Research shows that the CLOCK gene plays a major role as an activator of downstream elements in the pathway critical to the generation of circadian rhythms.

Takahashi used forward mutagenesis screening of mice treated with N-ethyl-N-nitrosourea to create and identify mutations in key genes that broadly affect circadian activity.

Mice homozygous for the mutation showed 27.3 hour periods, but eventually lost all circadian rhythmicity after several days in constant darkness.

PER and CRY proteins accumulate and dimerize during subjective night, and translocate into the nucleus to interact with the CLOCK:BMAL1 complex, directly inhibiting their own expression.

The binding of CLOCK-BMAL to an E-box promoter element activates transcription of clock genes such as per1, 2, and 3 and tim in mice.

It has been shown in mice that CLOCK-BMAL also activates the Nicotinamide phosphoribosyltransferase gene (also called Nampt), part of a separate feedback loop.

[18] WC1, an analog of CLOCK/BMAL1 found in fungal genomes, is a proposed candidate common ancestor predating the fungi-animal split.

[18] Another theory alternatively proposes the NPAS2 gene as the paralog of CLOCK that performs a similar role in the circadian rhythm pathway but in different tissues.

[20] Allelic variations within the Clock1a gene in particular are hypothesized to have effects on seasonal timing according to a 2014 study conducted in a population of cyprinid fishes.

[21] Polymorphisms in the gene mainly affect the length of the PolyQ domain region, providing an example of divergent evolution where species sharing an ecological niche will partition resources in seasonally variable environments.

[20][21] One 2017 study investigating the role of CLOCK expression in neurons determined its function in regulating transcriptional networks that could provide insight into human brain evolution.

When CLOCK activity was disrupted, increased neuronal migration of tissue in the neocortex was observed, suggesting a molecular mechanism for cortical expansion unique to human brain development.

[23] This region is well conserved between mice and humans and polymorphisms have been shown to affect mRNA stability, indicating allelic variants could disrupt normal circadian patterns in mammals leading to conditions such as insomnia or other sleep disorders.

Furthermore, the same researchers demonstrated that these mutant flies express low levels of PER and TIM proteins, indicating that Clock functions as a positive element in the circadian loop.

[25] The similar sequence between Jrk and its mouse homolog suggests common circadian rhythm components were present in both Drosophila and mice ancestors.

A recessive allele of Clock leads to behavioral arrhythmicity while maintaining detectable molecular and transcriptional oscillations.

This dominant-negative mutation results in a defective CLOCK-BMAL dimer, which causes mice to have a decreased ability to activate per transcription.

[31] Another single nucleotide polymorphism (SNP) in Clock, 3111C, associated with diurnal preference,[23] is also associated with increased insomnia,[32] difficulty losing weight,[33] and recurrence of major depressive episodes in patients with bipolar disorder.

[39] These results have led Colleen McClung to propose using Clock mutant mice as a model for human mood and behavior disorders.

REV-ERBα and RORα regulate Bmal by binding to retinoic acid-related orphan receptor response elements (ROREs) in its promoter.