Mapping of DDX11 genetic interactions defines sister chromatid cohesion as the major dependency.

DDX11/Chl1R is a conserved DNA helicase with roles in genome maintenance, DNA replication, and chromatid cohesion. Loss of DDX11 in humans leads to the rare cohesinopathy Warsaw breakage syndrome. DDX11 has also been implicated in human cancer where it has been proposed to have an oncogenic role and possibly to constitute a therapeutic target. Given the multiple roles of DDX11 in genome stability and its potential as an anticancer target, we set out to define a complete genetic interaction profile of DDX11 loss in human cell lines. Screening the human genome with clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA drop out screens in DDX11-wildtype (WT) or DDX11-deficient cells revealed a strong enrichment of genes with functions related to sister chromatid cohesion. We confirm synthetic lethal relationships between DDX11 and the tumor suppressor cohesin subunit STAG2, which is frequently mutated in several cancer types and the kinase HASPIN. This screen highlights the importance of cohesion in cells lacking DDX11 and suggests DDX11 may be a therapeutic target for tumors with mutations in STAG2.

ModelMatcher: A scientist-centric online platform to facilitate collaborations between stakeholders of rare and undiagnosed disease research.

Next-generation sequencing is a prevalent diagnostic tool for undiagnosed diseases and has played a significant role in rare disease gene discovery. Although this technology resolves some cases, others are given a list of possibly damaging genetic variants necessitating functional studies. Productive collaborations between scientists, clinicians, and patients (affected individuals) can help resolve such medical mysteries and provide insights into in vivo function of human genes. Furthermore, facilitating interactions between scientists and research funders, including nonprofit organizations or commercial entities, can dramatically reduce the time to translate discoveries from bench to bedside. Several systems designed to connect clinicians and researchers with a shared gene of interest have been successful. However, these platforms exclude some stakeholders based on their role or geography. Here we describe ModelMatcher, a global online matchmaking tool designed to facilitate cross-disciplinary collaborations, especially between scientists and other stakeholders of rare and undiagnosed disease research. ModelMatcher is integrated into the Rare Diseases Models and Mechanisms Network and Matchmaker Exchange, allowing users to identify potential collaborators in other registries. This living database decreases the time from when a scientist or clinician is making discoveries regarding their genes of interest, to when they identify collaborators and sponsors to facilitate translational and therapeutic research.

Mapping Synthetic Dosage Lethal Genetic Interactions in Saccharomyces cerevisiae.

Synthetic dosage lethality (SDL) is a type of genetic interaction that occurs when increasing the expression of a gene causes a fitness defect, such as lethality, in a specific mutant background but has little effect on fitness in a wild-type background. SDL genetic interactions discovered in model organisms such as the budding yeast, Saccharomyces cerevisiae , represent candidate genetic interactions that may be conserved in human cells. In some cases, SDL genetic interactions can be applied to study the biological implications of genes overexpressed in cancer and to discover potential anticancer therapeutic drug targets. Here, we provide a protocol for screening a query overexpression gene against ordered arrays of yeast mutant strains to identify mutations that sensitize yeast to increased dosage of a specific gene product. We outline applications and procedures for screening with an inducibly overexpressed wild-type gene, a common feature of cancer cells, or with an inducibly overexpressed gene carrying a dominant-negative missense mutation as a model of protein-inhibitor interactions. This high-throughput screening platform is adapted from synthetic genetic array (SGA) technology and enables the generation of large-scale SDL genetic interaction networks that can be applied to study gene/pathway function and to identify cross-species cancer-relevant processes.

Paralogous synthetic lethality underlies genetic dependencies of the cancer-mutated gene STAG2.

STAG2, a component of the mitotically essential cohesin complex, is highly mutated in several different tumour types, including glioblastoma and bladder cancer. Whereas cohesin has roles in many cancer-related pathways, such as chromosome instability, DNA repair and gene expression, the complex nature of cohesin function has made it difficult to determine how STAG2 loss might either promote tumorigenesis or be leveraged therapeutically across divergent cancer types. Here, we have performed whole-genome CRISPR-Cas9 screens for STAG2-dependent genetic interactions in three distinct cellular backgrounds. Surprisingly, STAG1, the paralog of STAG2, was the only negative genetic interaction that was shared across all three backgrounds. We also uncovered a paralogous synthetic lethal mechanism behind a genetic interaction between STAG2 and the iron regulatory gene IREB2 Finally, investigation of an unusually strong context-dependent genetic interaction in HAP1 cells revealed factors that could be important for alleviating cohesin loading stress. Together, our results reveal new facets of STAG2 and cohesin function across a variety of genetic contexts.

A nuclear proteome localization screen reveals the exquisite specificity of Gpn2 in RNA polymerase biogenesis.

The GPN proteins are a conserved family of GTP-binding proteins that are involved in the assembly and subsequent import of RNA polymerase II and III. In this study, we sought to ascertain the specificity of yeast GPN2 for RNA polymerases by screening the localization of a collection of 1350 GFP-tagged nuclear proteins in WT or GPN2 mutant cells. We found that the strongest mislocalization occurred for RNA polymerase II and III subunits and only a handful of other RNAPII associated proteins were altered in GPN2 mutant cells. Our screen identified Ess1, an Rpb1 C-terminal domain (CTD) prolyl isomerase, as mislocalized in GPN2 mutants. Building on this observation we tested for effects of mutations in other factors which regulate Rpb1-CTD phosphorylation status. This uncovered significant changes in nuclear-cytoplasmic distribution of Rpb1-GFP in strains with disrupted RNA polymerase CTD kinases or phosphatases. Overall, this screen shows the exquisite specificity of GPN2 for RNA polymerase transport, and reveals a previously unappreciated role for CTD modification in RNAPII nuclear localization.

Modeling DNA trapping of anticancer therapeutic targets using missense mutations identifies dominant synthetic lethal interactions.

Genetic screens can identify synthetic lethal (SL) interactions and uncover potential anticancer therapeutic targets. However, most SL screens have utilized knockout or knockdown approaches that do not accurately mimic chemical inhibition of a target protein. Here, we test whether missense mutations can be utilized as a model for a type of protein inhibition that creates a dominant gain-of-function cytotoxicity. We expressed missense mutations in the FEN1 endonuclease and the replication-associated helicase, CHL1, that inhibited enzymatic activity but retained substrate binding, and found that these mutations elicited a dominant SL phenotype consistent with the generation of cytotoxic protein-DNA or protein-protein intermediates. Genetic screens with nuclease-defective hFEN1 and helicase-deficient yCHL1 captured dominant SL interactions, in which ectopic expression of the mutant form, in the presence of the wild-type form, caused SL in specific mutant backgrounds. Expression of nuclease-defective hFEN1 in yeast elicited DNA binding-dependent dominant SL with homologous recombination mutants. In contrast, dominant SL interactions with helicase-deficient yCHL1 were observed in spindle-associated, Ctf18-alternative replication factor C (Ctf18-RFC) clamp loader complex, and cohesin mutant backgrounds. These results highlight the different mechanisms underlying SL interactions that occur in the presence of an inhibited form of the target protein and point to the utility of modeling trapping mutations in pursuit of more clinically relevant SL interactions.

A Multimodal Genotoxic Anticancer Drug Characterized by Pharmacogenetic Analysis in Caenorhabditis elegans.

New anticancer therapeutics require extensive in vivo characterization to identify endogenous and exogenous factors affecting efficacy, to measure toxicity and mutagenicity, and to determine genotypes that result in therapeutic sensitivity or resistance. We used Caenorhabditis elegans as a platform with which to characterize properties of the anticancer therapeutic CX-5461. To understand the processes that respond to CX-5461-induced damage, we generated pharmacogenetic profiles for a panel of C. elegans DNA replication and repair mutants with common DNA-damaging agents for comparison with the profile of CX-5461. We found that multiple repair pathways, including homology-directed repair, microhomology-mediated end joining, nucleotide excision repair, and translesion synthesis, were needed for CX-5461 tolerance. To determine the frequency and spectrum of CX-5461-induced mutations, we used a genetic balancer to capture CX-5461-induced mutations. We found that CX-5461 is mutagenic, resulting in both large copy number variations and a high frequency of single-nucleotide variations (SNVs), which are consistent with the pharmacogenetic profile for CX-5461. Whole-genome sequencing of CX-5461-exposed animals found that CX-5461-induced SNVs exhibited a distinct mutational signature. We also phenocopied the CX-5461 photoreactivity observed in clinical trials and demonstrated that CX-5461 generates reactive oxygen species when exposed to UVA radiation. Together, the data from C. elegans demonstrate that CX-5461 is a multimodal DNA-damaging anticancer agent.

The Canadian Rare Diseases Models and Mechanisms (RDMM) Network: Connecting Understudied Genes to Model Organisms.

Advances in genomics have transformed our ability to identify the genetic causes of rare diseases (RDs), yet we have a limited understanding of the mechanistic roles of most genes in health and disease. When a novel RD gene is first discovered, there is minimal insight into its biological function, the pathogenic mechanisms of disease-causing variants, and how therapy might be approached. To address this gap, the Canadian Rare Diseases Models and Mechanisms (RDMM) Network was established to connect clinicians discovering new disease genes with Canadian scientists able to study equivalent genes and pathways in model organisms (MOs). The Network is built around a registry of more than 500 Canadian MO scientists, representing expertise for over 7,500 human genes. RDMM uses a committee process to identify and evaluate clinician-MO scientist collaborations and approve 25,000 Canadian dollars in catalyst funding. To date, we have made 85 clinician-MO scientist connections and funded 105 projects. These collaborations help confirm variant pathogenicity and unravel the molecular mechanisms of RD, and also test novel therapies and lead to long-term collaborations. To expand the impact and reach of this model, we made the RDMM Registry open-source, portable, and customizable, and we freely share our committee structures and processes. We are currently working with emerging networks in Europe, Australia, and Japan to link international RDMM networks and registries and enable matches across borders. We will continue to create meaningful collaborations, generate knowledge, and advance RD research locally and globally for the benefit of patients and families living with RD.

Cross-Species Complementation of Nonessential Yeast Genes Establishes Platforms for Testing Inhibitors of Human Proteins.

Cross-species complementation can be used to generate humanized yeast, which is a valuable resource with which to model and study human biology. Humanized yeast can be used as an in vivo platform to screen for chemical inhibition of human protein drug targets. To this end, we report the systematic complementation of nonessential yeast genes implicated in chromosome instability (CIN) with their human homologs. We identified 20 human-yeast complementation pairs that are replaceable in 44 assays that test rescue of chemical sensitivity and/or CIN defects. We selected a human-yeast pair (hFEN1/yRAD27), which is frequently overexpressed in cancer and is an anticancer therapeutic target, to perform in vivo inhibitor assays using a humanized yeast cell-based platform. In agreement with published in vitro assays, we demonstrate that HU-based PTPD is a species-specific hFEN1 inhibitor. In contrast, another reported hFEN1 inhibitor, the arylstibonic acid derivative NSC-13755, was determined to have off-target effects resulting in a synthetic lethal phenotype with yRAD27-deficient strains. Our study expands the list of human-yeast complementation pairs to nonessential genes by defining novel cell-based assays that can be utilized as a broad resource to study human drug targets.

MRE11-RAD50-NBS1 promotes Fanconi Anemia R-loop suppression at transcription-replication conflicts.

Ectopic R-loop accumulation causes DNA replication stress and genome instability. To avoid these outcomes, cells possess a range of anti-R-loop mechanisms, including RNaseH that degrades the RNA moiety in R-loops. To comprehensively identify anti-R-loop mechanisms, we performed a genome-wide trigenic interaction screen in yeast lacking RNH1 and RNH201. We identified >100 genes critical for fitness in the absence of RNaseH, which were enriched for DNA replication fork maintenance factors including the MRE11-RAD50-NBS1 (MRN) complex. While MRN has been shown to promote R-loops at DNA double-strand breaks, we show that it suppresses R-loops and associated DNA damage at transcription-replication conflicts. This occurs through a non-nucleolytic function of MRE11 that is important for R-loop suppression by the Fanconi Anemia pathway. This work establishes a novel role for MRE11-RAD50-NBS1 in directing tolerance mechanisms at transcription-replication conflicts.

MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome.

Advances in long-read single molecule sequencing have opened new possibilities for 'benchtop' whole-genome sequencing. The Oxford Nanopore Technologies MinION is a portable device that uses nanopore technology that can directly sequence DNA molecules. MinION single molecule long sequence reads are well suited for de novo assembly of complex genomes as they facilitate the construction of highly contiguous physical genome maps obviating the need for labor-intensive physical genome mapping. Long sequence reads can also be used to delineate complex chromosomal rearrangements, such as those that occur in tumor cells, that can confound analysis using short reads. Here, we assessed MinION long-read-derived sequences for feasibility concerning: (1) the de novo assembly of a large complex genome, and (2) the elucidation of complex rearrangements. The genomes of two Caenorhabditis elegans strains, a wild-type strain and a strain containing two complex rearrangements, were sequenced with MinION. Up to 42-fold coverage was obtained from a single flow cell, and the best pooled data assembly produced a highly contiguous wild-type C. elegans genome containing 48 contigs (N50 contig length = 3.99 Mb) covering >99% of the 100,286,401-base reference genome. Further, the MinION-derived genome assembly expanded the C. elegans reference genome by >2 Mb due to a more accurate determination of repetitive sequence elements and assembled the complete genomes of two co-extracted bacteria. MinION long-read sequence data also facilitated the elucidation of complex rearrangements in a mutagenized strain. The sequence accuracy of the MinION long-read contigs (∼98%) was improved using Illumina-derived sequence data to polish the final genome assembly to 99.8% nucleotide accuracy when compared to the reference assembly.

The Chromosome Transmission Fidelity Assay for Measuring Chromosome Loss in Yeast.

The budding yeast Saccharomyces cerevisiae has served as an excellent model system for studying highly conserved biological pathways including pathways involved in genome transmission and maintenance. The Chromosome Transmission Fidelity (CTF) colony color assay was developed to assess chromosome instability (CIN) in yeast, by monitoring the loss or gain during cell division of an artificial chromosome fragment carrying a visual marker. The CTF assay monitors changes in chromosome number, allowing the detection of mutants that exhibit increased rates of chromosome nondisjunction or chromosome loss. In this article, we describe the SUP11-marker-based CTF assay system, and the methodologies for both qualitative analysis of mutants affecting chromosome transmission, and quantitative analysis for determining the types and rates of errors in chromosome transmission using half-sector analysis.

Model Organisms Facilitate Rare Disease Diagnosis and Therapeutic Research.

Efforts to identify the genetic underpinnings of rare undiagnosed diseases increasingly involve the use of next-generation sequencing and comparative genomic hybridization methods. These efforts are limited by a lack of knowledge regarding gene function, and an inability to predict the impact of genetic variation on the encoded protein function. Diagnostic challenges posed by undiagnosed diseases have solutions in model organism research, which provides a wealth of detailed biological information. Model organism geneticists are by necessity experts in particular genes, gene families, specific organs, and biological functions. Here, we review the current state of research into undiagnosed diseases, highlighting large efforts in North America and internationally, including the Undiagnosed Diseases Network (UDN) (Supplemental Material, File S1) and UDN International (UDNI), the Centers for Mendelian Genomics (CMG), and the Canadian Rare Diseases Models and Mechanisms Network (RDMM). We discuss how merging human genetics with model organism research guides experimental studies to solve these medical mysteries, gain new insights into disease pathogenesis, and uncover new therapeutic strategies.

Synthetic lethality and cancer.

A synthetic lethal interaction occurs between two genes when the perturbation of either gene alone is viable but the perturbation of both genes simultaneously results in the loss of viability. Key to exploiting synthetic lethality in cancer treatment are the identification and the mechanistic characterization of robust synthetic lethal genetic interactions. Advances in next-generation sequencing technologies are enabling the identification of hundreds of tumour-specific mutations and alterations in gene expression that could be targeted by a synthetic lethality approach. The translation of synthetic lethality to therapy will be assisted by the synthesis of genetic interaction data from model organisms, tumour genomes and human cell lines.

International Cooperation to Enable the Diagnosis of All Rare Genetic Diseases.

Provision of a molecularly confirmed diagnosis in a timely manner for children and adults with rare genetic diseases shortens their "diagnostic odyssey," improves disease management, and fosters genetic counseling with respect to recurrence risks while assuring reproductive choices. In a general clinical genetics setting, the current diagnostic rate is approximately 50%, but for those who do not receive a molecular diagnosis after the initial genetics evaluation, that rate is much lower. Diagnostic success for these more challenging affected individuals depends to a large extent on progress in the discovery of genes associated with, and mechanisms underlying, rare diseases. Thus, continued research is required for moving toward a more complete catalog of disease-related genes and variants. The International Rare Diseases Research Consortium (IRDiRC) was established in 2011 to bring together researchers and organizations invested in rare disease research to develop a means of achieving molecular diagnosis for all rare diseases. Here, we review the current and future bottlenecks to gene discovery and suggest strategies for enabling progress in this regard. Each successful discovery will define potential diagnostic, preventive, and therapeutic opportunities for the corresponding rare disease, enabling precision medicine for this patient population.

Canonical DNA Repair Pathways Influence R-Loop-Driven Genome Instability.

DNA repair defects create cancer predisposition in humans by fostering a higher rate of mutations. While DNA repair is quite well characterized, recent studies have identified previously unrecognized relationships between DNA repair and R-loop-mediated genome instability. R-loops are three-stranded nucleic acid structures in which RNA binds to genomic DNA to displace a loop of single-stranded DNA. Mutations in homologous recombination, nucleotide excision repair, crosslink repair, and DNA damage checkpoints have all now been linked to formation and function of transcription-coupled R-loops. This perspective will summarize recent literature linking DNA repair to R-loop-mediated genomic instability and discuss how R-loops may contribute to mutagenesis in DNA-repair-deficient cancers.

Dosage Mutator Genes in Saccharomyces cerevisiae: A Novel Mutator Mode-of-Action of the Mph1 DNA Helicase.

Mutations that cause genome instability are considered important predisposing events that contribute to initiation and progression of cancer. Genome instability arises either due to defects in genes that cause an increased mutation rate (mutator phenotype), or defects in genes that cause chromosome instability (CIN). To extend the catalog of genome instability genes, we systematically explored the effects of gene overexpression on mutation rate, using a forward-mutation screen in budding yeast. We screened ∼5100 plasmids, each overexpressing a unique single gene, and characterized the five strongest mutators, MPH1 (mutator phenotype 1), RRM3, UBP12, PIF1, and DNA2 We show that, for MPH1, the yeast homolog of Fanconi Anemia complementation group M (FANCM), the overexpression mutator phenotype is distinct from that of mph1Δ. Moreover, while four of our top hits encode DNA helicases, the overexpression of 48 other DNA helicases did not cause a mutator phenotype, suggesting this is not a general property of helicases. For Mph1 overexpression, helicase activity was not required for the mutator phenotype; in contrast Mph1 DEAH-box function was required for hypermutation. Mutagenesis by MPH1 overexpression was independent of translesion synthesis (TLS), but was suppressed by overexpression of RAD27, a conserved flap endonuclease. We propose that binding of DNA flap structures by excess Mph1 may block Rad27 action, creating a mutator phenotype that phenocopies rad27Δ. We believe this represents a novel mutator mode-of-action and opens up new prospects to understand how upregulation of DNA repair proteins may contribute to mutagenesis.

Overexpression screens identify conserved dosage chromosome instability genes in yeast and human cancer.

Somatic copy number amplification and gene overexpression are common features of many cancers. To determine the role of gene overexpression on chromosome instability (CIN), we performed genome-wide screens in the budding yeast for yeast genes that cause CIN when overexpressed, a phenotype we refer to as dosage CIN (dCIN), and identified 245 dCIN genes. This catalog of genes reveals human orthologs known to be recurrently overexpressed and/or amplified in tumors. We show that two genes, TDP1, a tyrosyl-DNA-phosphdiesterase, and TAF12, an RNA polymerase II TATA-box binding factor, cause CIN when overexpressed in human cells. Rhabdomyosarcoma lines with elevated human Tdp1 levels also exhibit CIN that can be partially rescued by siRNA-mediated knockdown of TDP1 Overexpression of dCIN genes represents a genetic vulnerability that could be leveraged for selective killing of cancer cells through targeting of an unlinked synthetic dosage lethal (SDL) partner. Using SDL screens in yeast, we identified a set of genes that when deleted specifically kill cells with high levels of Tdp1. One gene was the histone deacetylase RPD3, for which there are known inhibitors. Both HT1080 cells overexpressing hTDP1 and rhabdomyosarcoma cells with elevated levels of hTdp1 were more sensitive to histone deacetylase inhibitors valproic acid (VPA) and trichostatin A (TSA), recapitulating the SDL interaction in human cells and suggesting VPA and TSA as potential therapeutic agents for tumors with elevated levels of hTdp1. The catalog of dCIN genes presented here provides a candidate list to identify genes that cause CIN when overexpressed in cancer, which can then be leveraged through SDL to selectively target tumors.