Nuclear Receptor Rev-Erba: Up, Down, and All Around

Review

Nuclear receptor Rev-erba: up, down,

and all around

Logan J. Everett and Mitchell A. Lazar

Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Department of Genetics, and The Institute for

Diabetes, Obesity, and Metabolism, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA

Rev-erba is a nuclear receptor that links circadian the epigenome and the recruitment of the core transcrip-

rhythms to transcriptional control of metabolic path- tion machinery [19,20]. In particular, the nuclear corepres-

ways. Rev-erba is a potent transcriptional repressor sor complex consists of core proteins such as nuclear

and plays an important role in the core mammalian corepressor 1 (NCoR), which directly interacts with NR

molecular clock while also serving as a key regulator proteins in a conformation-dependent manner [21,22], and

of clock output in metabolic tissues including liver and epigenomic modifying enzymes such as histone deacety-

brown adipose tissue (BAT). Recent ﬁndings have shed lase 3 (HDAC3), which deacetylates histone protein tails to

new light on the role of Rev-erba and its paralog Rev- create a repressive chromatin environment [23]. Recent

erbb in rhythm generation, as well as additional regula- work on Rev-erba has revealed crucial and genome-wide

tory roles for Rev-erba in other tissues that contribute to interactions with speciﬁc components of the corepressor

energy expenditure, inﬂammation, and behavior. This complex in the liver [10]. These ﬁndings and their potential

review highlights physiological functions of Rev-erba implications on the mechanism of gene regulation by

and b in multiple tissues and discusses the therapeutic Rev-erba in liver and other tissues are discussed below.

potential and challenges of targeting these pathways in

Rev-erba is a dedicated repressor of transcription

human disease.

Rev-erba and b both lack the C-terminal helix that is

crucial for ligand-dependent recruitment of coactivators

Rev-erba: a nuclear receptor linking circadian rhythms

and transcriptional activation by NRs [19]. As a result,

and metabolism

Rev-erba is a potent repressor of transcription [24] and

Rev-erba (also known as NR1D1) is a member of the

interacts constitutively with the NR corepressor NCoR via

nuclear receptor (NR) superfamily of ligand-regulated

its C-terminal ligand-binding domain [25,26]. Rev-erba

transcription factors (TFs) [1]. It was discovered in

has also been shown to interact with the closely related

1989 and mapped to the reverse strand of the gene

corepressor silencing mediator of retinoid and thyroid

encoding another NR, thyroid hormone receptor a [2,3].

receptors (SMRT, also known as NCoR2), although this

A highly similar factor, Rev-erbb (also known as NR1D2),

requires distinct regions of the Rev-erba C terminus and

was identiﬁed in 1994 by multiple laboratories [4–7],

does not occur on DNA under conditions where NCoR is

although the functional connection between these two

bound to Rev-erba [27], potentially because the SMRT

paralogs is only now beginning to be understood. Recent

interaction is weaker [28]. NCoR has several short (<20

studies involving a combination of genetic, genomic, bio-

amino acids) nuclear receptor interaction domains referred

chemical, and pharmacological techniques have revealed

to as corepressor-NR (CoRNR) boxes [29–31]. Rev-erba

that Rev-erba and b play crucial roles in circadian

binds preferentially to the more N-terminal CoRNR1

rhythm generation [8,9] as well as in the normal function

[28] which forms an antiparallel b-sheet in the crystal

of many tissues [10–14]. Furthermore, the endogenous

structure of the complex [32].

ligand for Rev-erba is the metabolite heme [15,16] which

Molecular heme functions as a diffusible, saturable

is involved in mitochondrial respiration and cellular re-

ligand for Rev-erba and further stabilizes its interaction

dox balance [17]. Thus, Rev-erba also functions as a

with full-length, endogenous NCoR [15,16]. The structure

sensor for the metabolic state of the cell and likely

of heme bound to Rev-erbb shows that heme binds in a

entrains the clock to metabolic cues [15,18]. The emer-

prototypical NR ligand-binding pocket [33]. In addition to

gence of Rev-erba as a transcriptional link from circadian

serving as a heme-sensor, the oxidation state of the heme

rhythms to metabolism in multiple tissues is the main

iron may regulate Rev-erb activity [33], although this has

focus of this review.

not been universally observed [15,16] and future work is

NR proteins recruit coregulator complexes to speciﬁc

needed to clarify this important point.

genomic regions, and the complexes in turn impact upon

Rev-erba recruits NCoR/SMRT to the genome by bind-

Corresponding author: Lazar, M.A. ([email protected]).

ing to DNA in a sequence-speciﬁc manner, with the pre-

Keywords: Rev-erba; circadian rhythm; nuclear receptor; metabolism;

ferred binding site consisting of a classical NR half-site transcriptional regulation.

AGGTCA ﬂanked by an A/T-rich 5 sequence (typically

1043-2760/

the RORE because it is also bound by the retinoic acid

586 Trends in Endocrinology and Metabolism, November 2014, Vol. 25, No. 11

Review Trends in Endocrinology and Metabolism November 2014, Vol. 25, No. 11

receptor-related orphan receptor (ROR), which opposes

(A)

Rev-erba function by activating transcription [6,35–37]. Rev-erb Compeve

The Rev-erba DNA-binding domain (DBD) binds in the binding Target gene

acvaon

major groove of the AGGTCA core sequence, while a C-

ROR

terminal helical extension of the DBD makes minor groove

contacts with the A/T-rich sequence of the RORE [38]. RORE

Two Rev-erba molecules are required for a productive

interaction with NCoR [27]. This can occur as two Rev-erba AANTAGGTCA

monomers bound to independent ROREs or, more strongly

and cooperatively, as a dimer bound to a direct repeat of the

(B) Histone

RORE separated by 2 bp (referred to as the Rev-erb DR2 Ac deacetylaon

site, or RevDR2) [24,39]. ROR proteins are also capable of HDAC3

Heme

binding RevDR2 elements, primarily as a monomer, and Target gene

NCoR repression

drive transcriptional activation from these sites, similar to

their role at the RORE [40]. Notably, Rev-erba binds with Rev-erbα Rev-erbα

greater stability at RevDR2 sites, whereas ROR binds with

RORE RORE

greater stability at monomeric RORE sites [41], suggesting

that RevDR2 are dominantly controlled by Rev-erba. Rev-

erba binding conﬁgurations and mechanisms of transcrip-

tional regulation on DNA are shown in Figure 1. (C) Histone deacetylaon

Ac HDAC3

Circadian expression and function of Rev-erba

Shortly after its discovery, Rev-erba was implicated in

NCoR Target gene

aspects of metabolism including adipocyte differentiation repression

[42] and myogenesis [43]. However, mice engineered to α α

lack Rev-erba appeared relatively normal [44]. A major Rev-erb Rev-erb

breakthrough occurred in 1998 when Rev-erba emerged at RevDR2

the top of the list of transcripts whose expression oscillated

with a circadian rhythm in a cell-autonomous manner [45].

AANTAGGTCANNAGGTCA

In mice, Rev-erba mRNA levels were found to exhibit

robust circadian oscillation in multiple tissues TRENDS in Endocrinology & Metabolism

[37,46,47], and genetic ablation of Rev-erba in mice was

Figure 1. Binding configurations of Rev-erba. (A) At single RORE [retinoic acid

shown to shorten the period of behavioral rhythms by 0.5 h receptor-related orphan receptor (ROR) element] sites, Rev-erba can inhibit

transcription passively by competing for binding with ROR proteins. (B) Two

in the absence of daily light cues [48]. These studies also

Rev-erba proteins bound independently to separate RORE motifs can recruit

showed that Rev-erba was not strictly required for rhythm

nuclear corepressor 1 (NCoR) in a heme-dependent manner. The NCoR complex

generation, leading to the suggestion that it serves in an recruits histone deacetylase 3 (HDAC3), which deacetylates surrounding histone

tails and represses target gene transcription. (C) A Rev-erba dimer bound to a

auxiliary loop feeding back on the transcription of the core

RevDR2 (Rev-erb direct repeat separated by 2bp) motif can also recruit NCoR, with

clock component Bmal1 to stabilize circadian oscillations

similar regulatory effects as in (B).

[48]. Subsequent studies have implicated transcriptional

repression by Rev-erba in the rhythmicity of additional

circadian regulators, including Clock [49], Cry1 [50], Nﬁl3 b abrogated circadian gene expression in this system [8].

[51], and Npas2 [52]. Thus, Rev-erba is a highly connected Moreover, genetic ablation of both Rev-erba and b in adult

component of the molecular clock and has the potential to mice resulted in arrhythmic wheel-running behavior in

alter overall cellular oscillations through regulatory inter- both the presence or absence of light entrainment cues

actions with multiple genes. [9]. These ﬁndings demonstrate that Rev-erba and b are

necessary, although redundant, components of the core

Rev-erba in the core molecular clock: back-up by Rev- clock machinery. Rev-erba appears to be more important

erbb because its absence results in mild disruptions to circadian

Overexpression of Rev-erba in mouse liver suppressed 90% rhythms, in contrast to the relatively inconsequential loss

of cycling transcripts, suggesting a potentially broader of Rev-erbb alone [8,9,48].

impact on circadian rhythm than predicted by the deletion

of Rev-erba [53]. More recent studies on the simultaneous Rev-erba in liver: orchestrating an epigenomic rhythm

disruption of Rev-erba and b have now revealed an essen- and lipid metabolism

tial, although partially redundant, role for Rev-erba in The liver is a central tissue for whole-body metabolic

circadian rhythm generation. Rev-erba and b are homeostasis, and Rev-erba has long been known to regu-

expressed with very similar circadian patterns, with both late expression of the core clock gene Bmal1 in liver cells

proteins peaking in mouse liver at zeitgeber time (ZT) 10, [48,54]. Studies within the past decade have also shown an

although Rev-erba oscillation has a greater amplitude [8]. important role for Rev-erba in regulating whole-body me-

Depletion of either of the Rev-erb proteins has a minimal tabolism via the control of cholesterol and bile acid metab-

effect on the cell-autonomous circadian clock in mouse olism in liver [51,55] as well as regulating apolipoprotein

embryonic ﬁbroblasts, but loss of both Rev-erba and CIII [56,57]. More recently, the liver has become a key

587

Review Trends in Endocrinology and Metabolism November 2014, Vol. 25, No. 11

tissue for investigating the molecular mechanisms under- Rev-erba is similar to that in liver, peaking at ZT10. This is

lying gene regulation by Rev-erba and remains a central antiphase to the circadian rhythm of body temperature, and

tissue likely to mediate the effects of Rev-erba on whole- mice lacking Rev-erba were shown to have an attenuated

body metabolic homeostasis. nadir in temperature oscillation owing to derepression of

Rapid advances in genomic techniques have enabled uncoupling protein 1 (UCP1), a direct target of Rev-erba in

mapping of the complete set of binding sites, or ‘cistrome’, BAT [13]. Rev-erba was also shown to play a key role in

for Rev-erba in liver [10]. Rev-erba binds to thousands of defending body temperature against cold-challenge such

genomic locations at ZT10, when its expression is maximal, that mice either genetically lacking Rev-erba or at the

but to very few sites at ZT22, when its expression is nearly trough of normal Rev-erba expression are protected from

absent. Thus, the circadian expression of Rev-erba drives extreme cold.

its rhythmic binding genome-wide. Pathway analysis The marked effect of Rev-erba deletion on body temper-

revealed that Rev-erba-bound genes were strongly ature regulation suggests that Rev-erbb is not redundant

enriched in functions involved in lipid metabolism and, in BAT, although it remains to be determined whether the

correspondingly, Rev-erba null mice were found to have loss of both Rev-erbs would be even more dramatic, as in

hepatic steatosis [10]. liver. It should also be noted that strong Rev-erba binding

The liver Rev-erbb cistrome is very similar to that of was observed at the UCP1 gene in BAT but not in liver,

Rev-erba, and the binding sites for both Rev-erbs are highlighting the tissue speciﬁcity of the genomic binding of

highly enriched for the RORE and RevDR2 motifs [8]. Rev-erba that is readily apparent from cistromic compar-

Moreover, paralleling the partially redundant roles of isons of BAT and liver.

Rev-erba and b in the core clock, knockdown of Rev-erbb

in liver of Rev-erba null mice caused a further increase in Rev-erba in skeletal muscle: a promoter of energy

hepatic lipid content, although knockdown of Rev-erbb had expenditure

no effect on the liver of wild type mice [8]. Inducible genetic Skeletal muscle is a crucial peripheral tissue impacting

ablation of both Rev-erba and b in adult mice also dis- metabolic homeostasis, insulin sensitization, and glucose

rupted circulating glucose, triglyceride, and free-fatty acid disposal. A role for Rev-erba in skeletal myocytes was ﬁrst

levels, demonstrating an effect on whole-body metabolic shown using C2C12 cultured cells, and here Rev-erba

homeostasis [9]. Thus, similarly to their roles in the core repressed the expression of key genes required for muscle

clock, Rev-erba and b appear to be partially redundant in cell differentiation [43]. Rev-erbb was later shown to share

the maintenance of hepatic lipid homeostasis, with Rev- this role [68] as well as to regulate genes involved in lipid

erba being of greater importance. absorption in C2C12 myocytes [69]. Studies of Rev-erba in

In liver, genome-wide location analysis of the Rev-erba primary skeletal muscle found preferential expression in

corepressor components NCoR and HDAC3 revealed re- speciﬁc ﬁber types, and differential composition of muscle

markable similarity to the Rev-erba cistrome [10]. Thus, at ﬁber types in Rev-erba null mice [70]. Expression proﬁling

ZT10 NCoR and HDAC3 binding overlapped at the vast of NRs across multiple tissues found that Rev-erba mRNA

majority of the thousands of sites of Rev-erba binding, and is expressed in a circadian manner in mouse skeletal

the Rev-erba-dependent recruitment of HDAC3 generates muscle, with a phase similar to that in liver and adipose

a circadian rhythm of the epigenome at genome-wide Rev- [47].

erba binding sites [10]. Intriguingly, very little binding of More recently, overexpression of Rev-erba in C2C12

NCoR and HDAC3 was observed at ZT22, and this is quite myocytes was shown to increase mitochondrial content

surprising given that NCoR and HDAC3 are expressed and activity by modulating the AMP-activated protein

throughout the day and the NCoR complex can, in princi- kinase (AMPK) pathway, which also responds to changes

ple, be recruited to many other NRs and TFs [19,21,58]. in energy availability in the cell [12]. Similar ﬁndings were

The tight connection between the liver genomic binding of made in mouse skeletal muscle, and loss of Rev-erba

Rev-erba, NCoR, and HDAC3 reﬂects a shared function function was shown to reduce mitochondrial content and

because liver-speciﬁc ablation of NCoR or HDAC3 pheno- function, leading to impaired exercise capacity [12]. Sev-

copies the changes in gene expression and hepatic lipid eral genes involved in autophagy and mitophagy were

content resulting from the loss of Rev-erba and b [8,59,60]. shown to be direct targets of repression by Rev-erba in

However, the dedication of NCoR and HDAC3 to Rev-erba muscle, suggesting that Rev-erba inhibits autophagy of

seems to be liver-speciﬁc because recruitment of the core- mitochondria in this tissue, thereby increasing the mito-

pressor complex to the genome is not restricted to Rev-erba chondrial content and oxidative capacity of myocytes [12].

binding sites in macrophages [61–63]. These transcriptomic changes in muscle are not observed

in liver or BAT, and presumably reﬂect muscle-speciﬁc

Rev-erba in adipose tissue: fat storage and sites of Rev-erba recruitment to the genome, although this

thermogenesis requires further cistromic analyses. In addition, it remains

Studies of adipogenic cell lines have suggested that Rev- to be determined whether Rev-erba controls muscle mito-

erba is required for adipocyte differentiation [42,64,65]. chondrial content in a circadian manner.

However, adipose tissue mass is normal or even increased

in mice lacking Rev-erba [44,66], suggesting some redun- Rev-erba in endocrine pancreas function

dancy or compensation in vivo. Recently, a role for Rev-erba Recent studies in mice have demonstrated a role for

was identiﬁed in BAT [13], a major site of thermogenesis in Rev-erba in the function of both the insulin-producing

the body [67]. In mouse BAT the circadian expression of b cells and glucagon-producing a cells of pancreatic islets.

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Review Trends in Endocrinology and Metabolism November 2014, Vol. 25, No. 11

Rev-erba mRNA is expressed in islets and oscillates with a generation, via multiple regions of the brain. Interestingly,

circadian rhythm similar to that of liver [71–73]. Islets many of the behaviors observed in Rev-erba null mice

isolated at the time of peak Rev-erba expression have higher parallel human behaviors observed in bipolar disorder

levels of glucose-stimulated insulin secretion, and Rev-erba [78], which is also linked to Rev-erba by the molecular

regulates gene expression as well as insulin processing, actions of lithium [79] (discussed in the next section).

exocytosis, and proliferation in primary b cells and b cell

lines [73]. Rev-erba has also been shown to promote gluca- Targeting Rev-erba with small molecules

gon secretion from islet a cells [74]. Intriguingly, Rev-erba The activities of many NRs are regulated by direct binding of

was also found to regulate lipogenic genes in mouse islets speciﬁc lipophilic molecules, including both endogenous

[73], similarly to its role in liver [10], although the full set compounds and drugs, which target the ligand-binding

of genome-wide Rev-erba targets remains to be mapped in pocket of the NR protein [80]. The identiﬁcation of heme

the relevant islet cell types. Rev-erbb is also expressed as an endogenous ligand for Rev-erba and b [15,16] demon-

in both a cells and b cells [71], although its functional role strated that Rev-erb activity could be regulated by ligand

remains to be investigated. binding. The crystal structure of heme-bound Rev-erbb

identiﬁed the ligand-binding pocket [33], which is highly

Rev-erba in macrophages and inﬂammation conserved in Rev-erba and could also be the target of syn-

Rev-erba was ﬁrst shown to play a role in blocking proin- thetic ligands. Indeed, several synthetic agonists, deﬁned in

ﬂammatory signals in macrophages by repressing the Toll- the case of Rev-erb as molecules that increase corepressor

like receptor 4 (TLR4) gene, which triggers the innate interaction and repressive function, have been identiﬁed for

immune response to lipopolysaccharide (LPS) associated Rev-erba/b [81–85]. One study suggested that a Rev-erb

with Gram-negative bacteria [75]. More recently, Rev-erba agonist disrupts rhythmic wheel-running behavior and

was shown to mediate the circadian gating of the LPS- circadian hypothalamic gene expression in mice, and can

induced endotoxic response [11]. In human macrophages, also alter whole-body metabolism [82]. The same synthetic

pharmacological activation of Rev-erba led to a decrease in agonist mimicked muscle-speciﬁc overexpression of Rev-

production of the proinﬂammatory cytokine interleukin 6 erba in promoting exercise capacity and muscle mitochon-

(IL-6), whereas loss of Rev-erba increased IL-6. Thus, drial content [12]. These results suggest that Rev-erb

Rev-erba provides a crucial link between the circadian agonists could promote metabolic health, although the pleio-

clock and immune function through direct repression of tropic tissue-speciﬁc effects of Rev-erba, including disrup-

proinﬂammatory gene expression in macrophages. Recent tions to circadian rhythmicity [8,9,48] and increased

mapping of the macrophage Rev-erba and Rev-erbb thermogenesis upon loss of function [13], make Rev-erba

cistromes, together with the transcriptome changes in a complicated drug target. Thus, there is a need to examine

Rev-erba/b double-null macrophages, suggest that Rev- further the function of Rev-erbs and their synthetic ligands

erba controls gene expression in macrophages through in speciﬁc tissues, and extra attention must be paid to the

the repression of enhancer RNA transcription marked by time of day at which Rev-erba ligands are administered. It

lineage-determining TFs such as PU.1 [76]. may prove more effective to target Rev-erba or downstream

pathways in a tissue-speciﬁc manner.

Rev-erba in brain and behavior Rev-erba is also regulated at the level of protein stabili-

Rev-erba expression is circadian in the suprachiasmatic ty, as are other core components of the molecular clock [86].

nucleus (SCN) of the hypothalamus [48], which entrains E3 ligases, such as Arf-bp1 and Pam, ubiquitylate Rev-

other core clocks throughout the body [77], and this likely erba to promote its proteasomal degradation [87], and

controls the phase-advance in locomotor activity charac- glycogen synthase kinase 3 (GSK3) stabilizes Rev-erba

teristic of Rev-erba null mice [48]. Rev-erba null mice were through N-terminal phosphorylation [79]. Lithium, which

also shown to exhibit postnatal developmental delays in is commonly used in the treatment of bipolar disorder [88],

the cerebellum, although this delay was overcome and no inhibits phosphorylation by GSK3, thereby promoting the

cerebellar dysfunction was apparent in adults [44]. degradation of Rev-erba [79]. Subsequently, several

Recently, Rev-erba null mice were reported to exhibit human genetic studies reported associations between poly-

behavioral abnormalities, including novelty-induced hy- morphisms at the Rev-erba locus and responsiveness to

peractivity and impaired memory formation, suggesting lithium treatment among patients exhibiting bipolar dis-

abnormalities in hippocampal function as a result of order [89,90]. Thus, both the protein level and regulatory

alterations in dopaminergic tone [14]. They also exhibited activity of Rev-erba may be targeted with small molecules.

increased aggression, anxiety, and depression-associated

behaviors indicative of dysfunctions in the midbrain do- Concluding remarks and future perspectives

paminergic neurons [78]. Rev-erba expression in both the Within the past 5 years it has become clear that Rev-erba

hippocampus [14] and midbrain [78] is circadian, but with acts in a tissue-speciﬁc manner to regulate circadian

much lower amplitude than in the SCN or other tissues. rhythms as well as crucial metabolic, inﬂammatory, and

Rev-erba was shown to repress several common targets in behavioral functions (Figure 2), in some cases uniquely and

both hippocampus and midbrain, including tyrosine hy- in others redundantly with Rev-erbb. Thus, Rev-erba is a

droxylase, the rate-limiting enzyme in dopamine biosyn- key link between the core clock and numerous physiologi-

thesis [14,78]. Some of the additional behavioral changes cal processes, primarily studied in mice but likely to be

did not appear to be circadian, indicating that Rev-erba relevant to human health. Several open questions remain,

controls other behavioral functions, in addition to rhythm however, particularly with regard to the contributions of

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Acknowledgments

the E region of the orphan receptors. Nucleic Acids Res. 24, 4379–4386

Research on Rev-erb in the laboratory of M.A.L. is supported by National 27 Zamir, I. et al. (1997) Stoichiometric and steric principles governing

Institutes of Health (NIH) grant R01 DK45586. L.J.E. was supported by repression by nuclear hormone receptors. Genes Dev. 11, 835–846

NIH training grant F32 DK095526. 28 Hu, X. et al. (2001) Determinants of CoRNR-dependent repression

complex assembly on nuclear hormone receptors. Mol. Cell. Biol. 21,

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