MAP kinase regulation of cell-fate transitions in yeast

描述（由申请人提供）：在特定刺激下协调转录程序的机制是理解正常发育和体内平衡的核心。信息素诱导的出芽酵母向趋化营养或交配能力形式的转变是解剖这种调节的分子基础的模型。一个单一的有丝分裂原激活蛋白激酶（MAPK）级联介导这两种转变。该途径利用两个mapk， Fus3和Kss1，负向和正向调节Ste12和Tec1转录调控因子的活性。Ste12对基因表达的改变至关重要，而Tec1仅对趋化命运重要。低费洛蒙浓度诱导的趋化营养命运转变的遗传程序尚不明确。高、低费洛蒙对Fus3和Kss1激活谱的影响是不同的。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，以一种方式为细胞的一种或另一种分化程序做好准备。我们提出了一种多学科方法来比较这两种命运的调控网络和发育开关的分子基础：目的1。使用微阵列技术定义趋化营养生长的全局表达程序，并将其与交配分化的表达程序进行比较。提出了生物信息学方法来描述区分这两个程序的生理和表型特征和调节网络。目标2。a和b)将实验分析与计算建模相结合，量化Fus3和Kss1的正调控和负调控以及转录因子降解在介导替代程序之间切换中的时间控制的相对贡献。转录因子丰度和野生型和突变背景下干扰调节的代表性mrna的经验测量将用于测试模型的基本假设。c)将使用不同信息素诱导制度下的延时成像来测试信息素梯度是否加强了调节波动的反馈回路，从而减少了途径活动和命运决定的可变性。我们对信息素诱导的MAPK通路的理解，以及酵母遗传操作的便捷性，使我们能够辨别MAPK激活的幅度和时间的差异如何转化为不同的转录模式。由于MAPK通路的保守性，本文定义的调控范式将适用于人类中不同的MAPK介导的信号通路，这些信号通路是激素依赖性癌症、炎症性疾病和代谢紊乱发病机制的根源。丝裂原活化蛋白激酶（MAPK）途径控制正常细胞发育和功能。已知MAPK激活的振幅和时间的差异会影响细胞是否分裂和繁殖或发育成特定的细胞类型，如肝脏、骨骼或脑细胞。本研究利用模型遗传生物酿酒酵母的研究优势，确定MAPK激活不同模式导致不同发育命运的分子机制。这些发现将使我们更好地了解这些途径的异常调节是如何导致激素依赖性癌症、炎症性疾病和代谢性疾病（如II型糖尿病）的。

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.