How do VRI and PDP1 regulate circadian rhythms?

描述(申请人提供)：只要一晚睡眠受到干扰，我们就会意识到正常的睡眠/醒来节奏对身心健康有多么重要。这些节律是由内部生物钟(~24小时)控制的，我们从对果蝇的研究中了解到了大部分关于时钟是如何发挥作用的。事实上，第一个被发现的人类睡眠障碍基因是生物钟基因的同源基因，最先在果蝇中被发现和克隆。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，果蝇时钟是一个很好的系统，可以理解转录因子如何动态调节神经元功能，这在神经科学中具有广泛的普遍重要性。在这里，我们建议研究时钟转录因子如何控制时钟神经元的活动。我们重点研究了Vrille(VRI)和PDP1，它们构成了果蝇的第二个时钟反馈环。VRI和PDP1在维持反馈回路本身的稳健性方面发挥了作用，并且是通过调节黎明时达到峰值的基因，将时钟与节奏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。我们已经确定，在这个循环中存在额外的监管机构。其中一种调节因子是隐色素(CRY)，它以前被描述为昼夜节律的光感受器，但最近由我的实验室建立，在大多数时钟神经元中也起到转录抑制的作用。在这里，我们建议研究PDP1对不同时钟神经元的哭声调节。VRI和Pdp1表达的第二个调节因子可能是Stich1，它是哺乳动物Dec Clock蛋白的同源物，是有效的转录抑制因子，但尚未正式整合到钟表中。最后，确定哭声为零的等位基因将使我们能够测试大脑中哪个时钟神经元是哭声的抑制者。 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路径联系起来。我们已经开发了一种行为基因组学方法，其中来自纯化的起搏神经元的基因表达可以在整个基因组中进行分析。我们目前已经分析了一天中不同的时间，但我们将把这种分析扩展到不同的时钟突变，这些突变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联系起来。在我们最初的研究中，我们发现了一组基因，这些基因可能是起搏器神经元节律性活动和神经传递的基础。我们建议研究这些基因中的突变以寻找昼夜节律行为的缺陷。许多以前没有研究过的基因也受到严格的时钟调控，我们将通过在vri和Pdp1突变体中进行额外的基因芯片实验来进一步研究这些基因。

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.