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. We show that different BAF, PBAF and ncBAF subunits promote homologous recombination and are rapidly recruited to DSBs in a transcription-dependent manner. The PBAF and ncBAF complexes promote RNA polymerase II eviction near DNA damage to rapidly initiate transcriptional silencing, while the BAF complex helps to maintain this transcriptional silencing. Furthermore, ARID1A-containing BAF complexes promote RNaseH1 and RAD52 recruitment to facilitate R-loop resolution and DNA repair. Our results highlight how multiple SWI/SNF complexes perform different functions to enable DNA repair in the context of actively transcribed genes.

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. Using biochemical and single molecule chromatin fibre approaches, we show that G9a together with SUV39h1 induces chromatin compaction by accumulating the repressive modifications, H3K9me1/me2/me3, in the vicinity of stressed replication forks. This closed conformation is also favoured by the G9a-dependent exclusion of the H3K9-demethylase JMJD1A/KDM3A, which facilitates heterochromatin disassembly upon fork restart. Untimely heterochromatin disassembly from stressed forks by KDM3A enables PRIMPOL access, triggering single-stranded DNA gap formation and sensitizing cells towards chemotherapeutic drugs. These findings may help in explaining chemotherapy resistance and poor prognosis observed in patients with cancer displaying elevated levels of G9a/H3K9me3.

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, Drosophila, and yeast, indicating that this is a highly conserved response. The examination of histone deacetylase recruitment to chromatin after heat-shock identified SIRT1 as the major deacetylase subsequently enriched at gene-rich regions. Heat-induced SIRT1 recruitment was antagonized by chromatin remodeler SMARCAD1 depletion and, like hyperthermia, the depletion of the SMARCAD1 or combination of the two impaired DNA end resection and increased replication stress. Altered repair protein recruitment was associated with heat-shock-induced γ-H2AX chromatin changes and DSB repair processing. These results support a novel mechanism whereby hyperthermia impacts chromatin organization owing to H4K16ac deacetylation, negatively affecting the HR-dependent DSB repair.

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. The optimized Cancer-on-Chip (CoC) platform maintained viability and sustained proliferation of breast and prostate tumor slices for 7 days. No major changes in tissue morphology or gene expression patterns were observed within this time frame, suggesting that the CoC system provides a reliable and effective way to probe intrinsic chemotherapeutic sensitivity of tumors. The customized CoC platform accurately predicted cisplatin and apalutamide treatment response in breast and prostate tumor xenograft models, respectively. The culture period for breast cancer could be extended up to 14 days without major changes in tissue morphology and viability. These culture characteristics enable assessment of treatment outcomes and open possibilities for detailed mechanistic studies. SIGNIFICANCE: The Cancer-on-Chip platform with a 6-well plate design incorporating silicon-based microfluidics can enable optimal patient-specific treatment strategies through parallel culture of multiple tumor slices and diagnostic assays using primary tumor material.

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. A growing body of evidence correlates R-loop accumulation with changes in the epigenetic landscape. However, the role of chromatin modification and remodeling in R-loops homeostasis remains unclear. This review covers various mechanisms precluding R-loop accumulation and highlights the role of chromatin modifiers and remodelers in facilitating timely R-loop resolution. We also discuss the enigmatic role of RNA:DNA hybrids in facilitating DNA repair, epigenetic landscape and the potential role of replication fork preservation pathways, active fork stability and stalled fork protection pathways, in avoiding replication-transcription conflicts. Finally, we discuss the potential role of several Chro-Mates (chromatin modifiers and remodelers) in the likely differentiation between persistent/detrimental R-loops and transient/benign R-loops that assist in various physiological processes relevant for therapeutic interventions.

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. The spreading and inheritance of heterochromatin are believed to be dependent on preexisting H3K9 tri-methylation (H3K9me3), which is recognized by the histone methyltransferase Clr4/Suv39h via its chromodomain, to promote further deposition of H3K9me. However, the process involving the coupling of the "read" and "write" capabilities of histone methyltransferases is poorly understood. From an unbiased genetic screen, we characterize a dominant-negative mutation in histone H3 (H3G13D) that impairs the propagation of endogenous and ectopic heterochromatin domains in the fission yeast genome. H3G13D blocks methylation of H3K9 by the Clr4/Suv39h methyltransferase and acts in a dosage-dependent manner to interfere with the spreading and maintenance of heterochromatin. Our analyses show that the incorporation of unmethylatable histone H3G13D into chromatin decreases H3K9me3 density and thereby compromises the read-write capability of Clr4/Suv39h. Consistently, enhancing the affinity of Clr4/Suv39h for methylated H3K9 is sufficient to overcome the defects in heterochromatin assembly caused by H3G13D Our work directly implicates methylated histones in the transmission of epigenetic memory and shows that a critical density threshold of H3K9me3 is required to promote epigenetic inheritance of heterochromatin through the read-write mechanism.

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. We find that SMARCAD1 prevents accumulation of 53BP1-associated nucleosomes to preclude toxic enrichment of 53BP1 at the forks. In the absence of SMARCAD1, 53BP1 mediates untimely dissociation of PCNA via the PCNA-unloader ATAD5, causing frequent fork stalling, inefficient fork restart, and accumulation of single-stranded DNA. Although loss of 53BP1 in SMARCAD1 mutants rescues these defects and restores genome stability, this rescued stabilization also requires BRCA1-mediated fork protection. Notably, fork protection-challenged BRCA1-deficient naïve- or chemoresistant tumors require SMARCAD1-mediated active fork stabilization to maintain unperturbed fork progression and cellular proliferation.

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. Chromosomal regions such as subtelomeres that show defects in replication progression associate with the nuclear envelope in sap1 mutant cells. Moreover, high-resolution, genome-wide chromosome conformation capture (Hi-C) analysis revealed prominent contacts between telomeres and chromosomal arm regions containing replication origins proximal to binding sites for Taz1, a component of the Shelterin telomere protection complex. Strikingly, we find that Shelterin components are required for interactions between Taz1-associated chromosomal arm regions and telomeres. These analyses reveal an unexpected role for Shelterin components in genome reorganization in cells experiencing replication stress, with important implications for understanding the mechanisms governing replication and genome stability.

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. Importantly, we find that Fft3 suppresses turnover of histones at heterochromatic loci to facilitate epigenetic transmission of heterochromatin in cycling cells. Moreover, Fft3 also precludes nucleosome turnover at several euchromatic loci to prevent R-loop formation, ensuring proper replication progression. Our analyses show that overexpression of Clr4/Suv39h, which is also required for efficient replication through these loci, suppresses phenotypes associated with the loss of Fft3. This work uncovers a conserved factor critical for epigenetic inheritance of heterochromatin and describes a mechanism in which suppression of nucleosome turnover prevents formation of structural barriers that impede replication at fragile regions in the genome.

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. Our analyses of wild-type and mutant strains reveal key elements of chromosome architecture and genome organization. On chromosome arms, small regions of chromatin locally interact to form 'globules'. This feature requires a function of cohesin distinct from its role in sister chromatid cohesion. Cohesin is enriched at globule boundaries and its loss causes disruption of local globule structures and global chromosome territories. By contrast, heterochromatin, which loads cohesin at specific sites including pericentromeric and subtelomeric domains, is dispensable for globule formation but nevertheless affects genome organization. We show that heterochromatin mediates chromatin fibre compaction at centromeres and promotes prominent inter-arm interactions within centromere-proximal regions, providing structural constraints crucial for proper genome organization. Loss of heterochromatin relaxes constraints on chromosomes, causing an increase in intra- and inter-chromosomal interactions. Together, our analyses uncover fundamental genome folding principles that drive higher-order chromosome organization crucial for coordinating nuclear functions.

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. The Mtl1-Red1 core promotes degradation of mRNAs and noncoding RNAs and associates with different proteins to assemble heterochromatin via distinct mechanisms. Mtl1 also forms Red1-independent interactions with evolutionarily conserved proteins named Nrl1 and Ctr1, which associate with splicing factors. Whereas Nrl1 targets transcripts with cryptic introns to form heterochromatin at developmental genes and retrotransposons, Ctr1 functions in processing intron-containing telomerase RNA. Together with our discovery of widespread cryptic introns, including in noncoding RNAs, these findings reveal unique cellular strategies for recognizing regulatory RNAs and coordinating their functions in response to developmental and environmental cues.