Peter C. Stirling

We describe a 7 year‐old boy with severe neurodevelopmental disorder, medically‐refractory epileptic encephalopathy, hyperkinetic movement disorder, hypotonia, cerebral and cerebellar atrophy and hypomyelination who was found to have a heterozygous de novo TOP2B NM_001330700.2:c.187C>T, p.(His63Tyr) variant. This is the third individual reported with the H63Y variant and severe neurodevelopmental disorder. The phenotype associated with the H63Y variant differs greatly from the TOP2B‐related Immunodeficiency and Congenital Anomalies Spectrum (TICAS) syndrome that has been reported with heterozygous loss‐of‐function variants affecting the TOPRIM domain and adjacent regions of TOP2B. DNA topoisomerase II beta (TOP2B) is an essential enzyme that resolves DNA supercoils and knots to facilitate transcription and replication. To investigate the functional impact of this variant, we studied the homologous yeast variant, top2 H20Y (H20Y). Homozygous H20Y strains exhibited reduced viability, while heterozygous H20Y acted as a loss‐of‐function allele incapable of supporting growth. Overexpression of the chimeric human H63Y variant in yeast was lethal. Despite its dysfunction, H63Y retains partial enzymatic activity and appears to function as a dominant negative variant that induces DNA damage. Thus, this work establishes a novel gene‐disease association between TOP2B‐H63Y and a neurodevelopmental disorder we propose to name TOP2B‐related neurodevelopmental disorder. Further studies should elucidate the pathogenic mechanisms underlying H63Y‐associated phenotypes.

Drug-related UVA-induced photoreactions have been reported for several therapeutic compounds, including fluoroquinolones. CX-5461 is a clinical stage quinolone-derived anti-cancer small molecule with documented UVA-sensitizing activity. Here, we compared, by bulk and clonal whole-genome sequencing under extraneous light-protected conditions, the mutational signatures in human retinal pigment epithelial cells (RPE1) exposed to UVA, CX-5461, or co-exposed to UVA and CX-5461. Treatment with CX-5461 or UVA alone resulted in a low single-base mutation burden and background-like mutational profiles. In contrast, bulk sequencing of human cells co-exposed to UVA and CX-5461 had a markedly higher mutation burden characterized by T > A and T > C substitutions. Furthermore, single-cell clonal expansion and sequencing of CX-5461 alone, UVA alone, or CX-5461+UVA treatments confirmed that the pattern was only observed when cells were exposed to both UVA and CX-5461. The CX-5461+UVA-associated SBS signature we report arises only when CX-5461-treated cells are exposed to UVA, and is not observed when CX-5461-treated cells are shielded from light. We do not observe strong single-base mutagenic activity of CX-5461 alone, under light-protected conditions. Our data define a human SBS mutagenesis signature for CX-5461 potentiated UVA mutations and emphasize the need for appropriate controls and light-exposure precautions when studying SBS activity of known photosensitizer molecules.

The Cancer Dependency map project (DepMap) has created an unprecedented resource of siRNA and CRISPR genetic screens across more than 1000 cell line models. Numerous computational tools have been developed to analyze DepMap outputs and have revealed new genetic dependencies relevant to cancer. Here we extracted correlation information from the DepMap using curated gene sets of biological pathways to determine if this approach could reveal new high confidence candidates. Beginning with a published set of curated DNA repair proteins we determined correlations of query gene knockout fitness across all DepMap cell lines. As expected, this approach extracted many known genome stability genes and also suggested several additional candidates not previously linked to genome maintenance. To validate our analysis we used siRNA depletion for candidate genes and identified several factors whose depletion increases DNA damage or delay repair. Among these was WDR89. WDR89 relocalizes to the nucleolus in an ATM dependent manner upon DNA damage and physically interacts with a number of nucleolar proteins. Depletion of WDR89 also abrogates nuclear p53 accumulation upon irradiation, suggesting a role in p53 signaling. These data establish WDR89 as a new genome stability regulator and highlight the value of DepMap for predicting previously unknown biology.

While DNA:RNA hybrids contribute to multiple genomic transactions, their unscheduled formation is a recognized source of DNA lesions. Here, through a suite of systematic screens, we rather observed that a wide range of yeast mutant situations primarily triggering DNA damage actually leads to hybrid accumulation. Focusing on Okazaki fragment processing, we establish that genic hybrids can actually form as a consequence of replication-born discontinuities such as unprocessed flaps or unligated Okazaki fragments. Strikingly, such “post-lesion” DNA:RNA hybrids neither detectably contribute to genetic instability, nor disturb gene expression, as opposed to “pre-lesion” hybrids formed upon defective mRNA biogenesis, e.g., in THO complex mutants. Post-lesion hybrids similarly arise in distinct genomic instability situations, triggered by pharmacological or genetic manipulation of DNA-dependent processes, both in yeast and human cells. Altogether, our data establish that the accumulation of transcription-born DNA:RNA hybrids can occur as a consequence of various types of natural or pathological DNA lesions, yet do not necessarily aggravate their genotoxicity.

Chromatin structure and DNA accessibility are partly modulated by the incorporation of histone variants. H2A.Z, encoded by the non-essential HTZ1 gene in S. cerevisiae, is an evolutionarily conserved H2A histone variant that is predominantly incorporated at transcription start sites by the SWR1-complex (SWR1-C). While H2A.Z has often been implicated in transcription regulation, htz1Δ mutants exhibit minimal changes in gene expression compared to wild-type. However, given that growth defects of htz1Δ mutants are alleviated by simultaneous deletion of SWR1-C subunits, previous work examining the role of H2A.Z in gene expression regulation may be confounded by deleterious activity caused by SWR1-C when missing its H2A.Z substrate (apo-SWR1-C). Furthermore, as H2A.Z mutants only display significant growth defects in genotoxic stress conditions, a more substantive role for H2A.Z in gene expression may only be uncovered after exposure to cellular stress. To explore this possibility, we generated mRNA transcript profiles for wild-type, htz1Δ, swr1Δ, and htz1Δswr1Δ mutants before and after exposure to hydroxyurea (HU), which induces DNA replication stress. Our data showed that H2A.Z played a more prominent role in gene activation than repression during HU exposure, and its incorporation was important for proper upregulation of several HU-induced genes. We also observed that apo-SWR1-C contributed to gene expression defects in the htz1Δ mutant, particularly for genes involved in phosphate homeostasis regulation. Furthermore, mapping H2A.Z incorporation before and after treatment with HU revealed that decreases in H2A.Z enrichment at transcription start sites was correlated with, but generally not required for, the upregulation of genes during HU exposure. Together this study characterized the regulatory effects of H2A.Z incorporation during the transcriptional response to HU.

ARID1A is the core DNA-binding subunit of the BAF chromatin remodeling complex and is mutated in about 8% of all cancers. The frequency of ARID1A loss varies between cancer subtypes, with clear cell ovarian carcinoma (CCOC) presenting the highest incidence at > 50% of cases. Despite a growing understanding of the consequences of ARID1A loss in cancer, there remains limited targeted therapeutic options for ARID1A-deficient cancers. Using a genome-wide CRISPR screening approach, we identify KEAP1 as a genetic dependency of ARID1A in CCOC. Depletion or chemical perturbation of KEAP1 results in selective growth inhibition of ARID1A-KO cell lines and edited primary endometrial epithelial cells. While we confirm that KEAP1-NRF2 signalling is dysregulated in ARID1A-KO cells, we suggest that this synthetic lethality is not due to aberrant NRF2 signalling. Rather, we find that KEAP1 perturbation exacerbates genome instability phenotypes associated with ARID1A deficiency. Together, our findings identify a potentially novel synthetic lethal interaction of ARID1A-deficient cells.

Spatial compartmentalization is a key facet of protein quality control that serves to store disassembled or non-native proteins until triage to the refolding or degradation machinery can occur in a regulated manner. Yeast cells sequester nuclear proteins at intranuclear quality control bodies (INQ) in response to various stresses, although the regulation of this process remains poorly understood. Here we reveal the SUMO modification of the small heat shock protein Btn2 under DNA damage and place Btn2 SUMOylation in a pathway promoting protein clearance from INQ structures. Along with other chaperones, and degradation machinery, Btn2-SUMO promotes INQ clearance from cells recovering from genotoxic stress. These data link small heat shock protein post-translational modification to the regulation of protein sequestration in the yeast nucleus.

DDX11/Chl1R is a conserved DNA helicase with roles in genome maintenance, DNA replication, and chromatid cohesion. Loss of DDX11 in humans leads to the rare cohesinopathy Warsaw Breakage Syndrome. DDX11 has also been implicated in human cancer where it has been proposed to have an oncogenic role and possibly to constitute a therapeutic target. Given the multiple roles of DDX11 in genome stability and its potential as an anti-cancer target, we set out to define a complete genetic interaction profile of DDX11 loss in human cell lines. Screening the human genome with CRISPR guideRNA drop out screens in DDX11-WT or DDX11-deficient cells revealed a strong enrichment of genes with functions related to sister chromatid cohesion. We confirm synthetic lethal relationships between DDX11 and the tumour suppressor cohesin subunit STAG2, which is frequently mutated in several cancer types and the kinase HASPIN. This screen highlights the importance of cohesion in cells lacking DDX11 and suggests DDX11 may be a therapeutic target for tumours with mutations in STAG2.

Temperature‐sensitive (TS) alleles create tunable thermoswitches to deplete essential cellular activities and are used to dissect gene function. In their recent study, Link and colleagues (Schramm et al 2023) use a CRISPR‐based approach to systematically create TS alleles across essential genes in E. coli .