How do VRI and PDP1 regulate circadian rhythms?

描述（由申请人提供）：一晚的睡眠不安就足以让我们认识到正常的睡眠/觉醒节律对于身心健康有多么重要。这些节律由内部昼夜节律（约 24 小时）时钟控制，我们从果蝇研究中了解了有关时钟如何运作的大部分信息。事实上，第一个被识别的人类睡眠障碍基因是周期时钟基因的同源物，该基因首先在果蝇中被识别和克隆。作为一种简单的行为，昼夜节律提供了了解大脑如何控制行为的基本机制的机会。特别是，果蝇时钟是了解转录因子如何动态调节神经元功能的优秀系统，这是神经科学中具有广泛普遍重要性的主题。在这里，我们建议研究时钟转录因子如何控制时钟神经元活动。我们重点关注 Vrille (VRI) 和 PDP1，它们构成了第二个果蝇时钟反馈环路。 VRI 和 PDP1 在维持反馈环路本身的稳健性方面发挥着作用，并且是通过调节黎明时达到峰值的基因将时钟与节律输出联系起来的最下游因素。 在目标 1 中，我们提出了对 VRI/PDP1 时钟环路的详细分析。我们已经发现该循环中存在其他监管机构。隐花色素（Cryptochrome，CRY）是一种调节因子，它以前被认为是一种昼夜节律光感受器，但最近由我的实验室建立，它也可在大多数时钟神经元中充当转录抑制因子。在这里，我们建议研究 PDP1 在不同时钟神经元中的哭泣调节。 vri 和 Pdp1 表达的第二个调节因子可能是 Stich1，它是哺乳动物 Dec 时钟蛋白的同源物，是有效的转录抑制因子，但尚未正式安装到发条装置中。最后，cry null 等位基因的鉴定将使我们能够测试大脑中哪些时钟神经元 CRY 是抑制因子。 在目标 2 中，我们寻求扩展令人兴奋的发现，以帮助将时钟与输出路径连接起来。我们开发了一种行为基因组学方法，可以在整个基因组中分析纯化的起搏神经元的基因表达。我们目前已经分析了一天中的不同时间，但我们将把这种分析扩展到不同的时钟突变体，这些突变体对起搏器神经元的输出有已知的影响。因此，我们可以将基因表达模式与已知的行为输出关联起来。在我们的初步研究中，我们确定了一组可能是起搏神经元的节律性神经元活动和神经传递的基础的基因。我们建议研究这些基因的突变体以发现昼夜节律行为的缺陷。许多以前未研究的基因也受到严格的时钟调控，我们将缩小范围，通过 vri 和 Pdp1 突变体中的额外 GeneChip 实验进一步研究。

DESCRIPTION (provided by applicant): One night of disturbed sleep is all that it takes for us to realize how important normal sleep / wake rhythms are for mental and physical health. These rhythms are controlled by an internal circadian (~24hr) clock, and we have learnt most about how the clock functions from studies in Drosophila. Indeed, the first human sleep disorder gene identified is a homologue of the period clock gene, first identified and cloned in Drosophila. As a simple behavior, circadian rhythms provide the opportunity to understand fuindamental mechanisms of how brains control behavior. In particular, the Drosophila clock is an excellent system to understand how transcription factors dynamically regulate neuronal function, a topic of broad general importance in Neuroscience. Here, we propose to study how clock transcription factors control clock neuron activity. We focus on Vrille (VRI) and PDP1, which comprise the second Drosophila clock feedback loop. VRI and PDP1 have roles in maintaining the robustness of the feedback loops themselves, and are the most downstream factors that link the clock to rhythmic outputs by regulating genes that peak at dawn. In Aim 1, we propose a detailed analysis of the VRI/PDP1 clock loop. We have identified that additional regulators exist in this loop. One regulator is Cryptochrome (CRY), previously characterized as a circadian photoreceptor, but recently established by my lab to also function as a transcriptional represser in most clock neurons. Here we propose to study cry regulation by PDP1 in different clock neurons. A second regulator of vri and Pdp1 expression may be Stich1, a homologue of the mammalian Dec clock proteins, which are potent transcriptional repressors, but have not been formally fitted into the clockworks. Finally the identification of a cry null allele will allow us to test in which clock neurons in the brain CRY is a repressor. In Aim 2, we seek to extend exciting findings that should help link the clock to output pathways. We have developed a behavioral genomics approach in which gene expression from purified pacemaker neurons can be assayed across the whole genome. We have currently analyzed different times of day, but we will extend this analysis to different clock mutants with known effects on the outputs of pacemaker neurons. Thus we can correlate patterns of gene expression with known behavioral outputs. In our initial studies, we identified a group of genes which may underlie rhythmic neuronal activity and neurotransmission from pacemaker neurons. We propose to study mutants in these genes for defects in circadian behaviors. Many previously unstudied genes are also tightly clock-regulated, and we will narrow done which to study further through additional GeneChip experiments in vri and Pdp1 mutants.