Biology subjects

Kozik, Z.

Publications and source records attributed to Kozik, Z..

9 recordsLinked to original sources

bioRxiv2026

Mechanistic basis for the spatiotemporal assembly of the Bloom Syndrome complex on ultrafine DNA bridges

The BTRR (BLM/TOP3A/RMI1/RMI2) complex resolves DNA replication and recombination intermediates, as well as mitotic DNA intertwinements, yet how its diverse activities are regulated remains unclear. Here, we identify key determinants of stable assembly of the BTRR complex on ultrafine DNA bridges (UFBs). We reveal that the RMI1-RMI2 subcomplex acts as a molecular reader of hydrophobic-rich short linear motifs (SLiMs) embedded within PICH and FANCM, thereby providing a conserved mechanism for engaging distinct Superfamily2 DNA translocases during chromosome segregation and DNA repair. During UFB resolution, SLiM binding stabilises the PICH-BTRR complex assembly and supports essential interactions among BLM, PICH, and DNA. Persistent PICH-BTRR complex association post-mitosis would compromise interphase DNA damage responses and is prevented by active PICH nuclear exclusion. Our findings define the molecular basis of UFB-binding complex assembly and highlight its spatiotemporal control required to specialise BTRR complex functions.

cell biology↗

bioRxiv2026

BCOR mutations establish a persistent culture-adaptive state in human induced pluripotent stem cells

Human induced pluripotent stem cells (hiPSCs) are widely used for disease modelling and regenerative medicine, yet their utility depends on maintaining molecular integrity during long-term culture. BCOR (BCL6 Co-Repressor) mutations are among the most recurrent culture-acquired alterations in hiPSCs, but their functional consequences remain poorly understood. Here, we show that hiPSC BCOR mutations are predominantly truncating indels enriched in exon 7, defining a mutational landscape distinct from that observed in cancer. Multi-omics profiling reveals that BCOR loss drives widespread chromatin, transcriptomic and proteomic remodelling, with coordinated activation of developmental, pluripotency-associated and mitochondrial metabolism programmes. To facilitate routine surveillance, we develop a cost-effective TaqMan qPCR assay that accurately identifies BCOR-mutant hiPSCs across independent cell lines. Finally, we demonstrate that correction of BCOR mutation by CRISPR-Cas9 only partially restores the wildtype molecular state, highlighting the importance of early detection and monitoring of adaptive mutations in hiPSC cultures.

cell biology↗

bioRxiv2026

Citrobacter rodentium infection reveals preserved intestinal resilience in aged intestine

Ageing is generally associated with increased susceptibility to enteric infection and is widely considered to reflect progressive deterioration of intestinal function. However, whether increased susceptibility represents complete loss of intestinal resilience or a distinct host response to infection remains unclear. Using Citrobacter rodentium (CR) infection as a model of infectious colitis, we investigated how ageing shapes epithelial responses to enteric bacterial infection. Infection of aged mice with wild type CR (CRWT) resulted greater bacterial colonisation, deeper crypt localisation, impaired fluid-ion homeostasis and more severe colitis than young mice. Conversely, infection of aged mice with CRM12, a CR strain lacking 12 type III secretion system effectors, resulted in reduced epithelial colonisation, preservation of epithelial barrier function and fluid-ion homeostasis, restrained inflammatory responses, and markedly lower disease severity than CRWT infection. Deep quantitative proteomic analysis demonstrated that these divergent outcomes were accompanied by distinct adaptive molecular programmes within the aged intestinal epithelium. Together, our findings demonstrate that although aged mice exhibit increased susceptibility to enteric infection, the aged intestine retains the ability to mount distinct epithelial, inflammatory and molecular responses to pathogens with different virulence repertoires. Thus, increased susceptibility in aged mice should not be interpreted as complete loss of intestinal resilience.

microbiology↗

bioRxiv2026

High resolution interaction surface mapping by PRISMA reveals novel ARID1A interactions

The SWI/SNF chromatin remodelling complex controls proliferation and cell fate determination by regulating chromatin accessibility at promoters and enhancers, thereby modulating programs of gene expression, and has roles in DNA damage response, replication, splicing, and translation, and cell plasticity. The cBAF-exclusive subunit ARID1A acts as scaffold for the assembly of cBAF SWI/SNF complexes through its C-terminal globular domain and is the most frequently mutated SWI/SNF subunit in cancer. More than half of the ARID1A protein sequence contains regions of intrinsic disorder which are important for protein interactions, often mediated by short linear motifs. However, these interactions are notoriously difficult to study. Whilst hundreds of ARID1A interactions have been reported in the literature, their molecular basis remains obscure, and only a few have been explored functionally or mapped at an interface level. Here, we use a PRotein Interaction Screen on a peptide MAtrix (PRISMA) combined with quantitative mass spectrometry to identify novel ARID1A interactions and map amino acid residues and motifs that mediate interactions at high sequence resolution. The ARID1A PRISMA assay recapitulates binding of BAF subunits to ARID1A and detects the previously described binding of YAP1 transcriptional coactivator to a PPXY motif. Our PRISMA data reveals binding sites for transcriptional repressor SIN3A and identifies TOX4, CDK2 and CCNA2 as novel interactors. Mutation of a cell cycle-dependent CDK2 phosphorylation site in ARID1A leads to altered gene expression of microtubule factors and defects in cell proliferation. Our work underscores the utility of PRISMA to uncover weak or low abundance interactions that are not detectable by traditional affinity purification strategies. Together, our results characterise novel interactors and a new mode of regulation of ARID1A, and provide a useful resource to further explore mechanistic aspects of ARID1A function.

biochemistry↗

bioRxiv2025

Chromatin signalling pathways and FANCE amplification affect ATR inhibitor sensitivity in metastatic breast cancer

Metastatic breast cancer remains incurable, as many patients develop therapy resistance. Loss of ATM/p53 increases reliance on the ATR pathway, positioning ATR inhibitors (ATRi) as promising therapeutics. However, targeting a single pathway often leads to resistance due to tumour heterogeneity or alternative signalling mechanisms. Here, we combine chromatin-enrichment proteomics and phospho-proteomics with genome sequencing data in the ATRi-sensitive triple negative breast cancer (TNBC) cell line MDA-MB-453 to map adaptive responses to the ATR inhibitor AZD6738. We identify chromatin-associated activation of survival pathways, including AKT1, RPTOR (mTORC1), CDK4/5, and OGFR, alongside hyperphosphorylation of MKI67, SAFB2, and CHD4, indicating ATRi sensitivity. Complementary siRNA screening of DNA damage response (DDR) genes reveals that amplification of Fanconi anaemia (FA) pathway gene FANCE increases sensitivity to AZD6738. Analysis of breast cancer datasets highlights frequent FANCE amplification in metastatic patients, particularly in circulating tumour cells. Strikingly, pharmacological inhibition of the FA pathway (UBE2T/FANCL-IN-1) synergises with AZD6738. Together, our findings define adaptive resistance mechanisms to ATR inhibition and nominate FA pathway blockade as a rational combination strategy. Overall, our work provides fundamental insight into the complexity of DDR in metastatic breast cancer and offers a platform for mechanistic investigation, which can be exploited in cancer therapy.

cancer biology↗

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

The accessory type III secretion system effectors shape intestinal inflammatory infection outcomes

Injection of effectors via a type III secretion system (T3SS) is an infection strategy shared by various Gram-negative bacterial pathogens, many infecting mucosal surfaces. While individual T3SS effectors are well characterized, their network-level organization and the distinction between core and accessory effectors remain incompletely understood. Here, by systematically dissecting the T3SS effector network of the enteric mouse pathogen Citrobacter rodentium (CR) we identified a subset of 12 accessory effectors that, while dispensable for colonization, significantly alter infection outcomes. A strain lacking the accessory effectors (CRM12) remained virulent in susceptible mouse hosts yet resulted in reduced epithelial barrier damage, inflammation, and immune cell infiltration in resistant mice. Deep proteomic analysis specifically targeting CR-attached colonic epithelial cells revealed that, despite lacking 39% of its effector repertoire, infection with CRM12 results in similar changes to global protein expression as seen in mice infected with the wild-type strain, though key regulators of barrier integrity were differentially expressed. Using a host model with impaired barrier repair, we confirmed that accessory effectors shape infection outcomes without significantly impacting virulence. This study refines the concept of core and accessory effectors, providing a basis for further studies into effector-driven host adaptation.

microbiology↗

bioRxiv2025

IL-18 activates mucosal group 2 innate lymphoid cells following enteric bacterial infection

Group 3 innate lymphoid cells (ILC3s) play a major role in protecting against infection with the enteric mouse pathogen Citrobacter rodentium, used to model infections with enteropathogenic and enterohaemorrhagic Escherichia coli. ILC3s-secreted IL-22, shown to be indispensable for protection against C. rodentium infection, induces secretion of IL-18, antimicrobial peptides and nutritional immunity proteins as well as activation of tissue regeneration processes. While ILC2s have traditionally been associated with immune responses to helminth infection and allergic inflammation via the production of type 2 cytokines (e.g. IL-4, IL-5, IL-9 and IL-13), more recently they have been implicated in protection against Clostridium difficile and Helicobacter pylori infections. Here we show that colonic lamina propria ILC2s proliferate in response to C. rodentium infection and secrete IL-4, IL-5 and IL-13, which are involved in maintenance of the intestinal barrier function, tissue repair and mucus secretion. When stimulated with IL-18, colonic ILC2s from uninfected naive mice secreted type 2 cytokines. Injection of IL-18 binding protein (IL18BP), at 2- and 3-days post C. rodentium infection, blocked activation of ILC2s. These data show that ILC2s are activated in response to infection with an enteric Gram-negative pathogen, where stimulation with IL-18 plays a role in inducing proliferation and secretion of type 2 cytokines. Author SummaryWhile group 3 innate lymphoid cells (ILC3s) play a key role in protecting from bacterial infections, ILC2s are mainly associated with immune responses to helminth infection. Here we investigated if ILC2s are activated in responses to infection with the enteric mouse pathogen Citrobacter rodentium. We show that in infected mice, gut ILC2s expand and secreted type 2 cytokines. ILC2 isolated from uninfected mice were activated by IL-18. Consistently, administration of IL-18 binding protein into C. rodentium-infected mice inhibited ILC2 activation. These findings suggest that gut ILC2s are activated by Gram negative enteric pathogens, which is mediated in part by IL-18.

immunology↗

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↗