The genetics of circadian rhythms, sleep and health

Abstract

Circadian rhythms are 24-h rhythms in physiology and behaviour generated by molecular clocks, which serve to coordinate internal time with the external world. The circadian system is a master regulator of nearly all physiology and its disruption has major consequences on health. Sleep and circadian rhythm disruption (SCRD) is a ubiquitous feature in today's 24/7 society, and studies on shift-workers have shown that SCRD can lead not only to cognitive impairment, but also metabolic syndrome and psychiatric illness including depression (1,2). Mouse models of clock mutants recapitulate these deficits, implicating mechanistic and causal links between SCRD and disease pathophysiology (3-5). Importantly, treating clock disruption reverses and attenuates these adverse health states in animal models (6,7), thus establishing the circadian system as a novel therapeutic target. Significantly, circadian and clock-controlled gene mutations have recently been identified by Genome-Wide Association Studies (GWAS) in the aetiology of sleep, mental health and metabolic disorders. This review will focus upon the genetics of circadian rhythms in sleep and health.

Figure 1.

The mammalian molecular clock. The driving force of the mammalian molecular clockwork is the transcriptional drive provided by two proteins named ‘Circadian Locomotor Output Cycles Kaput’, CLOCK (CLOCK), which heterodimerises with ‘Brain muscle arnt-like 1’ (BMAL1). Bmal1 gene transcription produces a rhythmically produced BMAL1 protein that heterodimerises with a constitutively expressed CLOCK. The CLOCK-BMAL1 complex binds to E-box promoters driving rhythmic transcription of the Per1-3 and two Cryptochrome genes (Cry1, Cry2). The various PER and CRY proteins can complex (dimerise) with themselves to form PER-PER homo- or PER-CRY heterodimers. PER is phosphorylated by the kinase CK1 (Casein kinase 1 family of kinases) and other kinases earmarking it for degradation. However, the PER-CK1 complex allows the CRYs to bind to form a CRY-PER-CK1 complex which prevents further phosphorylation and degradation of PER in the cytoplasm. Within the complex of CRY-PER-CK1, CRY and PER are phosphorylated by other kinases which then allows the CRY-PER-CK1 complex to move into the nucleus and inhibit CLOCK-BMAL1 transcription of the Per and Cry genes forming the core negative limb of the transcriptional/translational feedback loop (TTFL). The CRY-PER-CK1 protein complex levels rise throughout the day, peak at dusk and decline to their lowest level the following dawn. The stability/degradation rate of the CRY-PER-CK1 complex in the nucleus and the resumption of CLOCK-BMAL1 mediated transcription is a key process in setting the period of the clock. It seems that CK1 and other kinases phosphorylate PER and target it for degradation, whilst at least two F-Box protein (FBXL3 and 11) target CRY proteins for degradation. The net result is that CRY and PER proteins fall to their lowest levels just before dawn. Light acts to up-regulate Per1 and Per2 transcription and this allows the entrainment of the molecular clockwork to the dawn/dusk cycle. An interlocked secondary TTFL directs alternating activation and repression of BMAL1 expression. This occurs via the nuclear receptors RORα (RAR-related orphan receptor alpha) and REV-ERBα, respectively, via binding at ROR elements (retinoic acid-related orphan receptor response elements/ROREs) in the Bmal1 promoter. Both Rorα and Rev-erbα have an E-box and are driven rhythmically via CLOCK-BMAL1 transcription. The rates of transcription and translation of these genes differ so that ROR peaks at dawn and REV-ERBα peaks at dusk and this action on the Bmal1 promoter ensures that BMAL1 levels rise at dusk, peak at dawn and then fall throughout the day to their low point just before dusk. In this way BMAL1 levels cycle in antiphase to those of CRY and PER. The Dec1 and Dec2 genes give rise to DEC1 and DEC2 proteins which inhibit CLOCK-BMAL1 transcription and constitute the tertiary TTFL, which reinforces the action of CRY-PER-CK1 inhibition on CLOCK-BMAL1 transcription. Finally, the presence of an E-box in the promoter of downstream clock target genes gives rise to overt circadian rhythms in physiology and behaviour. However, it is also known that many clock controlled genes do not possess an E-Box. As a result the nature of the circadian regulation in these genes remains uncertain.

Figure 2.

Light regulation of the molecular clockwork in mammals. The sequence of events that entrains the molecular clockwork of a SCN neurone to the solar day are summarised here and involve the following steps: (1) Light is detected by the photosensitive retinal ganglion cells (pRGCs) within the eye. This induces the release of neurotransmitters (glutamate and pituitary adenylate cyclase-activating polypeptide/PACAP) from the pRGC terminals which synapse with neurones in the ventral SCN. These neurotransmitters trigger a sequence of events that increase the levels of Calcium (Ca²⁺) and 3',5'-cyclic adenosine monophosphate (cAMP) within an SCN neurone. Calcium levels rise as a result of influx from the extracellular medium or release from internal stores. (2) Raised intracellular Ca²⁺ and cAMP activate two proteins: CREB-binding protein (CREB) through phosphorylation by Protein Kinase A (PKA) and CREB-regulated transcription coactivator 1 (CRTC1) by dephosphorylation, these work together and bind to a cAMP response element (CRE element) in the promoter of Per1, Per2 and Sik1. (3) CRE activation of the Per genes (+), leads to elevated Per mRNA and increased levels of PER1 and PER2 protein. Changed levels of PER 1 and 2 act to shift the molecular clockwork, advancing the clock at dawn and delaying the clock at dusk. However, Per mRNA and PER protein levels fall rapidly even if the animal remains exposed to light. As a result, the effects of light on the molecular clock are limited and entrainment is a gradual process requiring repeated shifting stimuli over multiple days. This phenomenon explains why we get jet-lag, the clock cannot move immediately to a new dawn/dusk cycle because there is a ‘brake’ on the effects of light on the clock. (4) The mechanism that provides this molecular brake is the production of SIK1 protein. SIK1 deactivates CRTC (-) by phosphorylation, so that it can no longer provide the co-transcriptional drive with CREB on the CRE promoter, and transcription largely stops. This negative feedback turns off Per1 and Per2 transcription and translation, limiting the effects of light on the clock. Sik1 mRNA and SIK1 protein levels also decline but more slowly than PER 1 and 2. The system then re-sets itself for possible light detection several hours later. Experiments on mice in which SIK1 has been suppressed show very rapid entrainment to simulated jet-lag. By limiting the shifting effects of light on the SCN, the circadian system of the animal is protected from abnormal light exposure at the wrong time of day. In addition, it may be important to buffer the effects of light on the SCN clock so that it is not pulled rapidly to a new phase, and in the process uncouple the SCN from the peripheral circadian network, resulting in internal desynchrony.

Figure 3.

The circadian control of metabolic pathways. Metabolism is under strong circadian control. Peripheral clocks (e.g. liver, pancreas, adipose tissue, etc.) are regulated by the SCN and in turn feedback upon the SCN. Light regulates the phase of the molecular clockwork in the SCN, whilst hormonal signals (e.g. insulin and glucocorticoids) and feeding/fasting behaviours that change the levels of glucose alter the phase of peripheral clocks. The molecular clockwork of both peripheral and SCN cells then interacts with the metabolic control systems. The molecular clock comprises a Per/Cry and Clock/Bmal1 feedback loop (See Figure 1). These genes and their protein products also control the expression of downstream transcription factors which in turn regulate metabolic target genes. General regulators include DBP (D site of albumin promoter (albumin D-box) binding protein), which binds to an upstream promoter in the insulin gene; HLF (Hepatic leukaemia factor), which regulates aspects of liver function; and TEF (Thyrotroph embryonic factor), involved in thyroid-stimulating hormone release. The circadian coordination of metabolism also involves members of the rev-erb (REV-ERB) receptor family, retinoic acid orphan receptors (ROR), PPARs (peroxisome proliferator-activated receptors) and other nuclear receptors (NR). Metabolic regulators, such as REV-ERBα and ROR, also participate directly in the clock mechanism by regulating Bmal1 transcription (See Figure 1). In addition, hepatic PPARα, which is activated by fatty acids, is regulated rhythmically by CLOCK and BMAL1 and is also regulated by glucocorticoids. These transcriptional regulators in-turn interact with genes associated with glycogen, fatty acid and triglyceride metabolism. Such target genes include: Glycogen synthase, involved in converting glucose to glycogen; HMG-CoA reductase which is the rate-controlling enzyme that produces cholesterol; CYP7A1 is a rate-limiting enzyme in bile acid synthesis; Acetyl-CoA carboxylase (ACC) and Fatty acid synthase (FAS) are involved in catalysing the synthesis of fatty acids. This regulation can be immensely complex, with multiple interlocking feedback loops between the clock and metabolic genes/proteins. For example, transcriptional regulation of rhythmic CYP7A1, is driven by DBP, the clock protein DEC2, and by nuclear receptors including PPARα. PPARα also regulates Rev-erbα expression in both liver and adipose cells, whilst ROR and Rev-erbα regulate lipid metabolism as well as being involved in Clock and Bmal1 expression.