Cell-fate determinants of yeast pseudohyphal growth

描述(申请人提供)：缺氮会导致酿酒酵母的二倍体细胞脱离营养生长并形成假菌丝。这些是杆状细胞的细丝，从菌落的外围辐射出来，扩散到琼脂培养基上。当酵母型(YF)细胞分裂并产生一个与其母亲具有不同发育命运的PH子代时，这种转变就发生了。这种不对称转换基本上与人类从多能干细胞产生不同血统的过程中发生的相同。在酵母中，就像在哺乳动物中一样，命运的改变需要多个转录因子控制的程序的协调。一些是对细胞外刺激的反应，另一些是关于关键命运决定因素不对称分离的细胞内在机制。 酿酒酵母Ash-1是一种GATA家族转录因子，与子代有差异分离，是PH转换所必需的。虽然Ash1不对称定位的机制已经被阐明，但关于它介导的确定PH命运的女儿特异性程序却知之甚少。这个程序很难用标准方法来研究，因为PH菌落是细胞类型的异质混合物。申请人建议开发使用绿色荧光蛋白(GFP)标记的策略，允许分析启动子功能以及单个活细胞中的蛋白质和mRNA动态。目前，细胞表面黏附蛋白FLO11是唯一已知的PH生长所必需的基因产物，其转录依赖于Ash1。目的(1)研究FLO11的转录调控。研究将确定Ash 1在该启动子上的依赖机制，并揭示Ash1如何与FLO11的信号依赖转录调节因子合作。目的(2)探讨FLO11在PH生长中的定位和功能。目的(3)提出了一种新的策略来产生子代特异的互补脱氧核糖核酸(CDNA)，用于阵列分析，以识别受Ash1控制的子代特异基因。 申请者为Ash1转录控制与信号依赖转录因子整合的机制建立的原则将广泛适用于GATA家族。特别是，Ash1是保守的，是YF-PH转换所必需的，而YF-PH转换是病原真菌毒力所必需的。因此，Errede博士定义的分子机制将直接适用于那些潜在的真菌致病性。此外，该项目有可能揭示真菌蛋白质，这些蛋白质将是药物干预的极佳候选对象。

DESCRIPTION (provided by applicant): Nitrogen deprivation causes diploid cells of the yeast S. cerevisiae to depart from vegetative growth and form pseudohyphae. These are filaments of rod-shaped cells that radiate from the periphery of a colony and spread over and into the agar medium. The transition arises when a yeast form (YF) cell divides and gives rise to a PH daughter that has a different developmental fate from its mother. This asymmetric switch is fundamentally the same as those that occur during the production of distinct lineages from pluripotent stem cells in humans. In yeast as in mammals, the fate change requires co-ordination of programs that are controlled by multiple transcription factors. Some are responsive to extracellular stimuli and others on cell inherent mechanisms for asymmetric segregation of the critical fate determining factors. S. cerevisiae Ash 1 is a GATA-family transcription factor that is differentially segregated to daughters and is essential for the PH transition. While the mechanism responsible for asymmetric localization of Ash1 has been elucidated, very little is known about the daughter-specific program that it mediates to establish the PH fate. This program has been difficult to study by standard methods because PH colonies are a heterogeneous mixture of cell-types. The applicant proposes to develop strategies using green fluorescent protein (GFP) labels that allow analyses of promoter function and protein and mRNA dynamics in individual living cells. Currently, the cell-surface adhesion protein, Flo11, is the only known gene product required for PH growth whose transcription is dependent on Ash1. Aim (1) focuses on Flo11 transcriptional regulation. Studies will define the Ash 1-dependent mechanism at this promoter and reveal how Ash1 collaborates with signal dependent transcriptional regulators of Flo11. Aim (2) addresses daughter-specific localization and functions of Flo11 in PH growth. Aim (3) proposes a novel strategy to generate daughter-specific complementary deoxyribonucleic acids (cDNAs) for use in array analyses to identify daughter-specific genes subject to Ash1 control. The principles the applicants establish for the mechanism by which Ash1 transcriptional control is integrated with that of signal dependent transcription factors will be broadly applicable to the GATA-family. In particular, Ash1 is conserved and required for the YF-PH transition that is essential for virulence of pathogenic fungi. Thus, the molecular mechanisms Dr. Errede defines will be directly applicable to those underlying fungal pathogenicity. Additionally, the project has the potential to reveal fungal proteins that would be excellent candidates as targets for drug intervention.