Leading Edge

Review

Nuclear Receptors, RXR,

and the Big Bang
Ronald M. Evans1,2,* and David J. Mangelsdorf1,3,*
1Howard Hughes Medical Institute
2The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
3The Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390, USA

*Correspondence: [email protected] (R.M.E.), [email protected] (D.J.M.)

http://dx.doi.org/10.1016/j.cell.2014.03.012

Isolation of genes encoding the receptors for steroids, retinoids, vitamin D, and thyroid hormone
and their structural and functional analysis revealed an evolutionarily conserved template for nu-
clear hormone receptors. This discovery sparked identification of numerous genes encoding
related proteins, termed orphan receptors. Characterization of these orphan receptors and, in
particular, of the retinoid X receptor (RXR) positioned nuclear receptors at the epicenter of the
‘‘Big Bang’’ of molecular endocrinology. This Review provides a personal perspective on nuclear
receptors and explores their integrated and coordinated signaling networks that are essential for
multicellular life, highlighting the RXR heterodimer and its associated ligands and transcriptional
mechanism.

Introduction ing, ligand binding, and transactivation domains (Giguère et al.,

The discovery of nuclear receptors has its historical roots in 1986; Green et al., 1986; Miesfeld et al., 1986). Importantly,
endocrinology and the identification of the lipophilic hormones access to the cDNAs enabled key experiments needed to test
that function as their ligands (Evans, 1988). Steroid and thyroid protein function, including mutagenesis of the receptor’s primary
hormones, along with vitamins A and D, were elucidated based structure to assess the importance of specific amino acids and
on their endocrine origin and the physiologic processes that characterization of the nucleotide code within the target gene’s
they regulate. Each of these small molecules is chemically promoter that allows gene-specific expression (Green and
distinct, and although there was initially no presumption that Chambon, 1987; Umesono and Evans, 1989; Umesono et al.,
they might have a shared signaling mechanism, initial biochem- 1988). As a result of this early work, transcriptional regulation
ical experiments revealed the presence of an intracellular by hormone-receptor complexes was shown to be a funda-
receptor that, upon ligand addition, activated transcription of mental process embedded in the circuitry of extracellular signal
tissue-specific sets of target genes (O’Malley, 1971; Yamamoto, transduction by lipophilic endocrine hormones and vitamins.
1985). Perhaps the most revolutionary finding to come from the clon-
The idea of a ‘‘Nuclear Receptor’’ that could directly translate ing of the first steroid receptors was the surprising discovery that
simple chemical changes into distinct physiologic effects per- dozens of other evolutionarily related proteins exist. Because
sisted for several decades, but the fundamental nature of this re- the associated small-molecule ligands were unknown, they
ceptor, its means for recognizing specific chemical ligands, its garnered the name ‘‘orphan’’ receptors (Giguere et al., 1988; Mil-
mode of interaction with the genome, and its mechanism for con- brandt, 1988; O’Malley, 1990; Wang et al., 1989). Of further phy-
trol of gene transcription were all beyond the limits of classic logenic significance, these orphan receptors were shown to be
biochemical analysis. Subsequent investigation into the mecha- conserved throughout metazoan evolution, although it should
nism of hormone action eventually led to the biochemical, be noted that nuclear receptors are absent in protozoans, fungi,
molecular, and genetic characterization of the genes encoding and plants (Markov and Laudet, 2011; Owen and Zelent, 2000;
the first steroid receptors in the mid-1980s (Mangelsdorf et al., Robinson-Rechavi et al., 2004).
1995).
Engine of Discovery
The Dawn of a Superfamily That there were orphan receptors immediately suggested the
The isolation of the first complete steroid receptor cDNAs, the existence of a host of previously unknown signaling pathways
glucocorticoid and estrogen receptors, was transformative regulated by a myriad of undiscovered ligands. The question
(Green et al., 1986; Hollenberg et al., 1985) (Figure 1). Compari- was how might one go about discovering such ligands? The
son of the sequences revealed a conserved evolutionary tem- answer came from a remarkable, innovative technological
plate, and it also permitted the delineation of the structural and achievement—the cotransfection assay (Giguère et al., 1986).
functional features that foreshadowed the emergence of a nu- The idea went like this: if the cDNA encoding the receptor was
clear receptor superfamily. Each sequence harbors DNA bind- sufficient to reconstitute a hormone response, then expression

Cell 157, March 27, 2014 ª2014 Elsevier Inc. 255

Figure 1. Nuclear Receptor Discovery Timeline
Schematic timeline showing landmark discoveries in the nuclear receptor field over the last three decades. The entries start from the cloning of the first steroid
hormone receptor cDNA to more recent ‘‘omic’’ findings. Inset on the right shows total publications for all nuclear receptors, as well as that for RXR and
heterodimer partners over time.

plasmids harboring the receptor’s cDNA could be cotransfected which receptors might mediate their signaling (Kliewer et al.,
with a hormone-responsive reporter gene to create a highly 1999). In comparison, the classic endocrinology methods that
defined two-component regulatory switch. With the switch flip- led to the isolation of thyroid hormone in 1914 required 3.5
ped ‘‘on’’ by hormone binding, the resulting powerful transcrip- tons of bovine thyroid gland (Kendall, 1917). Thus, the massive
tional response allowed rapid analysis of the receptor’s DNA shift in ligand screening sensitivity, as a consequence of receptor
and ligand binding domains, as well as ligand and target gene cloning and powerful reporter assays, could now unveil new and
specificity. The cotransfection assay was so versatile as a cell- unimagined signaling systems.
based means to study transcription that it quickly became the
mainstay of virtually every molecular biology laboratory, as well RXR and the Big Bang
as a pharmaceutical discovery tool. Applying the reverse endocrinology approach to the retinoid X
The cotransfection assay provided a quantitative and highly receptor (RXR) led to the identification of the first endogenous
efficient tool for screening, and as it was extremely sensitive to ligand for an orphan nuclear receptor (9-cis retinoic acid, a
small-molecule ligands, it became the mainstay of ‘‘deorphan- metabolite of vitamin A), establishing RXR as the founding mem-
ing’’ efforts. Early results showed that the DNA- and ligand-bind- ber of the ‘‘adopted’’ orphan class (Mangelsdorf et al., 1990;
ing domains of the receptors function autonomously, which led Heyman et al., 1992; Levin et al., 1992) (see Table 1 for receptor
to the idea that one could swap these domains between recep- nomenclature for the entire superfamily). The discovery of RXR
tors, and even other transcription factors, and still retain ligand- and its ligand resulted in the genesis of two prodigious concepts
dependent transactivation (Giguere et al., 1987; Green and in the nuclear receptor field. First, in revealing the existence of a
Chambon, 1987; Petkovich et al., 1987). With the cDNAs of the previously unknown signaling pathway, it provided a proof of
orphan receptors in hand, the cotransfection assay was instantly principle that precipitated a wave of research aimed at linking
adapted as an unbiased ligand screening method that could be other orphan receptors to specific ligands.
implemented even when the receptor’s DNA binding site (or Second, it led to the discovery of RXR heterodimerization with
response element) was not precisely known (Chawla et al., these adopted orphan receptors, thereby defining a novel
2001). This ‘‘reverse endocrinology’’ concept allowed for high- feature of multiple intertwined signaling pathways (Figure 2).
throughput screens of natural, synthetic, and xenobiotic ligands For example, one of the first uses of the cotransfection
that required less-than-micromolar concentrations to unearth assay identified interactions between an orphan receptor and

256 Cell 157, March 27, 2014 ª2014 Elsevier Inc.

compounds that promote peroxisome proliferation (fibrates and ready for subsequent rounds of activation. For this cyclic pro-
nafenopin) as the first xenobiotic ligand-receptor pair. The then- cess to occur, the ligands themselves must first be produced
named peroxisome proliferator-activated receptor (PPAR) was during the inductive phase and then eliminated to reset the bal-
ultimately shown to be one of a family of fatty acid receptors ance. Thus, like the second law of thermodynamics, for every
(Issemann and Green, 1990; Dreyer et al., 1992). PPARs were physiologic action, there is an equal, but opposite reaction.
the first class of orphan receptors shown to heterodimerize The unsung heroes in this dynamic process are the cyto-
with RXR (Kliewer et al., 1992). chrome P450s (CYPs), whose unique ability to add molecular
We refer to the discovery of heterodimerization as the ‘‘RXR oxygen to small molecules plays a key role in cholesterol, ste-
Big Bang’’ because its explosive impact gave rise to a wave of roid, and fatty acid metabolism (Baker, 2011; Zelko and Negishi,
discovery, including entirely new physiologic signaling pathways 2000). For example, all natural steroid hormones (glucocorti-
(Figure 2). The straightforward cotransfection assay yielded a coids, mineralocorticoids, progesterone, estrogens, and andro-
relatively familiar result—the receptor proteins are functional gens) are synthesized from cholesterol in the gonads and
heterodimers, and yet the complexity of regulation that has since adrenal glands when CYP11A1 (in the mitochondria) converts
emerged, under both normal and pathological conditions, is cholesterol to pregnenolone—the first reaction in steroidogene-
nothing short of astonishing. sis (Strushkevich et al., 2011; Hanukoglu, 1992). Several more
CYPs are specialized to advance pregnenolone metabolism to
What’s My Ligand? the individual hormonal products. This unique relationship be-
Classic endocrinology identified thyroid hormone and vitamins A tween steroid receptors and CYPs (as the ligand generators) ex-
and D as vital factors; however, these hormones bind their re- tends to virtually all nuclear receptors with endogenous ligands.
ceptors with high affinity, similar to the classic steroid hormones. Distinct CYPs play central roles in the production of vitamins A
In contrast, the reverse endocrinology strategy allowed for the and D for the RARs, RXRs, and VDR, as well for synthesis of oxy-
identification of lower-affinity endogenous ligands that were sterols for LXRs, bile acids for FXRs, and fatty acids and arach-
derived from dietary lipids (Kliewer et al., 1999). Furthermore, a idonic acid for PPARs (Blumberg and Evans, 1998; Kliewer et al.,
major role of the receptors for these lipid-derived ligands was 1998; McSorley and Daly, 2000; Ray et al., 1997; Russell, 2009).
to maintain the homeostasis of the ligands themselves. Thus, By virtue of their daily role in hormone synthesis, CYPs also
the natural ligands being identified turned out to be predictive take center stage in receptor ligand inactivation and clearance,
hallmarks of the physiologic pathways being regulated by their a process mostly driven in the liver by the phase I, II, and III
cognate receptors. For example, the finding that fatty acids are response, which are controlled in large part by the xenobiotic re-
endogenous ligands for PPARs led to the discovery that PPARs ceptors PXR and CAR. In addition to recognition of foreign com-
govern fatty acid metabolism. Likewise, the binding of choles- pounds (xenobiotics), these receptors also recognize and are
terol metabolites by LXR predicted its future role in controlling activated by endobiotic compounds: steroids, sterols, retinoids,
cholesterol metabolism. This key bit of fortune, though in retro- thyroid hormone, and bile acids (Kliewer et al., 1998; Blumberg
spect may appear obvious, was decidedly not so at the time. et al., 1998; Wei et al., 2000; Zelko and Negishi, 2000).
Because the simple chemical structure of the ligand could fore- In understanding the unique evolution of nuclear receptors as
tell the intrinsic function of the receptor, ligand discovery ligand-dependent transcription factors, it is of interest to note
became the clarion call of the field. Indeed, physiologic links that an important function of nuclear receptors is to autoregulate
were quickly established in the growing list of orphan receptors their own activity by governing the transcription of the specific
that were being adopted into the nuclear receptor superfamily. CYPs that, in turn, control the concentration of their cognate
These included fatty acid metabolism for the PPARs, sterol ho- ligands. Examples of this regulatory circuit include the feedback
meostasis for the liver X receptors (LXRs), bile acid homeostasis inhibition of steroid hormone synthesis via the hypothalamic pitu-
for the farnesoid X receptor (FXR), and endobiotic/xenobiotic itary target organ axis (Dallman et al., 1994; Seminara and Crow-
metabolism for pregnane X and constitutive androstane recep- ley, 2001), bile acid synthesis via the enterohepatic axis (de
tors (PXR and CAR) (Chawla et al., 2001; O’Malley, 1990). Of Aguiar Vallim et al., 2013), and the feedforward regulation of oxy-
further import, these discoveries elaborated the existence of a sterol, fatty acid, and vitamin D syntheses that are governed by
nuclear receptor superfamily and elucidated the unifying mech- their respective receptors (Chawla et al., 2001; Ory, 2004). Taking
anism by which chemically diverse classes of hormones and bio- this regulatory circuit one step further, the ‘‘make it and break it’’
logically active lipids coordinately regulate gene expression and process uses a special class of orphan nuclear receptors,
diverse physiological pathways. including SF-1, DAX-1, LRH-1, and SHP, to sequentially produce
and eliminate ligands for receptors (PXR and CAR) so that the ho-
Make It and Break It meostatic process can be maintained. Furthermore, given the
A defining feature of hormone action is the dynamic synthesis hydrophobic nature of nuclear receptor ligands, ligand transport
and subsequent degradation of receptor ligands. After all, this is coordinately regulated by the cognate receptor, including the
feature is what drives the recurring nature of the signaling circuits production of specific binding proteins and cellular transporters
within the physiologic process. Ligand synthesis and its subse- (Pardridge, 1981; Watanabe et al., 1991).
quent distribution deliver a body-wide hormonal response via
activation of receptor-mediated transcription, whereas ligand The Heterodimer Rule
degradation limits both duration and intensity of the response As transcription factors, nuclear receptors rely on DNA
by returning the system to its homeostatic baseline, where it is sequence-specific binding to transactivate their target genes.

Cell 157, March 27, 2014 ª2014 Elsevier Inc. 257

Table 1. Mammalian Nuclear Receptor Nomenclature and Ligands
Common Unified
Common Name Abbreviation Nomenclature Ligands
Androgen receptor AR NR3C4 androgens
Constitutive androstane receptor CAR NR1I3 xenobiotics
Chicken ovalbumin upstream promoter-transcription factor a COUP-TFa NR2F1
Chicken ovalbumin upstream promoter-transcription factor b COUP-TFb NR2F2
Chicken ovalbumin upstream promoter-transcription factor g COUP-TFg NR2F6
Dosage-sensitive sex reversal-adrenal hypoplasia congenital DAX-1 NR0B1
critical region on the X chromosome, gene 1
Estrogen receptor a ERa NR3A1 estrogens
Estrogen receptor b ERb NR3A2 estrogens
Estrogen related receptor a ERRa NR3B1
Estrogen related receptor b ERRb NR3B2
Estrogen related receptor g ERRg NR3B3
Farnesoid X receptor a FXRa NR1H4 bile acids
Farnesoid X receptor ba FXRb NR1H5
Germ cell nuclear factor GCNF NR6A1
Glucocorticoid receptor GR NR3CI glucocorticoids
Hepatocyte nuclear factor 4 a HNF4a NR2A1 [fatty acids]
Hepatocyte nuclear factor 4 g HNF4g NR2A2 [fatty acids]
Liver receptor homolog-1 LRH-1 NR5A2 [phospholipids]
Liver X receptor a LXRa NR1H3 oxysterols
Liver X receptor b LXRb NR1H2 oxysterols
Mineralocorticoid receptor MR NR3C2 mineralocorticoids and glucocorticoids
Nerve-growth-factor-induced gene B NGF1-B NR4A1
Neuron-derived orphan receptor 1 NOR-1 NR4A3
Nur-related factor 1 NURR1 NR4A2
Photoreceptor-cell-specific nuclear receptor PNR NR2E3
Peroxisome proliferator-activated receptor a PPARa NR1C1 fatty acids
Peroxisome proliferator-activated receptor b/d PPARb/d NR1C2 fatty acids
Peroxisome proliferator-activated receptor g PPARg NR1C3 fatty acids
Progesterone receptor PR NR3C3 progesterone
Pregnane X receptor PXR NR1I2 endobiotics and xenobiotics
Retinoic acid receptor a RARa NR1B1 retinoic acids
Retinoic acid receptor b RARb NR1B2 retinoic acids
Retinoic acid receptor g RARg NR1B3 retinoic acids
Reverse-Erb a REV-ERBa NR1D1 [heme]
Reverse-Erb b REV-ERBb NR1D2 [heme]
RAR-related orphan receptor a RORa NR1F1 [sterols]
RAR-related orphan receptor b RORb NR1F2 [sterols]
RAR-related orphan receptor g RORg NR1F3 [sterols]
Retinoid X receptor a RXRa NR2B1 9-cis retinoic acid and docosahexanoic acid
Retinoid X receptor b RXRb NR2B2 9-cis retinoic acid and docosahexanoic acid
Retinoid X receptor g RXRg NR2B3 9-cis retinoic acid and docosahexanoic acid
Steroidogenic factor 1 SF-1 NR5A1 [phospholipids]
Short heterodimeric partner SHP NR0B2
Tailless homolog orphan receptor TLX NR2E1
Testicular orphan receptor 2 TR2 NR2C1
Testicular orphan receptor 4 TR4 NR2C2
(Continued on next page)

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Table 1. Continued
Common Unified
Common Name Abbreviation Nomenclature Ligands
Thyroid hormone receptor a TRa NR1A1 thyroid hormones
Thyroid hormone receptor b TRb NR1A2 thyroid hormones
Vitamin D receptor VDR NR1I1 1a,25-dihydroxyvitamin D3 and lithocholic acid
Ligands in brackets indicate atypical ligands that, by structural studies, appear to be constitutively bound to their receptors.
a
FXRb exists only as a pseudogene in humans.

Utilizing a combination of approaches that included the cotrans- This plasticity allowed the heterodimer rule to expand from the
fection assay and the electrophoretic mobility shift assay, it was prototypical 3-4-5 rule to include DR1 through DR5 response
determined that the classic steroid receptors (GR, PR, AR, and elements selective for each RXR/receptor partner pair. Although
ER) bind as homodimers to response elements configured as in nature RXR heterodimers have adapted the ability to bind
palindromes composed of two hexad nucleotide sequences response elements that vary from the canonical direct repeats,
separated by three base pairs (Beato, 1991). the veracity of the general rule has been confirmed by numerous
In contrast, nonsteroid receptors (i.e., RAR, VDR, and TR), genome-wide binding analyses.
bind preferentially to response elements composed of two The crystal structures of the heterodimeric unit on DNA have
hexad half-sites arranged as tandem repeats (Koenig et al., elucidated the molecular basis for these interactions (Rastinejad
1987; Näär et al., 1991; Umesono et al., 1991). Further, the spec- et al., 1995; Chandra et al., 2008; Lou et al., 2014). The power of
ificity of each of these receptor/DNA interactions was shown to the direct repeat rule is that it offered every heterodimer a molec-
be encoded uniquely by the nucleotide spacing between the ular binding target even in advance of knowledge of its regulatory
two half-sites of the direct repeat. Thus, the response element function. Even so, with five different binding sites (six if we
for vitamin D was shown to be a direct repeat spaced by three include the single inverted repeat for RXR/FXR), there are still
nucleotides; for thyroid hormone, the spacing is four nucleo- more heterodimers than binding sites. Thus, as would be ex-
tides, and for retinoic acid, it is five nucleotides (an axiom that pected, individual sites can be the target of multiple receptor het-
became known as the 3-4-5 rule) (Figure 3) (Perlmann et al., erodimers. This promiscuity adds a layer of complexity that may
1993; Umesono et al., 1991). However, perhaps the most striking be explained, in part, by tissue-specific expression of individual
difference between these receptors and their steroid-binding, receptor complexes or perhaps features in the chromatin envi-
homodimeric counterparts was that the nonsteroid receptors ronment that help to ‘‘craft’’ specific binding environments.
bind DNA as part of a heterodimer in which their common partner Another interesting general property of the RXR heterodimer is
is RXR (Yu et al., 1991; Kliewer et al., 1992; Bugge et al., 1992; that, with only one or two special exceptions, the heterodimer
Leid et al., 1992; Marks et al., 1992; Zhang et al., 1992). There partners are all ligand dependent. Indeed, a curious evolutionary
are now known to be three RXRs (RXRa, RXRb, and RXRg), at oddity of the nuclear receptor superfamily has been the rather
least one of which is expressed in every cell in the body. The surprising finding that not all of the orphans have ligands that
three RXR proteins are highly conserved and functionally inter- bind in a reversible, regulatory fashion. For those orphan recep-
changeable both as heterodimer partners and as receptors for tors in which such ligands have not been forthcoming, structural,
9-cis retinoic acid (Mangelsdorf et al., 1992). biochemical, and physiological evidence indicate that the ligand
The paradigm of RXR heterodimerization quickly expanded binding pocket is either absent or occupied continually by a lipid
into the universe of orphan receptors that now included the (Wisely et al., 2002), phospholipid (Krylova et al., 2005; Li et al.,
PPARs, LXRs, FXR, PXR, and CAR. The success of this para- 2005), or hydrophobic molecule such as heme (Raghuram
digm was due in large part because it provided a simple but et al., 2007; Reinking et al., 2005; Yin et al., 2007). Consequently,
elegant solution to the evolution of target gene specificity. On these orphan receptors generally behave as constitutive activa-
its own, RXR was shown to function as a self-sufficient homo- tors or repressors of transcription. With respect to their DNA
dimer, binding to a direct repeat of half-sites separated by one binding, they also function predominantly as monomers or
nucleotide (i.e., a DR1 element). The DR1 sequence was ideally homodimers.
poised for evolution because it only required sequential intro- Of the 48 nuclear receptors in humans, approximately half of
duction of single nucleotides in the spacer between the two them fall into the subclass lacking traditional ligands. This obser-
DR1 half-sites to generate novel binding motifs (DR2, DR3, vation is of evolutionary interest, as this subclass comprises the
DR4, etc.). A key driver of that process was the innate structure most ancient members of the nuclear receptor superfamily, and
of the RXR ligand binding domain that permits it to adopt multiple homologs are found all the way down to the most primitive inver-
conformations and thereby dimerize with different nuclear re- tebrate species. Notably, the only invertebrate species in which
ceptors when bound to each of the direct repeat motifs (Rastine- ligand-dependent nuclear receptors have been discovered thus
jad et al., 1995; Chandra et al., 2008; Lou et al., 2014). In this way, far have been nematodes and arthropods. In C. elegans, for
each new heterodimer partner can bind to one of the half-sites, example, only one out of 284 nuclear receptors has been charac-
whereas RXR can continue to occupy the other because of the terized as ligand dependent (Motola et al., 2006; Taubert et al.,
flexibility of its dimerization domain (Perlmann et al., 1993). 2011). In Drosophila, which has 21 nuclear receptors, only two

Cell 157, March 27, 2014 ª2014 Elsevier Inc. 259

 Figure 2. The RXR Big Bang
The cloning of the RXRs as receptors for 9-cis
retinoic acid initiated an expanding wave of dis-
coveries that included (1) the ability of RXRs to
heterodimerize with numerous other nuclear re-
ceptors as a mechanism to control gene-specific
transcription; (2) the characterization of dietary
lipid metabolites (i.e., oxysterols, bile acids, fatty
acids, and xenobiotic lipids) as ligands for
RXR-partnered orphan receptors; (3) the elucida-
tion of novel endocrine and paracrine signaling
pathways mediated by RXR heterodimers, in-
cluding the fibroblast growth factors 1, 19, 21, and
23; and (4) the role of these receptors in diverse
developmental and metabolic pathways. BMR,
basal metabolic rate.

et al., 2000). From a pharmacologic

perspective, a number of potent synthetic
RXR ligands (called ‘‘rexinoids’’) have
also been described (Mukherjee et al.,
1997), and because of their ability to
simultaneously activate several hetero-
have been confirmed to have ligands (Fahrbach et al., 2012). One dimers, such panagonists may have utility against multiple ther-
of these is the ecdysteroid receptor, and it is of particular interest apeutic targets (Desvergne, 2007). Although the pharmacologic
because it functions as an obligate heterodimer with importance of RXR as a therapeutic target is clear, proof of the
ultraspiracle, the RXR homolog in insects (Oro et al., 1990; Yao physiologic importance of endogenous RXR ligands awaits ex-
et al., 1992). Thus, the appearance of RXR heterodimerization periments to effect genetic loss of ligand binding in both individ-
during evolution has been coincident with the dramatic expan- ual and combinations of each of the RXR isoforms.
sion of ligands for these receptors. In contrast to the lipid-sensing permissive partners, the
nonpermissive partners (e.g., TRs, VDR, and RARs) function pri-
The Silent Partner marily as hormone receptors and have endocrine ligands that are
An inherent characteristic of the receptor heterodimer is its under tight regulatory control. By silencing RXR activity and re-
potential to be activated by either the RXR ligand or the partner sponding only to their own ligands, these receptors permit tran-
receptor ligand. This structural reflection of dual-ligand regula- scriptional regulation that is directly proportional to the level of
tion can be divided into two categories: permissive and nonper- the hormone, thereby meeting the requirements of endocrine
missive heterodimers (Forman et al., 1995; Kurokawa et al., physiology (Shulman and Mangelsdorf, 2005).
1993). Permissive heterodimers are those that can be activated
by ligands of either RXR or its partner, whereas nonpermissive The Orphan Receptor Roadmap to Physiology: Engaging
heterodimers are those that can only be activated by the the Energy Vector
partner’s ligand while RXR is silent. An important regulatory Homeostasis is essentially a cooperative equilibrium among
feature of permissive receptor partners (PPARs, LXRs, FXR, cells, tissues, and organs that relies on a rigorous communica-
PXR, and CAR) is that the simultaneous presence of both RXR tion and sensory network as a means to sustain bodily function
and partner receptor ligands results in a cooperative, synergistic in all metazoans. In contrast, bacteria and yeast effectively live
response compared to that resulting from binding of only a single in their surrounding environment such that every cell must
receptor ligand (Leblanc and Stunnenberg, 1995). compete for limiting nutrients. In this context, collective cell pro-
Dual-ligand regulation of permissive heterodimers can have liferation usually continues until food sources are depleted and
both physiologic and pharmacologic consequences. It is of there is no need for a ‘‘physiologic mechanism,’’ as all cells are
physiologic relevance that all of the permissive receptor partners equal.
respond to dietary-derived lipids, whose levels can vary widely. However, in metazoan physiology, cells, tissues, and organs
Yet, because of their synergistic relationship with the RXR ligand, serve compartmentalized functions such that the internal envi-
even relatively small changes in these lipids can result in robust ronment is distinct from the external environment—thus, the
transcriptional activity and thus achieve a profound biological organism must have dynamic, adaptive, and codependent
response. Although endogenous RXR ligands have not been communication and metabolism at all times. Therefore, real-
confirmed in every tissue, it is conceivable that the presence of time control over each metabolic pathway is an essential feature
even trace amounts of such ligands would enable a synergistic of the homeostatic process. This modulation requires key regu-
response. With that in mind, it is of interest to note that, in addi- latory sensors to be in place that can monitor levels of specific
tion to 9-cis retinoic acid, other tissue-specific RXR ligands (e.g., metabolites and control rate-limiting nodes to reduce or enhance
docosahexanoic acid in brain) have been identified (de Urquiza specific routes of energy flux.

260 Cell 157, March 27, 2014 ª2014 Elsevier Inc.

 the liver transforms and delivers nutrients, and active tissues
burn nutrients, whereas adipose collects unused energy for
long-term storage without being a major consumer—effectively
representing an altruistic function.
Whereas the FXR/LXR/PPAR axis is on the front end of nutrient
acquisition, the xenobiotic receptors PXR and CAR are part of
the clearance system that allows the body to rid itself of toxic
dietary metabolites, drugs, and endobiotics (such as steroid,
Figure 3. The 3-4-5 Rule retinoid, and thyroid hormones) (Willson and Kliewer, 2002).
Receptors bind DNA as monomers to a single hexad motif, as homodimers to a The ability of these two receptors to be activated by hundreds
palindrome of two hexad motifs, or as RXR heterodimers to a tandem repeat of of compounds enables the transcription of a detoxification
the hexad motif. Heterodimers bind AGGTCA direct repeats (DRs) spaced by
three (DR3; vitamin D response element), four (DR4; thyroid response element) gene program that creates a clearance vector through which
or five (DR5; retinoic acid response element) nucleotides as described in the toxic body fluids are eliminated through the phase I, II, and III
3-4-5 rule. arms of the xenobiotic response.
During privation, when nutrients are limiting or absent, the
The RXR-partnered receptors play a defining role in this pro- entire energy vector is reversed, and the body now mobilizes
cess. During the fed state, the body switches on a nuclear recep- its stored energy reserves in adipose to use as fuel while at the
tor network that governs the uptake and storage of energy-rich same time suppressing unnecessary energy consuming path-
nutrients, as well as the protection from toxic dietary compo- ways. PPARa is a primary regulator of this adaptive response
nents (Vacca et al., 2011). For example, in the intestine, FXR is to starvation, where, in the liver, it senses the reversed flux of
activated by sensing the postprandial increase in bile acids fatty acids and activates a gene network to convert fatty acids
that both facilitate absorption of lipids as well as activate a com- into a usable energy source (i.e., ketone bodies) (Contreras
plex gene expression program to help control diet-linked micro- et al., 2013). Activation of PPARa also results in the production
bial inflorescence and intestinal inflammation (de Aguiar Vallim of the hepatokine FGF21, which sends a stress signal to other
et al., 2013). This process serves to create an ‘‘energy vector’’ sites in the body, thereby facilitating the body’s adaptation to
into the body while maintaining a barrier to the gut microbiome. an energy-deprived state (Potthoff et al., 2012).
In part, FXR creates the energy vector by inducing nutrient trans- Thus, unlike simple organisms in which the energy demands of
porters to bring food into the body and by activating postprandial each member of the population are approximately equal, in
liver metabolism, through the production of the intestinal hor- metazoans, such demands are highly variable. As direct sensors
mone FGF19, to help insulin build hepatic glycogen reserves of dietary lipids, bile acids, and their metabolites, RXR-partnered
and to recycle and maintain proper bile acid levels (Potthoff receptors are key in controlling the dynamics of energy flow and
et al., 2012). organ communication. This is summarized in Figure 4.
In concert with this process, activation of LXRs by dietary
cholesterol works together with insulin in the liver to promote Novel Therapeutics on the ‘‘Event Horizon’’
lipogenesis, further contributing to the energy vector by promot- An immediate implication that followed from the initial discovery
ing triglyceride production for delivery to peripheral tissues (Cal- of the receptors for the steroid and thyroid hormones and vita-
kin and Tontonoz, 2012). Activation of LXRs in both the gut and mins A and D was their potential as therapeutic targets. Indeed,
the liver also serves to maintain whole-body cholesterol homeo- drugs that target these receptors are among the most widely
stasis by upregulating a gene network that removes excess used and commercially successful. Thus, it should not be sur-
cholesterol from the body through the process of reverse prising that the RXRs and their orphan receptor partners might
cholesterol transport and hepatic catabolism of cholesterol become the next generation of relevant therapeutic targets.
into bile acids. To ensure that proper bile acid homeostasis Indeed, bexarotene and alitretinoin (RXRs), fibrates (PPARa),
and metabolic balance are maintained, activation of FXR in and thiazolidinediones (PPARg) are already approved drugs for
the intestine and liver activates an important feedback loop to treating cancer, hyperlipidemia, and type 2 diabetes, respec-
turn off LXR, inhibit further bile acid synthesis, and reset the tively (Moore et al., 2006). Looking out on the ‘‘event horizon’’
digestive system once the meal is finished. The second tier of of drug discovery, it is notable that FXR and LXR agonists are
this process is engaged in the periphery by the PPARs to either in development for treating nonalcoholic steatohepatitis and
consume or store excess nutrient lipids. In sensing the rising preventing atherosclerosis. Perhaps just as importantly, PXR
levels of fatty acids via enhanced energy flux from the liver, is now used routinely in the pharmaceutical industry to screen
PPARa and PPARd are activated to manage triglyceride and all new drug candidates for potentially dangerous drug-drug
fatty acid metabolism by promoting mitochondrial b-oxidation interactions.
and ATP production in muscle and heart (de Lange et al.,
2008; Madrazo and Kelly, 2008). The excess energy not Beyond the Big Bang
consumed is captured and stored in adipose tissue under the It is worth mentioning that another consequence of the RXR Big
control of PPARg in conjunction with FGF21 and FGF1 to pro- Bang has been the discovery of several other orphan receptors
mote adipose remodeling (Dutchak et al., 2012; Jonker et al., that integrate into the Ring of Physiology (Bookout et al., 2006;
2012; Tontonoz and Spiegelman, 2008). Thus, in a highly coop- Yang et al., 2006). The Nuclear Receptor Ring of Physiology is
erative and integrated fashion, the intestine acquires nutrients, a circular dendogram based on the unsupervised clustering of

Cell 157, March 27, 2014 ª2014 Elsevier Inc. 261

 vate target gene networks. Several studies have pointed to the
role pioneer factors play in collaborating with nuclear receptors
in a cell-type-specific manner (Heinz et al., 2010). In this way,
the same nuclear receptor is able to govern distinct transcrip-
tional networks in different cell types (Miranda et al., 2013).
Cistromes
In the past 20 years, we have learned that a major function of
RXR heterodimers is to act as a reversible switch, which, in the
absence of ligand, recruits a complex of factors (i.e., corepres-
sors) to target gene promoters to repress transcriptional output,
whereas, in the presence of the ligand, the heterodimer recruits
coactivator complexes to open up chromatin and turn up tran-
scription of target genes (O’Malley et al., 2012; Rosenfeld
et al., 2006). This view, although still largely correct, is only one
layer of the complexity of the process. To understand the
Figure 4. Metabolic Homeostasis and the Energy Vector complete logic of how these receptors function, one needs to
The figure illustrates that nutrients, such as sugar and fat, enter the body and
transition from probing single DNA-binding elements near the
are processed in a highly vectorial fashion. Vector 1: the gallbladder, pancreas,
and intestine are the most important digestive organs in the body. Detergent promoter to surveying chromatin interactions at the genome-
properties of bile acids both solubilize lipids to promote lipid absorption and, wide scale. The development of high-throughput techniques to
by activating FXR, to transiently induce hundreds of genes for nutrient trans- analyze these receptor-DNA interactions (e.g., chromatin immu-
port and suppression of microbial activity along with the release of FGF19 as a
hormonal signal to the liver. In the liver, PPARa is activated to break down fatty noprecipitation sequencing, ChIP-seq) has not only confirmed
acids and induce FGF21 as a hormonal signal to adipose (and other tissues in the structure-function rules for nuclear receptor response ele-
the body). LXR induces Cyp7a1 to convert cholesterol into bile acid and ments but has also led to a number of unexpected results. A
SREBP-1c activation for fatty acid synthesis. Vector 2: PPARd activation
during exercise triggers production of sugar and fat in the liver for delivery to
rather surprising finding is that the sum total of individual binding
muscle for both glycolytic and oxidative metabolism. Vector 3: unconsumed sites (cumulatively called a cistrome) for a single receptor in a
energy is sent to fat where, under the control of the PPARg-FGF1 and -FGF21 typical cell may comprise 10,000–25,000 sites within 250–
axis, nutrients are stored in adipose tissue. Vector 4: in response to demand, 1,000 genes (Tang et al., 2011). What do these many thousands
nutrients stored in visceral fat are sent to muscle or other tissues.
of binding events tell us about hormone signaling? Are they all
important, and if not, is there a way to decipher those that are
nuclear receptor mRNA expression in 39 mouse tissues that re- functional from those that are inactive decoys? Minimally, cistro-
vealed the relationship between nuclear receptor expression, mic analysis is valuable as it allows the positioning of RXR-heter-
function, and physiology (Bookout et al., 2006). Many of these re- odimers in relation to specific genes. Unlike steroid receptors
ceptors, such as the RORs and Rev-Erbs that govern circadian that enter the nucleus with ligand treatment, RXR heterodimers
clocks (Guillaumond et al., 2005) and the ERRs that govern remain nuclear (and bound to many of these sites) whether acti-
energy homeostasis (Giguère, 2008), are notable for their coordi- vated by ligand or not. In this context, RXR heterodimers are
nate regulation of physiologic networks that often intersect with unusual in the sense that a single bound factor at a single site
the RXR heterodimers. can cycle as both a positive and negative regulator, depending
Genomic Gymnastics—Dynamic Physiology from on the presence or absence of a ligand. An interesting general
Dynamic Genomes feature from cistromes is that motif analysis can only confirm
An unmet future goal of the nuclear receptor field and, indeed, of binding to known sites. Thus, divergent DNA binding elements
transcription factors in general, is understanding how they may be present, but difficult to identify. In addition, as at least
govern the transcriptional process. As ligand-dependent tran- 1/3 or more binding events have no clear hormone response
scription factors, nuclear receptors like the RXR heterodimers element; perhaps this fraction of chromatin bound nuclear re-
must be understood as a consequence of their ability to interact ceptors is present due to direct recruitment to other transcription
with the genome and promote chromatin remodeling that, via factors (tethering).
epigenetic changes, in turn modulate gene expression. This pro- Although cistromic analysis can uncover multiple cooperative,
cess must be dynamic, affecting elaborate networks of genes interactive, and synergistic relationships, it still represents only a
and global patterns of gene expression. As this regulation is hor- ‘‘snapshot’’ of a very dynamic and possibly short-lived process.
mone dependent, induction of this regulatory triad (DNA binding, Though fragmentary, significant evidence is accumulating that,
chromatin modulation, and transcriptional activation) must be in any one cell type, cistromes can expand and contract depend-
reversed when ligand levels recede. In essence, physiologic ing on developmental or environmental signals that trigger
complexity must arise from genomic complexity reflected by a expression or influx of transcription factors such as FOXa1
coordinated genome-wide interaction of the receptors with NF-kB, SMADs, and STATs into the nucleus. This process clearly
DNA. One key to understanding this physiologic complexity can expose large numbers of cryptic nuclear receptor binding
has come from the discovery of ‘‘pioneering factors’’ that func- elements. As just one example, in hepatic stellate cells, RXR/
tion as tissue-specific transcriptional programmers. By helping VDR heterodimers bind 6,281 sites in quiescent cells and
to guide cell lineage, pioneer factors set the genomic landscape 24,984 sites following LPS and TGFb-activation (Ding et al.,
through which signal-dependent transcription factors can acti- 2013). What triggers movement of RXR/VDR to new sites is not

262 Cell 157, March 27, 2014 ª2014 Elsevier Inc.

known but seems to be important as it allows nuclear receptors identities, regulation, and function of RXR and its heterodimeric
to be recruited to regions of genomic activity, even if these are partners since their original discovery more than 25 years ago.
only transient in nature. The ability to move ensures that hor- Much of this progress has been facilitated by striking advances
mone-responsive gene regulation can participate in the control in physiological techniques and model in vivo systems, as well
of all key gene networks in real-time as these networks are them- as in ‘‘omic’’ technologies. As these technologies have
selves being formed. Thus, the mechanism of ‘‘motional adapt- continued to advance, so have the critical informatic methods
ability’’ is important to explore as it underpins the rapid and to interrogate and compare the massive data being generated.
dynamic features of hormone signaling. Yet, although much has been learned about each of the adopted
Enhancers and Transcriptomes orphan RXR partners, it seems we have only begun to scratch
Although cistromes can be dynamic, they are still a type of the surface on a true understanding of their physiologic func-
‘‘genographical’’ homing mechanism that needs to be placed tions. The challenge represents a computational window into
in context of the enhancer. Enhancers represent the clustering the complexity of the physiologic process itself.
of cis-regulatory elements that can confer both positive and
negative regulation on target genes. In general, the average dis- Conclusions
tance of an enhancer from a promoter is 100 kb, greatly By taking a step back, in this perspective, we have tried to provide
strengthening the idea that the enhancer functions, in part, via a broad overview of the cumulative progress in understanding
DNA looping with the regulated promoter (Sanyal et al., 2012). nuclear receptor function in classic endocrinology and, more
Whether loops themselves are hormone inducible has not been importantly, to chart the expanding impact of the RXR ‘‘Big
established, and whether hormone repression involves loop Bang’’ on new biology. Physiology reflects integrated body-
disruption needs to be addressed. wide processes with receptors, metabolites, ligand, and conjoint
New techniques such as global run-on sequencing (GRO-seq) signaling molecules in a continual orchestration, much of which is
can measure near-instantaneous changes in expression in a sen- involved in the ‘‘trafficking’’ of nutrients throughout the body,
sitive and quantitative fashion. GRO-seq analysis unexpectedly along with hormonal signals that open and close portals to tis-
revealed that upstream enhancers produce short noncoding tran- sues, organs, and cells. Although many of these events are trace-
scripts known as eRNAs (Hah and Kraus, 2014). Recent work able in blood, a more complete understanding will require a much
(Lam et al., 2013) supports the idea that orphan nuclear receptors deeper insight into the brain as a specialized endocrine organ and
can dial up or down the transcriptional output of the eRNA in a into how it oversees what we now call physiologic integration.
fashion linked to the transcriptional activity of the downstream Nevertheless, great advances have been made and remarkable
gene itself (Hah et al., 2011). Although this is still very early days, insights gleaned into dozens of new physiologic pathways, as well
the idea that eRNAs are ligand responsive leads us to wonder as into pathology and treatment through modulation of nuclear re-
whether enhancer transcripts represent the initial level of hormon- ceptor function. Orphan nuclear receptors have now transitioned
al control of gene expression. Addressing this important question through the tipping point as therapeutic targets in the treatment
will undoubtedly comprise a major feature of future analysis. of human disease. Thus, the ‘‘Big Bang’’ is still expanding,
One other class of specialty components of the transcriptome enhancing our insight into a modern definition of physiology with
that deserves mention is the microRNAs, which, by virtue of hy- unlimited potential to uncover the secrets of the human condition.
bridizing with a complementary sequence, repress gene expres-
sion. Their relationship to RXR-heterodimer signaling is just now ACKNOWLEDGMENTS
coming to light. For example, in the case of muscle thyroid hor-
mone signaling, expression of a single miRNA represses type I We thank R. Yu, M. Downes, A. Atkins, S. Kliewer, and N. McKenna for discus-
myofiber formation to produce a fast muscle phenotype. Thus, sions and critical input, J. Simon for artwork, and C. Brondos and E. Ong for
administrative assistance. R.M.E. holds the March of Dimes Chair in Molecular
via their receptor-mediated expression, miRNAs can silence
and Developmental Biology at the Salk Institute and is supported by grants
large networks of genes and thereby play a key role in mediating from the National Institutes of Health (NIH) (DK057978, HL105278,
the hormonal response (Gan et al., 2013; van Rooij et al., 2007; DK090962, HL088093, ES010337 and CA014195), as well as the Helmsley
Williams et al., 2009). Future studies are needed to catalog and Charitable Trust, Samuel Waxman Cancer Research Foundation, and Ipsen/
characterize nuclear receptor-regulated miRNAs and their Biomeasure. D.J.M. is supported by the NIH (R01DK067158) and the
contribution to both tissue and systemic physiology. Robert A. Welch Foundation (grant I-1275). R.M.E. and D.J.M. are investiga-
tors of the Howard Hughes Medical Institute.
Although genome-wide DNA sequencing technology helped
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