Regulatory gene networks controlling carbon utilization in yeast

控制酵母碳利用的调控基因网络

• 批准号: RGPIN-2014-06406
• 负责人: Turcotte, Bernard
• 金额: $ 2.55万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=588984
• 关键词: Regulatory; gene; networks; controlling; carbon

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.

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。