Abstract
For almost a century, vitamin A has been known as a nutrient critical for normal development, differentiation, and homeostasis; accordingly, there has been much interest in understanding its mechanism of action. This review is about the discovery of specific receptors for the vitamin A derivative, retinoic acid (RA), which launched extensive molecular, genetic, and structural investigations into these new members of the nuclear receptor superfamily of transcriptional regulators. These included two families of receptors, the RAR isotypes (α, β, and γ) along with three RXR isotypes (α, β, and γ), which bind as RXR/RAR heterodimers to cis-acting response elements of RA target genes to generate a high degree of complexity. Such studies have provided deep molecular insight into how the widespread pleiotropic effects of RA can be generated.
Introduction
Up until the very end of 1987, it was not understood how vitamin A which had a well-defined role as the retinal chromophore in vision also exerted its effects on cell growth, differentiation, and pattern formation. The broad spectrum of developmental and homeostatic defects associated either with the vitamin A deficiency syndrome or hypervitaminosis A generated significant interest in determining its mechanism of action (Niederreither et al. 1996), and references therein). Cellular-binding proteins for vitamin A (retinol) and its active derivative, retinoic acid (RA), CRBP, and CRABP, had been discovered by that time and it had been proposed that CRABP might be involved in mediating the effects of RA on gene expression (Shubeita et al. 1987). But even according to Frank Chytil, a leader in the vitamin A field whose group first discovered these binding proteins, CRABPs action did not provide a satisfying explanation for the effects of RA on the regulation of gene expression (Chytil & Ong 1987). This group demonstrated that CRABP was apparently simply shuttling RA to unidentified binding components within the nucleus (Takase et al. 1986). The nature of these nuclear-binding components would soon be revealed as members of the steroid/thyroid receptor superfamily, marking the beginning of an important 35-year (and counting) campaign to understand how this key vitamin derived from plants would play such an important role in so many aspects of our molecular physiology, in both health and disease.
Steroid receptors, from biochemistry to molecular biology
In the period immediately leading up to the above discoveries, some very exciting advances were made in understanding the molecular nature of steroid hormone receptors, first conceptualized by Elwood Jensen in 1958 (Jensen 1958, 1991). After many years of interest in studying the biochemical nature of these steroid receptor proteins, the eventual purification of steroid receptor proteins for the glucocorticoid and estrogen receptors (Greene & Press 1986) enabled the isolation of cDNA clones encoding them (Govindan et al. 1985, Hollenberg et al. 1985, Weinberger et al. 1985a, b, Green et al. 1986). This work marked a major transition in the field of steroid hormone function, greatly accelerating our biochemical understanding of these receptors by adding molecular genetics approaches. In addition to the cDNA cloning and characterization of the so-named classical steroid receptor family members, other new members of this family were also identified, which because of similarities in deduced protein sequence and structural organization, were clearly ‘paralogously’ related, including the thyroid hormone receptor and its viral v-erbA counterpart found in the avian erythroblastosis virus genome (Krust et al. 1986, Benbrook & Pfahl 1987, Pfahl & Benbrook 1987, Thompson et al. 1987), and a related gene for which no function had been ascribed, found in hepatocellular carcinoma (de Thé et al. 1987). The cDNA cloning of these receptors revealed related regions that were similar in sequence to all members of this growing superfamily – most notably, a six domain structure (domains A–F), the central hallmark of which was the highly conserved stretch of 66–68 amino acids (domain C) which contain sequential pairs of conserved cysteine residues reminiscent of the zinc-binding fingers previously characterized in other transcription factors (Hollenberg et al. 1985, Green et al. 1986, Kumar et al. 1987). These early discoveries provided the basis for finding new and related genes corresponding to members of the steroid hormone and retinoic acid (RA) families.
Searching for a retinoic acid receptor
In setting out to identify the retinoic acid receptor, our premise was that a putative receptor for RA would be similar in structure to those of the steroid and thyroid hormone receptors already cloned and particularly that the nucleotide sequence encoding the DNA-binding domain would be conserved enough to be recognized with a cognate consensus oligonucleotide probe. Such a probe was constructed comprising a degenerate set of oligonucleotides derived from sequences from multiple receptors and used to screen two randomly primed cDNA libraries derived from the breast cancer cell lines MCF7 and T47D, which had previously been shown to respond to RA (Petkovich et al. 1987). A number of positive clones were obtained from the screening of these libraries, five of which turned out to contain a sequence sharing significant homology with the oligonucleotide consensus probe sequence. Furthermore, the DNA-binding domain sequence also shared 100% amino acid identity with the hORF (open reading frame) of a clone isolated from a human hepatocellular carcinoma. Thus, whichever ligand this new receptor was encoded for, it appeared that like for the thyroid hormone receptor, there could be multiple members in this subfamily.
Our first study, confirming that the ligand for this new receptor was indeed retinoic acid (RA), involved cloning this cDNA into a mammalian expression vector and producing the corresponding protein in HeLa cells. Extracts from these cells were used for ligand-binding assays. This approach confirmed that radiolabelled RA could bind avidly to the protein extract of transfected HeLa cells and that this binding could be prevented with unlabelled RA and, to some extent, with the retinoic acid precursor retinol but not by other ligands corresponding to those for steroid and thyroid hormones (Petkovich et al. 1987).
The next challenge was to demonstrate that this receptor could act to transduce the activity of retinoic acid (RA) binding into a transcriptional regulation event, that is, measuring some transcriptional output ‘event’ activated by the RA-bound receptor. Several different reporter gene constructs had been developed to demonstrate the transcriptional activity of the glucocorticoid receptor (GR) and the estrogen receptor (ER), such as the MMTV-Cat and the Vt-tk-Cat, based on studies demonstrating that MMTV contained a glucocorticoid responsive element (a GRE) and similarly that a functional estrogen responsive element (an ERE) had been identified in the vitellogenin promoter. However, at that time, nothing was known about the RA mechanism of action, in particular, what specific genes could be transcriptionally regulated and what a RA response element could look like. Nonetheless, elegant work from Stephen Green and Vijay Kumar, demonstrating that gene-specific regulations by the ER and the GR could be swapped by interchanging the 66-amino acid DNA binding domains of the receptors, provided us with a path forward (Green & Chambon 1987, Kumar et al. 1987). By making such a swap between the similarly highly conserved DNA-binding domains of the putative RA receptor with that of the ER, we constructed a hybrid receptor that conceivably could bind to RA (or whatever its cognate ligand might be) and activate the reporter gene constructed to evaluate the transcriptional activity of the ER. Our thinking at the time was that this approach could be developed for any novel ‘orphan’ receptor, to be used as a readout in the screening and purification of novel ligands from tissue extracts and chemical libraries.
The results of our DNA-binding domain swap between the receptors which became known as the retinoic acid receptor (RAR) and the ER resolved the transcriptional activity question spectacularly well. They clearly demonstrated that the RAR binding of the RA ligand resulted in an activated receptor that could trigger the transcriptional regulation of a gene promoter. They also provided a hint at what a retinoic acid response element (a RARE) could actually comprise. HeLa cells transfected with the vit-tk-CAT reporter gene exhibited some noticeable activity when these cells were treated with RA. Did the tk promoter also contain a RARE? Was it similar to the inverted repeat sequence to which the estrogen-bound ER could bind? These questions would have more surprising answers later as the identities of natural and synthetic RARS were uncovered (see below).
Nonetheless, at this instant, we had demonstrated that this newly discovered retinoic acid receptor (RAR) acting through a mechanism similar to that of steroid hormones was likely responsible for mediating the genomic effects of vitamin A on cell proliferation, differentiation, and pattern formation. Because a RAR, as well as a TR, could not be classified as a ‘steroid’ receptor per se, we coined the term ‘Nuclear Receptors’ with the expectation that this nuclear receptor superfamily would grow. The gene expression/transcription factor community first learned about the identification of the retinoic acid receptor in late fall of 1987, when our own data were first presented at a ‘Whitehead Symposium’. It was the first so-called ‘orphan receptor’ to be resolved. It was also at the same symposium that we learned that Vincent Giguère from Ron Evan’s group had made the same discovery. Our publication (Petkovich et al. 1987) appeared in Nature on December 10, 1987, closely followed 2 weeks later by that of Ron Evan’s group (Giguère et al. 1987) in Nature. This launched a ‘massive effort’ to uncover more concerning the molecular physiology of retinoic acid and additional possible ligand-inducible nuclear receptors. Intense international competition between very active molecular biology laboratories greatly accelerated the pace of additional discoveries.
More than one retinoic acid receptor
One of the obvious next steps was to settle the identity of the receptor fragment associated with hepatocellular carcinoma, which exhibited striking similarity with that of the RAR DBD. After a meeting over lunch at the ‘L’Ami Schutz’ in the beautiful ‘Petite France’ in Strasbourg with Huges de Thé and Anne Dejean from the Institut Pasteur (Paris), we agreed to collaborate in isolating and sequencing cDNAs corresponding to their hepatocellular gene fragment of interest.
Our aim was to use the DNA-binding domain ‘swap strategy’ to demonstrate its functionality.
This was a successful venture in uncovering the second member of the retinoic acid receptor family, RARβ (Brand et al. 1988). This finding raised two important questions: (1) how do multiple retinoic acid receptors work together to transduce the many effects of RA and (2) what was the implication of finding a RAR fragment associated with a tumor DNA mutation? The answer to the first question involved the characterization of additional RA receptor subtypes and their corresponding isoforms and later, three novel functionally related receptor species are known as retinoid-X-receptors (RXRs), followed by a ‘tour de force’ in the molecular dissection of each of the subtypes of this family (see below). The second question led to some exciting findings: Huges de Thé and collaborators (de Thé et al. 1987, 1990a ) uncovered a gene rearrangement possibly implicated in hepatocellular carcinoma (Dejean & de Thé 1990) and played a critical role in translocations creating fusion proteins specifically implicated in acute promyelocytic leukemia (de Thé et al. 1990a ).
The cloning of a second retinoic acid receptor (now named RARβ), leaving the first cloned receptor as RARα, also raised the obvious question of how many RARs might be encoded in the mammalian genome? The answer came from the cloning of a third member of the mammalian family, RARγ, from both murine and human sources (Krust et al. 1989, Zelent et al. 1989). This sparked a flurry of activity in cloning RARs from other species, mammalian, amphibian (Ragsdale et al. 1989), and teleost (White et al. 1994), as well as invertebrate chordates (Campo-Paysaa et al. 2015) and bilaterian ancestors (Albalat & Canestro 2009), among other species, underscoring the fundamental role that RA plays in the embryonic development of all chordate species and the evolutionary conservation of primitive machinery to precisely regulate gene expression in response to vitamin A derivatives.
RARs in embryonic development
With a three RAR family (α, β, and γ) characterized, and no evidence that additional subtypes of this family were encoded in the human or murine genomes, the next step was to identify their possible unique functions. Retinoic acid (RA), first considered to be a growth hormone, gained interest in view of its role in pattern formation, especially in developing and regenerating limbs. A brilliant series of studies conducted by Esther Ruberte, Pascale Dollé, Manuel Mark, and Cathy Mendelsohn in collaboration with Gillian Morris-Kay in UK mapped the expression of each of these RARs during the process of embryonic development (Dolle et al. 1989, 1990, Ruberte et al. 1990, 1993, Mark et al. 1991, Mendelsohn et al. 1994, Ruberte 1994). RARα appeared to be generally expressed, whereas the expression patterns of RARβ and RARγ were more restricted: RARβ in neurogenic and epithelial tissue, RARγ within mesenchymal condensations, osteochondrogenic lineages, and skin epithelium. However, the complexity of the RAR signal transduction system was only beginning to be unfolded.
Variations in the splicing pattern of the three RAR genes were found to generate multiple isoforms for every receptor subtype, each with their own restricted pattern of expression. Four different isoforms of RARβ were identified (RARβ1–4) (Zelent et al. 1991) and two isoforms each for RARα (RARα1 and 2) (Leroy et al. 1991a , b ) and RARγ (RARγ1 and 2) (Kastner et al. 1990). These variations resulted from alternative splicings of the N-terminal transcription of activation domain known as AF1 (see below). Exons encoding this region were found to be transcribed and spliced from different promoters in a cell-specific manner (Chambon 1994).
Similarly, two different isoforms were eventually identified for each of the three RXR subtypes. These isotypes resulted from different exon usage encoding distinct AF1 domains. Taken altogether, these discoveries showed that the ultimate effects of RA in different tissues were guided by other local factors dictating both the choice of the transcriptional promoter and also which of the AF1 domains would be spliced into the final RAR product. A better understanding of the function of these different isoforms came from an extensive molecular dissection of the retinoic acid receptor genes using homologous recombination. However, the identification of RXRs added a totally novel and unexpected dimension to gene regulation by all-trans and 9-cis RA.
RARs and RXRs – converging families
While the early studies aimed at characterizing the molecular expression patterns of the RARs were progressing, efforts in understanding the molecular interactions leading to gene transcriptional regulation by RARs revealed a new family of receptors, the so-named retinoid-X receptors (RXRs), playing a role not only in co-regulating RA responsive genes but also in genes controlled by other nuclear receptor family members. The initial discovery of a novel orphan receptor called H-2RIIBP, which became a member of the RXR family, was made by Hamada (Hamada et al. 1989) in Keiko Ozato’s group. At the time, it was unknown whether this nuclear receptor family member was a co-regulator of multiple signaling pathways including vitamin D, thyroid hormone, and retinoic acid. David Mangelsdorf in Ron Evan’s group identified RXRα as the first member of this subfamily and furthermore showed that this receptor, the ligand-binding domain of which shared little (<30%) amino acid identity with RARs, was still able to transduce an RA signal (Mangelsdorf et al. 1990), although strict binding to RA could not be demonstrated at the time. Three members of this family were eventually characterized, including that described by Hamada et al. (1989) which became RXRβ, and a third member named RXRγ (Mangelsdorf et al. 1992).
With the cloning of RXRα, it was clear that the retinoic acid signal transduction was by far more complex than initially anticipated. Some critical issues, that is, what could be the nature of the DNA sequence constituting a retinoic acid responsive element: does it differ between RARs and RXRs and are both receptors activated by the same ligand? The promiscuity of these nuclear receptors, in contrast to the homophilic steroid hormone receptors, became apparent upon the ‘growing’ evidence that RARs, TRs, VDRs, and RXRs were binding DNA as RXR heterodimers, and the demonstration that the geometric isomer of RA, that is 9-cis RA, was a ligand for RXRs (Heyman et al. 1992, Tate et al. 1994).
The sequence and nature of the DNA-binding motifs favored by RARs and RXRs were also actively investigated at that time. A series of naturally occurring and synthetic DNA response elements was characterized for TRs (Glass et al. 1987, Umesono et al. 1991), RARs (Vasios et al. 1989, 1991, de Thé et al. 1990b, Leroy et al. 1991b, Smith et al. 1991), and RXRs (Mangelsdorf et al. 1990, 1991, Rottman et al. 1991). These response elements consisted of a six-nucleotide core motif, AGG/TTCA, occurring in tandem as either a direct or inverted ‘repeat motif’.
Importantly, the spacing between the core motif sequence repeats appeared to have an important impact on receptor selectivity. Indeed, a systematic evaluation of the actual sequence orientation and spacing of the repeated motifs showed that RARs have a preference for 5 bp spaced motifs (Umesono et al. 1991), while TRs preferentially recognize motifs separated by 4 bp (Umesono et al. 1991), whereas RXRs favored a 1 bp spacing (Mangelsdorf et al. 1991). It was initially thought that RARs, TRs, and RXRs, like their steroid hormone receptor relatives, were bound as dimers to such direct repeated response elements (Glass et al. 1989, 1990, Lazar 1991). In this respect, it was also shown that the in vitro response element binding of RAR (Glass et al. 1990, Rottman et al. 1991) and TR (Murray & Towle 1989, Burnside et al. 1990) could be enhanced by adding components present in cellular nuclear extracts.
The nature of the binding component responsible for enhancing RAR binding to its response element was identified through a monumental effort by Mark Leid et al. who set out to purify RAR proteins in our laboratory for the purpose of characterizing their transcriptional activity and set the stage for protein crystallographic analysis of their structure (Leid et al. 1992). They demonstrated that as the protein purity increased, the DNA binding activity suddenly dropped off. However, by adding back cell extracts, they could restore the binding activity. As it turned out, the activity of the ‘restoring factor’ was none other than RXRβ, an astounding finding at that time. Furthermore, not only did RXR facilitate RAR binding but it was also required for the DNA binding of other nuclear receptors including TR and VDR. Eventually, other orphan receptors were added to this list, including PPARs, NGF1-B, LXR, and FXR (Leblanc & Stunnenberg 1995, Mangelsdorf & Evans 1995).
The potential significance of this convergence of receptor pathways was immediately realized, that is, RXR was a nexus connecting multiple hormone-regulated networks to coordinate their activities during vertebrate development and growth. Indeed, this raised the notion that in a cell containing limiting amounts of RXR, factors allowing one receptor subtype to form heterodimers more rapidly than another subtype formation would drive criticality a ‘winner-take-all’ decision-making process allowing one ligand to dominate over another in its influence on cell function. However, studies examining the interactions between receptor heterodimers and DNA did reveal that a ligand was not essential for DNA binding, but it rather played a role in whether a RAR/RXR complex would actively repress or activate the expression of a target gene. Furthermore, the number of spacer nucleotides between half-sites was defined for various RXR heterodimers, initially referred to as the ‘1–5 rule’: RXR–RXR and RAR–RXR (DR1), RXR–RAR (DR2), RXR–VDR (DR3), RXR–TR (DR4), and RXR–RAR (DR5) (Mangelsdorf & Evans 1995).
Another question was left unanswered: if the RAR DNA binding was co-dependent on their association with RXRs, they should necessarily be co-expressed in cells. The developmental expression patterns of the three mouse-retinoid X receptor genes were investigated and compared to those of retinoic acid receptor (RAR) genes (Dolle et al. 1994): RXRβ exhibited a ubiquitous expression at all developmental stages studied, while RXRα exhibited a diffuse expression at early developmental stages, which was enhanced in the epidermis and several other squamous epithelia at later stages. In contrast, RXRγ displayed a restricted expression in the myogenic lineage, initially in myotomes and subsequently in various differentiating muscles, including those of the face and limbs. RXRγ transcripts were also developmentally regulated in the otic epithelium, the retina, the pituitary, and thyroid glands. Furthermore, RXRγ was expressed in several discrete areas of the fetal CNS, namely in the diencephalon, the striatum, and in part of the ventral horns of the spinal cord. These findings showed that there was an extensive overlap between RARs and RXRs, consistent with the growing body of evidence suggesting that, in most instances, there are obligatory partners for driving RA-regulated events during embryogenesis (Dolle et al. 1994). However, their co-expression did not conclusively prove that the overlap in expression between RARs and RXRs was functionally relevant in vivo, which remained to be demonstrated through genetic analyses.
Dissecting the role of RARs and RXRs in embryological development
The severe developmental consequences of both vitamin A deficiency and RA teratogenicity, such as the limb-duplicating effects of retinoic acid exposure, are well known in developing and regenerating axolotl-limbs. Taken together with the wide developmental expression of all three RAR subtypes and isotypes, they supported a role for RA-regulated gene expression in controlling many aspects of embryogenesis. Were these effects mediated by RARs and RXRs together or independently? Knocking out each of the RARs, RXRs, and corresponding subtypes would be a massive undertaking, leading to the generation of a wealth of information regarding the role of retinoic acid in the regulation of gene networks critical for pattern formation and tissue maintenance. Comprehensive genetic knockout studies of the RARs, including both single and compound mutant mouse lines, indicated that the entire spectrum of defects previously associated with the fetal vitamin A deficiency (VAD) syndrome could be genetically fully recapitulated (Lohnes et al. 1994, Mendelsohn et al. 1994, Mark et al. 1995). Furthermore, RAR-deficient mouse models also revealed many abnormalities not previously associated with vitamin A function, including craniofacial, axial, and limb skeletal abnormalities. Whereas each of the single knockouts displayed limited defects and variable viability, the most profound defects were observed only in double RAR mutants, indicating that in the absence of a given RAR, the remaining RARs could still exhibit some redundancy (Lufkin et al. 1993, Lohnes et al. 1994, 1995, Mendelsohn et al. 1994, Mark et al. 1995, Labrecque et al. 1998).
RXRs involvement in developmental retinoid signaling had yet to be demonstrated, and more importantly, whether it was direct or indirect. RXRβ and RXRγ null mutant mice were shown to be viable without abnormalities typically associated with vitamin A deficiency or excess (Kastner et al. 1995, Krezel et al. 1996). However, ‘RXRα-null’ was embryonic lethal, exhibiting defects found in the fetal VAD syndrome (Kastner et al. 1995, Sucov et al. 1996). Furthermore, a preliminary analysis of a few RXRα/RARα and RXRα/RARγ compound mutants revealed a synergy between the effects of RXRα and RAR mutations: ocular and cardiovascular defects were observed in ‘combination’ knockouts, whereas such defects were not present in the corresponding single mutants (Kastner et al. 1995, Sucov et al. 1996). Shortly thereafter, phenotypic characterization of all combinations of RXR (α, β, or γ)/RAR (α, β, or γ) compound mutants was completed, showing that these various combinations of RXR/RAR mutations could synergistically recapitulate the majority of defects seen in RAR double mutants, thus providing strong genetic evidence that RXR/RAR heterodimers are functional in transducing the retinoid signal in vivo (Kastner et al. 1997).
Clearly, the highly pleiotropic effects of RA reflect combinatorial mechanisms through which multiple RXR/RAR heterodimers differentially transduce retinoid signals to selectively regulate the expression of networks of RA target genes, thereby shaping the axial and limb patterning of the early embryo and multiple aspects of organogenesis in later development.
Molecular anatomy of RARs and RXRs
DNA- and ligand-binding domains
While the molecular physiology of RARs and RXRs was explored in mouse genetic models, their molecular anatomy was also intensively investigated. Having six linearly arranged domains (A through F), each of them being functionally separable making it possible to dissect the individual domain functions and interactions with other components of the cell transcriptional machinery. For RARs, as previously demonstrated for other receptor family members (Chambon 1996), the DNA-binding domain region (C) encoded the ‘zinc-binding fingers’, while the ligand-binding domain region (E) was shown to bind its cognate RA ligand. Notably, domains C, D, and E were also shown to be involved in direct stabilization of heterodimer formation with RXRs (Chambon 1996, and references therein). For RXRs, whether 9-cis RA was the natural ligand required for their function was less obvious; indeed, other ligands have been proposed, such as DHA and other eicosanoids and species of RXR-related family members, like ‘usp’ in Drosophila or ‘zfRXRs-δ and -ε’ in zebrafish, do not bind to 9-cis RA (even though their zfRXRs α, -β, and -γ counterparts do so avidly) (Jones et al. 1995).
Transcriptional activation domains
In addition to the DNA- and ligand-binding domains of RARs, there are more discrete regions helping in mediating the recruitment of transcription factors; for example, the AF-1 activation function-1 exerts a transcriptional activation function encoded within the N-terminal A/B domain which is ligand-independent, while AF2 located within the C-terminal domain E, also involved in transcription factor recruitment, is ligand-dependent. Given that alternative splicing events determine which of the N-terminal regions is selected for different receptor subtypes, the AF1 domain will also be unique to different receptor isotypes (e.g. RARα1 vs RARα2).
Furthermore, although the AF1 can act autonomously, it has also been shown to synergize with the corresponding AF-2 from the same receptor subtype (Nagpal et al. 1992a, b, 1993). Thus, the activation potential of a given RAR may ultimately be determined by interactions between a common AF-2 and a cell type- and developmental stage-dependent isoform-specific AF-1, triggered by the presence of RA. RXRs were also shown to have functional AF-1 and AF-2 domains. However, the ligand-dependency was not implicit and RXRs may act in a permissive unliganded state.
Transrepressional activity
Studies aimed at investigating the nuclear receptor transcriptional activity of unliganded RAR and TR revealed a strong transrepression of the basal-level promoter activity of target genes in transfected cells. This repression was associated with the carboxy-terminal domains of the receptors that include the LBD (Chen & Evans 1995, Kurokawa et al. 1995, Perlmann & Vennstrom 1995, Zamir et al. 1996). A search for factors responsible for this repression led to the identification of nuclear receptor corepressor (N-CoR) and silencing mediator for RAR and TR. Both were shown to interact with unliganded receptors (Chen & Evans 1995) and N-CoR was shown to be associated with a RAR/RXR bound to a DR5 responsive element and to recruit factors that encode histone deacetylase (HDAC) activity involved in removing acetyl groups from lysine residues in histone N-termini, thus ‘tightening’ chromatin–DNA interactions within the vicinity of the target promoter. Interestingly, factors that are known for their gene repressor activity have also been shown to be recruited by RAR/RXR complexes including topoisomerase IIβ, polycomb group proteins, and calmodulin kinase IIγ (CaMKIIγ) (Chambon 2005, 1996).
Ligand-dependent derepression
N-CoR bound to RAR, as part of a typical DR5-bound heterodimer complex, is released upon binding of RA. This release is dependent on the integrity of the AF-2 AD core, an amphipathic alpha-helical core motif conserved in essentially all nuclear receptors and a key structural component involved in ligand-dependent conformational changes in the LBD (Chambon 1996, Perissi et al. 2004). Ligand binding also allows AF2 to recruit coactivator proteins which can help in mediating the changes in local chromatin structure that promote transcriptional activation. Numerous such RAR transcriptional co-factors have been characterized, some of which are RA-dependent coactivators (e.g. SRC 1–3, CBP/P300, GCN5, ADA3, PCAF) or corepressors, either ligand-independent (e.g. TBLR1, LSD1, CaMKIIγ) or ligand-dependent (e.g. TNIP1, RIF1, LCoR, RIP140) (Chakravarti et al. 1996, Chambon 1996, Voegel et al. 1996, Blanco et al. 1998, Brown et al. 2003, Fernandes et al. 2003, Hu et al. 2004, Perissi et al. 2004, Si et al. 2007, Lee et al. 2010). Some of these nuclear receptor-associated factors (such as CBP, p300, P/CAF), as well as some p160 coactivators, also inherently possess histone acetyltransferases which, in an action opposite to that of HDACs acetylate, thus ‘loosening’ the histone proteins grip on DNA, and facilitating transcription (Glass & Rosenfeld 2000, Westin et al. 2000). Taken altogether, these factors form multi-subunit complexes which modulate the ‘activity’ of histones and transcription factors. Thus, RXR/RAR heterodimers function in a context-dependent manner according to both the involved receptor isotypes and the gene-specific RARE to which they bind.
Structure and function of the RAR and RXR LBDs
The first liganded RAR structure to be elucidated was RARγ bound to all-trans RA, revealing a ligand-binding domain comprising a prototypical 12 α-helices (H1 to H12) and a short β-turn, arranged in three layers to form an antiparallel α-helical sandwich (Renaud et al. 1995). These studies showed that RA fits tightly in the RAR retinoic acid-binding pocket which is stabilized by extensive van der Waals contacts and a network of ionic and hydrogen bonds between the carboxyl group of RA and a conserved arginine in H5 (Renaud et al. 1995, Li et al. 2004).
The structure of the unliganded RXR LBDs was initially resolved by Bourguet et al. (1995a, b). Recalling that in the unliganded state, RAR generates transcriptional repression attributed to the formation of a corepressor complex causing tight local association of chromatin within the promoter region of RA target genes. Structural studies have shown that the corepressor complex is formed through interactions facilitated by a four-turn helical motif (LxxxIxxxIxxxF/Y) in the corepressor. Through this motif, the corepressor binds a hydrophobic surface of the RAR comprising helices H3 and H4, as well as an N-terminal extended β-strand (β1) which forms a specific antiparallel β-sheet with specific RAR residues.
What does change when RA enters the ligand-binding pocket? Comparison of the unliganded RXRα LBD and the ‘holo’ RARγ LBD suggested that RA binding to a predominantly hydrophobic pocket was ‘sealed-in’ by the folding over of the C-terminal helix H12, referred to as a ‘mousetrap mechanism’ (Bourguet et al. 2000a, b, Egea et al. 2000).
It is remarkable that the interactions between RA and the LBD can trigger such cascades of events, and although the simple act of binding has remained essentially unchanged over millions of years, its degree of sophistication has immensely increased.
In fact, not only does RA binding induce the repositioning of helix H12 but also causes a β-strand to α-helix transition that allows the formation of helix H11, which in turn disengages the corepressor complex, thus allowing the coactivator recruitment (le Maire et al. 2010). This induced helical structure presents a hydrophobic surface comprising the C-terminal part of helix H3, helix H4, and H12, which can be specifically recognized by either synthetic peptides or coactivator proteins containing short LxxLL motifs. In addition, the coactivator helix is thought to fold into a longer α-helix involving flanking residues which can form hydrophobic contacts with the newly formed hydrophobic groove and specific hydrogen bonds between a lysine at the C terminus of H3 and glutamate in H12, which together form a so-called ‘charge clamp’ (le Maire et al. 2010).
The next 35 years: understanding the molecular physiology and pathophysiology of RAR and RXR signaling and other endocrine signaling pathways in which RXRs are involved as heterodimeric partners
Our progress in understanding physiological processes, at the molecular and submolecular levels, is made, almost exclusively, in the wake of technological advances. Accordingly, the discovery of RARs was made possible because appropriate molecular biology tools became available. Starting from the simple sequencing of the first RAR cDNA, how retinoic acid could alter genetic programming in the cell became clearer. In a short span of time following this initial discovery, the genes encoding three RARs and their corresponding isotypes, along with those of the three heterodimeric RXR partners which, on their own can dimerize with a number of nuclear receptors belonging to the steroid/thyroid receptor superfamily, had been characterized.
Genetic knockout models were generated for individual and combined receptor subtypes and structural studies revealed satisfying mechanisms involved in retinoic acid signal transduction. All of these advances were in step with progress in technological advances, such as knockout mouse models and x-ray crystallography.
The next 35 years of exploration of RA molecular biology will also require additional tools. For example, the mechanisms associating RA-liganded RAR/RAR complexes and key components of the transcription machinery (e.g. TBP, TAF subunits of TFIID, and TFIIH), as well as transrepressors and transactivators, remain to be elucidated (Chambon 2005, Lonard & O’Malley 2006). Similarly, the role of subtype-specific AF-2 and AF-1 transactivation functions, and their corresponding co-activating partners, has yet to be defined. Efforts so far hint at a high degree of complexity and subtlety, not only between combinatorial partnerships between the multiple RAR and RXR subtypes but also between the multiple RXRs and their multiple endocrine receptor partners (e.g. TR, ER, AR, etc.) and the transcription co-factors that mediate their activities. More generally, consideration will be given to understanding connections between the vast networks of genes regulated by nuclear receptor heterodimeric combinations over the course of embryonic development and adult physiology. Genome-wide screens for RA-regulated genes have already revealed hundreds of response elements including non-canonical but verifiable RAREs, including those with DR0 and DR8 (Mendoza-Parra et al. 2011, Moutier et al. 2012). Similarly, there are potentially hundreds of transcription co-factors, modified by numerous post-translational modifications that will affect RARs instructions on gene regulation in a manner which is promoter-, cell status-, and tissue-dependent.
A key determinant of these dynamic interactions is also the availability of vitamin A, which may in severe nutritional VAD be limiting, or as in normal circumstances be determined by the balance between the enzymes which locally synthesize (e.g. Aldh1a1, Aldh1a2) or inactivate (CYP26A1, CYP26B1, CYP26C1) the RA RAR ligand (Abu-Abed et al. 2002, Niederreither et al. 2002, Ribes et al. 2007, Maclean et al. 2009, Pennimpede et al. 2010). RA production and catabolism are tightly spatiotemporally coordinated and autoregulated during developmental processes. Understanding how these regulatory loops change according to both availability of RA and the gradual changing of gene networks affected will help to provide links between differentiation and organogenesis.
Important methods to tackle these complex problems may already be in hand. Advances in gene-editing technologies will allow us to interrogate the impact of small alterations to RARs/RXRs or individual transcriptional components mediating RA control over gene expression, thus providing detailed insight into their impact on complex physiological and developmental processes. Advances in machine learning technology will enable the processing of huge data sets that these approaches will generate and help us to understand the functional relationships between RAR-regulated genes and ‘downstream’ physio(patho)logical processes.
Conclusion
Our progress in understanding retinoid biology at the molecular level has advanced significantly over the past 35 years built largely on the preceding important physiological studies completed since the molecular nature of vitamin A was discovered almost 100 years ago. The pleiotropy of this vitamin is well known, but the molecular mechanisms underlying its many specific effects remain undiscovered – awaiting the next stage of molecular dissection that will tease apart the roles of different combinations of heterodimers in various tissues of the organism at different stages of development. The work that we have outlined in this review has set the stage for the next generation of retinoid biologists to answer important questions regarding the molecular nature of our physiologies and within it, the role of vitamin A and its receptors.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.
Acknowledgements
The authors wish to acknowledge the critical contribution made by all past members of the Retinoic Acid Receptor Group (fellow researchers and technical experts) at the LGME (CNRS, INSERM and Faculté de Médecine of Strasbourg) and then at the IGBMC (built for us by Bristol-Myers-Squibb in Strasbourg-Illkirch), and thank all of those who around the world who collaborated or provided invaluable comments at various stages of this work.
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