Sifting through the regulatory weeds How to use diverse genetic screening strategies to enhance cancer research and drug development

Epigenetic gene regulation is crucial in cancer research owing to its dynamic and reversible properties, making epigenetic processes promising targets in gene editing, reprogramming, and cancer-drug discovery. However, epigenetic mechanisms are often cell-specific and context-dependent, requiring approaches that seamlessly collect statistically robust data in different cells. This thesis highlights the importance of diversifying large-scale genetic screens to decipher genotype–phenotype relationships that can further our understanding of gene regulation in cancer. By multiplexing Cas9 targeting in cells with multiple reporter genes integrated into the mouse genome, we show that target sequence and location explain most observed differences in mutation efficiency. Especially, one-nucleotide insertions – from staggered cuts – correlated with specific transcriptional, genomic, and epigenomic features. Furthermore, we used haploid genetic screens on HAP1 cells to identify novel Polycomb interactions. We found that disruption of the mitotic gene, NUMA1, made cells resistant to PTC-318, a small-molecule BMI1 inhibitor that causes mitotic arrest and death among NUMA1-proficient cells. In a separate study, independent HAP1 screens revealed that chromatin remodeler WSTF positively regulates H3K27me3 and is enriched in BMI1-inhibited cells. The findings suggest that WSTF may regulate an inverse relationship between PRC1 and PRC2 in which reduced levels of selected PRC2 proteins increase BMI1 levels. Finally, an shRNA screen with an epigenetics-focused library discovered that TRIM28 is a barrier to reprogramming somatic mouse cells, possibly by maintaining H3K9me3-associated endogenous retroviruses (ERVs). Together, the findings highlight the power of genomic screens for discovering previously unknown gene-regulatory properties that can enhance cancer research and drug discovery.