CRISPRi screen identifying epigenetic regulators of autophagic flux

Autophagy is a conserved self-digestion pathway essential for maintaining cellular homeostasis and catabolism. Under normal conditions, autophagic activity remains minimal but is rapidly upregulated in response to stresses such as nutrient deprivation. While the transcriptional and epigenetic activation of autophagy under nutrient deprived condition has been widely studied, less is known about the repression mechanisms of autophagy under normal conditions. Here, we identify PHF23 as an epigenetic repressor of autophagy through a CRISPR interference (CRISPRi) screening. PHF23 inhibits the expression of autophagy genes by recruiting the NuRD complex and reducing chromatin accessibility, particularly at enhancer regions. This repressive function of PHF23 requires an intact PHD domain and is attenuated under amino acid starvation or mTOR inhibition conditions. Notably, inhibiting PHF23 enhances the autophagic clearance of pathological protein aggregates including Tau and a1 antitrypsin Z variant (ATZ), underscoring its potential as a therapeutic target in diseases characterized by proteotoxic stress. Overall design: To systematically identify epigenetic regulators involved in the autophagy regulation, we conducted a CRISPR interference (CRISPRi) screen using a reporter cell line stably expressing the tandem fluorescent-tagged LC3B construct (mCherry-GFP-LC3B). We designed a CRISPRi sgRNA library targeting 613 epigenetic regulators with a total of 3,340 sgRNAs, and transduced it into the reporter cell line. Following transduction, cells were sorted using fluorescence-activated cell sorting (FACS) based on alterations in autophagic flux, and the distribution of sgRNAs was analyzed by next-generation sequencing.