Identification of DAF-16/FOXO Binding Partners in Caenorhabditis elegans by Immunoprecipitation and Mass Spectrometry

Abstract

The conserved transcription factor DAF-16/FOXO is a central hub in the regulation of stress responses and aging. It was first discovered as a protein activated by reduced insulin/IGF-like signaling (IIS) that would drive aging-preventive transcriptional outcomes. However, research from the last two decades has shown that its functions extend much further, with it responding to a broad spectrum of stress and deprivation-related stimuli and relaying them into optimal transcriptional outcomes that promote stress resistance, slow aging, and ultimately help the organism to survive each given threat. Evidence is mounting that DAF-16/FOXO is not self-sufficient in this important role but relies on numerous binding partners that help it achieve appropriate activity and target gene selectivity, with prominent examples being the chromatin remodeling complex SWI/SNF, the DAF-16-inhibitory protein HCF-1, or the transcription factor HLH-30/TFEB—just to mention a few. Here we present a protocol for the identification of such DAF-16/FOXO binding partners in Caenorhabditis elegans, comprising the large-scale growth and harvest of such animals, their lysis, and the eventual purification of DAF-16/FOXO, to yield samples that can be analyzed for co-purifying proteins und thus potential binding partners by tandem mass spectrometry.