Molecular Genetic Analysis of Fungal Circadian Rythms

描述（由申请人提供）：从细菌到人类的生物体都使用生物钟来控制日常的生化、生理和行为节律。生物钟影响人体生理，正常生物钟功能的破坏会导致各种健康问题。关于构成昼夜节律计时系统核心的振荡器，我们已经知道了相当多的信息。然而，将振荡器与其控制的活动连接起来的输出通路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，通路组分的突变或通路活性的改变与癌症有关。我们发现，在草神经孢子虫中，应激诱导的p38 MAPK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，一天中的时间信息从振荡器通过响应调节因子RRG-1或上游的MAPK信号通路传递，导致p38 MAPK节律性磷酸化。然后，Phospho-p38 MAPK向转录因子和其他效应分子发出信号，以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验证这一假设。首先，为了阐明ccgs受时钟调节的机制，我们将使用可用的突变体来确定哪些p38 MAPK通路组件是该通路时钟调节所必需的。我们还将确定该调节需要哪些振荡器组件。其次，再次使用可用的突变体，我们将确定在p38 MAPK下游发挥作用的转录因子，以调节ccg-1的节律性表达，ccg-1是由p38 MAPK途径调节的基因。这些结果将使我们第一次在任何生物体中描述ccg节律调节的完整机制。第三，为了研究生物钟对细胞信号通路的协同选择是否是控制基因节律表达的一般机制，我们将确定在神经孢子虫中，生物钟是只调节p38信号通路，还是调节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通路的节律性来确定时钟是否选择细胞信号通路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来促进昼夜节律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信号控制基因表达的机制，并可能为治疗由昼夜节律功能障碍和/或p38 MAPK通路失调引起的人类疾病提供新的治疗方法。

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.