Regulatory gene networks controlling carbon utilization in yeast

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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.