NSF Award
0114040
The research is on a newly discovered global response of yeast cells to the depletion of oxygen. Two families of "anaerobic genes"-i.e. genes expressed under anaerobic conditions, the "hypoxic genes" and the recently discovered DAN/TIR genes, are under the negative control of heme; heme also stimulates transcription of a large group of oxygen-induced genes. The DAN/TIR genes encode cell wall mannoproteins; they are induced at the same time that the normal cell wall proteins, Cwp1 and Cwp2 are down-regulated, leading to remodeling of the cell surface. Three of the Dan/Tir proteins are essential for anaerobic growth, indicating the importance of adaptation to oxygen depletion. Regulation of the hypoxic genes by the Rox1 and Mot3 repressors is now well understood. However, regulation of the eight DAN/TIR genes needs to be elucidated. This work should help define the pathway responsible for the reciprocal heme-mediated regulation of oxygen-induced and oxygen-repressed genes. Some factors play multiple roles in this pathway, providing an illuminating example of coordinately and reciprocally regulated gene expression system. For example Mot3, under the control of heme, represses the DAN/TIR genes, and simultaneously activates the CWP2 gene. The mechanism controlling the DAN/TIR genes is complex, involving three factors (the Mox proteins) needed for regulated anaerobic induction of the regulon and several repressors which serve to squelch basal expression in aerobic cells, when most of these genes are not expressed. In this project the mechanism by which heme controls the activity of Mox4, the keystone regulator of the DAN/TIR genes will be investigated. This binucleate zinc cluster protein is a transcriptional activator targeting a common anaerobic response element in the DAN/TIR promoters. It is controlled by two repressors, Mox1 and Mox2, in response to the regulatory signal provided by the heme molecule. The guiding hypothesis is that heme stimulates Mox1 and Mox2 to interact with Mox4 and block its activation function, through a repression domain which has been identified at the C-terminus. The structure and function of Mox4 as a target of heme repression will be analyzed by deletion mutagenesis and by testing fusions of sub-fragments of the protein to LexA, a DNA-binding protein. Subsequently the interaction of Mox4 with Mox1 and Mox2 will be analyzed by genetic and biochemical means, using co-immunoprecipitation analysis, in a strategy designed to test a series of possible mechanisms by which heme controls Mox4 activity. Finally the interaction will be studied in the setting of Mox4-DNA complexes.<br/> Yeast is a facultative organism capable of rapid adaptation to changes in nutrient and oxygen supply, and it has become our best model for the regulation of gene expression by oxygen in eukaryotic cells. The proposed research will greatly increase the value of this model as a teaching and research paradigm, significantly advancing our knowledge about a key topic in gene expression. Research on the oxygen regulation in yeast has intersected propitiously with the study of transcriptional control in yeast model systems. The regulation of oxygen-induced genes, e.g. the cytochrome c gene, was an early model for metabolic control of eukaryotic gene expression. This project will focus on the other side of oxygen regulation, how the adaptation of yeast cells to oxygen depletion is generated at the level of gene expression.
