Global gene disruption in human cells to assign genes to phenotypes

Insertional mutagenesis in a haploid background can disrupt gene function[superscript 1]. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones[superscript 1] enables rapid assessment of the spectrum of genes involved in the 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 responsible for tissue damage at distinct anatomical sites[superscript 2]. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT

Studying disease-linked phenotypes using haploid genetics

Although genes are unequivocally important for the development of both common and rare human diseases, the connection between the genotype (an individual’s genetic makeup) and phenotype (an individual’s observable traits) is often ill-defined. Even genetic disorders caused by a defect in only a single gene often manifest themselves differently in affected patients, indicating that the precise consequence of genetic mutations is often difficult to predict. This phenotype-genotype conundrum is a result of genes not functioning in isolation, but in complex genetic networks where gene function can be modulated by the action of other genes or the environment. A powerful approach to understand the genetic contribution to a phenotype is to generate mutations in model organisms. Large-scale mutation studies in yeast have illuminated some of the complex genetic architecture, but owing to technical constraints mutagenesis of human cells has long been unachievable. Central to this work is the application of haploid human mutant cells to study gene-environment and gene-gene interactions and their contribution to disease-relevant phenotypes. We describe a method for mapping mutations in pools of mutant cells following a phenotypic selection; typically mutants that survive exposure to otherwise noxious stimuli. This is applied to study the interactions between genes and the environment, focusing on a number of bacterial toxins that exploit the function of human genes in order to enter and cause damage to cells. In addition, the mechanism of resistance to multiple clinically-used drugs is examined. This same method is applied to identify host factors required by Rift Valley Fever Virus for infecting human cells. Further optimizations to this approach have made it possible not only to identify mutants which are favored under particular conditions, but also those which are under negative selection. We have applied this to define a set of around 2.000 genes required for cultured human cells to grow. This includes a number of genes which were uncharacterized, despite their essential function in basic cellular processes. The ability to identify essential genes furthermore enables examining gene essentiality specific to particular genetic backgrounds, referred to as genetic interactions. Mapping the essential genes in cell lines deficient for different genes uncovered a synthetic lethality network focused on the human secretory pathway. Comparable to yeast, human genes frequently engage in genetic interactions, which implies that many non-essential genes will become essential in the absence of other genes. The ability to efficiently map mutations in populations of fixed haploid cells is further leveraged to examine the genetic regulators of intracellular phenotypes. For a dozen of diverse biological traits, such as oncogenic pathway activation or organelle size, we have identified hundreds of genes that impact the measured phenotype. Based on this, we construct a preview of a phenotypic map of a human cell, and observe that pleiotropy is frequent for human genes, as close to half of the identified regulators affect multiple phenotypes. By iterative screens in genetic backgrounds, suppressors can be identified which restore the affected intracellular phenotypes. This enables the systematic dissection of a cellular process to gain mechanistic understanding

Studying disease-linked phenotypes using haploid genetics

Although genes are unequivocally important for the development of both common and rare human diseases, the connection between the genotype (an individual’s genetic makeup) and phenotype (an individual’s observable traits) is often ill-defined. Even genetic disorders caused by a defect in only a single gene often manifest themselves differently in affected patients, indicating that the precise consequence of genetic mutations is often difficult to predict. This phenotype-genotype conundrum is a result of genes not functioning in isolation, but in complex genetic networks where gene function can be modulated by the action of other genes or the environment. A powerful approach to understand the genetic contribution to a phenotype is to generate mutations in model organisms. Large-scale mutation studies in yeast have illuminated some of the complex genetic architecture, but owing to technical constraints mutagenesis of human cells has long been unachievable. Central to this work is the application of haploid human mutant cells to study gene-environment and gene-gene interactions and their contribution to disease-relevant phenotypes. We describe a method for mapping mutations in pools of mutant cells following a phenotypic selection; typically mutants that survive exposure to otherwise noxious stimuli. This is applied to study the interactions between genes and the environment, focusing on a number of bacterial toxins that exploit the function of human genes in order to enter and cause damage to cells. In addition, the mechanism of resistance to multiple clinically-used drugs is examined. This same method is applied to identify host factors required by Rift Valley Fever Virus for infecting human cells. Further optimizations to this approach have made it possible not only to identify mutants which are favored under particular conditions, but also those which are under negative selection. We have applied this to define a set of around 2.000 genes required for cultured human cells to grow. This includes a number of genes which were uncharacterized, despite their essential function in basic cellular processes. The ability to identify essential genes furthermore enables examining gene essentiality specific to particular genetic backgrounds, referred to as genetic interactions. Mapping the essential genes in cell lines deficient for different genes uncovered a synthetic lethality network focused on the human secretory pathway. Comparable to yeast, human genes frequently engage in genetic interactions, which implies that many non-essential genes will become essential in the absence of other genes. The ability to efficiently map mutations in populations of fixed haploid cells is further leveraged to examine the genetic regulators of intracellular phenotypes. For a dozen of diverse biological traits, such as oncogenic pathway activation or organelle size, we have identified hundreds of genes that impact the measured phenotype. Based on this, we construct a preview of a phenotypic map of a human cell, and observe that pleiotropy is frequent for human genes, as close to half of the identified regulators affect multiple phenotypes. By iterative screens in genetic backgrounds, suppressors can be identified which restore the affected intracellular phenotypes. This enables the systematic dissection of a cellular process to gain mechanistic understanding

BUB1 Is Essential for the Viability of Human Cells in which the Spindle Assembly Checkpoint Is Compromised

Summary: The spindle assembly checkpoint (SAC) ensures faithful segregation of chromosomes. Although most mammalian cell types depend on the SAC for viability, we found that human HAP1 cells can grow SAC independently. We generated MAD1- and MAD2-deficient cells and mutagenized them to identify synthetic lethal interactions, revealing that chromosome congression factors become essential upon SAC deficiency. Besides expected hits, we also found that BUB1 becomes essential in SAC-deficient cells. We found that the BUB1 C terminus regulates alignment as well as recruitment of CENPF. Second, we found that BUBR1 was not essential in SAC-deficient HAP1 cells. We confirmed that BUBR1 does not regulate chromosome alignment in HAP1 cells and that BUB1 does not regulate chromosome alignment through BUBR1. Taken together, our data resolve some long-standing questions about the interplay between BUB1 and BUBR1 and their respective roles in the SAC and chromosome alignment

Haploid genetic screens identify genetic vulnerabilities to microtubule‐targeting agents

The absence of biomarkers to accurately predict anticancer therapy response remains a major obstacle in clinical oncology. We applied a genome‐wide loss‐of‐function screening approach in human haploid cells to characterize genetic vulnerabilities to classical microtubule‐targeting agents. Using docetaxel and vinorelbine, two well‐established chemotherapeutic agents, we sought to identify genetic alterations sensitizing human HAP1 cells to these drugs. Despite the fact that both drugs act on microtubules, a set of distinct genes were identified whose disruption affects drug sensitivity. For docetaxel, this included a number of genes with a function in mitosis, while for vinorelbine we identified inactivation of FBXW7, RB1, and NF2, three frequently mutated tumor suppressor genes, as sensitizing factors. We validated these genes using independent knockout clones and confirmed FBXW7 as an important regulator of the mitotic spindle assembly. Upon FBXW7 depletion, vinorelbine treatment led to decreased survival of cells due to defective mitotic progression and subsequent mitotic catastrophe. We show that haploid insertional mutagenesis screens are a useful tool to study genetic vulnerabilities to classical chemotherapeutic drugs by identifying thus far unknown sensitivity factors. These results provide a rationale for investigating patient response to vinca alkaloid‐based anticancer treatment in relation to the mutational status of these three tumor suppressor genes, and could in the future lead to the establishment of novel predictive biomarkers or suggest new drug combinations based on molecular mechanisms of drug sensitivity

Genetic wiring maps of single-cell protein states reveal an off-switch for GPCR signalling

As key executers of biological functions, the activity and abundance of proteins are subjected to extensive regulation. Deciphering the genetic architecture underlying this regulation is critical for understanding cellular signalling events and responses to environmental cues. Using random mutagenesis in haploid human cells, we apply a sensitive approach to directly couple genomic mutations to protein measurements in individual cells. Here we use this to examine a suite of cellular processes, such as transcriptional induction, regulation of protein abundance and splicing, signalling cascades (mitogen-activated protein kinase (MAPK), G-protein-coupled receptor (GPCR), protein kinase B (AKT), interferon, and Wingless and Int-related protein (WNT) pathways) and epigenetic modifications (histone crotonylation and methylation). This scalable, sequencing-based procedure elucidates the genetic landscapes that control protein states, identifying genes that cause very narrow phenotypic effects and genes that lead to broad phenotypic consequences. The resulting genetic wiring map identifies the E3-ligase substrate adaptor KCTD5 (ref. 1) as a negative regulator of the AKT pathway, a key signalling cascade frequently deregulated in cancer. KCTD5-deficient cells show elevated levels of phospho-AKT at S473 that could not be attributed to effects on canonical pathway components. To reveal the genetic requirements for this phenotype, we iteratively analysed the regulatory network linked to AKT activity in the knockout background. This genetic modifier screen exposes suppressors of the KCTD5 phenotype and mechanistically demonstrates that KCTD5 acts as an off-switch for GPCR signalling by triggering proteolysis of Gβγ heterodimers dissociated from the Gα subunit. Although biological networks have previously been constructed on the basis of gene expression, protein-protein associations, or genetic interaction profiles, we foresee that the approach described here will enable the generation of a comprehensive genetic wiring map for human cells on the basis of quantitative protein states

Vasohibins encode tubulin detyrosinating activity

Tubulin is subjected to a number of posttranslational modifications to generate heterogeneous microtubules. The modifications include removal and ligation of the C-terminal tyrosine of ⍺-tubulin. The enzymes responsible for detyrosination, an activity first observed 40 years ago, have remained elusive. We applied a genetic screen in haploid human cells to find regulators of tubulin detyrosination. We identified SVBP, a peptide that regulates the abundance of vasohibins (VASH1 and VASH2). Vasohibins, but not SVBP alone, increased detyrosination of ⍺-tubulin, and purified vasohibins removed the C-terminal tyrosine of ⍺-tubulin. We found that vasohibins play a cell type-dependent role in detyrosination, although cells also contain an additional detyrosinating activity. Thus, vasohibins, hitherto studied as secreted angiogenesis regulators, constitute a long-sought missing link in the tubulin tyrosination cycle

Vasohibins encode tubulin detyrosinating activity

Tubulin is subjected to a number of posttranslational modifications to generate heterogeneous microtubules. The modifications include removal and ligation of the carboxy-terminal tyrosine of ⍺-tubulin. Whereas enzymes for most modifications have been assigned, the enzymes responsible for detyrosination, an activity observed forty years ago, have remained elusive. We applied a haploid genetic screen to find regulators of tubulin detyrosination. We identified SVBP, a peptide that regulates the abundance of Vasohibins (VASH1 and VASH2). Vasohibins, but not SVBP alone, increased detyrosination of ⍺-tubulin and purified Vasohibins removed the carboxy-terminal tyrosine of ⍺-tubulin. Vasohibins played a cell-type dependent role in detyrosination, but cells also contain an additional detyrosinating activity. Thus Vasohibins, hitherto studied as secreted angiogenesis regulators, constitute a long-sought missing link in the tubulin tyrosination cycle

A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

The Wee1 cell cycle checkpoint kinase prevents premature mitotic entry by inhibiting cyclin-dependent kinases. Chemical inhibitors of Wee1 are currently being tested clinically as targeted anticancer drugs. Wee1 inhibition is thought to be preferentially cytotoxic in p53-defective cancer cells. However, TP53 mutant cancers do not respond consistently to Wee1 inhibitor treatment, indicating the existence of genetic determinants of Wee1 inhibitor sensitivity other than TP53 status. To optimally facilitate patient selection for Wee1 inhibition and uncover potential resistance mechanisms, identification of these currently unknown genes is necessary. The aim of this study was therefore to identify gene mutations that determine Wee1 inhibitor sensitivity. We performed a genome-wide unbiased functional genetic screen in TP53 mutant near-haploid KBM-7 cells using gene-trap insertional mutagenesis. Insertion site mapping of cells that survived long-term Wee1 inhibition revealed enrichment of G(1)/S regulatory genes, including SKP2, CUL1, and CDK2. Stable depletion of SKP2, CUL1, or CDK2 or chemical Cdk2 inhibition rescued the gamma-H2AX induction and abrogation of G2 phase as induced by Wee1 inhibition in breast and ovarian cancer cell lines. Remarkably, live cell imaging showed that depletion of SKP2, CUL1, or CDK2 did not rescue the Wee1 inhibition-induced karyokinesis and cytokinesis defects. These data indicate that the activity of the DNA replication machinery, beyond TP53 mutation status, determines Wee1 inhibitor sensitivity, and could serve as a selection criterion for Wee1-inhibitor eligible patients. Conversely, loss of the identified S-phase genes could serve as a mechanism of acquired resistance, which goes along with development of severe genomic instability