Circadian Rhythm Proteins

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 Circadian Rhythm Proteins Background

Circadian rhythms in mammals

Tissue-level clocks

In mammals, circadian rhythms are organized in a hierarchical structure, in which inputs from the environment are processed separately by different tissue-level clocks. Light, the primary entraining factor, is directed towards the suprachiasmatic nucleus (SCN), a tissue in the brain that serves as the body’s master pacemaker. In the SCN, approximately 20, 000 cells communicate via intercellular coupling to determine a precise internal time, which coordinates rhythms in locomotor activity.

Peripheral tissues are responsible for responding to changes daily changes in food intake. Liver and adipose tissue, for instance, maintain robust circadian oscillations, which respond mainly to temperature and food resetting cues.

Such clocks are important to maintaining metabolic health, as knockout experiments demonstrate that compromised circadian in peripheral tissues leads to many disorders, including diabetes and obesity. Additionally, feeding cycles have been shown to have a profound impact on circadian oscillations in peripheral tissues. High-fat or out of phase feeding leads to a reprogramming of oscillatory genes and metabolites, often leading to metabolic disease.


Circadian oscillations at the single-cell level

Circadian rhythms are generated at the single-cell level through genetic regulatory networks with inherent time-delayed negative feedback. Transcription factors CLOCK and BMAL1, peaking in the early night, activate expression of EBOX genes period (Per1 and Per2) and cryptochrome (Cry1 and Cry2). Per and Cry protein products, PER and CRY, peaking during the early day, form a heterodimeric complex to cross the nuclear membrane. However, since PER is present in the cytoplasm in much lower quantities than CRY, it is stochiometrically-limiting for nuclear entry. Once inside the nucleus, the PER-CRY complex dissociates, and CRY represses CLOCK-BMAL1 mediated activation of EBOX genes, leading to rhythmic gene expression.

Additional components, namely the ROR and REV-ERB families of genes, add a layer of positive feedback to the clock regulatory loop. This addition results in more reliable clock oscillations, as the bistability induced by the positive feedback stabilizes the negative-feedback induced oscillations.


Molecular components of circadian rhythms

Much of the genome exhibits circadian expression with estimations from microarray studies showing that 10 to 50% of all genes exhibit rhythmic fluctuations; although the proportion of these directly affected by the core molecular clock is unknown, cycling in these transcripts may be largely due to factors downstream of the clock or due to behavioral rhythms. The core molecular clock is composed of both a positive and negative arm of transcriptional regulation. In mammals, the positive arm consists of the transcription factor brain and muscle aryl-hydrocarbon receptor nuclear translocator-like 1 (Bmal1), and the histone acetyl-transferase circadian locomotor output cycles kaput (Clock or its paralogue NPAS2), which heterodimerize and bind to an Ebox sequence in the promoter of clock controlled genes.

Some of the transcription factors that are positively regulated by the Clock/Bmal1 complex include the negative branch of circadian factors, including nuclear receptor subfamily 1, group D, member 1 (Rev-Erbα), Period (Per1, Per2), and Cryptochrome (Cry1, Cry2, Cry3). The nuclear receptor Rev-Erbα competes with the transcriptionally activating retinoic acid–related orphan receptor α (RORα) to bind to the ROR response element (RORE) in the Bmal1 promoter to repress its transcription in a complex with nuclear receptor co-repressor 1 (NCOR1). More recently, an RORE has also been described in the Clock promoter. The second negative arm of circadian rhythms is comprised of period and cryptochrome proteins that heterodimerize in the cytosol and then are shuttled back into the nucleus to directly inhibit the function of the Clock/Bmal1 complex.

There are several posttranslational mechanisms that control this circadian feedback system, including phosphorylation, ubiquitination, O-linked glycosylation, and acetylation. The nuclear abundance of period and cryptochrome is regulated by a phosphorylation by casein kinase 1ε (CK1ε) or AMPK, respectively, which tags them for polyubiquitylation by the E3 ubiquitin ligase complex β-transducin repeat containing protein (β-TrCP1), and F-box and leucine-rich repeat protein 3 (FBXL3), which leads to their subsequent degradation by the 26S proteasome. Ck1ε also plays an important role in nuclear translocation of the Per/Cry complex, and may be important in some of the inhibitory properties. Degradation of Per2, Clock and Bmal1 is countered by O-linked glycosylation to regulate circadian timing. Finally, acetylation of Bmal1 facilitates cryptochrome interaction with this complex to affect its repression.