Single cell RNA sequencing of mouse embryonic fibroblasts in autophagy processes

Autophagy, a catabolic process to remove unnecessary or dysfunctional cells, is triggered by various signals including nutrient starvation. Depending on the type of the nutrient deficiency, diverse sensing mechanisms and pathways are used for autophagy, suggesting subsequent nutrient dependent transcriptional regulation. Still, however, our knowledge about nutrient specific transcriptional regulation during autophagy is limited. To understand nutrient type dependent transcriptional mechanisms during autophagy, we performed single cell RNA sequencing (scRNAseq) for the mouse embryonic fibroblasts (MEFs) before and after applying glucose- (GS) as well as amino acid starvation (AAS). Trajectory analysis using scRNAseq identified sequential induction of potential transcriptional regulators for each deficiency condition. Gene regulatory rules inferred using TENET newly identified CCAAT/enhancer binding protein ? (C/EBP?) regulates autophagy processes specifically to AAS condition. Strikingly, knockdown of C/EBP? attenuated the autophagic process only in the AAS condition. Cell biological and biochemical studies validated that C/EBP? plays a switching role for ATF4 to activate autophagy genes under AAS, but not under GS. Together, our data identified C/EBP? as a previously unidentified key regulator under amino acid starvation-induced autophagy. Overall design: We performed single cell RNA sequencing (scRNAseq) for the mouse embryonic fibroblasts (MEFs) before and after applying glucose (GS) as well as amino acid starvation (AAS).