Genetic and phenotypic outputs of the circadian clock in Per3 knock-out mice and humanised Per3 knock-in mice

Context The circadian clock enables animals to synchronise their biology with the light/dark cycle. This allows animals to anticipate dawn and to prepare the body to emerge from sleep into alertness, a big evolutionary advantage with respect to prey detection or predator avoidance. In mammals, the master clock is in the suprachiasmatic nuclei (SCN) in the brain, which receive timekeeping signals from light detection in the eyes. The SCN sends out signals to subservient clocks in other organs and tissues to keep them synchronised. The oscillations of the circadian clock are created by specific clock genes. These are expressed into proteins, which interact in positive and negative feedback loops. In one central negative feedback loop, three groups of clock proteins called PER, CRY, and CK I combine into a 'timesome' in the cell's cytoplasm, and enter the nucleus together. Inside the nucleus, they inhibit the expression of the Per and Cry genes, preventing the reformation of PER and CRY protein. This eventually removes the feedback, and the cycle starts again. The time taken to complete this cycle determines the period of the clock, which in humans is just over 24 hours. In the cytoplasm, phosphate groups are added to the PER and CRY proteins by CK I (phosphorylation). This may cause the timesome to enter the nucleus faster, shortening the period of the clock. Our research has shown that human PER3 has a region to which many phosphate groups could potentially be added through phosphorylation. But humans have two versions of this region, one longer and one shorter. Because we carry two versions of each chromosome (apart from the sex chromosome), one from each parent, people may have two copies of one or one of each. The long and short versions have different numbers of phosphorylation sites. We have shown that the long form is associated with morning people, while the short form is associated with evening people and also a condition called delayed sleep phase syndrome (DSPS). Aims & Objectives Although mice have been used as a model animal for mammalian circadian studies, the mouse PER3 does not contain this phosphorylation region. We propose to investigate further the role of PER3 in the circadian clock. This will be done by studying mice in which the Per3 gene has been disrupted (so there is no PER3 protein) and also mice whose Per3 gene has been modified to resemble the two forms of the human gene. A comparison will then be made of how different genes are turned on or off in mice with a normal or humanised Per3 gene, and in knock-out mice which lack the Per3 gene altogether. The project will also investigate how this affects the output of the SCN through circadian behaviour and sleep. Potential Applications & Benefits This research will help us to understand more fully the role of PER3 in the circadian clock and how the phosphorylation can affect circadian function at the genetic, neural and behavioural levels. This should help us to understand why people have differences in morning and evening preference, and even why some of them suffer from sleep disorders such as DSPS. An association has also been found between human PER3 length and incidence of breast cancer. The results from the genetic work may identify genes that are involved in this association, and the mice with the human PER3 region added will provide a model system for further research in this area.

A variable number tandem repeat (VNTR) in the human PER3 gene associates with morning or evening preference and delayed sleep phase syndrome. The VNTR is present in humans in two alleles with four or five 54-nucleotide units but is absent in mice. Transgenic mice will be created which carry a humanised Per3 gene with either four or five repeat units. These knock-in mice will be compared with already existing Per3 knock-out mice and wild-type controls of the same genetic background (sv/129). Total RNA will be collected from suprachiasmatic nucleus (SCN), liver, kidney and heart of wild-type, Per3 knock-out and Per3 knock-in mice at three hourly intervals. From this expression profile, differentially labelled cDNA will be made from two time points from paired combinations of the mouse strains and will be hybridised to mouse genome microarray slides. The comparison of overall levels of gene expression between the strains will identify the potential roles of Per3 in the clock-controlled gene expression. Immunohistochemistry will be used with brain sections to examine the timing and localisation of clock proteins in the SCN and OVLT. QT-PCR with gene expression assays will be used to compare clock gene expression at different time points from various oscillating tissues between the different strains. These experiments will be carried out with animals in entrained conditions and after exposure to a phase-shifting light pulse. Locomotor activity and core body temperature will be monitored using telemetry. To establish the free-running period of locomotor activity and temperature, mice will be entrained to light/dark (LD) conditions for one week and then moved to DD for a two-week period. The acute phase-shifting effects of light will be assessed by administration of light pulses two hours after activity onset. This will be performed in free-running DD conditions after a period of LD entrainment.