Cellular Context of Coregulator and Adaptor Proteins Regulates Human Adenovirus 5 Early Region 1A-Dependent Gene Activation by the Thyroid Hormone Receptor

Xianwang Meng, Yong-Fan Yang, Xiemin Cao, Manjapra V. Govindan, Michael Shuen, Anthony N. Hollenberg, Joe S. Mymryk and Paul G. Walfish

Samuel Lunenfeld Research Institute of Mount Sinai Hospital (X.M., Y.-F.Y., X.C., P.G.W.) and Department of Medicine (P.G.W.), Endocrine Division, University of Toronto Medical School, Toronto, Ontario, Canada M5G 1X5; Centre de Recherche Hotel-Dieu de Québec Université Laval (M.V.G.), Québec, Canada G1R 2J6; Departments of Oncology (J.S.M.), Physiology and Pharmacology (J.S.M.), and Microbiology and Immunology (M.S., J.S.M.), The University of Western Ontario and London Regional Cancer Centre, London, Ontario, Canada N6A 4L6; and Thyroid Unit (A.N.H.), Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Dr. Paul G. Walfish, Mount Sinai Hospital, Endocrine Unit, 600 University Avenue, Suite 781, Toronto, Ontario, Canada M5G 1X5. E-mail: walfish{at}mshri.on.ca.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In mammalian cells, the human adenovirus type 5 early region 1A (E1A) oncoprotein functions as a thyroid hormone (TH)-dependent activator of the thyroid hormone receptor (TR). Interestingly, in the cellular context of the yeast Saccharomyces cerevisiae, E1A acts as a TR-specific constitutive coactivator that is down-regulated by TH. TH reduces the interaction of E1A with the TR in yeast but not HeLa cells. The N-terminal 82 amino acids of E1A are sufficient for coactivation in yeast and residues 4–29 are essential. In yeast, expression of the nuclear receptor corepressor (N-CoR) could down-regulate constitutive transcriptional activation of the TR by E1A, whereas expression of the glucocorticoid receptor interacting protein 1 (GRIP-1) coactivator reconstituted the E1A-induced pattern of enhanced TH-dependent gene activation by TR observed in mammalian cells. We further show that the mating type switching gene (SWI)/sucrose nonfermenting (SNF) gene chromatin remodeling complex is required for both TH/GRIP-1- and E1A-dependent coactivator function, whereas the general control nonrepressed protein (GCN5)/alteration/deficiency in activation protein (ADA2) components of the SPT, ADA, GCN5, acetylation (SAGA) transcriptional adaptor complex are required for TH/GRIP-1, but not E1A-dependent activation of the TR. Taken together, these studies demonstrate that the novel TR-specific coactivator function of E1A in yeast depends on the SWI/SNF chromatin remodeling complex and can be further influenced by changes in the cellular complement of transcriptional coregulatory proteins.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PROTEINS ENCODED BY the human adenovirus type 5 early region 1A (E1A) alter a wide variety of cellular processes involving transcription and chromatin remodeling (Refs. 1 and 2 and references therein). E1A exists primarily as one of two structurally homologous proteins of 243 and 289 amino acids in length (243R and 289R) (see Fig. 1AGo). Three highly conserved E1A regions designated CR1, CR2, and CR3 can induce multiple effects on transcription (1, 2, 3). Because different regions of E1A define important interactions with a number of cellular proteins, E1A has been used as a powerful tool to study cell growth, differentiation, and gene activation. Moreover, it was recently reported that E1A not only binds directly to the thyroid hormone receptor (TR) in vitro, but also enhances thyroid hormone (TH)-dependent gene activation by the chicken TR when transiently expressed in mammalian JEG cells (4).



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Figure 1. E1A Activates TRE-Dependent Transcription by Human TRß1 Constitutively, and This Transcriptional Activity Is Down-Regulated by TH

A, Schematic depiction of human adenovirus 5 E1A (E1A) proteins and deletion mutants. Shown are conserved regions (CR) 1, 2, and 3 of the 289R and 243R variants of full-length E1A, the truncated protein expressing only the N-terminal 82 residues, and the deletion mutants studied. B, The effect of the wild-type full-length E1A 289R or 243R protein on TRß1-dependent transcription. The YPH499 wild-type strain yeast was transformed with expression vectors for hTRß1 and E1A. The empty parental expression vector pRS315 was used as a negative control. Activation of transcription was monitored using a yeast ß-galactosidase (ß-gal) reporter containing a single copy of the chicken lysozyme (F2) T3 response element (TRE). Reporter gene responses were determined in the absence (open bars) or presence (solid bars) of T3 (10 -6 M). ß-gal activities were expressed as Miller units/mg protein. Data shown were pooled from three independent experiments and calculated as mean ± SE. C, The effect of the Gal4-DBD fused to full-length E1A289R (Gal4-E1A 289R) or the N-terminal 82 amino acids of E1A (Gal4-E1A 1–82) on hTRß1-dependent activation of transcription. The Gal4-DBD (Gal4) and Gal4-DBD fusion with the herpes simplex virus VP16 protein (Gal4-VP16) were used as controls. Activation of transcription was assayed as described in Fig. 1BGo. D, Gal4-E1A 1–82 induced gene activation by hTRß1 can be progressively down-regulated by increasing concentrations of either T3 or Triac. Yeast expressing Gal4-E1A 1–82, hTRß1, and the TRE-F2x1 reporter were treated with the indicated T3 or Triac concentrations (10-10 to 10-6 M). Activation of transcription was assayed as described in Fig. 1BGo. E, E1A 1–82-induced activation of a negative TRE. Experimental conditions were as described in Fig. 1BGo except that the negative TRE of the TRH gene was substituted for the positive chicken lysozyme (F2) TRE.

 
The TR is a member of the nuclear hormone receptor (NHR) family of transcription factors, which controls growth, development, and homeostasis (Ref. 5 and references therein). There are four known T3 binding TR isotypes, which have been named TR{alpha}1, TRß1, TRß2, and TRß3 (5, 6). As a hormone-regulated DNA-bound transcription factor, TR can either enhance or repress transcription by binding to thyroid hormone response elements (TREs) in the promoters of target genes (5, 6). The bimodal regulatory properties of TRs are regulated by crucial protein-protein contacts with basal transcription factors, transcriptional corepressor/coactivator proteins, as well as postreceptor multicomponent chromatin-remodeling protein complexes and chromatin structures (Refs. 7 and 8 and references therein).

In the absence of TH, TRs typically bind to DNA regions in gene promoters that contain positively regulated elements. In mammalian cells, transcription is silenced by the concurrent binding of nuclear corepressors (CoRs) such as N-CoR (nuclear receptor CoR) (9), SMRT (silencing mediator for retinoid receptors and TRs) (10) or TRACI/II (T3 receptor-associated corepressor I and II) (11). The binding of TH induces conformational changes in the TR that promote dissociation of TR-bound corepressors and the simultaneous recruitment of several coactivator/adaptor protein complexes. These hormone-dependent mammalian coactivators include p160 family members such as SRC-1 (steroid receptor coactivator-1), TIF2 (transcriptional intermediary factor 2), GRIP-1 (glucocorticoid receptor interacting protein 1) as well as the p300/CBP and PCAF (p300/CBP-associating factor) histone acetyltransferase (HAT) proteins (7, 8). Acetylation of nucleosomes in target genes by the histone acetyltransferases (HATs) that are recruited by DNA bound hormone activated NHRs is essential for the unfolding of chromatin and access of the transcriptional activation complex (12). In contrast, mobilization of histone deacetylases to the transcriptional complex promotes compaction of chromatin and silencing of transcription (13). Thus, histone acetylation and deacetylation can control TR-dependent activation or repression of transcription in eukaryotes through the regulation of chromatin unfolding and folding, respectively (Refs. 12 and 13 and references therein).

To reconstruct mechanisms of gene activation by TRs, we have used the budding yeast, Saccharomyces cerevisiae as a eukaryotic gene expression system (14, 15). In contrast to mammalian cells, S. cerevisiae is devoid of endogenous NHRs, p160 NHR coactivators, and the p300/CBP coregulator protein as well as the N-CoR and SMRT corepressor proteins (14, 15). However, the observation that TH-dependent activation of transcription by hTRß1 in yeast can be markedly enhanced by the expression of a p160 coactivator (i.e. GRIP-1 or SRC-1) indicates that the general transcription initiation machinery and chromatin-remodeling adaptor proteins in yeast have been evolutionarily conserved (14, 15). It is therefore likely that the use of a yeast model system will lead to critical insights into the potential mechanisms of transcriptional activation and repression regulated by TR and TH.

In the present report, we demonstrate that E1A functions in the yeast S. cerevisiae as a potent constitutive coactivator of TR-dependent gene activation and that TH down-regulates this effect. These transcriptional effects require an intact N terminus of E1A as well as an intact SWI/SNF chromatin-remodeling complex, but not the general control nonrepressed protein 5/alteration/deficiency in activation protein 2 (GCN5/ADA2) components of the SPT, ADA, GCN5, acetylation (SAGA) adaptor protein complex. We also show that E1A-induced constitutive gene activation of the TR in yeast can be down-regulated by expression of the mammalian corepressor N-CoR. Expression of the GRIP-1 coactivator in yeast reconstituted the E1A-induced pattern of enhanced TH-dependent gene activation by TR observed in mammalian cells. Taken together, our results demonstrate that the specific coactivator effects of E1A on TR function in yeast are dependent upon an intact SWI/SNF chromatin-remodeling complex and are differentially regulated by changes in the cellular context of mammalian NHR coregulatory proteins.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E1A Acts as a Specific Constitutive Coactivator of the TR in Yeast
To study the effects of E1A on gene transcription by human TR (hTR), full-length wild-type E1A 289R and 243R were coexpressed with hTRß1 and a reporter gene construct containing one copy of the chicken lysozyme F2 (F2x1) positive T3 response element (TRE). E1A 289R and 243R could strongly augment constitutive gene activation by the TR (Fig. 1BGo). When either full-length 289R or only the N-terminal 82 amino acids of E1A were fused to the yeast Gal4 DNA binding domain (Gal4-DBD), their constitutive coactivation of TR was 10-fold more potent (see Fig. 1Go, B and C). Neither the Gal4-DBD alone nor a Gal4-VP16 fusion protein exerted any constitutive coactivator effects on TR-mediated gene activation (Fig. 1CGo). Consequently, further experiments were performed using E1A fused to the Gal4-DBD. Surprisingly, the TR-dependent constitutive gene activation induced by E1A could be down-regulated by exposure to TH (Fig. 1Go, B and C). This down-regulation increased in a dose-dependent fashion with increasing TH concentrations, reaching basal levels of transcriptional activation at concentrations of 10-8 M for L-triiodothyroacetic acid (Triac) or 10-6 M T3 (Fig. 1DGo). Similar results were obtained with either the TRß2 or TR{alpha}1, or using reporter constructs containing single copies of other positively regulated TREs such as the rat GH gene PAL or a spaced direct repeat, DR+4 (data not shown). A similar pattern of gene activation and TH-induced repression was obtained using the negative TRE sequences of the TRH gene, except that the hTRß2 appeared more potent than the hTRß1 (Fig. 1EGo).

We also examined the effects of E1A on the function of several other NHRs on their cognate reporters in yeast, including retinoic acid receptor {alpha} or {gamma} or estrogen receptor {alpha} or ß subtypes. However, no E1A-induced constitutive activation of these NHRs was observed (data not shown). When the retinoid X receptor {gamma} (RXR{gamma}) was coexpressed with TR, we observed that E1A also enhanced the constitutive gene activation of the TR/RXR{gamma} heterodimer and that TH down-regulated this effect (Fig. 2AGo). Moreover, 9-cis-retinoic acid (9c-RA) had no such effect on the action of E1A on either the TR/RXR{gamma} heterodimer (Fig. 2AGo) or the RXR{gamma} homodimer (Fig. 2BGo). Overall, these observations indicate that E1A specifically activates constitutive TR-dependent transcription in yeast and is down-regulated by TH.



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Figure 2. E1A Enhances Transcription Dependent on the hTRß1 and RXR{gamma} Heterodimer, But Not Transcription by the RXR{gamma} Homodimer

A, Yeast strain YPH499 was transformed with expression vectors containing the designated nuclear receptor, its cognate hormone response element inserted into a ß-gal reporter construct, and Gal4-E1A 1–82. Transformed yeast cultures were incubated in the absence or presence of cognate ligands. Experimental conditions and assays were as described in Fig. 1BGo. hTRß1 and RXR{gamma} were coexpressed with the TRE-F2x1 reporter in the presence of either nil (open bar), 10-6 M T3 (solid bar), 10-6 M 9c-RA (hatched bar), or 10 -6 M T3 and 10-6 M 9c-RA (hatched bar). B, RXR{gamma} was coexpressed with the ApoA1x1 reporter and either nil (open bar) or 10-6 M 9c-RA (hatched bar).

 
E1A Residues 4–25 Are Essential for TR-Specific Constitutive Coactivator Function
To determine which regions of the E1A oncoprotein were essential for the induction of constitutive gene activation by the TR, we used several mutants of E1A containing small deletions (Fig. 1AGo). Mutants {Delta}4–25 and {Delta}26–35 could not support TR-dependent gene activation, whereas the mutant {Delta}30–49 functioned similarly to wild-type E1A (Fig. 3AGo). Lack of activation by the {Delta}4–25 and {Delta}26–35 mutants was not simply related to decreased protein expression as determined by Western blot analysis (Fig. 3BGo). Taken together, these studies identify an essential functional role for residues 4–29 of E1A in constitutive gene activation by the TR.



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Figure 3. Amino Acids 4–25 of E1A Are Essential for TR-Specific Gene Activation and Interaction with the TR

A, Specific N-terminal E1A sequences are essential for the gene activation by hTRß1. Wild-type Gal4-E1A 1–82 (WT) and mutant variants containing the indicated deletions ({Delta}4–25, {Delta}26–35, {Delta}30–49) of Gal4-E1A 1–82 were tested for their ability to coactivate hTRß1-dependent transcription. The Gal4-DBD (CTL) was used as a negative control. B, Level of expression of wild-type and E1A mutant proteins. Western blot analysis was performed using yeast extracts expressing wild-type E1A 1–82 or the two mutants ({Delta}4–25 and {Delta}26–35) using a mouse monoclonal antibody to E1A. C, E1A 289R binds to hTRß1 in vitro, and amino acids 4–25 of E1A are required for this interaction. 35S-labeled E1A 289R protein, or an otherwise identical mutant protein containing a deletion of residues 4–25 ({Delta}4–25), was incubated at 4 C for 30 min with bacterially synthesized GST (lane 2 or lane 6) or GST-hTRß1 (lanes 3 and 4 or lanes 7 and 8) in the absence or presence of T3 (10-5 M). Proteins were recovered with glutathione-Sepharose and analyzed by SDS-PAGE. Lanes 1 and 5 represent 8% of total input counts of 35S-labeled proteins used in the binding reactions.

 
To determine whether transcriptional activation of TR-dependent gene expression by E1A resulted from direct binding to the TR, a glutathione-S-transferase (GST) pull-down experiment was performed to compare the interaction of 35S-labeled in vitro transcribed and translated full-length wild-type E1A 289R and a 289R mutant lacking residues 4–25 ({Delta}4–25) with a GST-hTRß1 fusion protein. In contrast to GST alone, E1A bound the TR in the absence of TH (Fig. 3CGo, lanes 2 and 3). This interaction was slightly reduced in the presence of TH (Fig. 3CGo, lane 4). In contrast, the {Delta}4–25 mutant, which does not activate gene transcription by the TR, was unable to bind to GST-TRß1 in either the absence or presence of T3 hormone (Fig. 3CGo, lanes 7 and 8).

Effect of TH on E1A Binding to TR Depends on the Cellular Complement of Transcriptional Coregulatory Proteins
To determine whether E1A could interact with the TR in vivo, a yeast two-hybrid analysis was performed (Fig. 4AGo). The ligand binding domains (LBDs) of hTR{alpha}1 (residues 122–410) and hTRß (residues 189–476) were fused to LexA-DBD as bait and the N-terminal 82 residues of E1A were fused to the B42 activation domain (AD) as prey. A strong interaction between E1A and TR{alpha}1 (lanes 5 and 6; a 24-fold increase of ß-gal activity compared with bait alone) and TRß1 (lanes 9 and 10; a 22-fold increase of ß-gal activity compared with bait alone) was detected. Interestingly, this strong interaction in yeast could be markedly inhibited by the addition of TH. The {Delta}4–25 mutant did not interact with the TR in the yeast two-hybrid test (lanes 11 and 12), confirming that this region is essential for this binding. In contrast to E1A, human Sug1, a protein previously established to interact with the TR in the presence of TH (16), interacted efficiently with the TR{alpha}1 only in the presence of TH (lanes 15 and 16). These studies support the likelihood that in yeast, the down-regulation of E1A-activated transcription by TH in vivo is modulated by a TH-dependent dissociation of E1A from the TR.



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Figure 4. Differences in Eukaryotic Cell Type Regulate the in Vivo Interaction of E1A with the TR

A, Analysis of TR interaction with E1A using a yeast two-hybrid assay. LexA DBD fusions of the LBD portions of TR{alpha}1 (hTR{alpha}1 LBD-DBD) and TRß (hTRß LBD-DBD) were used as bait, and AD fusions of E1A 1–82 (E1A 1–82-AD) or Sug1 (Sug1-AD) were used as prey. Bait and prey plasmids were transformed in the yeast (EGY48) and then grown in selective galactose-supplemented medium overnight and assayed for ß-gal activity as described in Fig. 1BGo, in the absence (open bar) or presence (solid bar) of 10-7 M Triac. B, Analysis of TR interaction with E1A using a mammalian two-hybrid assay. HeLa cells were transiently transfected with the pG5CAT reporter plasmid in the absence or presence of 10-6 M T3, a vector expressing the Gal4-DBD fused to hTRß1, and a vector expressing either wild-type E1A 289R (pVP16-E1A 289R) or an E1A mutant lacking residues 4–25 (pVP16-E1A 289R {Delta}4–25). Cell lysates were analyzed for CAT activity as acetylated chloramphenicol (cpm) as described (31 ) and outlined in Materials and Methods.

 
To evaluate the effects of changes in the cellular background on E1A-TR interaction, we also performed a two-hybrid analysis in human HeLa cells (Fig. 4BGo). Full-length wild-type E1A 289R and the mutant {Delta}4–25 were expressed as fusions to the herpes simplex virus VP16 AD, and wild-type hTRß1 was expressed as a fusion to the Gal4-DBD. Similar to our findings in yeast, a strong interaction between E1A and TRß1 was detected (lane 3). Again, the E1A mutant {Delta}4–25 did not bind to TRß1 in these experiments (lane 5). However, in contrast to the yeast two-hybrid result (Fig. 4AGo, lanes 6 and 10), the interaction in HeLa cells of TRß1 with E1A in the presence of TH was maintained (Fig. 4BGo, lane 4). Taken together, these studies demonstrate the importance of the cellular context in determining whether TH will either suppress or enhance the binding of E1A to TR.

N-CoR and GRIP-1 Regulate E1A-Dependent Gene Activation by the TR
In mammalian cells, the N-CoR corepressor silences constitutive gene activation by the TR, and this activity is abrogated by a TH-induced dissociation of N-CoR from the TR (9). Therefore, it seemed likely that the presence of N-CoR in mammalian cells could modulate the effect of E1A on TR-dependent transcription. When we coexpressed full-length wild-type N-CoR with the N-terminal 82 residues of E1A fused to a Gal4-DBD in our yeast gene activation assay system, we observed a significant down-regulation of E1A-induced constitutive TR-dependent transcription (Fig. 5AGo).



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Figure 5. N-CoR Corepressor and GRIP-1 Coactivator Can Regulate E1A-Dependent Gene Activation by TR

A, Expression of the mammalian corepressor N-CoR inhibits Gal4-E1A 1–82-induced hTRß1-dependent transcription. The YPH499 yeast strain was transformed with the TRE-F2x1 reporter and vectors expressing N-CoR, TRß1, and Gal4-E1A 1–82. Growth and assay conditions were as described in Fig. 1BGo. B, Expression of the mammalian coactivator GRIP-1 in yeast can reconstitute the TR-dependent gene activation profile induced by E1A observed in mammalian cells. The experiment was performed as described in Fig. 5AGo, except that a vector expressing GRIP-1 was substituted for N-CoR. C, N-CoR and GRIP-1 differentially regulate the in vivo interaction of TR with E1A. A modified yeast two-hybrid assay was used (see Materials and Methods) to determine the effects of N-CoR and GRIP-1 on the interaction of TR with E1A in the absence (open bar) or presence of TH at a concentration of 10-7 M Triac (solid bar). The DY150 wild-type yeast strain was transformed with LexA fusions of the LBD region of the hTR{alpha}1 (hTR{alpha}1 LBD-DBD) as bait, the AD fusion of E1A 1–82 (E1A 1–82 AD) as prey, and the pSH1834 LexA-dependent ß-gal reporter. At the same time, a plasmid containing either N-CoR (panel A) or GRIP-1 (panel B) was coexpressed to determine their effect on the interaction of hTR{alpha}1 with E1A.

 
We have shown previously that the hormone-dependent NHR coactivator GRIP-1 dramatically enhances TH-dependent activation by the TR in yeast (14, 15). Expression of GRIP-1 did not alter E1A-dependent constitutive activation by the TR in yeast (Fig. 5BGo). Remarkably, the TH-induced down-regulation of E1A-dependent activation of the TR observed in yeast was reversed by GRIP-1, but not N-CoR (Fig. 5BGo), reconstituting the pattern of enhanced TH-dependent transcription induced by E1A in mammalian cells (4).

To determine whether the presence of N-CoR or GRIP-1 alters the interaction of the TR with E1A, we constructed a modified yeast two-hybrid assay in which multiple exogenous proteins were coexpressed in yeast. We observed that N-CoR and GRIP-1 reduced the constitutive interaction of the TR with E1A (Fig. 5CGo). However, GRIP-1, in contrast to N-CoR, appeared to markedly enhance the binding of E1A to the TR in the presence of TH (Fig. 5CGo). The latter finding is in accord with the observed property of GRIP-1 to function as a TH-dependent transcriptional coactivator in yeast (14, 15).

Differences in Adaptor Protein Complex Requirements for TH/GRIP-1 vs. E1A-Dependent Gene Activation by TR
The yeast Gcn5 protein serves as a highly conserved catalytic subunit of the multicomponent SAGA (SPT, ADA, GCN5, acetylation) transcriptional adaptor complex (Refs. 17 and 18 and references therein). Gcn5 thereby provides an essential in vivo HAT function for the SAGA complex and a molecular linkage between histone acetylation and gene activation (Refs. 12 and 13 and references therein). In addition, the yeast SWI/SNF chromatin-remodeling complex is a highly conserved eukaryotic multicomponent adaptor complex that provides an essential ATP-dependent function that is required by several sequence-specific transcriptional activators and the transcription of a subset of yeast genes (Ref. 19 and references therein).

As we have reported previously (15), disruption of the genes encoding the Gcn5 or Ada2 components of the SAGA complex has a profound effect on the ability of GRIP-1 to function as a TH-dependent activator of TRß1-dependent transcription in yeast (Fig. 6AGo). Similarly, TH/GRIP-1-dependent gene activation by the TR observed in wild-type strains was dramatically abrogated in mutant strains with disruptions of the genes encoding Swi2 (Fig. 6BGo) as well as Swi3 and Snf6 (Fig. 6CGo). Thus, the SAGA and the SWI/SNF complexes and their respective HAT and ATP-dependent chromatin-remodeling activities are required for TH/GRIP-1-dependent gene activation by the TR.



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Figure 6. Intact SAGA and SWI/SNF Adaptor Protein Complexes Are Essential for TH/GRIP-1-Dependent Gene Activation by TR

A, Deletion of the yeast GCN5 or ADA2 components of the SAGA complex impairs TH/GRIP-1-dependent gene activation by hTRß1. The PSY316 wild-type strain yeast or isogenic strains containing disruptions of either the Gcn5 ({Delta}gcn5) or Ada2 ({Delta}ada2) components of the SAGA complex were transformed with the TRE-F2x1 reporter and vectors expressing TRß1 and GRIP-1. Experimental and assay conditions were as described in Fig. 1BGo. B, Deletion of the yeast Swi2 component of the SWI/SNF complex abrogates TH/GRIP-1-dependent gene activation by TR. The DY150 wild-type yeast strain or an isogenic strain containing a disruption of the Swi2 ({Delta}swi2) component of SWI/SNF complex was transformed with the TRE-F2x1 reporter and vectors expressing TRß1 and GRIP-1. Experimental and assay conditions were as described in Fig. 1BGo. C, Deletion of yeast Swi3 and Snf6 components of the SWI/SNF complex impaired TH/GRIP-1-induced TRß-dependent transcription. Wild-type CY26 yeast strain or isogenic strains containing disruptions of either the Swi3 ({Delta}swi3) or Snf6 ({Delta}snf6) components of SWI/SNF complex were transformed with the TRE-F2x1 reporter and vectors expressing hTRß1 and GRIP-1. Experimental assay conditions were as described in Fig. 1BGo.

 
In contrast to their essential functional role in the TH/GRIP-1-dependent gene activation by TR (Fig. 6AGo), deletion of the Gcn5 or Ada2 components of the SAGA complex did not impair E1A-dependent gene activation. Indeed, the coactivation of TR-dependent gene activation by E1A was enhanced in these strains (Fig. 7AGo). Similar to their essential functional role in TH/GRIP-1-dependent gene activation (Fig. 6Go, B and C), disruption of the genes encoding the Swi2 (Fig. 7BGo), Swi3, or Snf6 (Fig. 7CGo) components of the SWI/SNF complex also dramatically impaired E1A-dependent gene activation by the TR. Taken together, these studies strongly support the notion that an intact SAGA and SWI/SNF adaptor protein complexes are required for TH/GRIP-1-dependent gene activation by TR, and that only the presence of an intact SWI/SNF adaptor protein complex is essential for E1A-dependent constitutive gene activation by the TR.



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Figure 7. An Intact SWI/SNF But Not SAGA Adaptor Complex Is Essential for E1A-Dependent Constitutive Activation by TR

A, Deletion of yeast Gcn5 or Ada2 components of the SAGA complex does not impair E1A-dependent gene activation by TR. The PSY316 wild-type yeast strain or isogenic strains containing disruptions of either the Gcn5 ({Delta}gcn5) or Ada2 ({Delta}ada2) components of the SAGA complex were transformed with the TRE-F2x1 reporter and vectors expressing TRß1 and E1A. Experimental and assay conditions were as described in Fig. 1BGo. B, Deletion of the Swi2 component of the SWI/SNF complex abrogates E1A-dependent gene activation by TR. The DY150 wild-type yeast strain and an isogenic strain containing a disruption of the Swi2 ({Delta}swi2) component of the SWI/SNF complex were transformed with the TRE-F2x1 reporter and vectors expressing TRß1 and E1A. Experimental and assay conditions were as described in Fig. 1BGo. C, Deletion of the Swi3 and Snf6 components of the SWI/SNF complexes abrogates E1A-dependent gene activation by TR. The wild-type CY26 strain or isogenic strains containing disruptions of either the Swi3 ({Delta}swi3) or Snf6 ({Delta}snf6) components of the SWI/SNF complex were transformed with TRE-F2x1 reporter and vectors expressing hTRß1 and E1A. Experimental and assay conditions were described in Fig. 1BGo.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
An important question in transcriptional regulation by TH is how TRs can either activate or silence the promoters of target genes. TH binding results in a conformational change in the TR (20, 21) that facilitates the dissociation of TR-bound corepressors such as N-CoR and SMRT, as well as the recruitment of coactivating proteins with intrinsic HAT activity such as the p160 coactivators (GRIP-1, SRC-1, transcriptional intermediary factor 2), p300/CBP, and PCAF. These crucial coactivators are believed to alter chromatin structure and influence the interaction of transcription factors with the general transcriptional machinery (Refs. 7 and 8 and references therein).

Using a yeast TRE gene activation assay, we report here that expression of either full-length human adenovirus type 5 E1A or just the N-terminal 82 residues of E1A greatly enhances constitutive reporter gene activation by hTRß1, hTRß2 (Fig. 1Go, B–E), and TR{alpha}1 (data not shown). These observations agree with another report demonstrating that E1A interacts with, and functions as a coactivator of, the chicken TR in JEG3 mammalian cells (4). In contrast to that study, we observed that activation of the TR by E1A in yeast can be decreased to basal levels by increasing concentrations of TH. Interestingly, a comparable constitutive activation by E1A and repression by TH was also achieved in our yeast coexpression assay regardless of whether the ß-galactosidase reporter gene contained a positive or a negative TRE sequence (Fig. 1Go, C and E).

This novel and potent coactivator function of E1A detected in yeast appears to be specific for the TR as it was not observed when other nuclear hormone receptor homodimers and their cognate HREs were substituted. Moreover, coexpression of TR as a TR/RXR{gamma} heterodimer does not substantially alter the constitutive coactivator function of TR or the ability of TH to down-regulate this effect even when 9c-RA was concurrently present (Fig. 2AGo). Presently, the precise reason for the specific targeting of the TRs by the human adenovirus type 5 E1A proteins remains to be elucidated. We speculate that it may relate to alterations in TH-regulated genes that are required for some aspect of the viral life cycle.

Our studies have determined that residues 4–29 of E1A are essential for its TR-specific constitutive in vivo coactivator function. Deletion of these residues abrogates in vivo gene activation by the TR (Fig. 3AGo), E1A binding to the TR in vitro (Fig. 3CGo), and the interaction of E1A with the TR in either yeast or HeLa two-hybrid analyses (Fig. 4Go, A and B). This sequence is not highly conserved between the E1A proteins of other human adenoviruses (3) and thus may represent a novel functional sequence present only in the E1A protein of human adenovirus type 5. Further characterization of this region may help identify cellular proteins that share a similar structure and, like E1A, can directly interact with unliganded TRs.

We observed that concentrations of TH that down-regulated E1A-induced constitutive in vivo transcriptional activation in yeast (Fig. 1DGo) also significantly decreased the binding of E1A to TRs when tested using a yeast two-hybrid assay (Fig. 4AGo). Indeed, the strong binding of E1A to TR{alpha}1 or TRß LBDs in yeast was almost completely abrogated by TH, which could account for the observed TH-dependent down-regulation of the constitutive gene activation observed in vivo (Fig. 1Go, B and C). In this respect, the strong direct in vivo interaction of E1A with TRs and its dissociation by TH resembles that observed for corepressor proteins such as N-CoR (9), except that E1A functions as a coactivator rather than a repressor of transcriptional activation by TR. A strong in vivo interaction of the TR with E1A was also documented when human HeLa cells were used (Fig. 4BGo). However, the results in HeLa cells differed strikingly from what was observed in yeast (Fig. 4AGo) because the addition of TH did not dissociate E1A from the TR (Fig. 4BGo). This suggests that the effects of TH on E1A-dependent gene activation by TR may be regulated by differences in the cellular context of coregulatory proteins.

The yeast S. cerevisiae is a primitive eukaryote that is devoid of mammalian nuclear hormone receptors, their corepressor proteins, CBP/p300, PCAF, and the p160 coactivators (14, 15). We therefore used the cellular context of yeast to examine the possibility that expression of individual mammalian coregulatory factors might reconstitute the effects of E1A on TR function observed in mammalian cells. Interestingly, coexpression of N-CoR down-regulated E1A-induced constitutive gene activation by TR in yeast (Fig. 5AGo), and transcriptional activation was enhanced rather than repressed by TH when GRIP-1 was coexpressed with E1A (Fig. 5BGo). Taken together, these studies demonstrate that the interaction of E1A with TR is regulated by TH and accessory coregulatory proteins. The differences between yeast and HeLa cells in the cellular context of coactivator and corepressor proteins can thus determine whether the TR activates or represses the transcription of target genes. The absence of mammalian corepressors and p160 coactivators in the yeast S. cerevisiae has facilitated the detection of a novel TR-specific constitutive coactivator function of E1A and the down-regulation of this in vivo effect by TH.

Interestingly, deletion of genes encoding multiple components of the SWI/SNF chromatin-remodeling complex abrogated E1A-induced constitutive gene activation by the TR (Fig. 7Go, B and C). This supports a primary role of the SWI/SNF complex in this pathway of constitutive gene activation by the TR. Previous observations that E1A can affect SWI/SNF activity (22) and that the SWI/SNF complex is required for transcriptional activation by the glucocorticoid receptor (23, 24, 25) add further support for a regulatory role of the SWI/SNF complex in gene activation by the TR. Presumably, E1A can recruit the SWI/SNF complex to TR-bound promoter elements, leading to a local unfolding of chromatin that facilitates constitutive gene transcription. However, a physical association of E1A with the SWI/SNF complex has not been demonstrated, and the exact component(s) of the complex targeted by E1A also remains to be identified. We also show that the SWI/SNF complex (Fig. 6Go, B and C) plays an essential role in TH/GRIP-1-dependent gene transcription in yeast. However, activation of TR-dependent transcription by TH/GRIP-1 differs from E1A in that the SAGA complex is essential for the former (Fig. 6AGo) but not for the latter (Fig. 7AGo). Intriguingly, although the N-terminal 82 residues of E1A can interact with the SAGA complex in yeast (26, 27), the interaction of E1A with this chromatin-remodeling adaptor complex is not essential for E1A to function as a coactivator of gene activation by TR.

The existence of endogenous mammalian coactivators with functional properties homologous to the viral E1A oncoprotein remains to be verified. Such proteins could potentially bind to TRs and interact with the SWI/SNF complex to form a constitutive gene activation complex that can be repressed by TH. It is conceivable that such putative coactivators could serve to regulate TR-dependent target gene function at different stages of mammalian embryogenesis and cellular development when TH and other TR-specific eukaryotic corepressors or coactivators have not yet been synthesized.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Yeast Strains and Media
The S. cerevisiae strain YPH499 (MAT{alpha}, ura3, lys2, ade2, trp1, his3, leu2) was used for most transformations. The GCN5 and ADA2 deletion strains and otherwise isogenic wild-type strain PSY316 (MAT{alpha}, ura3–52, ade2–101, leu2–3, 112, lys2, {Delta}trp1::his G, {Delta}his3–200) were described previously (19). The SWI3 and SNF6 deletion strains and otherwise isogenic wild-type strain CY26 (ura3–52, leu2-{Delta}1, his3-{Delta}200, trp1-{Delta}1, lys2–801, ade2–101) were kindly provided by C. Peterson (University of Massachusetts Medical Center, Worcester, MA). The SWI2 deletion strain and wild-type strain DY150 (ade2, can1, his3, leu2, trp1, ura3) were generously provided by D. Stillman (University of Utah Health Science Center, Salt Lake City, UT). The strain EGY48 (CLONTECH Laboratories, Inc., Palo Alto, CA) was used for the standard yeast two-hybrid experiments, and DY150 strain was used for the modified yeast two-hybrid experiments. For the yeast TRE reporter gene assays, transformants were grown in minimal medium (0.67% yeast nitrogen base/2% glucose) routinely supplemented with adenine and lysine (both at 40 mg/liter). For the modified yeast two-hybrid assay, the transformants were grown in the same minimal media, except for the substitution of 2% galactose for glucose. For the yeast two-hybrid assay, transformants were grown in minimal media (0.67% yeast nitrogen base/2% galactose) containing all the essential amino acids except for uracil, histidine, and tryptophan.

Assay of TR-Dependent Transcriptional Activation in Yeast
hTR, ß1-YEp46, hTRß1-YEp56, GRIP-1-PRS423, GST-hTRß1, and the ß-galactosidase reporter plasmids TRE-F2x1, TRE-DR4x1, TRE-PALx1 and ApoA1x1 for RXR{gamma} have been described previously (14, 15). HTRß2 was cloned into YEp90 (HIS3), hTR{alpha}1 into YEp46 (TRP1), and mRXR{gamma} into YEp56 (LEU2). The coding regions for wild-type full-length E1A289R or 243R were subcloned into the pRS315-GAL1 vector. The N-terminal 82 amino acids (E1A 1–82) were expressed as a fusion with the Gal4-DBD using the yeast expression vector pAS1 or the derivatives pAS1L and pAS1H, which carry TRP1-, LEU2-, and HIS3-selectable markers, respectively. Similarly, full-length E1A 289R was expressed as a fusion with the Gal4-DBD using pAS1H. E1A mutants containing the small in-frame deletions {Delta}4–25, {Delta}26–35, and {Delta}30-49 were generated by PCR and similarly cloned into pAS1L. The negative TRE sequence of the TRH gene was described previously (28, 29) and was subcloned into the pC2 (URA3) ß-galactosidase reporter plasmid to construct the negative TREx1. Full-length wild-type mouse N-CoR was subcloned into the YEp90 (HIS3)- or pRS423 (HIS3)-derived expression vectors.

The ß-galactosidase assays of transcriptional activation were performed after yeast transformants were isolated and grown in minimal medium supplemented with amino acids as required by methods previously described (15). Either T3 or Triac (Sigma Chemicals, Burlington, Ontario, Canada) were dissolved in a minimal volume of 0.1 M sodium hydroxide and further diluted with water. T3, Triac, or diluents were added 16 h before cells were harvested. All analyses were performed in three independent experiments and the data shown are calculated as mean ± SE Miller units/mg protein.

Western Blot Analysis
Extracts of yeast transformed with vectors expressing the Gal4-DBD fused to wild-type E1A 1–82 or the deletion mutants {Delta}4–25 and {Delta}26–35 were prepared as described previously (30). Western blots were performed using the anti-E1A monoclonal antibody M29 (generously provided by E. Harlow, Massachusetts General Hospital Cancer Center, Charlestown, MA).

GST Pull-Down Assays
GST, GST-hTRß1 (a gift from C. Glass, University of California, La Jolla, CA), and GST-E1A 1–82 were expressed in BL21 cells grown at 200 rpm at 30 C and induced with 0.25 mM isopropyl-ß-D-thiogalactopyranoside for 4 h. Full-length 35S-labeled hTRß1 and full-length E1A 289R were synthesized by in vitro transcription and translation using the TNT kit (Promega Corp., Madison, WI). Equivalent amounts of GST or GST-fusion proteins were used for in vitro binding assays as previously described (15).

Yeast Two-Hybrid Assay
The EGY48 yeast strain, LexA-parental bait vector (pEG202; HIS3), B42-parental prey vector (pJG4–5; TRP1), and LexA-dependent reporter plasmid pSH1834 (8 LexA operators in front of the LacZ gene; URA3) were purchased from CLONTECH Laboratories, Inc. A fragment of human wild-type TR{alpha}1 encoding amino acids 122–410 (generously provided by Dr. Joe Torchia, University of Western Ontario, Canada) was cloned into the EcoRI and SalI sites of pEG202 to construct a LexA-hTR{alpha}1 LBD fusion. The LexA-hTRß LBD fusion was a gift from Dr. P. Yen (National Institutes of Health, Bethesda, MD) and spans residues 189–476. Sequences encoding amino acids 1–82 of E1A or 1–82 with the {Delta}4–25 mutation were cloned into the EcoRI and XhoI sites of pJG4–5. A PCR product encoding full-length human Sug1 was cloned into the BamHI and XhoI sites of a modified version of pJG4–5 containing an expanded polylinker. EGY48 yeast transformed with reporter, bait, and prey plasmids was grown overnight in 2% galactose and minimal media containing all essential amino acids except uracil, histidine, and tryptophan in the presence or absence of Triac (10-7 M). Protein-protein interactions were monitored using the LacZ reporter gene and assayed as Miller units/mg protein (14, 15).

Modified Yeast Two-Hybrid Assay
A modified yeast two-hybrid assay system was constructed using the DY150 wild-type yeast strain to examine the effects of the exogenous NHR regulatory proteins N-CoR and GRIP-1 on coactivation by E1A. The LexA-dependent reporter plasmid pSH1834 (URA3) was used. The LexA-hTR{alpha}1 LBD fusion outlined in the yeast two-hybrid method was subcloned with its regulatory and terminator sequences into the commercial YEp1ac181 (LEU2) vector and was used as bait. pJG4–5 (TRP1) expressing amino acids 1–82 of E1A was used as prey. Either GRIP-1-pRS423 or N-CoR-Yep90 (HIS3) was coexpressed with the aforementioned components to evaluate its effects on the interaction between the TR and E1A. Transformations and reporter gene ß-galactosidase assays were performed as described above and reported previously (14, 15).

Mammalian Two-Hybrid Assays
The mammalian two-hybrid assay kit K-1601–1 was purchased from CLONTECH Laboratories, Inc. A cDNA fragment containing the human TRß1 open reading frame was ligated into the HindIII-XbaI site of the pM bait vector. The coding region for the wild-type E1A289R or full-length E1A containing the {Delta}4–25 mutation was ligated into the EcoRI-XhoI sites of the pVP16 prey vector. Plasmids were prepared and purified by CsCl gradient centrifugation. HeLa cells were grown on 100-mm plates in medium supplemented with 2% charcoal-treated fetal bovine serum. Cells were transfected at 70% confluency with a maximum of 10 µg DNA/plate with the pG5CAT reporter plasmid, pM and pVP16 derivatives, and 2 µg of the ß-galactosidase expression vector pcDNA1 LacZ (Invitrogen, Carlsbad, CA) by calcium phosphate precipitation as described previously (31). Protein-protein interactions in the absence (Nil) or presence of 10-6 M T3 were monitored by measuring chloramphenicol acetyltransferase (CAT) activity [assayed in a liquid scintillation counter as acetylated chloramphenicol (cpm)] using total cell extracts normalized to contain 10 U of ß-galactosidase activity as previously described (31).


    ACKNOWLEDGMENTS
 
We thank C. Glass for the GST-TRß1 fusion protein; P. Yen for the LexA-hTRß LBD plasmid; J. Torchia for the TR{alpha} LBD plasmid; E. Harlow for the E1A monoclonal antibody; C. L. Peterson and D. A. Stillman for SWI/SNF wild-type and mutant yeast strains; R. Y. Wang for technical assistance; and C. Walfish and N. Avvakumov for assistance in manuscript preparation.


    FOOTNOTES
 
This work was supported by a Canadian Institutes of Health Grant (MOP-49448), The Mount Sinai Hospital Foundation of Toronto and Department of Medicine Research Funds, The Julius Kuhl and the Temmy Latner/Dynacare Family Foundations, and Sensium Technologies Inc. (to P.G.W); a Canadian Institutes of Health Research Grant (MOP-14631) and a Canadian Institutes of Health Research Scholarship (to J.S.M.); and a joint Canadian Institutes of Health Research/London Regional Cancer Centre Studentship Award (to M.S.).

Abbreviations: AD, Activation domain; ADA2, alteration/deficiency in activation protein 2; CAT, chloramphenicol acetyltransferase; 9c-RA, 9-cis-retinoic acid; DBD, DNA binding domain; E1A, human adenovirus type 5 early region 1A; GCN5, general control nonrepressed protein 5; GRIP-1, glucocorticoid receptor interacting protein 1; GST, glutathione-S-transferase; HAT, histone acetyltransferase; hTR, human TR; LBD, ligand binding domain; N-CoR, nuclear receptor corepressor; NHR, nuclear hormone receptor; PCAF, p300/CBP-associating factor; RXR, retinoid X receptor; SAGA, SPT, ADA, GCN5, acetylation; SMRT, silencing mediator for retinoid receptors and TRs; SNF, sucrose nonfermenting gene; SRC-1, steroid receptor coactivator-1; SWI, mating type switching gene; SPT, suppressor of Ty; TH, thyroid hormone; TR; thyroid hormone receptor; TRE, thyroid hormone response element; Triac, L-triiodothyroacetic acid.

Received for publication August 22, 2002. Accepted for publication March 5, 2003.


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