Biology subjects

Crismani, W.

Publications and source records attributed to Crismani, W..

7 recordsLinked to original sources

bioRxiv2026

Fanconi Anaemia E3 Ligase complex activity is regulated by a druggable metabolite binding site in FANCX

The Fanconi anaemia (FA) DNA repair pathway is an emerging target for precision cancer therapy. Using a high-throughput FANCD2-monoubiquitination assay, we identified a class of small molecules, including MSG010, that inhibit the FA E3 ligase complex in vitro. Because these molecules, and the metabolite, palmitoyl-CoA, are known to engage allosteric drug and metabolite (ADaM) binding site in AMP-activated kinase (AMPK), we hypothesised that a similar pocket exists within the FA complex. Supporting this, long-chain, but not short-chain, fatty acyl-CoA molecules inhibited the FA E3 ligase complex activity, and sequence analysis revealed similarity between the AMPK ADaM site and a WD40 repeat in the FA subunit FANCX. Targeted mutagenesis of this FANCX region disrupted E3 ligase activity or abolished inhibition by MSG010, suggesting the presence of an ADaM-like site in FANCX. Moreover, MSG010 preferentially killed BRCA1-deficient cells in vitro. These findings identify a putative small-molecule binding site in the FA pathway that may be developed further to test for exploitation as anticancer therapeutics.

cancer biology↗

bioRxiv2025

Differential PARP inhibitor responses in BRCA1-deficient and resistant cells in competitive co-culture

Synthetic lethality describes a genetic relationship where the loss of two genes results in cell death, but the loss of one of those genes does not. Drugs used for precision oncology can exploit synthetic lethal relationships; the best described are PARP inhibitors which preferentially kill BRCA1-deficient tumours preferentially over BRCA1-proficient cells. New synthetic lethal targets are often discovered using genetic screens, such as CRISPR knockout screens. Here, we present a competitive co-culture assay that can be used to analyse drugs or gene knockouts with synthetic lethal effects. We generated new BRCA1 isogenic cell line pairs from both a triple-negative breast cancer cell line (SUM149) and adapted pre-existing non-cancerous BRCA1 isogenic pair (RPE). Each cell line of the isogenic pair was transformed with its own fluorescent reporter. The two-coloured cell lines of the isogenic pair were then grown together in the same vessel to create a more competitive environment compared to when grown separately. We used four PARP inhibitors to validate the ability to detect synthetic lethality in BRCA1-deficient cancer cells. The readout of the assay was performed by counting the fluorescently coloured cells after drug treatment using flow cytometry. We observed preferential targeting of BRCA1-deficient cells, by PARPi, at relative concentrations that broadly reflect clinical dosing. Further we reveal subtle differences between PARPi resistant lines compared to BRCA1-proficient cells. Here, we demonstrate the validation and potential use of the competitive assay, which could be extended to validating novel genetic relationships and adapted for live cell imaging.

genetics↗

bioRxiv2024

A high-resolution meiotic crossover map from single-nucleus ATAC-seq reveals insights into the recombination landscape in mammalian sperm

Meiotic crossovers promote correct chromosome segregation and the shuffling of genetic diversity. However, the measurement of crossovers remains challenging, impeding our ability to decipher the molecular mechanisms that are necessary for their formation and regulation. Here we demonstrate a novel repurposing of the single-nucleus Assay for Transposase Accessible Chromatin with sequencing (snATAC-seq) as a simple and high-throughput method to identify and characterise meiotic crossovers from sperm nuclei. We first validate the feasibility of obtaining genome-wide coverage from snATAC-seq by using ATAC-seq on bulk haploid mouse sperm, ensuring adequate variant detection for haplotyping. Subsequently, we adapt droplet-based snATAC-seq for crossover detection, revealing over 25,000 crossovers in F1 hybrid mice. Comparison between wildtype and a hyper-recombinogenic Fancm-deficient mutant mouse model confirmed an increase in crossover rates in this genotype, however a distribution which was unchanged. We also find that regions with the highest rate of crossover formation are enriched for DMC1 and PRDM9, with a subset that is further enriched for DMC1 in Fancm-deficient mice. Our findings demonstrate the utility of snATAC-seq as a robust and scalable tool for high-throughput crossover detection, offering insights into meiotic crossover dynamics and elucidating the underlying molecular mechanisms. It is possible that the research presented here with snATAC-seq of haploid sperm could be extended into fertility-related diagnostics.

genetics↗

bioRxiv2022

synapsis: A Bioconductor package to automate the analysis of meiotic double-strand break repair and crossover formation

SummaryImmunofluorescent staining is commonly used to generate images to characterise cytological phenotypes. The manual quantification of DNA double-strand breaks and their repair intermediates during meiosis using image data requires a series of subjective steps, from image selection to the counting of particular events per nucleus. Here we describe synapsis, a Bioconductor package, which includes a set of functions to automate the process of identifying meiotic nuclei and quantifying key double-strand break formation and repair events in a rapid, scalable and reproducible workflow, and compare it to manual user quantification. The software can be extended for other applications in meiosis research, such as incorporating machine learning approaches to categorise meiotic substages. Availability and implementationsynapsis can be freely downloaded and installed at: http://bioconductor.org/packages/release/bioc/html/synapsis.html R functions and further information can be found at https://gitlab.svi.edu.au/drr-public/synapsis Contactwcrismani@svi.edu.au

cell biology↗

bioRxiv2022

The FANCC-FANCE-FANCF complex is evolutionarily conserved and regulates meiotic recombination

At meiosis, programmed meiotic DNA double-strand breaks are repaired via homologous recombination, resulting in crossovers (COs). From a large excess of DNA double-strand breaks that are formed, only a small proportion gets converted into COs because of active mechanisms that restrict CO formation. The Fanconi anemia (FA) complex proteins AtFANCM, MHF1, and MHF2 were previously identified in a genetic screen as anti-CO factors that function during meiosis in Arabidopsis thaliana. Here, pursuing the same screen, we identify FANCC as a new anti-CO gene. FANCC was previously only identified in mammals because of low primary sequence conservation. We show that FANCC, and its physical interaction with FANCE-FANCF, is conserved from vertebrates to plants. Further, we show that FANCC, together with its subcomplex partners FANCE and FANCF, regulates meiotic recombination. Mutations of any of these three genes partially rescues CO-defective mutants, which is particularly marked in female meiosis. Functional loss of FANCC, FANCE, or FANCF results in synthetic meiotic catastrophe with the pro-CO factor MUS81. This work reveals that FANCC is conserved outside mammals and has an anti-CO role during meiosis together with FANCE and FANCF.

genetics↗

bioRxiv2022

Fancm regulates meiotic double-strand break repair pathway choice in mammals

Meiotic crossovers are required for accurate chromosome segregation and to produce new allelic combinations. Meiotic crossover numbers are tightly regulated within a narrow range, despite an excess of initiating DNA double-strand breaks. Here, we describe the tumour suppressor FANCM as a meiotic anti-crossover factor in mammals. Crossover analyses with single-gamete and pedigree datasets both reveal a genome-wide increase in crossover frequencies in Fancm-deficient mice. Gametogenesis is heavily perturbed in Fancm loss of function mice, which is consistent with the reproductive defects reported in humans with biallelic FANCM mutations. A portion of the gametogenesis defects can be attributed to the cGAS-STING pathway. Despite the gametogenesis phenotypes in Fancm mutants both sexes were capable of producing offspring. We propose that the anti-crossover function and role in gametogenesis of Fancm are separable and will inform diagnostic pathways for human genomic instability disorders.

genetics↗

bioRxiv2022

sgcocaller and comapr: personalised haplotype assembly and comparative crossover map analysis using single gametes

Profiling gametes of an individual enables the construction of personalised haplotypes and meiotic crossover landscapes, now achievable at larger scale than ever through the availability of high-throughput single-cell sequencing technologies. However, high-throughput single-gamete data commonly have low depth of coverage per gamete, which challenges existing gametebased haplotype phasing methods. In addition, haplotyping a large number of single gametes from high-throughput singlecell DNA sequencing data and constructing meiotic crossover profiles using existing methods requires intensive processing. Here, we introduce efficient software tools for the essential tasks of generating personalised haplotypes and calling crossovers in gametes from single-gamete DNA sequencing data (sgcocaller), and constructing, visualising, and comparing individualised crossover landscapes from single gametes (comapr). With additional data pre-possessing, the tools can also be applied to bulk-sequenced samples. We demonstrate that sgcocaller is able to generate impeccable phasing results for high-coverage datasets, on which it is more accurate and stable than existing methods, and also performs well on low-coverage single-gamete sequencing datasets for which current methods fail. Our tools achieve highly accurate results with user-friendly installation, comprehensive documentation, efficient computation times and minimal memory usage.

bioinformatics↗