Effect of GRP75 deficiency on gene expression in DN3 thymocytes

Mitochondria and endoplasmic reticulum contacts (MERCs) regulate multiple cellular processes including cell survival and differentiation. Based on the observations that MERCs were specifically enriched in the CD4-CD8- double negative (DN) stage, we studied their role in early thymocyte development. We found that T-cell-specific knockout of Hspa9, which encodes GRP75, a chaperone mediates MERC formation by assembling the IP3R-GRP75-VDAC complex, impaired DN3 thymocyte viability and resulted in thymocyte developmental arrest at the DN3-DN4 transition. Mechanistically, GRP75 deficiency induced mitochondrial stress, releasing mitochondrial DNA (mtDNA) into the cytosol and triggering the type I interferon (IFN-I) response. IFN-I pathway contributed to both the impairment of cell survival and DN3-DN4 transition blockage, while increased lipid peroxidation (LPO) played a major role downstream of IFN-I. Thus, our study reveals the essential role of GRP75-dependent MERCs in early thymocyte development and uncovers the governing facts of cellular survival and differentiation in the DN stage. Overall design: To inveatigate the role of GRP75 in early thymocyte development, we isolated DN3 (Lin-, CD4-, CD8-, CD44-, CD25+) thymocytes from 5-week-old WT and Hspa9 cKO mice. Then we performed gene expression profiling analysis using data obtained from RNA-seq of WT and Hspa9 cKO DN3 thymocytes.