The pathophysiology of obesity and type 2 diabetes mellitus is associated with abnormalities in endocrine signaling in adipose tissue and one of the key signaling affectors operative in these disorders is the nuclear hormone transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma). PPARgamma has pleiotropic functions affecting a wide range of fundamental biological processes including the regulation of genes that modulate insulin sensitivity, adipocyte differentiation, inflammation and atherosclerosis. To date, only a limited number of direct targets for PPARgamma have been identified through research using the well established pre-adipogenic cell line, 3T3-L1. In order to obtain a genome-wide view of PPARgamma binding sites, the pair end-tagging technology (ChIP-PET) was employed to map PPARgamma binding sites in 3T3-L1 preadipocyte cells. Coupling gene expression profile analysis with ChIP-PET, in a genome-wide manner, over 7700 DNA binding sites of the transcription factor PPARgamma and its heterodimeric partner RXR were identified during the course of adipocyte differentiation. The validation studies prove that the identified sites are bona fide binding sites for both PPARgamma and RXR and that they are functionally capable of driving PPARgamma specific transcription. The results strongly indicate that PPARgamma is the predominant heterodimerization partner for RXR during late stages of adipocyte differentiation. Additionally, PPARgamma/RXR association is enriched within the proximity of the 5' region of the transcription start site and this association is significantly associated with transcriptional up-regulation of genes involved in fatty acid and lipid metabolism confirming the role of PPARgamma as the master transcriptional regulator of adipogenesis. Evolutionary conservation analysis of these binding sites is greater when adjacent to up-regulated genes than down-regulated genes, suggesting the primordial function of PPARgamma/RXR is in the induction of genes. Functional validations resulted in identifying novel PPARgamma direct targets that have not been previously reported to promote adipogenic differentiation. In conclusion, the binding sites of PPARgamma and RXR during the course of adipogenic differentiation were identifed in 3T3L1 cells, and an important resource is provided for the study of PPARgamma function in the context of adipocyte differentiation (Hamza, 2009).

Mammalian RXR - Domain structure and function

A subset of nuclear receptors, including those for thyroid hormone (TR), retinoic acid, vitamin D3, and eicosanoids, can form heterodimers with the retinoid X receptor (RXR) on DNA regulatory elements in the absence of their cognate ligands. In a mammalian two-hybrid assay, recruitment of a VP16-RXR chimera by a Gal4-TRbeta ligand-binding domain fusion is enhanced up to 50-fold by thyroid hormone (T3). This is also observed with a mutant fusion, Gal4-TR(L454A), lacking ligand-inducible activation function (AF-2) and unable to interact with putative coactivators, suggesting that the AF-2 activity of TR or intermediary cofactors is not involved in this effect. The wild-type and mutant Gal4-TR fusions also exhibit hormone-dependent recruitment of RXR in yeast. Hormone-dependent recruitment of RXR is also evident with another Gal4-TR mutant, AHTm, which does not interact with the nuclear receptor corepressor N-CoR, suggesting that ligand-enhanced dimerization is not a result of T3-induced corepressor release. The interaction between RXR and TR is augmented by T3 in vitro, arguing against altered expression of either partner in vivo mediating this effect. It is proposed that ligand-dependent heterodimerization of TR and RXR in solution may provide a further level of control in nuclear receptor signaling (Collingwood, 1997).

The receptor for 9-cis-retinoic acid, retinoid X receptor (RXR), forms heterodimers with several nuclear receptors, including the receptor for all-trans-retinoic acid, RAR. Previous studies have shown that retinoic acid receptor can be activated in RAR/RXR heterodimers, whereas RXR is believed to be a silent co-factor. Efficient growth arrest and differentiation of the human monocytic cell line U-937 require activation of both RAR and RXR. The allosteric inhibition of RXR is not obligatory; RXR can be activated in the RAR/RXR heterodimer in the presence of RAR ligands. Remarkably, RXR inhibition by RAR can also be relieved by an RAR antagonist. Moreover, the dose response of RXR agonists differs between RXR homodimers and RAR/RXR heterodimers, indicating that these complexes are pharmacologically distinct. The AF2 activation domain of both subunits contributes to activation even if only one of the receptors is associated with ligand. These data emphasize the importance of signaling through both subunits of a heterodimer in the physiological response to retinoids and show that the activity of RXR is dependent on both the identity and the ligand binding state of its partner (Botling, 1997).

Thyroid hormone receptors (TR) function as part of multiprotein complexes that also include retinoid X receptor (RXR) and transcriptional coregulators. Both the Thyroid receptor CoR box and ninth heptad domains are required for interaction with RXR, and in turn, both domains are required for interaction with corepressor proteins N-CoR and SMRT. Remarkably, the recruitment of RXR to the repression-defective CoR box and ninth-heptad mutants via a heterologous dimerization interface restores both corepressor interaction and repression. The addition of thyroid hormone obviates the CoR box requirement for RXR interaction, provided that the AF2 activation helix at the C terminus of TR is intact. These results indicate that RXR differentially recognizes the unliganded and liganded conformations of TR and that these differences appear to play a major role in the recruitment of corepressors to TR-RXR heterodimers (Zhang, 1997).

A mouse mutation has been engineered that specifically deletes the C-terminal 18 amino acid sequence of the RXRalpha protein. This deletion (RXRalphaaf2o) corresponds to the last helical alpha structure (H12) of the ligand-binding domain (LBD), and includes the core of the Activating Domain of the Activation Function 2 (AF-2 AD core), which is thought to be crucial in mediating ligand-dependent transactivation by RXRalpha. The homozygous mutants, which die during the late fetal period or at birth, exhibit a subset of the abnormalities previously observed in RXRalpha-/- mutants, often with incomplete penetrance. In marked contrast, compound mutants bearing mutations in RXRalpha and RXRbeta or RXRalpha, RXRbeta and RXR gamma display a large array of malformations, which nearly recapitulate the full spectrum of the defects that characterize the fetal vitamin A-deficiency (VAD) syndrome and were previously found in RAR single and compound mutants, as well as in RXRalpha/RAR(alpha, beta or gamma) compound mutants. Analysis of RXRalphaaf2o/RAR(alpha, beta or gamma) compound mutants also reveals that they exhibit many of the defects observed in the corresponding RXRalpha/RAR compound mutants. Together, these results demonstrate the importance of the integrity of RXR AF-2 for the developmental functions mediated by RAR/RXR heterodimers, and hence suggest that RXR ligand-dependent transactivation is instrumental in retinoid signaling during development. In the presence of both RXR and RAR ligands, RXRalphaAF2o/RAR heterodimers are less efficient than wild-type heterodimers at providing stable occupancy of RA response elements. Since RXRalphaAF2o/RAR heterodimers bind RARE in vitro as efficiently as WT heterodimers, irrespective of ligand presence, this RXR AF2- and ligand-dependent enhancement of promoter occupancy most probably involves events occurring at the chromatin level (Mascrez, 1998).

The N-terminal A/B domain of RXRs contains an autonomous ligand-independent transcriptional activation function called AF-1, whereas the C-terminal, ligand-binding domain E, contains a ligand-dependent transcriptional activation function, AF-2. For each RXR, at least two isoforms exist, which differ in their N-terminal region. As to RXRalpha, the major isoform RXRalpha1 is widely expressed in embryos and adults, whereas RXRalpha2 and alpha3 are restricted to the adult testis. Furthermore, RXRalpha can be phosphorylated at several serine and threonine residues in its A/B domain. The AF-2 activity crucially depends upon a conserved amphipathic alpha helix (the AF-2 AD core), whose deletion in the mouse has revealed its requirement for a number of RA-dependent developmental events. However, little is known about the mechanisms through which AF-1 activates transcription or about the relevance of the A/B domain in the global activity of the receptor under physiological conditions in vivo. Depending on the promoter context, the AF-1 of a given RXR modulates the AF-2 activity in cultured cells. Thus, the transcriptional activity of a given RXR isoform may ultimately be determined, not only by its AF-2 activity, but also by its isoform-specific A/B domain (Mascrez, 2001 and references therein).

A mouse mutation has been engineered that specifically deletes most of the RXRalpha N-terminal A/B region, which includes the activation function AF-1 and several phosphorylation sites. The homozygous mutants (RXRalphaaf1^o), as well as compound mutants that further lack RXRß and RXRgamma, are viable and display a subset of the abnormalities previously described in RXRalpha-null mutants. In contrast, RXRalphaaf1^o/RAR^-/-(alpha, ß or gamma) compound mutants die in utero and exhibit a large array of malformations that nearly recapitulate the full spectrum of the defects that characterize the fetal vitamin A-deficiency (VAD) syndrome. Altogether, these observations indicate that the RXRalpha AF-1 region A/B is functionally important, although less so than the ligand-dependent activation function AF-2, for efficiently transducing the retinoid signal through RAR/RXRalpha heterodimers during embryonic development. Moreover, it has a unique role in retinoic acid-dependent involution of the interdigital mesenchyme. During early placentogenesis, both the AF-1 and AF-2 activities of RXRalpha, ß and gamma appear to be dispensable, suggesting that RXRs act as silent heterodimeric partners in this process. However, AF-2 of RXRalpha, but not AF-1, is required for differentiation of labyrinthine trophoblast cells, a late step in the formation of the placental barrier (Mascrez, 2001).

Mammalian RXR - allostery

Regulation of gene expression via allosteric control of transcription is one of the fundamental concepts of molecular biology. Studies in prokaryotes have illustrated that binding of small molecules or ligands to sequence-specific transcription factors can produce conformational changes at a distance from the binding site. These ligand-induced changes can dramatically alter the DNA binding and/or trans-activation abilities of the target transcription factors. In this work, analysis of trans-activation by members of the steroid and thyroid hormone receptor superfamily identifies a unique form of allosteric control, the phantom ligand effect. Binding of a novel ligand (LG100754) to one subunit (RXR) of a heterodimeric transcription factor results in a linked conformational change in the second noncovalently bound subunit of the heterodimer (RAR). This conformational change results in both the dissociation of corepressors and association of coactivators in a fashion mediated by the activation function of the non-liganded subunit. Without occupying the RAR hormone binding pocket, binding of LG100754 to RXR mimics exactly the effects observed when hormone is bound to RAR. Thus, LG100754 behaves as a phantom ligand (Schulman, 1997).

Heterodimerization is a common paradigm among eukaryotic transcription factors. The 9-cis retinoic acid receptor (RXR) serves as a common heterodimerization partner for several nuclear receptors, including the thyroid hormone receptor (T3R) and retinoic acid receptor (RAR). This raises the question as to whether these complexes possess dual hormonal responsiveness. A strategy was devised to examine the transcriptional properties of each receptor, either individually or when tethered to a heterodimeric partner. The intrinsic binding properties of RXR are masked in T3R-RXR and RAR-RXR heterodimers. In contrast, RXR is active as a non-DNA-binding cofactor with the NGFI-B/Nurr1 orphan receptors. Heterodimerization of RXR with constitutively active NGFI-B/Nurr1 creates a novel hormone-dependent complex. These findings suggest that allosteric interactions among heterodimers create complexes with unique properties. It is suggested that allostery is a critical feature underlying the generation of diversity in hormone response networks (Forman, 1995).

OR1 is a member of the steroid/thyroid hormone nuclear receptor superfamily that mediates transcriptional responses to retinoids and oxysterols. On a DR4 response element, an OR1 heterodimer with the nuclear receptor retinoid X receptor alpha (RXR alpha) conveys transcriptional activation in both the absence and presence of the RXR ligand 9-cis retinoic acid, the underlying mechanisms remaining unclear. The role of the respective carboxy-terminal activation domains (AF-2s) in the absence and presence of the RXR ligand has also been analyzed. The interaction of the RXR and OR1 ligand-binding domains unleashes a transcription activation potential that is mainly dependent on the AF-2 of OR1, indicating that interaction with RXR activates OR1. This defines dimerization-induced activation as a novel function of heterodimeric interaction and appears to be a mechanism for receptor activation not previously described for nuclear receptors. Activation of OR1 occurs by a conformational change induced upon heterodimerization with RXR (Wiebel, 1997).

Mutations of a single residue in the retinoid X receptor alpha (RXRalpha) ligand-binding pocket (LBP) generate constitutive, ligand-binding-competent mutants with structural and functional characteristics similar to those of agonist-bound wild-type RXR. Modelling of the mouse RXRalphaF318A LBP suggests that, like agonist binding, the mutation disrupts a cluster of van der Waals interactions that maintain helix H11 in the apo-receptor location, thereby shifting the thermodynamic equilibrium to the holo form. Heterodimerization with some apo-receptors (retinoic acid, thyroid hormone and vitamin D3 receptors) results in "silencing" of RXRalphaF318A constitutive activity, however, this efficiently contributes to synergistic transactivation within NGFI-B-RXR heterodimers. RAR mutants disabled for corepressor binding and/or lacking a functional AF-2 activation domain, do not relieve RXR "silencing." Not only RAR agonists, but also the RAR antagonist BMS614 induce conformational changes allowing RXR to exert constitutive (RXRalphaF318A) or agonist-induced (wild-type RXR) activity in heterodimers. Interestingly, the RXRalphaF318A constitutive activity generated within heterodimers in the presence of BMS614 requires the integrity of both the RXR and RAR AF-2 domains. These observations suggest that within RXR-RAR heterodimers, RAR can adopt a structure distinct from that of the active holo-RAR, thus allowing RXR to become transcriptionally responsive to agonists (Vivat, 1997).

Transactivation-defective retinoid X and thyroid hormone receptors have been used to examine mechanisms for hormonal activation. Activation and repression of transcription by retinoid X and thyroid hormone receptors are shown to be mediated by physically distinct and functionally independent regions of the hormone binding domain. Nevertheless, the ability of receptors to respond to hormone requires communication between both functional domains. Deletion of the hormone-dependent transactivation function of the retinoid X receptor, the common subunit of heterodimeric nuclear receptors, significantly impairs hormone-dependent transcription by retinoic acid, thyroid hormone, and vitamin D receptors. The results indicate that receptors do not exist in static off and on conformations but that hormone alters an equilibrium between inactive and active states (Schulman, 1996).

Mammalian RXR - interactions during transcriptional activation