Molecular Genetic Analysis of Fungal Circadian Rythms

描述(申请人提供)：从细菌到人类的生物体都使用生物钟来控制日常的生化、生理和行为节奏。这种时钟会影响人类的生理，而正常时钟功能的中断会导致各种健康问题。人们对构成昼夜节律计时系统核心的振荡器有相当多的了解。然而，连接振荡器和它们控制的活动的输出路径在很大程度上是未知的。在人类中，p38丝裂原活化蛋白激酶(MAPK)通路调节细胞增殖，而通路组件的突变或通路活性的改变与癌症有关。我们发现在粗枝脉孢菌中，胁迫诱导的p38MAPK通路被用作FRQ/WCC昼夜节律振荡器的输出通路。在非应激条件下，时间信息从振荡器通过反应调节因子RRG-1或上游的MAPK信号通路传递，导致有节奏的p38MAPK磷酸化。然后，磷酸化p38MAPK向转录因子和其他效应分子发出信号，调节基因表达的节奏。我们假设昼夜节律振荡器已经选择了细胞信号通路来控制时钟控制基因(CCGs)的表达，并提出了三个具体的目标来检验这一假说。首先，为了阐明CCGs受时钟调控的机制，我们将使用现有的突变体来确定哪些p38MAPK通路组件是时钟调控该途径所必需的。我们还将确定该法规需要哪些振荡器组件。其次，我们将再次利用已有的突变体，确定在p38MAPK下游发挥作用的转录因子(S)，以调控受p38MAPK途径调控的基因CCG-1的节律性表达。这些结果将使我们第一次在任何生物体中描述一种完整的CCG节律调节机制。第三，为了研究时钟选择细胞信号通路是否是控制节律性基因表达的一般机制，我们将确定时钟是否只调节脉孢子菌中的p38或p38和其他MAPK信号通路。最后，我们将通过确定时钟是否也调节哺乳动物p38途径的节律性，来检验通过时钟选择细胞信号通路是否是控制昼夜节律性的保守机制。由于已知p38激酶调节染色质结构和翻译，我们还将研究节律性p38 MAPK活性通过调节染色质重塑和翻译抑制而参与昼夜节律性的令人兴奋的可能性。总之，这些目标的结果将揭示昼夜节律振荡器通过输出通路发出信号来控制基因表达的机制，并可能为治疗由昼夜节律障碍和/或p38 MAPK通路错误调节引起的人类疾病提供新的方法。 与公共健康相关人类有一个内在的时钟，它调节睡眠/醒来周期等机制，并影响细胞对抗癌药物和其他药物的反应。我们发现真菌中的生物钟调节着p38丝裂原活化蛋白激酶活性的日常节律，p38丝裂原激活蛋白激酶在哺乳动物中起到抑制癌细胞形成的作用。了解时钟如何调节p38通路将有助于开发新的治疗方法来治疗由时钟功能障碍和/或p38 MAPK通路错误调节引起的人类疾病。

DESCRIPTION (provided by applicant): Organisms from bacteria to humans use a circadian clock to control daily biochemical, physiological, and behavioral rhythms. This clock affects human physiology, and disruptions of normal clock function can cause a variety of health problems. A considerable amount is known about the oscillators that form the core of the circadian timing system. However, the output pathways that connect oscillators to the activities they control are largely unknown. In humans, p38 mitogen-activated protein kinase (MAPK) pathways regulate cell proliferation, and mutations of pathway components, or alterations of pathway activity, are associated with cancer. We found that in Neurospora crassa the stress-induced p38 MAPK pathway is used as an output pathway from the FRQ/WCC circadian oscillator. Under non-stressed conditions, time-of-day information is passed from the oscillator through the MAPK signaling pathway at or upstream of the response regulator RRG-1, resulting in rhythmic p38 MAPK phosphorylation. Phospho-p38 MAPK then signals to transcription factors and other effector molecules, to regulate rhythms in gene expression. We hypothesize that circadian oscillators have co-opted cellular signaling pathways to control expression of clock-controlled genes (ccgs), and propose three specific aims to test this hypothesis. First, to elucidate the mechanism whereby ccgs are regulated by the clock, we will use available mutants to determine which p38 MAPK pathway components are necessary for clock regulation of the pathway. We will also determine which oscillator components are needed for this regulation. Secondly, again using available mutants, we will identify the transcription factor(s) that function downstream of the p38 MAPK to regulate rhythmic expression of ccg-1, a gene regulated by the p38 MAPK pathway. These results will allow us to describe, for the first time in any organism, a complete mechanism for rhythmic regulation of a ccg. Thirdly, to investigate whether co-opting cellular signaling pathways by the clock is a general mechanism for controlling rhythmic gene expression, we will determine if the clock regulates only p38, or p38 and other MAPK signaling pathways in Neurospora. Lastly, we will examine whether co-opting cellular signaling pathways by the clock is a conserved mechanism for controlling circadian rhythmicity by determining if the clock also regulates rhythmicity of the mammalian p38 pathway. Because the p38 kinases are known to regulate chromatin structure and translation, we will also investigate the exciting possibility that rhythmic p38 MAPK activity contributes to circadian rhythmicity by regulating chromatin remodeling and translational repression. Together, results from these aims will uncover the mechanisms by which circadian oscillators signal through the output pathways to control gene expression and may provide novel approaches for therapies to treat human diseases that result from circadian dysfunction and/or misregulation of the p38 MAPK pathway. PUBLIC HEALTH RELEVANCE Humans have an internal clock that regulates mechanisms such as the sleep/wake cycle and affects the response of cells to anti-cancer drugs and other pharmaceuticals. We found that the circadian clock in fungi regulates daily rhythms in the activity of p38 mitogen-activated protein kinase, which functions in mammals to suppress cancer cell formation. Understanding how the clock regulates the p38 pathway will facilitate efforts for developing new therapies for treating human diseases that result from clock dysfunction and/or misregulation of the p38 MAPK pathway.