123 research outputs found
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
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Application of CRISPR/Cas9 screening to study cancer drivers and to identify novel cancer vulnerabilities
The development of targeted therapies has had a significant impact on cancer survival rates. However, targeting cancers that are driven by loss of tumour suppressor genes remains a major challenge. One promising approach to treat these cancers is the exploitation of synthetic lethal interactions. Synthetic lethality describes an interaction between two genes, where loss of one gene alone does not affect viability but loss of both genes induces cell death. Inhibiting the synthetic lethal partner of a tumour suppressor gene should specifically kill tumour cells, and so these represent potential therapeutic targets. However, very few synthetic lethal interactions have been well-established.
The aim of this project was to systematically screen for synthetic lethal partners of known tumour suppressor genes. To do so, isogenic human induced pluripotent stem cell lines were generated, each carrying a loss-of-function mutation in a single tumour suppressor gene. These cells have a normal genetic background, thus making it simpler to accurately identify interactions. CRISPR/Cas9 technology was applied as it allows for large-scale, unbiased screening of genetic interactions. A genome-wide guide RNA library was prepared and implemented for knockout screening in the isogenic cell line panel. Analysis was performed to identify genes that were specifically essential for cell fitness/survival in the mutant lines. Particular focus was placed on four tumour suppressor genes that encode subunits of the PBAF/BAF complexes. Approximately 20% of human cancers harbour mutations in subunits of these complexes, so identifying dependencies associated with these could have broad therapeutic potential. Candidate synthetic lethal interactions with these genes were investigated using low-throughput assays in the stem cells and in a cancer cell line. The data obtained suggests that screening in stem cells produces highly variable results. Although potential vulnerabilities associated with all of the tumour suppressor genes were identified, further work is required to validate these and to assess the quality of the results.
In addition to genome editing, CRISPR/Cas9 has been adapted as a tool for controlling gene regulation. In collaboration with Dr Louise van der Weyden, I applied this technology to address another challenging area of cancer biology. Metastasis is the main cause of cancer mortality, yet we still have a poor understanding of the genes that control this process. Considering this, an in vivo CRISPR activation screen was performed to identify novel drivers of metastatic colonisation. A mouse melanoma cell line was transduced in vitro with a library designed to up-regulate expression of membrane proteins, which represent ideal drug targets. These cells were then used in an in vivo experimental metastasis assay. Enrichment of guide RNAs in the lungs was assessed to identify genes that increased pulmonary metastatic colonisation when activated. Candidate genes were selected using three analysis strategies, and hits from each were tested. Several genes were successfully validated using the experimental metastasis assay. The most robust hit was studied further to explore its potential as a therapeutic target.
Collectively, the work described in this thesis demonstrates how CRISPR/Cas9 screening can be applied in different model systems to study genes that drive cancer and to explore novel therapeutic strategies.I was funded by the Wellcome Sanger Institute and the MRC
Deciphering causal genetic determinants of red blood cell traits
Les études d’association pan-génomiques ont révélé plusieurs variants génétiques associés à des traits complexes. Les mesures érythrocytaires ont souvent fait l’objet de ce genre d’études, étant mesurées de façon routinière et précise. Comprendre comment les variations génétiques influencent ces phénotypes est primordial étant donné leur importance comme marqueurs cliniques et leur influence sur la sévérité de plusieurs maladies. En particulier, des niveaux élevés d’hémoglobine fœtal chez les patients atteints d’anémie falciforme est associé à une réduction des complications et une augmentation de l’espérance de vie. Néanmoins, la majorité des variants génétiques identifiés par ces études tombent à l’intérieur de régions génétiques non-codantes, augmentant la difficulté d’identifier des gènes causaux.
L’objectif premier de ce projet est l’identification et la caractérisation de gènes influençant les traits complexes, et tout particulièrement les traits sanguins. Pour y arriver, j’ai tout d’abord développé une méthode permettant d’identifier et de tester l’effet de gènes knockouts sur les traits anthropométriques. Malgré un échantillon de grande taille, cette approche n’a révélé aucune association. Ensuite, j’ai caractérisé le méthylome et le transcriptome d’érythroblastes différentiés à partir de cellules souches hématopoïétiques et identifié plusieurs gènes potentiellement impliqués dans les programmes érythroïdes fœtaux et adultes. Par ailleurs, j’ai identifié plusieurs micro-ARNs montrant des motifs d’expression spécifiques entre les stages fœtaux et adultes et qui sont enrichis pour des cibles exprimées de façon opposée. Finalement, j’ai identifié plusieurs variants génétiques associés à l’expression de gènes dans les érythroblastes (eQTL). Cette étude a permis d’identifier des variants associés à l’expression du gène ATP2B4, qui encode le principal transporteur de calcium des érythrocytes. Ces variants, qui sont également associés à des traits sanguins et à la susceptibilité à la malaria, tombent dans un élément d’ADN spécifique aux cellules érythroïdes. La délétion de cet élément par le système CRISPR/Cas9 induit une forte diminution de l’expression du gène et une augmentation des niveaux de calcium intracellulaires.
En conclusion, des échantillons de génotypages exhaustifs seront nécessaires pour étudier l’effet de gènes knockouts sur les traits complexes. Les érythroblastes montrent de grandes différences au niveau de leur méthylome et transcriptome entre les différents stages développementaux. Ces différences influencent potentiellement la régulation de l’hémoglobine fœtale et impliquent de nombreux micro-ARNs et régions régulatrices non-codantes. Finalement, l’exemple d’ATP2B4 montre qu’intégrer des études épigénomiques, transcriptomiques et des expériences d’édition de génome est une approche puissante pour caractériser des variants génétiques non-codants. Par ailleurs, ces résultats impliquent ATP2B4 dans l’hydratation des érythroblastes, qui est associé à la susceptibilité à la malaria et la sévérité de l’anémie falciforme. Cibler ATP2B4 de façon thérapeutique pourrait avoir un impact majeur sur ces maladies qui affectent des millions d’individus à travers le monde.Genome-wide association studies (GWAS) have revealed several genetic variants associated with complex phenotypes. This is the case for red blood cell (RBC) traits, which are particularly amenable to GWAS as they are routinely and accurately measured. Understanding RBC trait variation is important given their significance as clinical markers and modifiers of disease severity. Notably, increased fetal hemoglobin (HbF) production in sickle cell disease (SCD) patients is associated with a higher life expectancy and decreased morbidity. Nonetheless, most variants identified through GWAS fall in non-coding regions of the human genome, increasing the difficulty of identifying causal links.
The main goal of this project was to identify and characterize genes influencing complex traits, and in particular RBC phenotypes. First, I developed an approach to identify and test potential gene knockouts affecting anthropometric traits in a large sample from the general population, which did not yield significant associations. Then, I characterized the DNA methylome and transcriptome of erythroblasts differentiated ex vivo from hematopoietic progenitor stem cells (HPSC), and identified several genes potentially implicated in fetal and adult-stage erythroid programs. I also identified microRNAs (miRNA) that show specific developmental expression patterns and that are enriched in inversely expressed targets. Finally, I mapped expression quantitative trait loci (eQTL) in erythroblasts, and identify erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of RBCs. These genetic variants are associated with RBC traits and malaria susceptibly, and overlap an erythroid-specific enhancer of ATP2B4. Deletion of this regulatory element using CRISPR/Cas9 experiments in human erythroid cells minimized ATP2B4 expression and increased intracellular calcium levels.
In conclusion, large and comprehensive genotyping datasets will be necessary to test the role of rare gene knockouts on complex phenotypes. The transcriptomes and DNA methylomes of erythroblasts show substantial differences correlating with their developmental stages and that may be implicated in HbF production. These results also suggest a strong implication of erythroid enhancers and miRNAs in developmental stage specificity. Finally, characterizing the erythroid-specific enhancer of ATP2B4 suggest that integrating epigenomic, transcriptomic and gene editing experiments can be a powerful approach to characterize non-coding genetic variants. These results implicate ATP2B4 in erythroid cell hydration, which is associated with malaria susceptibility and SCD severity, suggesting that therapies targeting this gene could impact diseases affecting millions of individuals worldwide
Development of shRNA screens to identify effectors of three complex traits: neighbour suppression of tumour growth and proliferation and protection from lipotoxicity in β-cells.
RNA interference (RNAi) is a natural mechanism of cellular defence against exogenous double stranded RNA (dsRNA). The discovery of small dsRNA molecules which can be processed by the RNAi pathway in mammalian cells was one of the key advances in the study of functional genomics. These molecules can be designed to downregulate the expression of specific genes. Collections or libraries of dsRNA molecules targeting an extensive number of genes are now available. Using these libraries, numerous studies have implemented high-throughput screens for the study of molecular effectors of numerous phenotypes.
The process of designing an RNAi screen requires the consideration of several critical factors during both the experimental and analysis phases. The experimental screen should aim to reproduce the biological phenomenon studied as closely as possible by choosing an adequate model and screening conditions. Phenotype evaluation and assessment of knockdown effects need careful consideration. The results obtained from large-scale RNAi screens are often complex. An analysis pipeline should be implemented which integrates the biological basis of the phenomenon and facilitates the interpretation of the data.
This project designed and implemented an unbiased shRNA screen in two in vitro models relevant to carcinogenesis and diabetes. The first screen implemented used a model of neighbour suppression to study the molecular effectors of the response in tumorigenic cells to growth suppression cues from the surrounding tissue, a cellular interaction relevant in early tumorigenesis. The second screen studied two phenotypes relevant to diabetes: proliferation and resistance to lipotoxicity of β-cells in a reversibly immortalised cell line. An integrative analysis pipeline was also developed to apply network biology and functional enrichment analysis methods for the interpretation of the data obtained from both screens.Diabetes U
COMPUTATIONAL MODELING OF GENE REGULATION, GAMETE FORMATION, AND EMBRYO IMPLANTATION
DNA located in genes is transcribed into RNA which is translated into protein. The regulation of transcription and translation is carried out by several factors including a gene’s primary sequence, cis-regulatory elements (CREs) in non-coding DNA regions, epigenetic marks on the histones which compact DNA, and trans-binding factors (or proteins). The differential expression of a gene is crucial for establishing lineage-specific cell identity and phenotypic variability. Mutation or dysregulation may lead to natural variation within a population or aberrant gene expression and disease; trait-associated variation is known to be enriched in putative CREs, supporting their role in the origins of disease. Understanding the mechanisms by which CREs interact with one another and their cellular environment to regulate transcription may inform knowledge of biological pathways and provide a crucial foundation for developing new treatments. Further, because all DNA is passed to an offspring from their parents, it is important to understand not just the outcomes on expression due to coding and non-coding variation, but also how genetic material is passed to future generations. These dissertation chapters apply modeling approaches to large amounts of genetic and gene expression data in order to 1) better understand how the sequence and epigenetic makeup of CREs impact gene expression within hematopoiesis; 2) scan for selfish genetic elements which are preferentially passed to offspring within human sperm samples; and 3) predict implantation success for euploid embryos given gene expression profiles. Our models within Chapters 2-4 describe the impact of CREs within the blood cell lineage, connecting CREs to putative target genes, and establishing that the hematopoietic CREs were enriched for blood-trait associated genetic variation. Within Chapter 5, we find no compelling evidence of selfish genetic elements within a large sample of human sperm. Finally, within Chapter 6, we identify some genes which seem to impact the success of IVF embryo implantation by acting through regulation of translation
Guidescan Software For Improved Single And Paired Crispr Guide Rna Design Coupled With Computational Studies In Leukemia
CRISPR technology has revolutionized the field of genome engineering. CRISPR allows for the easy and efficient manipulation of virtually any genetic locus through a two-component system: a CRISPR endonuclease and guide RNA (sgRNA). These components form a complex that enacts double strand breaks in target DNA. The repair of the double strand break is the main mechanism by which genetic editing of a locus takes place. While the endonuclease cleaves target DNA, it is the sgRNA that specifies targets through complementary binding to a target site. Determining the specificity of sgRNAs to their target site represented a crucial challenge to the genome-engineering field. To facilitate the design of CRISPR libraries, we developed Guidescan, a software package that allowed for the customizable production of sgRNA databases that were guaranteed to match user-defined requirements for sgRNA uniqueness. Furthermore, several computational studies of leukemia are described in this thesis that illustrate different molecular actors and mechanisms through which a leukemic like disease, Myelodysplastic Syndrome, can progress towards leukemia, how leukemia hijacks a splicing protein to maintain its pathology, and finally, how a leukemia can develop resistance to a targeted therapy
Genetic Regulation Of Tmem106b In The Pathogenesis Of Frontotemporal Lobar Degeneration
Neurodegenerative diseases are an emerging global health crisis, with the projected global cost of dementia alone expected to exceed $1 trillion, or \u3e1% of world GDP, by 2018. However, there are no disease-modifying treatments for the major neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, frontotemporal lobar degeneration (FTLD), and amyotrophic lateral sclerosis. Therefore, there is an urgent need for a better understanding of the pathophysiology underlying these diseases. While genome-wide association studies (GWAS) have identified ~200 genetic variants that are associated with risk of developing neurodegenerative disease, the biological mechanisms underlying these associations are largely unknown. This dissertation investigates the mechanisms by which common genetic variation at TMEM106B, a GWAS-identified risk locus for FTLD, influences disease risk. First, using genetic and clinical data from thirty American and European medical centers, I demonstrate that the TMEM106B locus acts as a genetic modifier of a common Mendelian form of FTLD. Second, I investigate the role of increased TMEM106B expression levels, which have been reported both in FTLD patients and in individuals carrying the TMEM106B risk allele, in FTLD pathogenesis. I demonstrate that microRNA-132, the most dysregulated microRNA in a genome-wide screen of FTLD and control brains, directly represses TMEM106B expression in human cells, and likely contributes to the elevated TMEM106B levels seen in disease. I then combine statistical approaches, bioinformatics, and experimental approaches in order to functionally characterize all candidate GWAS causal variants at the TMEM106B locus. This approach identifies a noncoding variant, rs1990620, which affects CTCF-mediated long-range chromatin interactions between distal regulatory elements, as the likely causal variant responsible for altering TMEM106B expression levels and disease risk. These results provide a plausible mechanism by which TMEM106B genotype and expression levels influence FTLD risk and clinical progression, and provide a general framework for elucidating the biological mechanisms underlying a disease-associated risk locus. Such an approach will be necessary in order to translate the thousands of loci associated with disease risk by GWAS into mechanistic understanding and therapeutic advances
Into the blue...Using mouse models to uncover genes driving tumorigenesis and therapy resistance in human breast cancer
To improve cancer treatments, personalized medicine
approaches have aimed to identify exactly which mutations are driving tumor
development in a given patient and specifically target these mutations using
precision therapies. However, one of the main challenges of this approach is
identifying which mutations are true drivers, as tumors typically contain
many additional passenger mutations that do not actually contribute to tumor
development. Besides this, many patients often relapse after prolonged
treatment due to the emergence of acquired resistance, limiting the clinical
effectiveness of targeted treatments.
In this thesis, we focussed on using genetically engineered mouse models to
identify candidate cancer genes and therapy resistance mechanisms in two
different breast cancers: invasive lobular carcinoma (ILC) and
triple-negative breast cancer (TNBC). For ILC, we used transposon-based
insertional mutagenesis (TIM) to uncover several novel cancer genes driving
ILC development. Besides this, we also developed a novel computational
approach (IM-Fusion) for improving the discovery of cancer genes from TIM
screens and explored mechanisms of resistance in Fgfr2-driven ILC. For TNBC,
we used CRISPR-based iterative mouse modeling combined with comparative
oncogenomics to identify novel drivers of BRCA1-deficient TNBC. Finally,
using combined in-vivo/in-vitro screens, we identified Parg as a driver of
treatment resistance in BRCA2-deficient TNBC.
Divisions of Molecular Pathology and Molecular Carcinogenesis, Netherlands Cancer InstituteToxicolog
Identifying Genetic Dependencies and Potential Novel Therapeutic Targets for Osteosarcoma
With little recent improvement in osteosarcoma (OS) outcomes, identification of therapeutic targets is critical. RNA interference (RNAi) and drug screens using OS tumour cell lines (TCL) were used to identify novel genetic dependencies and validate tractable therapeutic targets. Cell viability data from RNAi screens in 18 OS TCL was integrated with whole-exome sequencing and protein expression data. Comparison with non-osteosarcoma TCL, demonstrated OS to be more reliant on skeletal morphogenesis pathways and FGFR1/2, with increased sensitivity to FGFR1 inhibitors. OS TCL positive for FGFR1 amplification and polysomy were significantly more sensitive to FGFR1 inhibitors than unknown or non-amplified OS TCL, providing further evidence for a clinical trial in an enriched population. Correlation of RNAi results with the presence of recurrent driver gene alterations revealed that sensitivity to selective silencing of DYRK1A was associated with deficiency of RB1. This finding was validated using RNAi in the OS TCL, an additional 34 breast TCL, and a DYRK1A kinase inactive model. Harmine, a DYRK1A inhibitor, resulted in greater apoptosis in an RB1 deficient OS TCL than in a RB1 wildtype model. DYRK1A has been identified as a protein interaction partner of RB1 and is pharmacologically tractable. Further work is necessary to mechanistically understand this synthetic lethality. The model system was also used as a tool to validate the potential role of BRCAness in OS, recently identified as a potential target in genomic studies. This determined the majority of OS TCL not to be profoundly sensitive to PARP inhibition. However, LM7 (an OS TCL) created by repeated pulmonary murine passage of SAOS2, demonstrated this acquired phenotype. Absence of RAD51 foci in LM7 in contrast to SAOS2, identifies this as a suitable, mechanistically relevant, tool for studying ‘BRCAness’ in OS. Integrated screens provided a framework for pre-clinical identification and validation of tractable therapeutic targets to facilitate translation into development of clinical trials
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