Temporal Formation of Distinct Thyroid Hormone Receptor Coactivator Complexes in HeLa Cells

Dipali Sharma and Joseph D. Fondell

Department of Physiology University of Maryland School of Medicine Baltimore, Maryland 21201


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thyroid hormone receptors (TRs) regulate transcription by recruiting distinct coregulatory complexes to target gene promoters. Coactivators implicated in ligand-dependent activation by TR include p300, the CREB-binding protein (CBP), members of the p160/SRC family, and the multisubunit TR-associated protein (TRAP) complex. Using a stable TR-expressing HeLa cell line, we show that interaction of TR with members of the p160/SRC family, CBP, and the p300/CBP-associated factor (PCAF) occurs rapidly (~10 min) following addition of thyroid hormone (T3). In close agreement with these observations, we find that TR is associated with potent histone acetyltransferase activity rapidly following T3-treatment. By contrast, we observe that formation of TR-TRAP complexes occurs significantly later (~3 h) post T3 treatment. An examination of the kinetics of T3-induced gene expression in HeLa cells reveals bimodal or delayed activation on specific T3-responsive promoters. Taken together, our data are consistent with the hypothesis that T3-dependent activation at specific target promoters may involve the regulated action of multiple TR-coactivator complexes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thyroid hormone receptors (TRs) are members of the steroid/nuclear receptor (NR) superfamily (1, 2) and mediate a vast array of biological responses including cellular metabolism, development, and differentiation (3). TRs (expressed as two subtypes, {alpha} and ß) have the dual ability to either activate or repress transcription on genes bearing TR-binding elements (TREs) (2). In general, TRs function as activators in the presence of thyroid hormone (T3) and repressors in the absence of T3, although T3-dependent repression has also been observed (2, 4, 5). The ability of TRs to regulate transcription has been linked to their ability to recruit distinct types of transcriptional coregulatory factors, termed coactivators and corepressors, to target promoters (6, 7).

Coactivators recruited by liganded TR and involved in transcriptional activation include members of the p160/SRC family such as SRC-1/NCoA-1, TIF2/GRIP1, and RAC3/pCIP/AIB1/ACTR/TRAM-1 (for reviews see Refs. 6, 7). While the precise mechanism of action of these proteins is still being defined, their ability to associate with histone acetyltransferases (HATs), such as CREB-binding protein (CBP)/p300 (8, 9, 10, 11, 12, 13) and p300/CBP-associated factor (PCAF) (14), and the presence of intrinsic HAT activity in some family members (9, 15) suggests a functional role in chromatin rearrangement. All members of the p160/SRC family have a centrally located NR-interaction domain containing multiple copies of a consensus leucine-rich motif, LXXLL (also termed NR box) (6, 7). Recent biochemical and crystallographic studies reveal that the surface of a single LXXLL motif directly contacts the highly conserved, ligand-dependent activation domain (AF-2) of NRs, thereby providing a molecular basis for NR-coactivator recruitment (16, 17, 18).

An alternative set of TR coactivators, termed the TR-associated protein (TRAP) complex, was first identified as a large multisubunit group of novel proteins that associate with TR in T3-treated HeLa cells (19). The ability of the TRAP complex to markedly stimulate TR-mediated transcription in vitro on naked DNA templates and in the absence of TATA-binding protein-associated factors suggested that TRAPs mediate a novel TR-coactivator pathway or activation step distinct from those mediated by SRC/p160 proteins and CBP/p300 and possibly involving a more direct influence on the basal transcription machinery (19, 20). Several, if not all, of the subunits of the TRAP complex have been identified in other large transcriptional coregulatory complexes including DRIP (21), NAT (22), SMCC (23), and CRSP (24). Furthermore, several TRAP subunits appear to be human homologs of yeast proteins found within "mediator," a large complex of nuclear proteins that interact with both transcriptional regulatory factors and RNA polymerase II (25). A single subunit of the TRAP complex, TRAP220, has been proposed to target and possibly anchor the entire TRAP complex to TR as well as other ligand-activated NRs (26). Interestingly, and analogous to the SRC/p160 proteins, TRAP220 contains two centrally located LXXLL motifs that are essential for physical and functional interactions with the AF-2 domain of TR (26, 27).

In this study, we examined the assembly kinetics of distinct types of TR-coactivator complexes in HeLa cells. We demonstrate that interaction between TR and members of the p160/SRC family, CBP, and PCAF occurs rapidly following T3 induction and that the resulting complexes possess potent levels of HAT activity. By contrast, formation of TR-TRAP complexes in HeLa cells occurs markedly later (~3 h) post T3-treatment. In examining the kinetics of T3-induced gene expression in HeLa cells, we observed that activation on selected T3-responsive promoters was bimodal or delayed with regard to T3 treatment. Collectively, these data suggest that T3-dependent activation of specific genes in HeLa cells may involve the regulated action of multiple TR-coactivator complexes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Differential Assembly Kinetics for Distinct TR-Coactivator Complexes in HeLa Cells
We recently generated a HeLa-derived cell line (termed {alpha}-2) that stably expresses a FLAG epitope-tagged human TR{alpha} and as such, renders the cells responsive to T3 (19). Previous studies demonstrated that transcriptionally active TR{alpha} in association with specific coactivators could be immunopurified from T3-treated {alpha}-2 cells using anti-FLAG antibodies (19, 20, 23, 28). In this study, we employed the {alpha}-2 line to examine the assembly kinetics of distinct types of TR-coactivator complexes as a function of T3 exposure. Toward this end, TR{alpha} was immunoprecipitated from {alpha}-2 cells cultured with T3 for different lengths of time ranging from 10 min to 18 h (Fig. 1Go). TR{alpha}containing protein complexes were then transferred to a membrane and probed by Western blot. We initially probed with specific antibodies representing the three different subtypes of the SRC/p160 family of coactivators (SRC-1, TIF2, and RAC3) (Fig. 1Go, A–D). Interestingly, TR{alpha} showed rapid interaction kinetics with all three members of the p160/SRC family as evidenced by significant levels of associated coactivator 10 min (SRC-1 and RAC3) and 20 min (TIF2) post T3 treatment (Fig. 1Go, A–C). Indeed, further experiments showed that interaction between TR{alpha} and SRC-1 or RAC3 occurs as quickly as 5 min post T3 induction (data not shown).



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Figure 1. Differential Temporal Assembly of Distinct TR-p160/SRC and TR-TRAP Complexes

A–D, TR{alpha} was immunoprecipitated from whole-cell lysate prepared from {alpha}-2 cells cultured with T3 for different lengths of time ranging from 10 min to 18 h (indicated above the lanes). TR{alpha}-associated protein complexes were then fractionated by SDS/PAGE, transferred to a membrane, and probed by Western blot. The blots were initially probed with specific antibodies against SRC-1 (panel A), TIF2 (panel B), and RAC3 (panels C and D). Each of the four blots (panels A–D) was stripped and successively reprobed with specific antibodies against TRAP220, and then TR{alpha}. In panel C, the blot was also probed with antibodies against RXR{alpha}. E, TR{alpha} was first immunoprecipitated from whole-cell lysates prepared from {alpha}-2 cells treated with T3 for different time intervals as indicated above the lanes. The precipitated protein complexes were then eluted from the anti-FLAG antibodies and reimmunoprecipitated with anti-TRAP220 antibodies coupled to protein A-sepharose. The resulting doubly immunoprecipitated complexes were then probed by Western blot with antibodies against either TRAP 220 or SRC-1 as indicated. Five micrograms of HeLa cell nuclear extract were loaded in the first lane as a positive control. F, Whole-cell lysate was prepared from {alpha}-2 cells cultured with T3 for different lengths of time ranging from 10 min to 18 h, fractionated by SDS/PAGE, and then transferred to membranes. The membranes were then probed by Western blot using specific antibodies against TR{alpha}, SRC-1, TIF2, RAC3, TRAP220, TRAP100, and PCAF as indicated on the right of the panel.

 
We next examined the assembly kinetics of the TR-TRAP coactivator complex. In view of TRAP220’s proposed role in targeting the entire TRAP complex to TR and other NRs via direct ligand-dependent NR binding, we first examined the time course of TR{alpha}-TRAP220 interactions. Using the same Western blots initially probed with the anti-p160/SRC antibodies (Fig. 1Go, A–C), we reprobed the membranes with specific antibodies against TRAP220. In contrast to the TR{alpha}-p160/SRC assembly time course, the interaction kinetics between TR{alpha} and TRAP220 were significantly slower as evidenced by the appearance of TRAP220 beginning 1–3 h post T3 treatment (Fig. 1Go, A–C). To verify that the kinetics of TR{alpha}-TRAP220 interaction accurately reflect those for TR{alpha}-TRAP complex assembly, we repeated the experiment using antibodies against TRAP100, a subunit of the TRAP complex that does not directly contact TR. As shown in Fig. 1DGo, the pattern of TR{alpha} coimmunoprecipitation with TRAP100 is nearly identical to the T3-induced timecourse of TR{alpha}-TRAP220 interactions (Fig. 1Go, A–C). The presence of retinoid X receptor (RXR) within the various TR-coactivator complexes was verified by probing the immunoblots with specific antibodies against RXR{alpha}. As evident in Fig. 1CGo, RXR{alpha} precipitates with TR{alpha} in both the presence and absence of ligand, thus demonstrating that coactivator complexes assemble with RXR-TR heterodimers.

To confirm that the TR{alpha}-containing SRC/p160 and TRAP coactivator complexes observed 3–18 h post T3 treatment are distinct entities and do not coexist within a single holocomplex, we performed double coimmunoprecipitation experiments (Fig. 1EGo). First, TR{alpha} was immunoprecipitated from {alpha}-2 cells cultured in T3 for different lengths of time. The precipitated protein complexes were then eluted off the anti-FLAG immunoaffinity resin using a FLAG peptide and subsequently reimmunoprecipitated with antibodies against TRAP220 coupled to protein A-sepharose beads. The resulting doubly precipitated proteins were then probed by Western blot with antibodies against either TRAP220 or SRC-1. As shown in Fig. 1EGo, SRC-1 did not coprecipitate with TRAP220, thus indicating that the interaction of SRC/p160 proteins vs. the interaction of the TRAP complex with TR{alpha} are separable molecular events that result in distinct TR{alpha}-coactivator complexes.

One possible explanation for the observed differential TR-coactivator interaction kinetics is that continuous treatment of {alpha}-2 cells with T3 changes the expression of either TR{alpha} or its specific coactivators. To address this possibility, Western blotting of cellular lysates prepared from {alpha}-2 cells exposed to T3 for different lengths of time was performed. As shown in Fig. 1FGo, no significant changes in protein expression levels were observed up to 18 h post T3 treatment for TR{alpha}, SRC-1, TIF2, RAC3, TRAP220, TRAP100, and PCAF. Taken collectively, these data indicate that the T3-induced kinetics of TR interaction with members of the p160/SRC family are rapid, while TR-TRAP complex assembly is significantly slower.

Rapid T3 Induction of TR-Associated HAT Activity
Members of the SRC/p160 family of coactivators are thought to function in part by associating with strong HAT coactivators such as CBP/p300 and PCAF and subsequently recruiting the HAT activity to NRs in a ligand-dependent manner (6, 7). To examine whether the rapidly induced TR{alpha}-p160/SRC complexes (Fig. 1Go, A–D) are associated with CBP and PCAF, we again immunoprecipitated TR{alpha} from {alpha}-2 cells cultured with T3, transferred the protein complexes to membranes, and then probed with specific antibodies against either CBP or PCAF. Similar to the TR{alpha}-p160/SRC interaction kinetics (Fig. 1Go, A–D), significant levels of both CBP and PCAF were associated with TR{alpha} rapidly following T3 treatment, thus suggesting that the kinetics of TR{alpha}-p160/SRC-CBP/PCAF complex formation are likewise fast (Fig. 2AGo). Furthermore, given that CBP/p300 is capable of direct ligand-dependent interactions with NRs (11), these data may additionally reflect the rapid formation of direct TR{alpha}-CBP/PCAF complexes.



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Figure 2. Rapid T3 Induction of TR-Associated HAT Activity

A, TR{alpha} was immunoprecipitated from nuclear extract prepared from {alpha}-2 cells cultured with T3 for different lengths of time (indicated above the lanes). TR{alpha}-associated protein complexes were then fractionated by SDS-PAGE, transferred to a membrane, and probed by Western blot with specific antibodies against CBP or PCAF as indicated. B, TR{alpha}-associated HAT complexes were immunoprecipitated from nuclear extract prepared from {alpha}-2 cells cultured with T3 for different lengths of time (indicated below the bars). The immune complexes were then subjected to a filter HAT assay in the presence of calf thymus histones and [3H]acetyl-coenzyme A (see Materials and Methods). The amount of HAT activity is quantitated as counts per min of 3H-acetate transferred from acetyl CoA to histones. C, Controls for the HAT filter assay. As a positive control, HAT activity associated with anti-CBP antibodies precipitated from HeLa nuclear extract ({alpha}-CBP + histones) is shown. As a negative control, HAT activity associated with anti-FLAG antibodies precipitated from HeLa extract ({alpha}-FLAG + histones) is shown. As a substrate specificity control, TR{alpha}-associated HAT complexes immunoprecipitated from {alpha}-2 cell extract, prepared from cells treated with T3 for 18 h, was incubated with [3H] acetyl-coenzyme A together with 25 mg BSA ({alpha}-FLAG + BSA) in place of calf thymus histones.

 
To verify that the HAT proteins associated with TR{alpha} are functionally active, we tested the TR{alpha}-coactivator complexes for specific HAT enzymatic activity. Using a filter binding assay, TR{alpha}-containing complexes immunoprecipitated from T3-cultured {alpha}-2 cells were tested for HAT activity in the presence of 3H-labeled acetyl CoA and calf thymus histones. Indeed, significant levels of HAT activity were detected almost immediately (10 min post T3 exposure) with nearly 90% of the maximal HAT activity detected after only 30 min T3 exposure (Fig. 2BGo). Thus, in close correlation with the interaction kinetics of TR{alpha} with the p160/SRC and CBP/PCAF proteins, these data demonstrate that TR{alpha} is associated with potent HAT activity rapidly following T3 exposure.

Kinetics of T3-Induced Gene Expression in {alpha}-2 Cells
The results shown in Figs. 1Go and 2Go indicate that in {alpha}-2 cells, the kinetics of T3-induced TR{alpha}-p160/SRC-CBP/PCAF complex assembly are remarkably fast while the kinetics of TR{alpha}-TRAP complex assembly are significantly slower. To begin to examine whether the differential formation of distinct TR{alpha}-coactivator complexes might be reflected at the level of gene expression, we transiently introduced T3-responsive reporter genes into {alpha}-2 cells and measured transcription after exposure to T3 for different lengths of time. As shown in Fig. 3Go, A–C, significant activation from three different luciferase reporters containing either synthetic TREs (palindromic or DR4) or a natural TRE (chick F2lysozyme element) was preceded by at least a 6-h lag period following T3 exposure.



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Figure 3. Kinetics of Transient T3-Induced Transcription in {alpha}-2 Cells

A –C, {alpha}-2 cells (2 x 105 cells) were transfected with 0.3 µg of either 2xT3RE-tk-Luc (panel A), 2xDR4-tk-Luc (panel B), or F2Lys-tk-Luc (panel C) together with 0.1 µg of the internal control plasmid pSV-ß-gal (see Materials and Methods). Cells were further incubated at 37 C for 24 h. During this period, T3 was added (10-7 M final) for the duration shown beneath the bars. Relative luciferase activities were determined from three independent transfections, although nearly identical results were obtained with multiple transfections. The data are presented as the mean ± the SD of the triplicated results.

 
To examine T3-dependent gene expression in {alpha}-2 cells under more physiologically relevant conditions, we measured endogenous T3-induced gene expression by Northern blot. Because wild-type HeLa cells express only trace levels of TR and typically do not exhibit T3-regulated gene expression, we first needed to identify endogenous genes in the {alpha}-2 line that are responsive to T3. With this objective, poly (A)+ mRNA was extracted from {alpha}-2 cells cultured for 24 h in the absence or presence of T3 and then hybridized with a panel of 12 different DNA probes representing genes previously shown to be regulated by T3 in other tissues (Fig. 4Go A and Materials and Methods). As shown in Fig. 4AGo, three genes were identified whose expression was markedly activated by T3: type 1 iodothyronine deiodinase (dio1), the proto-oncogene bcl-3, and spot 14.



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Figure 4. Kinetics of Endogenous T3-Induced Transcription in {alpha}-2 Cells

A, Poly (A)+ mRNA (2 µg) extracted from {alpha}-2 cells cultured for 24 h in the absence or presence of T3 was fractionated by electrophoresis in a 1% agarose-formaldehyde gel, transferred to a positively charged nylon membrane, and then hybridized with a panel of 12 different radiolabeled DNA probes representing genes previously shown to be regulated by T3 in other tissues (see Materials and Methods for details). B –D, Poly (A)+ mRNA was extracted from {alpha}-2 cells cultured with T3 for different lengths of time indicated above the lanes. Two micrograms of poly (A)+ mRNA from each time point were loaded into a 1% agarose-formaldehyde gel, fractionated by electrophoresis, transferred to a positively charged nylon membrane, and then hybridized with either dio1 (panel B), bcl-3 (panel C), or spot 14 (panel D) radiolabeled DNA probes. The blots were stripped and rehybridized with either ß-actin or sialyl transferase-radiolabeled DNA probes as internal controls.

 
To determine more precisely the T3-induced activation kinetics of these genes, poly (A)+ RNA was extracted from {alpha}-2 cells cultured with T3 for different lengths of time and then hybridized with either dio1, bcl-3, or spot 14 DNA probes. Activation of dio1 gene expression was relatively weak for up to 3 h post T3 treatment and then increased in a linear manner with maximal induction occurring 18 to 24 h post T3 exposure (Fig. 4BGo). T3-induced expression of bcl-3 exhibited an even more delayed pattern of induction with maximal activation occurring 6–18 h post T3 exposure (Fig. 4CGo). Consistent with earlier studies in rat liver (29), T3-induced activation of spot 14 transcription was bimodal, exhibiting a very modest peak of activation 1 h post T3 treatment and a second stronger peak at 18–24 h post T3 treatment (Fig. 4DGo). Thus, although TR{alpha} is associated with potent HAT activity minutes after T3 exposure (Fig. 2Go), significant T3-induced activation from the three endogenous genes examined here does not begin until several hours post-T3 treatment. While we cannot conclude that all T3-regulated genes exhibit similar activation kinetics, we were unable to identify in HeLa cells any other T3-responsive gene promoters, either transiently or endogenously, which exhibited a more rapid T3-induced activation. In sum, these results suggest that transcriptional activation from specific T3-responsive promoters may necessitate multiple functional steps involving the temporally regulated action of distinct coactivator complexes.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Recent studies have revealed a surprisingly large and complex array of coregulatory protein complexes that appear to enhance transcriptional activation by TR and other members of the NR superfamily (6, 7). In this study, we employed a HeLa-derived cell line stably expressing epitope-tagged TR{alpha} as a means of examining the cellular assembly kinetics of different TR-coactivator complexes in vivo. We found that interaction of TR with members of the p160/SRC family, and with the associated coactivators CBP/PCAF, occurs minutes after exposure to T3 and that the resulting complexes possess potent levels of HAT activity. By contrast, formation of TR-TRAP complexes in HeLa cells occurs markedly later following T3 treatment.

Our findings raise the question as to whether different types of TR-coactivator complexes might function in cooperation with one another, possibly during different functional steps of a common TR activation pathway. Given the temporal order of formation of TR-p160/SRC-CBP/PCAF complexes followed by TR-TRAP complexes, our results are suggestive of a sequential model of TR activation (6, 20). In this scenario, T3-activated TR might first recruit HAT activity to a promoter facilitating chromatin derepression. In a subsequent step, TR might recruit cofactors that more directly interface with the basal apparatus (e.g. TRAPs) and potentiate transcription initiation. Indeed, despite TR’s association with strong HAT activity in HeLa cells almost immediately after T3 exposure, significant T3-dependent transcription from three selected promoters did not occur until several hours post T3 treatment (Fig. 4Go), possibly implicating a multistep pathway involving distinct cofactors. While the significance of these transcription kinetics with regard to the different temporal assembly of TR-coactivator complexes is only correlative at this point, it should be noted that we were unable to identify any other T3-responsive gene promoters, either transiently or endogenously, that exhibited a more rapid T3-induced activation.

The kinetics of estrogen receptor (ER)-mediated gene expression in MCF-7 cells were recently shown to be significantly more rapid (30) than those reported here for TR{alpha}. Interestingly, these studies used chromatin immunoprecipitation assays to demonstrate that rapid and transient binding of SRC/CBP complexes to specific estrogen-responsive promoters precisely coincides with rapid ER-mediated transcriptional activation (30). These studies may therefore reflect temporal differences in the specific cofactor requirements for ER vs. TR{alpha}. Alternatively, the observed contrast in ER vs. TR{alpha} activation kinetics may reflect cell-specific differences in MCF-7 vs. HeLa cells or further reveal differences in the higher ordered chromatin structure of the dio1, bcl-3, and spot 14 gene promoters in HeLa cells vs. that for specific estrogen-responsive promoters in MCF-7 cells (30).

In addition to showing an ordered assembly of TR-HAT followed by TR-TRAP complexes between 0 and 3 h post T3 exposure, we also found that both types of complexes coexist in the nucleus between 3 and 18 h post T3 treatment. Thus, while current models of sequential NR activation propose that coactivators are successively exchanged with static chromatin-bound NRs, our data raise the possibility that entire TRcoactivator complexes are dynamically replaced at specific promoters by completely different TR-bound coactivator complexes. Furthermore, our findings are consistent with a TR activation pathway in which p160/SRC, CBP/PCAF, and TRAP coactivator complexes might function simultaneously at the same promoter (6). Indeed, it is interesting to note that the promoter regions for both the dio1 and spot 14 genes contain multiple TR binding elements (TREs) (31, 32). It is thus conceivable that different types of TR-coactivator complexes might be targeted to different TREs at a common promoter in a sequential or temporal fashion. Finally, the possibility also exists that TR-HAT and TR-TRAP complexes might function completely independently of one another via differential and temporal targeting to different T3-responsive promoters.

Another intriguing issue raised by our findings is the mechanistic nature of the delay in TR-TRAP complex assembly after T3 exposure. Previous studies have established that the TRAP complex, excluding TR, exists in a preassembled steady state complex (23, 26, 28). Given that the binding of p160/SRC proteins and TRAP220 with TR’s AF2 motif is mutually exclusive, one possible explanation for the delay is an initial greater T3-dependent TR affinity for the p160/SRC proteins than for the TRAP complex (33). Although we fail to detect a significant decrease in TR-p160/SRC complexes at the time when TR-TRAP complex formation is occurring (as might be predicted from this hypothesis), the situation here may be complicated by the steady state turnover of TR and TR cofactors, and by the dynamic formation of new complexes several hours post T3 exposure. In theory, a temporal delay in TR-TRAP assembly might also involve a regulatory posttranslational modification step (e.g. phosphorylation) of either TR, TRAP220, other TRAP subunits, or perhaps other regulatory factors that ultimately control TR interaction with the TRAP complex. Finally, TR-HAT complex assembly and action might be a regulatory prerequisite for TR-TRAP assembly. Future experiments will be aimed at investigating the molecular mechanisms regulating TR-TRAP complex formation and further examining whether different types of TR-coactivator complexes function at specific T3responsive promoters in a temporal fashion.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Preparation of Cellular Extracts and Coimmunoprecipitation
The HeLa-derived stably expressing FLAG-hTR{alpha} cell line {alpha}-2 (19) was routinely maintained in DMEM supplemented with 10% dialyzed FCS (Life Technologies, Inc., Gaithersburg, MD). After addition of T3 (10-7 M) for the duration indicated in the figures, whole cell lysates were prepared by scraping the cells (1 x 107 cells) in 1 ml of ice-cold buffer A [50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin]. The lysate was rotated 3600 for 1 h at 4 C followed by centrifugation at 12,000 x g for 10 min at 4 C to clear the cellular debris. Protein concentration was determined by Bradford assays. Proteins were either resolved directly in SDS-polyacrylamide gels after boiling in SDS sample buffer or subjected to immunoprecipitation (as outlined below) with the appropriate antibodies.

Preparation of nuclear extract was essentially as described previously (34). Briefly, {alpha}-2 cells in 15-cm culture dishes were collected for harvesting by gentle scraping in 1 ml ice-cold PBS and pelleting by centrifugation at 1200 rpm at 4 C. The PBS was aspirated and the cell pellet (5 x 10 7 cells) was washed once in PBS followed by resuspension in 1 ml of lysis buffer [10 mM Tris-Cl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40]. Nuclei were gently isolated by centrifugation at 4,000 rpm and resuspended in 200 µl of extraction buffer [20 mM Tris-Cl (pH 7.9), 0.42 mM KCl, 0.2 mM EDTA, 10% glycerol, 2 mM dithiothreitol, 0.1 mM phenylmethylsulfonylfluoride]. The resulting nuclear extracts were incubated on ice for 10 min and cleared by centrifugation at 10,000 rpm. Protein concentration of nuclear extracts was determined by Bradford assay.

For coimmunoprecipitation of FLAG-TR-coactivator complexes, 2.5 mg of whole cell lysate or 1 mg of nuclear extract were incubated with anti-FLAG antibodies coupled to agarose beads (20 µl packed volume) (M2 Affinity Resin; Sigma, St. Louis, MO), and the mixture rotated slowly at 4 C for 6–8 h. The beads were collected by gentle centrifugation and washed twice with 1.5 ml ice-cold buffer A. After the final wash, the precipitated TR-coactivator complexes were resuspended in SDS-sample loading buffer, fractionated by SDS-PAGE, and transferred to nitrocellulose membrane. Immuno-detection was performed by first blocking the membranes for 1 h in TBS buffer [20 mM Tris-Cl (pH 7.5), 137 mM NaCl, 0.05% Tween-20] containing 5% powdered milk followed by addition of the appropriate antibodies in TBS and incubating for 2 h at room temperature. Specifically bound primary antibodies were detected with peroxidase-coupled secondary antibodies and developed by enhanced chemiluminescence (ECL system, Amersham Pharmacia Biotech, Arlington Heights, IL) according to manufacturer’s instructions. Antibodies against TRAP220 and TRAP 100 were described earlier (37). Anti-TR{alpha}1 (FL-408), anti-RXR{alpha} (sc-774), anti RAC3 (C-20), and PCAF (C-16) were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against CBP (catalog no. 06–294) were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-SRC-1 (catalog no. MA1–840) was obtained from Affinity BioReagents, Inc. (Golden, CO). Anti-TIF2 was generously provided by Pierre Chambon (35).

For double immunoprecipitation assays, M2-agaroseimmunoprecipitated TR{alpha}-coactivator complexes were resuspended in 50 µl of BC100 buffer [20 mM Tris-Cl (pH 7.9), 20% glycerol, 100 mM KCL, 0.2 mM EDTA] containing 0.2 µg/µl FLAG peptide (N-DYKDDDDK–C), and the mixture was incubated at 4 C for 1 h. The beads were collected by centrifugation, and supernatant, containing the eluted receptorcoactivator complexes, was then subjected to a second round of immunoprecipitation using anti-TRAP220 antibody. Reactions containing anti-TRAP220 antibodies were further incubated with protein A-sepharose beads for an additional 1 h at 4 C. The resultant double immunoprecipitates were then pelleted by gentle centrifugation, washed four times with lysis buffer A, resuspended in SDS-sample loading buffer, fractionated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with specific antibodies.

For immunoprecipitation of HAT activity, nuclear extract was prepared as described above and incubated with either M2-affinity beads (20 µl packed volume) or anti-CBP antibodies (50 ng) for 2 h at 4 C with rocking. Reactions containing anti-CBP antibodies were further incubated with protein A-agarose beads for an additional 1 h at 4 C. The HAT complexes were then pelleted by gentle centrifugation, washed four times with lysis buffer A, and subjected to HAT filter assay (see below).

HAT Assay
HAT activity was assayed as described by Brownell and Allis (36). {alpha}-2 Cells were grown in 15-cm culture dishes and T3 was added as indicated in the figure legends. Nuclear extract preparation and immunoprecipitation of HAT complexes were performed as described above. The immune complexes were first equilibrated in HAT reaction buffer [50 mM Tris.Cl, pH 8.0, 10% (vol/vol) glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM EDTA, 10 mM butyric acid]. The reaction was initiated in a final volume of 30 µl HAT buffer containing 25 mg of calf thymus histones (Sigma type II A) and [3H]Acetyl-coenzyme A (50 nCi; 4.10 Ci/mmol, 152 GBq/mmol (Amersham Pharmacia Biotech), final concentration of 0.5 µM[rsqb]. The reactions were incubated for 30 min at 30 C, spotted onto P-81 filters (Whatman, Clifton, NJ), and then washed extensively with 50 mM sodium carbonate buffer (pH 9.2). [3H]Acetate incorporation was quantitated by liquid scintillation counting.

Transient Transfections
Transient transfections were performed using the Lipofectin reagent (Life Technologies, Inc.) as recommended by the manufacturer. One day before transfection, {alpha}-2 cells were seeded in 12-well plates at a density of 2 x 105 cells per well in DMEM containing 10% dialyzed FBS. A transfection mixture containing 0.3 µg T3-reporter plasmid [either 2xT3RE-tk-Luc, 2xDR4-tk-Luc, or F2Lys-tk-Luc] and 0.1 µg of the internal control plasmid pSV-ß-gal, together with Lipofectin reagent, was added to each well and incubated at 37 C in 5% CO2 for 3 h. One milliliter of DMEM containing 15% dialyzed FBS was added to the transfection mixture, and the cells were incubated at 37 C for 12 h. Transfected cells were then further incubated at 37 C for 24 h; during this period, T3 was added (10-7 M final) for the duration indicated in the figures. Cells were harvested with a cell lysis buffer supplied in a kit (Luciferase Assay System, Promega Corp., Madison, WI), and luciferase activity was determined by adding a commercial assay solution according to the manufacturer’s instructions (Promega Corp.) and then measuring in a Lumat LB 9507 luminometer (EG & G Wallace, Inc., Gaithersburg, MD). The ß-galactosidase activity of the lysed transfected cells (as above) was determined using a kit (ß-galactosidase Enzyme Assay System, Promega Corp.) according to the manufacturer’s instructions. The luciferase activity was normalized to the ß-gal activity and expressed as relative luciferase light units. The reporter constructs 2xDR4-tk-Luc or F2Lys-tk-Luc were kindly provided by Anthony Hollenberg; the 2xT3RE-tk-Luc plasmid has been described previously (37).

Preparation of mRNA and Northern Blots
Total RNA was prepared from {alpha}-2 cells using TRIZOL Reagent (Life Technologies, Inc.) following the instructions provided. The isolation of poly (A)+ RNA from purified total RNA was performed using Message maker reagent assembly (Life Technologies, Inc.) as instructed by the manufacturer. Two micrograms of poly (A)+ RNA were loaded per well and fractionated by electrophoresis in a 1% agarose-formaldehyde gel and subsequently transferred onto a positively charged nylon membrane (BrightStar-Plus, Ambion, Inc., Austin, TX) DNA probes were radiolabeled with {alpha}-32P-dCTP using a Random Primed DNA Labeling Kit (Roche Molecular Biochemicals, Indianapolis, IN). Northern blot hybridization contained 2 x 10 6 cpm/ml of radiolabeled probe and was performed using the Northern Max Northern blotting kit (Ambion, Inc.) following instructions by the manufacturer. Blots were exposed to scientific imaging film (Kodak, Rochester, NY) at -70 C.

The DNA probes used in the Northern blot assays were derived as follows: the human type 1 deiodinase gene probe is a 2.2-kb XhoI fragment excised from pL5Xhor (provided by P. Reed Larsen); the human mdm-2 probe is a 1-kb XbaI/BamHI fragment excised from pCGT-T7-hmdm2 (provided by Robert Freund); the probe for SERCA2 is a 1.6-kb EcoRI fragment excised from the pSERCA2 cDNA (provided by Wolfgang Dillman); the probe for malic enzyme is a 1-kb EcoRI fragment excised from pME6 (provided by Vera Nikodem); the probe for spot 14 is a 450-bp fragment excised from the 5'-end of the spot 14 cDNA (provided by Cary Mariash); the probe for human TRß is a 395-bp PCR product amplified from the 3'-end of the hTRß cDNA; probes for Na/K ATPase {alpha}1 and Na/K ATPase {alpha}2 were 280-bp and 230-bp PCR fragments, respectively, amplified from plasmids pGem9-MR{alpha}1 and pBSKS-MR{alpha}2 (provided by Dr. Shawn Robinson); the probes for glucose-6-phosphatase, bcl-3, and {alpha}-2,3-sialyl transferase genes were provided by Paul Yen as described previously (38); the ß-actin probe was provided by Dr. David Pumplin.


    ACKNOWLEDGMENTS
 
The authors thank Pierre Chambon, Wolfgang Dillman, Robert Freund, Anthony Hollenberg, P. Reed Larsen, Vera Nikodem, Cary Mariash, David Pumplin, Shawn Robinson, and Paul Yen for plasmids and antibodies. The authors thank Milan Bagchi, Howard Towle, Herb Samuels, and Paul Yen for helpful discussions.


    FOOTNOTES
 
Address requests for reprints to: Dr. Joseph D. Fondell, Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, Maryland 21201-1559. E-mail: jfond001{at}umaryland.edu

This work was supported by NIH Grant DK-54030–02 (to J.D.F.).

Received for publication July 10, 2000. Revision received August 18, 2000. Accepted for publication August 30, 2000.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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