GCN5 and ADA Adaptor Proteins Regulate Triiodothyronine/GRIP1 and SRC-1 Coactivator-Dependent Gene Activation by the Human Thyroid Hormone Receptor

Mordecai Anafi, Yong-Fan Yang, Nick A. Barlev, Manjapra V. Govindan, Shelley L. Berger, Tauseef R. Butt and Paul G. Walfish

Samuel Lunenfeld Research Institute (M.A., Y.-F.Y., P.G.W.) and Departments of Medicine, Pediatrics, and Otolaryngology (P.G.W.) University of Toronto Medical School Divisions of Head and Neck Oncology and Endocrinology of Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada
The Wistar Institute (N.A.B., S.L.B.) Philadelphia, Pennsylvania 19104
Centre de Recherche (M.V.G.) Hotel-Dieu de Quebec Université Laval Quebec G1R 2J6, Canada
LifeSensors Inc. (T.R.B.) Malvern, Pennsylvania 19355
Department of Biochemistry & Biophysics (T.R.B.) University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104-6509


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have used yeast genetics and in vitro protein-protein interaction experiments to explore the possibility that GCN5 (general control nonrepressed protein 5) and several other ADA (alteration/deficiency in activation) adaptor proteins of the multimeric SAGA complex can regulate T3/GRIP1 (glucocorticoid receptor interacting protein 1) and SRC-1 (steroid receptor coactivator-1) coactivator-dependent activation of transcription by the human T3 receptor ß1 (hTRß1). Here, we show that in vivo activation of a T3/GRIP1 or SRC-1 coactivator-dependent T3 hormone response element by hTRß1 is dependent upon the presence of yeast GCN5, ADA2, ADA1, or ADA3 adaptor proteins and that the histone acetyltransferase (HAT) domains and bromodomain (BrD) of yGCN5 must be intact for maximal activation of transcription. We also observed that hTRß1 can bind directly to yeast or human GCN5 as well as hADA2, and that the hGCN5387-837 sequence could bind directly to either GRIP1 or SRC-1 coactivator. Importantly, the T3-dependent binding of hTRß1to hGCN5387-837 could be markedly increased by the presence of GRIP1 or SRC1. Mutagenesis of GRIP1 nuclear receptor (NR) Box II and III LXXLL motifs also substantially decreased both in vivo activation of transcription and in vitro T3-dependent binding of hTRß1 to hGCN5. Taken together, these experiments support a multistep model of transcriptional initiation wherein the binding of T3 to hTRß1 initiates the recruitment of p160 coactivators and GCN5 to form a trimeric transcriptional complex that activates target genes through interactions with ADA/SAGA adaptor proteins and nucleosomal histones.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Regulation of transcription in eukaryotes is a multistep process that requires transcription factors to gain access to specific loci tightly packed in chromatin (1, 2). Nucleosomes are the fundamental repeat units of chromatin, and the nucleosome core consists of 1.75 turns of DNA wrapped around an octameric complex of histones comprising two H2A-H2B heterodimers and two H3- H4 heterodimers (3, 4). With the recognition that the transcriptional adaptor GCN5 (general control nonrepressed protein 5) serves as the catalytic subunit of the Tetrahyamena histone acetyltransferase (HAT) type A enzyme, a molecular basis for the linkage between histone acetylation and gene activation was discovered (5). Generally, activation of transcription is accompanied by enzymatic activation of HAT and acetylation of specific lysines of the core histones, thereby neutralizing their positive charges (2, 6). This leads to destabilization of the chromatin structure by ADA/GCN5 products and increases the accessibility of transcription factors to the nucleosomal DNA (7, 8). Thus, histone acetylation and deacetylation can control transcription in eukaryotes through the regulation of chromatin unfolding and folding, respectively (1, 2).

The yeast GCN5 transcriptional adaptor protein has been documented to have in its central structure several highly conserved HAT activational domains and an adjacent essential ADA2 interacting domain. These domains form the HAT catalytic subunit of at least two distinct multisubunit adaptor complexes regulating transcriptional activation by a number of acidic activators (8, 9, 10, 11, 12, 13, 14, 15). The first is the ADA trimeric complex, consisting of ADA2, GCN5, and ADA3 proteins (8, 9, 10, 11, 12, 13, 14). The second is the SAGA complex consisting of SPT, ADA, GCN5, and Acetylation adaptor complex (11, 12, 13). Both adaptor protein complexes were initially identified in yeast model systems. SPT 3, 7, 8, and SPT 20 (or ADA5) and ADA1 (15) are subunits within the SAGA complex (Ref. 13 and references therein). GCN5 provides an essential in vivo HAT function for the SAGA complex. Nucleosomal acetylation in vivo requires that the C terminus bromodomain (BrD) of yGCN5 be intact (Ref. 13 and references therein). Additionally, SPT components of the SAGA complex (13, 16) can interact with TATA-binding protein (TBP) and the TBP-activating factors (TAFIIs). Thus, both ADA and SAGA complexes have multiple, distinct transcription-related functions leading to interactions with the TATA box and facilitating acetylation of nucleosomal histones.

Several mammalian coactivator proteins have been identified through their ability to interact with class I and class II hormone-dependent nuclear receptors (NRs). These NR coactivators function as accessory factors distinct from general factors and transcriptional activators. Bona fide NR coactivator proteins SRC-1 (steroid receptor coactivator 1), TIF2 (transcription intermediary factor 2) GRIP1 (glucocorticoid receptor interacting protein 1) (Ref. 17 and references therein) and CBP interacting protein (pCIP) (18) function as p160 transcriptional coregulatory proteins distinct from general and transcription initiation apparatus factors by acting as hormone-dependent coactivator proteins recruited to NRs. NR coactivators such as SRC-1 (Refs. 19, 20 and references therein) and activator of retinoic acid receptor (ACTR) (21) also have intrinsic HAT activity. Furthermore, studies in mammalian cells have observed that thyroid (T3) hormone receptors (TRs) and retinoic acid receptor (RAR) NRs are activated by binding to the p300/cAMP response element binding protein (CBP), which in turn binds to the p300/CBP-associated factor (PCAF) and the p160 pCIP coactivator (19, 20, 21, 22, 23). The PCAF and p300/CBP components of this complex also have intrinsic HAT activity (19, 20, 21, 22, 23). These observations have favored the conclusion that recruitment of p160 coactivators and CBP/PCAF plays a central role in the regulation of transcriptional activation by RAR and other NRs by acetylation of nucleosomes to unfold chromatin and enhance contact with the general transcriptional machinery (19, 20, 24).

TRs are members of the Class II subclass of NRs. TRs function as hormone-regulated transcription factors that bind to enhancer regions in the promoter of target genes to control growth, development, and homeostasis (Refs. 25, 26 and references therein). TRs modulate gene expression by binding to enhancer regions containing hexameric (AGGTCA) core motifs, designated as T3 response elements (TREs) located at variable distances upstream from the transcription initiation site. TR/TRE interactions are hormone independent and are mediated through the highly conserved zinc fingers in the DNA binding domain (Refs. 25, 26 and references therein). Activation of transcription by T3 hormone and accessory proteins occurs by either direct or indirect interaction of the activation function 2 (AF-2) domain in the C terminus of TRs with downstream components of the transcription initiation apparatus assembled at the TATA-box (27, 28). TRs and RARs interact directly (29, 30) with basal transcription factors TFIIB and TBP/TFIID as well as with specific TAFs (Ref. 28 and references therein). However, the precise signaling pathways whereby DNA bound upstream transcriptional activators, such as TRs, increase the rate of transcription through linkage with accessory factors and the transcription initiation apparatus are not fully understood.

We have previously observed that in vivo activation of transcription by hTRß1 in yeast is relatively weak (31), but could be markedly enhanced by the presence of the p160 NR coactivator GRIP1 (32). Moreover, the observed reconstitution of T3-dependent activation of transcription by hTRß1 and {alpha}1, as well as RARs, in the presence of GRIP1 (32) also indicated that the yeast general transcription initiation machinery has been evolutionarily conserved. A search of the Saccharomyces cerevisiae genome has indicated that yeast is devoid of endogenous NRs, p160 NR coactivators, and p300/CBP adaptor proteins. Hence, the eukaryotic cellular context of the Baker’s yeast, S. cerevisiae, can be used to identify unique putative p300/CBP-independent adaptor/coactivator protein complexes regulating transcriptional activation by hTRß1. In the present report, we have used yeast genetics to examine the in vivo functional role of several components of the SAGA complex in mediating transcriptional activation by hTRß1 and a GRIP1 or SRC-1 p160 coactivator in the presence of T3. Parallel in vitro protein-protein interaction studies have been undertaken to determine whether hTRß1 and p160 coactivators could directly bind to several components of the ADA/SAGA adaptor protein complex. From these experimental approaches, we show that intact yeast GCN5 and ADA adaptor proteins are essential for T3-GRIP1/SRC-1 coactivator-dependent activation of transcription by hTRß1. GCN5 protein plays a central coregulatory role in the function of these adaptor complexes through its highly conserved central HAT catalytic subunit and a C-terminal BrD sequence. We show that either GRIP1 or SRC-1 can bind directly to the conserved C terminus of hGCN5 and enhance the binding of hGCN5 to hTRß1 in the presence of T3 hormone. We also show that hTRß1 binds to human ADA2 and that complementation with a LexA/hADA2 fusion protein can partially restore hTRß1-dependent transcriptional activation in a {Delta}ada2 yeast strain knockout. These in vivo and in vitro experiments suggest that the ADA/SAGA multisubunit adaptor complex can function as in vivo coregulators of the p160 coactivator/T3-dependent activation of transcription by hTRß1.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
yGCN5 Essential for T3/GRIP1 or SRC-1 Activation of Transcription by hTRß1
To determine the role of the yeast GCN5 adaptor protein in transcriptional activation by T3-liganded hTRß1 in the presence or absence of GRIP1 coactivator, we compared hTRß1 gene activation responses in wild-type and {Delta}gcn5 yeast knockout strains. As we have previously reported, T3-induced activation of transcription by hTRß1 in wild-type S. cerevisiae yeast is weak (31) but can be dramatically enhanced by the presence of the p160 coactivator GRIP1 (32). Compared with the wild-type-strain, the {Delta}gcn5 yeast knockout abrogated T3-GRIP1-dependent activation of hTRß1 (see Fig. 1AGo). Complementation with wild-type yeast GCN5 protein almost completely restored the impaired transcriptional activation responses in the {Delta}gcn5 yeast strain (see Fig. 1AGo). When the p160 hSRC-1 coactivator was substituted for GRIP1, essentially similar T3-dependent activation of transcription by hTRß1 was observed in both the wild-type and {Delta}gcn5 yeast strains (Fig. 1BGo). Interestingly, substitution of the {Delta}gcn5 knockout for the wild-type yeast strain detected a slightly greater impairment of T3-dependent transcription activation by hTRß1 in the presence of the GRIP1 p160 coactivator compared with SRC-1. These experiments have revealed that maximal T3/GRIP1/SRC-1 induced gene activation by hTRß1 is dependent upon the presence of the yGCN5 adaptor protein.



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Figure 1. Intact GCN5 and Its HAT Domains Are Essential for T3/GRIP1 or SRC-1 Mediated Gene Activation by hTRß1

A, T3/GRIP1320-1121-dependent activation of transcription in wild-type yeast (left panel) is compared with that of the {Delta}gcn5 yeast knockout (right panel). Transcription activation by full-length hTRß1 in the absence or presence of wild-type GRIP1 or yGCN5 was monitored using a yeast ß-gal reporter containing 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 10-6 M T3. ß-gal activities were expressed as Miller units/mg protein. Data shown were pooled from three independent experiments and calculated as mean ± SE. B, T3/SRC-1-dependent response of wild-type yeast (left panel) is compared with that of the {Delta}gcn5 knockout (right panel). Except for the substitution of full-length SRC-1 for GRIP1320-1121, the experimental details were as otherwise described in panel A. C, Mutations in HAT domains of GCN5 can modulate T3/GRIP1-dependent activation of transcription by hTRß1. Wild-type and several HAT domain mutants of GCN5 containing alanine substitutions of three adjacent amino acids in conserved HAT domains were analyzed for their transcriptional effects of a GRIP320-1121/TRE-F2 x 1 assay on the activation in wild-type yeast (left panel) or {Delta}gcn5 yeast strain (right panel). The triple alanine substitutions in yGCN5 HAT mutants were as follows: GY1 (a.a. 239–241), FAE (a.a.171–173), RGY (a.a. 186–188), and FKK (a.a. 221–223). Experimental conditions were otherwise as described in panel A.

 
Intact yGCN5 HAT Domains Essential for Gene Activation by hTRß1
It has been established that the conserved HAT domains of yGCN5 essential for GAL-VP16 transcriptional activation are localized to sequences 95–253 (10, 13, 33, 34). We therefore selected for study several yeast GCN5 mutants having alanine substitutions of three adjacent amino acids within these sequences. Previous experiments had documented that these mutants have varying degrees of impaired in vitro HAT enzymatic function (34). Interestingly, we observed that compared with the wild-type GCN5 replacement in the {Delta}gcn5 yeast strain, the GY1 mutant (239–241) documented to have the highest HAT function among the {Delta}gcn5 HAT mutants studied (34) also had the greatest in vivo transactivational function, while the mutant RGY (186–188) retained intermediate HAT function (see Fig. 1CGo). In marked contrast, substitution of the FAE (171–173) or the FKK (221–223) mutants devoid of HAT function substantially reduced transcriptional activation (Fig. 1CGo). These experiments performed in a yeast model system have demonstrated the essential functional role played by the conserved yGCN5-HAT domains in regulating of T3/GRIP1 coactivator-dependent transcriptional activation by hTRß1.

ADA/SAGA Adaptor Protein Components Essential for Gene Activation by hTRß1
In previous studies, yADA2 has been documented to bind to both yGCN5 and ADA3 to form an essential trimeric complex for maintaining transcriptional activation by the acidic activator VP16 (Refs. 10, 13 & references therein). Moreover, both yADA2 and yGCN5 have been shown to be essential for maintaining transcriptional activation by the acidic activator VP16 (10, 13, 33, 35, 36). To determine the in vivo functional role of yADA2 in transcriptional activation of hTRß1, the transcriptional responses of hTRß1 in the wild-type yeast strain were compared with those of the {Delta}ada2 knockout. As shown in Fig. 2AGo, substitution of the {Delta}ada2 mutant for wild-type yeast greatly impaired hTRß1 activation by T3 and GRIP1 coactivator, and the defect in transactivation could be almost fully restored when complemented with wild-type yADA2.



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Figure 2. Yeast ADA2, ADA3, and ADA1 Are Essential Adaptor Proteins for Activation of Transcription by hTRß1

A, The transcriptional responses in wild-type yeast (left panel) were compared with the {Delta}ada2 yeast knockout (right panel) in the absence or presence of wild-type yADA2. Activation of transcription was monitored using a ß-gal reporter containing a single copy F2-TRE x1. The experimental conditions were otherwise as described in Fig. 1AGo. B, The transcriptional responses of wild-type yeast (left panel) were compared with the {Delta}ada3 yeast knockout (right panel) in the absence or presence of wild-type yADA3. Experimental conditions were otherwise as described in Fig. 1AGo. C, The transcriptional response of wild-type yeast (left panel) was compared with the {Delta}ada1 yeast knockout strain (right panel) in the absence or presence of wild-type yADA1. Experimental conditions were otherwise as described in Fig. 1AGo.

 
To elucidate the functional role of other components of the ADA/SAGA adaptor protein complex, yeast strains containing {Delta}ada3 and {Delta}ada1 single knockouts were also studied. Deletions of either ADA3 (a trimeric component of the ADA complex) or ADA1 (a component of the SAGA complex) also dramatically reduced T3-dependent-GRIP1 activation of hTRß1 but could be effectively complemented in the yeast knockout strain by wild-type yADA3 and yADA1 protein replacements. (see Figs. 2BGo and 2CGo, respectively). Thus, single replacements of either wild-type GCN5, ADA2, ADA3, or ADA1 components were sufficient to complement transactivational function of a mutant yeast strain devoid of a specific adaptor protein. Taken together, these experiments have also documented that the SAGA adaptor protein complex can function as postreceptor cofactors that can regulate T3/GRIP1-dependent transcriptional activation of target genes by hTRß1.

hGCN5 and hADA2 Adaptor Proteins Do Not Complement Missing Yeast Homologs
To determine whether coexpression of human homologs of GCN5 and ADA2 could restore hTRß1 activation in yeast strains deleted of these adaptor proteins, complementation experiments using wild-type hGCN5 and hADA2 were performed. Neither a LexA/hGCN5 (Fig. 3AGo) nor a LexA/hADA2 (Fig. 3BGo) fusion protein could fully restore transcriptional activation by substituting a human protein for a deleted yeast homolog. However, the partial reconstitution of gene activation by the LexA/hADA2 fusion protein (Fig. 3BGo) supports the possibility that hADA2 is a potential regulator of hTRß1-dependent hormone action. In accord with these observations is the previously reported failure of human homologs to complement missing yeast GCN5 and ADA2 proteins in gene activation by VP16 (33). However, the partial restoration of gene activation by human and yeast GCN5 and ADA2 chimeric proteins (33) indirectly supports the possibility that human homologs of yeast adaptor proteins could play similar roles in regulating transcriptional activation. Together, these results suggest that the absence of essential interacting sequences in homologous human adaptor proteins accounts for their failure to maintain transcriptional activation by hTRß1 in a yeast model system.



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Figure 3. Complementation with Human ADA2 but Not Human GCN5 Can Partially Restore T3/GRIP1-Dependent Activation of Transcription by hTRß1

A, Compared with wild-type yeast (left panel) complementation of a {Delta}gcn5 yeast knockout (right panel) using a LexA/hGCN5 protein in the presence or absence of 10-6 M T3 is illustrated. Except for the substitution of LexA/hGCN5 fusion proteins for wild-type yGCN5, experimental conditions were otherwise as described for Fig. 1AGo. B, Compared with wild-type yeast (left panel), complementation of the {Delta}ada2 yeast knockout (right panel), the LexA/hADA2 fusion protein in the presence or absence of 10-6 M T3 is illustrated. Except for the substitution of the LexA/hADA2 fusion protein for the wild-type yADA2, experimental conditions were otherwise as described for Fig. 2AGo. C, Coexpressed hTRß1 protein can be detected in wild-type and mutant {Delta}gcn5 and {Delta}ada2 yeast strains. Using a polyclonal antibody to hTRß1, Western blot analyses were performed in yeast extracts containing coexpressed hTRß1 receptor. D, Coexpressed LexA/hGCN5 and LexA/hADA2 fusion proteins can be detected in wild-type and mutant yeast strains. Using LexA antibody, Western blot analyses were performed in yeast extracts containing either the LexA control or a LexA/hGCN5 or hADA2 fusion protein, respectively, coexpressed in the {Delta}gcn5 or {Delta}ada2 yeast knockout strain.

 
Human TRß1, GCN5, and ADA2 Proteins Expressed in Yeast Extracts
To evaluate the possibility that the observed defects in the transcriptional function of hTRß1 in ada/gcn5 mutant strains were the result of impaired transformation and expression of the hTRß1 yeast plasmid construct, Western blot analyses were performed on the yeast extracts containing the coexpressed proteins. Similar levels of hTRß1 expression were detected in both wild-type and ada/gcn5 mutant yeast strains (Fig. 3CGo). The preservation of hTRß1 expression in the yeast knockout strain indicated that the in vivo functional consequences observed in mutated ada/gcn5 yeast strains were directly related to the absence of the specific ADA/GCN5 adaptor protein components. Additionally, the expression in wild-type and mutant yeast strains of hADA2 and hGCN5 as LexA fusion proteins was also detected (Fig. 3DGo), thereby indicating that the failure of these proteins to complement gene activation by hTRß1 in yeast {Delta}ada2 or {Delta}gcn5 knockout strains was not caused by defective expression of their yeast plasmids.

hTRß1 Binds to Yeast and Human GCN5 as Well as Human ADA2
To determine which member(s) of the yeast ADA/GCN5 adaptor complex interacted with hTRß1, glutathione-S-transferase (GST) pull-down in vitro binding assays were performed. We observed that a full-length hTRß1-GST fusion protein specifically bound in the absence of T3 to full-length yGCN51-439 and the hGCN51-476 (the shorter spliced variant devoid of the extended N terminus present in the longer variant) as well as full-length hADA2 (see Fig. 4Go). However, we could detect no specific binding of the hTRß1-GST fusion protein to yADA2 and yADA3 (data not shown). In contrast to hGCN5 and hADA2, the central core sequences of GRIP1730-1121 bound to hTRß1 fused to GST only in the presence of T3 (see Fig. 4Go, top lane). Interestingly, the observed stronger protein-protein interaction between hTRß1 and hGCN5 compared with yGCN5 likely reflects a greater preference for binding interactions between human proteins. Nevertheless, our detection of the weaker but specific yGCN5 binding to the hTRß1 receptor is in accord with its discernible functional role in the regulation of reporter gene activation by hTRß1 and accounts for the feasibility of demonstrating in a yeast coexpression system the functional role of yGCN5 as a coregulator of gene activation by hTRß1.



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Figure 4. hTRß1 Interacts in Vitro with Human ADA2 and GCN5

Illustrated are the results of in vitro protein-protein interactions assays using bacterially synthesized GST or GST/hTRß1 and synthesized 35S-labeled in vitro translated proteins of GCN5, ADA2 and GRIP1730-1121 (TNT, Promega Corp.). GST alone or GST fusion proteins were incubated at 4 C with either T3 (1 µM) or vehicle (nil). Lane 1, Input of 35S-labeled proteins. ({approx}1% of total counts); lane 2, GST alone; lane 3, GST/hTRß1 and vehicle; lane 4, GST/hTRß1 and T3 hormone.

 
hGCN5387-837 Binds to GRIP1/SRC-1 and hTRß1
The established structural domains of yGCN5 (30) are depicted in Fig. 5AGo. The conserved carboxy terminus hGCN5387-837 fragment representing the longer spliced variant devoid of the N terminus sequences homologous to hPCAF but retaining HAT, ADA2, and the BrD[76% homology to the C-terminus of PCAF351-832 and 40% to yGCN520-439 (37, 38, 39)] was selected for in vitro binding interaction studies. The hGCN5387-837 fragment immobilized on a Ni agarose matrix was observed to bind directly to either 35S-labeled GRIP1 or SRC-1 (Fig. 5BGo, lanes 8 and 9). In agreement with GST pull-down results shown in Fig. 4Go, hTRß1 bound to the hGCN5387-837 fragment immobilized on the agarose matrix in the absence of T3 (lane 10). However, using densitometric analysis, we observed that hTRß1 binding to hGCN5 was enhanced {approx}7 fold by co-added T3 (lane 11) and was increased {approx}40 fold by T3 when either GRIP1 or SRC-1 p160 coactivator was present (Fig. 5BGo, lanes 13 and 15). Importantly, the {approx}35- to 40-fold increase in the in vitro binding of the hTRß1 to hGCN5 induced by T3 and either GRIP1 or SRC-1 p160 coactivators correlated with the in vivo increase of gene activation in the presence of wild-type yeast T3/GRIP1 (see Fig. 1AGo). These observations are in accord with the previously reported binding of PCAF (the hGCN5 long spliced variant homolog) to C-terminal sequences of SRC-1 (19, 24) and to an RAR/RXR heterodimer (23). Additionally, a PCAF1-428 mutant devoid of the C-terminal fragment was observed to be defective in 9-cis retinoic acid-dependent binding to a RAR/RXR heterodimer-RARE complex and activation of transcription in NIH3T3 cells (23), further supporting the notion that the carboxy-terminal domain of PCAF and its GCN5 homolog are required for maximal gene activation by TR and RAR.



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Figure 5. Intact GCN5 Carboxy Terminus Essential for Maximal Gene Activation by hTRß1

A, Schematic representation of the structural domains of yeast GCN5 [modified from Candau et al. (10 )]. B, Recombinant hGCN5387-837 was immobilized on Ni Agarose matrix (QIAGEN) to determine binding interactions with 35S-labeled in vitro translated proteins of SRC-1 or GRIP1 coactivators and hTRß1. Control binding experiments were performed using either in vitro translated reticulolysate lysate or matrix devoid of hGCN5387-837 recombinant protein to determine nonspecific 35S-labeled binding. [35S]Methionine-labeled proteins were synthesized in vitro using a commercial kit (TNT, Promega Corp.); lanes 1–3, 35S-labeled proteins GRIP1, SRC-1, and hTRß1. Input ({approx}10% of total counts); lane 4, reticulocyte lysate control; lanes 5–7, nonspecific binding controls of 35S-labeled proteins SRC-1, GRIP1, and TRß1, respectively; lanes 8–15, specific binding of recombinant hGCN5387-837 to each of the labeled proteins; lanes 10, 12,and 14, hTRß1 binding to hGCN5 in the absence of T3; lanes 11, 13, and 15, hTRß1 binding to hGCN5 in the presence of added T3 (10-6 M). C, Deletion of yGCN5 bromodomain ({Delta}Br) can substantially reduce T3/GRIP1-dependent activation of transcription by hTRß1. Complementation of the wild-type yeast strain (left panel) was compared with the yeast {Delta}gcn5 knockout strain (right panel) in the presence of wild-type yGCN5 or the mutant {Delta}BrD yGCN5. Experimental conditions were otherwise as described in Fig. 1AGo. D, Deletion of yeast GCN5 bromodomain ({Delta}BrD) can substantially impair T3/SRC-1 dependent gene activation by hTRß1. Except for the substitution of SRC-1 for GRIP1 as a p160 coactivator, experimental conditions were as described in Fig. 1BGo and Fig. 5CGo.

 
Intact yGCN5 BrD Essential for Maximal Gene Activation by hTRß1
Previous reports had identified an important role for the C-terminal BrD of yGCN5 in GAL4-VP16-induced transcriptional activation (36). Although an intact BrD of yGCN5 is not required for acetylation of free histones in vitro, it is essential for in vivo access of yGCN5 to nucleosomal histones (Ref. 13 and references therein). We therefore investigated, using previously studied (13, 33) yeast vectors containing either the mutant {Delta}BrD yeast GCN51-350 or the wild-type GCN51-439 protein, whether deletion of the highly conserved BrD of GCN5, while retaining intact HAT and ADA2 domains (Fig. 5AGo), would impair T3-dependent gene activation by hTRß1. Compared with wild-type yGCN5, the yGCN5 {Delta}BrD mutant was only partially active (65–80% less active than wild-type) in restoring transcriptional activation in a {Delta}gcn5 yeast knockout strain when either wild-type GRIP1 (Fig. 5CGo) or SRC-1 (Fig. 5DGo) p160 coactivator was present. As previously documented (13, 33), expression of either wild-type or {Delta}BrD mutant in the identical yeast expression plasmids, and the comparable maintenance of yeast colony growth by their identical auxotrophic nutritional marker, validated their efficient expression in a yeast model system. These experimental observations have documented the distinctive functional role of the yGCN5 BrD domain in mediating gene activation by hTRß1 in the presence of an intact HAT and ADA2 domain and has identified a novel functional role for the BrD in regulating T3/GRIP1-dependent gene activation by hTRß1. Moreover, these studies demonstrate that the BrD must be preserved to achieve maximal activation of transcription by intact HAT and ADA2 yGCN5 domains.

GRIP1-NR Box LXXLL Motif Mutants Decrease hTRß1 Binding to hGCN5
Our studies have established the important role of full-length wild-type p160 GRIP1/SRC-1 coactivators in the regulation of both in vivo activation of transcription (Fig. 1Go, A and B) and in vitro the formation of a transcriptional activation complex with the hGCN5 adaptor protein (Fig. 5BGo). Hence, further studies were undertaken to elucidate the functional effects induced by GRIP1 NR Box II and III LXXLL motif double alanine for leucine substitution mutants previously shown to regulate binding and gene activation for several hormone-dependent NR transcription activators (40). We first evaluated the effects of GRIP1/NR Box II and III mutants in modulating in vivo transcriptional activation using T3/GRIP1-F2 TRE yeast assay. In agreement with a previous study that used a yeast two-hybrid assay (40), the GRIP1 IIm mutant retained approximately 50% and the IIIm mutant approximately 90% of the transcriptional activation by hTRß1 obtained when wild-type GRIP1 was present. However, the substitution of the NR Box IIm + IIIm mutant for wild-type GRIP1 resulted in a marked (>90%) loss of in vivo T3-induced gene activation (Fig. 6AGo). We also confirmed that defects in transcriptional activation shown in Fig. 6AGo correlated with reductions in T3-dependent in vitro binding of hTRß1 to the GRIP1/GST fusions of NR Box II + IIIm >NR Box II > NR Box III (see Fig. 6BGo).



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Figure 6. Intact NR Box II and III LXXLL Motifs of GRIP1 Are Essential for Maximal Gene Activation and Binding Interactions with hTRß1 and hGCN5

A, Mutations in NR box LXXLL motifs of GRIP1 can impair T3-dependent activation of transcription by hTRß1. The effects on hTRß1-mediated transcriptional activation resulting from substitutions of NR box mutants II m (L693A, L694A), IIIm (L748A and L749A), or IIm + IIIm for wild-type GRIP1 are illustrated. Experimental conditions were as described in Fig. 1AGo. B, GRIP1 NR box mutants have impaired T3-dependent in vitro binding interactions with wild-type hTRß1. Bacterially synthesized GST fusion proteins were interacted with 35S-labeled hTRß1 in the absence (vehicle) or presence of T3 hormone (10-6 M). Lane 1, Input of 35S-labeled hTRß1 ({approx}10% of total counts); lane 2, GST alone; lanes 3 and 4, GST fusions of GRIP1730-1121 NR box wild-type; Lanes 5 and 6, GRIP1 NR box mutant II m; lanes 7 and 8, GRIP1 NR box mutant III m; lanes 9 and 10, GRIP1 NR box mutant II m + III m. Experimental conditions were as outlined in Fig. 4Go. C, Compared with wild-type GRIP1, the GRIP1 NR box IIm + IIIm mutant had impaired in vitro interactions with hGCN5 and hTRß1. Experimental details were as described in Fig. 5BGo. Lanes 1, 2, and 3, Input ({approx}20% of total counts) of S35-labeled protein. Lanes 4–10, Nonspecific binding to control Ni Agarose; lanes 11–14, specific binding to hGCN5 Ni Agarose; lane 12, specific binding to wild-type GRIP1 and 10-6 M T3; lane 14, specific binding to GRIP1 NR box mutant II m + III m and 10-6 M T3.

 
Since wild-type GRIP1 and SRC-1/p160 coactivators can bind to the hGCN5387-837 carboxy terminus in the absence of hormone and enhance binding to hGCN5 when T3 is present (see Fig. 5BGo), we determined the effects on GCN5387-837 binding when the GRIP1 NR box IIm + IIIm mutant was substituted. Compared with wild-type GRIP1, substitution of the GRIP1 NR Box IIm + IIIm mutant was associated with a marked reduction in direct T3 independent binding to the hGCN5387-837 fragment as well as a loss of the expected increase of hTRß1 in vitro binding to hGCN5 when 10-6 M T3 was present (see Fig. 6CGo, lanes 13 and 14). Taken together, these observations confirm that GRIP1 NR Box II + III LXXLL motifs must be intact to facilitate maximal T3-dependent recruitment of the hGCN5 to hTRß1.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A central question in the regulation of transcription by hTRß1 is how its carboxy terminus AF-2 domain links to the transcription initiation apparatus of target genes. Current schematic models have proposed the existence of interacting coactivators and adaptor proteins that facilitate hormone-dependent transcriptional activation by NRs. These interactions lead to the acetylation of nucleosomes to unfold chromatin and enhance linkage to the general transcriptional machinery of hormone-activated NRs bound to upsteam DNA response elements in target genes (1, 2, 27, 28). As schematically summarized (Fig. 7Go), our studies have provided experimental evidence indicating that the yeast GCN5 and ADA/SAGA proteins can function as important coactivators (adaptors) in the regulation of gene activation by hTRß1.



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Figure 7. Gene Activation by hTRß1 Can Be Regulated by Yeast and Human GCN5 and ADA/SAGA Adaptor Proteins

Depicted is a schematic model summarizing our experimental observations on the putative role of the ADA/SAGA adaptor protein complex and the central coactivator GCN5 in regulating gene activation by hTRß1 in a yeast model system. Our studies have shown that T3 hormone facilitates the recruitment of both GCN5 and GRIP1 coactivators to the hTRß1 receptor and the formation of a hormone-dependent transcriptional activation complex. This T3-liganded TRß1/GRIP1/GCN5 complex not only binds to the upstream DNA response element in [i.e. a T3 response element (TRE)], but also facilitates gene activation by interactions with several components of the ADA/SAGA complex and the TATA box. Illustrated are the separate but interdependent HAT, ADA2 binding, and bromodomain (BrD) functions of hGCN5 that must be intact for maximal gene activation. To promote the unfolding of chromatin and access of the transcriptional activation complex, it is postulated that the HAT catalytic unit of GCN5 acetylates H3 and H4 histones (13 ) to concurrently regulate the interaction of the BrD with the acetylated N terminus lysine residues of H3 and H4 histones (44 45 ). The ADA2 binding domain of GCN5 interacts with ADA2 and ADA3 to form the trimeric ADA complex which, in turn, binds to ADA1 and SPT components of the SAGA complex (13 ). Contact of the SPTs with TAFIIs and TBP/TFIID initiates transcription and increases RNA polymerase II. As schematically illustrated, these experimental observations also support the possibility that gene activation by T3 liganded hTRß1 can be initiated without a requirement for p300/CBP.

 
We have documented that several components within the multimeric ADA/SAGA complex are essential for in vivo T3/GRIP1 or SRC1-dependent transcriptional activation by hTRß1. The absence of either GCN5, ADA2, ADA3, or ADA1 yeast adaptor proteins abrogated T3-dependent transcriptional activation by hTRß1 (Figs. 1Go and 2Go). Mutational analyses of the HAT (Fig. 1CGo) and BrD (Fig. 5Go, C and D) domains of yeast GCN5 have indicated that preservation of each domain is essential for maximal transcriptional activation by hTRß1 in a yeast model system. When the yGCN5 BrD is intact, loss of HAT function (i.e. required for acetylation of H3 and H4 histones) impairs transcriptional activation in a yeast model system (Fig. 1CGo), whereas, when its HAT and ADA2 domains are intact, deletion of BrD substantially reduces transcriptional activation (Fig. 5Go, C and D). These findings document, for the first time, distinctive but interdependent functional roles for the HAT and BrD domains of GCN5 in hormone-induced transcriptional activation (Fig. 7Go, schematic illustration).

In this report, we have also observed that yeast and human GCN5 as well as human ADA2 transcriptional adaptor proteins can bind directly to the hTRß1 receptor (Fig. 4Go). Moreover, the conserved carboxy terminus of hGCN5387-837 (containing sequences with high homology to the HAT, ADA2, and BrD domain of yGCN5 and hPCAF) could also bind directly to GRIP1 or SRC1 coactivators (Figs. 5Go and 6Go). Importantly, the presence of T3 was not only essential for the binding of GRIP1 to hTRß1 (Figs. 4Go and 6Go) but also for the in vitro generation of a hGCN5/p160 coactivator/hTRß1 trimeric complex (Fig. 5BGo). Mutations in LXXLL motifs of GRIP1 NR box II and III (Fig. 6Go) and the absence of T3 (Figs. 5Go and 6Go) impaired in vitro formation of the trimeric complex. The marked transcriptional synergy induced by hTRß1 in the presence of T3 and GRIP1 could be directly correlated with the in vitro formation of this T3-dependent trimeric complex. To our knowledge, the observation of a direct in vitro binding interaction of hGCN5387-837 with hTRß1 that can be greatly augmented by T3 and GRIP1 or SRC1 has not been previously reported.

The possibility that yeast ADA/GCN5 adaptor proteins could play a role in p160 coactivator-dependent transcriptional activation by hormone-bound NRs has not been previously evaluated. Studies performed in the absence of a coexpressed p160 coactivator had previously shown that gene activation in yeast by the {tau}1c N terminus domain of the glucocorticoid receptor (GR) (41) as well as the full-length estrogen receptor and RXR (42) could be regulated by ADA adaptor proteins. GR also interacted with yADA2, and overexpressed hADA2 weakly enhanced activation of transcription by GR in HeLa cells (41). Additionally, a positive yeast two-hybrid hormone-dependent hormone-binding domain interaction of the estrogen receptor and RXR with yADA3 as well as a weaker binding to TR but not RAR was reported (42). In contrast, our report documents that the hTRß1 receptor can bind directly to GCN5 as well as hADA2 (Fig. 4Go), but not to yADA3 or yADA1 (data not shown). Since we have shown that TRs and RARs require GRIP1 (32) or SRC-1 coactivators (Fig. 1Go, A and B) for optimal hormone-dependent gene activation in a yeast model system, the current report has extended through yeast genetics our understanding of gene activation by a Class II hormone-dependent NR and demonstrated the importance of p160 coactivator interactions with human and yeast GCN5.

Human homologs of yeast ADA2, GCN5 (33) as well as ADA3 (43), have been identified. The hGCN5387-837 fragment selected for study in our experiment had 76% homology to hPCAF and 40% to yGCN5 (38, 39). Except for its N terminus, PCAF has high homology to yeast GCN5 (38, 39). PCAF interacts directly with RAR (22, 24) and RAR/RXR heterodimers (20, 23), and its C terminus is essential for RAR/RXR heterodimer function (23). It has been established that human and yeast GCN5 have the same H3 and H4 histone substrate specificity in the acetylation of nucleosomes (37). Similarly, the conserved HAT domains of mouse GCN5 and rat PCAF have similar substrate specificity in the acetylation of nucleosomes (43). Recently, the BrD of yGCN5 has been demonstrated to interact with the N terminus of H3 and H4 histones (44). Moreover, the hPCAF BrD has been discovered to consist of four amphipathic {alpha}-helix bundles with a left-handed twist and a hydrophobic pocket formed by two loops that can bind lysine-acetylated peptides of H3 and H4 histones (45). The in vitro observations in their report are in accord with our in vivo studies on the functional role of both the HAT and BrD domains of yGCN5 in hormone-dependent activation of transcription by hTRß1 (Figs. 1CGo and 5Go, C and D) These experimental findings support the intriguing possibility that we have identified a novel structure/function role for the highly conserved BrD of the GCN5/PCAF family of coactivators to promote maximal gene activation by hormone-bound NRs through essential protein-protein interactions of the BrD hydrophobic pocket with acetylated N-terminal lysine tails of H3 and H4 nucleosomal histones to unfold chromatin (Fig. 7Go).

The biological significance of the overlapping function of GCN5/PCAF adaptor complexes is currently unknown. Redundancy of signaling pathways may represent a protective biological mechanism for ensuring the preservation of essential functions by closely related proteins. It is also possible that each adaptor protein complex may be specifically expressed in different tissues to fulfill time-related transcriptional functions (39). Thus, alternatively spliced RNA transcripts may not only be reciprocally expressed in different tissues but could also have different functions (38, 39). The possibility of interrelated biological functions of GCN5 and PCAF has been supported by the detection of their ubiquitous and complementary expression in the mouse (39). Human and yeast ADA2 have conserved Cys-rich sequences with high similarity (~40%) to the CBP/p300 mammalian adaptor proteins (33). However, a search of the complete yeast genome (46) has revealed the absence of yeast proteins with significant homology to CBP and p300. Studies in HeLa cells have found that hPCAF and hGCN5 form multimeric complexes with hADA2 and hADA3, as well as several TAFIIs and other SAGA complex proteins, but not p300/CBP (43). Thus, our experimental findings in a yeast model system that is devoid of the p300/CBP and PCAF coactivators, has indicated that potent T3/GRIP1 or SRC-1 dependent gene activation by hTRß1 can be achieved in the absence of these mammalian coactivators.

We observed, however, that full-length hGCN5 or hADA2 could not complement transcriptional defects induced by deletion of a homologous yeast protein (Fig. 3Go, A and B). Previous observations on the failure of human homologs of yADA2 and yGCN5 to complement these missing wild-type yeast adaptor proteins (33), as well as the failure of the human TBP/TFIID to complement deleted yeast homologs (47, 48), suggest that interacting residues essential for yeast adaptor protein function have not been conserved in human homologs. Interestingly, the significant but only partial increase in transcriptional activation in a {Delta}ada2 yeast strain by a LexA-hADA2 fusion protein (see Fig. 3BGo) is in accord with the enhanced activation of transcription by GR when hADA2 was overexpressed in mammalian cells (41). Moreover, the ability of yeast and human chimeric proteins of GCN5 and ADA2 (33) or TBP (47) to achieve partial restoration of gene activation supports the possibility that human homologs of these adaptor proteins could have adaptor protein functional roles in gene activation by hormone-dependent NRs in humans. Nevertheless, subtle structure/function differences between yeast and human GCN5 and ADA adaptor proteins in the complementation of gene activation mediated by hTRß1 and p160 coactivators have been detected, and further studies on the function of these human homologs in mammalian cells systems will be required to validate their precise functional role in humans.

Although most affected families with a resistance to thyroid hormone (RTH) syndrome have been previously detected to have dominant negative mutations in the hormone-binding domains of TRß (Ref. 49 and references therein), approximately 10% of families with an RTH phenotype have been identified who do not have a discernible mutation in either TRß or TR{alpha}1 genes (50, 51). These observations are in accord with the speculation that postreceptor mutations in adaptor/coactivator proteins could also cause RTH syndromes (51). The detection of partial RTH in SRC-1-/-KO mice has demonstrated that impaired hormone action could be mediated by a mutation in a p160 coactivator and be accompanied by compensatory increases in TIF2/GRIP1 p160 coactivator (52). However, identifiable disturbances in thyroid function in humans with the Rubinstein-Taybi syndrome due to a p300/CBP mutation (53) have not been detected (54). Such observations suggest that either the p300/CBP adaptor protein plays no major physiological role in the maintenance of thyroid function in humans or that alternative signaling pathways with overlapping function do exist. Our experimental observations support the possibility that mutations in hGCN5 (or hPCAF homolog) as well as human homologs of other SAGA complex adaptor proteins could be potential postreceptor sites for defects in T3 hormone action. However, studies will be required in those RTH patients without detectable hTRß receptor mutations to determine whether defects in hGCN5 (hPCAF) or other ADA adaptor proteins could account for syndromes of resistance to hormone action in humans.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Yeast Strains
The Saccharomyces cerevisiae strain PSY316 (MAT{alpha}, Ura3–52, Ade2–101, Leu2–3, 112, Lys2, {Delta}trp1::his G, {Delta}his3–200) wild type or ada/gcn5 mutants (10, 35) was selected for these studies. GCN5, ADA2, ADA3, and ADA1 deletional mutants (10, 35, 36) were produced in the same genetic background of the PSY316 yeast strain.

Yeast Expression Vectors
Human TRß1 was cloned downstream of a CUP1 promoter into a 2 µ multicopy yeast vector containing either a Trp1 selectable marker (YEp 46) or a Leu2 selectable marker (YEp 56). GRIP1320-1121 was cloned into a 2 µ multicopy plasmid under the control of an alcohol dehydrogenase gene (ADH) promoter (PRS 423 vector) with a His3 auxotropic marker (32, 55). Full-lengh SRC-1 and GRIP1563-1121 wild-type or NR box leucine to alanine substitutions for IIm (L693A, L694A), IIIm (L748A, L749A), or combined IIm + IIIm as previously described (40) were cloned into a 2 µ yeast plasmid under the control of a ADH promoter (pGAD424 vector) containing a Leu2 auxotropic selection marker. The F2 enhancer element of the chicken lysozyme promoter was inserted into 2 µ multicopy pC2 reporter plasmid using Ura3 marker using a unique XhoI site upstream from a cytochrome C promoter (CYC1) linked to the Escherichia coli lacZ gene expressing ß-galactosidase (ß-gal) as previously described (31, 32). yADA2 pC98/Leu2 and LexA-hADA2 BTMN/Trp1 were under the control of an ADH promoter and a suitable Trp1 or Leu2 marker (33). yGCN5 PRS414/Trp1 and LexA/hGCN5 BTMN/TRp1 were under the control of an ADH promoter and Trp1 auxtrophic marker (33). Wild-type and mutated forms of the ADA/GCN5 complex were constructed in yeast ARS/CEN expression vectors under control of an ADH promoter and a Trp1 auxotropic marker as previously described (33). yADA1 and yADA3 proteins were cloned into the PDB20L ARS/CEN yeast expression vector with an ADH promoter and a Leu2 selectable marker as previously described (14, 15).

Yeast GCN5 Mutants
Using site-directed mutagenesis, yGCN5 HAT domain mutants were constructed within the highly conserved residues 95–280, which consisted of three adjacent amino acid alanine substitutions as previously described (34). Mutants selected for study and their corresponding sites of three adjacent amino acid alanine substitutions in yGCN5 were: FAE (171–173), RGY (186–188), FKK (221–223), and GYI (239–241). A yeast GCN5 truncation mutant devoid of the highly conserved C-terminal BrD350-439 sequences (yGCN51-350 {Delta}BrD) was constructed as previously described (13) and compared with wild-type yGCN51-439.

Analysis of NR Transcriptional Activation
The yeast transformants were isolated and grown in the appropriate minimal medium with added supplements as required. Cells were grown overnight with T3 at a final concentration of 1 µM, harvested, washed, resuspended in Z buffer, and lysed with glass beads (425–600 µm) before centrifugation. The supernatant was collected, and the protein concentration was determined by the Lowry method (56) using BSA as a standard. Twenty micrograms of protein were used for ß-gal assay, and transcriptional activities were expressed as Miller units/mg of protein (57). Data shown were pooled from three independent experiments and calculated as a mean ± SE.

Protein-Protein Interactions in Vitro
GST/full-length TRß1 (gift from C. Glass) and GST/GRIP1730-1121 and GST/GRIP1653-1121 wild-type and GRIP1 NR box double mutant fusion proteins (36) representing alanine for leucine substitutions in NR box IIm, NR box IIIm, or NR box II + IIIm (gifts from M. R. Stallcup) were used for in vitro pulldown interaction studies. GST fusion proteins bound to glutathione-Sepharose beads were analyzed for binding with 35S-labeled full-length proteins as previously described (32). In other in vitro protein-protein interaction studies, full-length GRIP11-1462 and GRIP1563-1121 wild-type and NR box II and III mutants, as well as full-length TRß1 and SRC-1 (gift from B. W. O’Malley) were [35S]methionine labeled by transcription and translation using the TNT kit (Promega Corp., Madison, WI). The human long GCN5 spliced variant cDNA SP64 construct (gift from Y. Nakatani) was digested with BglII restriction enzyme to isolate the GCN5387-837 C terminus fragment. This fragment was cloned in frame using the pRSETA vector (Invitrogen) for the synthesis of recombinant 6xHis tagged GCN5387-837 protein in a BL21p Lys host cell. Protein expression was under controlled conditions (30 C, 200 rpm) and induced with 0.25 mM IPTG for 4 h. To facilitate [35S]-Met labeling of the GRIP420-1232 fragment, a 5'-end ACCATG sequence was added by mutagenesis. 35S-labeled hTRß1, GRIP1, and SRC-1 were synthesized in vitro in a final volume of 12.5 µl. After synthesis, the lysate was diluted with 87.5 µl incubation buffer (10% glycerol, 100 mM KCl, 20 µM Tris/HCl, pH 7.9, 0.5% NP40, and 1% BSA without EDTA and dithiothreitol). The crude extract of hGCN5387-837 was thawed on ice and 10 µl of aliquots of the extracts were incubated with 10 µl of diluted [35S]methionine-labeled proteins in a final volume of 25 µl at 4 C for 10 min. The bound and free proteins were separated using Ni Agarose matrix (QIAGEN, Chatsworth, CA). Ten microliters of the packed volume of Ni Agarose were added to the reaction mixture and incubated at 4 C for 30 min with occasional agitation before washing three times with the incubation buffer containing 20 µM imidazole. The Ni Agarose-bound proteins were released by incubation with 25 µl of buffer containing 500 mM imidazole. Nonspecific [35S] protein binding was monitored using Ni Agarose matrix devoid of added hGCN5387-837. The eluate was diluted 1:1 with SDS sample buffer, and the samples were resolved on 10% or 12% acrylamide gels. Gels were treated with Enhance (NEN Life Science Products, Boston, MA), dried, and analyzed by autoradiography.


    ACKNOWLEDGMENTS
 
We thank R.Y. Wang, P. Rousseau, and S. Mori for technical assistance; C. Walfish, S. Ladak, and A. Chang for assistance in the preparation of the manuscript text and figures; L. Guarente for ADA1, ADA3 mutant yeast strains, and wild-type plasmid replacements; M. R. Stallcup for GRIP1 wild-type and mutant sequences as well as GST fusion proteins; C. Glass for the GST/hTRß1 fusion protein; Y. Nakatani for the hGCN5 long cDNA: and B.W. O’Malley for the SRC-1 cDNA.


    FOOTNOTES
 
Address requests for reprints to: Dr. Paul Walfish, 600 University Avenue, Suite 781, Toronto, Ontario M5G 1X5 Canada.

This work was supported by grants (to P.G.W.) from the Medical Research Council of Canada Grant MT-14798, The Samuel Lunenfeld Research Institute of Mount Sinai Hospital, The Mount Sinai Hospital Foundation, a Thyroid Foundation of Canada Fellowship (S. Mori), The Mount Sinai Hospital Department of Medicine Research Fund, and the Temmy Latner/Dynacare Foundation.

Received for publication April 30, 1999. Revision received January 26, 2000. Accepted for publication January 28, 2000.


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 INTRODUCTION
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 DISCUSSION
 MATERIALS AND METHODS
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