Biology subjects

Maman, Y.

Publications and source records attributed to Maman, Y..

4 recordsLinked to original sources

bioRxiv2026

Systematic Generation of Mutations at Topoisomerase II Cleavage Sites Enables Cancer Adaptation to Doxorubicin Therapy

Acquired resistance to chemotherapy remains a major cause of treatment failure. Here, we investigate the early events underlying the development of resistance to doxorubicin (Dox), one of the most widely used anticancer agents, during repeated drug exposure. Dox intercalates into DNA at sites influenced by chromatin structure and inhibits the DNA religation step of topoisomerase II (Top2), thereby inducing double-strand DNA breaks (DSBs). Here, we show that, in the subpopulation of cancer cells that survive initial Dox treatment, repair of Dox-induced DSBs leads to the accumulation of recurrent mutations at Top2 cleavage sites. These mutations prevent the formation of Dox-induced DSBs at the same loci upon subsequent exposures, effectively protecting the genome from Top2-mediated DNA damage and promoting the development of drug resistance. Importantly, the loss of Top2-dependent DNA cleavage and relaxation caused by these mutations increases the reliance of adapted cells on topoisomerase I (Top1), thereby sensitizing them to Top1 inhibitors. Together, our findings reveal a previously unrecognized mechanism of resistance based on the active generation of highly adaptive mutations, providing new insight into cancer evolution under therapeutic pressure.

cancer biology↗

bioRxiv2021

Immunoglobulin enhancers increase RNA polymerase stalling at somatic hypermutation target sequences

Somatic hypermutation (SHM) drives the genetic diversity of immunoglobulin (Ig) genes in activated B cells and supports the generation of antibodies with increased affinity for antigen. SHM is targeted to Ig genes by their enhancers (DIVACs; diversification activators), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase 2 (Pol2) and Pol2 occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol2 or production of full-length transcripts, indicating accumulation of stalled Pol2. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of single-stranded DNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol2 stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.

immunology↗

bioRxiv2020

Image-based pooled whole genome CRISPR screening for Parkin and TFEB subcellular localization

Genome-wide CRISPR screens have transformed our ability to systematically interrogate human gene function, but are currently limited to a subset of cellular phenotypes. We report a novel pooled screening approach for a wider range of cellular and subtle subcellular phenotypes. Machine learning and convolutional neural network models are trained on the subcellular phenotype to be queried. Genome-wide screening then utilizes cells stably expressing dCas9 (CRISPRi), photoactivatable fluorescent protein (PA-mCherry), and a lentiviral guide RNA (gRNA) pool. Cells are screened by microscopy and classified by artificial intelligence (AI) algorithms, which precisely identify the genetically altered phenotype. Cells with the phenotype of interest are photoactivated, isolated via flow cytometry, and the gRNAs are identified by sequencing. A proof-of-concept screen accurately identified PINK1 as essential for Parkin recruitment to mitochondria. A genome-wide screen identified factors mediating TFEB relocation from the nucleus to the cytosol upon prolonged starvation. Twenty of the sixty-four hits called by the neural network model were independently validated, revealing new effectors of TFEB subcellular localization. This approach, AI-Photoswitchable Screening (AI-PS) offers a novel screening platform capable of classifying a broad range of mammalian subcellular morphologies, an approach largely unattainable with current methodologies at genome-wide scale.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2019

ATM and PRDM9 Regulate SPO11-bound Recombination Intermediates During Meiosis

Meiotic recombination is initiated by genome-wide SPO11-induced double-strand breaks (DSBs) that are processed by MRE11-mediated release of SPO11. The DSB is then resected and loaded with DMC1/RAD51 filaments that invade homologous chromosome templates. In most mammals, DSB locations ("hotspots") are determined by the DNA sequence specificity of PRDM9. Here, we demonstrate the first direct detection of meiotic DSBs and resection in vertebrates by performing END-seq on mouse spermatocytes using low sample input. We find that DMC1 limits both the minimum and maximum lengths of resected DNA, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles in meiotic resection. Through enzymatic modifications to the END-seq protocol that mimic the in vivo processing of SPO11, we identify a novel meiotic recombination intermediate ("SPO11-RI") present at all hotspots. The SPO11-bound intermediate is dependent on PRDM9 and caps the 3 resected end during engagement with the homologous template. We propose that SPO11-RI is generated because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm-/- spermatocytes, SPO11-RI is reduced while unresected DNA-bound SPO11 accumulate because of defective MRE11 initiation. Thus in addition to their global roles in governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing.

molecular biology↗