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Molecular Endocrinology, doi:10.1210/me.2003-0065
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Molecular Endocrinology 18 (1): 13-25
Copyright © 2004 by The Endocrine Society

Androgen Receptor Corepressor-19 kDa (ARR19), a Leucine-Rich Protein that Represses the Transcriptional Activity of Androgen Receptor through Recruitment of Histone Deacetylase

Byung-Chul Jeong, Cheol Yi Hong, Soma Chattopadhyay, Jin Hee Park, Eun-Yeung Gong, Hyun-Jin Kim, Sang-Young Chun and Keesook Lee

Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea

Address all correspondence and requests for reprints to: Keesook Lee, Hormone Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea. E-mail: klee{at}chonnam.ac.kr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Androgen receptor (AR) that mediates androgen action is a crucial factor in male reproductive functions. Here, we report a novel AR corepressor ARR19 (androgen receptor corepressor-19 kDa), which has been isolated as a putative androgen-induced gene from murine testis. ARR19 encoding a leucine-rich protein is expressed only in male reproductive organs such as testis and prostate. ARR19 expression in the testis is developmentally regulated. Functional analysis conducted by the transient transfection of mammalian cells shows that ARR19 represses AR transactivation in a dose-dependent manner. Furthermore, yeast two-hybrid and glutathione S-transferase pull-down analyses reveal that ARR19 directly associates with AR through the N-terminal and leucine zipper-containing regions of ARR19 and the DNA binding-hinge domain of AR. Interestingly, ARR19 localized in the cytoplasmic compartment cotranslocates into the nucleus with AR upon androgen exposure. The ARR19 repression of AR transactivation is through the recruitment of histone deacetylase 4 (HDAC4) by ARR19. Overexpression of HDAC4 further inhibits the ARR19-repressed AR transactivation. In addition, ARR19 directly interacts with HDAC4 in vitro. Furthermore, DNA-protein complex immunoprecipitation assays reveal that HDAC4 is recruited to an androgen-regulated promoter through ARR19. Taken together, the results suggest that ARR19 may act as an AR corepressor in vivo and play an important role in male reproductive functions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ANDROGENS ARE IMPORTANT in male sexual differentiation and maturation and to the proper functioning of the male reproductive system including prostate and epididymis. Testosterone, the major physiological androgen, has been especially known to be essential for the initiation of spermatogenesis at puberty and its maintenance during adulthood (1, 2). Withdrawal of testosterone by hypophysectomy or ethane dimethanesulfonate (EDS) treatment in rats led to the failure of spermatogenesis (3, 4). The biological action of androgens is mediated through androgen receptor (AR), an androgen-dependent transcription factor. Mutations of AR causing its functional impairment may thus result in male infertility with complete or partial androgen insensitivity (5, 6, 7, 8).

AR belongs to the nuclear receptor superfamily that is a related group of ligand-inducible transcription factors (9, 10). Before exposure to androgens, AR is held in an inactive state being associated with specific heat shock proteins in the cytoplasm (11, 12). Upon ligand binding, AR undergoes conformational changes that facilitate its dissociation from the heat shock proteins and translocation to the nucleus, where it binds to the specific DNA sequence, called androgen responsive element (ARE), to regulate the transcription of target genes (13, 14). Like other members of the nuclear receptor superfamily, AR contains three distinct functional domains: the N-terminal activation function-1 (AF1) domain, the central DNA-binding domain (DBD), and the C-terminal ligand-binding domain (LBD) (15). A second, ligand-dependent activation function-2 (AF2) has also been identified in the C-terminal part of the LBD (16, 17), although the AF2 in AR is relatively weak compared with the AF1 (18).

A series of coregulators have been identified to associate with AR and regulate AR-mediated transcriptional activity (19). To modulate ligand-dependent transactivation of the nuclear receptors, coregulatorsare known to adopt diverse modes of action including direct interaction with basal transcription factors and covalent modification of histones and other proteins (20, 21, 22, 23). For example, AR coactivators, CREB-binding protein and steroid receptor coactivator-1, have been either copurified with the RNA pol II holoenzyme complex or shown to interact with TATA binding protein and transcription factor IIB (24, 25, 26), implicating their function in bridging between AR and the basal transcriptional machinery. On the other hand, several known AR coactivators such as CREB-binding protein, steroid receptor coactivator-1, and p300/CBP interacting protein (p/CIP) harbor inherent histone acetyltransferase activity that may modify mainly histones, resulting in chromatin structural perturbations to facilitate the subsequent recruitment of other transcriptional factors (27). In contrast, AR corepressors, which are less defined, may recruit histone deacetylase (HDAC) activity to the AR complex maintaining the chromatin structure, as do other nuclear receptor corepressors such as silencing mediator of retinoid and thyroid hormone receptor and nuclear receptor corepressor (N-CoR) (28, 29). This excludes functional interactions of the general transcriptional machinery and other transcription factors with the promoter.

In this study, we report the cloning and characterization of a novel AR corepressor ARR19 (androgen receptor corepressor-19 kDa). The ARR19 gene, encoding a leucine-rich and highly hydrophobic protein, is highly expressed in the testis and moderately in other male reproductive organs. ARR19 interacts directly with AR and inhibits its transactivation by cotranslocating into the nucleus in the presence of androgen. The ARR19-repression of AR transactivation involves the recruitment of HDAC4 by ARR19. The expression pattern of ARR19 in male reproductive organs and its function as an AR corepressor suggest that ARR19 may have an important role in male reproduction in vivo.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of ARR19 as a Putative Androgen-Induced Gene from Murine Testis
Administration of EDS, a cytotoxic compound, destroys Leydig cells in adult male rats, eventually resulting in the failure of spermatogenesis due to the deprivation of androgen (30). However, an exogenous supply of testosterone to the EDS-treated rats keeps the spermatogenesis maintained even in the absence of Leydig cells (31). To clone androgen-induced genes that may have a role in spermatogenesis, PCR-select cDNA subtraction was performed with the mRNA sample prepared from EDS-treated and testosterone-supplied rat testis against the sample from EDS-treated control rat testis. ARR19 was isolated as one of positive clones in the initial screening. Subsequent Northern blot analysis showed that ARR19 expression was down-regulated to 78% in EDS-treated testis, and up-regulated to 192% in EDS-treated and testosterone-supplied testis compared with that in the untreated testis (Fig. 1AGo). 3ß-Hydroxysteroid dehydrogenase, a Leydig cell-specific marker gene, was expressed only in the testis without EDS treatment, whereas nerve growth factor receptor (NGFR) expressed in Sertoli cells and negatively regulated by androgen was expressed in the EDS-treated control testis.



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  		Fig. 1. Isolation of ARR19 from Murine Testis

A, ARR19 expression is induced by testosterone-supplement after Leydig cell destruction. Northern blot analyses were performed with total RNAs from testes using indicated 32P-labeled mouse cDNAs as probes (top panel). 3ß-Hydroxysteroid dehydrogenase was used as a Leydig cell-specific marker. Nerve growth factor receptor (NGFR) was used as an indication of no androgen supplement. Expression of GAPDH was used as an internal control. The ARR19 mRNA signal was quantified and normalized by GAPDH mRNA level in each sample (bottom panel). EDS, EDS treatment; T, testosterone injection for 4 d. B, Complete cDNA and deduced amino acid sequences of the mouse ARR19. Nucleotides are numbered on the left and amino acids are numbered on the right. An LZ motif is shown by box with bold letters for the conserved leucine residues and the polyadenylation signal by underline. An upstream in-frame stop codon is indicated by double underline. C, Gene structure of ARR19. An alignment of the ARR19 gene on mouse chromosome 8. Boxes indicate exons (numbered), and heavy parts indicate coding regions. The position of the translation start in exon 2 is indicated. Exon and intron structure of the ARR19 gene is based on the alignment shown in the upper part.

 
A full-length ARR19 cDNA was cloned by screening a mouse testis cDNA library. Sequence analysis of the cDNA revealed a gene that has been previously cloned from a RIKEN full-length enriched library of mouse adult testis cDNA (chemokine-like factor; GenBank accession nos. NM-027022 and AK005603). However, to the best of our knowledge, the gene has not been further studied. The entire open reading frame of the mouse cDNA consists of 507 nucleotides, encoding a protein of 169 amino acids (Fig. 1BGo). Interestingly, the protein contains one leucine zipper (LZ) motif and is very hydrophobic, being composed of 67% hydrophobic residues, especially leucine, which alone accounts for 17% of the total number of residues (Fig. 1BGo). Mouse ARR19 showed 70% identity and 87% similarity with rat ARR19 in its amino acid sequence (data not shown). Comparison of the mouse cDNA sequence with its genomic sequence (Mouse Genome Resources, NCBI) has revealed that the ARR19 gene is located on mouse chromosome 8 and is composed of five exons (Fig. 1CGo).

ARR19 Is Expressed in Male Reproductive Organs, and Its Predominant Expression in the Testis Is Developmentally Regulated
To examine the expression pattern of ARR19, Northern blot analysis was performed with total RNAs from various mouse tissues. ARR19 was expressed in male reproductive organs, highly in the testis and moderately in other male reproductive organs such as prostate (Fig. 2AGo) and epididymis (data not shown). The results suggest that ARR19 is expressed in a tissue-specific manner.



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  		Fig. 2. Expression of ARR19 in Male Reproductive Organs

A, Northern blot analysis of total RNAs from adult mouse tissues. B, Northern blot analysis of total RNAs from testes at different developmental stages. Blots were hybridized with 32P-labeled mouse ARR19 cDNA (nucleotides 378–807) probe as described in Materials and Methods. The expression of 18S rRNA or GAPDH was used as an internal control.

 
The expression of ARR19 was also examined during mouse testis development. ARR19 mRNA was undetectable at d 7 and 14 but was present in a moderate amount at d 24 and further increased in intensity by d 33. Its mRNA level was then maintained to the adult stage (Fig. 2BGo). In situ hybridization analysis of the testis revealed that ARR19 was expressed within seminiferous tubules, most abundantly in germ cells (data not shown). The results demonstrate that the expression of ARR19 starts around puberty and is up-regulated throughout testis development to the adult stage.

ARR19 Inhibits the Transcriptional Activity of AR
Because a negative feedback mechanism has been reported for certain transcription factors, of which transcriptional activity is inhibited by or through the product of their own target gene (32, 33, 34, 35), we attempted to determine whether ARR19 could influence AR transactivation. Both AR and ARR19 expression plasmids, along with a reporter construct regulated by two AREs in front of E1b TATA sequence [pARE2-TATA-Luc (36)], were transiently transfected into CV-1 and HepG2 cells, and the effect of ARR19 coexpression was determined by the measurement of luciferase activity. As shown in Fig. 3Go, A and B, AR induced the expression of the reporter gene approximately 6- and 10-fold in CV-1 and HepG2 cells, respectively, in the presence of testosterone, and coexpressed ARR19 repressed this androgen-dependent AR transactivation in a dose-dependent manner in both cell lines. Interestingly, ARR19 also inhibited basal reporter activity in the absence of androgen. Because ARR19 did not affect the activity of an internal control reporter (data not shown), this could be a leaky phenomenon due to the overexpression of AR and ARR19, although their nuclear translocation and ARR19 recruitment to the promoter in the absence of androgen were not detected by the methods that we used. Otherwise, ARR19 might sequester some factor(s) to the cytoplasm, which is involved in the basal expression of the reporter.



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  		Fig. 3. Coexpression of ARR19 Inhibits the Transcriptional Activity of AR

CV-1 (A) and HepG2 (B and C) cells were transiently cotransfected with AR expression plasmid and ARE2-TATA-Luc or PSA-Luc reporter, along with increasing amounts of ARR19 encoding plasmid. LNCaP (D) cells were transiently cotransfected with PSA-Luc reporter (without AR expression plasmid), along with increasing amounts of ARR19 encoding plasmid. Total amounts of expression vectors were kept constant by adding appropriate amounts of the blank vector. Relative luciferase activity represents the percent of the level of activity with AR and reporter gene in the presence of the ligand (set as 100). All values represent the mean ± SE of at least three independent experiments.

 
The effect of ARR19 on a natural AR-regulated promoter was also examined using the prostate-specific antigen (PSA)-Luc reporter (37) containing 5.3 kb PSA promoter. In HepG2 cells, AR induced the reporter expression up to approximately 70-fold in the presence of testosterone, and ARR19 coexpression repressed the AR transactivation in a dose-dependent manner. In LNCaP cells expressing endogenous AR, ARR19 also showed the same repression of the transactivation activity of endogenous AR, which induced the expression of the reporter up to 9-fold in the presence of androgen. These results suggest that ARR19 may function as a corepressor of AR by modulating its transcriptional activity.

ARR19 Interacts with AR in Yeast
Because ARR19 repressed AR transactivation in mammalian cells, we investigated any physical interaction of ARR19 with AR using a yeast two-hybrid protein interaction assay. As shown in Fig. 4AGo, LexA-AR fusion protein itself showed weak androgen-dependent autonomous transactivation activity, whereas B42-ARR19 alone showed no activity (data not shown). The presence of both partners, however, induced strong activation of the LacZ reporter gene. This interaction was androgen dependent although weak interaction also occurred in the absence of the ligand. Furthermore, B42-AR fusion protein without any autonomous transactivation activity also interacted with LexA-ARR19. This occurred both in the absence and in the presence of testosterone although stronger induction of ß-galactosidase activity was shown in the presence of the ligand (Fig. 4BGo). On the other hand, other nuclear receptors such as retinoic acid receptor (RAR), retinoid X receptor (RXR), and estrogen receptor-{alpha} (ER{alpha}) did not interact with ARR19 in the presence or absence of their cognate ligands. These results suggest that ARR19 physically interacts with AR and has its binding specificity to AR.



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  		Fig. 4. ARR19 Specifically Interacts with AR in Yeast

ARR19 interacts with AR but not with RAR, RXR, and ER{alpha}. A, B42-ARR19 or B42 empty vector was cotransformed into yeast strain EGY-48 with LexA-AR, LexA-RAR, or LexA-RXR. B, LexA-ARR19 or LexA empty vector was cotransformed into the yeast strain with either B42-AR or B42-ER{alpha}. Cells were grown in liquid yeast culture medium with or without appropriate hormones (100 nM). The strength of the ARR19 interaction with receptors was determined by measuring ß-galactosidase activity. All values represent the mean ± SE of three independent colonies in triplicate.

 
To determine which region of the ARR19 protein was responsible for its interaction with AR, a yeast liquid ß-galactosidase assay was performed using B42 fusion protein of ARR19 truncated forms: N-terminal region (ARR19-N, residues 1–50), leucine-zipper domain (ARR19-LZ, residues 51–107), and C-terminal region (ARR19-C, residues 108–175) (Fig. 5AGo). AR interacted with both ARR19-N and ARR19-LZ as strongly as the full-length ARR19 and the positive control Sma- and Mad-related protein 3 (Smad3) (38), but not with ARR19-C (Fig. 5BGo). The results indicate that the ARR19 N-terminal region and the LZ domain are independently responsible for the interaction with AR.



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  		Fig. 5. Delineation of the Domains of ARR19 and AR that Mediate Their Interaction in Yeast

A, Schematic representation of the full-length ARR19 and different deletion mutants used in yeast two-hybrid assay. B, Mapping of ARR19 domain responsible for its interaction with AR. LexA-AR was coexpressed along with B42 fusions of the full-length ARR19 or ARR19 deletion mutants in the presence (+T) or absence (-T) of 100 nM testosterone. C, Schematic representation of the full-length AR and different deletion mutants used in yeast two-hybrid assay. D, Mapping of AR domain responsible for its interaction with ARR19. LexA-ARR19 was coexpressed along with B42 fusions of AR deletion mutants in the presence or absence of 100 nM testosterone. The ß-galactosidase activity of yeast cotransformed with LexA fusion (AR or ARR19) and B42-empty vector in the absence of ligand is set as 1. All values represent the mean ± SE of at least three independent colonies.

 
Involvement of the region of AR in the interaction with ARR19 was tested using LexA-ARR19 and B42-fusion protein of AR truncated forms: AF-1 domain (AF1, residues 1–537), DBD and hinge domain (DBDh, residues 539–654) and LBD domain (residues 656–994) (Fig. 5CGo). A strong interaction of ARR19 protein with AR-DBDh was observed irrespective of the presence of androgen (Fig. 5DGo). The results suggest that the DBDh domain of AR is the major contributor to the AR interaction with ARR19.

ARR19 Directly Interacts with AR in Vitro
Direct interaction between AR and ARR19 and the region(s) of each protein responsible for their interaction were assessed by glutathione-S-transferase (GST) pull-down experiments. [35S]methionine-labeled AR produced by in vitro translation was allowed to bind the GST fusion protein of ARR19-N, ARR19-LZ, ARR19-C, ARR19-n +LZ, and ARR19-LZ+C (Fig. 6AGo). Results showed that AR interacted with ARR19-N, ARR19-LZ, ARR19-n +LZ, and ARR19-LZ+C, but not with ARR19-C (Fig. 6BGo). The binding between AR and ARR19 was not significantly affected by androgen in the GST pull-down experiments. Similar observations have been reported for other nuclear receptor coregulators (38, 39, 40). Involvement of the region(s) of AR in the direct interaction with ARR19 was tested using [35S]methionine-labeled ARR19 and GST-fusion protein of AR truncated forms: N-terminal {tau}1 domain (AR-TAU1, residues 81–361), DBDh domain (AR-DBDh, residues 539–654), and LBD domain (AR-LBD, residues 656–994) (Fig. 6CGo). A specific interaction between ARR19 protein and GST-AR-DBDh was observed (Fig. 6DGo). These results are consistent with those from the yeast two-hybrid protein binding assays, suggesting that the N-terminal and the LZ-containing regions of ARR19 and the DBDh domain of AR mainly contribute to the interaction between AR and ARR19.



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  		Fig. 6. ARR19 Directly Interacts with AR in Vitro

A, Schematic representation of the full-length ARR19 and different deletion mutants used in GST pull-down assay. B, Interaction domain of ARR19 for AR. C, Schematic representation of the full-length AR and different deletion mutants used in GST pull-down assay. D, Interaction domain of AR for ARR19. Bacterially produced GST alone or GST fusion proteins were bound to glutathione-agarose beads and incubated with equivalent amounts of the 35S-labeled mouse AR in the presence (+T) or absence (-T) of 100 nM testosterone. Ten percent of the labeled protein used in the binding reaction was loaded as input.

 
ARR19 Interacts with AR in Vivo
Association of ARR19 with AR in vivo was investigated by coimmunoprecipitation analysis (Fig. 7Go). HeLa cells were transiently cotransfected with an AR expression plasmid together with either pEGFP-ARR19 or pEGFP vector and then treated with or without 10 nM testosterone. Immunoprecipitations of whole-cell extracts using anti-green fluorescent protein (GFP) antibody and subsequent Western blot analysis with anti-AR antibody revealed that AR was coimmunoprecipitated with ARR19, but only from the cells treated with testosterone. The immunoprecipitated material from the cells transfected with pEGFP empty vector retained no AR. The results suggest that ARR19 becomes associated with AR in cultured cells upon androgen treatment.



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  		Fig. 7. ARR19 Interacts with AR in Vivo

AR was coexpressed in HeLa cells with either GFP-ARR19 or GFP vector in the presence or absence of testosterone. A, Coimmunoprecipitations (IP) were carried out using an anti-GFP antibody, and Western blot analysis (WB) of the immunocomplexes was performed using anti-AR antibody. B, The whole-cell lysates were subject to Western blot analysis for AR expression. C, The whole-cell lysates were subject to Western blot analysis for the expression of GFP-ARR19 or GFP.

 
ARR19 Cotranslocates into the Nucleus with AR in an Androgen-Dependent Manner
Because the distribution of transcriptional coregulators may give an insight into the mechanism of their action, we examined the subcellular localization of ARR19 protein and its behavior under different cellular conditions. Fluorescence of cells overexpressing a full-length red fluorescent protein (RFP)-fused ARR19 protein alone revealed that ARR19 was predominantly localized in the cytoplasmic compartment being associated with cellular organelles (probably endoplasmic reticulum and Golgi apparatus), and testosterone treatment did not change its distribution (Fig. 8AGo, bottom panels). The GFP-fused ARR19 protein showed the same pattern of distribution as the RFP-fused ARR19 protein, suggesting its cytoplasmic localization as the nature of ARR19 protein (data not shown). In contrast, like the intact AR, the overexpressed GFP-fused AR protein was observed to translocate into the nucleus upon testosterone exposure (Fig. 8AGo, top panels).



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  		Fig. 8. ARR19 Colocalizes into the Nucleus with AR in the Presence of Androgen

A, CV-1 cells were transfected with either GFP-AR or RFP-ARR19 construct and cultured in the presence or absence of testosterone. GFP-fused AR and RFP-fused ARR19 proteins were detected with green and red fluorescence, respectively, using a fluorescent microscopy. B, CV-1 cells were cotransfected with the GFP-AR and RFP-ARR19 constructs and cultured in the presence or absence of testosterone. In fluorescent microscopy, the green and redfluorescence were merged using MetaFluor software. In confocal microscopy, images were captured and analyzed using Leica TCS NT software. C, CV-1 cells were transfected with GFP-AR alone or together with RFP-ARR19 and AR transcriptional activities in the presence of testosterone were measured by the expression of a reporter, luciferase. -T, without testosterone; +T, with testosterone.

 
Coexpression of the RFP-ARR19 protein with the GFP-AR, however, caused the ARR19 protein to cotranslocate into the nucleus with the AR upon ligand addition (Fig. 8BGo). In addition, confocal microscopy showed that the distribution patterns for ARR19 and AR in the nucleus overlapped extensively. Transient transfection assays in CV-1 cells indicated that the GFP-AR and RFP-ARR19 fusion proteins might function in an identical manner to their intact proteins, displaying the androgen-dependent transactivation and the repression of AR transactivation, respectively (Fig. 8CGo). Together, the results suggest that ARR19 protein cotranslocates into the nucleus with AR in an androgen-dependent manner, thus subsequently modulating AR transcriptional activity.

HDAC Is Involved in the ARR19 Repression of AR Transactivation
Because the repression of transcriptional activity of nuclear receptors by certain corepressors has been shown to involve HDAC activity (41), we tested whether the ARR19 repression of AR transactivation is also dependent on HDAC activity using the HDAC inhibitor trichostatin A (TSA). In HeLa cells, cotransfection of AR with ARR19 expression plasmid repressed AR transactivation as in other cell lines (Fig. 3Go), and the repression of AR activity was fully recovered with TSA treatment (Fig. 9AGo). The transactivation activity of liganded AR without ARR19 coexpression was affected a little, if any, by TSA treatment as previously reported (42). These results imply that HDAC is involved in the ARR19 repression of AR transactivation.



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  		Fig. 9. ARR19-Repression of AR Transactivation Involves HDAC Activity

A, AR expression plasmid alone or together with ARR19 was transfected into HeLa cells as in Fig. 4Go. After treating with 100 nM of testosterone in combination with or without 100 nM of TSA for 24 h, cells were harvested and monitored for luciferase activity. B, AR expression plasmid and HDAC1, -4, and -5 were cotransfected into HeLa cells together with or without ARR19 expression plasmid. Relative luciferase activity represents the percent of the level of activity with AR and reporter gene alone in the presence of testosterone (set as 100). Experiments were performed in duplicate at least three times. C, Direct interaction of ARR19 with HDAC4 in vitro. Bacterially produced GST alone or GST fusion proteins were bound to glutathione-agarose beads and incubated with equivalent amounts of the 35S-labeled HDAC4 produced by in vitro translation. Ten percent of the labeled protein used in the binding reaction was loaded as input.

 
To define further the involvement of HDAC in the ARR19 repression of AR transactivation, each of HDAC1, -4, and -5 expression plasmids along with ARR19 was cotransfected into HeLa cells, and its effect on ARR19 repression of AR transactivation was accessed. As shown in Fig. 9BGo, HDAC4 was able to further inhibit the ARR19-repressed AR transactivation, whereas HDAC1 and -5 were not. Expression of HDAC1, -4, or -5 alone did not significantly affect AR transactivation. The results suggest that HDAC4, but not HDAC1 and -5, plays a role in the ARR19 repression of AR transactivation.

To determine whether ARR19 can directly recruit HDAC4, we performed GST pull-down assays. [35S]methionine-labeled HDAC4 produced by in vitro translation was allowed to bind the GST fusion protein of ARR19-n +LZ, ARR19-LZ+C, or ARR19-C because the production of the full-length GST fusion of ARR19 was unsuccessful (Fig. 9CGo). HDAC4 interacted efficiently with both ARR19-n +LZ and ARR19-LZ+C, but not with ARR19-C. The results suggest that ARR19 may directly recruit HDAC4.

ARR19 and HDAC4 Are Recruited by AR to an Androgen-Regulated Promoter in Vivo
We also performed DNA-protein complex immunoprecipitation assays to determine whether ARR19 and HDAC4 are recruited by AR to an androgen-regulated promoter (Fig. 10Go). HeLa cells were cotransfected with AR and pEGFP-ARR19 expression plasmids, together with flag-tagged HDAC4 and linearized ARE2-TATA-Luc reporter, and treated with 100 nM testosterone. Cross-linked DNA fragments produced by sonication were immunoprecipitated with anti-AR, anti-GFP, or anti-flag antibody. The immunoprecipitates were then analyzed by PCR using pairs of specific primers spanning the ARE region of the reporter. Occupancy of ARE promoter by AR was detected whenever AR was expressed in the presence of the ligand. ARR19 and HDAC4 were also associated with the ARE promoter, but their associations were not detected unless AR and ARR19 were coexpressed in the presence of testosterone. No signal was detected from the control PCR for nonspecific immunoprecipitation with primers specific to the luciferase coding region, which is about 3.3 kb upstream of the ARE promoter in the pARE2-TATA-Luc reporter that was linearized at the SphI site located between the ARE promoter and luciferase coding region. The results suggest that ARR19, which is recruited to the androgen-regulated promoter by AR in the presence of androgen, recruits HDAC4 to inhibit androgen-regulated gene expression in vivo.



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  		Fig. 10. ARR19 and HDAC4 Are Recruited by AR to an Androgen-Regulated Promoter in Vivo

Indicated expression plasmids were cotransfected into HeLa cells together with linearized pARE2-TATA-Luc reporter. The cells were then treated either with or without 100 nM of testosterone. Cross-linked DNA fragments were immunoprecipitated with indicated antibodies and analyzed by PCR using pairs of specific primers spanning the ARE promoter of the reporter. A control PCR for nonspecific immunoprecipitation was performed with primers specific for the luciferase coding region.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The mechanisms by which androgen supports male germ cell development and reproductive functions are largely unknown, in contrast to the well established fact that androgen is essential for these functions. This is due, in part, to the fact that the identity of all but a few target genes of androgen in male reproductive organs remains undiscovered. In this study, we describe the isolation of ARR19 as a putative androgen-induced gene and its functional characterization as a novel corepressor of AR. Negative feedback regulations have been previously reported for several coregulatory proteins including receptor-interacting protein 140 (RIP140), SMRT/HDAC1-associated repressor protein (SHARP), short heterodimer partner (SHP) and glucocorticoid receptor (32, 33, 34, 35), each of which acts as a negative regulator of the relevant transcription factor that controls its expression. RIP140 and SHARP, for example, are both a target and an effector of RAR and ER, respectively. Further studies are required to determine whether the expression of ARR19 is directly regulated by androgen through AR, which may provide a negative feedback regulatory mechanism in androgen action.

Coregulators play a pivotal role in mediating gene expression by modulating the transcriptional activity of nuclear receptors. It has been suggested that some coregulatory molecules are expressed at a low level compared with the level of their binding partner of transcription factors (43, 44). This may imply that even a small change in the expression of coregulators may have a dramatic effect on the transcriptional activation of nuclear receptors. In light of this and the possible feedback regulation of ARR19 gene expression, it is quite reasonable for ARR19 to be moderately induced by androgen.

ARR19 is a leucine-rich protein containing a putative LZ motif. Proteins such as L7, NRIF3, and JEM-1, which contain a LZ-like motif, have been previously reported to function as a coregulator of nuclear receptors with their LZ motif as a protein interacting domain (45, 46, 47). Another property of ARR19 is its ability to translocate with AR in response to androgen. ARR19 is localized to cytoplasmic organelles in the absence of androgen but translocates into the nucleus upon exposure to androgen. However, this translocation occurs only when AR is coexpressed. Similarly, ß-catenin has been recently shown to cotranslocate into the nucleus with liganded AR (48, 49, 50). Other transcription factors and coregulators have been also reported to translocate within subcellular compartments in response to a certain signaling. For example, the N-CoR/TGF-ß-activated kinase 1 binding protein 2 (TAB2)/HDAC3-containing complex undergoes a nuclear to cytoplasmic translocation in response to IL-1ß signaling, whereas nuclear factor-{kappa}B undergoes a cytoplasmic to nuclear translocation in response to the same signaling (51). The molecular basis of IL-1ß-dependent export of the nuclear N-CoR turns out to be a MEKK1-dependent phosphorylation of TGF-ß-activated kinase 1 binding protein 2 (TAB2) that is physically associated with N-CoR in the nucleus. The detailed mechanism by which AR translocates ARR19 into the nucleus still requires further study.

Corepressors are generally thought to recruit HDAC activity. The HDAC inhibitor TSA caused the derepression of AR transactivation that was inhibited by ARR19, suggesting the involvement of deacetylation in the corepressor action. This was further supported by the facts that overexpression of HDAC4 led to further inhibition of the ARR19-repressed AR transactivation and that ARR19 was able to directly interact with HDAC4 in vitro. Furthermore, DNA-protein complex immunoprecipitation assays revealed that HDAC4 was recruited by AR through ARR19 to an AR-regulated promoter in vivo. The direct recruitment of HDAC by ARR19 is unique and inconsistent with the behavior of typical nuclear receptor corepressors that exert repressive effects by recruiting HDAC through Sin3A complexes (28, 29). Thus, the HDAC-interacting property of ARR19 may categorize it into a distinct group of negative regulators that are able to directly interact with HDACs. RIP140 has been previously reported to directly recruit HDACs to repress RAR transactivation (52).

In conclusion, we provide evidence that ARR19, the product of a putative androgen-induced gene, is a corepressor of AR. The regulation of AR transactivation by ARR19 may constitute an additional level of control for the fine tuning of cellular responses mediated by AR. In particular, the specific expression of ARR19 in male reproductive organs postulates its role in AR-regulated male reproduction. Further studies of ARR19, for example, with null mutants by knockout mouse approach may provide a strong insight into the physiological function(s) of ARR19 in male reproductive organs including the testis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
Plasmids for mammalian expression and in vitro translation of AR and ARR19 were constructed using a mammalian expression vector, pcDNA3 (Invitrogen, San Diego, CA). Mouse AR expression and pARE2-TATA-Luc reporter plasmids were previously described (53). PSA-Luc reporter construct (37) was kindly provided by Dr. C. J. Bieberich (University of Maryland Baltimore County, Baltimore, MD). All the LexA and B42 fusion vectors were constructed by cloning the indicated genes and their mutants in frame after LexA-DBD of p202PL and B42-AD of pJG4–5 (54), respectively. GST fusion constructs were made using pGEX4T (Amersham Pharmacia Biotech AB, Uppsala, Sweden). GFP-AR and -ARR19 and RFP-ARR19 were constructed using pEGFP-C1 and pDsRed-N1 vector (CLONTECH, Palo Alto, CA), respectively. Mammalian expression constructs of HDAC1, -4, and -5 were previously described (55, 56). HDAC4 was subcloned into a Not I-Xho I site of pBluescript II KS under the T7 promoter for in vitro translation.

PCR-Select cDNA Subtraction Cloning
Adult male Sprague Dawley rats (75–80 d old) were injected ip with EDS (75 mg/kg) in dimethylsulfoxide-water (1:3, vol/vol). At the same time the animals also received an injection of either 0.1 ml of vehicle oil or 25 mg testosterone esters (Sustanon; Organon Korea Ltd., Seoul, Korea). The latter treatment was repeated every 3 d. Animals were killed 4 d after the EDS injection. mRNA samples were isolated from EDS-treated control rat testis and EDS-treated/testosterone-supplied rat testis to prepare driver and tester cDNA, respectively. PCR-select cDNA subtraction was performed using PCR-Select cDNA Subtraction Kit (CLONTECH, Palo Alto, CA) according to the manufacturer’s instructions.

Screening of a Testis cDNA Library
A {lambda}ZAPII cDNA library of mouse testis was purchased from Stratagene, Inc. (La Jolla, CA) and screened according to the user manual. A total of 1.5 x 106 clones were screened. Briefly, phage particles on replica nylon membranes were denatured in 0.5 M NaOH-1.5 M NaCl and neutralized in 0.5 M Tris-HCl (pH 8.0)-1.5 M NaCl. After UV cross-linking, the filters were prehybridized and hybridized at 42 C in the presence of 50% formamide, 10% dextran sulfate, 5x standard sodium citrate (SSC), 1 mM EDTA, 10 µg/ml of denatured salmon sperm DNA, and 32P-labeled rat ARR19 cDNA probe (nucleotides 378–807). Filters were washed at 42 C for 20 min in 0.2x SSC and 0.2% sodium dodecyl sulfate (SDS) as a final stringency. The membranes were then exposed on Kodak x-ray film (Eastman Kodak, Rochester, NY) at 70 C. Secondary screening was repeated as described to isolate single pure phage plaques. After in vivo excision, the cDNA inserts were sequenced with the ABI PRISM sequence analyzer (PerkinElmer, Foster City, CA).

Northern Blot Analysis
Total RNA was extracted from dissected tissues using Tri reagent solution (Molecular Research Center, Inc., Cincinnati, OH). Total RNA (20 µg) was separated on a 1.2% denaturing agarose gel, transferred onto nylon membrane in 10x SSC, and then immobilized under UV light. After prehybridization, the membrane was hybridized at 42 C in a solution containing 50% formamide, 10% dextran sulfate, 5x SSC, 1 mM EDTA, 10 µg/ml of denatured salmon sperm DNA, and 32P-labeled mouse ARR19 cDNA probe (nucleotides 378–807). After washing at 65 C for 20 min in 0.2x SSC and 0.1% SDS as a final stringency, the membrane was exposed on Kodak x-ray film at 70 C. The membrane was then stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Transient Transfection Assay
CV-1, HepG2, and HeLa cells were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum. LNCaP cells (American Type Culture Collection, Manassas, VA) were maintained in RPMI 1640 medium (Life Technologies) supplemented with 10% fetal bovine serum. Twenty-four hours before transfection, cells were plated in 24-well plates and transfected with the indicated amount of expression plasmids, the reporter pARE2-TATA-Luc, and the control lacZ expression plasmid pCMVß using Superfect (QIAGEN, Chatsworth, CA). Total amounts of expression vectors were kept constant by adding appropriate amounts of the blank vector, pcDNA3. Twenty-four hours after transfection, the medium was replaced with fresh medium containing 10% charcoal-stripped serum and either 10 nM testosterone or vehicle. Cells were harvested 24 h after hormone addition, and luciferase and ß-galactosidase activities were assayed as described previously (57). The levels of luciferase activity were normalized to the lacZ expression.

Fluorescence
CV-1 cells were plated onto gelatin-coated coverslips the day before transfection. RFP-ARR19 alone, or together with GFP-AR, was transiently transfected using Effectene reagent (QIAGEN). After 16 h, transfected cells were fed with fresh medium with/without 100 nM testosterone and incubated for 24 h. For fluorescent microscopy, the coverslips were taken and analyzed directly in the presence of media using the Olympus 1x70 fluorescent microscope (Tokyo, Japan). The images were analyzed using MetaFluor software (Universal Imaging Corp., Downingtown, PA). For confocal microscopy, the cells on coverslips were washed with PBS, fixed with 3.7% formaldehyde for 5 min, mounted onto microscope slides, and examined using the Leica TCS NT laser-scanning microscope (Bensheim, Germany). Images were captured and analyzed using Leica TCS NT software.

Yeast Two-Hybrid Assay
Plasmids encoding LexA fusions and B42 fusions were cotransformed into Saccaromyces cerevisiae EGY48 containing the lacZ reporter plasmid, SH/18–34. The transformants grown on a plate of selective medium were then incubated in the same medium, but containing 2% galactose at 30 C for 3 h. An equal amount of cells were harvested, resuspended in a buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, and 50 mM 2-mercaptoethanol, pH 7.0), and lysed with 0.1% of SDS and 10% of chloroform at 30 C for 15 min. The liquid ß-galactosidase assays were carried out as described previously (58).

GST Pull-Down Assay
Escherichia coli BL21 cells transformed with pGEX-4T vector only or each of pGEX-4T-ARR19 fusions were grown at 37 C, and the synthesis of GST-fusion proteins was induced by addition of 0.2 mM isopropyl-ß-D-thiogalactopyranoside as a final concentration. The GST-fusion proteins were isolated with glutathione-Sepharose-4B beads (Amersham Pharmacia Biotech AB) and washed twice with PBS, and then incubated with [35S]methionine-labeled proteins produced by in vitro translation using the TNT-coupled transcription-translation system (Promega Corp., Madison, WI) under the conditions recommended by the manufacturer. Specifically bound proteins were eluted from the beads with 40 mM reduced glutathione in 50 mM Tris (pH 8.0), and then analyzed by SDS-PAGE and autoradiography.

Coimmunoprecipitation
In vivo coimmunoprecipitation assays were performed with HeLa cells transfected with 500 µg of pcDNA3-AR and 500 µg of either GFP-ARR19 or GFP empty vector. Transfected cells were cultured in the absence or presence of 100 nM testosterone for 24 h and harvested in a buffer containing 20 mM Tris-HCl at pH 8.0, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 0.5% Nonidet P-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 5 µg/ml pepstatin. Whole-cell lysates (400 µg) were incubated with anti-GFP monoclonal antibody (Molecular Probes, Eugene, OR) for 2 h at 4 C and further incubated for another 1 h after adding preequilibrated protein-A Sepharose beads (Amersham Pharmacia Biotech AB). The Sepharose beads were then washed three times with the same buffer at 4 C. Bound proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and subjected to Western blot analysis with anti-AR antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Proteins were detected using the ECL kit (Amersham Pharmacia Biotech AB).

DNA-Protein Complex Immunoprecipitation Assay
HeLa cells were cotransfected by using Effectene (QIAGEN) with pARE2-TATA-Luc linearized with SphI and flag-HDAC4 expression plasmid together with mouse AR and/or GFP-ARR19 expression vector. After 24 h transfection, cells were treated with or without 100 nM testosterone for 24 h. Cells were then cross-linked with 1% formaldehyde after washing with PBS, and DNA-protein complex immunoprecipitation assays were performed as chromatin immunoprecipitation assay, which was described previously (59). Immunoprecipitated DNA and input-sheared DNA were subjected to PCR using primer pairs (sense: 5'-CAGGTGCCAGAACATTTCTC-3' and antisense: 5'-GAGTTTTCACTGCATACGACG-3'), which amplify an approximately 400-bp region spanning the ARE promoter of the reporter. As a negative control, PCRs were performed using primer pairs (sense: 5'-GAAGGTTGTGGATCTGGATAC-3' and antisense: 5'-TTTCCGTCATCGTCTTTCCG-3'), which amplify an approximately 370-bp region spanning the C-terminal part of luciferase coding region of the reporter.


   ACKNOWLEDGMENTS
 
We thank Drs. C. J. Bieberich and J. W. Lee for providing PSA-Luc and the mammalian expression construct of HDACs, respectively.


   FOOTNOTES
 
This work was supported by a Korea Research Foundation Grant (KRF-2002-070-C0007) and a Hormone Research Center Grant (2002G0101).

Abbreviations: AF, Activation function; AR, androgen receptor; ARE, androgen response element; ARR19, androgen receptor corepressor-19 kDa; DBD, DNA-binding domain; DBDh, DBD and hinge domain; EDS, ethane dimethanesulfonate; ER, estrogen receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GST, glutathione-S-transferase; HDAC, histone deacetylase; LBD, ligand-binding domain; LZ, leucine zipper; NCoR, nuclear receptor corepressor; PSA, prostate-specific antigen; RAR, retinoic acid receptor; RFP, red fluorescent protein; RIP, receptor-interacting protein; RXR, retinoid X receptor; SSC, standard sodium citrate; TSA, trichostatin A.

Received for publication February 28, 2003. Accepted for publication October 15, 2003.


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Nuclear Receptors:   AR
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