One of the challenges facing researchers working on oxygen-regulated biological phenomenon, as well as medical practitioners attempting to minimize hypoxia-induced tissue damage, is gaining a fundamental understanding of the molecular mechanisms for gauging oxygen availability. In spite of significant progress in our understanding of the physiological response to hypoxia, the molecular components involved in sensing oxygen and the pathways for transducing this information are poorly understood. The long-term objective of our research is to delineate oxygen-sensing mechanisms and the signaling pathways involved in eliciting adaptive responses at the level of gene expression. In this revised proposal, we focus on determining the role that cytochrome c oxidase, and possibly other components of the mitochondrial respiratory chain, plays in oxygen-sensing pathways. A yeast model will be used to address the following specific aims.
Aim 1 : Identify the cis site(s) in the promoters of oxygen-responsive genes that confer(s) sensitivity to inhibitors and mutations in the respiratory chain. To address this aim, the promoter regions of several oxygen-responsive genes will be fused to the reporter gene LacZ and used to define the minimal-sized DNA fragment and essential nucleotides that confer sensitivity to inhibitors and mutations in the respiratory chain.
Aim 2 : Identify trans-acting factors that interact with these cis sites and other proteins involved in transducing the low oxygen signal from the mitochondrion to the nucleus. Once the cis site is defined, one-hybrid and two-hybrid screens will be used to identify the trans-acting factor(s) and other proteins responsible for mediating sensitivity to inhibitors and mutations in the respiratory chain. In addition, genome-wide transcript profiling analyses will be used to identify additional sensory and signaling proteins. These studies can be expected to provide novel findings and new insight into the crosstalk between the mitochondrion and nucleus that is involved in modulating the expression of oxygen-responsive genes and provide a general model of oxygen sensing in eukaryotic cells.
