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

背景生物钟使动物能够使它们的生物体与光/暗周期同步。这使得动物能够预测黎明，并使身体准备好从睡眠中醒来进入警觉状态，这在发现猎物或躲避捕食者方面是一大进化优势。在哺乳动物中，主时钟位于大脑的视交叉上核(SCN)，它接收眼睛中光探测到的计时信号。SCN向其他器官和组织中的从属时钟发出信号，以保持它们的同步。生物钟的振荡是由特定的时钟基因造成的。它们被表达成蛋白质，蛋白质在正反馈和负反馈循环中相互作用。在一个中央负反馈环路中，三组名为PER、CRY和CK I的时钟蛋白在细胞质中结合成一个“定时”蛋白，并一起进入细胞核。在细胞核内，它们抑制了Per和Cry基因的表达，阻止了Per和Cry蛋白的重组。这最终会移除反馈，循环再次开始。完成这个周期所需的时间决定了时钟的周期，而人类的时钟周期只有24小时多一点。在细胞质中，磷酸基团通过CK I(磷酸化)被添加到PER和CRY蛋白上。这可能会导致定时器更快地进入原子核，从而缩短时钟的周期。我们的研究表明，人类PER3有一个区域，许多磷酸基团可能通过磷酸化添加到该区域。但人类对这个区域有两个版本，一个更长，一个更短。因为我们携带每个染色体的两个版本(性染色体除外)，每个父母各一个，所以人们可能会有一个或一个的两个副本。长版本和短版本具有不同数量的磷酸化位点。我们已经证明，长型与早起的人有关，而短型与晚间的人有关，而且还与一种称为睡眠相延迟综合症(DSSPS)的疾病有关。目的与目的虽然小鼠已被用作哺乳动物昼夜节律研究的模型动物，但小鼠的PER3不包含该磷酸化区域。我们建议进一步研究PER3在生物钟中的作用。这将通过研究PER3基因被破坏的小鼠(因此没有PER3蛋白)和PER3基因被修改为与人类基因的两种形式相似的小鼠来实现。然后将比较具有正常或人源化PER3基因的小鼠和完全缺乏PER3基因的敲除小鼠的不同基因是如何打开或关闭的。该项目还将研究这如何通过昼夜行为和睡眠影响SCN的输出。潜在的应用和益处这项研究将帮助我们更充分地了解PER3在生物钟中的作用，以及磷酸化如何在遗传、神经和行为水平上影响昼夜节律功能。这应该有助于我们理解为什么人们在早晨和晚上的偏好上存在差异，甚至为什么他们中的一些人患有DPS等睡眠障碍。研究还发现，人类PER3长度与乳腺癌发病率之间存在关联。基因工作的结果可能会识别参与这种关联的基因，而添加了人类PER3区域的小鼠将为这一领域的进一步研究提供一个模型系统。

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.