Genome-wide CRISPR screen for PARKIN regulators reveals transcriptional repression as a determinant of mitophagy

PARKIN, an E3 ligase mutated in familial Parkinson’s disease, promotes mitophagyby ubiquitinating mitochondrial proteins for efficient engagement of the autophagy machinery. Specifically, PARKIN-synthesized ubiquitin chains represent targets for the PINK1kinase generating phosphoS65-ubiquitin (pUb), which constitutes the mitophagysignal. Physiological regulation of PARKINabundance, however, and the impact on pUb accumulation are poorly understood. Using cells designed to discover physiological regulators of PARKINabundance, we performed a pooled genome-wide CRISPR/ Cas9knockout screen. Testing identified genes individually resulted in a list of 53 positive and negative regulators. A transcriptional repressor network including THAP11 was identified and negatively regulates endogenous PARKINabundance. RNAseq analysis revealed the PARKIN-encoding locus as a prime THAP11 target, and THAP11 CRISPRknockout in multiple cell types enhanced pUb accumulation. Thus, our work demonstrates the critical role of PARKINabundance, identifies regulating genes, and reveals a link between transcriptional repression and mitophagy, which is also apparent in human induced pluripotent stem cell-derived neurons, a disease-relevant cell type.