Identification of New Cofactors of DAF-16

Insulin/IGF-1 signaling is highly conserved in various established model organisms and regulates early developmental decisions and important functions involved in stress resistance and aging of an organism. The Forkhead box O-class (FOXO) transcription factor DAF-16 determines the downstream gene expression in response to insulin/IGF-1 signaling in Caenorhabditis elegans. The transcriptional targets of DAF-16 include a large number of genes involved in aging, cellular stress responses (heat-shock and oxidative damage) and resistance to bacterial infection. Although all of these genes require DAF-16 for their transcription, subsets of these genes are regulated independently of each other. The goal of this proposal is to better understand at the molecular level how DAF-16 regulates aging and stress response. We hypothesize that the differences in the DAF-16 downstream gene expression are mediated through distinct sets of as yet unidentified cofactors that interact with DAF-16 complexes to differentially regulate the transcriptional potential of this FOXO transcription factor. The presented proposal seeks to utilize a reporter-based RNAi screen and mass spectrometry to identify cofactors and protein complexes of DAF-16. In the Dillin laboratory the nuclear protein SMK-1, a homologue of mammalian SMEK1, has been identified as a cofactor for DAF-16. It is required for life span extension by low insulin-like signaling and mediates certain forms of stress resistance. Using a reporter-based RNAi approach we will screen for SMK-1 like factors of DAF-16 and for activator- and repressor-class factors of DAF-16. To identify transcriptional complexes specific for the longevity function of DAF-16 with an alternative approach, we will use mass spectrometry to analyze immunoprecipitates of tagged versions of DAF-16 expressed in transgenic worms. Identified candidates interacting with DAF-16 will be compared with potential longevity factors obtained from the RNAi screen. Furthermore, we will use C. elegans based genetic analysis to verify and characterize the roles of identified proteins in insulin/IGF-1 signaling. We expect this work to expand the current understanding of the transcriptional mechanisms that regulate DAF-16 longevity and stress response functions.