Cells and organisms constantly experience harmful environmental stresses ranging from suboptimal temperatures to foreign substances such as nutrient contaminants or drugs. Oxidative stress occurs when reactive oxygen species (ROS), obligate and ubiquitous by-products of aerobic respiration, accumulate within the cell. This accumulation is toxic, and high ROS levels are hypothesized to cause and/or exacerbate diseases such as diabetes, cancers, and neurodegenerative disorders. MDT-15, a subunit of the Mediator complex, is a transcriptional coregulator which controls oxidative stress response gene programs in C. elegans. For example, SKN-1, the master regulator of oxidative stress responses in C. elegans, binds to MDT-15 and both proteins are necessary to activate SKN-1-dependent genes and to survive oxidative stress. Surprisingly, responses to the oxidative stressor tert-butyl hydroperoxide (tBOOH), which causes ROS accumulation, are SKN-1 independent. This suggests that additional, parallel mechanisms must exist that protect against excess ROS. We have discovered that the transcription factor NHR-49, which is required to express genes involved in lipid metabolism, including beta-oxidation and fatty acid desaturation, is also involved in this response pathway to induce tBOOH response genes such as fmo-2
. To identify new players acting in this pathway, I am performing a reverse genetic screen using RNAi. As readout, I am using a transcriptional reporter, composed of the GFP reporter fused to the promoter of fmo-2
. Targeted RNAi will identify which of the ~900 transcription factors, ~400 kinases and ~500 transcriptional coregulators are required for tBOOH-dependent induction of fmo-2
, and thus might map into the NHR-49 pathway. In addition, we have also begun to explore whether mdt-15
participate in other stress and defence responses, and/or whether they interact genetically with factors known to coordinate environmental adaptation.