help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2002-0340
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow NURSA Molecule Pages Link
Right arrow Reprints, Permissions and Rights
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hauksdottir, H.
Right arrow Articles by Privalsky, M. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hauksdottir, H.
Right arrow Articles by Privalsky, M. L.
Molecular Endocrinology 17 (3): 373-385
Copyright © 2003 by The Endocrine Society

Retinoic Acid Receptors ß and {gamma} Do Not Repress, But Instead Activate Target Gene Transcription in Both the Absence and Presence of Hormone Ligand

Herborg Hauksdottir, Behnom Farboud and Martin L. Privalsky

Section of Microbiology, Division of Biological Sciences, University of California at Davis, Davis, California 95616

Address all correspondence and requests for reprints to: Martin L. Privalsky, Section of Microbiology, Division of Biological Sciences, University of California Davis, 1 Shields Avenue, Davis, California 95616. E-mail: mlprivalsky{at}ucdavis.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Retinoic acid receptors (RARs) are important mediators of retinoid signaling in morphogenesis, development, and cell differentiation. Three major isotypes of RARs, denoted {alpha}, ß, and {gamma}, have been identified, each encoded by a distinct genetic locus. Although RAR{alpha}, RARß, and RAR{gamma} share many structural and functional features, these three isotypes are known to play unique, as well as overlapping, roles in physiology and development. We report here that the three RAR isotypes display different transcriptional properties in the absence of hormone ligand; under these conditions, RAR{alpha} is a strong repressor of target gene expression, whereas both RARß and RAR{gamma} fail to repress and instead are able to mediate substantial levels of hormone-independent transcriptional activation. These differing transcriptional properties appear to reflect the differing abilities of the three RAR isotypes to interact with the SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) corepressor protein: RAR{alpha} binds to SMRT strongly both in vitro and in vivo, whereas RARß and RAR{gamma} interact only weakly with SMRT. The ability to repress or to activate transcription in the absence of hormone maps predominantly to isotype-specific differences in the sequence of helix 3 within the hormone binding domain of the RARs, and the transcriptional properties of one isotype can be exchanged with that of another by exchanging portions of helix 3. The different transcriptional properties of RAR{alpha}, RARß, and RAR{gamma} in the absence of hormone contribute to the distinctive biological functions of these proteins and provide a rationale for the strong conservation of the three distinct isotypes during the vertebrate evolutionary radiation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NUCLEAR HORMONE RECEPTORS are a large family of ligand-regulated transcription factors that play important roles in organismal development, differentiation, and homeostasis (1, 2, 3, 4, 5, 6, 7, 8). The nuclear hormone receptors include the steroid receptors, thyroid hormone receptors, vitamin D receptors, peroxisome-proliferator activated receptors, retinoid X receptors, and retinoic acid receptors (RARs), all of which are defined by the presence of a characteristic central DNA binding domain joined to a more C-terminal, hormone-binding domain (1, 2, 3, 4, 5, 6, 7, 8). Nuclear hormone receptors regulate transcription by binding to DNA enhancer elements, referred to as hormone response elements, located within or adjacent to target gene promoters (9). Notably, many nuclear hormone receptors display bimodal transcriptional properties and can either repress or activate target gene transcription depending on the concentration and chemical nature of the hormone ligand, the nature of the target promoter, and the influence of additional signal transduction pathways operative in the cell (10, 11). These bipolar transcriptional responses reflect the ability of the nuclear hormone receptors to recruit a panel of auxiliary proteins, denoted corepressors and coactivators, which mediate the actual molecular events necessary for repression or activation of gene transcription (12, 13, 14, 15, 16, 17, 18, 19).

Nuclear hormone receptors typically bind to corepressors, such as SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) or its paralog, N-CoR (nuclear hormone receptor corepressor), in the absence of hormone agonist (10, 11). SMRT and N-CoR tether, in turn, additional components of the corepressor holocomplex, including TBL-1 (transducin ß-like protein-1) and a series of histone deacetylases (HDACS) that result in transcriptional repression (12, 13, 14, 15, 16, 17, 18, 19). Conversely, the binding of hormone agonist to a nuclear hormone receptor is believed to induce a conformational change that dissociates the corepressor complex and promotes the recruitment by the receptor of one or more coactivators, such as SRC-1 (steroid receptor coactivator protein-1)/GRIP-1(glucocorticoid receptor-interacting protein 1)/ACTR (activator of thyroid hormone and retinoic acid receptors), CBP (cAMP-responsive transcription factor binding protein)/p300, and the DRIP (vitamin D receptor interacting protein)/TRAP (thyroid hormone receptor associated protein) complexes, leading to target gene activation (10, 11). Corepressors and coactivators can modulate gene expression by multiple mechanisms, including covalent modifications of the chromatin complex and interactions with the general transcriptional machinery (20, 21, 22, 23, 24, 25, 26).

Three different loci encode retinoic acid receptors in vertebrates: RAR{alpha}, RARß, and RAR{gamma}. These three RAR isotypes (2, 3, 5) are similar but not identical in sequence and structure to one another, and they are expressed in distinctive tissue-specific and developmentally regulated patterns (2, 5, 27). Although gene disruption studies in mice have revealed some functional redundancy between the different RAR isotypes, each RAR isotype also performs unique functions in development and differentiation that cannot be replaced by the actions of the other isotypes; the use of isotype-selective retinoid ligands confirms these conclusions and provides additional evidence that each RAR isotype exerts a mixture of overlapping and specific actions in target gene regulation (28, 29, 30). Therefore, each of the RAR receptors plays a specific function in the organism that is likely to account for the high degree of conservation of the three isotypes throughout vertebrate evolution.

In contrast to their distinct biological functions in vivo, the different RAR isotypes display generally similar DNA and hormone binding properties in vitro (2, 3). However, we have previously noted that the RAR{alpha}, RARß, and RAR{gamma} isotypes do differ in terms of their ability to bind to the corepressor SMRT: RAR{alpha} efficiently binds to SMRT, whereas RARß and RAR{gamma} bind to SMRT much more weakly (31). The biological consequences of this isotype-specific corepressor interaction were not determined in our prior work, leading us to subsequently investigate the transcriptional regulatory properties of the different RAR isotypes. We report here that all three isotypes are able to activate reporter gene transcription in the presence of hormone agonist. Notably, however, although RAR{alpha} can repress target gene transcription in the absence of hormone, RARß and RAR{gamma} fail to repress and instead mediate transcriptional activation under the same conditions. The divergent transcriptional properties of RAR{alpha}, RARß, and RAR{gamma} in the absence of hormone parallel the ability of these isotypes to interact with SMRT and map primarily to isotype-specific amino acid differences in helix 3 within the hormone binding domain of these receptors. Only three amino acids differ between the relevant helix 3 domains of RAR, RARß, and RAR{gamma}: Ile 224, Asp 225, and Ser 234 in RAR{alpha} are replaced by Leu, Gly, and Ala, respectively, in RARß and RAR{gamma}. Notably, exchanging the amino acids in these three positions can exchange the ability of these receptors to bind to corepressor in vitro and to mediate transcriptional repression in transfected cells. We conclude that the different SMRT binding properties of RAR{alpha}, RARß, and RAR{gamma} result in distinct transcriptional regulatory properties that contribute to the divergent biological roles of these different isotypes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Unlike RAR{alpha}, the RARß Isotype Fails to Repress and Functions Instead to Activate Transcription in the Absence of Hormone
As noted in our prior study (31), RAR{alpha} and RARß displayed different abilities to bind to SMRT corepressor in a glutathione-S-transferase (GST)-pulldown/protein interaction assay: RAR{alpha} readily bound to an immobilized SMRT construct under these conditions, whereas RARß interacted with SMRT with only low efficiency (Fig. 1AGo, filled bars). This marked difference in the ability of RAR{alpha} and RARß to bind to GST-SMRT was maintained over a 10-fold range of receptor inputs and over a 12-fold range of GST-SMRT concentrations (data not shown). A similar difference was observed in the ability of RAR{alpha} and RARß to interact with SMRT in a mammalian two-hybrid assay (31). We therefore sought to investigate the effects of this isotype-specific corepressor recruitment on RAR-mediated transcriptional regulation in cells. We first tested whether RAR{alpha} and RARß differ in their abilities to repress transcription in hormone-striped culture medium. Figure 1BGo displays the results from a transient transfection of CV-1 cells. In the absence of hormone, RAR{alpha} repressed the expression of a luciferase reporter containing RAR-response elements (pTK-Luc-RARE3) compared with the same reporter lacking a response element (pTK-Luc empty; Fig. 1BGo, filled bars). In contrast to these results with RAR{alpha}, introduction of RARß did not repress the pTK-Luc-RARE3 reporter in the absence of hormone, but instead increased expression of the reporter to levels significantly above those observed in the absence of an RARE (Fig. 1BGo, filled bars). This difference in the transcriptional properties of the unliganded RAR{alpha} and RARß isotypes was observed over a range of expression vector concentrations (data not shown). As anticipated from prior reports (10, 11, 32), all-trans retinoic acid (ATRA), a pan-agonist for RARs, induced the dissociation of SMRT in vitro (Fig. 1AGo, open bars) and permitted both RAR{alpha} and RARß to function as strong transcriptional activators of the pTK-Luc-RARE3 reporter in transfected cells (Fig. 1BGo, open bars). The expression of a pCH110-lacZ reporter, used as an internal transfection control and lacking response elements, or of the pTK-Luc empty reporter itself, was not altered by expression of either RAR isotype, plus or minus hormone (Fig. 1BGo, and data not shown).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 1. Various RAR isotypes differ in their ability to bind to SMRT corepressor in vitro and to regulate reporter gene expression in cells. A, RAR{alpha}, but not RARß or RAR{gamma}, binds SMRT corepressor efficiently in vitro. Nonrecombinant GST, or a GST-SMRT fusion construct, was immobilized on a glutathione-agarose matrix and was incubated with radiolabeled RAR{alpha}, ß, or {gamma} as indicated. Incubations were performed both in the absence (filled bars) and presence (open bars) of 1 µM ATRA. Radiolabeled receptors remaining bound to the GST- or GST-SMRT matrix after repeated washing were eluted, were resolved by SDS-PAGE, and were quantified by PhosphorImager analysis. The amount of each receptor bound to the GST- or GST-SMRT matrix, expressed as a percentage of the input, is presented. The average of duplicate experiments and the SD are shown. B, Unlike RAR{alpha}, RARß and RAR{gamma} activate reporter gene expression in both the absence and presence of ATRA hormone. CV-1 cells were transiently transfected with a pSG5 vector containing the RAR{alpha}, ß, or {gamma} coding sequence, or an empty pSG5 expression vector (Vector), as indicated. Included in the transfection was a pTK-Luciferase reporter construct either lacking response elements (pTK-Luc empty) or containing three RAREs arranged in tandem (pTK-Luc RARE3). A pCH100-lacZ reporter was included in all transfections as an internal normalization control. The cells were subsequently incubated in the absence (filled bars) or presence (open bars) of 400 nM ATRA, harvested, and assayed for luciferase and ß-galactosidase activity. The relative luciferase activity was calculated as the ratio of absolute luciferase to ß-galactosidase. The average of duplicate experiments and the SD are shown.

 
We also examined the corepressor interaction and transcriptional regulatory properties of RAR{gamma}, the third major RAR isotype. RAR{gamma} interacted with SMRT corepressor relatively weakly (Fig. 1AGo) and did not repress, but rather enhanced expression of the pTK-Luc-RARE3 reporter in the absence of hormone (Fig. 1BGo, filled bars). As noted for RARß, this hormone-independent activation by RAR{gamma} required the presence of a functional response element in the reporter and was not detected for either the pTK-Luc empty reporter or the internal lacZ transfection control (Fig. 1BGo, and data not shown). In common with both RAR{alpha} and RARß, addition of ATRA further increased reporter gene activation by RAR{gamma} (Fig. 1BGo, open bars). We conclude that under the conditions used here, neither RARß nor RAR{gamma} is able to repress reporter gene expression; instead they mediate a hormone-independent activation of target gene expression. It should be noted that identical expression and reporter vectors were used for all three isotypes, that all three receptor isotypes induced approximately equal reporter activation in the presence of hormone (Fig. 1BGo), and that all three receptors were expressed at comparable levels in the transfected cells in the absence of hormone (although the levels of RARß were slightly increased in the presence of ATRA; Fig. 2Go).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 2. The different RAR isotypes are expressed at comparable levels in transfected cells in the absence of hormone. CV-1 cells were transfected with the various pSG5-RAR isotype constructs using the same protocol as in Fig. 1BGo. After 24 h, the cells were either maintained in hormone-free media (filled bars) or transferred to media containing 400 nM ATRA. After an additional 24 h, the cells were harvested and lysed, and the lysates were analyzed by SDS-PAGE and by immunoblotting using RAR-directed antisera. The resulting RAR bands were quantified by using an ECL plus detection system and Storm System image analysis. The relative expression levels of the different RAR isotypes, normalized to correct for differences in the immunoreactivity of the antisera toward the different isotypes (see Materials and Methods), are presented.

 
RARß Functioned as a Constitutive Transcriptional Activator in Multiple Cell Type and Promoter Contexts
Transient transfections of HeLa or COS-1 cells or use of a different reporter construct (M-pTK-Luc RARE2) produced essentially the same results as did the CV-1 and pTK-Luc-RARE3 system: in all cases, RARß failed to repress and instead mediated an activation of reporter gene expression in the absence of hormone ligand (data not shown). Notably, most vertebrate cells express RARs endogenously (2), and this includes the CV-1, COS-1, and HeLa cells used here. The presence of these endogenous RARs could be detected as an ATRA-dependent activation of the pTK-Luc-RARE3 construct that occurred in these cells even in the absence of an ectopically introduced RAR construct (note the effects of ATRA, open bars, on the pTK-Luc-RARE3 reporter when tested with the pSG5 vector-only control in Fig. 1BGo, right). Conversely, in the absence of ATRA, the level of endogenous RAR{alpha} in many of these cell lines appeared sufficient to repress the pTK-Luc-RARE3 reporter, with the consequence that the introduction of ectopic RAR{alpha} did not necessarily result in a further reduction of pTK-Luc-RARE3 reporter expression (compare the effects of pSG5-RAR{alpha} vs. pSG5 vector-only, Fig. 1BGo, filled bars). In support of this interpretation, when introduced without an ectopic RAR, the pTK-Luc empty reporter, which lacks response elements, displayed substantially higher levels of expression in the absence of hormone than did the otherwise identical pTK-Luc reporter containing RAR response elements (pTK-Luc-RARE3; Fig. 1BGo, right panel, closed bars). We used two approaches to circumvent this problem. We first used the L929 cell line that has previously been reported to be permissive for RAR{alpha}-mediated repression (33). We found that, in agreement with this previous report, the ectopic expression of RAR{alpha} caused a detectable repression of reporter gene transcription in the L929 cell line that was not seen when using an empty pSG5 vector control (Fig. 3AGo). Notably, RARß retained the same phenotype in the L929 cells as it displayed in our other cells lines: a lack of repression, and the converse ability to activate reporter gene expression even in the absence of ATRA (Fig. 3AGo). To more fully preclude the contribution of endogenous RARs (or other mammalian transcription factors that might promiscuously recognize our RARE reporter elements), we next repeated our transfection studies using a GAL4-DNA binding domain (DBD)-RAR fusion system. By replacing the RAREs in the reporter with a GAL-17mer recognition site and by substituting the native DNA binding domain of the RAR with that of the Saccharomyces cerevisae-derived GAL4DBD, this system produces a reporter that responds exclusively to the ectopic GAL4-RAR fusion proteins and is not subject to regulation by endogenous RARs. Using this method, a GAL4DBD-RAR{alpha} fusion mediated an unambiguous repression in the absence of hormone and a strong activation on addition of ATRA when compared with the pGAL4DBD vector control (Fig. 3BGo). In contrast, the GAL4DBD-RARß fusion failed to repress and instead activated the pTK-GAL-17mer reporter in the absence of hormone, and this activation was further stimulated by addition of ATRA (Fig. 3BGo). These results support our results with the native RARs and indicate that the transcriptional properties of RAR{alpha} and RARß diverge in the absence of hormone, with the former mediating target gene repression and the latter mediating activation. Furthermore, given the nature of the GAL4-DBD constructs, our results demonstrate that the receptor A/B domain, a region previously shown to contribute to transcriptional activation by many nuclear hormone receptors, is not required for the hormone-independent transcriptional activation properties of RARß.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 3. RAR{alpha} represses transcription in L929 cells, and in a GAL4DBD fusion assay system. A, In L929 cells, RAR{alpha} (but not RARß) represses transcription in the absence of hormone. A nonrecombinant pSG5 vector (vector) or a pSG5 vector encoding wild-type RAR{alpha}, wild-type RARß, the RAR{alpha}ß{alpha} chimera, or the RARß{alpha}ß chimera was introduced into L929 cells together with the pTK-Luc-ßRARE3 luciferase reporter. A pCH110 lacZ reporter was included as an internal normalization control. The cells were subsequently maintained in the absence of hormone and harvested, and the relative luciferase levels were determined as in Fig. 1BGo. The average of duplicate experiments and the SD are shown. B, When expressed as a GAL4-DBD-fusion, the unliganded RAR{alpha} (but not RARß) represses reporter gene transcription in CV-1 cells. CV-1 cells were transiently transfected with an expression vector either encoding no receptor, or encoding GAL4-DBD fusions of the hormone binding domains of RAR{alpha}, RARß, RAR{alpha}(ßH3), or RARß({alpha}H3), together with a pGL3-GAL4–17mer luciferase reporter vector. A pCH110 lacZ reporter was included as a internal control for transfection efficiency. Cells were subsequently incubated in the absence (top) or presence (bottom) of 400 nM ATRA, were harvested, and were analyzed for relative luciferase activity as in Fig. 1BGo. The average of duplicate experiments and the SD are shown.

 
Cotransfection of RXR as Heterodimer Partner Further Stimulates the Hormone-Independent Transcriptional Activation Properties of RARß
Many nuclear hormone receptors can heterodimerize with other members of the family, and these heterodimeric species are believed to play important roles in mediating the hormone response (3, 4, 9). Retinoid X receptor (RXR) is a particularly important heterodimer partner for many nuclear hormone receptors, and RXR/RAR heterodimers are known to bind DNA more strongly and display enhanced transcriptional regulatory properties, compared with homodimers of either parental receptor (3, 4, 9, 34). We therefore conducted additional transient transfection studies to determine whether RXR might alter the transcriptional properties of RARß. Transfecting an RXR expression construct alone had little or no effect on the pTK-Luc-RARE3 reporter in the absence of hormone (Fig. 4Go, top). When cotransfected, ectopic RXR slightly attenuated the ability of RAR{alpha} to repress in the absence of hormone, but greatly enhanced the ability of RARß to activate reporter gene expression under the same conditions (Fig. 4Go, top). As anticipated from the results of previous studies (3, 4, 9), cotransfecting the RXR heterodimer partner in the presence of ATRA enhanced transcriptional activation by both RAR{alpha} and RARß, with this effect being more pronounced for the RAR{alpha} isotype (Fig. 4Go, bottom). It should be noted that introduction of ectopic RXR also enhanced ATRA-mediated reporter gene activation by the endogenous RARs present in these CV-1 cells (Fig. 4Go, bottom; note the RXR-only lane). We conclude that ectopic RXR expression does not confer on RARß the ability to repress, but instead further enhances the hormone-independent transcriptional activation properties of the RARß isotype.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 4. Cotransfection of RXR enhances the hormone-independent transcriptional activity of RARß. A pSG5 expression vector encoding no receptor, wild-type RAR{alpha}, wild-type RARß, the RAR{alpha}ß{alpha} chimera, or the RARß{alpha}ß chimera was introduced into CV-1 cells in the presence or absence of a pSG5-RXR{alpha} expression vector, as indicated. The pTK-Luc-ßRARE3 luciferase and pCH100 lacZ reporters were also included in each transfection. The cells were subsequently incubated in the absence (top) or presence (bottom) of 400 nM ATRA, and the relative luciferase levels were determined as in Fig. 1BGo. The average of duplicate experiments and the SD are shown.

 
The Difference in Transcriptional Regulation by the Different Isotypes Maps Principally to Receptor Helix 3 Located within the Hormone Binding Domain
The different corepressor binding properties of RAR{alpha} and RARß map within a region encompassing helix 1, the {Omega} loop, and helix 3 in the hormone binding domain of these receptors (Fig. 5AGo and middle panel of B; Ref. 31). We next tested whether the same receptor region was also responsible for the differing transcriptional properties of these isotypes in our reporter gene assays. An RAR{alpha}ß{alpha} chimera that contained the helix 1-{Omega}-helix 3 region of RARß displayed the transcriptional properties of RARß in CV-1 transfection assays, whereas an RARß{alpha}ß chimera that contains the helix 1-{Omega}-helix 3 region of RAR{alpha} displayed the RAR{alpha} transcriptional phenotype (Fig. 5BGo, right, filled bars). Analogous results were obtained from transfections of L929 cells in the GAL4DBD-RAR fusion assay system and in the presence of ectopic RXR (Figs. 3Go and 4Go, and data not shown). Therefore, at this level of resolution, the failure of RARß to repress correlated with the failure of RARß to interact with corepressor. To more precisely identify the amino acid sequences that are responsible for the difference in repression by RAR{alpha} and RARß, we analyzed an additional series of chimeras in which helix 1, the {Omega} loop, or the relevant portions of helix 3 were individually swapped between the RAR{alpha} and RARß archetypes. These chimeras were first tested for their ability to activate or repress the pTK-Luc-RARE reporter. The RAR{alpha}(ßH1) and RAR{alpha}(ß{Omega}) chimeras displayed the transcriptional properties of the RAR{alpha} receptor, whereas the RAR{alpha}(ßH3) construct failed to repress and displayed a hormone-independent reporter gene activation resembling that of the RARß receptor (Fig. 6AGo, left). Reciprocally, the RARß({alpha}H1) and RARß({alpha}{Omega}) constructs retained most (but not all) of the hormone-independent transactivation properties of wild-type RARß, whereas the RARß({alpha}H3) chimera was greatly impaired in its ability to activate transcription in the absence of hormone (Fig. 6AGo, right). All constructs retained the ability to activate the reporter gene in the presence of ATRA (Fig. 6BGo). Therefore, we conclude that the divergent transcriptional properties of RAR{alpha} and RARß in the absence of hormone map primarily, but not exclusively, to differences in their helix 3 sequences, with the helix 1 and {Omega}-loop regions making a more minor, but still detectable, contribution. Notably, the divergent transcriptional properties of these different chimeras largely paralleled their ability to interact with SMRT corepressor: constructs possessing the RARß helix 3 interacted only weakly with SMRT in vitro, whereas constructs containing the RAR{alpha} helix 3 strongly interacted with SMRT in the absence of hormone (Fig. 6CGo, filled bars). The sequence of the helix 1 and the {Omega}-loop region also contributed, although in a minor fashion, to corepressor binding in that the RAR({alpha}H1) and RAR({alpha}{Omega}) chimeras displayed slightly elevated abilities to bind to SMRT when compared with RARß wild-type (Fig. 6CGo, and data not shown). Addition of hormone agonist (ATRA), as expected, caused dissociation of SMRT from all the RAR constructs tested (Fig. 6CGo, open bars). We extended these results to the GAL4DBD-RAR fusion/GAL-17mer reporter system (Fig. 3BGo). A GAL4DBD-RARß ({alpha}H3) chimera that contains the relevant portions of helix 3 of RAR{alpha} displayed the RAR{alpha} property of repressing reporter gene expression in the absence of hormone; the reciprocal GAL4DBD-RAR{alpha}(ßH3) chimera that contains the relevant helix 3 sequences derived from RARß exhibited the RARß property of activating reporter gene expression in the absence of hormone (Fig. 3BGo, top). Again, all constructs mediated strong transcriptional activation in the presence of ATRA (Fig. 3BGo, bottom). We conclude that the inability of RARß to interact efficiently with SMRT corepressor predominantly reflects isotype-specific amino acid differences in helix 3 and helps account for the inability of the RARß isotype to repress transcription in transfected cells.



View larger version (46K):
[in this window]
[in a new window]
  		Figure 5. The different transcriptional regulatory and SMRT corepressor binding properties of RAR{alpha} and RARß map to sequence differences within the hormone binding domain of these receptors. A, A comparison of the amino acid sequences of the hormone binding domains of RAR{alpha}, ß, and {gamma} is presented. A common numbering system, based on the sequence of RAR{gamma}, is used for ease of comparison. The regions corresponding to helix 1–12 in the crystallographic structure of the liganded RAR are indicated above the amino acid sequence, as is the position of amino acids 224, 225, and 234 that contribute to the differing corepressor and transcriptional regulatory properties of the different isotypes (see the text). B, Chimeric constructs of RAR{alpha} and RARß demonstrate that the corepressor binding and transcriptional regulatory properties of these isotypes map within the helix 1-{Omega} loop-helix 3 region of these receptors. Left, A schematic of the different receptor constructs used in our experiments. Middle, The ability of these different RAR constructs to bind to GST-SMRT in the absence of hormone is shown (expressed as percentage input bound). Right, The ability of these different RAR constructs to regulate transcription of the pTK-Luc-RARE3 reporter in the absence (filled bars) or presence (open bars) of 400 nM ATRA is shown; transfections were performed as in Fig. 1BGo. The average of duplicate experiments and the SD are shown.

 


View larger version (41K):
[in this window]
[in a new window]
  		Figure 6. The different transcriptional regulatory and SMRT corepressor binding properties of RAR{alpha} and RARß map to sequence differences within helix 3 of the hormone binding domain of these receptors. Different RAR{alpha} constructs bearing the helix 1 (H1), omega loop ({Omega}-loop), or N-terminal portion of helix 3 of RARß were created, as were the reciprocal constructs of RARß. A and B, The distinct transcriptional regulatory properties of RAR{alpha} and RARß map to helix 3. The various RAR chimeras were tested as pSG5 constructs for the ability to activate or repress transcription of the pTK-Luc-RARE3 reporter gene in transient transfections of CV-1 cells, as in Fig. 1BGo. The cells were subsequently incubated in the absence (A) or presence (B) of 400 nM ATRA, and the relative luciferase activity was determined as before. C, The distinct corepressor interaction properties of RAR{alpha} and RARß also map primarily to helix 3. The same chimeras were tested for the ability to bind to SMRT in vitro using a GST-pulldown assay. The amount of each RAR protein bound to the GST-SMRT construct in the absence (filled bars) or presence (open bars) of 400 nM ATRA is shown, expressed as a percentage of input. The average of duplicate experiments and the SD are shown in all cases.

 
Three Amino Acids that Differ in Helix 3 of RAR{alpha}, RARß, and RAR{gamma} Contribute to the Different Transcriptional Properties of These Three Different Isotypes
There are three amino acids that differ between RAR{alpha} and RARß within the relevant region of helix 3: Ile 224, Asp 225, and Ser 234 in RAR{alpha} are Leu, Gly, and Ala in RARß, respectively (Fig. 5AGo). We therefore mutated these amino acids, both individually and in pairs, to the corresponding sequence in the opposite isotype and tested the resulting receptors for their regulatory properties in transfection assays. Replacing the RARß amino acid at any one position with that of RAR{alpha} decreased the ability of RARß to induce reporter gene expression in the absence of hormone, with the G225D mutation having the most severe effect (Fig. 7Go, top). Certain double substitutions, such as the L224I/A234S mutation, conferred a more inhibitory effect on hormone-independent reporter gene activation by RARß than did either individual mutation (Fig. 7Go, top). Reciprocally, changing the amino acids in helix 3 of RAR{alpha} to the corresponding RARß sequence generated a more RARß-like phenotype; this was more evident for the I224L/D225G double and the complete-triple amino acid substitution than for any single mutation (Fig. 7Go, top). In contrast, point mutations in the helix 1 region (e.g. RAR{alpha} double mutant I190T, V193I or RARß double mutant T190I, I193V) and in the {Omega} loop region (RARß R221S) did not result in any significant change in the ability of the receptor to interact with corepressor in vitro or repress transcription in vivo (data not shown). All mutant clones were able to strongly activate reporter gene expression in the presence of ATRA (Fig. 7Go, bottom). We conclude that all three helix 3 amino acids that differ between RAR{alpha} and RARß, located at positions 224, 225, and 234 in the protein sequence, contribute to the lack of repression and to the constitutive activation properties of RARß. Notably, RAR{gamma}, which exhibits a constitutive transcriptional activation phenotype similar to that of RARß, also possesses an RARß amino acid sequence at these three helix 3 positions (Fig. 5AGo); changing these amino acids in RAR{gamma} to the RAR{alpha} sequence converted the former from a hormone-independent activator to a transcriptional repressor (data not shown), indicating that this same region of helix 3 operates to define the distinctive transcriptional regulatory properties of all three isotypes.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 7. All three amino acid differences between the relevant portions of helix 3 of RAR{alpha} and RARß contribute to the different transcriptional properties of these isotypes. Different RAR{alpha} constructs bearing single, double, or triple amino acid substitutions characteristic of the RARß sequence at codons 224, 225, or 234 were created by site-directed mutagenesis, as were the reciprocal substitution mutants of RARß. These different RAR mutants were then tested as pSG5 constructs for the ability to activate or repress transcription of the pTK-Luc-RARE3 reporter gene in transient transfections of CV-1 cells, as in Fig. 1BGo. The cells were incubated in the absence (top) or presence (bottom) of 400 nM ATRA, and the relative luciferase activity was determined as before. The average of duplicate experiments and the SD are shown in all cases.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In Contrast to RAR{alpha}, the RARß and RAR{gamma} Isotypes Do Not Repress, But Can Instead Activate Transcription in the Absence of Hormone
Although the different RAR isotypes have a significant degree of functional redundancy, each of the isotypes also mediates unique functions in the organism (2, 3, 5, 28, 29, 30, 35). The different RAR isotypes display very similar DNA and hormone binding properties in vitro (3). However, in a prior study, we reported that the abilities of RAR{alpha}, RARß, and RAR{gamma} to interact with SMRT corepressor are very different (31). In the current study, we examined whether these differences in corepressor interaction in vitro manifest as functional consequences in vivo. We report that there is a marked difference in the transcriptional properties of RAR{alpha}, RARß, and RAR{gamma} in the absence of hormone: RAR{alpha} appears unique in its ability to repress reporter gene expression, whereas both RARß and RAR{gamma} fail to repress and instead mediate a substantial level of transcriptional activation in the absence of ectopic hormone agonist. Moreover, there is a close correlation between the ability of the three RAR isotypes to repress in vivo and their ability to bind efficiently to SMRT corepressor in vitro. Nonetheless, all three RAR isotypes fully dissociate from SMRT, bind to coactivators, and mediate similar, strong reporter gene activation in the presence of an RAR hormone agonist, such as ATRA. These differences, seen in the absence of hormone, are likely to contribute to the physiological and developmental nonredundancy of the three different RAR isotypes and may help to account for the conservation of these unique isotypes during vertebrate evolution. The different abilities of the three RAR isotypes to recruit SMRT may also influence the ability of the corresponding RXR/RAR heterodimers to activate transcription in response to RXR agonists (36), suggesting an additional biological consequence of the differing corepressor interaction properties of the three different RAR isotypes.

The Ability of RAR{alpha} to Repress Transcription Is Obscured in Certain Cell Types, Apparently by the Presence of Endogenous Retinoid Receptors
Although RAR{alpha} efficiently binds to SMRT corepressor both in vitro and in vivo, prior studies have noted that the ability of RAR{alpha} to repress target genes in the absence of hormone varies in different cell types; in fact, the expression of ectopic RAR{alpha} in many cell lines does not produce an overt repression of reporter gene expression (33). In agreement with these studies, our analysis demonstrated that ectopic expression of RAR{alpha} repressed target gene expression most efficiently in L929 cells and had less of a repressive effect in several other cell lines. We noted that many of these same cell lines express high levels of endogenous RARs and exhibit an inherent ability to activate the pTK-Luc-RARE reporter in response to ATRA even in the absence of an experimentally introduced RAR. This suggested to us that the endogenous RAR{alpha} present in these cells may cause an inherent repression of the reporter gene, precluding the detection of any further effects of an ectopically introduced RAR{alpha}. Consistent with this hypothesis, the expression of a pTK-Luc reporter containing RARE sequences was suppressed in many cell lines compared with that of the same pTK-Luc reporter lacking RAREs, and this was observed even in the absence of an ectopically introduced RAR. To circumvent this experimental complexity, we created GAL4DBD fusions of the different RAR isotypes and tested their effects on a reporter gene construct engineered to be under the regulation of a GAL-17mer DNA site. The GAL-17mer reporter, unlike the pTK-Luc-RARE, displayed no retinoid responsiveness in the absence of an ectopic receptor construct, confirming that this reporter construct is not recognized by endogenous retinoid receptors. Using this experimental setup, we were able to detect unambiguous repression by GAL4DBD-RAR{alpha} in the absence of hormone, whereas the comparable GAL4DBD-RARß construct mediated significant reporter gene activation under the same conditions. As expected, addition of ATRA allowed both the GAL4DBD-RAR{alpha} and GAL4DBD-RARß fusions to activate the GAL-17mer reporter. We suggest that the prevalence of endogenous RARs in many cell lines has probably hampered prior experimental detection of the otherwise distinctive transcriptional properties of the different RAR isotypes.

We note that an alternative hypothesis might be advanced that certain of our cell lines may be able to produce retinoids intrinsically, and these intrinsic retinoids could serve to bind to, and obscure repression by, RAR{alpha} in the absence of ectopic ATRA. Although we cannot completely discount this hypothesis, we observed no evidence for an involvement of intrinsic retinoids when using the GAL-17mer reporter system, in which the GAL4DBD-RAR{alpha} construct functioned as a strong repressor in the absence of externally added ATRA.

The Different Transcriptional Properties of the Different RAR Isotypes Is Observed in Both the Absence and the Presence of an RXR Heterodimer Partner
RARs are believed to function primarily as RXR/RAR heterodimers in vivo (3, 4, 9, 34). The cell lines used in this study, as virtually all vertebrate cells, express significant levels of RXRs, and presumably these endogenous RXRs are available to form heterodimers with the ectopically introduced RARs studied here. It is difficult, however, to determine the extent of heterodimer formation in our transfections. We therefore investigated whether coexpressing RXR together with RAR{alpha} or RARß could alter the results of our transfection studies. Interestingly, cotransfection of RXR{alpha} did not confer transcriptional repression on RARß, but rather further enhanced the hormone-independent activation properties of this isotype. We suggest that although RXRs have been shown to have a detectable, if weak, ability to bind to SMRT corepressor in vitro, the strength of this interaction is too modest in vivo to confer repression by either RXR homodimers or the RXR/RARß heterodimers studied here.

The Different Abilities of RAR{alpha}, RARß, and RAR{gamma} to Repress Transcription Map Predominantly to a Region within Helix 3 of the Hormone Binding Domain of the Receptor
Our analysis of chimeric constructs created between RAR{alpha} and RARß demonstrated that the identity of the helix 1-{Omega} loop-helix 3 region of the hormone binding domain determines the ability of the receptor to repress or to activate transcription in the absence of hormone. Although the helix 1 and {Omega} loop contributed to this phenotype, the preponderance of the different activation/repression functions of unliganded RAR{alpha} and RARß mapped to amino acid differences within the helix 3 region of these isotypes. Simply replacing portions of helix 3 of RAR{alpha} with the corresponding sequence of RARß resulted in a loss of repression, whereas the reciprocal substitution of portions of helix 3 of RARß with the equivalent sequence of RAR{alpha} conferred the ability to repress reporter gene expression. All three of the amino acids that differ between RAR{alpha} and RARß within the relevant helix 3 domain, at postions 224, 225, and 234, contribute to some extent to this phenomenon. The same region of helix 3 also appears to play the same role in defining transcriptional properties of RAR{gamma}: RAR{gamma} possesses a helix 3 sequence identical to that of RARß, and in common with RARß; RAR{gamma} does not repress transcription, but instead activates reporter gene expression in the absence of hormone ligand.

How might helix 3 be exerting these transcriptional effects? We observed a close correlation between the ability of the different RAR isotypes to repress reporter gene expression in vivo and their ability to bind SMRT corepressor in vitro: RAR{alpha} binds corepressor strongly and represses, whereas RARß and RAR{gamma} bind corepressor comparatively weakly and activate rather than repress, in the absence of hormone. Helix 3 forms a portion of the corepressor interaction/docking surface on the nuclear receptor (37, 38, 39, 40). However, it is also well established that the conformation of receptor helix 12 also plays a major role in gating access of corepressors and coactivators to their docking surface on the nuclear hormone receptors (10, 11). A genetic analysis suggests that an interaction between helix 3 and helix 12 in RARß and RAR{gamma} stabilizes a closed helix 12 position in these isotypes and blocks access of corepressor to its docking site in the absence of hormone ligand, whereas the RAR{alpha} helix 3 sequence disrupts this helix 12 interaction, resulting in an exposed corepressor docking surface and corepressor binding (Farboud, B., H. Hauksdottir, Y. Wu, and M. L. Privalsky, submitted for publication). There is precedent for this proposal: for example, the helix 12 of Ultraspiracle (the Drosophila ortholog of RXR) and of the constitutively activated androstanol receptor (CAR) also assume a closed conformation in the absence of known ligand, although the structural basis for this phenomenon appears to differ for each receptor (41, 42, 43). We suggest that, unrestrained by corepressor binding, RARß and RAR{gamma} are able to recruit coactivators and to confer modest target gene activation even in the absence of hormone. The RAR{alpha} isotype, in contrast, is dependent on hormone agonist to release corepressor, recruit coactivator, and activate target gene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
No experimental animals were used in this research.

Molecular Clones
The human RAR{alpha}, RARß, and RAR{gamma} cDNA clones were obtained from P. Chambon (Institute of Genetics and Molecular and Cellular Biology, College of France, Illkirch Cedex, France). This particular isolate of RARß, originally obtained from a human hepatocellular carcinoma and denoted HAP, encodes a Met at position 416, whereas most other RARß isolates have been reported to encode a Leu in this position (44). Notably, this possible genetic polymorphism did not result in a difference in function in our assays, and we obtained comparable results when the relevant experiments were repeated with a construct in which Met 416 was replaced by a Leu (data not shown).

The subcloning of pSG5-RAR{alpha}ß{alpha} and pSG5-RARß{alpha}ß was described elsewhere (31). To create the pSG5-RAR{alpha}ß{alpha}{alpha} and pSG5-RARßß{alpha}ß chimeras, a SacI restriction site was introduced between codons 198 and 199 in pSG5-RARß and in pSG5-RAR{alpha}ß{alpha} by site-directed QuikChange mutagenesis (Stratagene, La Jolla, CA) to match a corresponding SacI site that occurs naturally in pSG5-RAR{alpha}. To avoid confusion arising from the different N-terminal lengths of the different RAR isotypes, we have adopted the convention of Ref. 45 and use in these descriptions an identical codon numbering system for all three isotypes based on that of RAR{gamma}. Subsequently, a SacI-BclI fragment from RAR{alpha}ß{alpha} was swapped with the SacI-BclI fragment from RAR{alpha} to create RAR{alpha}ß{alpha}{alpha}, representing an RAR{alpha} receptor with codons 187–233 derived from RARß. Likewise, a KpnI-SacI fragment from RARß{alpha}ß was swapped with a KpnI-SacI fragment from RARß to create RARßß{alpha}ß, creating a RARß receptor with codons 199–246 derived from the RAR{alpha} sequence. The RAR helix 1, {Omega} loop, and helix 3 chimeras were created by PCR overlap extension (46). The RAR{alpha}(ßH1) chimera represents an RAR{alpha} receptor with codons 183–200 derived from RARß sequence. Likewise, the RARß({alpha}H1) chimera is a RARß receptor wherein codons 183–200 have been replaced with the RAR{alpha} sequence. The RAR{alpha}(ß{Omega}) chimera represents an RAR{alpha} receptor with codons 201–223 replaced with RARß sequence; the RARß({alpha}{Omega}) chimera represents a receptor in which codons 201–223 in RARß have been replaced with RAR{alpha} sequence. In the RAR{alpha}(ßH3) chimera, codons 224–237 in RAR{alpha} have been replaced with RARß sequence, whereas in the RARß({alpha}H3) chimera, codons 224–237 have been replaced with the RAR{alpha} sequence. The pSG5-RAR{alpha}(S232A), pSG5-RARß(A225S), pSG5-RARß(R221S), pSG5-RAR{alpha}(IEKV->TEKI) and pSG5-RARß(TEKI->IEKV) mutants were created from the wild-type pSG5-RAR{alpha} and pSG5-RARß clones by oligonucleotide-mediated, site-directed QuikChange mutagenesis.

The GAL4DBD-RAR-LBD fusions were created as follows. The hormone binding domains of wild-type RAR{alpha}, RARß, RAR{gamma}, or the appropriate mutants were amplified by PCR. Subsequently, these sequences were excised as SmaI-BamHI restriction fragments and inserted into the corresponding sites in a pSG5-GAL4DBD mammalian expression vector (47). The M-pTK-Luc-RARE2 reporter vector was created as follows: the M-pTK-Luc vector (48) was digested with XhoI and SalI, and the direct-repeat-4 element was discarded. Two oligonucleotides were annealed (5'-TCGAGGGTAG GGTTCACCGA AAGTTCACTCT AGAGGGTTCA CCGAAAGTTC ACTCG-3' and 5'-TCGACGAGTG AACTTCGGTG AACCCTCTAG AGTGAACTTT CGGTGAACCC TACCC-3') to create a sequence containing three RARE elements with SalI/XhoI compatible ends. Subsequently, the annealed oligonucleotides were cloned into the SalI and XhoI sites on the vector. The pGL3-GAL-17mer reporter plasmid was created by annealing oligonucleotides containing two tandem GAL-17mer response elements with SalI/XhoI compatible ends (5'-TCGACCGGAG GACAGTCCTC CGGCC GGAGGACAGT CCTCCGG-3' and 5'-TCGACCGGAG GACTGTCCTC CGGCCGGAGG ACTGTCCTCCGG-3'). The annealed oligonucleotides were cloned into the SalI and XhoI sites of the pGL3-luciferase reporter vector (Promega Corp., Madison, WI). The pTK-Luc-RARE3 reporter, containing three copies of the RARE found within the human RARß promoter, was obtained from A. Dejean (Pasteur Institute, Paris, France).

Transient Transfections
Transient transfections were performed as previously described (49). Briefly, CV-1, HeLa, or L929 cells were plated at a density of 9 x 104 cells per well in 12-well tissue culture plates, were permitted to attach, and were transfected using either a LipofectAMINE plus (Figs. 2–5GoGoGoGo; Life Technologies, Inc., Gaithersburg, MD) or Effectin (Figs. 1Go, 6Go, 7Go; QIAGEN, Valencia, CA) protocol. Ten nanograms of the pSG5 expression plasmid (either an empty vector or encoding the appropriate RAR isotype), 200 ng of luciferase reporter (pTK-Luc empty, pTK-Luc-RARE3, M-pTK-Luc empty, M-pTK-Luc-RARE2, or pGL3-GAL-17mer-Luc), and 100 ng of a pCH110-lacZ reporter plasmid (used as an internal transfection normalization control) were introduced per well, plus sufficient pUC18 plasmid to adjust each transfection to 500 ng total DNA. After a 3-h incubation at 37 C in the transfection media, either ATRA or an equivalent amount of hormone-free ethanol carrier was added to each well, and the cells were incubated for an additional 24 h at 37 C. Cells were then harvested and lysed, and the ß-galactosidase and luciferase activities were detected as described previously (47).

RAR-Corepressor Binding Assays
Interactions between receptors and the SMRT corepressor were assayed in vitro by the use of a GST-pulldown protocol (50). GST or GST fused to TRAC-1 codons 406–769 (Ref. 47 ; corresponding to SMRT{alpha} codons 2109–25189 in Ref. 51) were produced in Escherichia coli and were purified by immobilization on a glutathione-agarose matrix (Sigma Chemicals, St. Louis MO). Radiolabeled RAR proteins were synthesized in a coupled transcription-translation system using the T7 promoter site within the pSG5 expression constructs (Promega Corp.). Protein binding reactions were performed as described previously (50). Briefly, radiolabeled receptor was incubated with the immobilized GST or GST-SMRT proteins for 1 h at 4 C, and unbound receptor was removed by four cycles of washing the glutathione agarose matrix with 1 ml each cycle of HEMG buffer. Any RARs remaining bound to the GST-SMRT matrix were then eluted by incubating the matrix with 10 mM glutathione in 50 mM Tris-Cl (pH 7.6), and the eluted protein was resolved by SDS-PAGE. Radiolabel was visualized and quantified by PhosphorImager analysis (Storm System, Molecular Dynamics, Inc., Sunnyvale CA).

Immunoblotting Analysis
CV-1 cells were transfected with pSG5 vectors expressing the RAR{alpha}, RARß, and RAR{gamma} isotypes using the transient transfection protocol detailed above. Two days after transfection, the cells were harvested by scraping and lysed in SDS-sample buffer, and the lysates were resolved by SDS-PAGE using a NuPage Tris-acetate buffer system (Invitrogen Inc., Carlsbad CA). The proteins were transferred by blotting to nitrocellulose membranes, and the membranes were blocked by incubation for 45 min in TBST buffer [10 mM Tris-Cl (pH 7.8), 150 mM NaCl, 0.1% Tween-20] plus 5% milk. The membranes were then washed in TBST buffer (2 x 5 min) and incubated for 1 h with rabbit polyclonal anti-RAR antisera diluted 1:1000 in 5 ml of TBST buffer plus 5% milk (antibody M454 from Santa Cruz Biotechnology, Inc., Santa Cruz CA). The membranes were then washed three times for 15 min in TBST buffer and incubated for 1 h with horseradish peroxidase-coupled, antirabbit IgG diluted 1:1000 in 5 ml of TBST buffer plus 5% milk. After additional washing in TBST, the RAR bands were visualized using ECL Plus (Amersham Pharmacia Biotech Biosciences, Piscataway, NJ) and quantified by scanning with the Storm System Imager using a chemiluminescence mode. Although capable of recognizing all three isotypes, the M454 antisera reacts preferentially with the RAR{gamma} isotype; we quantified these differences in M454 reactivity by using RAR isotypes synthesized to known levels by in vitro transcription and translation, and normalized the values obtained from immunoblots of transfected cells accordingly.


   ACKNOWLEDGMENTS
 
We thank P. Chambon for providing the RAR{alpha}, RARß, and RAR{gamma} clones, A. Dejean for the pTK-Luc-ßRARE3 reporter vector, and M. d. M. Vivanco Ruiz for the M-pTK-Luc reporter vector. We are also grateful to M. L. Goodson for the creation of the pGL3-GAL-17mer-Luc reporter vector, K. Naylor for assistance in developing the pSG5-RAR{alpha}ß{alpha}{alpha} and pSG5-RAR-ßß{alpha}ß constructs, and L. Liu for superb technical assistance.


   FOOTNOTES
 
This work was supported by Public Health Services/NIH Grant R01 DK-53528.

Abbreviations: ATRA, All-trans retinoic acid; DBD, DNA binding domain; GST, glutathione-S-transferase; N-CoR, nuclear hormone receptor corepressor; RAR, retinoic acid receptor; RARE, RAR response element; RXR, retinoid X receptor; SMRT, silencing mediator of retinoic acid and thyroid hormone receptor.

Received for publication September 28, 2002. Accepted for publication December 13, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Apriletti JW, Ribeiro RC, Wagner RL, Feng W, Webb P, Kushner PJ, West BL, Nilsson S, Scanlan TS, Fletterick RJ, Baxter JD 1998 Molecular and structural biology of thyroid hormone receptors. Clin Exp Pharmacol Physiol Suppl 25:S2–S11
  2. Chambon P 1996 A decade of molecular biology of retinoic acid receptors. FASEB J 10:940–954[Abstract]
  3. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839[CrossRef][Medline]
  4. Mangelsdorf DJ, Evans RM 1995 The RXR heterodimers and orphan receptors. Cell 83:841–850[CrossRef][Medline]
  5. Sucov HM, Evans RM 1995 Retinoic acid and retinoic acid receptors in development. Mol Neurobiol 10:169–184[Medline]
  6. Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  7. Yen PM 2001 Physiological and molecular basis of thyroid hormone action. Physiol Rev 81:1097–1142[Abstract/Free Full Text]
  8. Zhang J, Lazar MA 2000 The mechanism of action of thyroid hormones. Annu Rev Physiol 62:439–466[CrossRef][Medline]
  9. Glass CK 1996 Some new twists in the regulation of gene expression by thyroid hormone and retinoic acid receptors. J Endocrinol 150:349–357[Abstract/Free Full Text]
  10. Glass CK, Rosenfeld MG 2000 The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 14:121–141[Free Full Text]
  11. Privalsky ML 2001 Regulation of SMRT and N-CoR corepressor function. Curr Top Microbiol Immunol 254:117–136[Medline]
  12. Chen JD, Li H 1998 Coactivation and corepression in transcriptional regulation by steroid/nuclear hormone receptors. Crit Rev Eukaryot Gene Expr 8:169–190[Medline]
  13. Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:1167–1177[Abstract/Free Full Text]
  14. Koenig RJ 1998 Thyroid hormone receptor coactivators and corepressors. Thyroid 8:703–713[Medline]
  15. Lee H, Yen PM 1999 Recent advances in understanding thyroid hormone receptor coregulators. J Biomed Sci 6:71–78[CrossRef][Medline]
  16. McKenna NJ, O’Malley BW 2002 Combinatorial control of gene expression by nuclear receptors and coregulators. Cell 108:465–474[CrossRef][Medline]
  17. Ordentlich R, Downes M, Evans RM 2001 Corepressors and nuclear hormone receptor function. Curr Top Microbiol Immunol 254:101–116[Medline]
  18. Torchia J, Glass C, Rosenfeld MG 1998 Co-activators and co-repressors in the integration of transcriptional responses. Curr Opin Cell Biol 10:373–383[CrossRef][Medline]
  19. Xu L, Glass CK, Rosenfeld MG 1999 Coactivator and corepressor complexes in nuclear receptor function. Curr Opin Genet Dev 9:140–147[CrossRef][Medline]
  20. Ayer DE 1999 Histone deacetylases: transcriptional repression with SINers and NuRDs. Trends Cell Biol 9:193–198[CrossRef][Medline]
  21. Berger SL 2001 Molecular biology. The histone modification circus. Science 292:64–65[Free Full Text]
  22. Chen H, Tini M, Evans RM 2001 HATs on and beyond chromatin. Curr Opin Cell Biol 13:218–224[CrossRef][Medline]
  23. Jenuwein T, Allis CD 2001 Translating the histone code. Science 293:1074–1080[Abstract/Free Full Text]
  24. Muscat GE, Burke LJ, Downes M 1998 The corepressor N-CoR and its variants RIP13a and RIP13{Delta}1 directly interact with the basal transcription factors TFIIB, TAFII32 and TAFII70. Nucleic Acids Res 26:2899–2907[Abstract/Free Full Text]
  25. Turner BM 2000 Histone acetylation and an epigenetic code. Bioessays 22:836–845[CrossRef][Medline]
  26. Wong CW, Privalsky ML 1998 Transcriptional repression by the SMRT-mSin3 corepressor: multiple interactions, multiple mechanisms, and a potential role for TFIIB. Mol Cell Biol 18:5500–5510[Abstract/Free Full Text]
  27. Means AL, Gudas LJ 1995 The roles of retinoids in vertebrate development. Annu Rev Biochem 64:201–233[CrossRef][Medline]
  28. Ghyselinck NB, Dupe V, Dierich A, Messaddeq N, Garnier JM, Rochette-Egly C, Chambon P, Mark M 1997 Role of the retinoic acid receptor ß (RARß) during mouse development. Int J Dev Biol 41:425–447[Medline]
  29. Lohnes D, Mark M, Mendelsohn C, Dolle P, Decimo D, LeMeur M, Dierich A, Gorry P, Chambon P 1995 Developmental roles of the retinoic acid receptors. J Steroid Biochem Mol Biol 53:475–486[CrossRef][Medline]
  30. Wendling O, Ghyselinck NB, Chambon P, Mark M 2001 Roles of retinoic acid receptors in early embryonic morphogenesis and hindbrain patterning. Development 128:2031–2038[Abstract/Free Full Text]
  31. Wong CW, Privalsky ML 1998 Transcriptional silencing is defined by isoform- and heterodimer-specific interactions between nuclear hormone receptors and corepressors. Mol Cell Biol 18:5724–5733[Abstract/Free Full Text]
  32. Lin BC, Hong SH, Krig S, Yoh SM, Privalsky ML 1997 A conformational switch in nuclear hormone receptors is involved in coupling hormone binding to corepressor release. Mol Cell Biol 17:6131–6138[Abstract/Free Full Text]
  33. Baniahmad A, Dressel U, Renkawitz R 1998 Cell-specific inhibition of retinoic acid receptor-{alpha} silencing by the AF2/{tau} c activation domain can be overcome by the corepressor SMRT, but not by N-CoR. Mol Endocrinol 12:504–512[Abstract/Free Full Text]
  34. Kastner P, Mark M, Ghyselinck N, Krezel W, Dupe V, Grondona JM, Chambon P 1997 Genetic evidence that the retinoid signal is transduced by heterodimeric RXR/RAR functional units during mouse development. Development 124:313–326[Abstract]
  35. Kastner P, Mark M, Chambon P 1995 Nonsteroid nuclear receptors: what are genetic studies telling us about their role in real life? Cell 83:859–869[CrossRef][Medline]
  36. Germain P, Iyer J, Zechel C, Gronemeyer H 2002 Co-regulator recruitment and the mechanism of retinoic acid receptor synergy. Nature 415:187–192[CrossRef][Medline]
  37. Hu X, Lazar MA 1999 The CoRNR motif controls the recruitment of corepressors by nuclear hormone receptors. Nature 402:93–96[CrossRef][Medline]
  38. Hu X, Li Y, Lazar MA 2001 Determinants of CoRNR-dependent repression complex assembly on nuclear hormone receptors. Mol Cell Biol 21:1747–1758[Abstract/Free Full Text]
  39. Nagy L, Kao HY, Love JD, Li C, Banayo E, Gooch JT, Krishna V, Chatterjee K, Evans RM, Schwabe JWR 1999 Mechanism of corepressor binding and release from nuclear hormone receptors. Genes Dev 13:3209–3216[Abstract/Free Full Text]
  40. Perissi V, Staszewski LM, McInerney EM, Kurokawa R, Krones A, Rose DW, Lambert MH, Milburn MV, Glass CK, Rosenfeld MG 1999 Molecular determinants of nuclear receptor-corepressor interaction. Genes Dev 13:3198–3208[Abstract/Free Full Text]
  41. Billas IM, Moulinier L, Rochel N, Moras D 2001 Crystal structure of the ligand-binding domain of the ultraspiracle protein USP, the ortholog of retinoid X receptors in insects. J Biol Chem 276:7465–7474[Abstract/Free Full Text]
  42. Clayton GM, Peak-Chew SY, Evans RM, Schwabe JW 2001 The structure of the ultraspiracle ligand-binding domain reveals a nuclear receptor locked in an inactive conformation. Proc Nat Acad Sci USA 98:1549–1554[Abstract/Free Full Text]
  43. Dussault I, Lin M, Hollister K, Fan M, Termini J, Sherman MA, Forman BM 2002 A structural model of the constitutive androstane receptor defines novel interactions that mediate ligand-independent activity. Mol Cell Biol 22:5270–5280[Abstract/Free Full Text]
  44. de The H, Marchio A, Tiollais P, Dejean A 1987 A novel steroid thyroid hormone receptor-related gene inappropriately expressed in human hepatocellular carcinoma. Nature 330:667–670[CrossRef][Medline]
  45. Renaud JP, Rochel N, Ruff M, Vivat V, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the RAR-{gamma} ligand-binding domain bound to all-trans retinoic acid. Nature 378:681–689[CrossRef][Medline]
  46. Higuchi R 1990 Recombinant PCR. New York: Academic Press; 177–180
  47. Hong SH, David G, Wong CW, Dejean A, Privalsky ML 1997 SMRT corepressor interacts with PLZF and with the PML-retinoic acid receptor {alpha} (RAR{alpha}) and PLZF-RAR{alpha} oncoproteins associated with acute promyelocytic leukemia. Proc Nat Acad Sci USA 94:9028–9033[Abstract/Free Full Text]
  48. Vivanco Ruiz MM, Bugge TH, Hirschmann P, Stunnenberg HG 1991 Functional characterization of a natural retinoic acid responsive element. EMBO J 10:3829–3838[Medline]
  49. Hauksdottir H, Privalsky ML 2001 DNA recognition by the aberrant retinoic acid receptors implicated in human acute promyelocytic leukemia. Cell Growth Differ 12:85–98[Abstract/Free Full Text]
  50. Sande S, Privalsky ML 1996 Identification of TRACs (T3 receptor-associating cofactors), a family of cofactors that associate with, and modulate the activity of, nuclear hormone receptors. Mol Endocrinol 10:813–825[Abstract/Free Full Text]
  51. Ordentlich P, Downes M, Xie W, Genin A, Spinner NB, Evans RM 1999 Unique forms of human and mouse nuclear receptor corepressor SMRT. Proc Nat Acad Sci USA 96:2639–2644[Abstract/Free Full Text]

NURSA Molecule Pages Link:

Nuclear Receptors:   RARα  |  RARβ  |  RARγ  |  RXRα
Coregulators:   SMRT
Ligands:   all-trans-Retinoic acid



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
R. F. Gillespie and L. J. Gudas
Retinoic Acid Receptor Isotype Specificity in F9 Teratocarcinoma Stem Cells Results from the Differential Recruitment of Coregulators to Retinoic Acid Response Elements
J. Biol. Chem., November 16, 2007; 282(46): 33421 - 33434.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. T. Epping, L. Wang, J. A. Plumb, M. Lieb, H. Gronemeyer, R. Brown, and R. Bernards
A functional genetic screen identifies retinoic acid signaling as a target of histone deacetylase inhibitors
PNAS, November 6, 2007; 104(45): 17777 - 17782.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
B. A. Jonas, N. Varlakhanova, F. Hayakawa, M. Goodson, and M. L. Privalsky
Response of SMRT (Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor) and N-CoR (Nuclear Receptor Corepressor) Corepressors to Mitogen-Activated Protein Kinase Kinase Kinase Cascades Is Determined by Alternative mRNA Splicing
Mol. Endocrinol., August 1, 2007; 21(8): 1924 - 1939.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
P. Germain, P. Chambon, G. Eichele, R. M. Evans, M. A. Lazar, M. Leid, A. R. De Lera, R. Lotan, D. J. Mangelsdorf, and H. Gronemeyer
International Union of Pharmacology. LX. Retinoic Acid Receptors
Pharmacol. Rev., December 1, 2006; 58(4): 712 - 725.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. A. Shell, C. Hesse, S. M. Morris Jr., and C. Milcarek
Elevated Levels of the 64-kDa Cleavage Stimulatory Factor (CstF-64) in Lipopolysaccharide-stimulated Macrophages Influence Gene Expression and Induce Alternative Poly(A) Site Selection
J. Biol. Chem., December 2, 2005; 280(48): 39950 - 39961.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
C. Zechel
Requirement of Retinoic Acid Receptor Isotypes {alpha}, {beta}, and {gamma} during the Initial Steps of Neural Differentiation of PCC7 Cells
Mol. Endocrinol., June 1, 2005; 19(6): 1629 - 1645.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
S. Lee and M. L. Privalsky
Heterodimers of Retinoic Acid Receptors and Thyroid Hormone Receptors Display Unique Combinatorial Regulatory Properties
Mol. Endocrinol., April 1, 2005; 19(4): 863 - 878.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. L. Goodson, B. A. Jonas, and M. L. Privalsky
Alternative mRNA Splicing of SMRT Creates Functional Diversity by Generating Corepressor Isoforms with Different Affinities for Different Nuclear Receptors
J. Biol. Chem., March 4, 2005; 280(9): 7493 - 7503.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
H. Srinivas, D. M. Juroske, S. Kalyankrishna, D. D. Cody, R. E. Price, X.-C. Xu, R. Narayanan, N. L. Weigel, and J. M. Kurie
c-Jun N-Terminal Kinase Contributes to Aberrant Retinoid Signaling in Lung Cancer Cells by Phosphorylating and Inducing Proteasomal Degradation of Retinoic Acid Receptor {alpha}
Mol. Cell. Biol., February 1, 2005; 25(3): 1054 - 1069.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. A. Jonas and M. L. Privalsky
SMRT and N-CoR Corepressors Are Regulated by Distinct Kinase Signaling Pathways
J. Biol. Chem., December 24, 2004; 279(52): 54676 - 54686.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
B. Farboud and M. L. Privalsky
Retinoic Acid Receptor-{alpha} Is Stabilized in a Repressive State by Its C-Terminal, Isotype-Specific F Domain
Mol. Endocrinol., December 1, 2004; 18(12): 2839 - 2853.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. L. Abrams, J. Xu, C. Nativelle-Serpentini, S. Dabirshahsahebi, and M. B. Rogers
An Evolutionary and Molecular Analysis of Bmp2 Expression
J. Biol. Chem., April 16, 2004; 279(16): 15916 - 15928.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
S. Zaripheh, T. W.-M. Boileau, M. A. Lila, and J. W. Erdman Jr
[14C]-Lycopene and [14C]-Labeled Polar Products Are Differentially Distributed in Tissues of F344 Rats Prefed Lycopene
J. Nutr., December 1, 2003; 133(12): 4189 - 4195.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
A. D. Weston, B. Blumberg, and T. M. Underhill
Active repression by unliganded retinoid receptors in development: less is sometimes more
J. Cell Biol., April 28, 2003; 161(2): 223 - 228.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow NURSA Molecule Pages Link
Right arrow Reprints, Permissions and Rights
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hauksdottir, H.
Right arrow Articles by Privalsky, M. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hauksdottir, H.
Right arrow Articles by Privalsky, M. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals