Three Muscle-Specific DAF-16/FOXO Transcriptional Targets Activated by Reduced Insulin/IGF-1 Signaling

C. elegans insulin/insulin-like growth factor 1 signaling, IIS, affects diverse physiological processes through the DAF-16/FOXO transcription factor. Despite its ubiquitous presence in somatic cells, DAF-16's effects exhibit prevalent tissue specificity as well as tissue crosstalk. This implies that tissue-specific DAF-16 transcriptional programs contribute to functional diversity of IIS. To further investigate this possibility, we sought muscle-cell-specific DAF-16 transcriptional targets. Using fluorescence-activated cell sorting to enrich for body wall muscle cells from young hermaphroditic adults, we compared the muscle cell mRNA transcriptomes under conditions of high and low IIS activity, with and without DAF-16. We further analyzed DAF-16a's binding sites in muscle and intestine cells by chromatin-immunoprecipitation sequencing. Combined output of these analyses is 12 candidate DAF-16 targets enriched for muscle cells. Transcriptional and translational reporters for three out of the four top candidates - a secreted protein C54F6.5, a calcium-binding protein CEX-1/calexcitin, and a fatty acid metabolic enzyme MLCD-1/MCD - showed DAF-16-dependent activation specifically in body wall muscle cells. Notably, reporters for C54F6.5 and cex-1 exhibit DAF-16-independent, constitutive expression in non-muscle cells, explaining their low rank or absence from the DAF-16 target lists generated by whole-animal microarray or mRNA-sequencing analyses. These results highlight the need to examine FOXO targets in a cell-type-specific manner. Overall design: C. elegans of different genotypes (wild-type, daf-16(lf), daf-2(lf), daf-2(lf); daf-16(lf)) expressing GFP (green fluorescent protein) specifically in the body wall muscle cells were sorted with fluorescence-activated cell sorting. GFP+; PI- (propidium iodide negative) cells were collected as muscle cells. PI- cells were collected as all cells. mRNA-seq were performed on samples from four genotypes, all with both sorted 'muscle cell' and 'all cell' samples.