Discovering antiviral restriction factors and pathways using genetic screens

Research in the Hughes lab is supported by a grant from the Academy of Medical Sciences (SFB003/1028), a grant from Tenovus Scotland (T20/63), and The Wellcome Trust Institutional Strategic Support Fund (ISSF). Research in the Gray lab is supported Medical Research Council (MR/N001796/1) and the Biotechnology and Biological Sciences Research Council (BBS/E/D/20002172). C. E. J. is supported by a University of St Andrews Ph.D. scholarship.Viral infections activate the powerful interferon (IFN) response that induces the expression of several hundred IFN stimulated genes (ISGs). The principal role of this extensive response is to create an unfavourable environment for virus replication and to limit spread; however, untangling the biological consequences of this large response is complicated. In addition to a seemingly high degree of redundancy, several ISGs are usually required in combination to limit infection as individual ISGs often have low to moderate antiviral activity. Furthermore, what ISG or combination of ISGs are antiviral for a given virus is usually not known. For these reasons, and that the function(s) of many ISGs remains unexplored, genome-wide approaches are well placed to investigate what aspects of this response results in an appropriate, virus-specific phenotype. This review discusses the advances screening approaches have provided for the study of host defence mechanisms, including CRISPR/Cas9, ISG expression libraries and RNAi technologies.Publisher PDFPeer reviewe

Genetic predisposition for Multiple Myeloma. Identification and functional characterization of risk variants

Multiple myeloma (MM) is a blood malignancy originating from plasma cells. First-degree relatives of patients with MM have two- to four-fold higher risk of MM. However, the molecular basis remains largely unknown. This Ph.D. project aims to identify novel DNA sequence variants predisposing to MM through genome-wide association studies (GWAS) and, subsequently, characterize identified variants functionally.Article I describes a systematic study where we screened for causal gene-regulatory variants at 21 MM risk loci. Article II describes a Nordic GWAS identifying the SOHLH2 (13q13.3) as a novel MM risk locus. Article III describes a novel international meta-analysis of GWAS data totalling 10 906 cases and 366 221 controls, identifying twelve new risk variants for MM accounted for by nine loci: 5q35.2 CPEB4, 6p22.2 BTN3A2, 9q21.33 DAPK1, 10q24.33 STN1, 10q25.2 MXI1, 19p13.3 NFIC, 21q11.2, SAMSN1 and a rare variant at 13q13.1 BRCA2. Finally, in Article IV, we explore the possibility of identifying transcription factors that mediate allele-specific gene-regulatory effects through combined use of CRISPR/Cas9 screening and epistasis analysis of gene expression data.The work presented in this thesis provides new insight into the mechanisms underlying genetic predisposition for multiple myeloma

Investigating the role of schizophrenia-associated gene expression in the developing human brain using Machine Learning

Schizophrenia is a debilitating condition that affects 1% of the population, causes significant hardship and though there are treatments available they are characterised by several limitations. It is a complex mental disorder where some individuals show mild subclinical cognitive symptoms before psychosis onset in adolescence. The treatments available only target a portion of the symptoms and although extensive research has been conducted, a comprehensive understanding of the nature of schizophrenia remains elusive. Unlike other neurodevelopmental disorders, schizophrenia symptoms do not typically present themselves until adolescence. This study aimed to discover gene co-expression networks at multiple developmental stages to identify candidate therapeutic targets to better treat and manage schizophrenia. Recent genome-wide association studies have identified 145 genetic loci associated with schizophrenia. Allen Brain Atlas’s BrainSpan resource provides brain development data from neurotypical brains. Using this resource, it was possible to study the gene expression of 316 schizophrenia-associated genes, identified previously in a large-scale GWAS, across each of the developmental stages available in the Allen Brain Atlas. K means Clustering and a systems biology approach (WGCNA) was applied to these schizophrenia-associated genes at each developmental stage where modules within networks were created by grouping co-expressed genes. To facilitate biological interpretation of these modules co-expressed genes were visualised using Cytoscape and gene ontology pathway enrichment analysis was applied. We identified 21 hub genes using WGCNA. Of the 316 schizophrenia-associated genes, 27 modules were identified and 3 hub genes GPR52, INA, SATB2 were common in multiple developmental stages. Our results suggest that GPR52, INA, SATB2 represent candidate genes for future evaluation of their potential as therapeutic targets of schizophrenia. Additional hub genes included TRANK1 and ALMS1, genes which were previously identified as expression quantitative trait loci. Taken together our results add further evidence that these genes could be good candidates for further research as they may regulate several schizophrenia-related genes in their respective modules. Finally, our enrichment analysis implicated a role for positive regulation of macrophage proliferation and cellular response to catecholamine stimulus, and cellular response to diacyl bacterial lipopeptide at each developmental stage. The immune system and catecholamines, including dopamine, have long been associated with schizophrenia and our results provide further support for these hypotheses

Investigating the role of Schizophrenia-associated gene expression in the developing human brain using Machine Learning

Schizophrenia is a debilitating condition that affects 1% of the population, causes significant hardship and though there are treatments available they are characterised by several limitations. It is a complex mental disorder where some individuals show mild subclinical cognitive symptoms before psychosis onset in adolescence. The treatments available only target a portion of the symptoms and although extensive research has been conducted, a comprehensive understanding of the nature of schizophrenia remains elusive. Unlike other neurodevelopmental disorders, schizophrenia symptoms do not typically present themselves until adolescence. This study aimed to discover gene co-expression networks at multiple developmental stages to identify candidate therapeutic targets to better treat and manage schizophrenia. Recent genome-wide association studies have identified 145 genetic loci associated with schizophrenia. Allen Brain Atlas’s BrainSpan resource provides brain development data from neurotypical brains. Using this resource it was possible to study the gene expression of 316 schizophrenia-associated genes, identified previously in a large-scale GWAS, across each of the developmental stages available in the Allen Brain Atlas. K means Clustering and a systems biology approach (WGCNA) was applied to these schizophrenia-associated genes at each developmental stage where modules within networks were created by grouping coexpressed genes. To facilitate biological interpretation of these modules co-expressed genes were visualised using Cytoscape and gene ontology pathway enrichment analysis was applied. We identified 21 hub genes using WGCNA. Of the 316 schizophrenia-associated genes, 27 modules were identified and 3 hub genes GPR52, INA, SATB2 were common in multiple developmental stages. Our results suggest that GPR52, INA, SATB2 represent candidate genes for future evaluation of their potential as therapeutic targets of schizophrenia. Additional hub genes included TRANK1 and ALMS1, genes which were previously identified as expression quantitative trait loci. Taken together our results add further evidence that these genes could be good candidates for further research as they may regulate several schizophrenia-related genes in their respective modules. Finally, our enrichment analysis implicated a role for positive regulation of macrophage proliferation and cellular response to catecholamine stimulus, and cellular response to diacyl bacterial lipopeptide at each developmental stage. The immune system and catecholamines, including dopamine, have long been associated with schizophrenia and our results provide further support for these hypotheses

Investigation of conventional and unconventional protein secretion using galectin-3

Protein secretion is an essential process in cell biology. The conventional secretion pathway is well known, and involves proteins with a signal sequence being directed into the endoplasmic reticulum (ER). Once in the lumen of the ER, proteins are trafficked through the Golgi and remain separate from the cytosol throughout their journey to the outside of the cell. More recently, proteins without a signal sequence have been found outside the cell. These proteins are secreted unconventionally, and avoid the ER-Golgi route. For both pathways, some aspects remain unclear, particularly surrounding the regulation of protein secretion. Galectin-3, an unconventionally secreted protein, was used here to investigate both conventional and unconventional protein secretion. The unconventional protein secretion of galectin-3 is poorly understood. To address this, genetic manipulation of galectin-3 was used to gain insights into the mechanism of galectin-3 secretion. It was found that galectin-3 does not require unfolding or binding to cell surface glycoproteins to be secreted. Galectin-3 was also used to assess protein secretion using a genome-wide CRISPR screen. As galectin 3 binds to cell surface glycoproteins, galectin-3 on the cell surface was used to assess secretion of these glycoproteins. Hits were validated by a glycoprotein secretion assay. Using this pipeline, 93 hits were identified as involved in protein secretion, of which 51 were novel hits not previously associated with protein secretion. Many of the hits identified also resulted in a phenotype of altered Golgi morphology, and five of these were investigated further. Two novel hits that localised to the Golgi were identified, TMEM220 and GPR161. GPR161 likely mediates its effect on Golgi morphology by its interaction with golgin A5. This thesis presents new insights into both conventional and unconventional protein secretion. Insights into the mechanism for galectin-3 secretion were revealed. A novel Golgi-localised regulator of secretion, GRP161, was identified, with a mechanism for action suggested. Importantly, the list of 51 novel genes identified will serve as a useful resource for other researchers investigating protein secretion

Functional impact of inactivating mutations in epigenetic regulators in cancer

Cancer evolution is driven by selection acting on genetic and epigenetic diversity to promote the propagation of the fittest subpopulations. This phenomenon is shaped by the tumor microenvironment which is often characterized by stressful conditions. Epigenetic regulators are frequently mutated during the later stages of tumorigenesis, but the functional impact of their inactivation is poorly understood. In this thesis, I hypothesize that the disruption of the epigenetic regulatory network increases cell fitness in unfavorable environments and thus is selected over time. Through large-scale fitness assays in various cancer models, I demonstrate that epigenetic deregulation leads to a widespread stress-specific survival advantage. This effect is mediated by mutations in all layers of epigenetic regulation, is shared across different stress conditions and is cancer type independent. Then, I explore various cellular mechanisms that can underlie this stress-specific fitness advantage. Genetic diversity, transcriptional heterogeneity or phenotypic plasticity cannot explain the increased survival under stress, as revealed by a combination of reversible epigenetic inhibition, live-cell imaging and single-cell transcriptomics. On the contrary, epigenetically deregulated cells remain phenotypically inert (less responsive) under stress. Transcriptional profiling of cancer populations in hostile conditions, revealed significant alterations in fitness and growth-related signatures. Disruption of the epigenetic machinery results in a defective stress response, thus decreasing the probability of such cells to surpass a stressed threshold and ultimately die. This defective transcriptional rewiring underpins the inert phenotype that emerges upon epigenetic deregulation. Collectively, by investigating the effect of inactivating mutations in epigenetic regulators on cell fitness under environmental stress, I propose that phenotypic inertia is the favorable cellular trait that is selected over time. My findings provide a potential explanation for the widespread subclonal mutations affecting epigenetic regulators and have significant implications for cancer evolution.Open Acces

Molecular biology of the DExD-box helicases DDX49 and DDX52

The DExD-box family of helicases represents the largest family of helicases within eukaryotes and have been primarily associated with all aspects of RNA biology, including processes of mRNA synthesis, pre-mRNA processing and ribosome biogenesis. However, despite their key roles and associations with cancer and other diseases, the biochemical activity and function of many of the human forms of these helicases remain uncharacterised. Additionally, despite becoming popularly synonymous as ‘RNA helicases’, it has become clear in recent years that in addition to their canonical roles within RNA processing, many of these proteins are multi-functional and play important roles within processes of DNA repair, transcriptional regulation and viral immunity, amongst others. In this study we examine and characterise two poorly studied helicases, Probable ATP-dependent RNA helicases DDX49 and DDX52, which are both associated with several cancers and have previously identified connections within viral immunity and DNA repair, respectively. We hypothesised that both genes processed DNA substrates in addition to RNA substrates and by testing recombinant proteins within in vitro assays with several DNA substrates were successful in confirming this hypothesis, as well as identifying novel nuclease and annealing activities within DDX49 and DDX52, respectively. We also developed and optimised a CRISPR-Cas9 gene knockout system for mammalian cells and successfully generated and performed preliminary phenotyping of heterozygous U2OS cell lines. Finally, we explore and performed a comparative study of DDX49 with a potential homolog from Asgard Archaea, providing unexpected but novel insight into the biochemistry of the yeast protein Dbp8

Molecular biology of the DExD-box helicases DDX49 and DDX52

The DExD-box family of helicases represents the largest family of helicases within eukaryotes and have been primarily associated with all aspects of RNA biology, including processes of mRNA synthesis, pre-mRNA processing and ribosome biogenesis. However, despite their key roles and associations with cancer and other diseases, the biochemical activity and function of many of the human forms of these helicases remain uncharacterised. Additionally, despite becoming popularly synonymous as ‘RNA helicases’, it has become clear in recent years that in addition to their canonical roles within RNA processing, many of these proteins are multi-functional and play important roles within processes of DNA repair, transcriptional regulation and viral immunity, amongst others. In this study we examine and characterise two poorly studied helicases, Probable ATP-dependent RNA helicases DDX49 and DDX52, which are both associated with several cancers and have previously identified connections within viral immunity and DNA repair, respectively. We hypothesised that both genes processed DNA substrates in addition to RNA substrates and by testing recombinant proteins within in vitro assays with several DNA substrates were successful in confirming this hypothesis, as well as identifying novel nuclease and annealing activities within DDX49 and DDX52, respectively. We also developed and optimised a CRISPR-Cas9 gene knockout system for mammalian cells and successfully generated and performed preliminary phenotyping of heterozygous U2OS cell lines. Finally, we explore and performed a comparative study of DDX49 with a potential homolog from Asgard Archaea, providing unexpected but novel insight into the biochemistry of the yeast protein Dbp8