DNA sequence information is constantly threatened by damage. In the clinic, intentional DNA damage is often used to treat cancer. Cisplatin, a first-line chemotherapy used to treat millions of patients, functions specifically by generating physical links within DNA strands, blocking DNA replication, and killing dividing cells. To maintain genome integrity, organisms have evolved the capacity to repair, respond, or otherwise resist change to the DNA sequence through a network of genetically encoded DNA damage tolerance pathways. In chapter 1, I present advances in experimental design and current progress for a systems genetics approach, using Saccharomyces cerevisiae, to reveal relationships between cisplatin tolerance pathways. Additionally, recent efforts to sequence thousands of cancer genomes have revealed recurrent genetic changes that cause overexpression of specific cisplatin tolerance genes. In chapter 2, I present a submitted manuscript that models overexpression of an essential cisplatin tolerance gene. This study uses a systems genetics approach to reveal the genetic pathways that are essential for tolerating this perturbation, which ultimately led to mechanistic insights for this gene. Convenient genome engineering in Saccharomyces has made this organism an ideal model to develop systems genetics concepts and approaches. In chapter 3, I present a published manuscript that demonstrates a new approach to disrupting genes by making site-specific nonsense mutations. Importantly, this approach does not require cytotoxic double-strand DNA breaks and is applicable to many model organisms for disrupting almost any gene, which may advance systems genetics into new model organisms. Systems genetics provides a framework for determining how DNA damage tolerance pathways act together to maintain cellular fitness and genome integrity. Such insights may one day help clinicians predict which cancers will respond to treatment, potentially sparing patients from unnecessary chemotherapy.

Signal integration and transduction by cell-surface receptors is a complex, multi-layered process resulting in tight regulation of downstream mediators, which in turn elicit pre-defined native cellular responses. The modular architecture of transmembrane receptors provides a unique opportunity for engineering de novo sensor/effector circuits, enabling the development of custom cellular functions for research and therapeutic applications. The signal transduction module of most existing chimeric receptors consists of either native intracellular domains or effectors domains fused to non-programmable DNA binding proteins. Therefore, these receptors can only engage in natural signalling pathways or drive the expression of artificial, pre-integrated transgenes. By harnessing the programmability of a nuclease deficient CRISPR/Cas9 (dCas9) signal transduction module and leveraging the evolutionarily optimised ligand-sensing capacity of native receptors, I have created a novel class of dCas9-based synthetic receptors (dCas9-synR). I demonstrate that an optimised split dCas9-based core architecture and custom protease-based signal release mechanism can be standardised across multiple classes of extracellular domains to engineer receptor tyrosine kinase (RTK)-based and G-protein-coupled receptor (GPCR)-based chimeric receptors. dCas9-synRTK and dCas9-synGPCR integrate a broad variety of input signals (peptides, proteins, lipids, sugars) with highly specific and robust activation of any custom output transcriptional programme in an agonist dose-dependent manner. Finally, to showcase the therapeutic potential of dCas9-synRs, I used them to convert a pro-angiogenic signal into an anti-angiogenic response, deploy a chemokine/cytokine programme in response to tumour-enriched biomolecules, and induce insulin expression following glucose stimulation. The performance of dCas9-synRs and their unique versatility in redirecting the information flow makes them ideally suited to engineer designer cells capable of sensing specific disease markers and in turn drive various therapeutic programmes.

Mammalian neural stem cell (NSC) lines provide a useful experimental model for basic and applied research across stem cell and developmental biology, regenerative medicine and neuroscience. NSCs are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with functional genetic analyses. However, targeted genetic manipulations have not been reported for mammalian NSC lines. Here, we deploy the CRISPR/Cas9 technology and demonstrate a variety of diverse targeted genetic modifications in both mouse and human NSC lines such as: targeted transgene insertion at safe harbour loci; biallelic knockout of neurodevelopmental genes; knock-in of epitope tags and fluorescent reporters; and engineering of glioma driver mutations at endogenous proto-oncogenes. Leveraging these new optimised methods, we explored gene editing to model the earliest stages of paediatric gliomagenesis in primary human NSCs. Our data indicate that oncogenic mutations in histone H3.3 play a role in NSC transformation and may operate through suppression of replication induced senescence. By comparing cellular responses of NSC cultures from different compartments of the developing brain, we also identify differences in susceptibility to distinct H3.3 mutations that mirror the disease etiology. Altogether, our findings indicate that CRISPR/Cas9-assisted genome editing in NSC lines is a versatile tool to explore gene function in CNS development and cancer biology. Our project resulted in the creation of valuable human cellular models of paediatric gliomagenesis, which will allow us to further our understanding of the disease and carry out cell based drug discovery projects.

Remarkable progress has been made in our ability to design and produce synthetic DNA binding domains (TALE or Cas9-based), which can be further functionalized into synthetic transcription factors (sTFs). This technology is revolutionizing our ability to modulate expression of endogenous mammalian genes. Forced expression of cDNAs encoding transcription factors (TFs) is widely used to drive lineage conversions. However, this process is often inefficient and unreliable. Multiplex delivery of sTFs pool to activate endogenous master regulators and extinguish the expression profile of the host cell type could be a potential solution to this problem. We have developed a novel, simple TALE assembly method that enabled us to produce and screen large numbers of TAL effectors and compare their activity to dCas9-based TFs. During this process, we constructed many new functionally validated sTFs. Our ultimate goal is to test whether combining synthetic transcriptional activators and repressors can efficiently reprogram fibroblasts to NS cells or alternatively ‘program’ NS cell differentiation to neurons. We performed analyses of the transcriptome and chromatin accessibility of both fibroblasts and neural stem cells to unravel their core TF networks and their epigenetic state. This will allow us in the future the targeted design of sTFs and synthetic chromatin modifiers for specifically changing cell identity.

The Integrated Stress Response (ISR) is a conserved eukaryotic translational and transcriptional program implicated in mammalian metabolism, memory and immunity. Although mainly considered to be a protective mechanism, prolonged and severe ISR can result in cell death. The ISR is activated by diverse stress pathways converged on phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2) that inhibits the guanine nucleotide exchange activity of its partner eIF2B and attenuates overall rates of protein synthesis. Numerous mutations in eIF2B are linked to a fatal neurodegenerative disease of vanishing white matter. A new chemical inhibitor of the ISR (ISRIB), a bis-O-arylglycolamide, can reverse the attenuation of mRNA translation by phosphorylated eIF2 protecting mice from prion-induced neurodegeneration and traumatic brain injury. The work presented in this dissertation describes identification of mammalian eIF2B as a cellular target of ISRIB by implementing biochemical, biophysical, structural and chemogenetic methods. The herein reported cryo-electron microscopy-based structure of eIF2B uncovers a novel allosteric site on the translation factor capturing the ISRIB-binding pocket at the interface between its β and δ regulatory subunits. The extensive CRISPR/ Cas9-based screen for ISRIB-resistant and analogue-sensitive phenotypes revealed residues on the eIF2B dimer interface important for ISRIB binding. Based on the results reported in this dissertation along with the similar findings of others the potential molecular basis of ISRIB action, and its implication for the regulation of eIF2B's activity is broadly discussed. The identification of the ISRIB binding pocket away from the known interaction sites between eIF2B and eIF2 is also put into the context of a possible molecular mechanism of eIF2B's guanine exchange inhibition by phosphorylated eIF2. The work described in this dissertation provides new insight into the translational regulation and points to the importance of fine-tuning the activity of translation factors by small chemical molecules.

No description available.

GWAS (Genome wide association studies) have aided in the discovery of various novel variants associated with diabetes. However, a detailed study is required to uncover the role of these genes and to determine how their dysfunction affects pathophysiology. Previous work in the lab has been successful in establishing zebrafish as an efficient model to characterise the effects of these candidate genes. Consequently, efforts have been also made to establish zebrafish as an efficient model system for drug screening as well. The current POP (Proof of principle) study aims to find whether treatment with tolbutamide drug in zebrafish carrying MODY (Maturity onset diabetes of the young) mutations has the similar effects in humans. The study employed zebrafish carrying five (gck, hnf1a, hnf1ba, hnf1bb, pdx1) CRISPR induced MODY orthologues. The zebrafish larvae were supplemented with tolbutamide drug from 5dpf till 10dpf (day post fertilisation). At 10dpf, larvae were screened for various glycaemic traits, whole body glucose and lipids as well body size. CRISPR-CAS9- induced mutations were quantified using paired end sequencing. The results showed that treatment with tolbutamide had a significant effect on the hyperglycaemic outcome induced by hnf1bb, hnf1a, and pdx1 mutations which was in line with the known effects of the drug in humans. In conclusion, the POP study proved to be successful in leveraging zebrafish as an efficient model system for, in vivo characterisation of drugs and can likely help to identify novel targets for therapeutic interventions.

DNA-binding proteins bind to specific sequences to direct their activity to defined loci in the genome. Regulation of gene expression, for example, is dependent on the recognition of specific DNA sequences by transcription factors (TFs). These TFs receive input from cellular signals to control panels of genes to meet the needs of the cells. Critical to this function is the recognition and binding of TFs to the correct DNA sequence. The main focus of this thesis is to quantitatively determine how proteins, including TFs, distinguish DNA sequences, and to understand how DNA sequence affect their function. Primarily using the Glucocorticoid receptor (GR) as the model TF, I developed novel methods to measure the DNA binding specificity over long binding sites. These methods: 1) Distinguished the sequence specificity of GR and closely related androgen receptor (AR), which helped to both account for differential genomic localization between the two factors, and explained how GR can functionally substitute for AR in castration-resistant prostate cancer (Chapter II); 2) Explored the effect of DNA sequence on GR-regulated transcription through the specification of monomeric versus dimeric binding. Sequence motifs that bias GR binding toward the monomeric state were discovered (Chapter III); 3) Demonstrated a conserved role of intrinsic specificity in directing the degree of GR genomic occupancy in vivo in a fixed chromatin context (Chapter V); 4) Quantitatively modeled and decoupled the DNA binding and cleavage specificities of CRISPR-Cas9 system, providing a rapid pipeline to characterize the genome-editing reagents (Chapter IV). In summary, we showed here that DNA binding specificity is only the initial step in directing the activity of the bound protein. Beyond the affinity-based recruitment, DNA sequences can regulate the protein activity through alternative mechanisms, such as modulating the binding cooperativity, or directly serving as an allosteric ligand for protein function that is independent of DNA binding affinity.

Cells must employ a diversity of strategies to regulate the quantity and functionality of different proteins during development and adult homeostasis. Post-transcriptional regulation of gene transcripts by microRNAs (miRNAs) is recognized as an important mechanism by which the dosage of proteins is regulated. Despite this, the physiological relevance of direct regulation of an endogenous gene transcript by miRNAs in vivo is rarely investigated. PAX6 is a useful model gene for studying miRNA regulation directly. PAX6 is highly dosage-sensitive transcription factor that is dynamically expressed during development of the eye, nose, central nervous system, gut and endocrine pancreas, and is mutated in the haploinsufficiency disease aniridia. Several miRNAs have been implicated in regulating PAX6 in different developmental contexts. Notably, miR-7 appears to regulate Pax6 during specification of olfactory bulb interneurons in the ventricular-subventricular zone (V-SVZ) of the brain and during development of the endocrine pancreas. Here, we produced a bioinformatics tool to enable selective mutation of candidate microRNA recognition elements (MREs) for specific miRNAs while ensuring that new MREs are not inadvertently generated in the process. We then performed the first comprehensive analysis of the mouse Pax6 3’ untranslated region (3’UTR) to identify MREs that may mediate miRNA regulation of Pax6 and to identify miRNAs capable of interacting with the 3’UTR of Pax6. Using Pax6 3’UTR genetic reporter assay, we confirmed that two MREs for miR-7-5 located at 3’UTR positions 517 and 655 function together to regulate PAX6. We generated mice harbouring mutations in the Pax6 3’UTR that disrupt these miR-7-5p MREs, individually or in combination, to explore the biological relevance of miRNA regulation directly. PAX6 protein abundance was elevated in double miR-7-5p MRE mutants relative to wild type and single mutants in the ventral V-SVZ. However, this increase in PAX6 was not associated with an altered dopaminergic periglomerular neuron phenotype in the olfactory bulb. Our findings suggest that, in vivo, microRNA regulation can be mediated through redundant MRE interactions. This work also reveals that directly mutating predicted MREs at the genomic level is necessary to fully characterize the specific phenotypic consequences of miRNA-target regulation. / Graduate

Sturgeons are commonly known as living fossils or ancient giants that diverged from ancient pre-Jurassic teleost lineage approximately ~300 million years ago (Mya). Sturgeons' 85% species are listed as critically endangered in the International Union for Conservation of Nature (IUCN). Sturgeons' reproductive traits such as delay in sexual maturation and periodic interrupted spawning cycles make their rehabilitation more difficult. However, among sturgeon species, the sterlet (Acipenser ruthenus) has shortest sexual maturation period. Therefore, it can be used as a host in surrogate production in sturgeons. Dnd1 was discovered as germ-plasm specific maternal RNA that exclusively expresses in vertebrate germ-line. Various studies have confirmed that dnd1 protein is essential for Primordial Germ Cells (PGCs) migration; and disruption of the PGCs migration affects fish fertility. Dnd1 deficient PGCs in zebrafish transdifferentiate into somatic cells. Previously our colleagues used morpholino oligonucleotide to knock down dnd1 in sterlet to produce germ cell free host for surrogate production. CRISPR/Cas9, a cutting-edge genome editing technology is being used in different research fields; here we thus aimed to harness the power of aforementioned technology to knock out dnd1 in sterlet. No or less number of PGCs were observed in CRISPR/Cas9 injected embryos as compared to control group injected with FITC-dextran only in order to label PGCs. Furthermore, we compared three different sterilization techniques viz., CRISPR/Cas9 and morpholino oligonucleotide (MO) targeting dnd1 and ultraviolet irradiation to eliminate PGCs in sterlet. Our data showed higher hatching and survival rates in CRISPR/Cas9, UV irradiation, and MO knockdown groups, respectively. Interestingly, some embryos treated with CRISPR/Cas9 displayed malformations. We presume that malformations were due to off-target effects and/or due to double injections i.e., injection of CRISPR/Cas9 at animal pole to knock-out the dnd1 and FITC-dextran at vegetal pole. Taking advantages of Iron Oxide nanoparticles (IONs) applications in various burgeoning research fields, we opted to use them to label PGCs in sturgeons. We injected IONs combined with FITC-dextran into vegetal pole of sturgeon embryos, and have successfully labelled the PGCs. Injection of IONs in sturgeons did not affect hatching and survival rates of embryos. Interestingly at 5 dpf, significantly less number of FITC-dextran labelled PGCs in FITC-dextran/IONs injected group were observed when compared with PGCs that were labelled with FITC-dextran only. Less number of PGCs in IONs injected group presumably could be because of interference posed by IONs to PGCs during the course of their migration. This is first study of its kind where germ cells of any species have been labelled by using nanoparticles. In conclusions, this thesis provides information regarding role of Dnd1 protein as potential germ-cell molecular marker in various fish species, and its use for conservation of fish species. Dnd1 knockout sterlet can be potentially used as sterile host for surrogate production in sturgeons. Moreover, labelling of PGCs in sturgeons by using IONs can thus open new avenues to study interactions of nanoparticles with cells that will ultimately help in hyperthermia where cells/tissues are exposed to electromagnetic field increasing temperatures to activate their death. After insertion of IONs to PGC in sturgeon embryo, it could be possible to isolate PGC using a magnetic field or to apply hyperthermia for host sterilization purpose.