of targeting
to nuclei of transiently transfected CHO cells (panel A), was not
associated to a significant extent with the nuclear matrix (panel D).
This is consistent with results obtained with a GR DBD-ß-gal chimera
in stably transfected CHO cells (Fig. 1
). In contrast, a significant
portion of DBD-
2 protein that localized within nuclei of
ATP-depleted CHO cells (Fig. 3a
, panel B) was associated with a nuclear
matrix fraction as revealed by its resistance both to CSK buffer
extraction (Fig. 3a
, panel E) and a complete nuclear matrix preparation
(Fig. 3b
, panel B). The nuclear matrix of extracted cells was
visualized by costaining with the NuMA antibody (Fig. 3b
, panel A).
Thus, the
2 transactivation domain of rGR cooperates with the
receptors DBD to constitute a minimal NMTS.
The
2 transactivation domain alone is not sufficient for nuclear
matrix targeting as a Gal4 DBD-
2 chimera (Fig. 2
) expressed in
transiently transfected CHO cells did not efficiently associate with
nuclear matrix (Fig. 3c
). As will be shown later (see Fig. 7c
), the
Gal4 DBD-
2 chimera functions as an efficient transactivator in
transfected mammalian cells eliminating the possibility that its
inability to associate with the nuclear matrix is simply the result of
it adopting some aberrant protein structure. Thus, the Gal4 DBD, which
alone does not bind to the nuclear matrix (Fig. 3c
and Ref. 26), cannot
substitute for the rGR DBD to constitute a NMTS in combination with the
GR
2 transactivation domain.

View larger version (18K):
[in this window]
[in a new window]
|
Figure 7. Overexpression of hnRNP U Reduces the
Transactivation Activity of GR DBD Derivatives That Associate with the
Nuclear Matrix
a and b, CHO cells were transiently cotransfected with a luciferase
reporter plasmid containing a linked GRE (i.e.
TAT3-luc), a ß-gal reporter plasmid, and where indicated with GR DBD
expression plasmids (i.e. GR-DBD, GR-DBD- 2, or
GR-DBD- 2m in panel a, and GR-DBD or GR-DBD-VP16 in panel
b) and an hnRNP U cDNA expression plasmid. Relative luciferase
activities shown were normalized to correct for differences in transfection efficiency using ß-gal activity
measurements. Results shown are an average of three separate
determinations (± SD), each performed in duplicate. c,
Transient transfections performed and analyzed as described in panels a
and b, except that a Gal4 UAS-linked luciferase reporter
(i.e. (UAS)-TATA-Luc) was used along with Gal4 DBD or
Gal4 DBD- 2 expression plasmids. Results shown are an average of
three separate determinations (± SD), each performed in
duplicate.
|
|
Extensive mutagenesis of the rGR
2 domain has led to the
identification of two amino acids (i.e. Leu553 and Leu554),
which are highly conserved wtihin steroid receptors (31) that are
critical for its transactivation activity (31). As shown in Fig. 3a
(panel F), substitutions of leucines 553 and 554 with glycines in
DBD-
2m (Fig. 2
) abolished the nuclear matrix targeting
of this protein in ATP-depleted CHO cells. Substitution of a highly
conserved serine (31) located at position 573 of rGR with an alanine
residue (i.e. rGR DBD-
2 S573A, see Fig. 2
) also greatly
reduced
2 transactivation activity (31). Interestingly, nuclear
matrix targeting of the rGR DBD-
2 protein was not affected by the
S573A point mutation (Fig. 4
). Thus, the
transactivation and nuclear matrix-targeting activities of
2 are
separable and distinguished by point mutations at distinct, highly
conserved amino acids.
To provide a more quantitative assessment of GR-
2 nuclear matrix
binding, we performed Western blot analyses of various subcellular
fractions prepared from a suspension of transfected CHO cells. GRs in
whole-cell extracts, a deoxyribonuclease I (DNase I)-soluble fraction,
and a nuclear matrix pellet were detected, with the amount of
subcellular fraction loaded per lane corresponding to an equivalent
number of cells. When comparing nuclear matrix binding of GR
derivatives in separate samples, Western blots were costained with the
anti-NuMA antibody to provide an internal standard for nuclear matrix
recovery (Fig. 5a
). As shown in Fig. 5
, a
and b, approximately 30% of DBD-
2 was retained on the nuclear
matrix of ATP-depleted CHO cells, whereas only trace amounts
(i.e. <5%) of DBD and DBD-
2m proteins were
associated with the nuclear matrix. As shown in Fig. 5a
(lanes 2, 5,
and 8) DBD and DBD-
2m proteins were more readily
released from chromatin by DNase I digestion than DBD-
2. These
biochemical fractionations confirm the indirect immunofluorescence
analysis of in situ extracted cells and establish the
nuclear matrix-binding capability of the DBD-
2 protein.

View larger version (40K):
[in this window]
[in a new window]
|
Figure 5. Western Blot Analysis and Quantification of Nuclear
Matrix Binding of DBD- 2
a, CHO cells transiently transfected with DBD (lanes 13), DBD- 2
(lanes 46), and the DBD- 2 mutant (lanes 79) were depleted of ATP
and then subjected to subcellular fractionations. Each lane contains
protein amounts derived from equal numbers of cells. Whole-cell
extracts (lanes 1, 4, and 7), a DNase-released chromatin fraction
(lanes 2, 5, and 8), and a nuclear matrix pellet fraction (lanes 3, 6,
and 9) were subjected to separation by SDS-PAGE and GR derivatives
detected by Western blot analysis. A whole-cell extract from
nontransfected CHO cells was used as a negative control (lane 10).
Western blots were costained with an anti-NuMA antibody to provide an
internal standard for nuclear matrix recovery between individual
samples. b, Quantification of DBD (column 1), DBD- 2 (column 2), and
DBD- 2 mutant (column 3) binding to the nuclear matrix of
ATP-depleted CHO cells. The percent of GR derivative bound to the
nuclear matrix (NM), expressed relative to GR recovered in whole-cell
extracts (W), was determined from Western blot analysis (as shown in
panel a). The percentages shown represent a average of three separate
determinations.
|
|
The VP16 Transactivation Domain Can also Cooperate with the rGR DBD
to Constitute a NMTS
Mutations in the GR DBD diminish nuclear matrix binding (29). In
addition,
2 alone can not target a heterologous zinc finger DBD
(i.e. the yeast Gal 4p) to the nuclear matrix. Thus, it
appears that both the DBD and
2 domains of rGR must be intact to
constitute an efficient NMTS. Multiple mutations within the rGR DBD
that are expected to alter its DNA-binding properties have little
impact on nuclear matrix binding in ATP-depleted cells (data not
shown). Thus, the DNA-binding and nuclear matrix-targeting activities
of the GR DBD are distinct. In contrast, both nuclear matrix binding
and transactivation were dramatically reduced by a double point
mutation within
2. Is the
2 transactivation domain unique in its
ability to impart nuclear matrix binding to the rGR DBD? To address
this question we made use of a chimera (i.e. DBD-VP16) that
contained the Herpes simplex virus VP16 transactivation domain (amino
acids 412454) linked carboxyl terminal to the rGR DBD. As shown in
Fig. 6
, the DBD-VP16 protein was found to
associate with the nuclear matrix of ATP-depleted CHO cells. Thus,
either a homologous (i.e.
2) or heterologous
(i.e. from VP16) transactivation domain linked carboxyl
terminal to the rGR DBD can constitute an effective NMTS.

View larger version (89K):
[in this window]
[in a new window]
|
Figure 6. The VP16 Transactivation Domain Can Also Cooperate
with the rGR DBD to Constitute a NMTS
CHO cells transiently transfected with a DBD-VP16 transactivation
domain chimera were subjected to an ATP depletion and either fixed
directly (A and B) or fixed after a nuclear matrix preparation (C and
D). The GR-VP16 chimera was detected using the BuGR2 anti-GR
antibody.
|
|
The RNA-Binding Nuclear Matrix Protein, hnRNP U, Interferes with
the Transactivation Activity of GR Derivatives That Can Associate with
the Nuclear Matrix
The presence of a specific NMTS within the rGR suggests that the
nuclear matrix may possess a unique receptor(s) for this signal.
Specific "acceptor" proteins have been hypothesized to direct
receptors to the nuclear matrix (28), but such nuclear matrix
"receptors" for the steroid receptors have not been definitively
identified (32). For example, although Spelsberg and colleagues (32)
have identified a nuclear matrix protein (i.e. RBF-1) that
interacts with progesterone receptor, the specific receptor domain
responsible for RBF-1 binding has not been identified. Recently,
Renkawitz and co-workers (33) demonstrated an association between human
GR and the RNA-binding nuclear matrix protein hnRNP U. In addition to
the use of biochemical assays to establish a GR/hnRNP U interaction, a
cotransfection assay was used to establish a functional interaction
between these two proteins in vivo (33). Specifically, in
this cotransfection assay, overexpression of hnRNP U was found to
inhibit GR transactivation from a transiently transfected template
(33). Since transiently transfected templates are not associated with
the nuclear matrix, the results obtained in this cotransfection assay
most likely reflect the titration of GR from transfected templates by
virtue of its direct or indirect association with hnRNP U.
The segment of the human GR LBD that was shown to functionally
associate with hnRNP U (i.e. amino acids 488777) in
cotransfection assays possesses the
2 domain [i.e. amino
acids 525556 (9)]. We therefore examined whether the minimal NMTS
that we identified within rGR, which included
2, functionally
interacted with hnRNP U using an analogous cotransfection paradigm. CHO
cells were cotransfected with GR DBD expression plasmids, a
glucocorticoid response element (GRE)-linked luciferase reporter
plasmid [i.e. TAT3-Luc (14)], and an expression plasmid
encoding full-length hnRNP U cDNA (33). All of our cotransfection
experiments also included an expression plasmid for the bacterial
ß-gal gene to provide an internal control for transfection
efficiency. As shown in Fig. 7a
, cotransfection of hnRNP U cDNA inhibited the transactivation activity
of GR DBD-
2 by 50%. The GR DBD and the DBD-
2m, both
of which exhibited some transactivation activity compared with TAT3-Luc
alone, were unaffected by hnRNP U (Fig. 7a
). Thus, hnRNP U effects were
selective for GR DBD derivatives that exhibited nuclear
matrix-targeting activity.
To further corroborate the relationship between hnRNP U effects on
transactivation and nuclear matrix targeting, we examined whether other
GR derivatives with alternative nuclear matrix-targeting ability were
affected by hnRNP U. While cotransfection of hnRNP U cDNA reduced the
transactivation activity of GR DBD-VP16, which possessed nuclear
matrix-targeting activity (Fig. 7b
), transactivation mediated by the
Gal4 DBD-
2 chimera was insensitive to cotransfected hnRNP U (Fig. 7c
). Thus, minimal GR NMTSs that possess either a homologous or
heterologous transactivation domain functionally interact with hnRNP U,
while separated GR domains that lack NMTS activity (i.e. DBD
or
2) are insensitive to hnRNP U effects.
 |
DISCUSSION
|
---|
The association of various transcription factors with the nuclear
matrix is dynamic and subjected to cell type-specific, cell cycle,
developmental, and hormonal regulation (23, 24, 25, 26). Despite advances in
our understanding of regulated subnuclear trafficking, the identity of
targeting signals that direct proteins to distinct subnuclear
compartments is limited. Unique sequences have been identified that
direct DNA methyltransferase to sites of DNA replication (34) and RS
domain-splicing factors to speckle domains (35). The only reported NMTS
was recently identified within the leukemia- and bone-related
AML/CBF-
transcription factor, AML-1B (26).
Previous studies from our group and others have
suggested that a specific NMTS exists within steroid receptor proteins,
but the precise identity of this signal remained undefined. In
particular, the DBD of human (30) and rat (29) GR, and human androgen
receptor [AR (30)], was implicated in nuclear matrix targeting. We
show herein that the rGR DBD, while necessary, is not sufficient to
target receptors to the nuclear matrix. Rather, the DBD, along with the
2 transactivation domain linked in its native configuration,
constitutes a minimal NMTS for rGR. The
2 domain does not target a
heterologous zinc finger DBD (i.e. from the yeast Gal4p) to
the nuclear matrix when linked to its carboxyl terminus and thus
differs from AML-1B NMTS, which has the capacity to target Gal4p to the
matrix (26). It is not known whether homotypic pairing between the rGR
DBD and
2 domain is strictly required for nuclear matrix targeting,
but this should become apparent from analysis of steroid receptor
chimeras possessing swapped DBD and
2 domains.
The analysis of two
2 mutants that possess minimal transactivation
activity established that the transactivation and nuclear
matrix-targeting activities of
2 are separable. Thus, while the
L553G/L554G double-point mutation abolished both transactivation and
nuclear matrix-targeting activities of
2, the S573A mutation
affected transactivation but not the matrix-targeting function of
2.
Similarly, the NMTS within the AML-1B protein is closely associated
with its transactivation domain, and its nuclear matrix-targeting and
transactivation activities can be uncoupled (26). In a model of the rGR
2 domain based upon the crystal structure of the thyroid hormone
receptor
LBD, L553 and L554 are predicted to be located within a
different
-helix than S573 (31). Thus, distinct surfaces of
2 may
be used to either promote nuclear matrix targeting or interact
productively with coactivators or components of the basal transcription
machinery.
The amino acid sequence of the
2 domain is highly conserved among
other steroid receptors, but not other members of the nuclear receptor
superfamily (31). In particular, the amino acids of rGR that are
particularly important for the nuclear matrix-targeting activity of
2 (i.e. L553 and L554) are either perfectly conserved or
contain a conservative valine substitution at one position (31).
Although the nuclear matrix- targeting properties of other steroid
receptor
2 domains have not been strictly tested, a
carboxyl-terminal deletion of human AR that removes its
2 domain
reduces its association with the nuclear matrix (30). Given the high
degree of conservation between the
2 domains of steroid receptors,
the role of this domain in targeting of all steroid receptors to the
nuclear matrix may not be unexpected.
Do other steroid receptor transactivation domains contribute to nuclear
matrix targeting? In our ATP-depletion paradigm, rGRs lacking
2 but
containing amino-terminal 556 amino acids, were also capable of binding
to the nuclear matrix (29). This suggests that a separate domain within
the amino terminal half of rGR can also function, either alone or in
concert with the GR DBD, to constitute a NMTS. Although the
1/enh2
transactivation domain that resides within steroid receptor
amino-terminal domains is unrelated to
2 in amino acid sequence, its
role in nuclear matrix targeting of receptors cannot be excluded.
Interestingly, the amino-terminal domain of steroid receptors has been
found to influence their DNA- and nuclear-binding affinity (36), as
first revealed by the phenotype of amino-terminal-deleted (37)
nti (i.e. increased nuclear
transport) mutant GRs (38). The fact that mutations within the rGR
amino terminus (37, 38), DBD (29, 30, 39), and LBD (this report)
distinctly impact subnuclear trafficking of the receptors highlights
the importance of considering the appropriate targeting of receptors
when evaluating the phenotypes of receptor transactivation mutants.
Although the
2 domain requires the GR DBD to constitute a NMTS, its
function in nuclear matrix targeting can be supplied by a heterologous
transactivation domain from VP16 of HSV. Both
2 and the
transactivation domain of VP16 have been postulated to form an
amphipathic
-helix. The NMTS of AML-1B has not been modeled in the
same manner but contains a hydrophobic segment interspersed between two
regions rich in hydrophilic amino acids (26). While the
2 and VP16
transactivation domains may share some general structural features,
they differ in the extent of their acidic amino acid character. The
VP16 transactivation domain was initially defined as an acidic
transactivation domain (40), although both hydrophobic and acidic amino
acids have important contributions to its transactivation activity
(41). In contrast, point mutations within four of five acidic amino
acids within the rGR
2 domain have little to no impact on its
transactivation activity (31). As mentioned above, complete loss of
2 transactivation activity results from the mutation of two
conserved leucine residues (31).
How might the NMTS of GR function? The hinge region of steroid
receptors, which includes the
2 domain, has been shown to interact
with the general transcription factor TAFII30 in vitro (42)
and a novel antagonist-specific transcriptional coactivator L7/SPA
in vivo (43). It is unclear whether these interactions are
relevant to the nuclear matrix targeting function of
2. The
interaction between the GR DBD and components of the SNF/SWI complex
(44, 45) may play some role in directing receptors to the matrix as a
fraction of two human SNF/SWI homologs, i.e. hBRM and BRG1
proteins, is associated with the nuclear matrix (46). There may be
multiple nuclear matrix proteins that participate in binding of steroid
receptors utilizing either components of the NMTS that we have
identified or other potential NMTS within receptor amino- or
carboxyl-terminal domains.
Using a cotransfection assay to assess functional interactions, we have
identified at least one nuclear matrix protein, hnRNP U, that may play
a role in targeting GR to the matrix. rGR DBD derivatives that
associate with the nuclear matrix due to a linked
2 domain or VP16
transactivation domain functionally interact with hnRNP U. However,
both the rGR DBD and
2 domain alone, which do not bind to the
matrix, were insensitive to hnRNP U effects in transfected cells. Thus,
the functional interaction between rGR DBD derivatives and hnRNP U was
correlated with nuclear matrix binding of the receptor DBD. Since we
have not determined whether the
2 domain is solely responsible for
functional interactions between hnRNP U and the LBD of GR (33), we
cannot exclude the possibility of associations between hnRNP U and
other regions of the LBD. Any contributions of the receptor
amino-terminal domain to nuclear matrix binding (29) are unlikely to be
mediated by hnRNP U (33).
In addition to its role in RNA processing (47), hnRNP U specifically
binds to matrix/scaffold attachment regions (MARs/SARs) (48, 49). Given
this activity, hnRNP U has also been designated scaffold attachment
factor-A (SAF-A) (48). It is tempting to speculate that the GR NMTS,
through its interaction with hnRNP U/SAF-A, may serve to target the
receptor to regions of the matrix that include target genes poised to
respond to activated receptors and associated coactivators.
Considerable attention has recently been focused on the direct or
indirect role of nuclear receptor transcriptional coactivators in the
modification of histones and resulting alterations in chromatin
structure (50, 51). Curiously, the majority of nuclear histone
acetyltransferase activity appears to be associated with the nuclear
matrix (52). Thus, the appropriate subnuclear compartmentalization of
transactivators, such as nuclear receptors, and their partner
coactivators (7, 8) may play more of an essential role in
transcriptional activation than previously appreciated.
 |
MATERIALS AND METHODS
|
---|
Plasmids
Figure 2
depicts the structures of most plasmids that were used
in this study. The DBD-ß-gal expression plasmid possesses the rGR DBD
(i.e. amino acids 407545) fused at its carboxyl terminus
to the bacterial ß-galactosidase (ß-gal) gene (53). Amino acids
407556 of the rGR were fused to the transactivation domain of the
Herpes simplex virus VP16 protein (amino acids 413454) to generate
the expression plasmid DBD-VP16 (kindly provided by J. A.
Iniguez-Lluhi and K. R. Yamamoto). Expression plasmids encoding
the rGR DBD and a linked
2-transactivation domain were described
previously (31). These plasmids possess rGR DBD sequences from amino
acid 407545 (i.e. DBD) or 407574 (i.e.
DBD-
2, DBD-
2m, and DBD-
2(S573A)). In
DBD-
2m, leucines 553 and 554 are substituted by glycine
residues while in DBD-
2(S573A) serine 573 is substituted by an
alanine (31). The pM2 plasmid (54) was used to express the yeast
transcription factor Gal4 DBD in mammalian cells and also used to
generate a Gal4 DBD-rGR
2 chimera. In this construct, the GR
2
segment was linked to the carboxyl terminus of the Gal4 DBD. The hnRNP
U cDNA expression plasmid (33) and luciferase reporter plasmids
including linked GREs {i.e. TAT3-Luc (14) or Gal4 upstream
activating sequence (UAS) [i.e.
(UAS)4-TATA-Luc; (55)] have been described previously.
Cell Culture
Chinese Hamster Ovary (CHO) fibroblasts were maintained in DMEM
(GIBCO-BRL, Grand Island, NY) supplemented with 10% FBS (Irvine
Scientific, Santa Ana, CA). CHO cells stably transfected with the
DBD-ß-gal expression plasmid were maintained in DMEM supplemented
with 10% FBS plus 200 µg/ml G418 (GIBCO-BRL). Cellular ATP pools
were depleted upon culturing cells in 2-deoxyglucose and sodium azide
as described previously (29). Typically, CHO cells were treated with 10
mM sodium azide and 6 mM deoxyglucose for 90
min. This treatment has been found to cause minimal cell damage and
permits complete recovery of ATP levels upon removal of sodium azide
and glucose replenishment (29).
Stable and Transient Transfections
For stable transfections, a plasmid encoding the bacterial
neomycin resistance gene was cotransfected with the DBD-ß-gal plasmid
using the calcium-phosphate precipitation method (29). Stable
transfectants were selected and maintained in G418-containing medium.
For subcellular fractionations either in situ or in
suspension, CHO cells grown on 22 x 22 glass coverslips in 35-mm
petri plates or on 60-mm tissue culture plates, respectively, were
transfected using lipofectamine as recommended by the supplier
(GIBCO-BRL). For cotransfections with luciferase reporters, the calcium
phosphate precipitation method was used as previously described (29).
One microgram of indicated luciferase reporter plasmids was included
along with 0.5 µg each of GR and hnRNP U expression plasmids. In
addition, each transfection reaction included 0.3 µg of a ß-gal
expression plasmid [i.e. CMV-ßgal (56)] to provide an
internal control for transfection efficiency between separate samples.
The total amount of DNA transfected per plate of cells was kept
constant using herring sperm DNA (Sigma Chemical Co., St. Louis, MO)
when needed. Luciferase assays were performed with equivalent amounts
of total cell-lysate protein (56). All luciferase activity values were
normalized to ß-gal activity measured in the same lysates (56).
Indirect Immunofluorescence
Indirect immunofluorescence assays were carried out as described
previously (29). BuGR2, a mouse monoclonal antibody recognizing an
epitope adjacent to the rGR DBD (57, 58), was used to detect GR in most
experiments after methanol fixation. In some cases, a rabbit anti-GR
polyclonal antibody was used to detect GR (Affinity BioReagents, Inc.,
Neshanic, NJ). Gal4 DBD and Gal4 DBD-
2 proteins were detected using
an anti-Gal4 DBD mouse monoclonal antibody (Clontech Laboratories,
Inc., Palo Alto, CA). Where indicated, fixed cells were incubated with
either a rabbit polyclonal or mouse monoclonal anti-ß-gal antibody
(Sigma) to detect ß-gal chimeras. The NuMA nuclear matrix protein was
detected with the Ab-1 anti-NuMA mouse monoclonal antibody (Oncogene
Science Inc., Uniondale, NY). DAPI (Sigma) was used to visualize DNA in
fixed cells. Either fluorescein isothiocyanate- or rhodamine-conjugated
goat anti-mouse or anti-rabbit IgG (Boehringer Mannheim Corp.,
Indianapolis, IN) was used as a secondary antibody.
Nuclear Matrix Preparation and Subcellular Fractionation
For in situ extractions (29), cells grown on
coverslips were washed three times with ice-cold PBS and then treated
for 5 min with ice-cold CSK buffer [10 mM
piperazine-N,N'-bis(2-ethanesulfonic acid), pH 6.8, 100
mM NaCl, 300 mM sucrose, 3 mM
MgCl2, 1 mM EGTA, 4 mM vanadyl
riboside complex, 0.5% Triton X-100, and protease inhibitors]. A
complete nuclear matrix preparation was obtained by subjecting CSK
buffer-extracted cells to a DNase I digestion and ammonium sulfate
extraction (29). Analogous extractions were also performed to prepare
nuclear matrices and other subcellular fractions from cells in
suspension (29).
Western Blots
Western blot analysis was used to detect GR in various
subcellular fractions (29). This included GR released by DNase I
digestion and receptors that remained in the nuclear matrix pellet. In
each case, GR levels were compared in fractions obtained from
equivalent amounts of transfected cells. GR was also visualized in
whole-cell extracts (29) prepared from equivalent amounts of cells.
When comparing the relative amounts of GR in different nuclear matrix
preparations, Western blots were costained with the anti-NuMA Ab-1
antibody to provide an internal standard for nuclear matrix recovery
(29).
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. J. A. Iniguez-Lluhi, M.-J. Tsai, and K.
R. Yamamoto for kind gifts of DNA.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Donald B. DeFranco, Department of Biological Sciences, University of Pittsburgh, 5th and Ruskin Streets, Pittsburgh, Pennsylvania 15260. E-mail: dod1{at}vms.cis.pitt.edu
This study was supported by National Institute of Health Grants
CA-43037 (to D.B.D.), CA-65463 (to R.H.G.), and DK-43093 (to M.R.S.), a
predoctoral fellowship from the Andrew Mellon Foundation (to Y.T.), the
Deutsche Forschungsgemeinschaft Grant Re 433/93 (to R.R.), and Fonds
der Chemischen Industrie (to R.R.).
Received for publication March 20, 1998.
Revision received May 27, 1998.
Accepted for publication June 4, 1998.
 |
REFERENCES
|
---|
-
Evans RM 1988 The steroid and thyroid hormone receptor
superfamily. Science 240:889895[Medline]
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz
G, Umesono K, Blumberg B, Kastner P, Mark L, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835839[Medline]
-
Tsai MJ, OMalley BW 1994 Molecular mechanisms of action of
steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451486[CrossRef][Medline]
-
Jonat C, Rahmsdorf HJ, Park K-K, Cato ACB, Gebel S, Ponta H,
Herrlich P 1990 Antitumor promotion and antiinflammation:
down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone.
Cell 62:11891204[Medline]
-
Schüle R, Rangarajan P, Kliewer S, Ransone LJ, Bolado
J, Yang N, Verma IM, Evans RM 1990 Functional antagonism between
oncoprotein c-Jun and the glucocorticoid receptor. Cell 62:12171226[Medline]
-
Miner JN, Yamamoto KR 1991 Regulatory crosstalk at composite
response elements. Trends Biochem Sci 16:423426[CrossRef][Medline]
-
Horwitz KB, Jackson TA, Bain DA, Richer JK, Takimoto GS, Tung
L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:11671177[Abstract]
-
Perlmann T, Evans RM 1997 Nuclear receptors in Sicily: all in
the famiglia. Cell 90:391397[Medline]
-
Hollenberg SM, Evans RM 1988 Multiple and cooperative
trans-activation domains of the human glucocorticoid
receptor. Cell 55:899906[Medline]
-
Tora L, White J, Brou C, Tasset D, Webster N, Scheer E,
Chambon P 1989 The human estrogen receptor has two independent
nonacidic transcriptional activation functions. Cell 59:477487[Medline]
-
Danielian PS, White R, Lees JA, Parker MG 1992 Identification
of a conserved region required for hormone dependent transcriptional
activation by steroid hormone receptors. EMBO J 11:10251033[Abstract]
-
Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP,
a novel mouse protein that serves as a transcriptional coactivator in
yeast for the hormone-binding domains of steroid receptors. Proc Natl
Acad Sci USA 93:49484952[Abstract/Free Full Text]
-
Almlof T, Gustafsson J, Wright A 1997 Role of hydrophobic
amino acid clusters in the transactivation activity of the human
glucocorticoid receptor. Mol Cell Biol 17:934945[Abstract]
-
Iniguez-Lluhi JA, Lou DY, Yamamoto KR 1997 Three amino acid
substitutions selectively disrupt the activation but not the repression
function of the glucocorticoid receptor N terminus. J Biol Chem 272:41494156[Abstract/Free Full Text]
-
Segraves WA 1991 Something old, some things new: the steroid
receptor superfamily in Drosophila. Cell 67:225228[Medline]
-
Noris JD, Fan D, Kerner S, McDonnell DP 1997 Identification of
a third autonomous activation domain within the human estrogen
receptor. Mol Endocrinol 11:747754[Abstract/Free Full Text]
-
Spector DL 1993 Macromolecular domains within the cell
nucleus. Annu Rev Cell Biol 9:265315[CrossRef]
-
He D, Nickerson JA, Penman S 1990 Core filaments of the
nuclear matrix. J Cell Biol 110:569580[Abstract]
-
Fey EG, Bangs P, Sparks C, Odgren P 1991 The nuclear matrix:
defining structural and functional roles. CRC Rev Eukaryot Gene
Expression 1:127144
-
Robinson SI, Nelkin BD, Vogelstein B 1982 The ovalbumin gene
is associated with the nuclear matrix of chicken oviduct cells. Cell 28:99106[Medline]
-
Ciejek E, Tsai M, OMalley BW 1983 Actively transcribed genes
are associated with the nuclear matrix. Nature 306:607609[Medline]
-
Jackson DA, Cook PR 1985 Transcription occurs at the
nucleoskeleton. EMBO J 4:919925[Abstract]
-
Van Wijnen AJ, Bidwell JP, Fey EG, Penman S, Lian JB, Stein
JL, Stein GS 1993 Nuclear matrix association of multiple
sequence-specific DNA binding activities related to SP1, ATF, CCAAT,
C/EBP, OCT-1, and AP-1. Biochemistry 32:83978402[Medline]
-
Bidwell JP, van Wijnen AJ, Fey AG, Dworetzky S, Penman S,
Stein JL, Lian JB, Stein GS 1993 Osteocalcin gene promoter-binding
factors are tissue-specific nuclear matrix components. Proc Natl Acad
Sci USA 90:31623166[Abstract]
-
Sun J-M, Chen HY, Davie JR 1994 Nuclear factor 1 is a
component of the nuclear matrix. J Cell Biochem 55:252263[Medline]
-
Zeng C, van WiJnen AJ, Stein JL, Meyers S, Sun W, Shopland L,
Lawrence JB, Penman S, Lian JB, Stein GS, Heibert SW 1997 Identification of a nuclear matrix targeting signal in the leukemia and
bone-related AML/CBF-alpha transcription factors. Proc Natl Acad Sci
USA 94:67466751[Abstract/Free Full Text]
-
Barrack ER, Coffey DS 1980 The specific binding of estrogens
and androgens to the nuclear matrix of sex responsive tissues. J
Biol Chem 255:72657275[Abstract/Free Full Text]
-
Barrack ER 1987 Localization of steroid receptors in the
nuclear. In: Clark CR (ed) Steroid Hormone Receptors. Their
Intracellular Localisation. Ellis Horwood Ltd., Chichester, UK, pp
86127
-
Tang Y, DeFranco DB 1996 ATP-dependent release of
glucocorticoid receptors from the nuclear matrix. Mol Cell Biol 16:19892001[Abstract]
-
van Steensel B, Jenster G, Damm K, Brinkmann AO, van Driel R 1995 Domains of the human androgen receptor and glucocorticoid receptor
involved in binding to the nuclear matrix. J Cell Biochem 57:465478[Medline]
-
Milhon J, Lee S, Kohli K, Chen D, Hong H, Stallcup MR 1997 Identification of amino acids in the
2 region of the mouse
glucocorticoid receptor that contribute to hormone binding and
transcriptional activation. Mol Endocrinol 11:17951805[Abstract/Free Full Text]
-
Spelsberg TC, Lauber AH, Sandhu AP, Subramaniam MA 1996 Matrix acceptor site for the progesterone receptor in the avian c-myc
gene promoter. Recent Prog Horm Res 51:6396[Medline]
-
Eggert M, Michel J, Schneider S, Bornfleth H, Baniahmad A,
Fackelmayer FO, Schmidt S, Renkawitz R 1997 The glucocorticoid receptor
is associated with the RNA-binding nuclear matrix protein hnRNP U.
J Biol Chem 272:2847128478[Abstract/Free Full Text]
-
Leonhardt H, Page AW, Weier H-U, Bestor TH 1992 A targeting
sequence directs DNA methyltransferase to sites of DNA replication in
mammalian nuclei. Cell 71:865873[Medline]
-
Hedley ML, Amrein H, Maniatis T 1995 An amino acid sequence
motif sufficient for subnuclear localization of an arginine/serine-rich
splicing factor. Proc Natl Acad Sci USA 92:1152411528[Abstract]
-
Danielsen M, Northrop JP, Jonklaas J, Ringold GM 1987 Domains
of the glucocorticoid receptor involved in specific and nonspecific
deoxyribonucleic acid binding, hormone activation, and transcriptional
enhancement. Mol Endocrinol 1:816822[Abstract]
-
Dieken ES, Meese EU, Miesfeld RL 1990 nti
glucocorticoid receptor transcripts lack sequences encoding the amino
terminal transcriptional modulatory domain. Mol Cell Biol 10:45744581[Medline]
-
Yamamoto KR, Gehring U, Stampfer MR, Sibley CH 1976 Genetic
approaches to steroid hormone action. Recent Prog Horm Res 32:332[Medline]
-
Tang Y, Ramakrishnan C, Thomas J, DeFranco DB 1997 A role for
HDJ-2/HSDJ in correcting subnuclear trafficking, transactivation and
transrepression defects of a glucocorticoid receptor zinc finger
mutant. Mol Biol Cell 8:795809[Abstract]
-
Triezenberg SJ, Kingsbury RC, McKnight SL 1988 Functional
dissection of VP 16, the transactivator of herpes simplex virus
immediate early gene expression. Genes Dev 2:718729[Abstract]
-
Cress WD, Triezenberg SJ 1991 Critical structural elements of
the VP16 transcriptional activation domain. Science 251:8790[Medline]
-
Jacq X, Brou C, Lutz Y, Davidson I, Chambon P, Tora L 1994 Human TAFII30 is present in a distinct TFIID complex and is required
for transcriptional activation by the estrogen receptor. Cell 79:107117[Medline]
-
Jackson TA, Richer JK, Bain DL, Takimoto GS, Tung L, Horwitz
KB 1997 The partial agonist activity of antagonist-occupied steroid
receptors is controlled by a novel hinge domain-binding coactivator
L7/SPA and the corepressors N-coR or SMRT. Mol Endocrinol 11:693705[Abstract/Free Full Text]
-
Yoshinaga SK, Peterson CL, Herskowitz I, Yamamoto KR 1992 Roles of SWI1, SWI2, and SWI3 proteins for transcriptional enhancemnet
by steroid receptors. Science 258:15981604[Medline]
-
Muchardt C, Yaniv M 1993 A human homologue of
Saccharomyces cerevisiae SNF2/SWI2 and Drosophila
brm genes potentiates transcriptional activation by the glucocorticoid
receptor. EMBO J 12:42794290[Abstract]
-
Reyes JC, Muchardt C, Yaniv M 1997 Components of the human
SWI/SNF complex are enriched in active chromatin and are associated
with the nuclear matrix. J Cell Biol 137:263274[Abstract/Free Full Text]
-
Dreyfuss G, Matunis MJ, Pinol-Roma S, Burd CG 1993 hnRNP
proteins and the biogenesis of mRNA. Annu Rev Biochem 62:289321[CrossRef][Medline]
-
von Kries JP, Buck F, Stratling WH 1994 Chicken MAR binding
protein p120 is identifcal to human heterogeneous nuclear
ribonucleoprotein (hnRNP) U. Nucleic Acids Res 22:12151220[Abstract]
-
Romig H, Fackelmayer FO, Renz A, Ramsperger U, Richter A 1992 Characterization of SAF-A, a novel nuclear DNA-binding protein from
HeLa cells with high affinity for nuclear matrix/scaffold attachment
DNA elements. EMBO J 11:34313440[Abstract]
-
Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y 1996 The transcriptional coactivators p300 and CBP are histone
acetyltransferases. Cell 87:953959[Medline]
-
Brownell JE, Zhou J, Ranalli T, Kobayashi R, Edmondson DG,
Roth SY, Allis CD 1996 Tetrahymena histone acetyltransferase A: a
homolog to yeast Gcn5p linking histone acetylation to gene activation.
Cell 84:843851[Medline]
-
Hendzel MJ, Sun J, Chen H, Rattner JB, Davie JR 1994 Histone
acetyltransferase is associated with the nuclear matrix. J Biol
Chem 269:2289422901[Abstract/Free Full Text]
-
Picard D, Yamamoto KR 1987 Two signals mediate
hormone-dependent nuclear localization of the glucocorticoid receptor.
EMBO J 6:33333340[Abstract]
-
Sadowski I, Bell B, Broad P, Hollis M 1992 Gal4 fusion vectors
for expression in yeast or mammalian cells. Gene 118:137141[CrossRef][Medline]
-
Spencer TE, Jenster G, Burcin MM, Allis CD, Zhou J, Mizzen CA,
McKenna NJ, Onate SA, Tsai SY, Tsai MJ, OMalley BW 1997 Steroid
receptor co-activator 1 is a histone acetyltransferase. Nature 389:194198[CrossRef][Medline]
-
Chandran UR, Attardi B, Friedman R, Zheng Z-w, Roberts JL,
DeFranco DB 1996 Glucocorticoid repression of the mouse
gonadotropin-releasing hormone gene is mediated by promoter elements
that are recognized by heteromeric complexes containing glucocorticoid
receptor. J Biol Chem 271:2041220420[Abstract/Free Full Text]
-
Gametchu B, Harrison RW 1984 Characterization of a monoclonal
antibody to the rat liver glucocorticoid receptor. Endocrinology 114:274288[Abstract]
-
Rusconi S, Yamamoto KR 1987 Functional dissection of the
hormone and DNA binding activities of the glucocorticoid receptor
protein. EMBO J 6:13091315[Abstract]