How do VRI and PDP1 regulate circadian rhythms?

描述（由申请人提供）：一个晚上的睡眠不安是我们意识到正常的睡眠/觉醒节律对身心健康是多么重要所需要的。这些节奏是由内部的昼夜节律（~ 24小时）时钟控制的，我们已经从果蝇的研究中了解了大部分关于时钟的功能。事实上，第一个被发现的人类睡眠障碍基因是第一个在果蝇中被发现和克隆的周期时钟基因的同源物。作为一种简单的行为，昼夜节律提供了理解大脑如何控制行为的基本机制的机会。特别是，果蝇时钟是一个很好的系统，以了解转录因子如何动态调节神经元功能，在神经科学中广泛的普遍重要性的主题。在这里，我们打算研究时钟转录因子如何控制时钟神经元的活动。我们专注于Vrille（VRI）和PDP 1，其中包括第二果蝇时钟反馈回路。VRI和PDP 1在维持反馈回路本身的鲁棒性方面发挥作用，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，我们对VRI/PDP 1时钟环路进行了详细分析。我们已经确定，额外的监管机构存在于这个循环。其中一个调节器是隐花色素（CRY），以前被描述为昼夜光感受器，但最近由我的实验室建立，在大多数时钟神经元中也起着转录抑制剂的作用。在这里，我们建议在不同的时钟神经元中研究PDP 1对cry的调节。vri和Pdp 1表达的第二个调节因子可能是Stich 1，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，但还没有正式安装到时钟装置中。最后，cry无效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的CRY是阻遏物。 在目标2中，我们寻求扩展令人兴奋的发现，这些发现应该有助于将时钟与输出通路联系起来。我们已经开发了一种行为基因组学方法，其中可以在整个基因组中测定来自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相关联。在我们最初的研究中，我们确定了一组基因，这些基因可能是节律性神经元活动和起搏神经元神经传递的基础。我们建议研究这些基因的突变体在昼夜节律行为的缺陷。许多以前未研究的基因也是严格受时钟调控的，我们将通过在vri和Pdp 1突变体中进行额外的基因芯片实验来进一步研究。

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.