MAP kinase regulation of cell-fate transitions in yeast

描述（由申请人提供）：协调转录程序响应特定刺激的机制是理解正常发育和稳态的核心。信息素诱导的芽殖酵母转化为化养型或交配型是一个模型，解剖这种调节的分子基础。一个单一的丝裂原活化蛋白激酶（MAPK）级联介导这两个转变。该途径利用两个MAPK，Fus3和Kss1，负和正调节活性的Ste12和Tec1转录调节因子。Ste12是必不可少的基因表达的变化，建立两个命运的基础，而Tec1是重要的，只有化能营养的命运。低浓度信息素诱导的化能营养命运转换的遗传程序仍然不确定。Fus3和Kss1激活配置文件的差异影响高与低信息素。我们假设，它们的激活和拮抗调节作用的剂量依赖性差异控制Ste12和Tec1的活性和降解的方式，准备一个或其他分化程序的细胞。我们提出了一个多学科的方法来比较两种命运的调控网络和发展开关的分子基础：目的1。利用微阵列技术定义化能营养生长的全局表达程序，并与交配分化的全局表达程序进行比较。生物信息学的方法，提出了描绘的生理和表型的签名和监管网络，区分这两个程序。目标二。a和B）将联合收割机实验分析与计算建模相结合，以量化Fus3和Kss1的正调控和负调控以及转录因子降解对介导替代程序之间的转换的时间控制的相对贡献。在野生型和突变体背景中干扰调控的转录因子丰度和代表性mRNA的经验测量将用于测试模型的基本假设。c）不同信息素诱导方案下的延时成像将用于测试信息素梯度是否加强调节波动的反馈循环，从而减少途径活动和命运决定的变异性。我们的信息素诱导的MAPK途径的理解和易于遗传操作与酵母允许辨别的差异的幅度和时间的MAPK激活转化为不同的转录模式。由于MAPK途径的保守性，本文定义的调控范例将适用于人类中不同的MAPK介导的信号传导途径，这些途径是激素依赖性癌症、炎性疾病和代谢紊乱的发病机理的根源。丝裂原活化蛋白激酶（MAPK）通路控制正常的细胞发育和功能。已知MAPK激活的幅度和时间的差异会影响细胞是否分裂和繁殖或发育成专门的细胞类型，如肝细胞、骨细胞或脑细胞。该方案充分利用了模式遗传生物S.酿酒酵母，以确定翻译成不同的发育命运的MAPK激活的不同模式的分子机制。这些发现将使我们更好地了解人类这些途径的异常调节如何导致激素依赖性癌症，炎症性疾病和代谢紊乱，如II型糖尿病。

DESCRIPTION (provided by applicant): Mechanisms that coordinate transcriptional programs in response to specific stimuli are central to understanding normal development and homeostasis. The pheromone-induced transition of budding yeast to either a chemotrophic or mating competent form is a model for dissecting the molecular basis of this regulation. A single mitogen activated protein kinase (MAPK) cascade mediates both transitions. This pathway utilizes two MAPKs, Fus3 and Kss1, that both negatively and positively regulate activity of the Ste12 and Tec1 transcriptional regulators. Ste12 is essential for the changes in gene expression that underlie establishment of both fates while Tec1 is important only for the chemotrophic fate. The genetic program for the chemotrophic fate transition induced by low pheromone concentration is still undefined. Fus3 and Kss1 activation profiles are differentially affected at high vs. low pheromone. We hypothesize that the dose-dependent differences in their activation and their antagonistic regulatory roles control Ste12 and Tec1 activity and degradation in a manner that prepares cells for one or the other differentiation program. We propose a multidisciplinary approach to compare the regulatory networks for the two fates and the molecular basis of the developmental switch: Aim 1. Define the global expression program for chemotrophic growth using microarray technology and compare it to that for mating differentiation. Bioinformatic approaches are proposed to delineate the physiological and phenotypic signatures and regulatory networks that distinguish the two programs. Aim 2. a and b) Combine experimental analyses with computational modeling to quantify the relative contributions from positive and negative regulation by Fus3 and Kss1 and the temporal control of transcription factor degradation on mediating the switch between alternative programs. Empirical measurements of transcription factor abundance and representative mRNAs in wild-type and mutant backgrounds that perturb regulation will be used to test the underlying hypotheses of the model. c) Time-lapse imaging under different pheromone induction regimes will be used to test whether a pheromone gradient reinforces feedback loops that regulate fluctuations and thereby reduce variability in pathway activity and fate determination. Our understanding of the pheromone induced MAPK pathway and the ease of genetic manipulations available with yeast allow discernment of how differences in the amplitude and timing of MAPK activation translate into different transcriptional patterns. Because of the conservation of MAPK pathways, the regulatory paradigms defined here will apply to different MAPK mediated signaling pathways in humans that are at the root of the pathogenesis of hormone dependent cancers, inflammatory diseases, and metabolic disorders. PUBLIC HEALTH RELEVANCE Mitogen activated protein kinase (MAPK) pathways control normal cellular development and function. Differences in the amplitude and timing of MAPK activation are known to affect whether cells divide and multiply or develop into a specialized cell types, such as liver, bone, or brain cells. This proposal exploits advantages of studies with the model genetic organism, S. cerevisiae, to define the molecular mechanisms that translate different patterns of MAPK activation into different developmental fates. The findings will give us a better understanding of how aberrant regulation of these pathways in humans leads to hormone dependent cancers, inflammatory diseases, and metabolic disorders such as Type II diabetes.