Systemic approaches using single cell transcriptome reveal that C/EBPγ regulates autophagy under amino acid starved condition.

Autophagy, a catabolic process to remove unnecessary or dysfunctional organelles, is triggered by various signals including nutrient starvation. Depending on the types of the nutrient deficiency, diverse sensing mechanisms and signaling pathways orchestrate for transcriptional and epigenetic regulation of autophagy. However, our knowledge about nutrient type-specific transcriptional regulation during autophagy is limited. To understand nutrient type-dependent transcriptional mechanisms during autophagy, we performed single cell RNA sequencing (scRNAseq) in the mouse embryonic fibroblasts (MEFs) with or without glucose starvation (GS) as well as amino acid starvation (AAS). Trajectory analysis using scRNAseq identified sequential induction of potential transcriptional regulators for each condition. Gene regulatory rules inferred using TENET newly identified CCAAT/enhancer binding protein γ (C/EBPγ) as a regulator of autophagy in AAS, but not GS, condition, and knockdown experiment confirmed the TENET result. Cell biological and biochemical studies validated that activating transcription factor 4 (ATF4) is responsible for conferring specificity to C/EBPγ for the activation of autophagy genes under AAS, but not under GS condition. Together, our data identified C/EBPγ as a previously unidentified key regulator under AAS-induced autophagy.