Genome wide mapping of stress-response and ageing pathway regulators

Stress and molecular stress responses are inextricably linked to many age-related diseases. To gain insight into the genetic regulation of the stress response machinery it is essential to uncovergene expression regulators on a genome wide scale. Here we report the uncovering of heat-shock regulators by exploring natural variation of genome wide gene transcription in C. elegansusing two fully sequenced recombinant inbred populations derived from wild types Bristol N2 and CB4856. The first population is a genetic mosaic of the two parental alleles (RILs). The second is a population of introgression lines (ILs), where each line contains only a single locus of CB4856 in an N2 background. Variation in gene expression levels waslinked to genomic loci (eQTL) inworms grown at 20oC for their entire life and worms exposed to a 2 hour 35oC heat-shock at the end of their life. This allowed us to identify novel regulatory loci affecting multiple transcriptsunder heat-stress conditions. These regulatory loci were verified using ILs. The outcomes of these experiments allowed us to identify candidate regulators by transposing our data overWormnet. Next, these candidate genes were validated by phenotyping knock-out mutants. Theregulatory locus affecting most genes upon heat-shock was found on the top left arm of chromosome IV. This locus (IV: 1.27-1.93 Mb) affects the expression of 315 genes and coincides with a QTL affecting survival after heat-shock. Subsequently we could confirm the effect of this locus on 182 genes by the IL expression profiles. Transposing the eQTL results on Wormnet identified 44 candidate genes that potentially regulate the heat-stress response, including cmk-1andegl-4. In the final experiment the transcription profiles under heat-shock were measured in44 mutants. The transcripts were investigated for differentially expressed genes compared to N2. These differentially expressed genes were investigated for overlap with eQTLs mapping (close)to the physical position of the mutant. This approach revealed four genes involved in the heatstress response of which cmk-1as a main regulator of the transcriptional response to heat-stress. This implies that heat-shock response requires cmk-1activity which is essential for normal thermo-sensory behavior. Together, our results provide a framework for using natural variation to identify stress-pathway regulators.