Rev-ErbA alpha

Rev-Erb alpha (Rev-Erbɑ), also known as nuclear receptor subfamily 1 group D member 1 (NR1D1), is one of two Rev-Erb proteins in the nuclear receptor (NR) family of intracellular transcription factors. In humans, REV-ERBɑ is encoded by the NR1D1 gene, which is highly conserved across animal species.

Rev-Erbɑ plays an important role in regulation of the core circadian clock through repression of the positive clock element Bmal1. It also regulates several physiological processes under circadian control, including metabolic and immune pathways. Rev-Erbɑ mRNA demonstrates circadian oscillation in its expression, and it is highly expressed in mammals in the brain and metabolic tissues such as skeletal muscle, adipose tissue, and liver.

Discovery
Rev-Erbɑ was discovered in 1989 by Nobuyuki Miyajima and colleagues, who identified two erbA homologs on human chromosome 17 that were transcribed from opposite DNA strands in the same locus. One of the genes encoded a protein that was highly similar to chicken thyroid hormone receptor, and the other, which they termed ear-1, would later be described as Rev-Erbɑ. The protein was first referenced by the name Rev-Erbɑ in 1990 by Mitchell A. Lazar, Karen E. Jones, and William W. Chin, who isolated Rev-Erbɑ complementary DNA from a human fetal skeletal muscle library. Similar to the gene in rats, they found that human Rev-Erbɑ was transcribed from the strand opposite human thyroid hormone receptor alpha (THRA, c-erbAα).

Rev-Erbɑ was first implicated in circadian control in 1998, when Aurelio Balsalobre, Francesca Damiola, and Ueli Schibler demonstrated that expression of Rev-Erbɑ in rat fibroblasts showed daily rhythms. Rev-Erbɑ was first identified as a key player in the transcription translation feedback loop (TTFL) in 2002, when experiments demonstrated that Rev-Erbɑ acted to repress transcription of the Bmal1 gene, and Rev-Erbɑ expression was controlled by other TTFL components. This established Rev-Erbɑ as the link between the positive and negative loops of the TTFL.

Genetics and evolution
The NR1D1 (nuclear receptor subfamily 1 group D member 1) gene, located on chromosome 17, encodes the protein REV-ERBɑ in humans. It is transcribed from the opposite strand of the human thyroid hormone receptor alpha (THRA, c-erbAα) so that NR1D1 and THRA cDNA are complementary on 269 bases. The gene consists of 7,797 bases with 8 exons, forming only 1 splice variant. The NR1D1 promoter itself contains a REV-ERB response element (RevRE), which allows for regulation of gene expression both through autoregulation and regulation by retinoic acid receptor-related orphan receptor alpha (RORɑ), another nuclear receptor transcription factor. NR1D1 also contains an E-box at its promoter, which allows for regulation by BMAL1. In humans, NR1D1 (REV-ERBɑ) is highly expressed in the brain and metabolic tissues, including skeletal muscle, adipose tissue, and the liver.

Genomic analysis suggests that the NR1D1 gene was present in the most recent common ancestor of all animals, with orthologs present in 378 species tested, including chimpanzees, dogs, mice, rats, chickens, zebrafish, frogs, and fruit flies. Comparison to the rat ortholog, Nr1d1, indicates high conservation in the DNA binding and carboxy-terminal domains, as well as conservation of transcription of c-erbA alpha-2 and Rev-Erbɑ on opposite strands. In humans, NR1D1 has only one paralog, NR1D2 (REV-ERBβ), which is located on chromosome 3 and likely arose from a duplication event. However, both NR1D1 and NR1D2 are members of the nuclear receptor family, indicating they share common ancestry. As such, NR1D1 is functionally related to other nuclear receptor genes, such as peroxisome proliferator activated receptor delta (PPARD) and retinoic acid receptor alpha (RARA). Furthermore, studies have shown that the NR1D1/THRA genetic locus is genetically linked to the RARA gene.

Protein structure
The human NR1D1 gene produces a protein product (REV-ERBα) of 614 amino acids. REV-ERBα has 3 major functional domains, including a DNA-binding domain (DBD) and a ligand-binding domain (LBD) at the C-terminus, and a N-terminus domain which allows for activity modulation. These three domains are a common feature of nuclear receptor proteins.

The Rev-Erb proteins are unique from other nuclear receptors in that they do not have a helix in the C-terminal that is necessary for coactivator recruitment and activation by nuclear receptors via their LBD. Instead, Rev-Erbα interacts via its LBD with Nuclear Receptor Co-Repressor (NCoR) and another closely related co-repressor Silencing Mediator of Retinoid and Thyroid Receptors (SMRT), although the interaction with NCoR is stronger due to its structural compatibility. Heme, an endogenous ligand of Rev-Erbα, further stabilizes the interaction with NCoR. The repression by Rev-Erbα also requires interaction with the class I histone deactylase 3 (HDAC3) - NCoR complex. The catalytic activity of HDAC3 is activated only when it complexes with NCoR or SMRT, so Rev-Erbα must interact with this complex in order for gene repression to occur via histone deacetylation. It is still unknown whether other HDACs play a role in the function of Rev-Erbα. Rev-Erbα recruits the NCoR-HDAC3 complex through binding a specific DNA sequence commonly referred to as RORE due to its interaction with the transcriptional activator Retinoic Acid Receptor-related Orphan Receptor (ROR). This sequence consists of an "AGGTCA" half-site preceded by an A/T sequence.. Rev-Erbα binds in the major groove of this sequence via its DBD domain, which contains two C4-type zinc fingers. Rev-Erbα can repress gene activation as a monomer through competitive binding at this RORE site, but two Rev-Erbα molecules are required for interaction with NCoR and active gene repression. This can occur by two Rev-Erbα molecules binding separate ROREs or as a stronger interaction through binding a response element that is a direct repeat of the RORE (RevDR2).

In mice, it has been shown that the N-terminal regulatory domain contains an important site for phosphorylation by casein kinase 1 epsilon (Csnk1e), which aids in proper localization of Rev-Erbα, and furthermore, that this domain is necessary for activation of the gap junction protein 1 (GJA1) gene.

Circadian oscillator
Rev-Erbα has been proposed to coordinate circadian metabolic responses. Circadian rhythms are driven by interlocking transcription/translation feedback regulatory loops (TTFLs) that generate and maintain these daily rhythms, and Rev-Erbα is involved in a secondary TTFL in mammals. The primary TTFL features transcriptional activator proteins CLOCK and BMAL1 that contribute to the rhythmic expression of genes within this loop, notably per and cry. The expression of these genes then act through negative feedback to inhibit CLOCK:BMAL1 transcription. The secondary TTFL, featuring Rev-Erbα working in conjunction with Rev-Erbβ and the orphan receptor RORα, is thought to strengthen this primary TTFL by further regulating BMAL1. RORα shares the same response elements as Rev-Erbα but exerts opposite effects on gene transcription; BMAL1 expression is repressed by Rev-Erbα and activated by RORα. CLOCK:BMAL1 expression activates the transcription of NR1D1, encoding the Rev-Erbα protein. Increased Rev-Erbα expression in turn, represses transcription of BMAL1, stabilizing the loop. The oscillating expression of RORα and Rev-Erbα in the suprachiasmatic nucleus, the principal circadian timekeeper in mammals, leads to the circadian pattern of BMAL1 expression. The occupancy of the BMAL1 promoter by these two receptors is key for proper timing of the core clock machinery in mammals.

Metabolism
Rev-erbα plays a role in the regulation of whole body metabolism through controlling lipid metabolism, bile acid metabolism, and glucose metabolism. Rev-Erbα relays circadian signals into metabolic and inflammatory regulatory responses and vice versa, although the precise mechanisms underlying this relationship are not entirely understood.

Rev-erbα regulates the expression of liver apolipoproteins, sterol regulatory element binding protein, and the fatty acid elongase elovl3 through its repressional activity  In addition, the silencing of Rev-erbα is associated with the reduction of fatty acid synthase, a key regulator of lipogenesis. Rev-erbα deficient mice exhibit dyslipidemia due to elevated triglyceride levels and Rev-erbα polymorphisms in humans have been associated with obesity. Rev-erbα also regulates adipogenesis of white and brown adipocytes. Rev-Erbα transcription is induced during the adipogenic process, and over-expression of Rev-erbα enhances adipogenesis. Researchers have proposed that Rev-erbα's role in adipocyte function may affect the timing of processes such as lipid storage and lipolysis, contributing to long term issues with BMI control. Rev-erbα also regulates bile acid metabolism by indirectly down-regulating Cyp7A1, which encodes the first and rate controlling enzyme of the major bile acid biosynthetic pathway.

Rev-erbα plays both indirect and direct roles in glucose metabolism. BMAL1 heavily influences glucose production and glycogen synthesis, thus through the regulation of BMAL1, Rev-erbα indirectly regulates glucose synthesis. More directly, Rev-erbα's expression in the pancreas regulates the function of α-cells and β-cells, which produce glucagon and insulin, respectively.

Muscle and cartilage
Rev-erbα plays a role in myogenesis through interaction with the transcription complex Nuclear Factor-T. It also represses the expression of genes involved in muscle cell differentiation and is expressed in a circadian manner in mouse skeletal muscle. Loss of Rev-erbα function reduces mitochondrial content and function, leading to an impaired exercise capacity. Over-expression leads to improvement.

This protein has also been implicated in the integrity of cartilage. Out of all known nuclear receptors, Rev-erbα is the most highly expressed in osteoarthritic cartilage. One study found that in patients with osteoarthritis has reduced Rev-erbα levels compared to normal cartilage. Research on rheumatoid arthritis (RA) has implicated the potential for treatment with Rev-erbα agonists to RA patients due to their suppression of bone and cartilage destruction.

Immune system
Rev-erbα contributes to the inflammatory response in mammals. In mouse smooth muscle cells, the protein up-regulates expression of interleukin 6 (IL-6) and cyclooxygenase-2. In humans, it controls the lipopolysaccharide (LPS) induced endotoxic response through repressing toll-like receptor (TLR-4), which triggers the immune response to LPS. In the brain, Rev-erbα deletion causes a disruption in the oscillation of microglial activation and increases the expression of pro-inflammatory transcripts.

Many immune and inflammatory proteins exhibit circadian oscillatory behavior, and research has shown that Rev-erbα deficient mice no longer exhibit these oscillations, notably in IL-6, IL-12, CCL5, and CXCL1, and CCL2. Rev-erbα has also been implicated in the development of group 3 innate lymphoid cells (ILC3), which play a role in regulating intestinal health and are responsible for lymphoid development. REV-ERBα promotes RORγt expression, and RORγt is required for ILC3 expression. Rev-erbα is highly expressed in ILC3 subsets.

Mood and behavior
Rev-erbα has been implicated in the regulation of memory and mood. Rev-erbα knockout mice are deficient in short term, long term, and contextual memories, showing deficits in the function of their hippocampus. In addition, Rev-erbα has been proposed to play a role in the regulation of midbrain dopamine production and mood-related behavior in mice through repression of tyrosine hydroxylase gene transcription. Dopamine related dysfunction is associated with mood disorders, notably major depressive disorder, seasonal affective disorder, and bipolar disorder. Genetic variations in human NR1D1 loci are also associated with bipolar disorder onset.

Rev-erbα has been proposed as a target in the treatment of bipolar disorder through lithium, which indirectly regulates the protein at a post-translational level. Lithium inhibits glycogen synthase kinase (GSK 3β), an enzyme that phosphorylates and stabilizes Rev-erbα. Lithium binding to GSK 3β then destabilizes and alters the function of Rev-erbα. This research has been implicated in the development of therapeutic agents for affective disorders, such as lithium for bipolar disorder.