Chapter 7 Nuclear Receptor Repression

Nuclear Receptor Repression: Regulatory Mechanisms and Physiological Implications M. David Stewart* and Jiemin Wong{ *Department of Genetics, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA {

Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China

I. Introduction ................................................................................. II. Corepressors ................................................................................ A. Ligand‐Independent Corepressors ................................................ B. Ligand‐Dependent Corepressors .................................................. III. Types of NR Repression.................................................................. A. Repression by Unliganded Receptors............................................. B. Repression by Antagonist‐Bound Steroid Receptors .......................... C. Repression by Agonist‐Bound Receptors ........................................ IV. Molecular Mechanisms of Transcriptional Repression ............................ A. Histone Deacetylation................................................................ B. Histone Methylation .................................................................. C. Chromatin Assembly/Remodeling ................................................. D. DNA Methylation ..................................................................... V. Physiological Functions of NR‐Mediated Repression.............................. A. Physiological Function of Repression by Unliganded TR .................... B. TR‐Mediated Repression in the Regulation of Amphibian Metamorphosis ................................................... C. Repression by PPARg ................................................................ D. Repression Mediated by NCoR and SMRT ..................................... E. Repression Mediated by RIP140................................................... VI. Concluding Remarks ...................................................................... References...................................................................................

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The ability to associate with corepressors and to inhibit transcription is an intrinsic property of most members of the nuclear receptor (NR) superfamily. NRs induce transcriptional repression by recruiting multiprotein corepressor complexes. Nuclear receptor corepressor (NCoR) and silencing mediator of retinoic and thyroid receptors (SMRT) are the most well characterized corepressor complexes and mediate repression for virtually all NRs. In turn, corepressor complexes repress transcription because they either contain or associate with chromatin modifying enzymes. These include histone deacetylases, histone Progress in Molecular Biology and Translational Science, Vol. 87 DOI: 10.1016/S1877-1173(09)87007-5

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H3K4 demethylases, histone H3K9 or H3K27 methyltransferases, and ATP‐dependent chromatin remodeling factors. Two types of NR‐interacting corepressors exist. Ligand‐independent corepressors, like NCoR/SMRT, bind to unliganded or antagonist‐bound NRs, whereas ligand‐dependent corepressors (LCoRs) associate with NRs in the presence of agonist. Therefore, LCoRs may serve to attenuate NR‐mediated transcriptional activation. Somewhat unexpectedly, classical coactivators may also function as ‘‘corepressors’’ to mediate repression by agonist‐bound NRs. In this chapter, we will discuss the various modes and mechanisms of repression by NRs as well as discuss the known physiological functions of NR‐mediated repression.

I. Introduction Nuclear receptors (NRs) are ligand‐dependent transcription factors that play pivotal roles in a variety of key metabolic and developmental processes. Their ligands are lipophilic molecules such as steroid hormones, thyroid hormone, retinoic acid, vitamin D, fatty acids, fatty acid derivatives, and xenobiotics. These molecules can diffuse through the plasma membrane and interact with NRs located in either the nuclear or cytosolic compartments. NRs are composed of five functionally distinct domains referred to as A/B, C, D, E, and F. The A/B N‐terminal domain varies greatly between NRs and contains the activation function 1 (AF1). AF1 mediates ligand‐independent transactivation for some NRs. The C region contains a sequence‐specific DNA‐binding domain (DBD) consisting of two zinc fingers. The D domain is a variable hinge region separating the DBD from the ligand‐binding domain (LBD) which comprises region E. The D domain usually contains nuclear import/export sequences. Region E also contains the ligand‐dependent AF2. Finally, the sequence and function of the C‐terminal F domain varies greatly among NRs. NRs are well known for their hormone‐dependent transcriptional activation function. However, NRs also have capacity to repress transcription both in the absence or presence of their ligands. Studies over last decade or two have begun to reveal the molecular mechanisms and physiological relevance of the NR repression function. Important for this discussion of repression by NRs, a subset of NRs including thyroid hormone receptors (TR) and retinoic acid receptors (RAR) are constitutively nuclear and bind to their target genes in the absence of their cognate hormones. These receptors exhibit a potent transcriptional repression function in the absence of hormones. The repression activity could be mapped to their LBD and is transferable.1 It is this property that led to the identification of various proteins termed ‘‘corepressors’’ that preferentially interact with and participate in repression by unliganded receptors.

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The most notable corepressors for NRs are nuclear receptor corepressor (NCoR) and the highly similar silencing mediator of retinoic and thyroid receptors (SMRT). Subsequent molecular and structural analyses have revealed that NCoR and SMRT interact with the NR LBD through one or more corepressor nuclear receptor (CoRNR) boxes.2–4 The CoRNR box consists of the sequence (L/I)XX(I/V)I or LXXX(I/L)XXX(I/L) where X is any amino acid. This interaction is regulated via ligand‐induced conformational change of the LBD. In the absence of ligand, the LBD conformation is such that it prefers interaction with the corepressor proteins containing CoRNR boxes. Upon interaction with agonist, the LBD alters its conformation such that it prefers association with coactivator proteins containing the sequence LXXLL termed the NR box.5 In addition to the corepressors that associate with and mediate repression by unliganded NRs, there is a class of corepressors that contain one or more NR boxes and therefore specifically associate with agonist‐bound receptors. A well studied example is RIP140, which was first identified as an agonist‐ dependent estrogen receptor alpha (ERa) AF2 domain‐interacting protein and thought to be a coactivator.6 These ‘‘ligand‐dependent’’ corepressors may serve to attenuate NR‐mediated transcriptional activity. Given that transcription is a complex multistep process involving a large number of general transcriptional factors and cofactors, in principle a corepressor protein may repress transcription by interfering with any of the steps. This idea is consistent with the large number of corepressors identified so far. Noticeably, corepressors often contain or associate with one or more histone modifying activities that actively inhibit transcription though covalent modifications to the core histones. These modifications include deacetylation, demethylation of H3K4, and methylation of H3K9 or H3K27. Repressive histone modifications can attenuate transcriptional activity either by creating a local repressive heterochromatin environment or by preventing the binding of coactivators or basal machinery. In addition to repression by unliganded NRs, it has become clear that agonist‐bound NRs can also repress transcription of target genes. This phenomenon is in general termed transrepression. The best example of this is the ability of the glucocorticoid receptor (GR) to inhibit expression of proinflammatory genes via direct interaction with NFkB.7,8 We will focus this chapter on the molecular mechanisms of corepressor function. Since recent interesting new work using mouse genetics has shed light on the role of NR‐corepressor interactions in the regulation of gene expression by NRs, we will also discuss the physiological functions of NR repression as well as their disease associations.

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II. Corepressors Transcriptional repression by NRs is mediated by a class of coregulatory proteins termed corepressors. Thus, we will first discuss corepressors in detail and then describe how they are utilized in the various modes of NR‐mediated repression. Simply put, corepressors are molecules that have the ability to inhibit transcription and also interact with NRs. They usually exist in large multiprotein complexes that couple multiple enzymatic, NR binding, chromatin binding, and regulatory activities into a single functional unit. The best studied NR‐corepressor complexes are NCoR and SMRT. Other corepressors exist and may interact with NRs in a cell‐specific or gene‐specific manner. These include Sin3A, Alien, and SUN‐CoR. Additionally, there exists an expanding class of agonist‐bound corepressors, including Hairless, LCoR, MTA1, PRAME, REA, and RIP140.

A. Ligand‐Independent Corepressors Ligand‐independent corepressors are those that bind NRs in the absence of agonist. They contain one or more CoRNR boxes, which interact with the AF2 of the LBD. Upon binding of agonist, ligand‐independent corepressors dissociate from the NR allowing association of NR box‐containing coregulators, typically coactivators. This is the ‘‘canonical’’ molecular mechanism of gene regulation by type II NRs, which bind DNA constitutively, repressing in the absence and activating transcription in the presence of ligand. NCoR and SMRT are the most well characterized ligand‐independent corepressors. They are ubiquitously expressed proteins that mediate repression by numerous NRs as well as other transcription factors. Their molecular mechanisms of action have been extensively studied for the NRs for which they are named: TR and RAR, but their mechanism of action is applicable to all NRs with which they interact. The core NCoR/SMRT protein complex consists of NCoR/SMRT, Transducin b‐like 1/related 1 (TBL1/TBLR1), Histone deacetylase 3 (HDAC3), and G‐protein pathway suppressor 2 (GPS2).9,10 NCoR and SMRT essentially serve as platforms for complex assembly. They bind NRs and associate with each of the other complex subunits. Interaction with NRs is mediated by three CoRNR box‐containing receptor interaction domains (RIDs) located in their C‐termini. HDAC3, as the name implies, is a histone deacetylase and the only deacetylase identified in the core NCoR/SMRT complex. It has activity toward acetylated lysines on all core histones.11 HDAC3 requires interaction with NCoR/SMRT for potent enzymatic activity; thus, the region of NCoR/SMRT that binds and

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activates HDAC3 is referred to as the deacetylase activating domain (DAD).12 TBL1 and TBLR1 are highly related proteins and members of the WD40 superfamily. They directly interact with the N‐terminal repression domain of NCoR/SMRT (RD1) and bind deacetylated histone H4.13 In this manner, they may function to stabilize the corepressor complex on chromatin and thus facilitate repression. Interestingly, TBL1/TBLR1 has also been reported to mediate degradation of NCoR/SMRT by the proteasome during hormone‐ dependent transcriptional activation.14 Little is known of the function of GPS2 other than its ability to inhibit the Jun N‐terminal kinase (JNK) pathway. Direct association with NCoR/SMRT is required for this activity.10 It may function in NR‐mediated transrepression, which will be discussed later in this chapter. While purification of NCoR/SMRT core complex revealed the absence of Sin3A corepressor complex, both NcoR and SMRT have been reported to interact with Sin3A or a component of the Sin3A corepressor complex.15–17 The mammalian SIN3 complex consists of SIN3A/B, HDAC1/2, RbAP46/48, SAP18/30, and SDS3.18–22 The SIN3 complex may be recruited to target genes either by direct interaction with a transcription factor (i.e., Mad1) or indirectly through interaction with NCoR or SMRT.23 NCoR associates with SIN3A/B complexes via the adapter protein SAP30. SMRT only associates with SIN3A complexes and does so through direct interaction with SIN3A.17 Thus, SIN3 corepressor complexes may also contribute to NR‐mediated repression through interaction with NCoR/SMRT. SIN3 complexes are very similar in composition to that of NCoR/SMRT. They both have large proteins that form the molecular scaffold (SIN3A/B, NCoR/SMRT). They both have WD40 superfamily members that stabilize the complexes on chromatin via interaction with core histones (RbAP46/48, TBL1/TBLR1). And they both repress transcription by histone deacetylase subunits (HDAC1/2, HDAC3). Although not components of the core complex, SIN3 also associates with the histone H3K9 methyltransferase ESET/SETDB1 and the ATP‐dependent chromatin remodeling complex SWI/SNF.24,25 Therefore, the ability of NCoR/SMRT to associate with SIN3 complexes may draw additional chromatin modifying enzymes to participate in NR‐mediated transcriptional repression. The corepressor Alien was originally identified as a corepressor for TR, but has since been found to associate with DAX1, COUP‐TFI, COUP‐TFII, AR, and VDR.26–29 Alien is a highly conserved and widely expressed protein. A gene knockout study indicates that it is required for mouse early embryonic development.30 It contains autonomous transcriptional repression activity and represses transcription through both HDAC‐dependent and ‐independent pathways. The HDAC‐dependent pathway involves recruitment of the HDAC1/2‐containing SIN3A complex.31 The HDAC‐independent pathway consists of repression via creation of a compact nucleosome environment and is mediated though recruitment of the nucleosome assembly factor NAP1.32

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In addition to aforementioned histone deacetylase‐dependent mechanism, recent studies indicate that histone methylation and demethylation also contribute to repression by NRs. SUV39H1, a histone H3K9 methyltransferase, has been reported to interact with TR.33 In vitro, this interaction is insensitive to hormone. However, addition of T3 led to dissociation of SUV39H1 from TR in vivo. SUV39H1 enhances TR repression in a H3K9 methyltransferase activity‐dependent manner. Another H3K9 methyltransferase G9a was shown to interact with and participate in repression of CYP7A1 gene by the small heterodimer partner (SHP).34 LSD1 is the first identified histone lysine demethylase that specifically demethylates mono‐ and dimethyl‐H3K4.35 Interestingly, while LSD1 was shown to function as a coactivator for AR and ER, it seems to serve as a corepressor for orphan receptor TLX (also called NR2E1).36,37 LSD1 interacts directly with TLX and potentiates TLX repression through its H3K4 demethylase activity.

B. Ligand‐Dependent Corepressors Unlike ligand‐independent corepressors, ligand‐dependent corepressors (LCoRs) interact with the LBD of agonist‐bound NRs and may contain one or more NR boxes typically found in coactivators. Therefore, this class of corepressors may function to attenuate the activity of agonist‐bound NRs. The list of LCoRs includes hairless (Hr), LCoR, receptor‐interacting protein 140 (RIP140), metastasis associated factor 1 (MTA1), preferentially expressed antigen in melanoma (PRAME), and repressor of estrogen activity (REA). We will briefly mention the best studied of these factors as they have been extensively reviewed elsewhere. RIP140 was the first LCoR identified. It was first identified as an agonist‐ dependent ERa AF2 domain‐interacting protein and thought to be a coactivator.6 It was subsequently found to associate with a number of other NRs.38 Later it was found to have transrepression activity and could negatively regulate agonist‐bound NRs by competing with coactivators for NR binding.39 In addition, RIP140 has been shown to inhibit transcription by means of distinct repression domains that function by both HDAC‐dependent and ‐independent mechanisms.40,41 RIP140 is widely expressed and has critical roles in adipose biology and female reproductive physiology, specifically oocyte release during ovulation.42,43 Similar to RIP140, LCoR was first identified for its ligand‐dependent interaction with ERa, but subsequently was found to associate with many NRs.44 The LCoR gene is broadly expressed in embryonic and adult tissues. LCoR also exhibits both HDAC‐dependent and ‐independent repression. Like most corepressors, LCoR associates with HDAC activity. Specifically, it directly interacts with HDAC3, 6, and 10. Interestingly, its transrepression activity for

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ERa and GR is sensitive to deacetylase inhibitors; whereas, its transrepression activity for progesterone receptor (PR) and vitamin D receptor (VDR) is not. Thus, LCoR exhibits an example of HDAC‐independent repression. The HDAC‐independent repression of LCoR is mediated through its association with CtBP1/2.44 CtBP associates with many transcription factors and is a component of large multiprotein complexes containing histone modifying enzymes. Specifically, CtBP directly interacts with polycomb group (PcG) proteins containing methyltransferase activity for histone H3K27.45 Methylation of H3K27 is well known to be a repressive histone modification. Hr is unique in that it contains both NR and CoRNR boxes; therefore it functions as both a ligand‐independent and LCoR. TR was the first NR found to interact with Hr and for TR, Hr functions as a typical ligand‐independent corepressor.46 Hr associates with the TR LBD in the absence of ligand via its CoRNR box and represses transcription. It also brings HDAC activity via direct interaction with HDAC1, 3, and 5. In the presence of thyroid hormone, Hr dissociates from TR. Unlike NCoR and SMRT, which are ubiquitously expressed, Hr is selectively expressed mainly in the skin and brain.47 Therefore, it may function as a tissue‐specific corepressor for TR. In contrast, Hr is a LCoR for RAR‐related orphan receptor (ROR) and VDR.48,49 It associates with the AF2 domain of these receptors via its two NR boxes. Hr is unable to bind these receptors in the absence of agonist, because its CoRNR motifs are selective for TR. Thus, in tissues where it is expressed, like the brain and skin, Hr attenuates the transcriptional activity of ROR and VDR in response to ligand.

III. Types of NR Repression A. Repression by Unliganded Receptors Type II NRs are those that constitutively reside in the nucleus and bind to their DNA recognition sequences. These NRs include all the isoforms of TR, RAR, VDR, and PPAR. In the absence of ligand, these receptors interact with corepressor complexes and actively repress expression of their target genes. In the presence of ligand, the corepressors are displaced and coactivators are recruited. Repression by type II NRs is generally mediated by ligand‐ independent corepressors, like NCoR and SMRT, which interact with the unliganded NR LBD via CoRNR boxes (Fig. 1A). Certain orphan NRs are constitutive repressors. Therefore, in any tissue in which they are expressed, they bind to response elements and suppress expression of their target genes. Examples include COUP‐TFI, COUP‐TFII, DAX1, EAR2, germ cell nuclear factor (GCNF), SHP, TLX, TR2, and TR4. Like type II NRs, the repression activity of these orphan receptors is mediated by

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A HDAC3

CoRNR box-containing corepressors recruited by unliganded NR

NCoR/SMRT

TR RXR

B HDAC3

CoRNR box-containing corepressors recruited by antagonist-bound NR

NCoR/SMRT 4-HT ER

4-HT ER

C HDAC 1,3,5

NR box-containing corepressors recruited by agonist-bound NR

Hr

GRIP1

Agonist-bound NR represses trancription via classical coactivators

E2 ERa

Vit D VDR RXR

cJun NFκB

FIG. 1. Types of nuclear receptor repression. (A) Repression by unliganded NRs (typical of type II NRs). (B) Repression by antagonist‐bound NRs. The antagonist‐bound LBD favors interaction with corepressors. (C) Repression by agonist‐bound NRs. Two examples are given. Ligand‐dependent corepressors may be recruited by agonist‐bound NRs. Alternatively, classical coactivators may repress transcription by agonist‐bound NRs.

corepressor complexes like NCoR and SMRT. However, since these receptors have no known ligand, they are always maintained in the repressive conformation leading to constitutive repression.

B. Repression by Antagonist‐Bound Steroid Receptors Steroid hormone receptors in general reside in cytoplasm and associate with chaperones in the absence of hormones. Binding of hormones triggers conformational changes that lead to dissociation of cytoplasmic chaperones, nuclear translocation, DNA binding, and ultimately activation of target genes. However, it is well established that antagonist‐bound steroid receptors can lead

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to repression of gene expression (Fig. 1B). Indeed, various antagonists for ER, GR, AR, and PR have been explored for various therapeutic purposes. It is generally believed that different antagonists or partial antagonists can induce the LBD of a NR to adopt different conformations. This different conformation in turn dictates the association of a spectrum of coactivators and corepressors and subsequent transcriptional activation or repression. Since this type of repression has been extensively reviewed, we will not discuss this in detail here.

C. Repression by Agonist‐Bound Receptors While most studies on agonists focus on transcriptional activation, agonist‐ bound receptors can also induce transcriptional repression. One of the best known cases is the negative feedback repression of thyrotropin (TSH) in the pituitary as part of the endocrine hypothalamus–pituitary–thyroid feedback loop.50 The repression function of agonist‐bound receptors are well supported by recent numerous microarray studies. For example, while 17b‐estradiol treatment of MCF7 cells induces activation of a large number of genes, a similar number of genes are repressed.51 Similar results have been reported for TR, VDR, and GR.52–57 Whereas the mechanism whereby agonist‐bound receptors activate gene expression has been extensively studied, little is known about how agonist‐ bound receptors induce transcriptional repression. In the case of feedback repression of TSH by liganded TRb, several working models have been proposed, including (1) direct binding of TR to a negative TRE that leads to the repressive effect of liganded TR, (2) liganded TR associates with the target gene through protein–protein interactions with other transcription factors or cofactors and interferes with transcriptional activation, or (3) squelching effects by sequestering coactivators essential for transcriptional activation.58–62 Each of these mechanisms is supported by some experimental evidence, yet none of these have fully explained the repressive feedback of TSH gene expression by TR. Similarly, recent studies on vitamin D‐induced transcriptional repression of several negative VDR target genes have begun to reveal diverse modes for repression.63,64 Nevertheless, histone deacetylation induced by histone deacetylase corepressors appears to facilitate vitamin D‐induced transcriptional repression via the VDR. ERa, classically a ligand‐induced transcriptional activator, has been reported to function in an opposite manner to regulate expression of the TNFa gene.65 For TNFa, unliganded ERa acted as a coactivator for c‐Jun and NFkB. In the presence of estradiol, ERa repressed transcription of TNFa in a manner dependent on recruitment of the ‘‘coactivator’’ GRIP1. Thus, the authors conclude that GRIP1 can function as either a coactivator or corepressor depending on the context of the gene promoter. Figure 1C illustrates some

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of the modes of repression by agonist‐bound NRs. Taken together, the mechanisms behind agonist‐induced transcriptional repression are probably more complex than those of agonist‐induced transactivation and will be an important area for future study.

IV. Molecular Mechanisms of Transcriptional Repression In principle, NRs and corepressors that they interact with may repress transcription by interfering with any step(s) along the multistep process of transcription. Indeed, NRs have been reported to inhibit transcription by various mechanisms including interfering with the binding of transcription factors to DNA, competition for heterodimer partner RXR, interaction with general transcriptional factors such as TATA‐binding protein (TBP). However, accumulative evidence points to key roles of histone modifications and chromatin remodeling in mediating repression by NRs. In fact, many corepressor proteins are either histone modifying enzymes or function to recruit histone modifying enzymes that deposit repressive marks on chromatin (Fig. 2). Here, we will elaborate on these chromatin related repression mechanisms.

A. Histone Deacetylation By far the most heavily studied molecular mechanism of gene repression is histone deacetylation. It is well known that hypoacetylated histones are associated with gene silencing and heterochromatin. Furthermore, every NR

Histone H3K9 methyltransferases

Histone deacetylases

DNA methyltransferases

ATP-dep. chromatin remodeling factors

NR NR

Histone H3K27 methyltransferases

Histone H3K4 demethylases

FIG. 2. Molecular mechanisms of inhibiting gene expression by NRs. Regardless of the type of NR‐mediated repression (unliganded, antagonist‐bound, agonist‐bound), specific types of chromatin modifying enzymes are recruited to inhibit transcription.

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corepressor associates with one or more HDACs (i.e., HDAC3 for NCoR/ SMRT). Chromatin immunoprecipitation (ChIP) experiments performed on NR target gene promoters have unanimously found that in the absence of ligand the gene is hypoacetylated and becomes hyperacetylated in the presence of agonist. So, histone acetylation is a major mechanism through which NRs mediate transcriptional repression, but how does histone deacetylation actually repress transcription? First, acetylated histones repel internucleosomal interactions.66–68 Internucleosomal interactions are responsible for higher order chromatin structure, which sterically inhibits assembly of transcription machinery. Thus, histone deacetylation would promote the formation of higher order repressive chromatin. Second, acetylation of lysine neutralizes its positive charge thereby loosening the association between histones and DNA.69,70 This theoretically allows histones to be more easily displaced from promoter regions thereby allowing transcription factors access to the promoter. Third, acetylated lysine is the molecular substrate for the bromodomain.71 Bromodomains are found in numerous coactivators and ATP‐dependent chromatin remodeling complexes and function to stabilize their association with their target chromatin template (i.e., target gene promoter). Thus, recruitment of HDAC‐containing corepressor complexes by NRs inhibits transcription through multiple mechanisms.

B. Histone Methylation Histone methylation plays critical roles in regulating chromatin structure and function. Strong evidence indicates that histone methylation also plays important roles in NRs‐induced transcriptional repression. In this regard, CARM1 and PRMT1 were both identified as coactivators for NRs.72–74 CARM1 methylates histone H3 on R2, R17, and R26, whereas PRMT1 primarily methylates histone H4 on R3. Consistent with methyl‐H3K4 being a mark for transcriptional activity, the H3K4 methyltransferase MLL has been identified as a component of the ASC2 coactivator complex.75,76 In contrast, histone methyltransferases (HMTs) with specific activity for H3K9 or H3K27 are autonomous transcriptional repressors. SUV39H1, an H3K9‐specific HMT, was found to associate with unliganded TR.33 Thus, unliganded TR represses transcription through a combination of histone deacetylation mediated by HDAC3‐containing NCoR/SMRT complexes and H3K9 methylation mediated by SUV39H1. Another H3K9 HMT, G9a, was shown to mediate repression by the small heterodimer partner SHP.34,77 Enhancer of zeste homolog 2 (EZH2) is a polycomb protein with histone H3 K27 methyltransferase activity. Conflicting data have been reported for EZH2 as to its function for ER. In one report, EZH2 was shown to activate ER transcription, whereas in the

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other report EZH2 was identified as an REA‐interacting protein that functioned as an ER corepressor.78,79 Thus, the function of EZH2 for ER may be cell type and context dependent. LSD1 is the first identified histone lysine demethylase.35 In vitro purified LSD1 exhibits histone demethylase activity toward only mono‐ and dimethylated H3K4. LSD1 is part of the large protein complex that also contains CoREST and HDAC1/2.80 Given its association with HDAC1/2 and H3K4 demethylase activity, it is not surprising that knockdown of LSD1 by RNAi caused an increase of H3K4 methylation and concomitant derepression of target genes. Consistent with this notion, LSD1 serves as a corepressor for orphan receptor TLX (also called NR2E1).37 LSD1 interacts directly with TLX and potentiates TLX repression through its H3K4 demethylase activity. Interestingly, LSD1 was also identified as an AR‐interacting protein that facilitated AR transactivation.36 This functional switch is brought by a change in its demethylase specificity. Upon interaction with AR, LSD1 was shown to switch its specificity from methyl‐H3K4 to methyl‐H3K9. At this stage, the molecular mechanism that underlies this specificity switch is not clear. In addition to above specific examples, evidence exists for a general involvement of histone methylation in governing ligand‐dependent activation by NRs. It was shown that different H3K9 methyltransferases bring in repressive epigenetic marks and impose gene‐specific gatekeeper functions that prevent unliganded NRs or other transcription factors from binding to their target genes in the absence of ligands or other stimulating signals.81 Histone demethylases including LSD1 thus are required to reverse the repressive epigenetic marks and allow the ligand‐ and signal‐dependent transcriptional activation to take place. This study highlights the functional significance of histone methylation/demethylation in regulation of gene expression.

C. Chromatin Assembly/Remodeling ATP‐dependent chromatin remodeling factors utilize ATP hydrolysis to alter chromatin structure through mechanisms including nucleosome assembly/disassembly, sliding, and conformational changes. While SWI/SNF, a prototype ATP remodeling complex, generally functions as coactivator for NRs, a SNF2H‐containing remodeling complex was reported to be required for efficient repression by unliganded TR.82 In this case, the NCoR/HDAC3 complex and SNF2H complex function cooperatively. Mechanistically, NCoR/HDAC3 generates deacetylated histone H4 tails to which the SNF2H complex binds and induces nucleosomal reorganization. This mechanism of repression through chromatin reorganization is not unique to SNF2H, as corepressor Alien has also been reported to recruit a histone chaperone NAP1 for chromatin assembly.32 NAP1 can assemble or

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disassemble nucleosomes in a DNA replication‐independent manner. However, when tethered to DNA via the heterologous DBD of yeast Gal4, it preferentially represses transcription. Direct interaction of Alien with NAP1 enhances its nucleosome assembly activity. In addition, Alien can directly bind the core histones H3 and H4. Thus, the combination of stable chromatin association of NAP1 via the histone binding property of Alien combined with enhanced nucleosome assembly activity imparted by direct interaction with Alien provides a perfect environment for NAP1 to assemble nucleosomes in a manner that inhibits transcriptional activity. ATP‐dependent chromatin remodeling activity was also shown to participate in repression by the orphan receptor SHP.83 In this study, SHP directly interacted with Brm‐containing SWI/SNF remodeling complexes. Dominant negative ATPase mutants prevented repression. For the CYP7A1 gene, SHP‐ induced repression was mediated by both mSin3A‐HDAC1/2 and SWI/SNF. This is another example of the use of ATP‐dependent chromatin remodeling factors for transcriptional repression. The role of yet another chromatin assembly/remodeling protein Acf1 in NR repression has recently been demonstrated84 and is discussed in detail in Chapter 6.

D. DNA Methylation DNA methylation is a common mechanism for long‐term gene silencing. Since NRs can induce acute transcriptional repression, DNA methylation in general has not been considered as a mechanism for NR‐mediated repression. However, there is evidence for the involvement of DNA methylation in transcriptional repression induced by NRs. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by excess promyelocytes and deficiency in cells of the myeloid lineage. The most common genetic cause of APL is t(15;17) translocation, which generates the leukemia‐promoting PML–RARa fusion protein.85,86 Contrary to full‐length RAR, PML–RARa maintains its interaction with SMRT and NCoR at physiological levels of RA.87,88 For this reason, the fusion protein functions as a constitutive repressor of RAR target genes, blocking differentiation and resulting in leukemogenesis. In addition to recruiting the SMRT/NCoR corepressor complexes, PML–RAR can induce hypermethylation of target genes by recruiting DNA methyltransferases.89 DNA hypermethylation contributes to the leukemogenic potential of PML–RAR. In another study, the corepressor RIP140 was reported to direct both histone and DNA methylation to silence Ucp1 expression in white adipocytes.90 As a recent genome wide study revealed an intimate correlation between histone methylation and DNA methylation,91 perhaps DNA methylation is more commonly involved in transcriptional repression than we previously thought.

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V. Physiological Functions of NR‐Mediated Repression Regardless whether repression is mediated by unliganded or liganded receptors, transcriptional repression is an integral function of all NRs. However, the physiological function of repression is difficult to assess and underappreciated. For example, while it is well established that unliganded TR is a potent repressor, whether this repression activity is physiologically relevant is still a subject of debate. Much of our knowledge concerning the physiological function of NR‐mediated repression comes from knockout studies of corepressor proteins. However, corepressors can mediate repression for both NR and non‐NR transcription factors. Here, we will summarize some recent progress in this area.

A. Physiological Function of Repression by Unliganded TR For most NRs, it is difficult to discern the physiological roles of repression from activation. For TRb, the mutations in the LBD that result in reduced hormone binding are responsible for the syndrome of resistance to thyroid hormone (RTH). RTH is characterized by elevated serum levels of T4 and T3, inappropriately ‘‘normal’’ or elevated serum TSH concentrations, diffuse goiter and varying manifestations of hypothyroidism.92 The RTH‐associated TRb mutations identified so far represent a range of reduced hormone‐binding activities. These TRb mutants maintain association with corepressors SMRT and NCoR at physiological levels of T3 and therefore behave as constitutive repressors. In a sense, the RTH syndrome exemplifies the pathological effects of transcriptional repression by unliganded TRb. Another example of a pathological link to unliganded TR is the v‐Erb A oncoprotein from the avian erythroblastosis virus (AEV). The v‐Erb A protein is a derivative of c‐Erb A, an avian TRa. Compared to c‐Erb A, v‐Erb A was found to sustain a series of alterations including an N‐terminal fusion of viral ‘‘gag’’ sequence, deletion of the C‐terminal AF2 and 13 internal point mutations.93,94 As a consequence, v‐Erb A binds constitutively and with higher affinity to the corepressors SMRT and NCoR.95 This repression function of v‐ Erb A in turn blocks the terminal differentiation of erythroid cells and contributes to its oncogenic activity. Despite the findings described above, it is still unclear if repression by unliganded TR is of physiological significance under normal developmental and growth conditions. In fact, mice with null mutations in all TR isoforms actually exhibit far less severe phenotypes than mice made congenitally

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hypothyroidism by treatment with thyrostatic drugs. These results imply that transcriptional repression by apo‐ or unliganded TRs are responsible for the detrimental effects of hypothyroidism.

B. TR‐Mediated Repression in the Regulation of Amphibian Metamorphosis Xenopus laevis, the African clawed frog, is a model for amphibian development. Frogs begin life as tadpoles and undergo metamorphosis to become air‐ breathing adults. Metamorphosis in frogs is under the master control of thyroid hormone. Although it is unclear if repression by unliganded TR is of physiological relevance in mammals, elegant studies in amphibian frogs have demonstrated a critical role for repression by unliganded TRs in regulating the timing and precise programs of metamorphosis. A dual function model has been proposed for regulation of amphibian development.96 Tadpoles express TRa in virtually all cell types, but do not produce thyroid hormone. Thus, TRa is bound by NCoR/SMRT complexes and mediates silencing of genes involved in metamorphosis.97,98 During metamorphosis, thyroid hormone is produced and TRb becomes expressed at high levels. In this state, TRa and TRb are bound by coactivators and activate expression of prometamorphic genes. As expected, gene activation correlates with dissociation of NCoR/SMRT, binding of coactivators, and histone hyperacetylation. This system not only represses genes that might adversely affect premetamorphic development, but also primes cells to be responsive to thyroid hormone so that they can induce metamorphosis at the appropriate time. The above dual function model is further supported by studies using dominant negative or positive transgenes. First, tadpoles expressing a dominant negative TR, which can bind thyroid hormone but cannot recruit coactivators, are unable to undergo metamorphosis even upon thyroid hormone treatment.99 Second, tadpoles expressing a dominant negative SRC3 also cannot undergo metamorphosis.100 Thus, both corepressor release and coactivator recruitment are required to induce the metamorphic program. Furthermore, expression of a dominant positive TR, which activates TR reporter genes independently of thyroid hormone, causes precocious metamorphosis.101 Additionally, expression of a dominant negative NCoR consisting of only its RID in tadpoles causes derepression of TR target genes and accelerated development.102 Thus, TR‐mediated repression is essential for proper premetamorphic development and regulating the timing of metamorphosis.

C. Repression by PPARg PPARg is a master regulator of adipogenesis. Its repression activity has been linked to maintenance of the preadipocyte cell type or in other words repression by PPARg prevents differentiation of preadipocytes to adipocytes.

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PPARg homozygous null mice die during embryogenesis due to placental defects. Following rescue of the placental phenotype by tetraploid complementation, PPARg null mice survive to term but then manifest defects in adipogenesis.103 White adipose tissue‐specific reduction of PPARg (via genetic deletion of exon 2) results in growth retardation, lipodystrophy, and hyperlipidemia.104,105 Under normal dietary conditions, PPARg activity prevents mobilization of fatty acids and stimulates adipogenesis. However, upon caloric restriction, repression of PPARg target genes prevents fat storage and promotes lipolysis. Thus, at least one of the physiological functions of PPARg‐mediated repression is to allow for the mobilization of fat stores during caloric restriction. In mice, mobilization of fatty acids from white adipose tissue is dependent on the PPARg corepressors, NCoR, and Sirt1 (mammalian ortholog of yeast Sir2).106 Sirt1 represses PPARg target genes, including those involved in fat storage, via direct interaction with NCoR. Caloric restriction induces expression of Sirt1.107 Thus, upon food withdrawal, Sirt1 interacts with PPARg to repress target genes involved in lipogenesis. Accordingly, mice heterozygous for a null mutation in Sirt1 have a limited ability to mobilize fatty acids upon food withdrawal. NIH 3T3‐L1 fibroblasts are a well characterized model of adipogenesis. In the absence of ligand, PPARg represses the adipogenic program in these cells in an NCoR‐dependent manner.108 Additionally, Sirt1 overexpression prevents and Sirt1 knockdown enhances adipogenesis in this model system. In primary adipocytes, increased expression of Sirt1 stimulates lipolysis. Thus, PPARg is a master regulator of adipogenesis and fatty acid metabolism, influencing both the commitment to adipocyte differentiation and the balance between lipogenesis and lipolysis. In this manner, the physiological function of PPARg‐mediated repression is to maintain the preadipocyte cell type and stimulate fatty acid breakdown.

D. Repression Mediated by NCoR and SMRT In general, the physiological functions of NR‐mediated repression have been difficult to address, although some recent studies have begun to shed light on this topic. As discussed above, repression by NRs is mediated by corepressors. Thus, corepressor knockout models have been generated to assess the functional significance of repression. However, most, if not all, NR corepressors also mediate the repressive actions of non‐NR transcription factors. These experiments provide excellent in vivo demonstration of the physiological function of individual corepressors, but fail to precisely address the role of NR‐mediated gene repression.

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Two recent studies have used gene targeting in mouse embryonic stem cells to create alleles of NCoR or SMRT with mutations in their NR interaction domains.109,110 The gene products of these alleles cannot interact with NRs, but it is assumed that they retain their ability to interact with non‐NR transcription factors. The phenotype of the SMRT RID mutant mice was interesting in that homozygous animals did not exhibit embryonic lethality, which was the phenotype of the SMRT null mutation. Instead animals appeared grossly normal and fertile; however, they did exhibit metabolic defects. Homozygous mutants exhibited a 20% decrease in respiration, a 70% increase in adiposity, increased blood glucose, glucose intolerance, and insulin resistance. For NCoR, the RID was conditionally deleted in the liver and was used to test the physiological importance of the NCoR–TR interaction. These mice also appeared grossly normal and fertile. When these mice were pharmacologically induced to be hypothyroid, repression of TR target genes was not observed. Thus, NCoR is critical for TR‐mediated repression in the liver. Interestingly, the authors also found increased expression of many TR target genes compared with wild type controls. These data suggest that NCoR regulates the transcriptional activity of liganded TR and supports an equilibrium model in which corepressors and coactivators compete for the NR LBD.

E. Repression Mediated by RIP140 RIP140 is a corepressor for most, if not all, NRs. The gene encoding RIP140 is expressed in virtually all tissues and subject to regulation by steroid hormones, retinoic acid, and vitamin D. It appears to primarily function in the regulation of energy homeostasis. Accordingly, highest gene expression is found in adipose, muscle, and liver. Studies of homozygous null RIP140 mice (Nrip1 / ) revealed the major function of this corepressor is to regulate metabolic gene expression.42 Nrip1 / mice exhibit a 70% reduction in total body fat, including almost complete absence of subcutaneous fat. White adipocytes were approximately 70% smaller; therefore, these mice have a limited capacity to store triglycerides and cholesterol esters. Additionally, these mice exhibit a 30% increase in respiration owing to an increase in the ratio of slow oxidative to fast twitch muscle fibers. These alterations in adipogenesis and muscle physiology have the ‘‘positive’’ effect of resistance to diet‐induced obesity. Unfortunately, Nrip1 / females are infertile due to an ovulation defect.43 The Nrip1 / phenotype is similar to that of PGC1a overexpression, indicating the main physiological function of this corepressor is to mediate the repression function of PPARg.

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VI. Concluding Remarks To conclude we would like to emphasize the fact that the ability to associate with corepressors and inhibit transcription is an intrinsic property of most members of the NR superfamily. While this is quite apparent for type II and orphan NRs, which constitutively bind DNA and repress transcription in the absence of ligand, it is also true for steroid receptors. Generally speaking, in the absence of hormone, steroid receptors (AR, ER, PR, MR, and GR) reside in the cytoplasm. Upon hormone binding, they translocate to the nucleus, bind DNA, and activate transcription. However, steroid receptors will also associate with corepressors upon binding antagonists. This highlights the fact that CoRNR box‐containing corepressors and NR box‐containing coactivators compete for the LBD of NRs. The equilibrium is shifted in either direction depending on the nature of the ligand. A prime example of this is the cell‐ type specific effects of the mixed agonist/antagonist tamoxifen on association of ERa with coactivators or corepressors.111 NRs mediate transcriptional repression by recruiting multiprotein corepressor complexes. In turn, corepressor complexes repress transcription because they either contain or associate with chromatin modifying enzymes. The best known repressive chromatin modifying enzymes are HDACs. However, we tried in this chapter to emphasize other repressive chromatin modifications brought about by corepressors. These include methylation of histone H3K9 and H3K27 and demethylation of H3K4. Additionally, several corepressor complexes associate with ATP‐dependent chromatin remodeling factors, which in this setting serve to assemble nucleosomes and compact local chromatin. Finally, it is important to note that we are far from understanding the physiological functions of NR‐mediated repression. We have reviewed the few studies that have used mouse models to address this topic and pointed out that it is inherently difficult to separate the effects of NR‐mediated repression from activation. In the future, we expect to see more attempts to determine the precise role of NR‐mediated repression using mouse genetics.

Acknowledgments We apologize to colleagues whose original work could not be cited owing to space constraints. The work in J.W.’s laboratory is supported by a grant from National Science Foundation (90919025) and a grant from The Ministry of Science and Technology (2009CB918402). M.D.S. is supported by NCI training grant CA009299.

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