Regulatory gene networks controlling carbon utilization in yeast

这项建议旨在以酿酒酵母为模式生物来研究调控基因网络。在酿酒酵母中，葡萄糖是首选碳源，发酵是产生能量的主要途径，即使在好氧条件下也是如此。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，需要转换为呼吸作用。酿酒酵母也可以利用其他不可发酵的碳源，如乳酸或甘油。从发酵到呼吸的转变导致了基因表达的大规模重新编程。例如，当转向乙醇时，糖异生和乙醛酸循环的基因表达增加，相反，在这些条件下，一些发酵基因的表达减少。 调节这一过程的一个重要角色是Snf1激酶。它在低糖条件下被激活，导致许多底物的磷酸化，其中包括DNA结合蛋白，如Mig1，Cat8，Sip4和RDS2。Mig1是一种转录抑制因子，在被Snf1磷酸化后被失活，从而使靶基因(如CAT8)失活。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控制与非发酵碳源利用相关的各种基因的表达。例如，这些因素控制PCK1的表达，编码磷酸烯醇式丙酮酸羧激酶(一种糖异生的必需酶)。 除了RDS2，我们还发现了与非发酵碳源的使用有关的其他调控因子：锌簇蛋白ERT1、Gsm1和Rop1。例如，ERT1的全基因组定位分析(CHIP-CHIP)表明，它与Cat8、RDS2和Sip4有共同和不同的靶点。我们的结果还表明，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，每个因素似乎也有不同的目标。除ERT1和RDS2外，当从葡萄糖转变为乙醇时，其他因子的表达增加。我们还有证据表明，另外两个锌簇蛋白Sut1和Ume6是糖异生的上游调节因子。 我们建议用Gsm1、Rop1、Sut1、Ume6完成我们的芯片研究，并将结果与表达谱研究相关联。将进行免疫共沉淀实验以确定特定因子的合作伙伴(S)。我们还将进行动力学实验。我们将在RNA和蛋白质水平上测量这些因子在从发酵到呼吸的转变过程中的表达。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来干扰网络，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转录和非发酵碳源对其调控的实验。 总之，酿酒酵母是一种被广泛研究的模式生物。然而，我们的结果表明，碳利用的监管比预期的要复杂得多。拟议的工作将使我们更好地了解这一进程。重要的是，我们的建议是基于一个高度易处理的系统，该系统为研究调控因子网络提供了一个极好的模型，而调控因子网络是生物学各个方面的基础。最后，我们的研究应该产生对生物技术应用有用的见解，例如生产乙醇作为生物燃料。

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.