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Molecular Endocrinology, doi:10.1210/me.2002-0308
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Molecular Endocrinology 17 (5): 908-922
Copyright © 2003 by The Endocrine Society

Transcriptional Activation by Thyroid Hormone Receptor-ß Involves Chromatin Remodeling, Histone Acetylation, and Synergistic Stimulation by p300 and Steroid Receptor Coactivators

Kathleen C. Lee1, Jiwen Li1, Philip A. Cole, Jiemin Wong2 and W. Lee Kraus2

Department of Molecular Biology and Genetics (K.C.L., W.L.K.), Cornell University, Ithaca, New York 14853; Department of Molecular and Cellular Biology (J.L., J.W.), Baylor College of Medicine, Houston, Texas 77030; Department of Pharmacology and Molecular Sciences (P.A.C.), The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; and Department of Pharmacology (W.L.K.), Weill Medical College of Cornell University, New York, New York 10021

Address all correspondence and requests for reprints to: W. Lee Kraus, Department of Molecular Biology and Genetics, Cornell University, 465 Biotechnology Building, Ithaca, New York 14853. E-mail: wlk5{at}cornell.edu; or Jiemin Wong, Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: jwong{at}bcm.tmc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transcriptional regulation by heterodimers of thyroid hormone receptor (TR) and the 9-cis retinoid X receptor (RXR) is a highly complex process involving a large number of accessory factors, as well as chromatin remodeling. We have used a biochemical approach, including an in vitro chromatin assembly and transcription system that accurately recapitulates ligand- and activation function (AF)-2-dependent transcriptional activation by TRß/RXR{alpha} heterodimers, as well as in vitro chromatin immunoprecipitation assays, to study the mechanisms of TRß-mediated transcription with chromatin templates. Using this approach, we show that chromatin is required for robust ligand-dependent activation by TRß. We also show that the binding of liganded TRß to chromatin induces promoter-proximal chromatin remodeling and histone acetylation, and that histone acetylation is correlated with increased TRß-dependent transcription. Additionally, we find that steroid receptor coactivators (SRCs) and p300 function synergistically to stimulate TRß-dependent transcription, with multiple functional domains of p300 contributing to its coactivator activity with TRß. A major conclusion from our experiments is that the primary role of the SRC proteins is to recruit p300/cAMP response element binding protein-binding protein to hormone-regulated promoters. Together, our results suggest a multiple step pathway for transcriptional regulation by liganded TRß, including chromatin remodeling, recruitment of coactivators, targeted histone acetylation, and recruitment of the RNA polymerase II transcriptional machinery. Our studies highlight the functional importance of chromatin in transcriptional control and further define the molecular mechanisms by which the SRC and p300 coactivators facilitate transcriptional activation by liganded TRß.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE MOLECULAR ACTIONS of thyroid hormone (T3) are mediated through thyroid hormone receptors (TR{alpha} and TRß). TRs belong to the nuclear receptor (NR) superfamily and play important roles in development, differentiation, homeostasis, and tumorigenesis through their ability to regulate gene expression (1). TRs function as heterodimers with the 9-cis retinoic acid receptor (RXR) and, in the absence of hormone, the heterodimers bind to thyroid hormone response elements (TREs) and actively repress transcription (2, 3). In contrast to unliganded TRs, ligand-bound TRs function as transcriptional activators. Ligand-dependent activation by TR/RXR heterodimers requires an intact activation domain residing at the carboxyl terminus of the TR ligand-binding domain [known as activation function (AF)-2], as well as cellular coactivators (4).

Many coactivators have been implicated in T3-dependent activation, including the steroid receptor coactivator (SRC) family of proteins, p300/cAMP response element binding protein-binding protein (CBP), p300/CBP associated factor (PCAF), and the mediator-like TR associated proteins (TRAP)/vitamin D receptor-interacting proteins (DRIP)/SRB/mediator-containing cofactor complex (SMCC) (hereafter referred to as the TRAP complex; for reviews, see Refs. 5, 6, 7, 8, 9). The SRC family contains three highly related and possibly functionally redundant proteins referred to herein under the unified nomenclature SRC-1, SRC-2, and SRC-3 (5). The SRCs interact directly with liganded NRs and serve as adapter molecules to recruit other coactivators such as p300/CBP. Furthermore, some investigators have found certain SRCs (i.e. SRC-1 and SRC-3) to possess a weak intrinsic acetyltransferase (AT) activity (10, 11), although others have been unable to detect this activity (12, 13). p300/CBP and PCAF are potent acetyl transferases that can acetylate histones and a variety of transcription-related factors (14, 15), as well as interact with components of the basal transcriptional machinery (16, 17). p300/CBP is recruited to liganded NRs indirectly via the SRCs (5, 7), whereas PCAF is part of a multipolypeptide complex that can interact directly with liganded NRs and p300/CBP (18, 19). The TRAP complex is a multipolypeptide coactivator complex that interacts with liganded NRs via the TRAP220 subunit and may play a role in recruiting RNA polymerase II to the promoter (8, 20, 21). Recent chromatin immunoprecipitation (ChIP) experiments have demonstrated that after the binding of ligand, TRß first recruits SRC proteins and p300, resulting in histone acetylation, followed by the TRAP complex (22). Thus, coactivators facilitate transcriptional activation through at least two distinct, but not mutually exclusive, mechanisms: 1) acetylation of histones to facilitate the relief of chromatin-mediated repression; and 2) recruitment of the basal transcriptional machinery.

The packaging of DNA into chromatin represses gene expression (23) and specific biochemical mechanisms have been shown to relieve this repression (24). These include 1) acetylation of the positively charged amino-terminal tails of core histones, which is thought to loosen nucleosome structure and/or disrupt the formation of higher order chromatin structures (25, 26), as well as create new factor binding sites on the histone tails (27); and 2) ATP-dependent chromatin remodeling by complexes such as SWI/SNF, which use the energy of ATP hydrolysis to alter nucleosome structure and/or facilitate nucleosome mobility (24).

The role of chromatin remodeling in TR-mediated transcription has been well established (28). For example, previous studies have provided strong evidence that transactivation by liganded TRß/RXR{alpha} heterodimers appears to involve both HATs and ATP-dependent chromatin remodeling complex(es) (29, 30). However, additional studies have shown that histone acetylation itself is insufficient to fully activate the T3-regulated TSH{alpha} promoter (31). Thus, both chromatin remodeling and covalent histone modifications are important for the function of transcriptional activators such as liganded TRs.

Although it is clear that transcriptional activation by TRs involves a number of distinct coactivators, how those coactivators function together to facilitate TR-dependent transcription is not clear. Both the SRC coactivators and TRAP complex interact directly with liganded TRs (22, 32). It is not yet clear whether the SRC proteins and TRAP complex represent two distinct activation pathways or if they work in a sequential manner to facilitate activation by NRs (20). However, the SRC proteins and p300/CBP appear to function together to form an activation pathway. p300 is indirectly recruited to liganded TRß during the activation process through interactions with SRC proteins, as illustrated by the fact that p300 does not exhibit strong direct binding to the receptor (33) and that deletion of the SRC interaction domain in p300 greatly diminishes the coactivator activity of p300 with both TRß and estrogen receptor (ER; Refs. 33 and 34).

A biochemically defined in vitro chromatin-based transcription system is useful for addressing a number of questions related to the molecular mechanisms of transcriptional regulation. We have now established a TRß-dependent, T3-responsive in vitro transcription system using chromatin templates, HeLa cell nuclear extract, and purified recombinant receptor proteins and coactivator proteins. Using this system, we show that TRß/RXR{alpha} heterodimers induce promoter-proximal disruption of nucleosomal arrays upon binding to chromatin and that acetylation of nucleosomal histones enhances T3-dependent activation. We also demonstrate that both recombinant SRC and p300 proteins facilitate T3-dependent activation and that the primary role of SRC coactivators is to recruit p300. Finally, we show that multiple functional domains in p300 are critically important for its coactivator activity with TRß. Together, our results demonstrate how distinct coactivators function with liganded TRß to overcome chromatin-mediated transcriptional repression.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
T3-Independent Induction of Chromatin Remodeling by TRß/RXR{alpha} Heterodimers in Vitro
Recombinant Xenopus and human TRß and RXR{alpha} proteins (xTRß, xRXR{alpha}, hTRß, and hRXR{alpha}), including a transcriptionally inactive xTRß mutant (TRßm) containing a deletion of the last nine amino acids of the AF-2 activation domain, were purified to at least 85% purity in all cases and to near homogeneity in most cases (Fig. 1AGo and data not shown). In the functional studies described herein, no significant differences between the Xenopus and human receptor proteins were observed. Thus, we show only one example for each experiment and refer to the receptors collectively as TRß and RXR{alpha} for simplicity. Gel mobility shift assays (Fig. 1BGo) revealed that both TRß and TRßm bound efficiently to a consensus DR4 TRE (5'-AGGTCAnnnnAGGTCA-3') as TRß/RXR{alpha} heterodimers. Thus, the purified recombinant receptors are competent for DNA binding. The DR4 TRE was selected for the experiments described herein due to its selectivity for TRß/RXR{alpha} heterodimers over TRß homodimers (i.e. TRß/RXR{alpha} heterodimers bind to the DR4 TRE approximately 20-fold more efficiently than TRß homodimers; Refs. 35, 36, 37).



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  		Figure 1. Recombinant TRß/RXR{alpha} Heterodimers Bind to Chromatin and Induce Chromatin Remodeling

A, Purified recombinant RXR{alpha}, TRß, and TRßmut (TRßm) proteins were analyzed by SDS-PAGE with staining using Coomassie Brilliant Blue R-250. The purified receptors are marked by arrows, and the positions of molecular weight markers are indicated. B, Purified recombinant TRß and TRßm proteins bind to a TRE (DR4) element primarily as heterodimers with RXR{alpha} (receptor-DNA shifted complexes are indicated by an arrow). The purified TRß proteins were mixed in 1:1 molar ratio with RXR{alpha} proteins and used for gel shift analysis. C, T3-independent binding of TRß/RXR{alpha} to chromatin assembled in vitro. 4xTRE-pS2 (100 ng) was assembled into chromatin with or without receptors (15 nM each) as indicated. The DNase I-primer extension footprinting experiments were carried out in duplicate. A schematic diagram of the 4xTRE-pS2 template is shown (side) and the positions of the TREs are indicated. The major hypersensitive sites flanking the TREs are indicated by the black arrows. Note that no significant protection is observed in lanes with TRß or RXR{alpha} alone. D, TRß/RXR{alpha} heterodimers induce localized, T3- and AF-2-independent chromatin remodeling as revealed by an MNase array disruption assay using the template 4xTRE-TK assembled into chromatin in vitro. A schematic diagram of the 4xTRE-TK template is shown (top). The positions of the subnucleosomal (sub), mono-, di-, tri-, and tetranucleosomal fragments are indicated. The filter was hybridized with a TRE probe (top panel), stripped, and then rehybridized with a control probe (bottom panel).

 
To characterize further the recombinant receptors, we tested whether TRß/RXR{alpha} could bind to TREs in chromatin by deoxyribonuclease (DNase) I footprinting (Fig. 1CGo). Using the 4xTRE-pS2 template assembled into chromatin in vitro, we assessed the binding of RXR{alpha} (lane 2), TRß (lane 3), or TRß/RXR{alpha} heterodimers (lanes 4 and 5). Each binding reaction was partially digested with DNase I and the resulting DNA products were analyzed by primer extension. Addition of TRß/RXR{alpha} heterodimers resulted in the protection of all four TRE sites in both the absence (lane 4) and presence of T3 (lane 5), consistent with previous studies showing that TRß/RXR{alpha} can bind to TREs in chromatin and repress transcription in a hormone-independent manner (29). These results also illustrate the importance of heterodimerization with RXR{alpha} for efficient binding of TRß to the DR4 TRE in chromatin (compare lanes 3 and 4).

Next, we performed micrococcal nuclease (MNase) array disruption assays to determine if TRß/RXR{alpha} heterodimers could induce chromatin remodeling. Hybridization using a TRE-specific probe revealed that the addition of TRß/RXR{alpha} heterodimers in the absence of T3 led to chromatin remodeling, evidenced by a substantial loss of a defined nucleosomal ladder (Fig. 1DGo, TRE probe). Importantly, chromatin remodeling was localized to the promoter-proximal region, as shown in subsequent experiments with the same blots using a control probe that hybridizes about 2 kb downstream of the TRE (Fig. 1CGo, control probe; note the intact MNase ladder). Subnucleosomal DNA fragments, representing nucleosome-free DNA fragments protected by the binding of TRß/RXR{alpha} heterodimers, were detected in all samples containing receptors (Fig. 1DGo, TRE probe, "Sub") but were not detected using the control probe. Heterodimers containing the transcriptionally inactive TRßm also induced efficient chromatin remodeling (Fig. 1DGo), demonstrating that the AF-2 domain is dispensable for the chromatin remodeling activity. Subsequent experiments comparing chromatin remodeling induced by TRß/RXR{alpha} heterodimers in the presence or absence of ligand indicated that addition of T3 had no effect on the extent of chromatin remodeling (data not shown). In addition, chromatin remodeling was observed regardless of whether the TRß/RXR{alpha} heterodimers were added during or after chromatin assembly (data not shown). Together, the footprinting and nucleosome disruption assays indicate that the binding of TRß/RXR{alpha} heterodimers to chromatin induces promoter-proximal chromatin remodeling, even with transcriptionally inactive receptors (i.e. unliganded or AF-2 mutant).

Transcriptional Repression and Activation by TRß/RXR{alpha} Heterodimers in Vitro
The addition of either TRß/RXR{alpha} or TRßm/RXR{alpha} heterodimers to unassembled ("naked") 4xTRE-TK DNA template led to a strong repression of transcription (Fig. 2AGo, lanes 1–5). This repression required the presence of TREs, as a control template lacking TREs was only marginally affected (data not shown). Although preincubation of the receptors with T3 relieved repression by unliganded TRß/RXR{alpha} (compare lanes 4 with 5), no activation above the basal level was observed (compare lanes 1 with 5). In contrast, preincubation with T3 failed to relieve repression by TRßm/RXR{alpha} (lane 3). Thus, an intact AF-2 activation domain is not required for basal repression by unliganded TRß but is required for the relief of repression in the presence of T3.



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  		Figure 2. Transcriptional Activation by TRß/RXR{alpha} with Naked DNA and Chromatin Templates

A, Comparison of transcriptional activation by TRß/RXR{alpha} on naked DNA (left panel) and chromatin templates (right panel) using in vitro transcription assays with the 4xTRE-TK template. In lanes where the addition of T3 is indicated, TRß/RXR{alpha} or TRßm/RXR{alpha} heterodimers (15 nM) were preincubated with 1 µM T3 for 20 min before being added to the transcription reactions. The transcripts were detected by primer extension. The level of transcription in the absence of ligand and receptors (lanes 1 and 6) was arbitrarily designated as 1. B, The addition of both TRß and RXR{alpha} is required for efficient ligand-dependent transcription with chromatin templates in vitro. In vitro chromatin assembly and transcription experiments were performed as described in panel A using the 4xTRE-E4 template. TRß, RXR{alpha}, and were added as indicated. C, Western blot of HeLa cell nuclear extract demonstrating the presence of RXR{alpha}. Two different volumes of the extract were analyzed as indicated. Aliquots of recombinant RXR{alpha} and MDA-MB-231 breast cancer cell nuclear extract were run in adjacent lanes for comparison.

 
Several important differences were observed when the template was assembled into chromatin and used for in vitro transcription with TRß/RXR{alpha}. First, as expected, basal levels of transcription were dramatically reduced when compared with the naked DNA template (about 50- to 100-fold; Fig. 2AGo, compare lanes 1 and 6). Furthermore, a strong T3-dependent activation was observed (Fig. 2AGo, compare lanes 6 and 10). This activation required an intact AF-2 activation domain because TRßm/RXR{alpha} failed to activate under the same conditions (Fig. 2AGo, lanes 6–8). Thus, this in vitro system accurately recapitulates T3-dependent activation and the requirement for an intact AF-2 domain. T3-dependent activation with chromatin templates is not specific to the thymidine kinase (TK) promoter as both the human pS2 and adenovirus E4 promoters gave similar results (see below). Interestingly, repression by unliganded TRß/RXR{alpha} was not observed with the chromatin templates, most likely due to the fact that chromatin assembly itself repressed transcription to a marginally detectable level.

Significant contributions of TRß homodimers to the receptor-dependent transcriptional effects shown in Fig. 2AGo are unlikely for the following reasons. First, as mentioned above, TRß homodimers do not bind strongly to the DR4 TRE in our reporter templates (35, 36, 37). Second, RXR{alpha} is required for efficient binding of TRß to the DR4 TRE-containing chromatin templates used in our in vitro transcription studies, as shown in Fig. 1CGo. To examine this issue more directly, we performed in vitro transcription experiments with chromatin templates using TRß in the presence or absence of RXR{alpha} (Fig. 2BGo). As noted above, the addition of recombinant TRß/RXR{alpha} heterodimers gave a robust T3-dependent transcriptional response in this assay (lane 7). The addition of TRß without RXR{alpha} also gave a T3-dependent transcriptional response, but the magnitude of the effect was about 2.5-fold less than that observed with TRß/RXR{alpha} heterodimers (lane 6). Why would the addition of TRß alone give a transcriptional response when TRß alone does not bind to DR4 TRE with chromatin templates? This is likely due to the presence of RXR{alpha} (and possibly other RXR subtypes) in the HeLa cell nuclear extracts that we used for the in vitro transcription studies (see the Western blot in Fig. 2CGo). Because the footprinting assays were performed in the absence of the HeLa cell nuclear extract, the effects of endogenous HeLa cell RXR{alpha} were not observed. Taken together, the available data indicate that the transcriptional responses that we observed are due to TRß/RXR{alpha} heterodimers and not TRß homodimers.

Histone Acetylation Enhances TRß-Mediated Transcription
The levels of histone acetylation in vivo are ultimately determined by the balance of HAT and histone deacetylase (HDAC) activities. Our in vitro chromatin assembly and transcription system contains both endogenous HATs and HDACs, as well as acetyl-coenzyme A (CoA; Kraus, W. L., unpublished observations). To test the effect of histone acetylation on T3-dependent activation in vitro, we used a specific HDAC inhibitor, trichostatin A (TSA), to block deacetylation and increase the overall levels of histone acetylation. As shown in Fig. 3AGo, the addition of TSA (1 µM) to the chromatin assembly reaction led to an approximately 2-fold increase in bulk histone H4 acetylation (compare lanes 1 and 4), whereas addition of liganded or unliganded TRß/RXR{alpha} alone had no effect (lanes 2 and 3). In vitro transcription experiments demonstrated that the addition of TSA enhanced T3-dependent activation by about 2.5-fold (Fig. 3BGo, compare lanes 4 and 8). The addition of TSA also enhanced transcription in the absence of receptors (lanes 1, 2, 5, and 6), indicating that increased acetylation of bulk histones has a general stimulatory effect on both basal and TRß-mediated transcription.



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  		Figure 3. Promoter-Targeted Histone Acetylation Enhances Transcriptional Activation by Liganded TRß/RXR{alpha}

A, TSA treatment enhances the level of histone acetylation in chromatin. Reactions were identical to the transcription assays described in Fig. 2BGo except that 1 µM of TSA was added to the reaction in lane 4. Histone acetylation was analyzed by Western blotting using an anti-acetylated H4 antibody. B, TSA treatment enhances T3-dependent activation. Chromatin templates were assembled with or without TSA (1 µM) as in (A) and used for transcription assays. The relative transcription was quantified using a PhosphorImager and represents the average results from two independent experiments. C, Liganded TRß/RXR{alpha} induces a localized acetylation of histones H4 and H3 as assessed by in vitro ChIP assays. A schematic diagram of the 4xTRE-TK construct and the locations of the oligonucleotide probes used for slot blot hybridization are shown (top). Chromatin assembly and incubation with TRß/RXR{alpha} and HeLa cell nuclear extracts were as described in (A). ChIP assays were performed with anti-acetylated H4 and antiacetylated H3 antibodies with subsequent analysis by sequential slot-blot hybridization using a promoter region probe (TATA) and a control probe, as indicated. The data shown are averaged from three independent experiments are standardized to the input signal for each experiment (see representative input at right).

 
To assess whether liganded TRß/RXR{alpha} is able to induce localized chromatin acetylation through the recruitment of coactivators with intrinsic HAT activities, we used an in vitro ChIP assay (Fig. 3CGo). 4xTRE-TK was assembled into chromatin in the presence or absence of receptors, ligand, and TSA, as indicated. The chromatin templates were then used in reactions under transcription conditions, except that ribonucleotide 5'-triphosphates were omitted to avoid complications due to potential effects of transcriptional elongation on acetylation. After extensive MNase digestion, the chromatin was immunoprecipitated with antibodies specific for the acetylated forms of either histone H4 or H3, and the coimmunoprecipitated DNA was analyzed by slot-blot hybridization. The presence of liganded TRß/RXR{alpha} led to about a 3-fold enhancement of H4 and H3 acetylation in the promoter region, as evidenced by an enrichment of DNA in lanes 8 and 12 compared with lanes 5 and 9, respectively, with a probe specific for the TATA box (top panel). This ligand- and receptor-dependent enhancement of H4 and H3 acetylation was localized to the promoter region, as a control probe located about 2 kb downstream of the promoter showed little, if any, increase above the basal level (bottom panel). In contrast, TSA treatment led to about a 7.5-fold increase in acetylation in both the proximal and distal locations (lanes 6 and 10), indicating that the hyperacetylation of chromatin induced by TSA is not a targeted event. Thus, liganded TRß/RXR{alpha} can target histone acetylation to T3-activated promoters, presumably through hormone-dependent recruitment of HATs such as p300/CBP and PCAF.

SRC Proteins and p300 Synergistically Stimulate TRß-Dependent Transcription in Vitro
Next, we examined whether the addition of purified recombinant SRC-3 and p300 could stimulate TRß-dependent activation in vitro. Full-length FLAG-tagged SRC-3 protein was purified from microinjected Xenopus oocytes (Fig. 4AGo) and full-length his6-tagged p300 protein was purified from baculovirus-infected Sf9 cells (Fig. 4CGo). As shown in Fig. 4BGo, the addition of increasing amounts of SRC-3 stimulated TRß-dependent activation approximately 3.5-fold in the presence of T3. Similar results were also obtained when purified recombinant SRC-1 and SRC-2 proteins were used under similar conditions (data not shown). As shown in Fig. 4DGo, the addition of p300 stimulated TRß-dependent activation approximately 4-fold in the presence of T3. Thus, both SRC proteins and p300 are able to stimulate TRß-dependent activation with chromatin templates in vitro.



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  		Figure 4. Recombinant SRC-3 and p300 Enhance TRß-Dependent Transcription with Chromatin Templates in Vitro

A, Purified SRC-3 was analyzed by SDS-PAGE with staining using Coomassie Brilliant Blue R-250. B, Addition of SRC-3 stimulates T3-dependent transcription with chromatin templates. 4xTRE-TK (see schematic, top) was assembled into chromatin and used for in vitro transcription in the presence of TRß/RXR{alpha}, T3, and increasing amounts of SRC-3 protein (1.25 nM, 2.5 nM, and 5 nM), which was added to the reactions in lanes 4 through 6 after chromatin assembly was complete. C, Purified p300 was analyzed by SDS-PAGE with staining using Coomassie Brilliant Blue R-250. D, Addition of p300 stimulates T3-dependent transcription with chromatin templates. 4xTRE-pS2 (see schematic, top) was assembled into chromatin and used for in vitro transcription in the presence of TRß/RXR{alpha}, T3, and p300 protein (5 nM), which was added to the reactions in lanes 4–6 after chromatin assembly was complete.

 
To further elucidate the molecular mechanisms by which SRC and p300 stimulate TRß-dependent activation, we investigated potential synergistic interactions between the two coactivators. Recombinant full-length SRC-3 protein and an SRC-2 protein containing only the receptor interaction domain (RID) and p300/CBP-interaction domain (PID; amino acids 624-1130; Figs. 5Go, A and B) were added to in vitro transcription experiments with TRß/RXR{alpha} and p300. Note that the amount of p300 was reduced relative to the experiments in Fig. 4Go to expose potential synergism with the SRCs. With the E4 promoter, p300 and the SRCs individually stimulated TRß-dependent transcription about 10-fold and 2- to 3-fold, respectively (Fig. 5CGo, top; lanes 2–5). When added together, p300 and the SRCs acted synergistically, producing about a 20-fold increase in TRß-dependent transcription relative to the same conditions without exogenously-added coactivators [Fig. 5CGo, top; compare lanes 3, 5, and 7 for p300 and SRC-3; lanes 3, 4, and 6 for p300 and SRC-2(RID/PID)]. With the pS2 promoter, p300 stimulated TRß-dependent transcription about 2-fold, but the SRCs had little or no effect when added alone (Fig. 5CGo, bottom; lanes 2–5). However, when added together, p300 and SRC gave about a 3-fold increase in TRß-dependent transcription with the pS2 promoter [Fig. 5CGo, bottom; see lanes 3, 5, and 7 for p300 and SRC-3; lanes 3, 4, and 6 for p300 and SRC-2(RID/PID)]. Interestingly, the effects of SRC-2(RID/PID) were generally similar to the effects observed with full-length SRC-3 (Fig. 5CGo, compare lanes 4 and 5, and 6 and 7), indicating that the receptor and p300/CBP interaction domains of SRC-2 are sufficient to mediate coactivator activity. Together, our results indicate that p300 and SRC proteins function synergistically to stimulate TRß-mediated transcription with chromatin templates, and that a central role for SRC is to recruit p300 to the liganded receptor.



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  		Figure 5. SRC and p300 Synergistically Stimulate Ligand-Dependent Transcription by TRß/RXR{alpha}

A, Generalized schematic diagram of the SRC coactivators, including the following functional domains: basic helix-loop-helix (bHLH) domain, Per/Arnt/Sim (PAS) domain, RID, PID, and the glutamine (Q)-rich region. Also indicated are regions that bind PCAF and CARM1. SRC-2(RID/PID) contains only amino acids 624-1130 of SRC-2. B, Purified SRC-2(RID/PID) and SRC-3 were analyzed by SDS-PAGE with staining using Coomassie Brilliant Blue R-250. C, Effects of the addition of SRC and p300 on TRß-mediated transcription with chromatin templates. 4xTRE-E4 and 4xTRE-pS2 (see schematics at right) were assembled into chromatin and used for in vitro transcription in the presence of TRß/RXR{alpha} (4.5 nM), T3 (1 µM), p300 (0.5 nM), SRC-3 (5 nM), and SRC-2(RID/PID) (5 nM), as indicated. Note that the p300 amount was reduced compared with the experiments in Fig. 4Go to show synergism with the SRCs. The RNA products were analyzed by primer extension.

 
Multiple Domains of p300 Are Required for Its Coactivator Activity with TRß
p300/CBP contains multiple functional domains, including a bromodomain, three cys/his (CH)-rich regions, an acetyltransferase domain, and an SRC interaction domain (Fig. 6AGo). The bromodomain is found in many chromatin- and transcription-related factors and is believed to be important in histone binding and other protein-protein interactions (38, 39, 40). The CH3 region is a protein interaction domain that interacts with a variety of factors, including PCAF (15), transcription factor IIB (TFIIB) (41), and RNA polymerase II (42). To further explore the role of p300 in TRß-mediated transcription, we used a set of previously characterized p300 mutants (Fig. 6AGo; Ref. 34). The mutants included: 1) a bromodomain deletion ({Delta}Bromo), 2) an AT mutant (MutAT2), 3) a CH3 region deletion ({Delta}CH3), and 4) an SRC interaction domain deletion ({Delta}SRC). The purified mutant p300 proteins (Fig. 6BGo) were used in TRß-dependent transcription reactions with the 4xTRE-pS2 template assembled into chromatin. The use of these mutants, in conjunction with the additional experimental approaches shown in Figs. 7Go and 8Go, allowed us to address the role of p300-SRC interactions and p300 HAT activity in TRß-mediated transcription, as well as questions about the other functional domains in p300.



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  		Figure 6. Multiple Functional Domains of p300 Are Important for Stimulating TRß-Dependent Transcription with Chromatin Templates

A, Schematic diagrams of wild-type and mutant p300 proteins used in this study showing the various functional domains of p300: bromodomain (Bromo), AT domain, CH-rich region 3 (CH3), and the glutamine (Q)-rich region. Also indicated are regions that bind the adenovirus E1A protein, PCAF, TFIIB, and RNA polymerase II (pol II). B, Purified wild-type and mutant p300 proteins were analyzed by SDS-PAGE with staining using Coomassie Brilliant Blue R-250. C, Effects of the addition of wild-type and mutant p300 proteins (5 nM) on TRß-mediated transcription with chromatin templates. 4xTRE-pS2 (see schematic at bottom) was assembled into chromatin and used for in vitro transcription as described in Fig. 4CGo. D, Graphical representation of the results in (C) shown in comparison with the results from similar experiments performed with ER{alpha}. Only the contributions from the exogenously added recombinant p300 proteins are included in the results. Each bar represents the mean + the SEM from three independent experiments.

 


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  		Figure 7. p300-SRC Interactions Are Required for Full Transcriptional Activation by TRß

A, Polypeptide inhibitors reduce p300 stimulated TRß-activated transcription. The template 4xTRE-E4 (see schematic Fig. 5CGo) was assembled into chromatin and used for in vitro transcription in the presence of TRß/RXR{alpha} (4.5 nM), T3 (1 µM), and p300 (5 nM) and GST peptide (225 nM) where indicated. Wild-type (W) or mutant (M) versions of GST-SRC2(PID) and GST-p300(SID) (225 nM) were added as indicated before transcription. B, Graphical representation of results shown in (A). Each bar represents the mean + the SEM from four experiments with the level of transcription in the presence of liganded receptor set at 100%.

 


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  		Figure 8. Both p300 and PCAF HAT Activities Contribute to TRß-Mediated Transcription with Chromatin Templates

The 4xTRE-TK reporter (see schematic, top) was assembled into chromatin and used for in vitro transcription in the presence of TRß/RXR{alpha}, T3, and increasing amounts of either H3-CoA-20 (a PCAF-selective HAT inhibitor) or Lys-CoA (a p300/CBP-selective HAT inhibitor) as indicated (+, 2 µM; ++, 4 µM; +++, 8 µM). The transcription and primer extension assays were essentially as described in Fig. 1Go except that HeLa nuclear extracts were preincubated with the indicated amounts of the HAT inhibitors for 10 min on ice before addition to the transcription assays. The level of transcription in the absence of ligand and receptors (lane 1) was designated as 1.

 
Deletion of the SRC interaction domain dramatically reduced the ability of p300 to enhance TRß-dependent transcription (Fig. 6CGo, compare lanes 4 and 8). This result further supports the conclusion that recruitment of p300 to TRß via the SRCs is essential for p300 coactivator activity with TRß. Deletion of either the bromodomain or the CH3 region also led to a substantial reduction in p300 activity (Fig. 6CGo, compare lanes 4, 5, and 7), indicating that both domains are important for p300 coactivator activity with TRß. The p300 MutAT2 protein, which has about 1% of wild-type HAT activity (34), showed a 50% reduction in coactivator activity with TRß. Thus, p300 HAT activity is also needed for full coactivator activity with TRß (see additional experiments presented below that address this issue further). For comparison, the results from multiple experiments with TRß were quantified and plotted vs. results from similar experiments using ER{alpha} (Fig. 6DGo). It is clear from this comparison that multiple p300 functional domains are required for maximal coactivator activity with both TRß and ER{alpha} with chromatin templates.

Inhibition of p300-SRC Interactions or p300 HAT Activity Inhibits TRß-Mediated Transcription
To evaluate further the role of p300-SRC interactions and p300 HAT activity in TRß-mediated transcription, we used previously characterized polypeptide inhibitors of p300-SRC interactions and chemical inhibitors of HAT activity in chromatin transcription experiments with TRß. First, to block p300-SRC interactions, we used a set of glutathione-S-transferase (GST)-fused polypeptides containing either the PID of SRC-2 or the SRC interaction domain (SID) of p300. These polypeptides, which have previously been shown to be potent inhibitors of ER{alpha}-mediated transcription (43), specifically and competitively block interactions between the endogenous SRC proteins in the HeLa cell transcription extract and exogenously-added p300 (43). Both GST-SRC-2(PID) and GST-p300(SID) inhibited the enhancement of TRß-mediated transcription by p300 (Fig. 7AGo, compare lanes 2 and 3 with lanes 4 and 5; Fig. 7BGo). The inhibitory effects were not observed with mutant versions of the same polypeptides ("Mut") that fail to bind their cognate partners (Fig. 7AGo, compare lanes 4 and 5 with lanes 6 and 7; Fig. 7BGo). These results complement our results with the p300{Delta}SRC mutant protein (Fig. 6CGo) and illustrate further the critical role that p300-SRC interactions play in TRß-mediated transcription with chromatin templates.

Second, to explore further the role of p300 HAT activity in TRß-mediated transcription, we used Lys-CoA, a chemical inhibitor that selectively blocks p300/CBP HAT activity (44, 45, 46). Lys-CoA is at least 200-fold more potent at blocking p300/CBP HAT activity than the HAT activities of GCN5, PCAF, Mof, and Esa1 (Cole, P. A., unpublished observations). For comparison, we also used H3-CoA-20, a chemical inhibitor that selectively blocks PCAF HAT activity (44). As shown in Fig. 8Go, increasing amounts of either inhibitor caused a reduction in TRß-mediated transcription, although the p300/CBP-selective Lys-CoA was a more effective inhibitor than the PCAF-selective H3-CoA-20. These results suggest a role for both p300/CBP and PCAF HAT activities in TRß transcriptional activity, although nonspecific effects of these inhibitors cannot be ruled out. The results with Lys-CoA are in agreement with our results using the p300 HAT mutant (Fig. 6Go), further supporting a role for p300 HAT activity in TRß-mediated transcription. Differences in the magnitude of the effects with the chemical inhibitor and the p300 mutant may be due to promoter-specific effects (pS2 vs. TK) or the fact that the Lys-CoA targets the endogenous p300 in the transcription extract, whereas the p300 HAT mutant is an addition to the endogenous levels of p300. Nonetheless, both approaches indicate an important role for p300 HAT activity in TRß-mediated transcription. Furthermore, the possible contribution of PCAF HAT activity to TRß-mediated transcription may also provide an explanation for the lack of an absolute requirement for p300 HAT activity under certain promoter contexts (Fig. 6Go).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have used an in vitro chromatin assembly and transcription system that accurately recapitulates ligand- and AF-2-dependent transcriptional activation by TRß/RXR{alpha} heterodimers to study TRß-mediated transcription with chromatin templates. Together, our results demonstrate that a biochemical approach that includes chromatin is a useful way to study the mechanisms of TRß-mediated transcription. Below, we highlight the most significant results from our studies.

Chromatin Remodeling by TRß/RXR{alpha} Heterodimers in Vitro
In a previous report, we showed that TRß/RXR{alpha} heterodimers induce an extensive, localized disruption of chromatin only in the presence of T3 in Xenopus oocytes (47). In contrast, we show herein that TRß/RXR{alpha} heterodimers induce a localized disruption of chromatin in a T3-independent manner (Fig. 1DGo). In fact, other receptors, such as ER{alpha}, progesterone receptor, and retinoic acid receptor (RAR)-{alpha}/RXR{alpha}, also induce ligand-independent remodeling in this in vitro system (data not shown; Refs. 48 and 49). The discrepancy between these two systems is most likely due to differences in the concentrations or types of ATP-dependent chromatin remodeling factors present in Xenopus oocytes and the Drosophila embryo extracts used for our in vitro chromatin assembly experiments. Drosophila embryo extracts are highly enriched for ATP-dependent chromatin remodeling factors such as nucleosome remodeling factor (NURF), chromatin accessibility complex (CHRAC), and ATP-utilizing chromatin assembly and remodeling factor (ACF) (Refs. 50, 51, 52), which are capable of inducing nucleosome sliding without nucleosome displacement (24). The chromatin remodeling induced by TRß/RXR{alpha} in our in vitro system (Fig. 1DGo) most likely reflects the binding of TRß/RXR{alpha} heterodimers to nucleosome-free TREs produced by the actions of chromatin remodeling factors in the absence of specific recruitment. Although TRß/RXR{alpha}-mediated chromatin remodeling in vitro is ligand independent, it is likely to be a prerequisite for subsequent transcriptional activation (53). This idea is consistent with a recent report demonstrating that the efficient binding of RAR{alpha}/RXR{alpha} heterodimers to chromatin requires ATP-dependent chromatin remodeling factors (54) and that the ATP-dependent chromatin remodeling factor NURF stimulates transcription activation by the synthetic activator GAL4-VP16 (55).

Role of Histone Acetylation in TRß-Mediated Transcription
The inclusion of the HDAC inhibitor TSA in the in vitro system increased the acetylation of histones over the whole population of nucleosomes on the plasmid templates (Fig. 3CGo). The increased acetylation was correlated with a 2- to 3-fold increase in ligand-dependent transcription by TRß/RXR{alpha} (Fig. 3BGo). However, increased acetylation was also correlated with increased transcription in the absence of receptor and by unliganded TRß/RXR{alpha}, consistent with the idea that acetylation of histones has a general positive effect on transcription (Fig. 3BGo). Thus, although increased histone acetylation is likely to enhance TRß transcriptional activity, the effects of TSA are not specific. In contrast, TRß-mediated histone acetylation in our in vitro ChIP assay was specific for the promoter region (Fig. 3CGo). These results are consistent with recent reports that liganded RAR{alpha} and ER{alpha} are able to induce histone acetylation at the promoters of hormone target genes in mammalian cells (56, 57) and in biochemical assays (43, 54). In agreement with recently reported ChIP data (22), our results indicate that liganded TRß/RXR{alpha}, once bound to TRE elements in chromatin, can recruit HATs such as p300/CBP and PCAF, which in turn acetylate the adjacent nucleosomes. The acetylation of nucleosomes in the promoter region helps to relieve chromatin-mediated repression and facilitate transcriptional activation.

With regard to the specific HAT enzymes involved in TRß-mediated transcription, our results with the p300 HAT mutant and the chemical inhibitor Lys-CoA indicate that p300 HAT activity (and possibly CBP HAT activity, as well) is required for full transcriptional activation by TRß with chromatin templates (Figs. 6Go and 8Go). These results are in agreement with previous studies using Xenopus oocytes in which the HAT activity of p300 was shown to be required for the stimulation of T3-dependent activation by TRß/RXR{alpha} (33). Our results with the chemical inhibitor H3-CoA-20 (Fig. 8Go) suggest a role for PCAF HAT activity in TRß-dependent transcription with chromatin templates as well, although nonspecific effects with the H3-CoA-20 inhibitor cannot be ruled out (Ref. 44 ; and Cole, P. A., unpublished observations). Interestingly, we found little evidence for a contribution of the putative SRC HAT activity in TRß-mediated transcription, as a fragment of SRC-2 containing only the receptor and p300/CBP interaction domains gave activity similar to that of full-length SRC proteins (Fig. 5Go and data not shown). Together, our results indicate that specific coactivators and their associated enzymatic actions on nucleosomal histones play important roles in transcriptional regulation by TRß.

A Critical Role for SRC-p300 Interactions in TRß-Mediated Transcription
Our studies with TRß indicate that p300 is recruited indirectly to promoter-bound TRß through its interaction with SRC proteins. This conclusion is supported by the following results. First, we showed that purified recombinant SRC-3 and p300 synergistically enhance TRß-mediated transcription with chromatin templates (Fig. 5CGo). Second, a fragment of SRC-2 containing only the receptor and p300/CBP interaction domains was able to synergize with p300 and was functionally equivalent to full-length SRC-3 (Fig. 5CGo). Third, a p300 mutant lacking the SRC interaction domain (p300{Delta}SRC) was unable to function as a coactivator for TRß/RXR{alpha} (Fig. 6DGo). Finally, polypeptide inhibitors that directly interfere with TRß-SRC-p300 interactions blocked the ability of p300 to stimulate TRß-mediated transcription. Thus, a primary role for the SRC proteins is to recruit p300/CBP to liganded receptors, and the TRß-SRC-p300/CBP pathway constitutes one pathway for the activation of T3-dependent transcription by TRß. These conclusions apply to other nuclear receptors and are further supported by some recent cell-based studies (12, 22, 33, 58), as well as in vitro transcription analyses (43, 48, 59).

Multiple Domains in p300 Contribute to Its Coactivator Activity with TRß
Both p300 and CBP are multifunctional proteins that stimulate the transcriptional activity of many different transcriptional activators, including NRs (17). Our results indicate that in addition to the SRC-interaction domain discussed above, both the bromodomain and CH3 region are critically important for p300 coactivator activity with TRß. We have previously shown that both of these domains are critical for p300 coactivator activity with a variety of transcriptional activators, including ER{alpha}, nuclear factor-{kappa}B p65, and Gal4-VP16 (34). The bromodomain is found in many chromatin- and transcription-related factors and is believed to be important in chromatin binding and/or protein-protein interactions (38, 39, 40). We have recently shown that the p300 bromodomain mediates the stable interaction of p300 with chromatin and is important for p300 nucleosomal HAT activity (34, 40). The p300 CH3 region has been found to interact with proteins such as TFIIB and RNA polymerase II (41, 42). Thus, the loss of coactivator activity for p300{Delta}CH3 could be explained by its inability to interact with or recruit components of the basal transcriptional machinery.

Together, our results suggest a multiple step pathway for transcriptional regulation by liganded TRß, including chromatin remodeling, recruitment of bridging and HAT coactivators (e.g. SRCs and p300/CBP, respectively), targeted histone acetylation, and recruitment of the RNA polymerase II transcriptional machinery. Multiple step pathways may be a universal feature for transcriptional activation by NRs (60). The absence of transcriptional activation above basal levels with naked DNA (Fig. 2AGo), as well as the requirement for ligand and an intact AF-2 domain (both of which facilitate coactivator recruitment) for activation with chromatin templates (Fig. 2BGo) suggest that coactivators such as SRC and p300 function by alleviating the repressive effects of chromatin at the promoter. Our results suggest that, for TRß/RXR{alpha} heterodimers, this can be achieved through targeted histone acetylation and specific contacts with the transcriptional machinery.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Expression and Reporter Plasmids
The bacterial expression constructs for Xenopus RXR{alpha} (pET15b-xRXR{alpha}) and TRß (pET15b-xTRß) have been described before (61). The bacterial expression construct for xTRßm with a deletion of the last nine amino acids of the AF-2 domain was generated by replacing the wild-type xTRß in the pET15b-xTRß with the xTRßm1 from the pSP64-TRm1 plasmid described previously (47). The 4xTRE-pS2 reporter template is the same as 2xERE-pS2 (62) except that the EREs have been replaced with four TREs. Both the 4xTRE-E4 and 4xTRE-TK reported templates were constructed by inserting four TREs upstream of the adenovirus E4 promoter in pIE0 or the herpes simplex virus TK promoter in pTK-CAT, respectively. The sequence of the DR4 TRE used in our studies is 5'-GATATCAGGTCATTTCAGGTCAGCATGC-3'.

Expression and Purification of Recombinant Proteins
Purification of bacterially expressed his6-tagged Xenopus TRß, TRßm, and RXR{alpha} was by nickel nitrilotriacetic acid (Ni-NTA) agarose chromatography (QIAGEN, Valencia, CA) followed by Mono S chromatography (Amersham Pharmacia Biotech, Arlington Heights, IL). The receptors were eluted from the Mono S column with a salt gradient from 100–500 mM KCl. His6-tagged human TRß and RXR{alpha} were prepared from baculovirus-infected Sf9 cells by Ni-NTA agarose chromatography as described before for p300 (63). Full-length SRC-3 containing an amino terminal FLAG tag was prepared from microinjected Xenopus oocytes as described (48). Bacterially expressed his-tagged SRC-2(RID/PID; amino acids residues 624-1130) was purified by Ni-NTA agarose chromatography as described before (43). Wild-type and mutant his6-tagged p300 proteins were prepared from baculovirus-infected Sf9 cells by Ni-NTA agarose chromatography as described before (34, 63). GST-tagged wild-type and mutant polypeptide inhibitors [SRC-2(PID), SRC-2(PID)Mut, p300(SID), p300(SID)Mut] were purified as previously described (43). All purified recombinant proteins were evaluated by SDS-PAGE with staining using Coomassie Brilliant Blue R-250.

In Vitro Chromatin Assembly and Transcription
Chromatin assembly reactions were performed with an S-190 chromatin assembly extract derived from Drosophila embryos as previously described (48, 63). TRß/RXR{alpha} or TRßm/RXR{alpha} proteins were added to the chromatin assembly reactions either at the beginning of chromatin assembly or after the assembly reactions were complete. SRC and p300 proteins, as well as the polypeptide inhibitors, were added after chromatin assembly, before the addition of transcription extract. In vitro transcription reactions were performed with HeLa cell nuclear extracts that were prepared essentially by the method of Dignam et al. (64). Transcription reactions were set up under conditions described previously (43, 48, 63). The reactions were performed in duplicate, but single samples from each experiment are shown in the figures. The data were analyzed and quantified with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Each experiment was run a minimum of two separate times, but more typically three or more separate times, to ensure reproducibility.

DNase I Footprinting, Micrococcal Nuclease Array Disruption Assays, and ChIP
DNase I-primer extension footprinting to examine the binding of TRß/RXR{alpha} to chromatin was performed as described previously (65). Analysis of TRß/RXR{alpha}-induced chromatin remodeling by MNase array disruption assays was performed essentially as described (48, 65). For the ChIP assays, reactions were set up as for the transcription assays except that ribonucleotide 5'-triphosphates were not added. In the experiments with addition of TSA, no acetyl-CoA was added due to the presence of acetyl-CoA in the Drosophila S-190. The chromatin was then digested extensively with MNase (10 U/reaction for 10 min at room temperature). The ChIP assays were performed essentially as described (54), except that the DNA in the immunoprecipitated fractions was recovered directly by phenol/chloroform extraction and ethanol precipitation. The immunoprecipitated DNA was then analyzed by slot-blot hybridization with 32P-labeled oligo probes as indicated.


   ACKNOWLEDGMENTS
 
We thank Carl Wu and Jim Kadonaga for assistance with the Drosophila chromatin assembly extracts, Tory Manning for the p300 proteins, and Mi Young Kim for the SRC-2(RID/PID) and GST-polypeptide inhibitor constructs.


   FOOTNOTES
 
This work was supported by NIH postdoctoral fellowship F32-DK-59702 (to K.C.L.), NIH Grants R01-DK-56324 and R01-DK-58679 (to J.W.), and NIH Grant R01-DK-58110 and a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund (to W.L.K.).

1 K.C.L. and J.L. are equal contributors. Back

2 J.W. and W.L.K. are equal contributors. Back

Abbreviations: AF, Activation function; AT, acetyltransferase; CBP, cAMP binding protein-binding protein; CH, cys/his; ChIP, chromatin immunoprecipitation; CoA, coenzyme A; ER, estrogen receptor; DNase, deoxyribonuclease; DRIP, vitamin D receptor-interacting proteins; GST, glutathione-S-transferase; HAT, histone acetyltransferase; HDAC, histone deacetylase; MNase, micrococcal nuclease; NR, nuclear receptor; PID, p300/CBP interaction domain; RID, receptor interaction domain; RXR, 9-cis retinoic acid receptor; SID, SRC interaction domain; SRC, steroid receptor coactivator; TK, thymidine kinase; TR, thyroid hormone receptor; TRßm, transcriptionally inactive xTRß mutant; TRAP, TR-associated proteins; TRE, thyroid hormone response element; TSA, trichostatin A.

Received for publication September 4, 2002. Accepted for publication February 3, 2003.


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NURSA Molecule Pages Link:

Nuclear Receptors:   TRβ  |  RXRα  |  ERα
Coregulators:   P/CAF  |  CBP  |  p300  |  GRIP1  |  AIB1
Ligands:   Thyroid hormone



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