ARNTL

History

The Arntl gene was originally discovered in 1997 by two groups of researchers, John B. Hogenesch et al. in March and Ikeda and Nomura in April as part of a superfamily of PAS domain transcription factors. The ARNTL protein, also known as MOP3, was found to dimerize with MOP4, CLOCK, and hypoxia-inducible factors. The names BMAL1 and ARNTL were adopted in later papers. One of ARNTL protein's earliest discovered functions in circadian regulation was related to the CLOCK-BMAL1 (CLOCK-ARNTL) heterodimer, which would bind through an E-box enhancer to activate the transcription of the gene encoding vasopressin. However, the gene's importance in circadian rhythms was not fully realized until the knockout of the gene in mice showed complete loss of circadian rhythms in locomotion and other behaviors.

Structure

The BMAL1 protein contains fours domains according to its crystallographic structure: a bHLH domain, two PAS domains called PAS-A and PAS-B, and a trans-activating domain. The dimerization of CLOCK:BMAL1 proteins involves strong interactions between the bHLH, PAS A, and PAS B domains of both CLOCK and BMAL1 and forms an asymmetrical heterodimer with three distinct protein interfaces. The PAS-A interactions between CLOCK and BMAL1 involves an interaction, in which an α-helix of CLOCK PAS-A and the ß-sheet of BMAL1 PAS-A, and an α-helix motif of the BMAL1 PAS-A domain and the ß-sheet of CLOCK PAS-A. CLOCK and BMAL1 PAS-B domains stack in a parallel fashion, resulting in the concealment of different hydrophobic residues on the ß-sheet of BMAL1 PAS-B and the helical surface of CLOCK PAS-B, such as Tyr 310 and Phe 423. Key interactions with specific amino acid residues, specially CLOCK His 84 and BMAL1 Leu125, are important in the dimerization of these molecules.

Circadian

The protein encoded by the Bmal1 gene in mammals binds with a second bHLH-PAS protein via the PAS domain, CLOCK (or its paralog, NPAS2) to form a heterodimer in the nucleus. Via its BHLH domain, this heterodimer binds to E-box response elements in the promoter regions of Per (Per1 and Per2) and Cry genes (Cry1 and Cry2). This binding upregulates the transcription and translation of PER1, PER2, CRY1 and CRY2 proteins.

After the PER and CRY proteins have accumulated to sufficient levels, they interact by their PAS motifs to form a large repressor complex that travels into the nucleus to inhibit the transcriptional activity of the CLOCK:BMAL1 heterodimer This inhibits the transcription of Per and Cry genes, and causes protein levels of PER and CRY drop. This transcription, translation negative feedback loop (TTFL) is modulated in the cytoplasm by phosphorylation of PER proteins by casein kinase 1ε or δ (CK1 ε or CK1 δ), signaling these proteins for degradation by the 26S proteasome. SIRT1 also regulates PER protein degradation by inhibiting transcriptional activity of the BMAL1:CLOCK heterodimer in a circadian manner through deacetylation. The degradation of PER proteins prevents the formation the large protein complex, and thus prevents the inhibition of transcriptional activity of the BMAL1:CLOCK heterodimer. The CRY protein is also signaled for degradation by poly-ubiquination from the FBXL3 protein, also preventing the inhibition of the CLOCK:BMAL1 heterodimer. This allows transcription of the Per and Cry genes to resume. It has also been observed in the TTFL loop of nocturnal mice that transcription levels of the Bmal1 gene peak at CT18, during the mid-subjective night, anti-phase to the transcription levels of Per, Cry, and other clock control genes, which peak at CT6, during the mid-subjective day. This process occurs with a period length of approximately 24 hours and supports the notion that this molecular mechanism is rhythmic.

Regulation of Bmal1 activity

In addition to the circadian regulatory TTFL loop described above, Bmal1 transcription is regulated by competitive binding to the retinoic acid-related orphan receptor response element-binding site (RORE) within the promoter of Bmal1. The CLOCK/BMAL1 heterodimer also binds to E-box elements in promotor regions of Rev-Erbα and RORα/ß genes, upregulating transcription and translation of REV-ERB and ROR proteins. REV-ERBα and ROR proteins regulate BMAL1 expression through a secondary feedback loop and compete to bind to Rev-Erb/ROR response elements in the Bmal1 promoter, resulting in BMAL1 expression repressed by REV-ERBα and activated by ROR proteins. Other nuclear receptors of the same families (NR1D2 (Rev-erb-β); NR1F2 (ROR-β); and NR1F3 (ROR-γ)) have also been shown to act on Bmal1 transcriptional activity in a similar manner.

Several posttranslational modifications of BMAL1 dictate the timing of the CLOCK/BMAL1 feedback loops. Phosphorylation of BMAL1 targets it for ubiquitination and degradation, as well as deubiquitination and stabilization. Acetylation of BMAL1 recruits CRY1 to suppress the transactivation of CLOCK/BMAL1. The sumoylation of BMAL1 by small ubiquitin-related modifier 3 signals its ubiquitination in the nucleus, leading to transactivation of the CLOCK/BMAL1 heterodimer. CLOCK/BMAL1 transactivation, is activated by phosphorylation by casein kinase 1ε and inhibited by phosphorylation by MAPK. Phosphorylation by CK2α regulates BMAL1 intracellular localization and phosphorylation by GSK3B controls BMAL1 stability and primes it for ubiquitination.

Other functions

The Arntl gene is located within the hypertension susceptibility loci of chromosome 1 in rats. A study of single nucleotide polymorphisms (SNPs) within this loci found two polymorphisms that occurred in the sequence encoding for Arntl and were associated with type II diabetes and hypertension. When translated from a rat model to a human model, this research suggests a causative role of Arntl gene variation in the pathology of type II diabetes. Recent phenotype data also suggest this gene and its partner Clock play a role in the regulation of glucose homeostasis and metabolism, which can lead to hypoinsulinaemia, or diabetes, when disrupted. In regards to other functions, another study shows that the CLOCK/BMAL1 complex upregulates human LDLR promoter activity, suggesting the Arntl gene also plays a role in cholesterol homeostasis. In addition, Arntl gene expression, along with that of other core clock genes, were discovered to be lower in patients with bipolar disorder, suggesting a problem with circadian function in these patients. Arntl, Npas2, and Per2 have also been associated with seasonal affective disorder in humans. Lastly, Arntl has been identified through functional genetic screening as a putative regulator of the p53 tumor suppressor pathway suggesting potential involvement in the circadian rhythms exhibited by cancer cells.

Species distribution

Along with mammals such as humans and mice, orthologs of the Arntl gene are also found in fish (AF144690.1), birds (Arntl), reptiles, amphibians (XI.2098), and Drosophila (Cycle, which encodes a protein lacking the homologous C-terminal domain, but still dimerizes with the CLOCK protein). Unlike the mammalian Artnl, however, the drosophila Cycle (gene) is constitutively expressed instead of circadian regulated, meaning that it is present in relatively constant amounts. In humans, three transcript variants encoding two different isoforms have been found for this gene. The importance of these transcript variants is unknown.

Knockout studies

The Arntl gene is an essential component within the mammalian clock gene regulatory network. It is a point of sensitivity within the network, as it is the only gene whose single knockout in a mouse model generates arrhythmicity at both the molecular and behavioral levels. In addition to defects in the clock, these Arntl null-mice also have reproductive problems, are small in stature, age quickly, and have progressive arthropathy that results in having less overall locomotor activity than wild type mice. However, recent research suggests that there might be some redundancy in the circadian function of Arntl with its paralog Bmal2.

Interactions

Arntl has been shown to interact with: