Physiological ReviewPhenotyping of PER3 variants reveals widespread effects on circadian preference, sleep regulation, and health
Introduction
Disruption of sleep and circadian rhythms is prominent in mental and physical diseases. Inter-individual variation in sleep and circadian rhythmicity and risk for physical and mental diseases is in part explained by genetic variation. Here we review how variation in the PERIOD3 (PER3) gene contributes to inter-individual differences in sleep and circadian rhythmicity phenotypes and disease risk. We pay attention to the wide range of phenotypic associations and attempt to understand how these associations may be connected through common pathways.
PER3 is a molecular component of the circadian clock and is a member of the protein-binding family that contain PAS (PER-ARNT-SIM) domains which enable protein dimerization. It binds with other PERIOD and CRYPTOCHROME (CRY) proteins in the negative limb of the transcriptional/translational feedback loop and inhibits the expression of core clock genes and clock-controlled genes by the heterodimer transcription factor CLOCK/BMAL1 which binds to promoter region E-box motifs (Fig. 1) [1]. Recently, electron microscopy has been used with mouse liver cell extracts to show that PER3 protein forms part of a mature cytoplasmic multi-globular complex with PER1, PER2, CRY1, CRY2, casein kinase 1 delta (CK1δ) and that this complex migrates to the nucleus to bind to and inhibit CLOCK/BMAL1 [2].
Three Per paralogues exist in most vertebrates and have likely evolved via two genome duplication events from a single ancestral gene [3]. After such evolutionary events, duplicated genes are commonly lost if they confer no diverse functional adaptation, or are retained if accumulating genetic variation provides a selective advantage. Because PER3 exists in humans, this implies that functions associated with it have been positively selected and maintained, unlike PER4 which has been lost from the genome [3].
PER1 or PER2 are essential for normal circadian function but PER3 alone cannot drive the central circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus [4]. The absence of PER3 has only minor effects on behaviour driven by the SCN clock [5]. However, in peripheral tissues, circadian period and phase are disrupted in Per3 knock out (KO) mice [6]. Thus, the evolutionary selective pressure to retain the duplicated Per3 gene is unlikely to have derived from its role within the central, hypothalamic clock, but more likely came from novel functions in peripheral clocks and associated phenotypes. These phenotypes include diurnal preference, sleep homeostasis, circadian rhythm sleep–wake disorders (CRSWDs), cognitive performance, light sensitivity, mental disorders, and cancer. Thus, although PER3 has often been neglected as a circadian clock gene, there is a wealth of data that links it with physiological and health phenotypes. The question remains, what are the characteristics of PER3 and the underlying mechanisms that give rise to this?
In mice, robust expression levels of Per3 have been found in the CNS including the ventromedial hypothalamic nucleus, gyrus dentatus, arcuate nucleus and medial amygdaloid nucleus, with moderate expression levels in the cingulate cortex, hippocampal pyramidal cells, cerebellar cortex and the nucleus tractus solitarius [7]. Per3 is also strongly expressed in peripheral mouse tissues, including in heart, lung, liver, skeletal muscle, kidney and testis [7], [8]. Robust, rhythmic expression of Per3 occurs in the SCN and the organum vasculosum lamina terminalis (OVLT), with peak expression at circadian time (CT) 4 and 8, respectively (CT0 = subjective dawn) [7]. The peak of rhythmic expression of Per3 in peripheral mouse tissues and certain CNS regions (liver, skeletal muscle, testis, arcuate nucleus, ventromedial hypothalamic nucleus, and retina) appears shifted relative to the SCN to between CT9 and 21 [7], [8]. Of interest, Per3 expression in the rat sleep-related ventrolateral preoptic nucleus (VLPO) is 12 h out of phase with its expression in the SCN [9].
The role of PER3 in the SCN may be redundant but its contribution to peripheral clocks is more significant. In Per3 KO mice, the period and phase of tissue explants from pituitary, liver, lung, adrenal, oesophagus, aorta, thymus, arcuate complex, and gonadal adipose were significantly shorter and/or advanced compared to wild type mice [6], [10]. Period length was also shorter in fibroblast, adipocyte, and hepatocyte cell cultures where Per3 had been silenced [11]. SCN explants from Per3 KO mice showed only a small reduction in luciferase reporter period [11], which were similar to previous reports for Per3 KO locomotor activity period [5]. However, when explanted SCN cells were dissociated they showed a substantially shorter luciferase reporter period in Per3 KO compared to wild type (25.58 ± 0.12 h vs. 27.23 ± 0.24, mean ± SEM) [11]. All these findings underline a potential prominent role for PER3 in the periphery, but also in the SCN, where its importance only becomes apparent when the coupling between pacemaker neurons in the intact tissue is removed.
Unlike Per1 and Per2, Per3 expression in the SCN is not induced by light [7], [8]. Nevertheless, high levels of rhythmic expression of Per3 were found in the mouse retina and specifically in photoreceptors [8], [12], as well as in rat pineal [13]. In human tissues the highest levels of PER3 are recorded in retina, thyroid and pineal, while in the mouse the highest levels are found in the salivary and lacrimal glands, the pituitary and adrenal glands, and many compartments of the eye including the retina (BioGPS GeneAtlas, biogps.org). Thus, in addition to its wide expression throughout the body (Fig. 1), PER3 is also specifically present in tissues involved with light-dependent phenotypes. This may be related to some of the light-dependent phenotypes observed in Per3 KO mice [14] and also in humans [15], *[16] (see below).
In humans, assessment of the time course of PER3 across the 24-h day has been carried out by targeted quantitative polymerase chain reaction (PCR) in white adipose tissue [17] and blood cells (e.g., [18], [19]), and by genome-wide microarray transcriptomic profiling in hair follicles [20], keratinocytes [21], post-mortem brain tissue [22], [23], [24], bone cell cultures [25], and whole blood in different sleep–wake conditions [26], [27], [28]. A novel machine learning-based approach has also been used to order samples with no time stamps to form a time series showing robust expression of PER3 also in human liver biopsies [29]. An analysis of a subset of these human datasets, together with 16 mouse time series data from different tissues and conditions revealed that PER3 is the third most robustly expressed clock gene (after NR1D1 and NR1D2), being rhythmic in 94% of mouse tissue time series and 86% of the few human tissues investigated (Fig. 2) [30]. These studies all confirm PER3 as ranking among the top rhythmically expressed genes in peripheral tissues. The peak of PER3 expression in human non-brain tissues is reported to be around relative clock time 6–10 (∼4–8 am; Fig. 2), while in the brain PER3 expression is around relative clock time 4–6 (∼4–6 hours after onset of pseudoday; Fig. 2), although given the sampling resolution it remains to be firmly established whether these differences are robust. In a study of prefrontal cortex post-mortem samples from young and old individuals, core clock gene expression was found to be phase-advanced and reduced in amplitude in older individuals except for PER3, which remained unchanged [24].
A recent detailed analysis of transcription factor units within the human PER3 transcription regulatory region has revealed three E-box and two D-box motifs [31]. CLOCK/BMAL1 and DBP (D-box binding PAR BZIP transcription factor), independently bind to the E box and D box motifs, respectively, to drive expression of PER3. By removing different combinations of the motifs from the PER3 regulatory region, Matsumura et al. showed that the motifs contributed differentially to the overall amplitude and phase of expression, and that their combined activation led to robust, high amplitude expression. This observation likely accounts for the consistent observation in the literature of robust PER3 expression.
Section snippets
PER3 polymorphisms
The study of sequence variation within a gene and genotype/phenotype associations is a powerful way to identify functional characteristics of the encoded protein. The human PER3 sequence together with a catalogue of its polymorphisms was published soon after it was first isolated in mice [32]. Compared with PER1 and PER2, PER3 is highly polymorphic [33] and subsequently many more coding and non-coding polymorphisms have been found that associate with diverse phenotypes and disease (see Table 1).
The PER3 VNTR
Because the PER3 VNTR could change protein phosphorylation levels in addition to tertiary protein structure and interactions with binding partners, this led to the hypothesis that the VNTR would cause functional changes in PER3 that would be associated with measurable individual phenotypic differences [35]. This hypothesis was validated through the association of the 5-repeat allele with morning diurnal preference and its lower than expected frequency within patients suffering from delayed
PER3 VNTR and sleep homeostasis
In the first electroencephalogram (EEG) sleep study comparing 14 PER34/4 vs. 10 PER35/5 participants in their twenties, several sleep parameters were investigated at baseline and during recovery sleep following sleep deprivation (SD) [62]. At baseline, PER35/5 individuals fell asleep quicker and had more slow wave sleep (SWS) than PER34/4 individuals. Rapid eye movement (REM) sleep and total sleep time did not differ between the genotypes. Quantitative analyses of the EEG revealed more slow
Effects of PER3 VNTR on cognition depend on cognitive domain, sleep pressure and circadian phase
Several studies have indicated that PER3 VNTR genotype may influence the effects of sleep loss on cognitive function. Young PER35/5 participants performed significantly worse in cognitive tasks than PER34/4 during SD [62]. This effect was most pronounced when performance was assessed in the late-night and early-morning hours, i.e., at a time of high sleep need when the circadian timing system does not promote wakefulness [70]. Because no differences in core physiological markers of circadian
Genetic variation in PER3 modulates the impact of light
Exposure to light is the main synchroniser of human circadian rhythms [70]. In humans, light also conveys a stimulating signal that acutely increases alertness, improves some aspects of cognitive performance, and modulates cognitive brain responses during wakefulness and affects subsequent sleep intensity [70]. Animal and human experiments have shown that these effects are most likely mediated through a pathway involving intrinsically photosensitive retinal ganglion cells (ipRGC) expressing the
PER3 and mental disorders and their symptoms
In this section, we will discuss papers that have investigated associations between PER3 and mental disorders, or symptoms indicative of mental disorders. Many of the data were collected prior to the publication of the Diagnostic and Statistical Manual of Mental Disorders fifth edition (DSM-V; 2013), and therefore use the term ‘mood disorder’, even though in DSM-V mood disorders are separated into ‘depressive and related disorders’ and ‘bipolar and related disorders’. Wherever possible we have
PER3 and cancer
The cell cycle controls cell division and proliferation. Uncontrolled cell proliferation can lead to cancer. The cell cycle is regulated by the interaction of cyclins with cyclin-dependent kinases and phosphatases and these complexes act as checkpoints for different steps of the cycle. The circadian clock and the cell cycle are coupled such that the expression and post-translational modification of many elements at different points in the cell cycle are regulated by the molecular clock. The
Conflict of interest
The authors report no conflicts of interest with respect to the content of this review.
Acknowledgements
The author's research is funded by the BBSRC (UK), BB/F022883, BB/E003672/1, BSS/B/08523, AFOSR (USA), FA9550-08-1-0080, Royal Society Wolfson Merit award (UK), WM120086, FNRS (Belgium), MIS F.4513.17, FMRE (Belgium), WELBIO, University of Liège, and SNF (Switzerland), #310030-130689. The opinions presented in this review are those of the authors. We thank Emma Laing for preparing Fig. 2 and colleagues at the Surrey Sleep Research Centre for collaborations.
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