Regulatory gene networks controlling carbon utilization in yeast

本研究旨在以酿酒酵母为模式生物研究调控基因网络。在酿酒酵母中，葡萄糖是首选的碳源，发酵是能量产生的主要途径，即使在有氧条件下也是如此。然而，当葡萄糖变得稀缺时，发酵过程中产生的乙醇被用作碳源，需要转变为呼吸作用。其他不可发酵的碳源，如乳酸或甘油，也可以被酿酒酵母利用。从发酵到呼吸的转变导致基因表达的大量重编程。例如，糖异生和乙醛酸循环基因的表达增加，在转向乙醇时观察到，相反，在这些条件下，一些发酵基因的表达减少。Snf1激酶是调控这一过程的重要分子。它在低葡萄糖条件下被激活，导致许多底物磷酸化，包括DNA结合蛋白，如Mig1， Cat8， Sip4和Rds2。Mig1是一种转录抑制因子，在被Snf1磷酸化后，它会失活，从而导致靶基因（如CAT8）的抑制。Adr1是参与乙醇利用的基因的转录激活因子。Cat8、Sip4和Rds2是锌簇蛋白家族的成员。Cat8、Rds2和Sip4控制各种参与非发酵碳源利用的基因的表达。例如，这些因子控制PCK1的表达，编码磷酸烯醇丙酮酸羧激酶（糖异生的必需酶）。*除了Rds2，我们还确定了其他参与使用不可发酵碳源的调节因子：锌簇蛋白Ert1， Gsm1和Rop1。例如，Ert1的全基因组定位分析（ChIP-chip）显示其与Cat8、Rds2和Sip4具有共同和不同的靶点。我们的研究结果还表明，Rop1是PCK1的负调控因子。值得注意的是，Adr1、Cat8、Rds2、Sip4、Ert1和Gsm1都与PCK1基因的启动子结合。然而，每种因素似乎也有不同的目标。除Ert1和Rds2外，其他因子的表达随着葡萄糖向乙醇的转变而增加。我们也有证据表明，另外两个锌簇蛋白，Sut1和Ume6，是糖异生的上游调节因子。*我们建议完成Gsm1、Rop1、Sut1、Ume6的ChIP-chip研究，并将结果与表达谱研究相关联。将进行共免疫沉淀实验，以确定给定因子的伙伴。我们也会做动力学实验。我们将测量从发酵到呼吸过程中这些因子在RNA和蛋白质水平上的表达。我们还将通过使用携带编码这些因子的基因的单或双缺失的菌株干扰网络来确定这些因子在调节彼此表达方面的相对贡献。最后，提出了旨在更好地了解线粒体DNA转录及其通过不可发酵碳源调控的实验。*综上所述，酿酒葡萄球菌是一种被广泛研究的模式生物。然而，我们的研究结果表明，碳利用的调控比预期的要复杂得多。拟议的工作将有助于更好地理解这一过程。重要的是，我们的建议是基于一个高度可处理的系统，为研究生物学各方面基础的调节因子网络提供了一个极好的模型。最后，我们的研究应该产生对生物技术应用有用的见解，例如乙醇作为生物燃料的生产。

This proposal is aimed at studying regulatory gene networks using the yeast Saccharomyces cerevisiae as a model organism. In S. cerevisiae, glucose is the preferred carbon source and fermentation is the major pathway for energy production, even under aerobic conditions. However, when glucose is becoming scarce, ethanol produced during fermentation is used as a carbon source, requiring a shift to respiration. Other non-fermentable carbon sources, such as lactate or glycerol, can also be used by S. cerevisiae. The shift from fermentation to respiration results in massive reprogramming of gene expression. For example, increased expression of genes for gluconeogenesis and the glyoxylate cycle is observed upon a shift to ethanol and, conversely, expression of some fermentation genes is reduced under these conditions. * An important player for the regulation of this process is the Snf1 kinase. It becomes activated under low glucose conditions resulting in the phosphorylation of a number of substrates that include DNA binding proteins such as Mig1, Cat8, Sip4 and Rds2. Mig1 is a transcriptional repressor that, following phosphorylation by Snf1, is inactivated allowing derepression of target genes such as CAT8. Adr1 is a transcriptional activator of genes involved in the utilization of ethanol. Cat8, Sip4 and Rds2 are members of the family of zinc cluster proteins. Cat8, Rds2 and Sip4 control expression of various genes involved in the utilization of non-fermentable carbon sources. For example, these factors control the expression of PCK1, encoding phosphoenolpyruvate carboxykinase (an essential enzyme for gluconeogenesis).* In addition to Rds2, we have identified additional regulators involved in the use of non-fermentable carbon sources: the zinc cluster proteins Ert1, Gsm1 and Rop1. For example, genome-wide localization analysis (ChIP-chip) of Ert1 showed that it has common and distinct targets with those of Cat8, Rds2 and Sip4. Our results also show that Rop1 is a negative regulator of PCK1. Remarkably, Adr1, Cat8, Rds2, Sip4, Ert1, and Gsm1 all bind to the promoter of the PCK1 gene. However, each factor also appears to have distinct targets. With the exception of Ert1 and Rds2, expression of the other factors is increased upon a shift from glucose to ethanol. We also have evidence that two additional zinc cluster proteins, Sut1 and Ume6, are upstream regulators of gluconeogenesis.* We propose to complete our ChIP-chip studies with Gsm1, Rop1, Sut1, Ume6, and correlate the results with expression profiling studies. Co-immunoprecipitation experiments will be performed to identify partner(s) of a given factor. We will also perform kinetics experiments. We will measure the expression of these factors at the RNA and protein levels during the shift from fermentation to respiration. We will also determine the relative contribution of these factors in regulating each other's expression by perturbing the network using strains carrying single or double deletions of genes encoding these factors. Finally, experiments aimed at better understanding transcription of mitochondrial DNA and its regulation by non-fermentable carbon sources are proposed.* In summary, S. cerevisiae is a model organism that has been extensively studied. However, our results show that the regulation of carbon utilization is much more complex than anticipated. The proposed work will provide a better understanding of this process. Importantly, our proposal is based on a highly tractable system that provides an excellent model for studying networks of regulatory factors that are fundamental to all aspects of biology. Finally, our studies should yield insights useful for biotechnological applications, such as the production of ethanol as a biofuel.