MAP kinase regulation of cell-fate transitions in yeast

描述(由申请人提供)：协调转录程序以响应特定刺激的机制是理解正常发育和动态平衡的核心。信息素诱导的萌芽酵母向营养或交配能力形式的转变是剖析这一调控的分子基础的一个模型。单一的丝裂原活化蛋白激酶(MAPK)级联介导了这两种转变。该途径利用两个MAPK，Fus3和Kss1，负向和正向调节STE12和Tec1转录调节因子的活性。STE12对于决定这两种命运的基因表达变化是必不可少的，而Tec1只对化学营养命运重要。低信息素浓度诱导的趋化命运转移的遗传程序尚不清楚。Fus3和Kss1的激活谱在高信息素和低信息素时受到不同的影响。我们假设，STE12和Tec1的激活及其拮抗调节作用的剂量依赖性差异控制着STE12和Tec1的活性和降解，从而为细胞的一个或另一个分化程序做好准备。我们提出了一种多学科的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.利用微阵列技术定义趋化生长的全球表达程序，并将其与交配分化的表达程序进行比较。生物信息学方法被提出来描述区分这两个程序的生理和表型特征以及调控网络。目的2.a和b)将实验分析和计算模型相结合，以量化Fus3和Kss 1的正负调控以及转录因子降解的时间控制在调节可选程序之间的切换方面的相对贡献。对转录因子丰度和野生型和突变型背景中干扰调控的代表性mRNAs的经验测量将用于检验该模型的基本假设。C)将使用不同信息素诱导机制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环，从而减少了途径活动和命运确定的可变性。我们对信息素诱导的MAPK途径的理解以及酵母可用的遗传操作的简便性使我们能够辨别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，这里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，这些通路是激素依赖型癌症、炎症性疾病和代谢紊乱发病的根源。与公共健康相关的丝裂原活化蛋白激酶(MAPK)通路控制着正常的细胞发育和功能。众所周知，MAPK激活的幅度和时间的不同会影响细胞的分裂和增殖，或者发展成一种特殊的细胞类型，如肝、骨或脑细胞。这一建议利用对模式遗传有机体酿酒酵母的研究优势，来确定将不同的MAPK激活模式转化为不同的发育命运的分子机制。这些发现将使我们更好地理解这些途径在人类中的异常调节如何导致激素依赖型癌症、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.