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Molecular Endocrinology 12 (9): 1420-1431
Copyright © 1998 by The Endocrine Society

The DNA-Binding and {tau}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
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 receptor’s 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 {tau}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 {tau}2 are separable, as transactivation mutants were identified that either inhibited or had no apparent effect on matrix targeting of {tau}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
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {tau}1/enh2 transactivation domain of steroid receptors (9, 13, 14). Finally, a third transactivation domain, {tau}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 {tau}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
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1BGo). 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. 1EGo, 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. 1Go, A and D) and 4,6-diamidino-2-phenylidole (DAPI) (Fig. 1Go, 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.



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  		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 (A–C) or subjected to a nuclear matrix preparation before extraction (D–F). 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 {tau}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 2Go 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 407–574) possesses an intact DBD linked in its natural context to the {tau}2 transactivation domain (31).



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  		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. 3aGo, the DBD of rGR (i.e. amino acids 407–545), 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. 1Go). In contrast, a significant portion of DBD-{tau}2 protein that localized within nuclei of ATP-depleted CHO cells (Fig. 3aGo, panel B) was associated with a nuclear matrix fraction as revealed by its resistance both to CSK buffer extraction (Fig. 3aGo, panel E) and a complete nuclear matrix preparation (Fig. 3bGo, panel B). The nuclear matrix of extracted cells was visualized by costaining with the NuMA antibody (Fig. 3bGo, panel A). Thus, the {tau}2 transactivation domain of rGR cooperates with the receptor’s DBD to constitute a minimal NMTS.



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  		Figure 3. The {tau}2 Transactivation Domain and DBD of rGR Constitute a Minimal NMTS

(a) DBD [amino acids (a.a.) 407–545, A and D], DBD-{tau}2 (a.a. 407–574, panels B and E), and DBD-{tau}2 mutant (a.a. 407–574, L553G/L554G, panels C and F) were transiently transfected into CHO cells. Transfected cells were depleted of ATP and either fixed directly (A–C) or extracted with CSK buffer containing 0.5% Triton X-100 before fixation (D–F). BuGR2 was used to detect all GR derivatives. (b) CHO cells transiently expressing DBD-{tau}2 were depleted of ATP and subsequently subjected to an in situ nuclear matrix preparation. DBD-{tau}2 was detected using a polyclonal anti-GR antibody (B) while the nuclear matrix (A) and DNA (C) were visualized by anti-NuMA antibody and DAPI staining, respectively. (c) The {tau}2 transactivation domain of GR cannot target a linked Gal4 DBD to the nuclear matrix. CHO cells transiently transfected with the Gal4 DBD or a Gal4 DBD-{tau}2 chimera were depleted of ATP and either directly fixed (A and C) or subjected to a nuclear matrix preparation before fixation (B and D). Gal4 DBD derivatives were detected using a monoclonal anti-Gal4 DBD antibody.

 
The {tau}2 transactivation domain alone is not sufficient for nuclear matrix targeting as a Gal4 DBD-{tau}2 chimera (Fig. 2Go) expressed in transiently transfected CHO cells did not efficiently associate with nuclear matrix (Fig. 3cGo). As will be shown later (see Fig. 7cGo), the Gal4 DBD-{tau}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. 3cGo and Ref. 26), cannot substitute for the rGR DBD to constitute a NMTS in combination with the GR {tau}2 transactivation domain.



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  		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-{tau}2, or GR-DBD-{tau}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-{tau}2 expression plasmids. Results shown are an average of three separate determinations (± SD), each performed in duplicate.

 
Extensive mutagenesis of the rGR {tau}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. 3aGo (panel F), substitutions of leucines 553 and 554 with glycines in DBD-{tau}2m (Fig. 2Go) 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-{tau}2 S573A, see Fig. 2Go) also greatly reduced {tau}2 transactivation activity (31). Interestingly, nuclear matrix targeting of the rGR DBD-{tau}2 protein was not affected by the S573A point mutation (Fig. 4Go). Thus, the transactivation and nuclear matrix-targeting activities of {tau}2 are separable and distinguished by point mutations at distinct, highly conserved amino acids.



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  		Figure 4. The Nuclear Matrix-Targeting and Transactivation Activities of {tau}2 Are Separable

CHO cells transiently transfected with GR DBD-{tau}2 (S573A) were subjected to an ATP-depletion and either fixed directly (Fix, panels A and B) or fixed after a nuclear matrix preparation (NM, panels C and D). The GR DBD-{tau}2 (S573A) protein was detected using the BuGR2 anti-GR antibody (B and D) while DNA was visualized by DAPI staining (A and C).

 
To provide a more quantitative assessment of GR-{tau}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. 5aGo). As shown in Fig. 5Go, a and b, approximately 30% of DBD-{tau}2 was retained on the nuclear matrix of ATP-depleted CHO cells, whereas only trace amounts (i.e. <5%) of DBD and DBD-{tau}2m proteins were associated with the nuclear matrix. As shown in Fig. 5aGo (lanes 2, 5, and 8) DBD and DBD-{tau}2m proteins were more readily released from chromatin by DNase I digestion than DBD-{tau}2. These biochemical fractionations confirm the indirect immunofluorescence analysis of in situ extracted cells and establish the nuclear matrix-binding capability of the DBD-{tau}2 protein.



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  		Figure 5. Western Blot Analysis and Quantification of Nuclear Matrix Binding of DBD-{tau}2

a, CHO cells transiently transfected with DBD (lanes 1–3), DBD-{tau}2 (lanes 4–6), and the DBD-{tau}2 mutant (lanes 7–9) 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-{tau}2 (column 2), and DBD-{tau}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, {tau}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 {tau}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 {tau}2. Is the {tau}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 412–454) linked carboxyl terminal to the rGR DBD. As shown in Fig. 6Go, the DBD-VP16 protein was found to associate with the nuclear matrix of ATP-depleted CHO cells. Thus, either a homologous (i.e. {tau}2) or heterologous (i.e. from VP16) transactivation domain linked carboxyl terminal to the rGR DBD can constitute an effective NMTS.



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  		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 488–777) in cotransfection assays possesses the {tau}2 domain [i.e. amino acids 525–556 (9)]. We therefore examined whether the minimal NMTS that we identified within rGR, which included {tau}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. 7aGo, cotransfection of hnRNP U cDNA inhibited the transactivation activity of GR DBD-{tau}2 by 50%. The GR DBD and the DBD-{tau}2m, both of which exhibited some transactivation activity compared with TAT3-Luc alone, were unaffected by hnRNP U (Fig. 7aGo). 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. 7bGo), transactivation mediated by the Gal4 DBD-{tau}2 chimera was insensitive to cotransfected hnRNP U (Fig. 7cGo). 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 {tau}2) are insensitive to hnRNP U effects.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
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-{alpha} 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 {tau}2 transactivation domain linked in its native configuration, constitutes a minimal NMTS for rGR. The {tau}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 {tau}2 domain is strictly required for nuclear matrix targeting, but this should become apparent from analysis of steroid receptor chimeras possessing swapped DBD and {tau}2 domains.

The analysis of two {tau}2 mutants that possess minimal transactivation activity established that the transactivation and nuclear matrix-targeting activities of {tau}2 are separable. Thus, while the L553G/L554G double-point mutation abolished both transactivation and nuclear matrix-targeting activities of {tau}2, the S573A mutation affected transactivation but not the matrix-targeting function of {tau}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 {tau}2 domain based upon the crystal structure of the thyroid hormone receptor {alpha} LBD, L553 and L554 are predicted to be located within a different {alpha}-helix than S573 (31). Thus, distinct surfaces of {tau}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 {tau}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 {tau}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 {tau}2 domains have not been strictly tested, a carboxyl-terminal deletion of human AR that removes its {tau}2 domain reduces its association with the nuclear matrix (30). Given the high degree of conservation between the {tau}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 {tau}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 {tau}1/enh2 transactivation domain that resides within steroid receptor amino-terminal domains is unrelated to {tau}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 {tau}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 {tau}2 and the transactivation domain of VP16 have been postulated to form an amphipathic {alpha}-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 {tau}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 {tau}2 domain have little to no impact on its transactivation activity (31). As mentioned above, complete loss of {tau}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 {tau}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 {tau}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 {tau}2 domain or VP16 transactivation domain functionally interact with hnRNP U. However, both the rGR DBD and {tau}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 {tau}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
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
Figure 2Go 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 407–545) fused at its carboxyl terminus to the bacterial ß-galactosidase (ß-gal) gene (53). Amino acids 407–556 of the rGR were fused to the transactivation domain of the Herpes simplex virus VP16 protein (amino acids 413–454) 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 {tau}2-transactivation domain were described previously (31). These plasmids possess rGR DBD sequences from amino acid 407–545 (i.e. DBD) or 407–574 (i.e. DBD-{tau}2, DBD-{tau}2m, and DBD-{tau}2(S573A)). In DBD-{tau}2m, leucines 553 and 554 are substituted by glycine residues while in DBD-{tau}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 {tau}2 chimera. In this construct, the GR {tau}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-{tau}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 433/9–3 (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.


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 MATERIALS AND METHODS
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