Biology subjects

Ging, K.

Publications and source records attributed to Ging, K..

3 recordsLinked to original sources

bioRxiv2024

TFAP2C and HNRNPK control mTOR cell metabolism and prion propagation

Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) is a limiting factor for prion propagation. However, little is known about the function of hnRNP K except that it is essential to cell survival. Here, we performed a synthetic-viability CRISPR ablation screen to identify epistatic interactors of HNRNPK. We found that deletion of Transcription Factor AP-2{gamma} (TFAP2C) suppressed the death of hnRNP K-depleted LN-229 and U-251 MG cells, whereas its overexpression hypersensitized cells to hnRNP K loss. HNRNPK ablation decreased cellular ATP, downregulated genes related to lipid and glucose metabolism, and enhanced autophagy. Co-occurrent deletion of TFAP2C reversed these effects, restoring transcriptional balance and alleviating energy deficiency. We linked HNRNPK and TFAP2C interaction to mTOR signaling, observing that HNRNPK ablation inhibited mTORC1 activity through downregulation of mTOR and Rptor, while TFAP2C overexpression enhanced mTORC1 downstream functions. In prion-infected cells, TFAP2C activation reduced prion levels and countered the increased prion propagation caused by HNRNPK suppression. Short-term pharmacological inhibition of mTOR also elevated prion levels and partially mimicked the effects of HNRNPK silencing. Our study identifies TFAP2C as a genetic interactor of HNRNPK, implicates their roles in mTOR metabolic regulation, and establishes a causative link between these activities and prion propagation.

cell biology↗

bioRxiv2024

Direct and indirect regulation of β- glucocerebrosidase by the transcription factors USF2 and ONECUT2

Mutations in the GBA gene, which encodes the lysosomal enzyme {beta}-glucocerebrosidase (GCase), are the most prevalent genetic susceptibility factor for Parkinsons disease (PD). However, only approximately 20% of carriers develop the disease, suggesting the presence of genetic modifiers influencing the risk of developing PD in the presence of GBA mutations. Here we screened 1,634 human transcription factors (TFs) for their effect on GCase activity in cell lysates of the human glioblastoma line LN-229, into which we introduced the pathogenic GBA L444P variant via adenine base editing. Using a novel arrayed CRISPR activation library, we uncovered 11 TFs as regulators of GCase activity. Among these, activation of MITF and TFEC increased lysosomal GCase activity in live cells, while activation of ONECUT2 and USF2 decreased it. Conversely, ablating USF2 increased GBA mRNA and led to enhanced levels of GCase protein and activity. While MITF, TFEC, and USF2 affected GBA transcription, ONECUT2 was found to control GCase trafficking by modulating the guanine exchange factors PLEKHG4 and PLEKHG4B. Hence, our study provides a systematic approach to identifying modulators of GCase activity, expands the transcriptional landscape of GBA regulation, and deepens our understanding of the mechanisms involved in influencing GCase activity.

molecular biology↗

bioRxiv2022

Robust and Versatile Arrayed Libraries for Human Genome-Wide CRISPR Activation, Deletion and Silencing

Arrayed CRISPR libraries extend the scope of gene-perturbation screens but require large numbers of efficacious sgRNA-expressing vectors. Using a newly invented liquid-phase plasmid cloning methodology, we constructed genome-wide arrayed libraries for human gene ablation (19,936 plasmids), activation, and epigenetic silencing (22,442 plasmids). At least 76% of each plasmid preparation encoded an intact array of 4 non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. We achieved perturbation efficacies of 75-99%, 76-92% and up to 10,000x in deletion, silencing and activation experiments, respectively. Upon conversion into massively parallel lentiviral vectors, an arrayed activation screen of 1,634 human transcription factors yielded 11 novel regulators of the cellular prion protein PrPC. Furthermore, a screen using a pooled version of the ablation library identified 5 novel modifiers of autophagy that went undetected with either of two 1sgRNA libraries. The CRISPR libraries described here represent a powerful resource for the targeted perturbation of human protein-coding genes.

genomics↗