Single-cell transcriptogenomics reveals transcriptional exclusion of ENU-mutated alleles.
ABSTRACT: Recently, great progress has been made in single cell genomics and transcriptomics. Here, we present an integrative method, termed single-cell transcriptogenomics (SCTG), in which whole exome sequencing and RNA-seq is performed concurrently on single cells. This methodology enables one to track germline and somatic variants directly from the genome to the transcriptome in individual cells. Mouse embryonic fibroblasts were treated with the powerful mutagen ethylnitrosourea (ENU) and subjected to SCTG. Interestingly, while germline variants were found to be transcribed in an allelically balanced fashion, a significantly different pattern of allelic exclusion was observed for ENU-mutant variants. These results suggest that the adverse effects of induced mutations, in contrast to germline variants, may be mitigated by allelically biased transcription. They also illustrate how SCTG can be instrumental in the direct assessment of phenotypic consequences of genomic variants.
Project description:Functional annotation of every gene in the mouse genome is a herculean task that requires a multifaceted approach. Many large-scale initiatives are contributing to this undertaking. The International Knockout Mouse Consortium (IKMC) plans to mutate every protein-coding gene, using a combination of gene trapping and gene targeting in embryonic stem cells. Many other groups are performing using the chemical mutagen ethylnitrosourea (ENU) or transpon-based systems to induce mutations, screening offspring for phenovariants and identifying the causative mutations. A recent paper in BMC Research Notes by Arnold et al. presents data from an ENU-based mutagenesis project that provides not only some of the first phenotype-genotype information for a large number of genes, but also a trove of information, all publicly available, that demonstrates the specificity and efficiency of ENU mutagenesis.
Project description:Hereditary forms of iron-deficiency anemia, including animal models, have taught us much about the normal physiologic control of iron metabolism. However, the discovery of new informative mutants is limited by the natural mutation frequency. To address this limitation, we have developed a screen for heritable abnormalities of red blood cell morphology in mice with single-nucleotide changes induced by the chemical mutagen ethylnitrosourea (ENU). We now describe the first strain, fragile-red, with hypochromic microcytic anemia resulting from a Y228H substitution in the ferrireductase Steap3 (Steap3(Y288H)). Analysis of the Steap3(Y288H) mutant identifies a conserved motif required for targeting Steap3 to internal compartments and highlights how phenotypic screens linked to mutagenesis can identify new functional variants in erythropoiesis and ascribe function to previously unidentified motifs.
Project description:Approximately 10 % of the population worldwide suffers from hearing loss (HL) and about 60 % of persons with early onset HL have hereditary hearing loss due to genetic mutations. Highly efficient mutagenesis in mice with the chemical mutagen, ethylnitrosourea (ENU), associated with relevant phenotypic tools represents a powerful approach in producing mouse models for hearing impairment. A benefit of this strategy is to generate alleles to form a series revealing the full spectrum of gene function in vivo. It can also mimic the range of human mutations and polymorphisms for HL. In the course of a genome ENU mutagenesis program, we selected a new mouse model for hearing defect based on a dysmorphological screen. We identified by gene mapping the mutation responsible for this phenotype and characterized it at the histological level of the inner ear and evaluated the vestibule by following the recommendations of the standard operating procedures, IMPReSS. We have identified and characterized a new recessive allele of the otogelin gene, Otog (vbd/vbd) , due to a homozygous one base pair substitution at the splice donor site of intron 29. This mutation leads to a frame-shift and a premature stop codon. We observed a decrease in the amount of sensory cells in the maculae of Otog (vbd/vbd) mice as well as an apparent drastically decreased density to almost absence of the otoconial membrane. Compared to Otog (tm1Prs) and twister, the two other existing otogelin alleles, the detailed analysis of Otog (vbd/vbd) revealed that these mice share some common behavioural characteristics either with Otog (tm1Prs) or twister whereas the fine vestibular phenotype and the hearing defect are different. Our results emphasize the importance of detecting and characterizing a new allele of a gene in order to get comprehensive information about the gene function.
Project description:The immune system exerts a critical function as it recognizes and eliminates transformed or neoplastic cells, a process also referred to as immunosurveillance. NK cells play a particularly important role in that they are able to recognize tumor cells via "missing-self"-i.e., the absence of major histocompatibility complex Class I on target cells. Moreover, recent studies suggest that NK cells also participate in the onset and regulation of adaptive immune responses. The exact molecular pathways by which this occurs, however, remain poorly understood. To obtain further insight into the genes that are required for self-induced immune responses via NK cell-mediated cell death, our laboratory initiated a forward genetic approach using N-ethyl-N-nitrosourea (ENU) as a mutagen. Specifically, we tested the ability of NK cells from G3 ENU germline mice to recognize missing-self target cells and induce CD8+ T-cell responses following immunization with irradiated tumor cells. Here we present two ENU germline mutants, designated Ace and Chip, that are defective in the recognition of ?-2 microglobulin-deficient target cells, yet exhibit improved clearance of B16 melanoma cells in vivo. Coarse mapping and whole genome sequencing of the Chip mutation revealed a missense mutation causing a T'A amino acid substitution in the highly conserved third immuno-receptor tyrosine-based switch motif of CD244 (2B4). The forward genetic approach described here promises to reveal important insight into critical genes that are required for host responses involved in anticancer immunity.
Project description:<h4>Background</h4>With the advent of sequence-based approaches in the mutagenesis studies, it is now possible to directly evaluate the genome-wide pattern of experimentally induced DNA sequence changes for a diverse array of organisms. To gain a more comprehensive understanding of the mutational bias inherent in mouse ENU mutagenesis, this study describes a detailed evaluation of the induced mutational pattern obtained from a sequence-based screen of ENU-mutagenized mice.<h4>Results</h4>Based on a large-scale screening data, we derive the sequence-based estimates of the nucleotide-specific pattern and frequency of ENU-induced base replacement mutation in the mouse germline, which are then combined with the pattern of codon usage in the mouse coding sequences to infer the spectrum of amino acid changes obtained by ENU mutagenesis. We detect a statistically significant difference between the mutational patterns in phenotype- versus sequence-based screens, which presumably reflects differential phenotypic effects caused by different amino acid replacements. We also demonstrate that the mutations exhibit strong strand asymmetry, and that this imbalance is generated by transcription, most likely as a by-product of transcription-coupled DNA repair in the germline.<h4>Conclusion</h4>The results clearly illustrate the biased nature of ENU-induced mutations. We expect that a precise understanding of the mutational pattern and frequency of induced nucleotide changes would be of practical importance when designing sequence-based screening strategies to generate mutant mouse strains harboring amino acid variants at specific loci. More generally, by enhancing the collection of experimentally induced mutations in unambiguously defined genomic regions, sequence-based mutagenesis studies will further illuminate the molecular basis of mutagenic and repair mechanisms that preferentially produce a certain class of mutational changes over others.
Project description:Trinucleotide mutational signatures extracted from cancer genomes provide clues useful in understanding the roles of mutagens and mutagenic mechanisms in cancer development. The lack of a simple method for genome-wide analysis of alterations induced by mutagens hampers the identification of trinucleotide signatures of mutagen exposure and evaluation of their relationships with human cancers. Here, we describe a novel approach to facilitate analysis of chemically induced mutations in bacterial cells by detection of increased frequencies of base substitutions after mutagen exposure, using paired-end overlapping next-generation sequencing. DNA samples from Salmonella typhimurium strain TA100, exposed to three alkylating agents, ethylnitrosourea (ENU), methylnitrosourea (MNU), and ethyl methansulphonate (EMS), were analysed. The G:C?>?A:T mutation frequency was increased in all samples, whereas A:T base pair substitution frequencies were increased specifically in samples exposed to ENU, consistent with previous reports. Mutation patterns in the context of 96 possible trinucleotide formats in these samples exhibited a sharp peak corresponding to an NpCpY consensus sequence, which is similar to the mutational signature of alkylating agents in human cancer. These results indicate that our approach can be useful in facilitating the understanding of mechanisms underlying chemical mutagenicity and for identification of unknown causal mutagens in human cancer.
Project description:ENU mutagenesis is a powerful method for generating novel lines of mice that are informative with respect to both fundamental biological processes and human disease. Rapid developments in genomic technology have made the task of identifying causal mutations by positional cloning remarkably efficient. One limitation of this approach remains the mutation frequency achievable using standard treatment protocols, which currently generate approximately 1-2 sequence changes per megabase when optimized. In this study we used two strategies to attempt to increase the number of mutations induced by ENU treatment. One approach employed mice carrying a mutation in the DNA repair enzyme Msh6. The second strategy involved injection of ENU to successive generations of mice. To evaluate the number of ENU-induced mutations, single mice or pooled samples were analyzed using whole exome sequencing. The results showed that there is considerable variability in the induced mutation frequency using these approaches, but an overall increase in ENU-induced variants from one generation to another was observed. The analysis of the mice deficient for Msh6 also showed an increase in the ENU-induced variants compared to the wild-type ENU-treated mice. However, in both cases the increase in ENU-induced mutation frequency was modest.
Project description:<h4>Background</h4>Mice harbouring gene mutations that cause phenotypic abnormalities during organogenesis are invaluable tools for linking gene function to normal development and human disorders. To generate mouse models harbouring novel alleles that are involved in organogenesis we conducted a phenotype-driven, genome-wide mutagenesis screen in mice using the mutagen N-ethyl-N-nitrosourea (ENU).<h4>Methodology/principal findings</h4>ENU was injected into male C57BL/6 mice and the mutations transmitted through the germ-line. ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads. From 52 pedigrees screened 15 were detected with anomalies in one or more of the structures/organs screened. Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1.<h4>Conclusions/significance</h4>In this study we have generated mouse models in which the anomalies closely mimic those seen in human disorders. The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.
Project description:Forward genetics in model organisms has boosted our knowledge of the genetic bases of development, aging, and human diseases. In this experimental pipeline, it is crucial to start by inducing a large number of random mutations in the genome of the model organism to search for phenotypes of interest. Many chemical mutagens are used to this end because most of them display particular reactivity properties and act differently over DNA. Here we report the use of N-ethyl-N-nitrosourea (ENU) as a mutagen in the fission yeast Schizosaccharomyces pombe As opposed to many other alkylating agents, ENU only induces an S N 1-type reaction with a low s constant (s = 0.26), attacking preferentially O2 and O4 in thymine and O6 deoxyguanosine, leading to base substitutions rather than indels, which are extremely rare in its resulting mutagenic repertoire. Using ENU, we gathered a collection of 13 temperature-sensitive mutants and 80 auxotrophic mutants including two deleterious alleles of the human ortholog ATIC. Defective alleles of this gene cause AICA-ribosiduria, a severe genetic disease. In this screen, we also identified 13 aminoglycoside-resistance inactivating mutations in APH genes. Mutations reported here may be of interest for metabolism related diseases and antibiotic resistance research fields.
Project description:Although the Factor V Leiden (FVL) gene variant is the most prevalent genetic risk factor for venous thrombosis, only 10% of FVL carriers will experience such an event in their lifetime. To identify potential FVL modifier genes contributing to this incomplete penetrance, we took advantage of a perinatal synthetic lethal thrombosis phenotype in mice homozygous for FVL (F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/-) to perform a sensitized dominant ENU mutagenesis screen. Linkage analysis conducted in the 3 largest pedigrees generated from the surviving F5L/L Tfpi+/- mice ('rescues') using ENU-induced coding variants as genetic markers was unsuccessful in identifying major suppressor loci. Whole exome sequencing was applied to DNA from 107 rescue mice to identify candidate genes enriched for ENU mutations. A total of 3,481 potentially deleterious candidate ENU variants were identified in 2,984 genes. After correcting for gene size and multiple testing, Arl6ip5 was identified as the most enriched gene, though not reaching genome-wide significance. Evaluation of CRISPR/Cas9 induced loss of function in the top 6 genes failed to demonstrate a clear rescue phenotype. However, a maternally inherited (not ENU-induced) de novo mutation (Plcb4R335Q) exhibited significant co-segregation with the rescue phenotype (p = 0.003) in the corresponding pedigree. Thrombosis suppression by heterozygous Plcb4 loss of function was confirmed through analysis of an independent, CRISPR/Cas9-induced Plcb4 mutation (p = 0.01).