CRISPR Genome-scale CRISPR/Cas9 screening reveals the role of PSMD4 in colibactin-mediated cell cycle arrest

Colibactin is a genotoxic secondary metabolite produced by selective Enterobacteriaceae strains that populate the mammalian intestine and produces specific mutational signature in human colonocytes. However, the host pathways involved in colibactin response remain unclear. To address this gap, we performed global transcriptomics and genome wide CRISPR/Cas9 knockout screens and RNA sequencing utilizing live pks+ bacteria and synthetic colibactins. From our genome-wide screens we identified 20 enriched genes with a MaGECK score > 2.0 in both screens, including proteasomal subunits (e.g., PSMG4, PSMD4), RNA processing factors (e.g., SF1, PRPF8), and RNA polymerase III (e.g., CRCP), and validated the role of PSMD4 in colibactin sensitization. PSMD4 knockout ameliorated reductions in cell viability and G2/M cell cycle arrest after 742 exposure. Conversely, PSMD4 knockout did not affect the amount of gammaH2AX foci after 742 exposure. Consistent with these observations, PSMD4-/- cells had a significantly higher colony formation rate and reduced colony size than control cells after 742 exposure. These findings suggest that PSMD4 regulates cell-cycle arrest following colibactin-induced DNA damage, and that cells with deficiencies in these pathways may continue to replicate despite DNA damage, potentially increasing the risk of malignant transformation.

Colibactin（大肠杆菌素）是一类由定殖于哺乳动物肠道的特定肠杆菌科菌株产生的基因毒性次级代谢产物，可在人类结肠细胞中诱导特异性突变特征。然而，宿主响应Colibactin的相关通路仍未明确。为填补这一研究空白，本研究利用活pks+细菌与合成型Colibactin，开展了全局转录组学分析、全基因组CRISPR/Cas9敲除筛选与RNA测序（RNA Sequencing）实验。通过全基因组筛选，我们在两次筛选中均鉴定出20个MaGECK评分大于2.0的富集基因，包括蛋白酶体亚基（如PSMG4、PSMD4）、RNA加工因子（如SF1、PRPF8）以及RNA聚合酶III相关基因（如CRCP），并验证了PSMD4在Colibactin致敏中的作用。敲除PSMD4可缓解经742处理后细胞活力的下降与G2/M期细胞周期阻滞。与之相反，敲除PSMD4并不会影响经742处理后细胞内γH2AX焦点的数量。与上述观察结果一致，经742处理后，PSMD4基因敲除（PSMD4-/-）细胞的集落形成率显著高于对照细胞，而集落尺寸则更小。上述研究结果表明，PSMD4可调控Colibactin诱导的DNA损伤后的细胞周期阻滞过程；携带此类通路缺陷的细胞即便存在DNA损伤仍可继续复制，这可能会增加恶性转化的风险。