Enhancing quantitative imaging to study DNA damage response: A guide to automated liquid handling and imaging.

Laboratory automation and quantitative high-content imaging are pivotal in advancing diverse scientific fields. These innovative techniques alleviate the burden of manual labour, facilitating large-scale experiments characterized by exceptional reproducibility. Nonetheless, the seamless integration of such systems continues to pose a constant challenge in many laboratories.

Different SWI/SNF complexes coordinately promote R-loop- and RAD52-dependent transcription-coupled homologous recombination.

The SWI/SNF family of ATP-dependent chromatin remodeling complexes is implicated in multiple DNA damage response mechanisms and frequently mutated in cancer. The BAF, PBAF and ncBAF complexes are three major types of SWI/SNF complexes that are functionally distinguished by their exclusive subunits. Accumulating evidence suggests that double-strand breaks (DSBs) in transcriptionally active DNA are preferentially repaired by a dedicated homologous recombination pathway.

Dynamic de novo heterochromatin assembly and disassembly at replication forks ensures fork stability.

Chromatin is dynamically reorganized when DNA replication forks are challenged. However, the process of epigenetic reorganization and its implication for fork stability is poorly understood. Here we discover a checkpoint-regulated cascade of chromatin signalling that activates the histone methyltransferase EHMT2/G9a to catalyse heterochromatin assembly at stressed replication forks.

Heat-induced SIRT1-mediated H4K16ac deacetylation impairs resection and SMARCAD1 recruitment to double strand breaks.

Hyperthermia inhibits DNA double-strand break (DSB) repair that utilizes homologous recombination (HR) pathway by a poorly defined mechanism(s); however, the mechanisms for this inhibition remain unclear. Here we report that hyperthermia decreases H4K16 acetylation (H4K16ac), an epigenetic modification essential for genome stability and transcription. Heat-induced reduction in H4K16ac was detected in humans, , and yeast, indicating that this is a highly conserved response.

A Microfluidic Cancer-on-Chip Platform Predicts Drug Response Using Organotypic Tumor Slice Culture.

Optimal treatment of cancer requires diagnostic methods to facilitate therapy choice and prevent ineffective treatments. Direct assessment of therapy response in viable tumor specimens could fill this diagnostic gap. Therefore, we designed a microfluidic platform for assessment of patient treatment response using tumor tissue slices under precisely controlled growth conditions.

R-Loops and Its Chro-Mates: The Strange Case of Dr. Jekyll and Mr. Hyde.

Since their discovery, R-loops have been associated with both physiological and pathological functions that are conserved across species. R-loops are a source of replication stress and genome instability, as seen in neurodegenerative disorders and cancer. In response, cells have evolved pathways to prevent R-loop accumulation as well as to resolve them.

Spreading and epigenetic inheritance of heterochromatin require a critical density of histone H3 lysine 9 tri-methylation.

Heterochromatin assembly requires methylation of histone H3 lysine 9 (H3K9me) and serves as a paradigm for understanding the importance of histone modifications in epigenetic genome control. Heterochromatin is nucleated at specific genomic sites and spreads across extended chromosomal domains to promote gene silencing. Moreover, heterochromatic structures can be epigenetically inherited in a self-templating manner, which is critical for stable gene repression.

SMARCAD1-mediated active replication fork stability maintains genome integrity.

The stalled fork protection pathway mediated by breast cancer 1/2 (BRCA1/2) proteins is critical for replication fork stability. However, it is unclear whether additional mechanisms are required to maintain replication fork stability. We describe a hitherto unknown mechanism, by which the SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily-A containing DEAD/H box-1 (SMARCAD1) stabilizes active replication forks, that is essential to maintaining resistance towards replication poisons.

Shelterin components mediate genome reorganization in response to replication stress.

The dynamic nature of genome organization impacts critical nuclear functions including the regulation of gene expression, replication, and DNA damage repair. Despite significant progress, the mechanisms responsible for reorganization of the genome in response to cellular stress, such as aberrant DNA replication, are poorly understood. Here, we show that fission yeast cells carrying a mutation in the DNA-binding protein Sap1 show defects in DNA replication progression and genome stability and display extensive changes in genome organization.

SNF2 Family Protein Fft3 Suppresses Nucleosome Turnover to Promote Epigenetic Inheritance and Proper Replication.

Heterochromatin can be epigenetically inherited in cis, leading to stable gene silencing. However, the mechanisms underlying heterochromatin inheritance remain unclear. Here, we identify Fft3, a fission yeast homolog of the mammalian SMARCAD1 SNF2 chromatin remodeler, as a factor uniquely required for heterochromatin inheritance, rather than for de novo assembly.

Cohesin-dependent globules and heterochromatin shape 3D genome architecture in S. pombe.

Eukaryotic genomes are folded into three-dimensional structures, such as self-associating topological domains, the borders of which are enriched in cohesin and CCCTC-binding factor (CTCF) required for long-range interactions. How local chromatin interactions govern higher-order folding of chromatin fibres and the function of cohesin in this process remain poorly understood. Here we perform genome-wide chromatin conformation capture (Hi-C) analysis to explore the high-resolution organization of the Schizosaccharomyces pombe genome, which despite its small size exhibits fundamental features found in other eukaryotes.

Mtr4-like protein coordinates nuclear RNA processing for heterochromatin assembly and for telomere maintenance.

The regulation of protein-coding and noncoding RNAs is linked to nuclear processes, including chromatin modifications and gene silencing. However, the mechanisms that distinguish RNAs and mediate their functions are poorly understood. We describe a nuclear RNA-processing network in fission yeast with a core module comprising the Mtr4-like protein, Mtl1, and the zinc-finger protein, Red1.