Although the genes essential for life have been identified in less complex model organisms, their elucidation in human cells has been hindered by technical barriers. We used extensive mutagenesis in haploid human cells to identify approximately 2000 genes required for optimal fitness under culture conditions. To study the principles of genetic interactions in human cells, we created a synthetic lethality network focused on the secretory pathway based exclusively on mutations. This revealed a genetic cross-talk governing Golgi homeostasis, an additional subunit of the human oligosaccharyltransferase complex, and a phosphatidylinositol 4-kinase β adaptor hijacked by viruses. The synthetic lethality map parallels observations made in yeast and projects a route forward to reveal genetic networks in diverse aspects of human cell biology.

SummaryThe spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show here that chromatin loop size can be increased and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin’s DNA release factor WAPL restricts this loop extension and also prevents looping between incorrectly oriented CTCF sites. We reveal that the SCC2/SCC4 complex promotes the extension of chromatin loops and the formation of topologically associated domains (TADs). Our data support the model that cohesin structures chromosomes through the processive enlargement of loops and that TADs reflect polyclonal collections of loops in the making. Finally, we find that whereas cohesin promotes chromosomal looping, it rather limits nuclear compartmentalization. We conclude that the balanced activity of SCC2/SCC4 and WAPL enables cohesin to correctly structure chromosomes.

Although platinum-based drugs are widely used chemotherapeutics for cancer treatment, the determinants of tumor cell responsiveness remain poorly understood. We show that the loss of subunits LRRC8A and LRRC8D of the heteromeric LRRC8 volume-regulated anion channels (VRACs) increased resistance to clinically relevant cisplatin/carboplatin concentrations. Under isotonic conditions, about 50% of cisplatin uptake depended on LRRC8A and LRRC8D, but neither on LRRC8C nor on LRRC8E. Cell swelling strongly enhanced LRRC8-dependent cisplatin uptake, bolstering the notion that cisplatin enters cells through VRAC. LRRC8A disruption also suppressed drug-induced apoptosis independently from drug uptake, possibly by impairing VRAC-dependent apoptotic cell volume decrease. Hence, by mediating cisplatin uptake and facilitating apoptosis, VRAC plays a dual role in the cellular drug response. Incorporation of the LRRC8D subunit into VRAC substantially increased its permeability for cisplatin and the cellular osmolyte taurine, indicating that LRRC8 proteins form the channel pore. Our work suggests that LRRC8D-containing VRACs are crucial for cell volume regulation by an important organic osmolyte and may influence cisplatin/carboplatin responsiveness of tumors.

Viruses and Congenital Disorders Mutations in genes involved in α-dystroglycan O-linked glycosylation result in posttranslation modifications associated with the congenital disease Walker-Warburg syndrome (WWS). This cellular modification is also required for efficient Lassa virus infection of cells. Jae et al. (p. 479 , published online 21 March) screened for genes involved in O-glycosylation that affected Lassa virus infection and identified candidates involved in glycosylation. Individuals from different pedigrees exhibiting WWS had unique mutations among genes identified in the genetic screen. Thus, comprehensive forward genetic screens can be used to define the genetic architecture of a complex disease.

Insertional mutagenesis in a haploid background can lead to complete disruption of gene function1. Here we generate a population of human cells that contain insertions in >98% of their expressed genes. We established Phenotypic Interrogation via Tag Sequencing (PhITSeq) as a method to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of selected cells rather than individual clones provides a rapid assessment of the spectrum of genes involved in phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic gram-negative bacteria that cause tissue damage at distinct anatomical sites2. We identified 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. While related CDTs may share host factors, they also exploit unique host factors yielding a characteristic profile for each CDT.