Biology subjects

Vergara, X.

Publications and source records attributed to Vergara, X..

5 recordsLinked to original sources

bioRxiv2025

Megabase-scale detachment of genome-nuclear lamina interactions is required for efficient repair of double stranded breaks

Lamina-associated domains (LADs) are large genomic regions that contact the nuclear lamina (NL). Double-strand breaks (DSBs) in LADs are known to be repaired more slowly and with different pathway preferences compared to other chromatin contexts. However, little is known about the chromatin changes at LADs that occur during DSB repair. Here, we report that a single DSB inside a LAD can cause detachment from the NL over several megabases. This profound spatial rearrangement is transient and reverts within 48 hours. Preventing this detachment slows down repair kinetics and renders repair incomplete, indicating that NL detachment is required for efficient repair of DSBs in LADs. NL detachment is dependent on {psi}H2AX and ATM, while it is antagonized by DNAPKcs activity. Remarkably, {psi}H2AX also antagonizes NL interactions at chromosome ends. Taken together, our data indicate that {psi}H2AX accumulation in LADs induces large scale rewiring of genome-NL interactions, allowing for efficient repair of DSBs.

cell biology↗

bioRxiv2023

Chromatin context-dependent effects of epigenetic drugs on CRISPR-Cas9 editing

The efficiency and outcome of CRISPR/Cas9 editing depends on the chromatin state at the cut site. It has been shown that changing the chromatin state can influence both the efficiency and repair outcome, and epigenetic drugs have been used to improve Cas9 editing. However, because the target proteins of these drugs are not homogeneously distributed across the genome, the efficacy of these drugs may be expected to vary from locus to locus. Here, we systematically analyzed this chromatin context-dependency for 160 epigenetic drugs. We used a human cell line with 19 stably integrated reporters to induce a double-stranded break (DSB) in different chromatin environments. We then measure Cas9 editing efficiency and repair pathway usage by sequencing the mutational signatures. We identified 67 drugs that modulate Cas9 editing efficiency and/or repair outcome dependent on the local chromatin environment. For example, we find a subset of histone deacetylase inhibitors that improve Cas9 editing efficiency throughout all types of heterochromatin (e.g., PCI-24781), while others were only effective in H3K27me3-marked regions (e.g., Vorinostat). In summary, this study reveals that most epigenetic drugs alter CRISPR editing in a chromatin-dependent manner, and provides a detailed guide to improve Cas9 editing more selectively at the desired location. HIGHLIGHTSO_LIA screen identifies dozens of drugs that alter Cas9 editing in a chromatin context-dependent manner C_LIO_LIMany HDAC inhibitors boost Cas9 editing efficiency throughout all types of heterochromatin C_LIO_LIThe DNMT inhibitor Decitabine completely blocks resection-dependent repair across the genome C_LI

genomics↗

bioRxiv2022

Widespread chromatin context-dependencies ofDNA double-strand break repair proteins

DNA double-strand breaks are repaired by multiple pathways, including non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ). The balance of these pathways is dependent on the local chromatin context, but the underlying mechanisms are poorly understood. By combining knockout screening with a dual MMEJ:NHEJ reporter inserted in 19 different chromatin environments, we identified dozens of DNA repair proteins that modulate pathway balance dependent on the local chromatin state. Proteins that favor NHEJ mostly synergize with euchromatin, while proteins that favor MMEJ generally synergize with distinct types of heterochromatin. BRCA2 is an example of the former, which is corroborated by chromatin-dependent shifts in mutation patterns of BRCA2-/- cancer genomes. These results uncover a complex network of proteins that regulate MMEJ:NHEJ balance in a chromatin context-dependent manner. ONE SENTENCE SUMMARYA multiplexed screen reveals how dozens of proteins sense the local chromatin context to tune the balance between two DNA repair pathways.

molecular biology↗

bioRxiv2020

Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance

DNA double-strand break (DSB) repair is mediated by multiple pathways, including classical non-homologous end-joining pathway (NHEJ) and several homology-driven repair pathways. This is particularly important for Cas9-mediated genome editing, where the outcome critically depends on the pathway that repairs the break. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a newly developed multiplexed reporter assay in combination with Cas9 cutting, we systematically measured the relative activities of three DSB repair pathways as function of chromatin context in >1,000 genomic locations. This revealed that NHEJ is broadly biased towards euchromatin, while microhomology-mediated end-joining (MMEJ) is more efficient in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 shifts the balance towards NHEJ. Single-strand templated repair (SSTR), often used for precise CRISPR editing, competes with MMEJ, and this competition is weakly associated with chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance, and guidance for the design of Cas9-mediated genome editing experiments.

molecular biology↗

bioRxiv2019

Transcription-induced formation of extrachromosomal DNA during yeast ageing

Extrachromosomal circular DNA (eccDNA) facilitates adaptive evolution by allowing rapid and extensive gene copy number variation, and is implicated in the pathology of cancer and ageing. Here, we demonstrate that yeast aged under environmental copper accumulate high levels of eccDNA containing the copper resistance gene CUP1. Transcription of CUP1 causes CUP1 eccDNA accumulation, which occurs in the absence of phenotypic selection. We have developed a sensitive and quantitative eccDNA sequencing pipeline that reveals CUP1 eccDNA accumulation on copper exposure to be exquisitely site specific, with no other detectable changes across the eccDNA complement. eccDNA forms de novo from the CUP1 locus through processing of DNA double-strand breaks (DSBs) by Sae2 / Mre11 and Mus81, and genome-wide analyses show that other protein coding eccDNA species in aged yeast share a similar biogenesis pathway. Although abundant we find that CUP1 eccDNA does not replicate efficiently, and high copy numbers in aged cells arise through frequent formation events combined with asymmetric DNA segregation. The transcriptional stimulation of CUP1 eccDNA formation shows that age-linked genetic change varies with transcription pattern, resulting in gene copy number profiles tailored by environment.

molecular biology↗