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Molecular Endocrinology, doi:10.1210/me.2005-0065
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Molecular Endocrinology 20 (1): 56-64
Copyright © 2006 by The Endocrine Society

Mechanism of Action of Hic-5/Androgen Receptor Activator 55, a LIM Domain-Containing Nuclear Receptor Coactivator

M. D Heitzer and D. B. DeFranco

Department of Cell Biology and Physiology (M.D.H.) and Department of Pharmacology (D.B.D.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Address all correspondence and requests for reprints to: D. B. DeFranco, Department of Pharmacology, University of Pittsburgh School of Medicine, 200 Lothrop Street, Pittsburgh, Pennsylvania 15261. E-mail: dod1{at}pitt.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hic-5/androgen receptor (AR) coactivator 55 (ARA55) is a group III LIM domain protein that functions as a nuclear receptor coactivator. In the present study, we examined the mechanism by which Hic-5/ARA55 potentiates glucocorticoid receptor (GR) transactivation in the A1–2 derivative of T47D breast cancer cells. Hic-5/ARA55 is an important component of GR-coactivator complexes in A1–2 cells because ablation of Hic-5/ARA55 expression by RNA interference-mediated silencing reduced GR transactivation. As shown by chromatin immunoprecipitation (ChIP) assays, Hic-5/ARA55 is recruited to glucocorticoid-responsive promoters of the mouse mammary tumor virus, c-fos, and p21 genes in response to glucocorticoid treatment. Results from sequential ChIPs established that Hic-5/ARA55 associates with GR-containing complexes at these promoters. We also used sequential ChIPs to examine Hic-5/ARA55 interactions with other well-characterized nuclear receptor coactivators and detected transcriptional intermediary factor 2, receptor-associated coactivator 3, cAMP response element binding protein-binding protein, and p300 within Hic-5/ARA55 complexes on the mouse mammary tumor virus promoter in hormone-treated cells. Ablation of Hic-5/ARA55 expression resulted in reduction of both transcriptional intermediary factor 2 and p300 recruitment to glucocorticoid-responsive promoters. Hic-5/ARA55 is also associated with the corepressor, nuclear receptor corepressor, on glucocorticoid-responsive promoters in cells not exposed to glucocorticoids. These results suggest that Hic-5/ARA55 is required for optimal GR-mediated gene expression possibly by providing a scaffold that organizes or stabilizes coactivator complexes at some hormone-responsive promoters.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE NUCLEAR RECEPTOR family of transcription factors, including the glucocorticoid receptor (GR), is a diverse group of proteins that regulate target gene expression through a variety of mechanisms. Once in the nucleus, ligand-bound nuclear receptors are recruited to target gene promoters either through direct binding to hormone response elements or association with other promoter-bound transcription factors (1). Coactivator complexes are assembled onto receptor-bound promoters and stimulate nuclear receptor-mediated transcription either through direct interactions with the basal transcription machinery or by inducing local chromatin remodeling (2, 3, 4, 5, 6, 7). Some coactivators possess enzymatic activity such as histone acetyltransferase (HAT) and methyltransferase activities that act directly on histone proteins to affect chromatin structure, whereas others that lack such activities function to recruit chromatin-modifying enzymes to active promoters.

Recently, a focal adhesion-associated protein, hydrogen peroxide-inducible clone-5 [Hic-5/androgen receptor (AR) coactivator 5(ARA55)], was identified as a nuclear receptor coactivator by yeast two-hybrid analyses (8, 9). For GR, a specific Hic-5/ARA55 binding region has been identified, the {tau}2 transactivation domain located in its hinge region, whereas a Hic-5/ARA55-interacting domain for AR has only been broadly localized to its ligand-binding domain (8, 9). Hic-5/ARA55 belongs to the group III LIM domain protein family, containing four LIM domains at its carboxyl terminus (10). Group III LIM domain-containing proteins, including Hic-5/ARA55, are predominately localized at focal adhesions (11). However, some group III LIM domain proteins such as zyxin, lipoma-preferred partner, thyroid interacting protein partner (Trip6), paxillin, and Hic-5/ARA55 can also be found in the nucleus (8, 12, 13, 14, 15). When localized within the nucleus, group III LIM domain proteins may directly affect gene transcription through their interaction with several families of transcription factors (8, 13, 16, 17). For example, a nuclear isoform of Trip6 acts as a coactivator for activator protein 1 (AP-1) and nuclear factor-{kappa}B (NF-{kappa}B)-mediated gene expression (14). Additionally, FHL2 (four and a half LIM-only protein), a member of the LIM-only family of proteins, is an AR-specific coactivator, working in conjunction with the HAT-containing p300 coactivator (18).

In addition to its enhancement of GR and AR transactivation, Hic-5/ARA55 is also a coactivator for Sp1 (8, 19). In this case, Hic-5/ARA55 forced into the nucleus with an added heterologous nuclear localization signal (NLS) influences p21 expression by interacting directly with Smad3 (Sma and Mad related protein 3) and indirectly with Sp1 and p300 (19). The mechanism of Hic-5/ARA55 action on nuclear receptor transactivation is not known although analogous to other LIM domain proteins (FHL2), it may act as a scaffold to recruit coactivators to steroid-responsive promoters (20). At focal adhesion complexes, Hic-5/ARA55, as well as other group III LIM domain proteins, links various intracellular signaling modules to plasma membrane receptors that respond to various extracellular signals including growth factors and the extracellular matrix (21, 22).

In this report, we examine the mechanism of Hic-5/ARA55 action as a coactivator, focusing exclusively on endogenous Hic-5/ARA55 in the A1–2 derivative of T47D breast cancer cells and not relying on artificial enhancement of its nuclear localization. Single and sequential chromatin immunoprecipitation (ChIP) assays reveal an association of Hic-5/ARA55 with GR and various coactivators on viral and cellular glucocorticoid-responsive promoters. Furthermore, short interfering RNA (siRNA)-mediated ablation experiments established Hic-5/ARA55 in maintaining the assembly of coactivator complexes required for efficient glucocorticoid-induced transcription. Thus, Hic-5/ARA55 may function as a steroid receptor coactivator as an adaptor protein, recruiting or stabilizing histone acetyltransferase-containing complexes at steroid-responsive promoters.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hic-5/ARA55 Is Localized to a Glucocorticoid-Responsive Promoter
Although an interaction between Hic-5/ARA55 and GR has been revealed in yeast two-hybrid assays, the relevance of this association to the coactivator activity of Hic-5/ARA55 toward GR is unknown (8, 9). To determine the mechanism by which Hic-5/ARA55 serves as a GR coactivator, we assessed whether Hic-5/ARA55 was bound to the glucocorticoid-responsive mouse mammary tumor virus (MMTV) promoter, using ChIP assays. A1–2 cells, a T47D cell derivative that contains an integrated MMTV-luciferase gene, were grown in medium containing steroid-depleted serum for 2 d before initiating hormone treatments. After a 1-h ethanol-vehicle or dexamethasone (Dex) (100 nM) treatment, GR and Hic-5/ARA55 recruitment to the MMTV promoter was analyzed by ChIP analysis using antibodies specific for each (Fig. 1Go). As expected, there was a Dex-dependent localization of GR to the MMTV promoter, but not the coding region of the luciferase gene. Hic-5/ARA55 was also recruited to the MMTV promoter in the presence of Dex. These results provide the first demonstration of endogenous Hic-5/ARA55 binding to a specific promoter in vivo and the hormone-dependent recruitment of Hic-5/ARA55 to a nuclear receptor-responsive promoter.



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  		Fig. 1. Binding of Hic-5/ARA55 to the MMTV Promoter

A, Soluble chromatin was prepared from A1–2 cells treated with EtOH-vehicle or Dex (100 nM) for 1 h. Protein-bound DNA complexes were immunoprecipitated with antibodies against GR or Hic-5/ARA55. After cross-link reversal, purified DNA was amplified with primers specific for the MMTV promoter (left panels) or the coding region of the luciferase gene (right panels). PCR products in the input panel were amplified using diluted chromatin that was not immunoprecipitated. A rabbit IgG was used to detect any nonspecific immunoprecipitated DNA. Gel shown of PCR products is representative of three separate experiments. B, Relative changes in GR and Hic-5/ARA55 recruitment to the MMTV promoter were calculated based on semiquantitative results from the original images. Values represent percent of sample input after subtraction of IgG control ± SD for three separate experiments. *, P < 0.05, significantly different from the mean value of ethanol controls; bars, SD values.

 
GR and Hic-5/ARA55 Co-Occupy a Glucocorticoid-Responsive Promoter in Vivo
To determine whether GR and Hic-5/ARA55 are contained within a stable complex at GR-responsive promoters, we performed sequential immunoprecipitations followed by ChIP analysis (Fig. 2Go). A1–2 cells were grown and treated as in Fig. 1Go, and the diluted chromatin was first immunoprecipitated using antibodies specific for GR. After extensive washes, the precipitates were reimmunoprecipitated using antibodies specific for Hic-5/ARA55. As shown in Fig. 2Go, GR-containing chromatin segments of the MMTV promoter also contain Hic-5/ARA55. These data demonstrate that GR and Hic-5/ARA55 can co-occupy the MMTV promoter in vivo and that this association is enhanced upon Dex treatment. The sequential immunoprecipitation reduced the detection of GR in chromatin isolated from cells not treated with hormone. However, Hic-5/ARA55 was still detected in MMTV chromatin isolated from untreated cells. Thus, Hic-5/ARA55 may play a role in both basal and hormone-regulated transcription from the MMTV promoter.



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  		Fig. 2. Co-occupancy of GR and Hic-5/ARA55 on the MMTV Promoter

Soluble chromatin was prepared as in Fig. 1Go. GR-bound DNA complexes were immunoprecipitated (indicated as 1st ChIP), eluted, and reimmunoprecipitated with GR- and Hic-5/ARA55-specific antibodies. After cross-link reversal, purified DNA was amplified with primers specific for the MMTV promoter. PCR products in the input panel were amplified using diluted chromatin that was not immunoprecipitated. A rabbit IgG was used to detect any nonspecific immunoprecipitated DNA. Gel shown of PCR products is representative of two separate experiments.

 
GR and Hic-5/ARA55 Associate with Endogenous Glucocorticoid-Responsive Promoters in Vivo
To establish whether both GR and Hic-5/ARA55 are recruited to endogenous promoters, we initially performed RT-PCR analysis to identify endogenous glucocorticoid-responsive genes in A1–2 cells. Glucocorticoid induction of p21 expression has been demonstrated in A1–2 cells (23). Although the mechanism responsible for hormone effects on p21 expression is not known, p21 may be a target of Hic-5/ARA55 action because overexpression or forced nuclear retention of Hic-5/ARA55 resulted in increased p21 and c-fos expression in human immortalized fibroblasts (24, 25). To test whether Hic-5/ARA55 played a role in glucocorticoid induction of these genes in A1–2 cells, we used ChIP assays to assess promoter occupancy of the p21 and c-fos genes.

A1–2 cells were grown in medium containing charcoal dextran-stripped fetal bovine serum (FBS). After 2 d, the cells were treated with ethanol or 100 nM Dex for 10 h followed by RT-PCR analysis. As shown in Fig. 3AGo, we confirmed by the use of RT-PCR that both p21 and c-fos mRNAs were induced by Dex in A1–2 cells. Furthermore, GR association with the c-fos and p21 promoters was enhanced by Dex treatment (Fig. 3BGo). Importantly, sequential ChIP experiments demonstrated that Hic-5/ARA55 was also a component of GR-containing chromatin at these endogenous promoters. Thus, endogenous Hic-5/ARA55 is included within GR complexes at the promoter of endogenous genes whose transcription is regulated by glucocorticoids.



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  		Fig. 3. Association of GR and Hic-5/ARA55 with the Chromatin of Endogenous Promoters in Vivo

A, Glucocorticoid-responsive gene expression in A1–2 cells was analyzed by RT-PCR. A1–2 cells were treated with EtOH-vehicle or Dex (100 nM) for 10 h. RNA from A1–2 cells was isolated to determine the relative expression of p21, c-fos, and GAPDH mRNAs. The reverse transcriptase reaction was carried out with 0.5 µg total RNA. Parallel reactions performed without reverse transcriptase to control for the presence of contaminant DNA did not generate specific PCR products for any primers (data not shown). Gel shown of PCR products is representative of two separate experiments. B, Chromatin reimmunoprecipitation analysis was performed as in Fig. 2Go using c-fos or p21 promoter-specific primers. Gels shown of PCR products are representative of two separate experiments.

 
Reduced Expression of Hic-5/ARA55 Results in Decreased GR Transactivation
It has been established that overexpression of Hic-5/ARA55 increases GR-mediated transcription (8). However, these types of experiments do not reveal whether endogenous Hic-5/ARA55 is necessary for GR activity. Thus, we used a siRNA approach to ablate Hic-5/ARA55 expression in A1–2 cells and assess the impact on GR transactivation. A1–2 cells were analyzed for Hic-5/ARA55 expression in A1–2 cells by Western blot analysis after transfection with either a control green fluorescent protein siRNA or Hic-5/ARA55 siRNA. Densitometric analysis revealed that there was approximately 60% less Hic-5/ARA55 in cells transfected with the Hic-5/ARA55 siRNA as compared with control (Fig. 4AGo). Two other distinct Hic-5/ARA55 siRNAs tested were less effective at reducing endogenous Hic-5/ARA55 expression. Nonetheless, the reduction in Hic-5/ARA55 expression in A1–2 cells by the most effective siRNA was sufficient to generate reduced GR transactivation, as measured by ligand-dependent luciferase expression (Fig. 4BGo). Furthermore, this reduction in GR activity was not due to decreased GR expression in siHic-5/ARA55-transfected cells (Fig. 4AGo). We also analyzed glucocorticoid-induced c-fos and p21 mRNA expression after transfection with Hic-5/ARA55 siRNA (Fig. 4CGo). Although there was no reduction in c-fos expression, there was a 30% reduction in p21 expression upon silencing of Hic-5/ARA55. Because the silencing of Hic-5/ARA55 expression was not completely effective, we cannot conclude that Hic-5/ARA55 is essential for GR activity. However, these results indicate that endogenous Hic-5/ARA55 contributes to optimal GR transactivation from a subset of promoters in A1–2 cells.



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  		Fig. 4. Effects of RNA Interference-Mediated Silencing of Hic-5/ARA55 on GR Transactivation

A, A1–2 cells were transfected with green fluorescent protein (negative control) or human Hic-5/ARA55 RNA interference constructs. After 48 h, cells were harvested, lysed, and analyzed for Hic-5/ARA55, GR, and GAPDH protein levels by Western blotting. B, A1–2 cells were transfected with RNA interference constructs as in panel A. After 48 h, the cells were treated with EtOH-vehicle or 1 nM Dex for 24 h, after which luciferase activity was measured. Data shown are mean of three separate experiments. C, A1–2 cells were transfected as in A. After 48 h, the cells were treated with 1 nM Dex for 8 h. RT-PCRs were then performed as in Fig. 3AGo. Gel shown of PCR products is representative of three separate experiments. *, P < 0.005, significantly different from the mean value of siGFP controls; bars, SD values. siGFP, Short interfering green fluorescent protein; siHic-5, short interfering Hic-5; RLU, relative light units.

 
Hic-5/ARA55 Interaction with Coactivators on GR-Responsive Promoters
Because Hic-5/ARA55 does not possess HAT or methyltransferase activity, it may not modify histones directly. However, Hic-5/ARA55 may be involved in recruiting other chromatin-modifying coactivators. To determine whether Hic-5/ARA55 interacts with other coactivator proteins at glucocorticoid-responsive promoters, we performed sequential ChIPs, first using antibodies specific for Hic-5/ARA55 followed by reimmunoprecipitation with anti-steroid receptor coactivator 1 (SRC-1), anti-transcriptional intermediary factor 2 (TIF-2), anti-receptor-associated coactivator 3 (RAC3), anti-CREB-binding protein (CBP), or anti-p300 antibodies. As shown in Fig. 5Go, TIF-2, RAC3, CBP, and p300, but not SRC-1 were associated within Hic-5/ARA55-containing chromatin at the MMTV promoter in glucocorticoid-treated A1–2 cells. Interestingly, whereas TIF-2, RAC3, and CBP were detected within Hic-5/ARA55-containing chromatin complexes at the c-fos promoter, neither p300 nor SRC-1 was detected in Hic-5/ARA55 chromatin complexes (Fig. 5BGo). p300 is recruited to the c-fos promoter in response to glucocorticoids (Fig. 5BGo), but must exist in a separate coactivator complex that does not include stably associated Hic-5/ARA55. These results indicate that Hic-5/ARA55 is located within a complex of coactivator proteins at glucocorticoid-responsive promoters and that recruitment of p300 to Hic-5/ARA55-associated chromatin complexes may be promoter specific.



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  		Fig. 5. Co-occupancy of Hic-5/ARA55 and Coregulators on the MMTV and c-fos Promoters in Vivo

Chromatin reimmunoprecipitations were performed as in Fig. 2Go. Hic-5/ARA55-bound DNA complexes were eluted and reimmunoprecipitated with the specified coregulator-specific antibodies. Purified DNA was amplified using MMTV (A and C) or c-fos (B)-specific primers. Gels shown of PCR products are representative of three separate experiments.

 
Because Hic-5/ARA55 was present on the chromatin of glucocorticoid-responsive promoters in the absence of hormone stimulation, we analyzed its possible interaction with corepressor-containing complexes. Using sequential ChIP, we first immunoprecipitated Hic-5/ARA55-containing complexes followed by reimmunoprecipitation with nuclear receptor corepressor (NcoR)-specific antibodies. As shown in Fig. 5CGo, the association of NCoR with Hic-5/ARA55 at the MMTV promoter is higher in cells not exposed to hormone. Thus, Hic-5/ARA55 can be contained within both coactivator and corepressor complexes at an individual promoter.

Reduced Promoter Recruitment of TIF-2 and p300 after Silencing Hic-5/ARA55 Expression
Because Hic-5/ARA55 is made up of multiple protein interaction motifs, it may function as an adaptor protein at hormone-responsive promoters, interacting with multiple coregulator proteins or stabilizing the coactivator complex. To assess the necessity of Hic-5/ARA55 in coactivator recruitment to glucocorticoid-responsive genes, we performed ChIP assays in cells after silencing Hic-5/ARA55 expression. As shown in Fig. 6Go, after ablation of Hic-5/ARA55 expression, TIF-2 and p300 recruitment to the MMTV promoter in response to glucocorticoids was reduced. Interestingly, there was increased p300 recruitment in the absence of hormone upon silencing Hic-5/ARA55 expression. The corresponding reduction of GR transactivation (Fig. 4Go) and coactivator recruitment (Fig. 6Go) that results from partial Hic-5/ARA55 ablation demonstrates the critical role of Hic-5/ARA55 in maintaining the assembly of coactivator complexes required to bring about efficient glucocorticoid-induced transcription.



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  		Fig. 6. Coactivator Recruitment on the MMTV Promoter following Ablation of Hic-5/ARA55

A1–2 cells were transfected as in Fig. 4Go with vectors expressing siRNA for Hic-5/ARA55 or green fluorescent protein. After 72 h, ChIP analysis was performed as in Fig. 1Go. After cross-link reversal, purified DNA was amplified with primers specific for the MMTV promoter. Gel shown of PCR products is representative of two separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Coactivators are ligand-dependent steroid receptor-interacting proteins that enhance steroid receptor-dependent gene expression (5). Many coactivators have been identified and classified into subfamilies consisting of similar members. For example, the p160 family of coactivators, designated SRCs consists of SRC-1 (or NcoA1), SRC-2 (or TIF-2, GR-interacting protein 1), and SRC-3 (or p300/CBP/cointegrator-associated protein, RAC3, acetyltransferase, or amplified in breast cancer 1) (26, 27, 28, 29, 30, 31, 32). These coactivators share conserved regions termed "nuclear receptor interaction domains," which permit interactions with a broad range of nuclear receptors. Other coactivators that are distinct from the p160 family are components of large complexes, such as the vitamin D-interacting proteins and thyroid receptor-associated proteins (33, 34). A number of other potential coactivators, including the various AR activators (ARAs), may utilize unique mechanisms to impact nuclear receptor transactivation (35, 36).

Here, we show that endogenous Hic-5/ARA55 is recruited to both stably integrated viral and endogenous glucocorticoid-responsive promoters in response to hormone treatment. Furthermore, GR and Hic-5/ARA55 co-occupy these promoters within stable complexes that can be recovered by sequential ChIPs. We also demonstrated an association between Hic-5/ARA55 and coactivator-containing complexes at glucocorticoid-responsive promoters using sequential ChIPs. Using gel shift assays and coimmunoprecipitations, Shibanuma et al. (19) showed that Hic-5/ARA55 containing a heterologous NLS regulated p21 expression by interacting with Smad3, but not p300 or Sp1 directly in transfected cells. Whereas these results imply that Hic-5/ARA55 association with p300 may be cell type or promoter specific, it may not be appropriate to compare the function of endogenous Hic-5/ARA55 in our case with transfected, overexpressed Hic-5/ARA55 that contains a heterologous NLS to force robust nuclear retention. However, promoter-specific recruitment of p300 to endogenous Hic-5/ARA55 chromatin complexes was observed in A1–2 cells, corroborating the results obtained with exogenously introduced NLS-Hic-5/ARA55 constructs.

Interestingly, for the promoter in which p300 in not a component of Hic-5/ARA55 complexes (i.e. the c-fos promoter), partial ablation of Hic-5/ARA55 is not sufficient to impact glucocorticoid-regulated transcription. Although we cannot rule out the possibility that more complete ablation of Hic-5/ARA55 would hinder glucocorticoid-induced transcription from the c-fos promoter, Hic-5/ARA55 may only exert an essential function to maintain the stability of distinct subsets of coactivator complexes (i.e. p300-containing). Partial Hic-5/ARA55 ablation also blocks peroxisome proliferator-activated receptor-{gamma}-induced expression of a select set of epithelial-specific genes (37). Although the differential recruitment of coactivators was not examined in this case, these results highlight the importance of Hic-5/ARA55 in impacting gene-specific effects of nuclear receptors.

LIM domain-containing proteins have been shown to either positively or negatively influence gene expression under certain cellular contexts. For example, Trip6, a group III LIM domain protein, is able to both enhance and repress AP-1 and NF-{kappa}B-regulated promoters by assembling different complexes in response to cellular signals (14). Upon TPA or TNF{alpha} treatment, Trip6 is recruited to the ColI or IL-8 promoters, enhancing AP-1 and NF-{kappa}B-mediated gene expression, respectively. However, upon glucocorticoid treatment, Trip6 represses AP-1 and NF-{kappa}B action by tethering GR to specific promoters (14). This indicates that specific signaling events can determine whether Trip6 activates or represses specific gene expression. Additionally, four and a half LIM-only protein (FHL2) is also a LIM domain-containing protein that can both enhance or repress gene expression. FHL2 was initially identified as an AR-specific coactivator but was later reported to influence ß-catenin-mediated gene expression (18, 20). The mechanism by which FHL2 coactivates ß-catenin-mediated transcription is due, in part, to a physical interaction with CBP/p300, resulting in increased acetylation of ß-catenin by CBP/p300 (18). Acetylation of ß-catenin may enhance its interaction with TCF4, thereby increasing ß-catenin-mediated gene expression (38). Interestingly, FHL2 represses ß-catenin-mediated gene expression in muscle cells, resulting in myogenic differentiation (39).

Although there have been no reports indicating a repressive function for Hic-5/ARA55 on nuclear receptor-mediated gene expression, ChIP analysis consistently revealed Hic-5/ARA55 binding to the MMTV promoter in the absence of hormone. The enhanced association of NCoR with Hic-5/ARA55 complexes at the MMTV promoter in non-hormone-treated cells suggests that Hic-5/ARA55 may, in fact, participate in maintaining low basal levels of transcription from this promoter. However, Hic-5/ARA55 is not essential for limiting MMTV transcription in the absence of hormone as its partial ablation does not lead to enhanced transcription. Although we have observed enhanced basal transcription from a steroid hormone-regulated gene upon Hic-5/ARA55 ablation in a prostate cell line, definitive demonstration of Hic-5/ARA55 participation in transcriptional repression will require more thorough analysis.

Because Trip6, FHL2, and Hic-5/ARA55 do not possess an obvious catalytic domain that is responsible for their coactivation properties, their mechanism of coregulator function has remained undefined. However, they may serve as adaptor molecules, either recruiting or stabilizing promoter-specific protein complexes. LIM proteins are well recognized for their roles as molecular adaptors, functioning in stabilizing higher order protein complexes at either focal adhesion complexes or promoter sequences. For example, Hic-5/ARA55 and paxillin interact with multiple focal adhesion-associated proteins such as vinculin and focal adhesion kinase (40). Furthermore, in addition to nuclear receptors, Hic-5/ARA55 functions as a coactivator for Sp1, enhancing p21 expression (19). Although a direct interaction between Sp1 and Hic-5/ARA55 has not been detected, Hic-5/ARA55 has been found to associate with Smad3. Also, a LIM 4 deletion mutant of Hic-5/ARA55 interfered with the coactivation properties of p300, suggesting a functional interaction between these coactivators. siRNA ablation experiments reported here establish that Hic-5/ARA55 is required for the stable association of p300 and TIF-2 with the MMTV promoter. Thus, Hic-5/ARA55 may stabilize select protein complex formation at GR-responsive promoters by serving as an adaptor molecule.

The recent demonstration of Hic-5/ARA55 involvement in PPAR{gamma}-induced epithelial cell differentiation program illustrates the importance of adaptor coactivators that lack enzymatic activity in assembling functional coregulator complexes on distinct promoters. More detailed analysis of this novel family of nuclear receptor coactivators may unlock multiple nuclear receptor gene networks that utilize LIM domain-containing adaptors to organize gene-specific coactivator assemblies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Antibodies
Antibodies used in this study included: anti-GR (Affinity Bioreagents, Inc., Golden, CO); anti-Hic-5 (BD Transduction Laboratories, Los Angeles, CA); antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CSA-335; Stressgen, Victoria, British Columbia, Canada); anti-NCoR (06–892 Upstate Biotechnology, Lake Placid, NY); anti-GR (H-300); anti-p300 (C-20); anti-SRC-1 (M 341); and anti-TIF-2 (M 343) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Cell Culture and Transient Transfection
The A1–2 cells were derived from the T47D human mammary carcinoma cells line by stable transfection with pGRneo and MMTV-LTR-luc plasmids as previously described (41). Thus, these cells contain exogenous copies of stably integrated rat GR and a glucocorticoid-responsive luciferase reporter. The A1–2 cells were routinely maintained in modified Eagle’s medium at 37 C under 5% CO2. The media were supplemented with 100 µg/ml penicillin-streptomycin, 10% FBS, 10 mM HEPES, 2 mM glutamine, and 160 µg/ml G418. Cells were seeded on 12-well cell culture dishes at a density of 1.5 x 105 cells per well for 24 h before transfection. Transfections were performed using Opti-MEM (Life Technologies, Gaithersburg, MD) and Lipofectamine transfection reagent (Invitrogen Technology, Carlsbad, CA) according to manufacturer’s instructions. After 3 h, fresh medium was added to the cells and hormone treatments were initiated where relevant.

Western Blot Analysis
Cell lysates were collected in RIPA buffer [10 mM Tris (pH 8), 1 mM EDTA, 0.5 mM EGTA, 140 mM NaCl, 1% Triton X-100, 0.1% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonylfluoride, and protease inhibitors] and were boiled in Sample Buffer (62.5 mM Tris, pH 6.8; 2% SDS; 10% glycerol; 5% 2-mercaptoethanol; 0.001% bromophenol blue) for 5 min. Proteins were then separated on 7.5% SDS-PAGE and transferred to PVDF transfer membrane (Bio-Rad Laboratories, Hercules, CA) in transfer buffer (20% methanol, 48 mM Tris, 39 mM glycine, and 1.3 mM SDS) at 15 V for 30 min. Membranes were then incubated in blocking buffer (5% dry milk in Tris-buffered saline, pH 7.4) for 2 h to overnight. Next, the membranes were incubated with Hic-5-specific antibodies diluted 1:1000 in blocking solution for 2 h at room temperature. After extensive washing, the membranes were then probed with horseradish peroxidase-conjugated goat antimouse IgG antibodies (Santa Cruz Biotechnology) diluted in blocking solution for 1 h. Finally, the membranes were washed and developed using the Renaissance Western Blot Chemiluminescence Reagent (NEN Life Sciences Products, Boston, MA) according to the manufacturer’s instructions. The membranes were stripped with Re-Blot Plus Strong Solution following the manufacturer’s instructions (Chemicon International, Temecula, CA) and reprobed for GAPDH as a loading control. Where indicated, quantification of scanned images was performed using the NIH Image software.

RNA Interference
RNA interference plasmids were constructed following the manufacturer’s guidelines (pSilencer hygro, Ambion, Austin, TX). Template sequence corresponding to amino acids 129–135 in human Hic-5/ARA55 (5'-GAAAAGACCCAGCCTCCCT-3') were annealed to form double-stranded DNA and inserted into the p2.1-U6 plasmid after enzymatic digestion by HindIII and BamHI. The green fluorescent protein template sequence was used as a positive control during the plasmid construction and a negative control for transfection analyses.

Luciferase Assays
Luciferase activity in cell-free lysates was measured using a Victor3 1420 multilabel reader (PerkinElmer, Wellesley, MA). Cells were washed with PBS and lysed in Reporter Lysis Buffer (Promega Corp., Madison, WI) followed by a freeze and thaw incubation to ensure proper cell lysis. The lysate was incubated with luciferase assay reagent followed by a 10-sec relative luciferase unit measurement. Luciferase activity was normalized by total protein concentration as measured by Bradford assays (Bio-Rad). All experiments were performed three or more times.

Chromatin Immunoprecipitation
A1–2 cells were grown to 80–90% confluence in medium supplemented with charcoal dextran-treated FBS 24 h. The cells were treated with EtOH-vehicle or 100 nM Dex for 1 h, cross-linked with 1% formaldehyde at room temperature for 30 min, lysed, and sonicated as previously described (42). Chromatin fragments were immunoprecipitated with specific antibodies overnight at 4 C. After immunoprecipitation, 30 µl of protein A and G sepharose (Upstate, Lake Placid, NY) was added and the incubation was continued for 1 h. After extensive washing, precipitates were eluted with reverse cross-linking buffer (1% SDS and 0.1 M NaHCO3) at 65 C for 4 h followed by a proteinase K treatment for 1 h at 45 C. Eluted DNA was isolated using the QIAquick PCR purification kit (QIAGEN, Valencia, CA). PCRs were performed using the Platinum PCR Supermix (Invitrogen Life Technologies, Carlsbad, CA), 2 µl of DNA, and 40 cycles of amplification. Primers for the MMTV promoter were: forward primer, 5'-GCGGTTCCCAGGGCTTAAGT-3'; and reverse primer, 5'-CCATTTTACCAACAGTACCG-3'. Primers for the c-fos promoter were: forward primer, 5'-TCCCAGCAGTCGAGGTATTC-3'; and reverse primer, 5'-GGTCAGTTCGGGATGACAAG-3'. Primers for the p21 promoter were: forward primer, 5'-GGTGTCTAGGTGCTCCAGGT-3'; and reverse primer, 5'-GCACTCTCCAGGAGGACACA-3'. Primers for the luciferase gene were: forward primer, 5'-CCAGGGATTTCAGTCGATGT-3'; and reverse primer, 5'-AATCTGACGCAGGCAGTTCT-3'. PCR products were resolved on a 12% polyacrylamide gel and visualized with ethidium bromide. Semiquantitation was done using densitometric analysis of the resolved gels using the Kodak Imaging System. Data points were subtracted for background and normalized to the input data.

For sequential ChIPs, complexes immunoprecipitated with either anti-GR, anti-Hic-5, or anti-p300 were eluted by incubation with 10 mM dithiothreitol for 30 min at 37 C and diluted 1:50 in ChIP Dilution Buffer (20 mM Tris-HCl, pH 8.1; 2 mM EDTA; 150 mM NaCl; 1% Triton X-100), followed by reimmunoprecipitation with either isotype control Ab, anti-Hic-5, or various coactivators or corepressor antibodies.

RT-PCR
Total RNA was isolated from A1–2 cells using the RNAqueous RNA isolation kit (Ambion, Austin, TX) following the manufacturer’s instructions. For RT-PCR, 1 µg of RNA was incubated with 100 µl of reaction mix containing 25 mM MgCl2, 25 mM deoxynucleotide triphosphates (PerkinElmer), 10x PCR II Buffer (Life Technologies), 40 U/µl RNAsin RNase inhibitor (Promega), 45 µM random hexamers (Integrated DNA Technologies, Coralville, IA), 200 U/µl Superscript reverse transcriptase (Life Technologies), and nuclease-free water (Ambion). Parallel reactions were performed without reverse transcriptase to control for the presence of contaminant DNA. The samples were incubated at 25 C for 10 min, at 48 C for 30 min, and at 95 C for 5 min followed by 4 C for 5 min to inactivate the reverse transcriptase.

For amplification, a PCR containing a cDNA aliquot along with AmpliTaq Gold DNA polymerase in a volume of 25 µl was used according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). Primers used for gene expression analysis were: c-fos: forward primer, 5'-TGCCAACTTCATTCCCACGGT-3'; and reverse primer, 5'-TAGTTGGTCTGTCTCCGCTTG-3'; p21: forward primer, 5'-GCGACTGTGATGCGCTAATGG-3'; and reverse primer, 5'-TCCCAACTCATCCCGGCCTC-3'; GAPDH: forward primer, 5'-CATCACCATCTTCCAGGAGCGAGA-3'; and reverse primer, 5'-GTCTTCTGGGTGGCAGTGATGG-3'. Thermocycling conditions involved an initial denaturation step at 95 C for 12 min followed by 28–30 cycles at 95 C for 30 sec and 56 C for 30 sec and 72 C for 30 sec. Specific PCR amplification products were separated on a 12% PAGE and detected by EtBr staining. Experiments were performed with duplicates for each data point.


   ACKNOWLEDGMENTS
 
We thank Dr. Trevor Archer for providing the A1–2 cells.


   FOOTNOTES
 
This work was supported, in part, by National Institutes of Health Grant CA 43037.

First Published Online September 1, 2005

Abbreviations: AP-1, Activator protein 1; AR, androgen receptor; ARA, AR activator; CBP, cAMP response element-binding protein (CREB)-binding protein; ChIP, chromatin immunoprecipitation; Dex, dexamethasone; FBS, fetal bovine serum; FHL2, four and a half LIM-only protein; GR, glucocorticoid receptor; HAT, histone acetyltransferase; MMTV, mouse mammary tumor virus; NcoR, nuclear receptor corepressor; NF-{kappa}B, nuclear factor-{kappa}B; NLS, nuclear localization signal; RAC3, receptor-associated coactivator 3; SDS, sodium dodecyl sulfate; SRC-1, steroid receptor coactivator-1; SDS, sodium dodecyl sulfate; siRNA, small interfering RNA; Smad3, Sma and Mad related protein 3; SRC, steroid receptor coactivator; TIF-2, transcription intermediary factor-2; Trip6, thyroid interacting protein partner.

Received for publication January 27, 2005. Accepted for publication August 22, 2005.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   GR
Coregulators:   ARA55  |  TRIP6  |  CBP  |  p300  |  FHL2  |  SRC-1  |  GRIP1  |  AIB1  |  NCOR
Ligands:   Dexamethasone



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