Circadian clock

A secondary loop also exists in the mammalian clock: transcription of the BMAL1, CLOCK, and NPAS2 genes is activated by Retinoid-related orphan receptor-alpha (RORA) and RORC (ROR-gamma) paralogs and repressed by NR1D1 (REV-ERBA), all of which are targets of regulation by BMAL1:CLOCK/NPAS2 and all of which compete for the same ROR regulated elements (RREs, ROREs) in the BMAL1 and CLOCK promoters and in the promoters of many other genes (reviewed in Duez and Staels 2008). This mutual control forms a reinforcing loop of the circadian clock (reviewed in Kim et al. 2022).

BMAL1 can form heterodimers with either CLOCK or NPAS2, which appear to act redundantly but may show different tissue specificity (inferred from mouse homologs in Landgraf et al. 2016, Reick et al. 2001, Peng et al. 2021). After translation in the cytosol, BMAL1 and CLOCK (and probably NPAS2) are phosphorylated, heterodimerize and enter the nucleus (reviewed in Lee et al. 2001, Hirano et al. 2016). The phosphorylated BMAL1:CLOCK heterodimers bind E-box elements (consensus CANNTG) in the promoters of target genes (inferred from mouse homologs in Gekakis et al. 1998) and recruit coactivators to enhance transcription of the target genes. Genes activated by BMAL1:CLOCK are expressed diurnally. DBP is among the genes activated by phosphorylated BMAL1:CLOCK/NPAS2 and binds D-box elements in the promoters of many circadian-regulated genes to increase the amplitude of rhythmic expression (inferred from mouse homologs in Yamaguchi et al. 2000).

The PER genes (PER1, PER2, PER3) and CRY genes (CRY1, CRY2) are also among those activated by BMAL1:CLOCK and BMAL1:NPAS2 (reviewed in Cox and Takahashi 2019). PER gene and CRY gene mRNAs accumulate during the morning and the proteins accumulate during the afternoon. PER and CRY proteins form complexes in the cytosol and these are bound by kinases CSNK1D or CSNK1E, which phosphorylate PER and CRY proteins (reviewed in Narasimamurthy and Virshup 2021). Phosphorylation of the PER:CRY:kinase complex is required for its translocation into the nucleus. CDK5 phosphorylates PER2 at serine residue 396 (serine-394 of the mouse orthologue) in a diurnal fashion. This phosphorylation facilitates interaction with CRY1 and nuclear entry of the PER2-CRY1 complex. Within the nucleus the phosphorylated PER:CRY:kinase complexes bind BMAL1:CLOCK heterodimers, inhibiting their transactivation activity and causing dissociation of BMAL1:CLOCK from DNA (inferred from mouse homologs Cao et al. 2021). This reduces transcription of the target genes of BMAL1:CLOCK during the afternoon and evening.

During the night PER:CRY complexes are polyubiquitinated and degraded by the 26S proteasome, allowing the cycle to begin again (reviewed in Abdalla et al. 2022). PER:CRY complexes traffic out of the nucleus into the cytosol due to the nuclear export signal of PER proteins (inferred from mouse homologs in Vielhaber et al. 2001). In the cytosol, phosphorylated PER proteins are bound by Beta-TrCP1, a F-box component of a cytosolic SCF E3 ubiquitin ligase complex that polyubiquitinylates the PER proteins. Also in the cytosol, CRY proteins are bound by FBXL21, another F-box component of a SCF E3 ubiquitin ligase complex that polyubiquitinylates the CRY proteins (inferred from mouse homologs in Hirano et al. 2013, Yoo et al. 2013). In the nucleoplasm, CRY proteins are bound by FBXL3, a F-box component of a SCF E3 ubiquitin ligase complex that polyubiquitinylates CRY proteins (inferred from mouse homologs in Busino et al. 2007). FBXL21 appears to antagonize FBXL3, though the mechanism is not clear.

The core circadian clock both regulates metabolism and receives inputs from metabolism (reviewed in Bass and Takahashi 2010, Laothamatas et al. 2023). BMAL1:CLOCK,NPAS2 transcriptionally activate thousands of genes, including PPARA, which regulates lipid metabolism (inferred from mouse homologs in Oishi et al. 2005), and NAMPT (inferred from mouse homologs in Nakahata et al. 2009), which is involved in NAD+ metabolism. Retinoid-related orphan receptors (RORs) and NR1D1 also regulate numerous genes, including CPT1A (inferred from mouse homologs in Lau et al. 2004) involved in fatty acid catabolism and SREBF1 (inferred from mouse homologs in Lau et al. 2008) involved in cholesterol biosynthesis. Metabolic inputs include NAD+, which is a substrate in the deacetylation of BMAL1 by SIRT1 (inferred from mouse homologs in Ramsey et al. 2009), and AMP, which regulates the phosphorylation of CRY1 by AMPK, causing enhanced degradation of CRY1 (inferred from mouse homologs in Lamia et al. 2009). Additional metabolic inputs are provided through CLOCK acetylase activity, GSK3beta kinase activity, and casein kinase II (CK2) kinase activity (reviewed in Laothamatas et al. 2023).

The circadian clock is cell-autonomous and some, but not all cells of the body exhibit circadian rhythms in metabolism, gene transcription (reviewed in Albrecht et al. 2012), and cell division (reviewed in Farshadi et al. 2020). The suprachiasmatic nucleus (SCN) in the hypothalamus is the major clock in the body and receives its major input from light (via retinal neurons) and a minor input from nutrient intake (reviewed in Bechtel 2024). The SCN and other brain tissues determine waking and feeding cycles and influence the clocks in other tissues by hormone secretion and nervous stimulation. Independently of the SCN, other tissues such as liver receive inputs from signals from the brain and from nutrients.

Relationships between the circadian clock and metabolism, disease, and drug efficacy are extensive and are the subject of active investigation (reviewed in Cederroth et al. 2019, Battaglin et al. 2021, Laothamatas et al. 2023, Zhuang et al. 2024).

Mice are often used as model organisms for studying the mammalian circadian clock, however some differences exist between mouse clocks and human clocks. For example, peak expression of core circadian genes such as CRY1, CRY2, PER1, PER2, PER3, and NR1D1 occurs about 6 hours earlier in humans than in mice.