Stephen Jackson

Loss-of-function mutations in the OCRL gene, which encodes the phosphatidylinositol [PI] 4, 5-bisphosphate [PI (4, 5) P2] 5-phosphatase OCRL, cause defective endocytosis and proximal tubule dysfunction in Lowe syndrome and Dent disease 2. The defect is due to increased levels of PI (4, 5) P2 and aberrant actin polymerization, blocking endosomal trafficking. PI 3-phosphate [PI (3) P] has been recently identified as a coactivator with PI (4, 5) P2 in the actin pathway. Here, we tested the hypothesis that phosphoinositide 3-kinase (PI3K) inhibitors may rescue the endocytic defect imparted by OCRL loss, by rebalancing phosphoinositide signals to the actin machinery. The broad-range PI3K (phosphoinositide 3-kinase) inhibitor copanlisib and class IA p110a PI3K (phosphoinositide 3-kinase) inhibitor alpelisib reduced aberrant actin polymerization in OCRL-deficient human kidney cells in vitro. Levels of PI 3, 4, 5-trisphosphate, PI (4, 5) P2, and PI (3) P were all reduced with alpelisib treatment, and siRNA knockdown of the PI3K catalytic subunit p110a phenocopied the actin phenotype. In a humanized OcrlY/-mouse model, alpelisib reduced endosomal actin staining while restoring stress fiber architecture and levels of megalin at the plasma membrane of proximal tubule cells, reflected by improved endocytic uptake of low molecular weight proteins in vivo. Thus, our findings support the link between phosphoinositide lipids, actin polymerization, and endocytic trafficking in the proximal tubule and represent a proof-of-concept for repurposing alpelisib in Lowe syndrome/Dent disease 2.

DNA-protein crosslinks (DPCs) are highly toxic DNA lesions arising from a variety of endogenous and exogenous genotoxins including chemotherapeutic drugs. DPCs can be enzymatic or non-enzymatic in nature, with non-enzymatic DPCs arising from endogenous aldehydes such as formaldehyde constituting a potentially significant source of endogenous DNA damage. Mechanistic studies of non-enzymatic DPC repair have been hampered by the fact that any chromatin-associated protein could feasibly become crosslinked to DNA, making it challenging to assess the impact of genomic context on DNA induction and repair by conventional methods such as ChIP-seq. Here, we describe DPC-seq, a protocol by which researchers can purify and sequence genomic DNA that bore DPCs, where sequencing read coverage can be used to infer DPC occupancy at specific genomic loci.

Genomic instability arising from defective responses to DNA damage or mitotic chromosomal imbalances can lead to the sequestration of DNA in aberrant extranuclear structures called micronuclei (MN). Although MN are a hallmark of ageing and diseases associated with genomic instability, the catalogue of genetic players that regulate the generation of MN remains to be determined. Here we analyse 997 mouse mutant lines, revealing 145 genes whose loss significantly increases (n = 71) or decreases (n = 74) MN formation, including many genes whose orthologues are linked to human disease. We found that mice null for Dscc1, which showed the most significant increase in MN, also displayed a range of phenotypes characteristic of patients with cohesinopathy disorders. After validating the DSCC1-associated MN instability phenotype in human cells, we used genome-wide CRISPR–Cas9 screening to …

The nucleoside analog decitabine (or 5-aza-dC) is used to treat several hematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC induces covalent DNA methyltransferase 1 DNA-protein crosslinks, leading to DNA hypomethylation. However, 5-aza-dC’s clinical outcomes vary, and relapse is common. Using genome-scale CRISPR/Cas9 screens, we map factors determining 5-aza-dC sensitivity. Unexpectedly, we find that loss of the dCMP deaminase DCTD causes 5-aza-dC resistance, suggesting that 5-aza-dUMP generation is cytotoxic. Combining results from a subsequent genetic screen in DCTD-deficient cells with identification of the DNMT1-DPC-proximal proteome, we uncover the ubiquitin and SUMO1 E3 ligase, TOPORS, as a new DPC repair factor. TOPORS is recruited to SUMOylated DNMT1-DPCs and promotes their degradation. Our study suggests that 5-aza-dC-induced DPCs cause cytotoxicity when DPC repair is compromised, while cytotoxicity in wild-type cells arises from perturbed nucleotide metabolism, potentially laying the foundations for future identification of predictive biomarkers for decitabine treatment.

Combination treatment, the mainstay of cancer therapy, often fails because treatment interactions evoke complex resistance mechanisms that are hard to predict. Designing combination therapy to prevent treatment resistance is especially challenging for rare cancers. Here, we introduce RECODR: a computational pipeline that tracks how genes change their transcriptome context across cancer development and drug treatment conditions. By applying RECODR to a genetically modified mouse model of choroid plexus carcinoma, a rare brain tumour of young children, we identified patterns of transcriptome evolution, cellular heterogeneity and treatment targets that emerged as tumours were initiated and resisted combination treatment. This enabled the prediction of treatment resistance mechanisms and the design of highly effective therapeutic protocols that avoided treatment failure. RECODR can describe complex and dynamic changes in normal and diseased tissues that could be applied to multimodal data from a variety of settings, mitigating treatment resistance across cancers and other diseases.

Covalent DNA–protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA–protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study …

Pooled CRISPR-Cas9 genetic knockout screens are powerful high-throughput tools for identifying chemo-genetic, synthetic-lethal and synthetic-viability interactions and are used as a key step towards identifying disease-modifying knockout candidates and informing drug design and therapeutic regimens. CRISPR guide libraries are commercially available for purchase and have been widely applied in different cell lines. However, discrepancies between the genomes used to design CRISPR libraries and the genomes of the cells subjected to CRISPR screens lead to loss of signal or introduction of bias towards the most conserved genes. Here, we present an algorithm, EXOme-guided Reannotation of nuCleotIde SEquences (Exorcise), which uses sequence search and CRISPR target annotation to adapt existing CRISPR libraries to user-defined genomes and exomes. Applying Exorcise on 55 commercially available CRISPR-spCas9 knockout libraries for human and mouse, we found that all libraries have mis-annotations, and that design strategy affects off-target effects and targeting accuracy relative to a standard reference sequence. In simulations on synthetic data, we modelled common mis-annotations in CRISPR libraries and found that they adversely affected recovery of the ground truth for all genes except for those with the strongest signals. Finally, we reanalysed DepMap and DDRcs CRISPR screens with Exorcise annotations and found that strong hits were retained, and lower-confidence hits were strengthened. Use of Exorcise on DepMap with exomes inferred from transcriptomic expression data demonstrated that cell-line-aware …

Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here, we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a new method for the genome-wide mapping of DNA-protein adducts, DPC-seq, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB-or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at UV-induced DNA lesions, are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS.