During normal cell development, many genes are activated and repressed, usually through epigenetic mechanisms. These are modifications of the DNA and protein within the nucleus that result in changes in gene expression without alteration in DNA sequence. Key proteins for epigenetic modifications are the histone proteins bound to DNA in the nucleus. The best-characterised epigenetic complexes that modify histone proteins are the polycomb group proteins (PcG), comprising polycomb repressive complexes 1 (PRC1) and 2 (PRC2). The repressive modifications generated by these complexes can be removed, and the blocked genes reactivated, by enzymes that are the subject of this project. PRC1 repressive marks are removed by deubiquitinases USP12, USP16 and BAP1, whereas PRC2 marks are removed by demethylases KDM6A, KDM6B and potentially UTY. During the development of cancer, the regulation of many genes becomes abnormal, allowing the cells to escape normal growth restrictions. In this thesis, the expression of this set of chromatin-modifying enzymes in a leukaemia cell line was investigated. The FANTOM consortium has been helping to understand patterns of gene expression for over 10 years. The FANTOM4 dataset described changes in gene expression and promoter usage during differentiation of the THP-1 acute monocytic leukaemia cell line, using CAGE (Cap Analysis of Gene Expression) technology. This human monocyte-like cancer cell line can be stimulated with phorbol esters to halt proliferation and differentiate into macrophages. However, the FANTOM4 time course did not capture detailed mechanisms of regulatory factors in macrophage differentiation due to sparse time points and low read coverage. The main aim of this project was therefore to repeat the time course with tighter time points and deeper sequencing of the transcriptome to develop a very precise picture of sequential activation of gene expression, transcription start site (TSS) usage and the activity of enhancers during the transition from proliferating monocytes to differentiated macrophage phenotype of the THP-1 leukaemia cell line, using CAGE. The focus of this research was on the chromatin-modifying enzymes, but other key cell cycle and macrophage genes have also been examined. The differentiation time course was repeated in triplicate. RNA was extracted and CAGE libraries generated for 18 time points, including the 6 originally studied in FANTOM4. Sequencing results were analysed and normalised using bioinformatics tools. It was shown that analysing 8 samples on one Illumina HiSeq 2500 lane yielded enough read coverage to detect activity from even low expression TSSs, such as those associated with enhancer activity. Clusters of genes which were up- and downregulated at different time points during the differentiation process were identified and characterised. CAGE results for key genes encoding chromatin modifying enzymes and macrophage markers were validated by qRT-PCR. There was a rapid increase of histone demethylase KDM6B mRNA once differentiation was initiated. Histone deubiquitinase USP12 mRNA was also upregulated early in the process. Histone deubiquitinase BAP1 mRNA shows an interesting cyclic regulation pattern which was not seen in the more limited samples of FANTOM4. These interesting chromatin-modifying enzymes and their close paralogues (deubiquitinases USP12, USP16 and BAP1, together with demethylases KDM6A, KDM6B and UTY) were investigated by bioinformatics and genetic tools. USP16 knockout THP-1 cell line was successfully created using CRISPR-Cas9 and its ability to differentiate into macrophages was examined using cell cycle analysis and CAGE sequencing. The USP16 knockout cell line, along with siRNA knock downs of USP12, USP16 and BAP1, was also compared to wildtype THP-1 differentiation using CAGE. Unfortunately, creating other mutant THP-1 cell lines was unsuccessful due to low THP-1 viability after single cell sorting. Investigating KDM6A, KDM6B and UTY using bioinformatics showed that UTY and KDM6A gene expression is positively correlated and this is disrupted in cancer samples. Gene expression and sequence comparison suggested that KDM6A and UTY are coregulated and may act in a similar way in histone demethylation. In summary, the results in this thesis show the transcriptomic changes as the leukaemia cell line ceases proliferation and commences differentiation. Detailed examination suggests that histone modifications are important in the transition between proliferation and differentiation and provide better understanding of regulatory factors in macrophage differentiation and leukaemia.

Ten-Eleven-Translocation (TET) proteins form a family of enzymes responsible for active DNA demethylation by oxidation of 5-methylcytosine. TET proteins play a key role in genomic reprogramming in vitro and in vivo. Although TET proteins are expressed in embryonic stem cells (ESCs), their role in regulating pluripotency remains unclear. In addition, the mechanisms by which TET proteins are recruited to chromatin are largely unknown. To visualise TET protein dynamics during pluripotency and differentiation, the endogenous Tet1/2/3 alleles were fused to epitope tags in ESCs using CRISPR/Cas9. Characterisation of these cell lines showed that TET1 is the highest expressed TET protein in both naïve and primed pluripotent cells. In contrast, TET2 is expressed heterogeneously in ESCs and marks cells with a high self-renewal capacity. To assess the function of Tet genes in pluripotent stem cells, the endogenous Tet1/2/3 ORFs were removed using CRISPR/Cas9. Comparative analysis of single and combined Tet gene knockout ESC lines indicated that Tet1 and Tet2, but not Tet3, play redundant roles to promote loss of pluripotency. Furthermore, Tet-deficient cells retained a naïve morphology in differentiating conditions, suggestive of a LIF-independent self-renewal phenotype. To characterise physiological TET1 protein-protein interactions, TET1 protein partners were identified in ESCs by mass spectrometry and co-immuno-precipitations. This revealed that TET1 interacts with multiple epigenetic and pluripotency-related factors in ESCs. Moreover, detailed characterisation of the interaction between TET1 and NANOG identified three regions of TET1 involved in protein-protein interactions that are conserved in evolution. To investigate TET1 chromatin binding in ESCs, both at the molecular and cellular levels, TET1 was characterised by ChIP-seq analysis and live imaging experiments. Interestingly, TET1 is targeted to chromatin by two different mechanisms, involving distinct protein regions. The interaction with multiple protein partners, including NANOG, might enable TET1 to be targeted to specific chromosomal locations. Additionally, TET1 has the unusual ability to bind mitotic chromatin through its N-terminus, independently of its interaction with NANOG. Together these analyses provide a new understanding of the role of TET proteins in pluripotent cells, as well as a detailed map of TET1 residues involved in protein-protein interactions and mitotic chromatin binding.

Production of large numbers of desirable human cell types in the laboratory is one of the major goals of stem cell research. Current experimental approaches have focused on the strategy of recapitulating the events of normal embryogenesis in culture, by treating cells - either tissue stem cells or pluripotent stem cells (iPS/ES cells) - with cocktails of growth factors, matrix proteins or pharmacological agents. This is challenging and often requires weeks or months of elaborate cell culture regimes. An alternative approach is the forced expression of master regulatory transcription factors; this can bypass developmental programs and drive conversion to the target cell type. Each of these strategies is inefficient and unreliable. Recently a new opportunity has arisen to exploit synthetic transcription factors (sTFs) to program and reprogram cell fate. To create such sTFs the CRISPR/Cas9 system is repurposed through tethering of catalytically dead Cas9 to various transcriptional regulatory effector domains (e.g. VP16, KRAB). In this thesis, we have explored sTFs as tools to reset transcriptional regulatory networks in neural stem cells and mouse embryonic fibroblasts. We tested transcriptional activation of key neural lineage target genes (e.g Olig2, Sox10 and Nkx6.2). We designed and validated a series of sTFs that could effectively activity these. We have found that activation of Sox10 by dCas9-VP160 in mouse neural stem cells can increase the amount of arising oligodendrocyte and oligodendrocyte precursors cells during the differentiation. The activity of sTFs strongly depends on cellular context: i.e. a specific sTF might work well in one cell type but not another. Importantly, these biological barriers are not easily overcome by increasing the strength of the sTF - either through levels or types of effector domains used. Our data inspecting single cells suggests that multiplex delivery of sTFs can indeed cooperate by both increasing the number of cells that activated the gene of interest and increasing the level of transcriptional activation in a given cell. To fully exploit these new technologies, we therefore developed a new construction pipeline that allows easy and efficient assembly of multiple sTFs. Using this approach, we were able to successfully activate three different target genes from a single expression plasmid (Olig2, Sox10 and Nkx6.2) in fibroblasts. These sTFs we able to force fibroblast transdifferentiation towards oligodendrocyte lineage. Future studies will explore further how to exploit these sTFs to augment or replace current reprograming strategies.

The haematopoietic phosphatase PTPN22 is a key regulator in balancing immune responses between self-reactivity and tolerance. PTPN22 downregulates T cell signaling and harbors the non-HLA genetic variation most strongly associated with autoimmune disease in humans, the single nucleotide polymorphism R620W. The effect of this mutation is currently controversial due to confounding results in mouse and human models. The polymorphism is linked to increased susceptibility to autoimmunity in both human and mouse models, although the latter does depend on genetic background. However, mouse data clearly shows that the polymorphism has a loss-of-function effect on T cell signalling, whereas studies in human models largely demonstrate a gain-of-function effect for R620W. A confounding issue in human studies is that they depend on comparison of T cells from distinct individuals, on protein over-expression, or on RNA interference, techniques for which it is difficult to control for genetic and environmental variables, changes in stoichiometry, and off-target effects or incomplete knockdown, respectively. We aimed to create isogenic human cell lines with mutations in PTPN22 at the genomic level to alleviate the complications inherent in analysing human data. In addition to autoimmune pathogenesis, we are interested in the role of PTPN22 in a cancer setting. Because PTPN22 has a strong suppressive effect on T cell responses to weak affinity antigen, which encompass most tumour antigens, we postulated that knocking out PTPN22 may better enable T cells to kill tumour cells. Furthermore, we have shown that PTPN22 knockout (KO) leads to increased IL-2 expression in mouse T cells, and that this effect is protective against TGF-β mediated suppression, a common driver of T cell inhibition in the tumour microenvironment. T cell transfer experiments in mice showed that PTPN22 KO T cells are indeed more effective at reducing tumour size. Based on these findings, we aim to determine whether PTPN22 KO in human cells confers a similar effect on signaling. To investigate the effects of PTPN22 KO on human T cell signaling, we used CRISPR gene-editing to target PTPN22 in a Jurkat cell line. By combining this technique with lentiviral transduction of a specific T cell receptor, we generated human cell lines which are genetically identical, save for specific alterations to PTPN22, and which can be stimulated with strong or weak cognate antigen. We found that PTPN22 KO Jurkat cells develop an enhanced activation phenotype upon stimulation, including increased IL-2 expression. Additionally, PTPN22 KO Jurkat cells show enhanced Erk signalling following stimulation with weak affinity antigen, but this difference is lost as stimulus strength increases. CRISPR technology has presented the opportunity to create novel models of PTPN22 signalling in the context of human T cell lines. The data from these lines suggests that, unlike the R620W mutation, complete loss of PTPN22 has a comparable effect in human and mouse T cells. In conjunction with our previous findings, these results suggest that knocking out PTPN22 may lead to signalling alterations that improve adoptive T cell cancer therapy.

Osteosarcoma is the most common primary malignancy of bone. 5-year survival has remained stable at around 60-70% for 40 years. However, a number of patients will suffer from recurrent and/or metastatic disease representing a large unmet clinical need. CSPG4 is a transmembrane protein which is expressed on a number of progenitor cells and tumour types. Preliminary work had found CSPG4 present in osteosarcoma tumour samples. In this study, CSPG4 mRNA and protein expression was demonstrated in clinical samples and model cell lines. CSPG4 mRNA is overexpressed in osteosarcoma samples compared to mature osteoblast cells, the putative cell of origin for osteosarcoma. In a cohort of patients, CSPG4 protein expression was found on 86% of samples. Furthermore, CSPG4 expression was demonstrated in U2OS, MG63, HOS, HOS-MNNG and 143B osteosarcoma cell lines. CSPG4 protein expression was successfully deleted in 143B cells using CRISPR/Cas9 technology. Two stable CSPG4-negative cell lines were produced. CSPG4 expression was then reintroduced into negative cell lines, as well as the parental 143B cell line. This created a panel of 6 cell lines with differing CSPG4 expression. Furthermore, siRNA treatment of U2OS, MG63, 143B and U87MG cell lines reduced CSPG4 expression. These cells provided another panel with varying CSPG4 expression for in vitro investigation. In vitro experiments failed to demonstrate a role for CSPG4 in osteosarcoma tumorigenesis. The CRISPR/Cas9 cell panel found that CSPG4 expression did not influence cell proliferation, adhesion and spreading on fibronectin or collagen-I, cell migration, chemosensitivity or anchorage-independent growth. Similarly, the siRNA cell panel found that CSPG4 expression did not influence cell proliferation or anchorage-independent growth. In vivo experimentation did not demonstrate a role for CSPG4 in mediating osteosarcoma tumour growth or metastatic spread. Treatment with a sc-Fv antibody fragment failed to demonstrate specific toxicity of CSPG4-positive cell lines. These results indicate that CSPG4 plays no role in osteosarcoma tumour cell behaviour. However, due to its wide expression pattern it represents a viable therapeutic option for drug targeting.

abstract: Chromatin is the dynamic structure of proteins and nucleic acids into which eukaryotic genomes are organized. For those looking to engineer mammalian genomes, chromatin is both an opportunity and an obstacle. While chromatin provides another tool with which to control gene expression, regional density can lead to variability in genome editing efficiency by CRISPR/Cas9 systems. Many groups have attempted to de-silence chromatin to regulate genes and enhance DNA's accessibility to nucleases, but inconsistent results leave outstanding questions. Here, I test different types of activators, to analyze changes in chromatin features that result for chromatin opening, and to identify the critical biochemical features that support artificially generated open, transcriptionally active chromatin. I designed, built, and tested a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin with the aims of 1) activating repressed transgenes in mammalian cells and 2) reversing the inhibitory effects of closed chromatin on Cas9-endonuclease activity. Pioneer factors are unique in their ability to bind DNA in closed chromatin. In order to repurpose this natural function, I designed SPiFs from a Gal4 DNA binding domain, which has inherent pioneer functionality, fused with chromatin-modifying peptides with distinct functions. SPiFs with transcriptional activation as their primary mechanism were able to reverse this repression and induced a stably active state. My work also revealed the active site from proto-oncogene MYB as a novel transgene activator. To determine if MYB could be used generally to restore transgene expression, I fused it to a deactivated Cas9 and targeted a silenced transgene in native heterochromatin. The resulting activator was able to reverse silencing and can be chemically controlled with a small molecule drug. Other SPiFs in my panel did not increase gene expression. However, pretreatment with several of these expression-neutral SPiFs increased Cas9-mediated editing in closed chromatin, suggesting a crucial difference between chromatin that is accessible and that which contains genes being actively transcribed. Understanding this distinction will be vital to the engineering of stable transgenic cell lines for product production and disease modeling, as well as therapeutic applications such as restoring epigenetic order to misregulated disease cells. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2019

abstract: The research question explored in this thesis is how CRISPR mediated editing is influenced by artificially opened chromatin in cells. Closed chromatin poses a barrier to Cas9 binding and editing at target genes. Synthetic pioneer factors (PFs) are a promising new approach to artificially open condensed heterochromatin allowing greater access of target DNA to Cas9. The Haynes lab has constructed fusions of enzymatic chromatin-modifying domains designed to remodel chromatin and increase Cas9 editing efficiency. With a library of PFs available, this research focuses on analyzing the behavior of Cas9 in chromatin that has been artificially opened by PFs. The types and frequency of INDELs (insertions & deletions) were determined after non-homologous end joining (NHEJ) in PF and Cas9-treated cells using quantitative Sanger sequencing and Synthego’s ICE software. Furthermore, NOME-seq analysis was carried out to map nucleosome position in PF and Cas9 treated cells. Although this experiment was unsuccessful, the heat map generated with data obtained from Synthego ICE predicts a possible presence of nucleosome in the vicinity suggesting that perhaps a fully open chromatin state was not achieved. Linear Regression analysis with certain assumptions confirms that with the increase in distance downstream of cut-site, the editing frequency decreases exponentially. Nevertheless, further experimental work should be carried out to investigate this hypothesis. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2019

Las agrupaciones de repeticiones CRISPR (clustered regularly interspaced short palindromic repeats) junto con las proteínas Cas (CRISPR associated) forman los sistemas CRISPR-Cas que constituyen una barrera adaptativa a la entrada de material genético en Bacteria y Archaea. Esta tesis se centra en los sistemas CRISPR-Cas de Escherichia coli, y en especial en el perteneciente al subtipo I-F. En el presente trabajo se ha analizado la secuencia de la región CRISPR I-F de una variedad de cepas de E. coli, lo cual ha permitido una descripción pormenorizada de los componentes del sistema y de su diversidad. La ausencia de genes cas I-F (codificantes de las proteínas Cas I-F) en la mayor parte de las cepas analizadas, cuestiona la relevancia de estos sistemas a nivel de la especie, aun cuando la invariable conservación de repeticiones sugiere que incluso los sistemas aparentemente vestigiales deben desempeñar un papel importante. Por otro lado, análisis in silico de los loci CRISPR-Cas I-E y I-F han permitido reconstruir la historia evolutiva de ambos sistemas y poner de manifiesto la existencia de dos variantes de genes cas asociados al subtipo I-E que podrían diferir en aspectos particulares de su mecanismo de acción. Finalmente, estudios de expresión y análisis de interferencia del sistema I-F de la cepa E. coli LF82 han revelado que dicho sistema es activo en condiciones de crecimiento óptimas en el laboratorio, siendo responsables de una reducción significativa en la eficiencia de transformación del plásmido portador de la secuencia diana (denominada protoespaciador). Dicha actividad de interferencia está muy influenciada por la identidad de motivos de secuencia adyacentes a la región protoespaciadora. Sorprendentemente, la mayor interferencia se observó para los motivos que difieren de aquellos normalmente asociados a los protoespaciadores nativos de este sistema. Esta discrepancia entre motivos implica que el sistema I-F analizado supone una barrera parcialmente tolerante con la entrada de DNA foráneo. Probablemente, esta restricción en la eficacia del sistema es consecuencia de un compromiso entre la prevención de infección por virus líticos y la adquisición de material genético potencialmente beneficioso.

The multisystemic tumors characteristic of the monogenic neoplastic diseases, tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), share common signaling aberrations upon the loss of heterozygosity in either the TSC1 or TSC2 genes. However, their physical manifestations are vastly different and can generally be classified as being either neurological (TSC) or mesenchymal (TSC & LAM; referred to herein as LAM for simplicity) in origin. In this study, I present a comprehensive stem cell model of LAM utilizing multiple TSC2 knockout (TSC2-/-) pluripotent stem cell lines differentiated to the putative cell of origin for mesenchymal tumors, neural crest cells (NCCs). TSC2-/- NCCs faithfully recapitulate LAM phenotypes and temporal RNA-seq analysis of neural and neural crest differentiation was performed to model disease pathogenesis. Analysis revealed immediate activation of stress response signaling resulting in protein aggregation and lysosome and autophagosome accumulation upon neuralization in TSC2-/- cells. This resulted in acute and lasting effects specific to neural progenitor cells (NPCs), that are transient and ameliorated in NCCs. These lineage-specific effects resulted in selective sensitization of NPCs to cell death via proteasome inhibition, suggesting a potential therapeutic avenue for neurological TSC, but not LAM. Thus, a genome-wide CRISPR knockout screen was performed in TSC2-/- NCCs. Analysis of synthetic lethal genes reveals pathways previously targeted for LAM, but provides gene-level resolution to the vulnerable nodes within these pathways. Importantly, 18 novel gene targets were identified that display synthetic lethality to TSC2-/- cells with high specificity. 3 genes within this list were targetable using commercially available small molecule inhibitors, one of which, FGFR1, shows highly selective lethal targeting of TSC2-/- NCCs. Importantly, this model system, paired with the expansive resource of transcriptomic and synthetic lethal data, serves as a foundation for the development of next generation treatment strategies for LAM, and potentially the entire spectrum of TSC manifestations.

The impact that mosquitoes and vector-borne diseases have on humans is vast and continues to grow with our expanding global interactions, such as international travel and shipping, so the need for effective vector controls is imperative. Aedes aegypti is a species of mosquito that spreads some of the most common vector-borne diseases, including zika virus, dengue fever, chikungunya, and yellow fever. A. aegypti have yet to be successfully contained, so they are favorable targets for implementing these new vector-control techniques. A review of scientific literature was performed from 1965 to present, timeline was constructed of studies on A. aegypti and their diseases, with inclusion criteria of techniques like bacterial controls and genome editing. Bacterial controls, such as using an endosymbiont like Wolbachia, can result in sterilization of mosquitoes as well as inhibiting the ability for mosquitoes to be infected by pathogens. Genome editing techniques involve CRISPR and gene drives, allowing the manipulation of certain genes to decrease fitness or susceptibility of pathogens. Combining newly discovered genes that play a role in sterilization with the introduction of sterilizing Wolbachia bacteria could result in a more effective method for controlling A. aegypti. Neither technique is known to be entirely effective on its own, but research indicates that highly effective vector-controls could be developed by combining aspects from both fields.