Biology subjects

van Eijk, P.

Publications and source records attributed to van Eijk, P..

4 recordsLinked to original sources

bioRxiv2025

Inducible estrogen receptor alpha in normal breast epithelial cells demonstrate estrogen receptor-dependent DNA damage

BackgroundSignaling by estrogen-receptor alpha (ER) plays a major role in breast cancer initiation Investigations of the mechanism of DNA damage mediated by ER signaling are carried out in breast cancer cell lines due to the lack of ER+ normal human breast epithelial cells lines (HBEC). Defining the mechanisms by which ER induces DNA damage and initiates tumorigenesis requires normal HBECs that express ER, demonstrate estrogenic responses, and are amenable to long term propagation in culture. MethodsWe utilized lentiviral expression of an inducible ER construct to generate four HBEC lines (HBEC-ESR1). We studied these cells for ER-dependent responses using a luciferase reporter, endogenous gene expression and proliferation assays. RNA-Seq was performed to characterize the ER-mediated transcriptomic patterns in the four HBEC lines. ER mediated DNA double strand breaks (DSBs) were analyzed using {gamma}H2AX immunofluorescence. ResultsExpression and functional activation of ER were observed in all HBEC-ESR1 lines, whereas proliferation in response to 17{beta}-estradiol (E2) was observed in 3 of the cell lines. Proliferative responses were due to intrinsic signaling within the HBECs as conditioned media from the cells failed to cause proliferation. A total of 682 genes were differentially expressed at 24h following treatment with 10nM E2 with 43% of these genes were also observed in ER+ breast cancer cell lines (MCF7 or T47D). Gene-set enrichment analysis identified differential expression of genes in ER signaling pathways and DNA repair pathways in E2-treated cells. E2-induced ER signaling also increased {gamma}H2AX foci in 3 of the 4 cell lines. Levels of DSBs were increased by inhibition of the non-homologous end-joining (NHEJ) and homologous recombination (HR) pathways. DSBs were also increased in MCF10A-ESR1 cells heterozygous for the BRCA1185delAG mutation causing a truncated protein. ConclusionsInducible expression of ER in immortalized HBECs recapitulate transcriptional, replicative and DNA damage responses. Increased DSBs in MCF10A-ESR1 cells with heterozygous mutation of BRCA1 indicate haploinsufficiency and the potential for increased genetic instability due to ER signaling.

cancer biology↗

bioRxiv2024

The Role of BAZ2-dependent Chromatin Remodeling in Suppressing G4 DNA Structures and Associated Genomic Instability

DNA G-quadruplexes (G4s) are secondary structures with significant roles in regulating genome function and stability. Dysregulation of the dynamic formation of G4s is linked to genomic instability and disease, but the underlying mechanisms are not fully understood. In this study, we conducted a screen of chromatin-modifying enzymes and identified nine potential inhibitors of G4 formation, including seven that were not previously characterized. Among these, we highlight the role of BAZ2 chromatin remodelers as key suppressors of G4 DNA and G4-related genome instability. Depletion of BAZ2 subunits led to increased G4 formation, especially at transcriptional regulatory elements. BAZ2B was found to associate with G4 loci, suggesting that it plays a direct role in suppressing G4s. While BAZ2-deficient cells exhibited modest genomic instability, treatment with the G4-stabilizing ligand BRACO19 exacerbated double-strand breaks (DSBs), highlighting its utility as a tool to study G4-dependent genome instability. DSB profiling using INDUCE-seq uncovered distinct breakage patterns around G4s, further underscoring the impact of G4s on genome integrity. Notably, we found that within G4s, G repeats were more susceptible to DSBs than loops. These results establish BAZ2 chromatin remodeling complexes as direct regulators of G4 dynamics and provide new insights into G4-dependent genome instability.

molecular biology↗

bioRxiv2020

Precision digital mapping of endogenous andinduced genomic DNA breaks by INDUCE-seq

Understanding how breaks form and are repaired in the genome depends on the accurate measurement of the frequency and position of DNA double strand breaks (DSBs). This is crucial for identification of a chemicals DNA damage potential and for safe development of therapies, including genome editing technologies. Current DSB sequencing methods suffer from high background levels, the inability to accurately measure low frequency endogenous breaks and high sequencing costs. Here we describe INDUCE-seq, which overcomes these problems, detecting simultaneously the presence of low-level endogenous DSBs caused by physiological processes, and higher-level recurrent breaks induced by restriction enzymes or CRISPR-Cas nucleases. INDUCE-seq exploits an innovative NGS flow cell enrichment method, permitting the digital detection of breaks. It can therefore be used to determine the mechanism of DSB repair and to facilitate safe development of therapeutic genome editing. We discuss how the method can be adapted to detect other genomic features.

genomics↗

bioRxiv2018

Nucleosome remodelling at origins of Global Genome-Nucleotide Excision Repair occurs at the boundaries of higher-order chromatin structure

Repair of UV-induced DNA damage requires chromatin remodeling. How repair is initiated in chromatin remains largely unknown. We recently demonstrated that Global Genome Nucleotide Excision Repair (GG-NER) in chromatin is organized into domains around open reading frames. Here, we identify these domains, and by examining DNA damage-induced changes in the linear structure of nucleosomes, we demonstrate how chromatin remodeling is initiated during repair. In undamaged cells, we show that the GG-NER complex occupies chromatin at nucleosome free regions of specific gene promoters. This establishes the nucleosome structure at these genomic locations, which we refer to as GG-NER complex binding sites (GCBSs). We demonstrate that these sites are frequently located at genomic boundaries that delineate chromasomally interacting domains (CIDs). These boundaries define domains of higher-order nucleosome-nucleosome interaction. We show that efficient repair of DNA damage in chromatin is initiated following disruption of H2A.Z-containing nucleosomes adjacent to GCBSs by the GG-NER complex.

genomics↗