Rev-Erb alpha (Rev-ErbÃÂ), 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-ERBÃÂ is encoded by the NR1D1 gene, which is highly conserved across animal species.
Rev-ErbÃÂ 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-ErbÃÂ 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.
Rev-Erbàwas 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-ErbÃÂ. The protein was first referenced by the name Rev-Erbàin 1990 by Mitchell A. Lazar, Karen E. Jones, and William W. Chin, who isolated Rev-Erbàcomplementary DNA from a human fetal skeletal muscle library. Similar to the gene in rats, they found that human Rev-Erbàwas transcribed from the strand opposite human thyroid hormone receptor alpha (THRA, c-erbAñ).
Rev-ErbÃÂ was first implicated in circadian control in 1998, when Aurelio Balsalobre, Francesca Damiola, and Ueli Schibler demonstrated that expression of Rev-ErbÃÂ in rat fibroblasts showed daily rhythms. Rev-ErbÃÂ was first identified as a key player in the transcription translation feedback loop (TTFL) in 2002, when experiments demonstrated that Rev-ErbÃÂ acted to repress transcription of the Bmal1 gene, and Rev-ErbÃÂ expression was controlled by other TTFL components. This established Rev-ErbÃÂ as the link between the positive and negative loops of the TTFL.
The NR1D1 (nuclear receptor subfamily 1 group D member 1) gene, located on chromosome 17, encodes the protein REV-ERBàin humans. It is transcribed from the opposite strand of the human thyroid hormone receptor alpha (THRA, c-erbAñ) 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 (RORÃÂ), another nuclear receptor transcription factor. NR1D1 also contains an E-box at its promoter, which allows for regulation by BMAL1. In humans, NR1D1 (REV-ERBÃÂ) 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-Erbàon opposite strands. In humans, NR1D1 has only one paralog, NR1D2 (REV-ERBò), 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.
The human NR1D1 gene produces a protein product (REV-ERBñ) of 614 amino acids. REV-ERBñ 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-Erbñ 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-Erbñ, further stabilizes the interaction with NCoR. The repression by Rev-Erbñ 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-Erbñ 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-Erbñ. Rev-Erbñ 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-Erbñ binds in the major groove of this sequence via its DBD domain, which contains two C4-type zinc fingers. Rev-Erbñ can repress gene activation as a monomer through competitive binding at this RORE site, but two Rev-Erbñ molecules are required for interaction with NCoR and active gene repression. This can occur by two Rev-Erbñ 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-Erbñ, and furthermore, that this domain is necessary for activation of the gap junction protein 1 (GJA1) gene.
Rev-Erbñ 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-Erbñ 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-Erbñ working in conjunction with Rev-Erbò and the orphan receptor RORñ, is thought to strengthen this primary TTFL by further regulating BMAL1. RORñ shares the same response elements as Rev-Erbñ but exerts opposite effects on gene transcription; BMAL1 expression is repressed by Rev-Erbñ and activated by RORñ. CLOCK:BMAL1 expression activates the transcription of NR1D1, encoding the Rev-Erbñ protein. Increased Rev-Erbñ expression in turn, represses transcription of BMAL1, stabilizing the loop. The oscillating expression of RORñ and Rev-Erbñ 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.
Rev-erbñ plays a role in the regulation of whole body metabolism through controlling lipid metabolism, bile acid metabolism, and glucose metabolism. Rev-Erbñ relays circadian signals into metabolic and inflammatory regulatory responses and vice versa, although the precise mechanisms underlying this relationship are not entirely understood.
Rev-erbñ 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-erbñ is associated with the reduction of fatty acid synthase, a key regulator of lipogenesis. Rev-erbñ deficient mice exhibit dyslipidemia due to elevated triglyceride levels and Rev-erbñ polymorphisms in humans have been associated with obesity. Rev-erbñ also regulates adipogenesis of white and brown adipocytes. Rev-Erbñ transcription is induced during the adipogenic process, and over-expression of Rev-erbñ enhances adipogenesis. Researchers have proposed that Rev-erbñ'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-erbñ 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-erbñ plays both indirect and direct roles in glucose metabolism. BMAL1 heavily influences glucose production and glycogen synthesis, thus through the regulation of BMAL1, Rev-erbñ indirectly regulates glucose synthesis. More directly, Rev-erbñ's expression in the pancreas regulates the function of ñ-cells and ò-cells, which produce glucagon and insulin, respectively.
Rev-erbñ 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-erbñ 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-erbñ is the most highly expressed in osteoarthritic cartilage. One study found that in patients with osteoarthritis has reduced Rev-erbñ levels compared to normal cartilage. Research on rheumatoid arthritis (RA) has implicated the potential for treatment with Rev-erbñ agonists to RA patients due to their suppression of bone and cartilage destruction.
Rev-erbñ 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-erbñ 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-erbñ deficient mice no longer exhibit these oscillations, notably in IL-6, IL-12, CCL5, and CXCL1, and CCL2. Rev-erbñ 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-ERBñ promotes RORót expression, and RORót is required for ILC3 expression. Rev-erbñ is highly expressed in ILC3 subsets.
Rev-erbñ has been implicated in the regulation of memory and mood. Rev-erbñ knockout mice are deficient in short term, long term, and contextual memories, showing deficits in the function of their hippocampus. In addition, Rev-erbñ 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-erbñ 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 3ò), an enzyme that phosphorylates and stabilizes Rev-erbñ. Lithium binding to GSK 3ò then destabilizes and alters the function of Rev-erbñ. This research has been implicated in the development of therapeutic agents for affective disorders, such as lithium for bipolar disorder.