Biology subjects

Downs, J. A.

Publications and source records attributed to Downs, J. A..

5 recordsLinked to original sources

bioRxiv2025

ARID1A stabilises non-homologous end joining factors at DNA breaks induced by the G4 ligand pyridostatin

ARID1A is a subunit of the BAF chromatin remodelling complex that is frequently mutated in cancer. It is challenging to predict how ARID1A loss impacts cancer therapy response because it participates in many different cellular pathways. G quadruplex (G4) binding ligands, such as pyridostatin, have shown anticancer effects, but the pathways and genetic determinants involved in the response to G4 ligands are still not fully understood. Here, we show that ARID1A deficient cells are selectively sensitive to pyridostatin when compared with isogenic controls. Sensitivity to pyridostatin was apparent in ovarian and colorectal cancer cell line models, and in vivo studies suggest that G4 ligands hold promise for treating ARID1A deficient cancers. While we find that ARID1A impacts on pyridostatin-induced transcriptional responses, we find that pyridostatin-mediated toxicity in ARID1A-deficient cells is driven by defective DNA repair of topoisomerase-induced breaks. We show that ARID1A-deficient cells are unable to efficiently accumulate non-homologous end joining proteins on chromatin following pyridostatin exposure. These data uncover a role for ARID1A in the cellular response to G4 ligands, and link remodelling to G4 ligand-induced transcriptional and DNA damage responses.

molecular biology↗

bioRxiv2025

Chromosome-Specific Aneuploidy Engineering via dCas9-Induced Centromeric Chromatin Relaxation

Aneuploidy, the gain or loss of chromosomes, is prevalent in both normal and disease conditions, however, experimental approaches to engineer and study aneuploidy remain limited, leaving its functional significance under-characterized. Here, we present CRISPR-Taiji (CRISPRt), an efficient method for inducing chromosome-specific mis-segregation and aneuploidy generation across all 24 human chromosomes via dead Cas9 (dCas9)-induced centromeric chromatin relaxation. Using CRISPRt with scRNA-seq, we generated the first comprehensive transcriptomic alteration landscape of nearly all autosomal aneuploidies at chromosome-arm resolution. This genotype-phenotype map provides causal evidence linking recurrent aneuploidies in clear cell renal cell carcinoma (ccRCC) to molecular and clinical phenotypes observed in patient tumors. Notably, chromosome 3(p) loss, the ccRCC initiating event, specifically drives strong interferon signaling activation, offering novel insights into ccRCC tumorigenesis and immune modulation. Overall, we establish CRISPRt as a simple, efficient and scalable approach for chromosome-specific aneuploidy engineering and characterization in preclinical models to advance aneuploidy research across diverse biological contexts.

genomics↗

bioRxiv2024

The mitotic CIP2A-TOPBP1 axis facilitates mitotic pathway choice between MiDAS and MMEJ

Mitotic DNA double-strand breaks (DSBs) accumulate in response to replication stress or BRCA1/2 deficiency posing a significant threat to genome stability as repair by non-homologous end-joining (NHEJ) and homologous recombination (HR) is inactivated in mitosis. Mitotic cells instead rely on the mechanisms of microhomology mediated end-joining (MMEJ) and mitotic DNA synthesis (MiDAS). However, how these pathways are regulated in mitosis remains unknown. Here we reveal the CIP2A-TOPBP1 complex facilitates recruitment of SMX complex components to mitotic chromatin marked by CIP2A, through a CDK1-dependent interaction between TOPBP1 BRCT 1/2 and SLX4 phospho-threonine1260, that drives MiDAS. Furthermore, CIP2A promotes the recruitment of Pol{theta} to facilitate mitotic MMEJ. This defines a mechanistic framework for mitotic DSB repair, where simultaneous disruption of MiDAS and MMEJ pathways underpins the synthetic lethality observed in BRCA1/2-deficient cells following CIP2A depletion. These findings provide critical insights into mitotic DNA repair and highlights therapeutic opportunities in HR deficient tumours.

molecular biology↗

bioRxiv2024

PBRM1 directs PBAF to pericentromeres and protects centromere integrity

The specialised structure of the centromere is critical for effective chromosome segregation, but its repetitive nature makes it vulnerable to rearrangements. Centromere fragility can drive tumorigenesis, but protective mechanisms preventing fragility are still not fully understood. The PBAF chromatin remodelling complex is frequently misregulated in cancer, but its role in cancer is still not fully characterized. Here, we identify PBAF as a protector of centromere and pericentromere structure with profound consequences for genome stability. A conserved feature of isogenic cell lines lacking PBRM1, a subunit of PBAF, is compromised centromere and pericentromere integrity. PBAF is present at these regions, and the binding pattern changes when PBRM1 is absent. PBRM1 loss creates a dependence on the spindle assembly checkpoint, which represents a therapeutic vulnerability. Importantly, we find that even in the absence of any perturbations, PBRM1 loss leads to centromere fragility, thus identifying a new player in centromere protection.

molecular biology↗

bioRxiv2023

A Recombinant Approach For Stapled Peptide Discovery YieldsInhibitors of the RAD51 Recombinase

Stapling is a macrocyclisation method that connects amino acid side chains of a peptide to improve its pharmacological properties. We describe an approach for stapled peptide preparation and biochemical evaluation that combines recombinant expression of fusion constructs of target peptides and cysteine-reactive divinyl-heteroaryl chemistry, as an alternative to solid-phase synthesis. We then employ this workflow to prepare and evaluate BRC-repeat-derived inhibitors of the RAD51 recombinase, showing that a diverse range of secondary structure elements in the BRC repeat can be stapled without compromising binding and function. Using X-ray crystallography, we elucidate the atomic-level features of the staple moieties. We then demonstrate that BRC-repeat-derived stapled peptides can disrupt RAD51 function in cells following ionising radiation treatment.

biochemistry↗