TFAP2C and HNRNPK control cellular bioenergetic metabolism and prion propagation [CRISPR screen]

Heterogenous Nuclear Ribonucleoprotein K (HNRNPK) is a limiting factor for prion propagation. However, little is known about the function of hnRNP_K other that it is essential to cell survival. Here, we performed a synthetic-viability CRISPR ablation screen to identify functional epistatic interactors of HNRNPK. We found that deletion of Transcription Factor AP-2γ (TFAP2C) mitigated the survival of hnRNP_K-depleted LN-229 and U-251MG cells, whereas its overexpression hypersensitized cells to the loss of hnRNP_K. HNRNPK ablation induced downregulation of genes related to lipid and glucose metabolism, decreased cellular ATP and enhanced catabolism through modulation of the mTOR and AMPK pathways. Conversely, TFAP2C overexpression promoted mTOR anabolic activity, and its deletion countered the energetic crisis resulting from HNRNPK ablation. We then found that TFAP2C overexpression reduced prion propagation in wild-type cells and neutralized prion accumulation in HNRNPK-suppressed cells. We conclude that TFAP2C and HNRNPK are genetic interactors controlling cell metabolism, bioenergy and prion propagation. We conducted a genome-wide CRISPR ablation screen for synthetic lethal/viable interactions of HNRNPK in LN-229 cells. Cells were transduced with a lentivirus-packaged Brunello library using an MOI of 0.3, maintaining a coverage of approximately 1000 cells/sgRNA throughout the screen. After 24 hours (Day 1), 280 million cells were collected as a baseline reference to assess library distribution. The remaining cells were selected for 7 days. On Day 7, cells were further transduced with CRISPR knockout guides targeting HNRNPK or with non-targeting (NT) control guides. After 7 additional days under selection, cells were harvested on Day 14 for genomic DNA extraction and sequencing. The screen was repeated in two biological replicates. Differential gene enrichment analysis was performed using EdgeR, identifying significant sgRNAs (log2 fold change ≤ -1 or ≥ 1, FDR < 0.01).