I. INTRODUCTION

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Small lipophilic molecules such as steroid and thyroid hormones or the active forms of vitamin A (retinoids) and vitamin D play an important role in the growth, differentiation, metabolism, reproduction, and morphogenesis of higher organisms and humans. Most cellular actions of these molecules are mediated through binding to nuclear receptors that act as ligand-inducible transcription factors. Almost two decades have gone by since the cloning of the first nuclear receptor for a steroid hormone. Since then, other nuclear hormone receptors were rapidly cloned and their target sequences on DNA identified. Our knowledge on regulation of gene expression by nuclear receptors has grown spectacularly during the last years, mainly due to the realization that not only the interaction of the receptors with DNA was important for transcriptional responses, but also that many coregulators (coactivators and corepressors) were crucial in transmitting the hormonal signal to the transcriptional machinery. On the other hand, crystal structures of ligand-binding domains of nuclear receptors have been solved, and this has allowed the definition of the structural basis for their transcriptional functions. Another major breakthrough in the study of nuclear receptors has been the targeted disruption of receptor genes in mice that allows an analysis of the relevance of particular receptors and receptor isotypes on mammalian physiology and development. These studies, which have shown the complexity of the mechanisms by which hormones elicit their role in vivo, and the existence of both redundant and specific mechanisms for particular receptor isoforms are not described in this review.

Cloning of the receptors for steroid and thyroid hormones demonstrated that they share an extensive homology, and this observation led to a search for new proteins with similar structure. During the course of the last decade, the identification and characterization of close to 40 vertebrate receptors has led to the discovery of new hormonal responses and to the novel concept of "reverse endocrinology" in which the characterization of the receptor precedes the study of its physiological function. Regulatory ligands for many of these receptors have not yet been identified, and they have been called "orphan receptors." In the last years ligands have been found for several of these orphan receptors. Some of these ligands are products of lipid metabolism, and it is now known that compounds such as fatty acids, leukotrienes, prostaglandin and cholesterol derivatives, bile acids, pregnanes, or even benzoate derivatives can regulate gene expression through their binding to nuclear receptors. Therefore, as opposed to classic hormones, other ligands are intracellularly originated as metabolic products, which may explain why their role as regulators of nuclear receptors was not previously identified by physiological experimentation. Many other orphan receptors may have a still unidentified ligand, but others may act in a constitutive manner or could be activated by other means, i.e., phosphorylation (Fig. 1). That orphan receptors also play key roles in development, homeostasis, and disease has been proven by targeted deletion in mice and by their association with different diseases including atherosclerosis, cancer, diabetes, or lipid disorders. These findings have opened new strategies for treatment of these diseases, and orphan receptors at this point, together with the search for new agonist and antagonist ligands for classical receptors, constitute important targets for drug discovery.

View larger version (33K): [in this window] [in a new window]   Fig. 1. Mechanism of action of nuclear receptors. Left: the ligand can be generated in three different ways: 1) an active ligand or hormone is synthesized in a classical endocrine organ and enters the cell, 2) the ligand may be generated from a precursor or prohormone within the target cell, and 3) the ligand may be a metabolite synthesized within the target cell. The unliganded receptor may have a nuclear location. However, some steroid receptors are cytoplasmic in the absence of ligand due to their association with a large multiprotein complex of chaperones, including Hsp90 and Hsp56. Ligand binding induces dissociation of the complex and nuclear translocation. Once in the nucleus, the receptors regulate transcription by binding, generally as dimers, to hormone response elements (HREs) normally located in regulatory regions of target genes. Right: alternative ligand-independent pathways for activation of nuclear receptors exist. Some receptors may be constitutively active, and the activity of others is modulated by other means, for instance, phosphorylation mediated by hormones and growth factors that stimulate diverse signal transduction pathways.

The goal of this work is to review the progress in the field of transcriptional regulation by nuclear receptors. We start by describing the domain structure of nuclear receptors and the characterization of DNA hormone response elements to which they bind in general as homo- or heterodimers. A brief description of the existence of mechanisms involved in non-DNA binding-dependent regulation by cross-talk with other signal transduction pathways follows. Some of the problems facing this field are the elucidation of mechanisms of transcriptional activation, mechanisms of transcriptional repression, and characterization of coactivator and corepressor complexes that are described with more detail.

To limit the references to a reasonable number, it is impossible to make a comprehensive analysis of all that has been published on nuclear receptors signaling. Instead, we try to highlight the more recent discoveries and to summarize the present knowledge on the mechanisms by which nuclear receptors regulate gene expression. To facilitate understanding we include a table that summarizes the more representative mammalian receptors, and we have created figures that schematically explain the mechanisms involved in transcriptional regulation. Because it is not be possible to cite all relevant articles, we are including many up-to-date reviews on specific topics. We apologize to our colleagues when an original reference is not mentioned due to lack of space.

	II. THE NUCLEAR RECEPTOR SUPERFAMILY

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Nuclear receptors are grouped into a large superfamily and are thought to be evolutionarily derived from a common ancestor. A list of classical and orphan hormone receptors and their ligands is shown in Table 1. Evolutionary analysis of the receptors has led to a subdivision in six different subfamilies (145). One large family is formed by thyroid hormone receptors (TRs), retinoic acid receptors (RARs), vitamin D receptors (VDRs) and peroxisome proliferator-activated receptors (PPARs) as well as different orphan receptors. Ligands for some of these receptors have been recently identified (see Table 1). The second subfamily contains the retinoid X receptors (RXRs) together with chicken ovalbumin upstream stimulators (COUPs), hepatocyte nuclear factor 4 (HNF4), testis receptors (TR2) and receptors involved in eye development (TLX and PNR). RXRs bind 9-cis-retinoic acid and play an important role in nuclear receptor signaling, as they are partners for different receptors that bind as heterodimers to DNA. Ligands for other receptors have not been identified, whereas long-chain fatty acid acyl-CoA thioesters may be endogenous ligands for HNF4. The third family is formed by the steroid receptors and the highly related orphan receptors estrogen-related receptors (ERRs). The fourth, fifth, and sixth subfamilies contain the orphan receptors NGFI-B, FTZ-1/SF-1, and GCNF, respectively (for a recent comprehensive review in function and recently identified ligands for nuclear orphan receptors see Ref. 84). Most subfamilies appear to be ancient since they have an arthropod homolog, with the exception of steroid receptors that have no known homologs. It has been suggested that the ancestral receptors were constitutive homodimeric transcription factors that evolved to independently acquire the ability to bind a ligand and to heterodimerize. However, the possibility that the ancestral receptor was ligand dependent and that mutations changed the ligand-binding specificity or led to loss of ligand binding during evolution cannot be ruled out.

Like other transcriptional regulators, nuclear receptors exhibit a modular structure with different regions corresponding to autonomous functional domains that can be interchanged between related receptors without loss of function. A typical nuclear receptor consists of a variable NH₂-terminal region (A/B), a conserved DNA-binding domain (DBD) or region C, a linker region D, and a conserved E region that contains the ligand binding domain (LBD). Some receptors contain also a COOH-terminal region (F) of unknown function. A scheme of a nuclear receptor is shown in Figure 2. The receptors also contain regions required for transcriptional activation. The hypervariable A/F region of many receptors contains an autonomous transcriptional activation function, referred to as AF-1, that contributes to constitutive ligand-independent activation by the receptor. A second transcriptional activation domain, termed AF-2, is located in the COOH terminus of the LBD, but unlike the AF-1 domain, the AF-2 is strictly ligand dependent and conserved among members of the nuclear receptor superfamily (see sect. VA).

View larger version (16K): [in this window] [in a new window]   Fig. 2. Schematic representation of a nuclear receptor. A typical nuclear receptor is composed of several functional domains. The variable NH2-terminal region (A/B) contains the ligand-independent AF-1 transactivation domain. The conserved DNA-binding domain (DBD), or region C, is responsible for the recognition of specific DNA sequences. A variable linker region D connects the DBD to the conserved E/F region that contains the ligand-binding domain (LBD) as well as the dimerization surface. The ligand-independent transcriptional activation domain is contained within the A/B region, and the ligand-dependent AF-2 core transactivation domain within the COOH-terminal portion of the LBD.

This modulatory region is the most variable both in size and sequence and in many cases contains an AF-1 domain. Multiple receptor isoforms generated from a single gene by alternative splicing or by the use of alternative promoters diverge in their A/B regions in most cases. This is the case for the TR isoforms TR1 and TR2 or for the various isoforms generated from the RAR genes, which are identical in their DBD and LBD, but differ in their NH₂-terminal regions. The A/B domain shows promoter- and cell-specific activity, suggesting that it is likely to contribute to the specificity of action among receptor isoforms and that it could interact with cell type-specific factors. On the other hand, the modulatory domain is the target for phosphorylation mediated by different signaling pathways, and this modification can significantly affect transcriptional activity (for a review see Ref. 243). There are several reports indicating that RARs and other receptors can be phosphorylated by cyclin-dependent kinases and that this phosphorylation is important for ligand-dependent and -independent transactivation (222, 223, 259). Furthermore, other nuclear receptors such as the estrogen receptors (ERs) are phosphorylated at serine or threonine residues by the mitogen-activated protein kinase (MAPK) in vitro, and in cells treated with growth factors that stimulate the Ras-MAPK cascade, and this phosphorylation enhances transcriptional activity (131, 197). A specific tyrosine phosphorylation site located at the COOH-terminal region of the receptor is involved in ligand-independent activity and may be a target for a different signaling pathway (285). A strong AF-1 domain in PPAR is also modulated by phosphorylation by MAPK, and this phosphorylation enhances transcriptional activity (124). However, phosphorylation of the A/B domain of PPAR by the same kinase negatively regulates its transcriptional functions. Interestingly, this modification reduces ligand binding to the receptor, showing that binding can be regulated by intramolecular communication between the modulatory domain and the COOH-terminal LBD (244). MAPK-dependent phosphorylation of the RXR can also alter biological actions of a partner receptor (250).

The DBD, the most conserved domain of nuclear receptors, confers the ability to recognize specific target sequences and activate genes (Fig. 3). The DBD contains nine cysteines, as well as other residues that are conserved across the nuclear receptor superfamily and are required for high-affinity DNA binding. This domain comprises two "zinc fingers" that span ~60-70 amino acids and a COOH-terminal extension (CTE) that contains the so-called T and A boxes. In each zinc finger, four of the invariable cysteines coordinate tetrahedrically one zinc ion, and both zinc finger modules fold together to form a compact, interdependent structure as determined by nuclear magnetic resonance and crystallographic studies (160, 239). Amino acids required for discrimination of core DNA recognition motifs are present at the base of the first finger in a region termed the "P box," and other residues of the second zinc finger that form the so-called "D box" are involved in dimerization. The core DBD contains two -helices: the first one beginning at the third conserved cysteine residue (the recognition helix) binds the major groove of DNA making contacts with specific bases, and the second one that spans the COOH terminus of the second zinc finger forms a right angle with the recognition helix (see Fig. 3). The nuclear magnetic resonance structure of the RXR DBD identified a third helix in the CTE that packs against helix 1 (148).

View larger version (27K): [in this window] [in a new window]   Fig. 3. The DNA binding domain of the nuclear receptors. A diagram of the two zinc fingers and the COOH-terminal extension (CTE). In the zinc fingers, four conserved cysteines coordinate a zinc ion. Other conserved residues are shown and designated by the corresponding letter. Helix 1 contains P box residues involved in the discrimination of the response element. Residues in the second zinc finger labeled as D box form a dimerization interface. The CTE contains the T and A boxes critical for monomeric DNA binding. As shown in the bottom panel, helix 1 and helix 2 cross at right angles to form the core of the DBD that recognizes a hemi-site of the response element. [Bottom panel from Glass (87). Copyright the Endocrine Society.]

The LBD is a multifunctional domain that, in addition to the binding of ligand, mediates homo- and heterodimerization, interaction with heat-shock proteins, ligand-dependent transcriptional activity, and in some cases, hormone reversible transcriptional repression. The LBDs contain two well-conserved regions: a "signature motif" or Ti and the COOH-terminal AF-2 motif responsible for ligand-dependent transcriptional activation (294).

The crystal structures of the LBDs of multiple nuclear receptors have been solved. These studies have demonstrated that overall structures of the different receptors are rather similar, suggesting a canonical structure for the different members of the nuclear receptor superfamily (for a review see Ref. 178). Figure 4 shows a schematic representation of the crystal structure of a receptor LBD. The LBDs are formed by 12 conserved -helical regions numbered from H1 to H12. A conserved -turn is situated between H5 and H6. However, PPAR is unique in its overall structure and contains an extra helix designed H2', and the VDR contains a poorly structured insertion between helices H1 and H3 for which no functional role has been defined (221). The LBDs are folded into a three-layered, antiparallel helical sandwich. A central core layer of three helices is packed between two additional layers to create a cavity, the ligand-binding pocket, which accommodates the ligand. This domain is mainly hydrophobic and is buried within the bottom half of the LBD. Contacts with the ligand can be extensive and include different structural elements through the LBD. The size of the ligand binding pocket varies among the different receptors, being for instance very large in PPAR, which allows binding of very differently sized ligands (273). Several differences are evident when comparing unliganded and ligand-bound receptors. The liganded structures are more compact than the unliganded ones, demonstrating that upon ligand binding the receptors undergo a clear conformational change.

View larger version (33K): [in this window] [in a new window]   Fig. 4. Schematic drawing of the nuclear receptor ligand-binding domain (LBD). On the left, the LBD from the crystal structure of the unliganded RXR is shown. On the right, the ligand-bound LBD of the RAR is shown. Cylinders represent -helices that are numbered from 1 to 12. Note the different position of the COOH-terminal helix 12 that contains the core AF-2 domain in both situations. [From Wurtz et al. (294), reprinted by permission from Nature, Macmillan Magazines Ltd.]

Nuclear receptors regulate transcription by binding to specific DNA sequences in target genes known as hormone response elements or HREs. These elements are located in regulatory sequences normally present in the 5'-flanking region of the target gene. Although often the HREs are found relatively close to the core promoter, in some cases they are present in enhancer regions several kilobases upstream of the transcriptional initiation site. The analysis of a large number of naturally occurring as well as synthetic HREs revealed that a sequence of 6 bp constitutes the core recognition motif. Two consensus motifs have been identified: the sequence AGAACA is preferentially recognized by steroid class III receptors, whereas AGG/TTCA serves as recognition motif for the remaining receptors of the superfamily (17). It should be noted that these motifs represent consensus idealized sequences and that naturally occurring HREs can show significant variation from the consensus. Although some monomeric receptors can bind to a single hexameric motif, most receptors bind as homo- or heterodimers to HREs composed typically of two core hexameric motifs. For dimeric HREs, the half-sites can be configured as palindromes (Pal), inverted palindromes (IPs), or direct repeats (DRs).

Steroid hormone receptors typically bind to palindromes of the AGAACA sequence separated by three nucleotides, with the exception of the ERs that recognize the consensus AGGTCA motif with the same configuration. On the basis of the analysis of glucocorticoid receptor/ER chimeras, the first zinc finger has been identified as the one responsible for the discrimination of the DNA motif (91). Further studies have shown that mutation of three residues in the P box, which are identical in the glucocorticoid, progesterone, androgen, and mineralocorticoid receptors that recognize the same HRE, was sufficient to switch the sequence recognized by glucocorticoid receptors and ERs. Furthermore, cocrystal structures of receptor DBDs with DNA have shown that P box residues, which are contained within the recognition helix 1 of the DBD, were indeed involved in interaction with specific bases of the recognition motifs (for a detailed review on the interaction of receptors with the HREs, see Ref. 87).

In contrast to steroid receptors that almost exclusively recognize palindromic elements, nonsteroidal receptors can bind to HREs with different configurations (Fig. 5). In this case, the arrangement as well as the spacing between the motifs are determinant to confer selectivity and specificity. Some of these response elements are capable of mediating transcriptional responses to more than one ligand. This is the case of the palindromic element AGGTCATGACCT that confers regulation by both thyroid hormones and retinoic acid (271). As a consequence, both ligands can control overlapping gene networks as demonstrated by the regulation of the rat growth hormone gene by the two hormones via a common HRE (19). Similarly, IPs can also mediate transcriptional responses to both ligands as well as to vitamin D. However, a careful analysis of natural and synthetic HREs has shown that the most potent HREs for nonsteroid receptors are configured as DRs. Analysis of variably spaced DRs suggested that the length of the spacer region was an important determinant of the specificity of hormonal responses. Thus DRs separated by 3, 4, and 5 bp (i.e., DR3, DR4, and DR5) mediate preferential regulation by vitamin D, thyroid hormone, and retinoic acid, respectively (183, 272). The subsequent demonstration that DR1 serves as the preferred HRE for the RXR or for the PPAR and that RARs can also activate transcription through a DR2, expanded the model from a 3-to-5 rule to a 1-to-5 rule (reviewed in Ref. 163). Furthermore, a DR0 sequence can also act as a receptor binding site, and widely spaced DRs can act as promiscuous response elements for different nonsteroid receptors and even for ERs (132). The configuration of the preferred HREs for different classical and orphan receptors has been included in Table 1.

More recent results have shown that in addition to spacing, small differences in the half-site sequence and the sequence of the flanking extension of the response elements also appear to be important parameters in determining receptor binding efficiency (162).

Several orphan nuclear receptors can bind DNA with high affinity as monomers (84). For monomeric HREs, a single AGG/TTCA half-site is preceded by a 5'-flanking A/T-rich sequence. Monomeric nuclear receptors utilize the CTE of the LBD to recognize that sequence (37, 85, 86, 289). The "A box" in this region is critical for the recognition of the amino acids at positions 1 and 2 of the core recognition motif (290). The third helix formed by the CTE can make extensive contacts with the minor groove of DNA and effectively extends the surface contact of the receptor DBD to beyond the consensus half-site recognition sequence providing additional receptor-DNA contacts in monomeric sites necessary for specific and high-affinity binding. Although other nuclear receptors generally do not bind with high affinity to DNA as monomers, it is likely that residues of the A and T boxes in the CTE also contribute to sequence specificity and affinity of binding to DNA.

Steroid receptors almost exclusively bind as homodimers to the HRE. Two steroid hormone receptor monomers bind cooperatively to their response elements, and dimerization interfaces have been identified both in the LBD and in the DBD. In ER, the dimer interface in the LBD contains residues from helices 7, 8, and 9 as well as the loop between helices 8 and 9 but is dominated by a conserved hydrophobic region at the NH₂ terminus of helix 10/11 (260). Other nuclear receptors use similar dimer interfaces because the corresponding residues are highly conserved in different receptors and have been implicated in dimerization by mutagenesis. It has been recently shown that the PPAR/RXR heterodimer is asymmetric and that the heterodimeric interface is composed of conserved motifs that form a coiled-coil along helix 10 with additional charge interactions from helices 7 and 9 (80). In contrast, the RAR/RXR heterodimer is not asymmetrical (30).

Palindromic DNA repeats impose a symmetrical structure that results in a head-to-head arrangement of the DBDs with each DBD of the homodimer making analogous contacts with one half-site. Crystallographic analysis of the DBD of the glucocorticoid receptor DBD-DNA complexes has demonstrated that the dimerization interface in the DBD involves amino acids of the D box. The formation of this interface is responsible for the selection of the spacing distance between the two halves of the palindrome, but it does not appear to function as an effective dimerization interface in the absence of DNA since isolated DBDs do not dimerize in solution (100, 274).

Although several nonsteroidal nuclear receptors also bind DNA as homodimers, many nonsteroidal receptors bind to their HREs preferentially as heterodimers. In this case, the RXR is the promiscuous partner for different receptors (33, 135, 150, 163, 165, 307, 317). Typical heterodimeric receptors such as TR, RAR, or VDR can bind to their response elements as homodimers, but heterodimerization with RXR strongly increases the efficiency of DNA binding and transcriptional activity.

Some monomeric receptors (for instance NGFI-B) can also form heterodimers with RXR, and the heterodimers then recognize DRs rather than the monomeric extended sequence. Homodimeric receptors such as COUP-TF can also form heterodimers with RXR (84, 87). Furthermore, there are receptors that can bind as monomers, homodimers, and heterodimers to different response elements. In Table 1, the receptors that bind as monomers, homodimers, and heterodimers to their HREs are indicated.

Because DRs are inherently asymmetric, heterodimeric complexes may bind to them with two distinct polarities. Indeed, it has been established that on DR3, DR4, and DR5, RXR occupies the upstream half-site, and the heterodimeric partner (e.g., VDR, TR, or RAR) occupies the downstream motif (141, 151, 201, 235, 312). RAR/RXR heterodimers can bind to DR1 elements, and under these conditions, the heterodimer exhibits no response to RAR activating ligands. Interestingly, a reversed polarity is found in the case of a RAR/RXR heterodimer bound on a DR1 element in which RXR occupies the 3' half-site (140). However, this orientation not always results in inactivity, since the PPAR/RXR heterodimer activates transcription by binding to DR1 elements with the same polarity (68).

A two-step model for heterodimeric binding to DNA has been proposed. First, RXR would form heterodimers in solution with its partner through their dimerization interfaces contained in the LBDs, and in a second step, the DBDs would be able to bind with affinity to the DNA (163). The ability of heterodimeric receptors to bind to palindromes, IPs, and DR elements implies that the DBDs must be rotationally flexible with respect to the LBD dimerization interface (see Fig. 5). In contrast to the head-to-head arrangement of steroid receptor DBDs on DNA, the X-ray structure of the RXR/TR heterodimer reveals a polar head-to-tail assembly of the two proteins on a DR4, with RXR indeed occupying the upstream motif (212). Selective binding of heterodimers to their appropriate DRs appears to be a consequence of a cooperative dimer interaction within the DBDs. In a DR, a different region of the DBD of each receptor is used to create the dimerization interface. The heterodimeric DBD interface that is responsible for the cooperative binding of RXR/RAR to DR5 elements involves the D box of RXR and the tip of the RAR first zinc finger (311). Similar interfaces would be used for binding of RXR/TR to a DR4 (201). A second type of dimerization surface, which specifically implicates the RAR T box and the second zinc finger of RXR, determines selective binding of RXR/RAR to DR2 elements (312). The same type of dimerization interface (RXR T box and second zinc finger) is responsible for the cooperative binding of RXR homodimers to DR1 elements. In all cases the DBD contributing the second zinc finger has to be positioned 5' to its cooperatively bound partner resulting in polarity of the heterodimer. In the case of RAR/RXR bound to the DR1 element with the reverse polarity, the heterodimeric partners associate in a DNA-dependent manner using the T box of RXR and the second zinc finger of RAR. The protein-DNA contacts, the dimerization interface, and the DNA curvature in the RAR/RXR complex are different from those of the RXR homodimer bound to the same element (213).

View larger version (43K): [in this window] [in a new window]   Fig. 5. Binding of receptors to the hormone response elements (HREs). Receptors can bind as monomers, homodimers, or RXR heterodimers to DNA. Dimerization is mediated by a strong dimerization interface (composed of hydrophobic heptad repeats) present in the LBD, and cooperative binding of receptor dimers is facilitated by a DNA-dependent interface that forms between the DBDs. Steroid receptors bind as homodimers to palindromic elements spaced by three nucleotides in a symmetrical way. Monomeric binding requires the half-core motif preceded by a 5'-flanking A/T-rich sequence. Heterodimers can recognize diverse HREs in which half-core motifs can be arranged as palindromes (Pal), direct repeats (DRs), or inverted palindromes (IPs). The ability of binding to these different motifs implies that the DBDs can rotate with respect to the LBDs that are held together through the dimerization interface.

Theoretically, four different states of heterodimer occupancy can be predicted: both receptors unoccupied, only RXR occupied, only the partner receptor occupied, and both receptors occupied. However, three types of heterodimeric complexes exist: unoccupied heterodimers, nonpermissive heterodimers that can be activated only by the partner's ligand but not by an RXR ligand alone (77, 140), and permissive heterodimers that can be activated by ligands of either RXR or its partner receptor and are synergistically activated in the presence of both ligands (117, 135, 287) (Fig. 6). Nonpermissive heterodimers include RXR/TR, RXR/VDR, or RXR/RAR heterodimers. In the nonpermissive heterodimers the ligand-induced transcriptional activities for RXR are suppressed when complexed with VDR, TR, and RAR, and the formation of the heterodimer actually precludes binding of ligand to RXR. Thus, in these instances, RXR is said to be a "silent partner." However, in the case of RXR/RAR, although a RXR ligand alone cannot activate the heterodimer, binding of the RAR ligand allows the subsequent binding of the ligand of RXR, that then enhances the transcriptional response to the RAR ligand (174).

View larger version (25K): [in this window] [in a new window]   Fig. 6. Permissive and nonpermissive heterodimers. In nonpermissive heterodimers, such as RXR/RAR, heterodimerization precludes binding of the RXR ligand. Binding of ligand to the RAR moiety causes receptor activation and allows binding of the RXR ligand resulting in synergism. Permissive heterodimers, such as PPAR/RXR, can be activated by ligands of either RXR or its partner receptor and are synergistically activated in the presence of both ligands.

PPAR/RXR, FXR/RXR, or NGFI-B/RXR are permissive heterodimers. The LXR-RXR complex also belongs to the class of permissive heterodimers as demonstrated by the finding that an RXR ligand stimulates the transcriptional activity of the heterodimer (286, 287). However, RXR occupies the upstream half-site of the HRE, a polarity that inhibits binding of the ligand to RXR in other heterodimers, demonstrating that permissivity does not depend exclusively on the polarity of the heterodimer. Interestingly, stimulation by 9-cis-retinoic acid requires the LXR but not the RXR AF-2 domain, demonstrating that binding of the RXR ligand results in a conformational change in LXR that leads to transcriptional activation. This phenomenon has been referred to as the "phantom ligand" effect (286). A synthetic retinoid specific for RXR also behaves as a phantom ligand and mimics exactly the effects of an RAR ligand without occupying the RAR ligand binding pocket (238).

The ligands could play a role in dimerization and binding to DNA. For instance, thyroid hormone inhibits binding of homodimers but not heterodimers to DNA, thus promoting formation of heterodimeric complexes on the HRE (219). In contrast 9-cis-retinoic acid in some instances can increase binding of RXR homodimers to a DR1 (318), which can lead to unavailability for heterodimer formation with other receptors and to decreased levels of transcription for genes depending on heterodimers.

The above-mentioned observations demonstrate that RXR plays a dual role in nuclear receptor signaling. On one hand, this receptor binds to a DR1 as a homodimer and activates transcription in response to 9-cis-retinoic acid (164), and on the other hand serves as a heterodimer partner for other nuclear receptors. Experiments with knock-out mice have clearly shown that the RXR/RAR heterodimer is responsible for different biological effects of retinoids on development (129, 130). However, there is still no evidence of a role for RXR in signaling by other heterodimeric receptors and, in fact, double TR-RXR knock-outs do not have a stronger phenotype than that shown by the TR knock-out alone (14).

	III. TRANSACTIVATION AND TRANSREPRESSION

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Although most of the attention has been focused on transcriptional activation by binding of nuclear receptors to positive HREs, nuclear receptors can also repress gene expression in a ligand-dependent manner. In some cases repressive effects may be due to passive inhibition, which can occur due to competition for DNA sites with other transactivators or to formation of transcriptionally inactive heterodimers. However, there are also the so-called "negative HREs" that bind the receptors and mediate negative regulation by the ligand. These elements have been identified for glucocorticoids in the proopiomelanocortin gene (POMC) and for thyroid hormones in the thyrotropin (TSH) and thyrotropin-releasing hormone (TRH) genes, indicating an important role of these sites in feedback mechanisms in the pituitary (28, 38, 70, 71, 106). In the case of the TRs, several other negative elements have been identified. Some of these elements have been shown to preferentially bind a TR homodimer in the absence of hormone, and a RXR/TR heterodimer in the presence of 3,3',5-triiodothyronine (T₃). However, other negative HREs essentially bind only heterodimers both in the presence and absence of ligand. A rather common finding is that on negative HREs the unoccupied receptor increases transcription and the ligand reverses this stimulation. Although at the present time the properties of the negative HREs are not totally understood, location of the element may a play a role. Negative HREs are generally very close to the transcription initiation sites, and some are positioned downstream of the TATA box (20, 199, 226) or even have an unusual location at the 3'-untranslated region (24). This sequence can have properties that depend on its localization, exhibiting negative responses only when placed downstream of the transcription initiation site, suggesting that the HRE could affect the transcriptional activity of the target gene by regulating the rate of release of RNA polymerase II from the promoter.

In addition to ligand-dependent gene activation and inhibition, a subset of nuclear receptors represses basal transcription in the absence of ligand when bound to a positive HRE. This silencing activity is due to the binding of corepressors to the unliganded receptors and is reviewed in detail in section VI.

Although as described in section IIC, the orientation and spacing of the half-sites can determine selective transcriptional responses to nuclear receptors, specificity is not total, and some HREs can bind different heterodimers with high affinity. However, only a subset of receptor DNA binding elements function as response elements. As described above, the heterodimer RAR/RXR binds to a DR1 in a transcriptionally inactive form and antagonizes the response mediated by the active RXR homodimers (140). Equally, VDR/RXR can bind retinoic acid and thyroid hormone response elements in a transcriptionally inactive form, and under these circumstances vitamin D can inhibit the response to those ligands (81, 120). However, although competition for DNA binding by transcriptionally inactive VDR/RXR heterodimers may contribute to this inhibitory response, mutants lacking the A/B domain and the DNA-binding domain also display a dominant negative activity, suggesting that titration of coactivators could be involved in the inhibitory effect of vitamin D (121). Similarly, mutant or truncated transcriptionally inactive receptors in some syndromes of hormone resistance can compete binding of wild-type receptors to DNA, presenting a dominant-negative activity and reducing hormone-mediated transcriptional responses.

In the case of heterodimeric receptors, competition for limiting concentrations of RXR may also represent a mechanism for modulating transcriptional responses to several partner receptors (12). Thus COUPs can act as transcriptional repressors antagonizing activation mediated by different receptors, and this antagonism may involve competition for DNA binding sites, competition for RXR, and formation of inactive complexes with other receptors (269). An unusual receptor, the small heterodimer partner (SHP), lacks a typical DBD and can heterodimerize with different nuclear receptors leading to inhibition of binding to DNA and transcriptional inactivation (122, 240, 241).

Nuclear receptors can also modulate gene expression by mechanisms independent of binding to an HRE. Thus they can alter expression of genes that do not contain an HRE through positive or negative interference with the activity of other transcription factors, a mechanism generally referred to as "transcriptional cross-talk" (90). The ERs utilize protein-protein interactions to enhance transcription of genes that contain AP-1 sites (83). The AP-1 complex that is composed of dimers of Jun family proteins and preferently of Jun/Fos heterodimers plays an important role in cell proliferation. ER and ER have been shown to signal in opposite ways at AP-1 sites. ER activates transcription in the presence of estradiol, whereas with ER estradiol inhibits AP-1-dependent transcription. Furthermore, antiestrogens can act as agonists of ER action at AP-1 sites. This is particularly evident in the case of ER, which enhances AP-1-dependent transcription in the presence of antiestrogens but not estrogens (194).

One of the best known examples of the cross-talk between nuclear receptors and AP-1 complexes is the finding that several receptors, such as TR, RAR, or GR, can act as ligand-dependent transrepressors of AP-1 (Jun/Fos) activity, and reciprocally, that AP1 can inhibit transactivation by nuclear receptors (203). It is believed that many of the antiproliferative effects of ligands of nuclear receptors could be mediated by their anti-AP-1 activity. Similarly, some nuclear receptors, specifically GR, can also mutually interfere with NF-B activity, which could be involved in the anti-inflammatory and immunosuppressive effects of glucocorticoids.

In some cases the cross-talk between the receptors and AP-1 can involve binding to a "composite element" that can bind both the receptors and the AP-1 complex, and depending on the composition of the AP-1 complexes they can either cooperate or antagonize transcription by nuclear receptors (for a review see Ref. 203). However, the receptors can negatively regulate target gene promoters that carry AP-1, NF-B, or CREB binding sites, without binding to these DNA elements themselves. It was originally proposed that the receptors directly contact the basic leucine zipper region of c-Jun or the rel homology domain of the p65 subunit of NF-B and that this interaction inhibits binding to their corresponding cognate sites (291). However, more recent evidence suggested that competition for common transcriptional mediators could be involved in the antagonism observed (90; see also sect. VC). Additional mechanisms have been suggested, including an induction of the Ik-B factor that sequesters NF-B in the cytoplasm (5), or an inhibition of the Jun-NH₂-terminal kinase (JNK) activity by the receptors that would prevent phosphorylation of c-Jun (35).

A most interesting finding is that receptor-mediated transactivation and transrepression can be separated: mutations that impair transactivation, retain their ability to antagonize AP-1 or NF-B activity. Interestingly, it has been possible to generate synthetic ligands of GR and retinoid receptors that dissociate transactivation from transrepression (217). These ligands are largely devoid of the ability to activate target genes containing HREs, but they retain in vivo anti-inflammatory or antiproliferative activity (53, 159, 275). These "dissociated" ligands have a large potential as pharmacological tools in the treatment of a variety of diseases including cancer and inflammatory diseases.

That transrepression plays a very important role in vivo has been demonstrated in a "knock-in" mouse in which the wild-type glucocorticoid receptor has been replaced by a mutant receptor containing a substitution in the DBD that results in a dimerization-defective receptor (GR^dim). This mutation allows transrepression, but the mutant receptor no longer binds with high affinity to the glucocorticoid response element. Whereas GR "knock-out" mice die at birth as a result of a failure in lung maturation, the GR^dim survives despite impairment of several physiological functions of glucocorticoids (214).

The cross-talk between nuclear receptors and other signaling pathways is not restricted to the transcriptional antagonism described above (198). Phosphorylation of nuclear receptors provides an important link between signaling pathways. As already stated in section IIA, multiple kinases activated by extracellular signals that bind to surface receptors, including for instance MAPKs, cell cycle-dependent kinases (CDKs), casein kinase, and protein kinase A, affect receptor activity through phosphorylation events (243). Depending on the receptor and in the residue involved, in some cases phosphorylation can inhibit ligand-dependent activation by nuclear receptors due to a reduction in ligand binding or in DNA binding affinity. However, in other cases, the receptors can be activated in the absence of its cognate ligand by phosphorylation through signals originated in membrane receptors.

Contrary to the antiproliferative effects of some nuclear receptor ligands, ovarian hormones stimulate growth of breast cancer cells. It has been reported that estrogens activate the Src/Ras/MAPK signal transduction pathway and that this cross-talk could be crucial for their growth-promoting effect in these cells. MAPK activation occurs very rapidly and is receptor mediated, but appears to represent a nongenomic action of the steroid (172). A direct interaction of ER with c-Src could be involved in this phenomenon, and the progesterone receptor (PR) that does not interacts with c-Src can activate this pathway by association with ER (173).

A novel mechanism of cross-talk between nuclear receptors, specifically VDR, and transforming growth factor- (TGF-) has been recently reported (300). Smad3, one of the proteins downstream in the TGF- signaling pathway, was found to act as a coactivator for VDR by forming a complex with a nuclear receptor coactivator. These interactions are potentially important in the control of cell proliferation and differentiation by vitamin D and the growth factor.

	IV. RECEPTOR-INTERACTING PROTEINS

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Promoters transcribed by RNA polymerase II are recognized by two types of transcription factors: the basal or general transcription factors (GTFs) that interact with the core promoter elements, and the sequence-specific transcription factors, among which nuclear receptors are included, which generally interact with sequences located further upstream. The core promoter may contain the TATA box close to an initiator sequence that spans the transcriptional start site where the RNA polymerase II binds. Most of the factors involved in formation of the transcriptional initiation complex have been characterized. In addition to RNA polymerase II (which is composed of at least 12 subunits), these include TFIID, TFIIB, TFIIA, TFIIF, TFIIE, and TFIIH (for a review see Ref. 288).

TFIID, whose binding to the promoter is thought to be a rate-limiting step in transcriptional initiation, is composed of TBP (or TATA binding protein) and TBP-associated factors (TAF_IIs) forming several complexes (18, 224). TBP is a highly conserved protein that binds to the minor groove of DNA over the TATA region causing a drastic bend of DNA and also contacts the largest subunit of RNA polymerase II. TFIID is comprised of at least two different subpopulations, one containing TAF_II250, TAF_II135, TAF_II100, and TAF_II28, present in all TFIID complexes, and the other containing additional TAFs, such as TAF_II30, TAF_II20, or TAF_II18. After TFIID binding to DNA, recruitment of TFIIB is a critical step in the formation of the preinitiation complex. TFIIB contacts DNA upstream and downstream of the TATA box on the concave side of the bend induced by TFIID binding. It was formerly believed that the preinitiation complex was assembled in an ordered fashion, with binding of TFIID to the TATA box followed by sequential binding of TFIIB, the polymerase, and other factors. An alternative to the sequential recruitment of individual GTFs is the existence of performed complexes, including the RNA polymerase II and GTFs, that could be directly recruited to the promoter by sequence-specific transcription factors. These complexes, that contain RNA polymerase II, TFIIB, TFIIH, TFIIF, SRBs (supressor of RNA polymerase B), and several other proteins, have been isolated from yeast and mammalian cells and are termed the holoenzyme (45, 92). The current hypothesis is that transcription factors will finally cause their effect on gene expression by influencing the rate of assembly of these complexes to the regulated promoter.

As with other transcriptional regulatory proteins, one aspect of the mechanisms by which nuclear receptors affect the rate of RNA polymerase II-directed transcription likely involves the interaction of receptors with components of the transcription preinitiation complex. This interaction may be direct, or it may occur indirectly through the action of coregulators (coactivators and corepressors, see sects. V and VI) which act as bridging factors. Nuclear receptors seem to be able to interact with several components of the general transcriptional machinery. It has been shown that TBP can interact with several receptors and that overexpression of TBP enhances ligand-dependent transactivation in transfection assays (22, 227, 237). TAF_IIs have been also identified as potential targets for hormone receptors. Thus TAF_II30 is required for transactivation by ER (116), whereas expression of TAF_II135 strongly potentiates transcriptional stimulation by RAR, TR, or VDR, but does not affect the responses to ER or RXR (171). Therefore, TAF_IIs can act as coactivators of nuclear receptors. An interaction with TFIIB has been well documented for TR and VDR as well as for other receptors (8, 27, 167).

Although the functionality of direct protein to protein interactions of receptors with the basal transcriptional machinery is yet to be determined, it is likely that these interactions could cause the recruitment of the basal components to the promoter and the enhancement of transcription.

In natural promoters HREs are located close to recognition sequences for other transcription factors, and interaction between the receptors and these factors, which can result in functional synergism or repression, can play an important role in determining transcriptional rates. Early observations demonstrated that HREs can synergize with many different transcription factors in artificial promoters (236). In some cases HREs have been shown to be dependent on cooperative interactions with adjacent transcription factors. Such interactions may serve to restrict a hormonal response to cell types that express the appropriate set of transcription factors. Expression of pituitary genes appears to be a good example of these interactions. Transcription of the growth hormone and prolactin genes is stimulated by a number of ligands for nuclear receptors, and this stimulation requires binding of the pituitary-specific homeodomain factor GHF-1/Pit-1 to its recognition sites in the promoters (40, 55, 65, 262). A direct protein to protein interaction between the receptors and these factors appears to be involved in this synergism (40, 195). Similarly, on the mouse mammary tumor virus (MMTV) promoter, the transcription factors NF-1 and Oct-1 are required for a normal induction by glucocorticoids or progesterone, and a direct interaction of GR and PR with Oct-1 has been described (31).

	V. NUCLEAR RECEPTOR COACTIVATORS

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Early studies suggested that the most COOH-terminal part of nuclear receptors, termed the AF-2 domain (13, 62, 72, 281), was involved in ligand-dependent transactivation "in vivo," and mutation analysis has shown that this region is also involved in transcriptional interference. This domain possesses a high homology over a very short region from which the consensus motif XE ( being a hydrophobic amino acid) can be derived, preceded by a loop of length varying from 8 to 12 amino acids that is variable in sequence and composition. The region comprising the conserved sequence adopts an amphipathic -helical conformation with the two well-conserved pairs of hydrophobic residues pointing toward the core of the LDB and negatively charged residues exposed on its surface (313). This motif is conserved in most members of the nuclear receptor superfamily, with the exception that it is absent in Rev-erbA and the viral oncogene v-erbA, contains a conservative substitution of aspartic for glutamic acid in COUP-TF, and a positive charged amino acid substitutes for the highly conserved central glutamic acid residue in NGFI-B. This residue is important for transactivation but is not required for ligand binding, and its mutation in different receptors generates dominant-negative mutants that are transcriptionally silent (72). Remarkably, one of the "hot spots" for mutations in the TR gene that cause the syndrome of generalized resistance to thyroid hormone, maps to the COOH-terminal region (46, 137), and mutations in this region of PPAR are associated with severe insulin resistance, diabetes mellitus, and hypertension (15).

Although the COOH-terminal region, that is located in helix 12 of the LBD, contains the core AF-2 activity, this domain comprises other dispersed elements brought together upon ligand binding. One such element is a region whose sequence is also extremely well conserved. This region, which has been called the nuclear receptor "signature motif," encompasses the COOH-terminal half of helix 3, helix 4, and the loop between them. Mutations in this region affect neither ligand binding nor dimerization, but impair ligand-dependent transactivation. Specifically, a highly conserved lysine in the COOH terminus of helix 3 that is exposed to the solvent in the receptor crystals is important for transcriptional activity of several receptors (104, 119). Furthermore, natural mutations in the signature region have been identified in patients with androgen insensitivity syndrome (208) and also in thyroid hormone-resistant patients (57). Crystal structure of nuclear receptors has provided an explanation for the importance of the COOH-terminal AF-2 domain and this residue in ligand-dependent transactivation. The most striking difference observed in the receptors upon ligand binding is the position of helix 12, which contains the core AF-2 domain. Helix 12 projects away from the body of the LBD in unliganded RXR (29). However, in liganded receptors, this helix moves in a "mouse-trap" model being tightly packed agains helix 3 or 4 and making direct contacts with the ligand (216, 280) (see Fig. 4). Because both the charged residues in helix 12 and residues in the signature region, including the lysine residue in helix 3, are contiguous and exposed in the surface of the LBD, they probably generate a hydrophilic surface responsible for coactivator interactions (178). Reinforcing this model, it has been recently demonstrated that in ER LBD bound to the antagonists raloxifen or dihydroxytamoxifen the position of helix 12 is different from that shown by the agonist-bound LBD (32, 245). In the antagonist-bound receptor, helix 12 is rotated and shifted with respect to its position when bound to estrogen. As a result, helix 12 lies in a groove formed by helix 5 and the COOH-terminal end of helix 3. This position overlaps with the surface of coactivator interaction, thus precluding coactivator binding and consequently transcriptional activity.

Initial biochemical studies demonstrated that several proteins interact with the nuclear receptors. The most abundant of these were proteins of a molecular mass of 140 and 160 kDa (p140 and p160) designated as ER-associated proteins (ERAPS) (98), receptor-interacting proteins (RIPs) (41, 42), glucocorticoid receptor interacting proteins (GRIPs) (74), or TR-associated proteins (TRAPs) (75). A potential role for these proteins as coactivators for the nuclear receptors was suggested by the ligand dependence for their interaction with the receptors and by the finding that they failed to interact with transcriptionally inactive receptor mutants or with antagonist-bound receptors. Different cloning strategies have led to the identification of numerous receptor-interacting proteins. Some of them have been demonstrated to play a role as bona fide receptor coactivators, whereas others could play different roles in modulating nuclear receptor function (for recent reviews, see Refs. 88, 89, 169, 220, 264, 297). To date, the following families of coactivators have been characterized.

The first coactivator, identified using a yeast two-hybrid screen of a human B-lymphocyte library using PR as bait, was SRC-1 (192). This protein interacts with the receptor in an agonist and AF-2-dependent manner and acts as a prototypic coactivator for different nuclear receptors including other steroid receptors such as GR or ER, and nonsteroid group II receptors such as VDR, PPAR, TR, or RXR, stimulating the transcriptional activity of the corresponding ligands both in mammalian cells and in yeasts. In parallel studies, the mouse homolog of SRC-1 was identified by screening bacteriophage-based expression libraries with the LBD of ER in the presence of estrogen and was denominated NCoA-1 (265). This protein was highly related to human SRC-1 at the COOH terminus but encoded an extended NH₂ terminus, suggesting that the initially identified SRC-1 was either a partial clone or a splice variant of the full-length protein.

Immunoprecipitation experiments showed that SRC-1/NCoA-1 only accounted partially for the p160 proteins, suggesting that other coactivators with the same size might also exist. This was demonstrated by the cloning of a second set of p160 coactivators (SRC-2), termed TIF-2 in humans (278) and GRIP-1 or NCoA-2 in mice (107, 265). Truncated versions of these proteins exhibit dominant negative activity and can inhibit ligand and coactivator responses (108, 118). Both types of coactivators share not only considerable sequence similarity, but also many functional characteristics. Apart from interacting with various receptors and enhancing ligand-dependent transcriptional responses, they are also capable of relieving squelching, showing that they constitute common limiting factors recruited by the liganded receptors (278). Loss of function studies using microinjected antibodies against the coactivators also suggest that they are required for nuclear receptors function. Furthermore, these coactivators contain two major transactivation domains that retain their activity when fused with the DBD of the yeast GAL4 activator.

A third member of the p160 family of proteins was subsequently characterized. It was independently isolated as p/CIP in mice (265) and ACTR, AIB1, RAC3, or TRAM-1 in humans (4, 48, 152, 257). This coactivator has been generically named SRC-3. Although many properties of this coactivator are similar to those of the other p160 proteins, a major difference is that it also enhances the transcriptional activity of a number of different transcription factors including signal transducers and activators of transcription (STAT-1) and cAMP response element binding protein (CREB) (265). It should be noted that although SRC-1 was initially considered as a nuclear receptor-specific coactivator, more recently it has been demonstrated that it can also function as a coactivator for NF-B, serum response factor, or p53 (134, 149) and that it is even required for muscle cell differentiation mediated by the helix-loop-helix transcription factor MEF-2 (54).

The three members of the p160 family of coactivators show a sequence similarity of 40%. Conservation is maximal in their NH₂-terminal domains that contain the nuclear localization signal, and bHLH and PAS domains. These domains mediate protein to protein homo- and heterodimeric interactions, suggesting that these coactivators could interact with other PAS proteins. A serine/threonine-rich region and a COOH-terminal glutamine-rich region are also well conserved in these coactivators, which contain three nuclear receptor-interacting domains (see sect. VD) in their central region. Both activation domains are also located at the COOH terminus. The stronger transactivation domain is indistinguishable from the region of interaction with the cointegrator CREB binding protein (CBP), and a weaker transactivation domain located in the far COOH terminus of the coactivators has been recently shown to interact with an arginine methyltransferase (47, 97). The p160 coactivators possess histone acetyltransferase activity that maps also to the COOH-terminal region (48, 253). A diagram of the structure of a p160 coactivator is shown in Figure 7.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Domains of p160 and CBP/p300 families of receptor coactivators. General features based on sequence homology. p160 coactivators contain a basic helix-loop-helix (bHLH) motif and a Per-Arnt-Sim (PAS) homology region at the NH2 terminus. The nuclear receptor interacting domain (RID) contains three LXXLL motifs indicated by asterisks. Two activation domains (AD1 and AD2) with an intervening glutamine-rich (Q) region are located at the COOH terminus. Below the sequence, brackets refer to the functional region histone acetyltransferase (HAT) activity and to the regions of interaction with CBP/p300, the HAT PCAF or the arginine methyltransferase CARM1. In CBP/p300 the RID, indicated by an asterisk, is located in the NH2 terminus. CBP/p300 also contains the KIX domain (of interaction with CREB), and a bromodomain (Br) as well as three zinc finger regions (C/H1, C/H2, and C/H3) of interaction with various transcription factors and components of the basal transcriptional machinery. The HAT domain, as well as the regions of interaction with PCAF and p160 coactivators are also indicated.

A possible application derived from the ligand-dependent recruitment of coactivators by the receptors is the identification of new ligands. An assay termed coactivator-dependent receptor ligand assay (CARLA) using SRC-1 has served for the identification of naturally occurring fatty acids and metabolites as well as hypolipidemic drugs as bona fide ligands for PPARs (139). This technique, which only identifies agonist ligands, is also applicable to the identification of ligands for orphan receptors.

PPAR coactivator-1 (PGC-1), which was isolated in a yeast two-hybrid screen using a PPAR fragment as the bait and a brown fat cDNA library, was demonstrated to interact with this receptor, as well as with other members of the nuclear receptor superfamily. PGC-1 is a coactivator that plays a major role in the regulation of adaptive thermogenesis, an important component of energy homeostasis (207). PGC-1 mRNA expression is dramatically elevated upon cold exposure of mice in both brown fat and skeletal muscle, two key thermogenic tissues. PGC-1 greatly increases the transcriptional activity of PPAR and the thyroid hormone receptor on the uncoupling protein (UCP-1) promoter. PGC-1 also stimulates mitochondrial biogenesis and respiration in muscle cells through an induction of uncoupling protein 2 (UCP-2) and through regulation of the nuclear respiratory factors (NRFs), which are transcription factors that regulate genes involved in mitochondrial DNA replication and transcription (293). PGC-1 has been shown to have a low inherent transcriptional activity when it is not bound to a transcription factor. The docking of PGC-1 to PPAR stimulates an apparent conformational change in PGC-1 that permits binding of SRC-1 and the cointegrator CBP/p300, resulting in a large increase in transcriptional activity. Thus transcription factor docking can serve to switch on the activity of coactivators (206).

A surprising finding has been the identification of an RNA coactivator for steroid receptors (144). This RNA, denominated SAR, works exclusively through the NH₂-terminal AF-1 domain and can be detected in a large complex of 600-700 kDa which contains several proteins and specifically SRC-1. It has been suggested that SAR might serve as part of a ribonucleoprotein scaffold through which SRC-1 is recruited, and whether or not this RNA could possess intrinsic catalytic activity is still unknown.

To date, many other proteins have been demonstrated to enhance transactivation by nuclear receptors. A list of these proteins with a description of their characteristics can be found in excellent recent reviews (169, 220) and references therein. Some of these proteins such as E6-AP, ARA70, NCoA62, or NRIF3 interact with the receptors in a ligand-dependent manner and require the AF-2 domain. However, other coregulators, including p68, PGC-1, or PGC-2 interact with the AF-1 domain. Other coactivators such as TLS, Trip-1/Sug-1, or TSC-2 could be involved in protein degradation pathways, RNA stability, or nuclear transport. Future studies will surely clarify the role of each protein in transcriptional regulation by nuclear receptors. Recent studies also suggest that cell-specific coactivators may play an important role in gene-specific transcriptional activation. In addition, some coactivators exhibit a relative preference for a determined group of nuclear receptors. For instance, ARA70 specifically enhances androgen receptor transcriptional responses (304), and FHL2, which has a unique tissue-specific expression pattern, selectively increases the transcriptional activity of this receptor, but not that of other nuclear receptors, in an agonist- and AF-2-dependent manner (180).

C.  Cointegrators

1.  CBP/p300

CBP and p300 are large evolutionary conserved proteins that serve coactivator roles for different types of transcription factors. CBP was originally identified on the basis of its association with CREB in response to cAMP-mediated phosphorylation (142), and the highly related protein p300 was isolated by its interaction with the viral E1A protein (73). Further studies have demonstrated that CBP/p300 interacts with a large variety of transcription factors including AP-1, myoD, Jun, Fos, NF-B, Pit-1, STATs, and Ets and serves a coactivator role for these factors potentiating their transcriptional activity (247). The finding that these proteins can function as coactivators for different transcription factors has led to the notion that they serve as cointegrators of extracellular and intracellular signaling pathways. CBP/p300 also interacts with TBP, TFIIB, or YY1 and might serve to link the receptors to the basal transcriptional machinery.

In vitro studies, coimmunoprecipitation experiments, and yeast and mammalian two-hybrid assays have also demonstrated an interaction of different nuclear receptors with CBP. This interaction is ligand dependent and AF-2 dependent, and CBP/p300 appears to function as an essential coactivator for the receptors (43, 126). This conclusion comes from the observations that overexpression of CBP/p300 potentiates ligand-dependent transcriptional activation by different nuclear receptors and that, more importantly, microinjection of anti-CBP antibodies blocks ligand-dependent activation by GR, RAR, and RXR. In addition, retinoic acid-dependent transcription is markedly blunted in fibroblasts from p300 knock-out mice (303), supporting the notion that CBP/p300 are key components of hormonal regulation of transcription in vivo.

The interaction between CBP and the receptors maps to the NH₂ terminus of the coactivator (126) (Fig. 7). CBP/p300 contains several other functional domains, including the CREB interaction domain (KIX) to which other transcription factors such as Jun or Myb also associate, and the three zinc finger regions (C/H1, C/H2, and CH/3) that bind many other factors. The histone acetylase PCAF, an ortholog of yeast GCN5, also associates with the CH/3 region. A bromodomain is present between KIX and the second zinc finger, and a domain exhibiting intrinsic histone acetyltransferase (HAT) activity is found between the bromodomain and the third zinc finger (11, 190). Removal or mutation of the HAT domain results in loss of function for many transcription factors, indicating the importance of this activity.

CBP/p300 not only directly binds the nuclear receptors, but also associates with the p160 family of coactivators through a different, COOH-terminal, region (126, 265, 277). This interaction has been identified both in vivo and in vitro and provides the receptors with two different ways of interacting with CBP/p300, one through a direct interacion with the NH₂-terminal domain, and other through interaction with the p160 coactivators. As different regions of CBP are involved in interaction with receptors and coactivators, it is possible that they may form a ternary complex. That these complexes are indeed formed in the cells is suggested by the finding that CBP synergizes with SRC-1 in PR- and ER-mediated transactivation (249). As in the case of CBP, microinjection of anti-p/CIP antibodies blocks ligand-dependent activation by different receptors, and activity can only be restored when both p/CIP and CBP expression vectors are coinjected (265). These results, as well as coimmunoprecipitation experiments demonstrating that a significant portion of endogenous CBP/p300 associates with p/CIP, also suggest that they form a functional complex.

ACTR also interacts with PCAF (26, 138), the first identified mammalian HAT. Thus ACTR might independently interact with both CBP/p300 and their associated factor PCAF, serving as a docking platform to bridge this protein complex to DNA-bound nuclear receptors. There is also evidence that PCAF is a nuclear receptor coactivator. It has been shown that retinoid receptors directly recruit PCAF from mammalian cell extracts in a ligand-dependent manner and that increased expression of PCAF leads to enhanced retinoid-dependent transcription. Direct interaction of PCAF with multiple receptors suggests that its recruitment may be a universal property of nuclear receptors. In contrast to CBP/p300 and the p160 coactivators which require the AF-2 receptor domain for binding, the receptor DBD has a critical role in binding to PCAF, although other regions of the receptors may have auxiliary roles. PCAF binds directly and independently to both nuclear receptors (26, 138) and CBP (302). These interactions may occur sequentially with PCAF first associating with receptors followed by interaction with CBP/p300. Ligand binding may stimulate independent recruitment of both coactivators to the receptor dimers, which is then followed by a cooperative multipoint interaction between these molecules (26). It has also