Project description:Cancers harbouring loss-of-function (LOF) alterations in tumour suppressor genes lack targeted therapies, thus alternative means to characterise gene function and identify vulnerabilities in these cancer cells are required. Here, we map the in silico genetic networks of KMT2D, a frequently mutated tumour suppressor gene, to demonstrate its utility in uncovering novel functional associations and vulnerabilities in cancer cells with tumour suppressor gene LOF alterations. In silico KMT2D networks revealed associated with histone modification, DNA replication, metabolism, and immune response. We identified synthetic lethal (SL) candidates encoding exising therapeutic targets. Analysing patient data from The Cancer Genome Atlas (TCGA) and the Personalized OncoGenomics Project (NCT021556210), we showed dysregulated pathways associated with SL candidates and elevated immune checkpoint response markers in KMT2DLOF cases, bringing forth evidence supporting KMT2D as a biomarker for immune checkpoint inhibitors. Our study presents a framework for identifying targetable vulnerabilities in cancers with tumour suppressor gene alterations.
Project description:Phenotype-driven forward genetic experiments are among the most powerful approaches for linking biology and disease to genomic elements. Although widely used in a range of model organisms, positional cloning of causal variants is still a very laborious process. Here, we describe a novel universal approach, named fast forward genetics that combines traditional bulk segregant techniques with next-generation sequencing technology and targeted genomic enrichment, to dramatically improve the process of mapping and cloning multiple mutants in a single experiment. In a two-step procedure the mutation is first roughly mapped by ‘light’ sequencing of the bulk segregant pool, followed by genomic enrichment and deep-sequencing of the mutant pool for the linked genomic region. The latter step allows for simultaneous fine-mapping and mutation discovery. We successfully applied this approach to three Arabidopsis mutants, but the method can in principle be applied to any model organism of interest and is largely independent of the genome size. Moreover, we show that both steps can be performed in multiplex using barcoded samples, thereby increasing efficiency enormously. Inducible overexpression of the RETINOBLASTOMA-RELATED (RBR-OE) gene in Arabidopsis roots causes the complete differentiation of stem cells and premature differentiation of daughter cells, leading to a full exhaustion of the primary root meristem. In order to identify regulators of RBR function in cell differentiation, RBR-OE plants in the Columbia background (Col0) were treated with EMS mutagenesis and a set of genetic suppressors of RBR-OE, which restores root growth capacity, were isolated. In this study, we used one the identified suppressor lines, which segregated as a recessive mutation. Mapping populations were generated by outcrossing to Ler ecotype. Seedlings from the F2 population were grown for 15 days post germination (dpg). A pool of 60 seedlings each with a clear suppressor phenotype (homozygous for suppressor mutation) and of 60 seedlings showing RBOE phenotype (Heterozygous for the suppressor mutation) were prepared and genomic DNA was isolated with the RNeasy Plant Mini Kit from QIAGEN according to manufacturer's protocol. The other two, mutants 136 and 193 were obtained in fluorescence based mutant screen and a QCmarker based mutagenesis, respectively. Mutants were generated by chemical mutagenesis (EMS) in Colombia (Col) genetic background. Mutants were subsequently crossed to the Landsberg (Ler) ecotype to create the mapping populations. Bulk-segregant pools of about 200 mutant as well as wild-type plants were generated for every mutant line.
Project description:50 phenotypic plants were isolated from backcross F2 generation, and DNA was extracted and mixed for high-throughput sequencing analysis,Then SNP sites in the restorer mutants were compared with their parents
Project description:Post-transcriptional modifications are important for transfer RNAs (tRNAs) to be efficient and accurate in translation on the ribosome. The m1G37 modification on a subset of tRNAs in bacteria are generated by a conserved methyltransferase TrmD and is essential for bacterial growth. Previous studies showed that m1G37 has an important role in preventing translational frameshifting and also that this modification is coupled with aminoacylation of tRNAs for proline. Here we performed suppressor screening to isolate a mutant E. coli cell that lacks TrmD but is viable, and the whole-genome sequencing revealed several mutations on prolyl-tRNA synthetase (ProRS) gene conferring cell viability in the absence of TrmD. Biochemical assays confirmed uncoupling of m1G37 modification and aminoacylation, and cell-based assays uncovered the critical role of m1G37 in supporting Wobble decoding.
Project description:Nonstop extension or stop-loss mutations in the von Hippel Lindau (VHL) tumor suppressor gene are recurrently found in kidney cancer patients as observed from the NonStopDB database. We observed that presence of this nonstop extension mutation caused loss of the protein via the ubiquitin-proteasome pathway. Stabilization of the nonstop extended VHL with proteasome inhibitor Bortezomib revealed that the mutant protein was preferentially translated from an upstream alternative start site compared to the wild-type. The objective of this study was to identify proteins bound to the VHL mRNA that could be responsible for the differential start site selection. For this, we employed the in vivo RNA-affinity purification method to pull down wild-type and nonstop mutant VHL mRNAs along with their bound proteins after cross-linking using UV irradiation and pull-down with raPOOLs, which are a pool of biotinylated DNA oligonucleotides reverse complementary to the VHL mRNA. The RNA-protein complexes were then pulled down by streptavidin beads, the bound proteins were isolated and subjected to mass-spectrometry based identification.
Project description:PauA2 plays an essential role in spermine catabolism and that exogenous spermine exerts a bactericidal effect on the ΔpauA2 mutant of P. aeruginosa. Not only subjected to growth inhibition by spermine, the pauA2 mutant without a functional γ-glutamylpolyamine synthetase PauA2 became more sensitive to β-lactam antibiotics in human serum. To explore PauA2 as a potential target of drug development, suppressors of the pauA2 mutant were isolated from selection plates containing spermine. These suppressors share common changes in various phenotypes. Genome resequencing of a representative suppressor revealed a unique mutation at the phoU gene, and a constitutive expression of the Pho regulon as evidenced by measurements of transcriptome analysis.
Project description:In the yeast saccharoryces cerevisiae, RPA49 full-deletion mutants show a strong growth defect at 30°C and are unable to grow at 25°C. However, spontaneous suppressors that restore growth at 25°C are observed. We attend to identified new suppressor alleles after UV treatment. To mapped the genomic location of suppressor allele we have used a derivative of the so called \\"Genetic Interaction Mapping\\" (GIM) method (Decourty et al, 2008). Briefly, we have mated a yeast strain carrying the deletion of rpa49 and the a suppressor allele (query strain) with the pool of all haploid deletion mutants from the Euroscarf yeast gene deletion project. All deletions in strains from the Euroscarf collection are flanked by 2 unique 20 bases pair DNA tag (so-called Up-Tag and DN-tag) that allow detection on oligonucleotide micro-array. After sporulation in mass, spores carrying the rpa49 deletion, the suppressor allele and euroscarf deleted genes were selected and grow in a competition culture for 10 generations. As selected spores result from genetic re-association of allele from tested strains and euroscarf strain, all strains carrying a deleted genes from the euroscarf collection that is genetically link to the suppressor allele will be depleted in the culture. We next extract genomic DNA , amplified and labelled the tags. The relative quantity of each deletion tags in experiments with strain carrying suppressor alleles were estimated in a two color array experiment relative to the quantity of the tags in parallel experiments using two different wild-type strains.
Project description:It is a TMT10Plex proteomics dataset used for relative protein and phosphoprotein quantification in the manuscript entitled "Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function". It contains both non-phosphorylated (used for identification and quantification of total proteomes) and phospho enriched (used for identification and quantification of phosphoproteins) dataset.