Project description:We undertook a unbiased genome-wide haploid genetic screen to identify new components in interferon lambda signaling. In addition, we performed a genome-wide screen to identify genes that repress spontaneous activation of interferon stimulated genes in the absence of interferon. Both of these screens were performed using a HAP1 cell line containing GFP reporter under the transcriptional regulation of the Interferon-Stimulated Response Element from IFIT2. We also overexpressed IL28RA (IFNLR1) in this cell line, in order to sensitize the cells to type III interferon
Project description:Autophagy is a conserved degradative process that promotes cellular homeostasis under stress conditions. Under nutrient starvation autophagy is largely non-selective, promoting indiscriminate breakdown of cytosolic components. Conversely, selective autophagy is responsible for the specific turnover of damaged organelles. We hypothesized that selective autophagy may be regulated by distinct upstream signaling from starvation induced autophagy to promote organelle turn-over. To address this question, we conducted kinome-wide CRISPR screens using the DsRed-IRES-GFP-p62 reporter line to identify distinct signaling pathways responsible for the regulation of basal autophagy, starvation-induced autophagy, and two types of selective autophagy, ER-phagy and pexophagy. The Brunello kinome library was designed to enhance on-target activity while minimizing off-target effects, ensuring the effectiveness and efficiency of our screens. These parallel screens identified established and novel autophagy shared regulators under these conditions, as well as kinases specifically required for ER-phagy or pexophagy. More specifically, CDK11A and NME3 were further characterized to be selective ER-phagy regulators. Meanwhile, PAN3 and CDC42BPG were identified as activator or inhibitor of pexophagy, respectively. Collectively, these datasets provide the first comparative description of the kinase signaling specificity, separating regulation of selective autophagy and bulk autophagy.
Project description:Comparative haploid genetic screens to annotate common and specialized genes required for the biogenesis of individual GPI anchored proteins. SEC62 and SEC63 were required for proper PrP targeting, and were dispensable for CD59. CD59 however, required a GPI side chain modification for maturation, in addition to the aspartyl intramembrane cleaving protease: SPPL3.
Project description:Mammalian haploid embryonic stem cells (haESCs) provide new possibilities for large-scale genetic screens because they bear only one copy of each chromosome. However, haESCs are prone to spontaneous diploidization through unknown mechanisms. Here, we report that a small molecule combination could restrain mouse haESCs from diploidization by impeding exit from naïve pluripotency and by shortening the S-G2/M phases. Combined with 2i and PD166285, our chemical cocktail could maintain haESCs in the haploid state for at least five weeks without fluorescence-activated cell sorting (FACS) enrichment of haploid cells. Taken together, we established an effective chemical approach for long-term maintenance of haESCs, and highlighted that proper cell cycle progression was critical for the maintenance of haploid state.