The epigenetic contribution to phenotype is now well established. Studies over the past decade have shown that proteins that are able to establish and propagate epigenetic modifications are essential for mammalian development. Some of the genes involved in these processes have been identified, but the roles of many remain unknown. The mutagenesis screens for modifiers of position effect variegation in Drosophila suggest that there are over 200 genes that are able to modify epigenetic variegation. We emulated this screen in the mouse to identify mammalian modifiers of a variegating transgene. The screen aimed to identify novel genes involved in epigenetic reprogramming, and to generate mouse models to study the impact of disruption to the epigenome. Inbred male mice carrying a variegating GFP transgene expressed in erythrocytes were mutagenised with ENU. Offspring were screened by flow cytometry and in the initial rounds of mutagenesis, 11 dominant mutant lines were identified. These lines were called MommeDs (Modifiers of murine metastable epialleles, dominant). This thesis describes the mapping and phenotypic characterisation of three Momme lines: MommeD7, MommeD8 and MommeD9. The MommeD9 mutation enhances variegation and was mapped to a 3.4 Mb interval on Chromosome 7. A mutation in a 5? splice site was found in the Trim28 gene. Analysis of Trim28 mRNA and protein in heterozygotes showed that the mutant allele was null. Homozygotes die before mid-gestation. Heterozygotes are viable but display variable and complex phenotypes, including infertility, obesity, behavioural abnormalities and premature death. Obese MommeD9 mice have liver steatosis, impaired glucose tolerance and other indicators of metabolic syndrome. This phenotype has not previously been reported for mice haploinsufficient for Trim28. There is considerable variability of phenotypes among inbred MommeD9 heterozygotes, which suggests a role for epigenetics in phenotypic noise or “intangible variation”. MommeD8 is a semi-dominant enhancer of variegation. Some homozygotes are viable but some die around birth. Viable homozygotes weigh less than wildtype littermates and have increased CpG methylation at the GFP transgene enhancer element. The mutation was mapped to a 4 Mb interval on chromosome 4. Extensive candidate gene sequencing failed to find a mutation and so DNA from mutant and wildtype individuals were sequenced across the entire linked interval by 454 Sequencing technology. MommeD8 individuals carry two point mutations, one is intergenic and the other lies in an intron of the Ppie gene. Analysis of Ppie mRNA in heterozygotes and homozygotes shows that mutants have reduced transcript levels, suggesting that a deficiency in Ppie causes the increased silencing of GFP. The Ppie gene has not been reported to be involved in epigenetic reprogramming and little is known about its function. Mice heterozygous for MommeD7 have a marked increase in expression of GFP. Heterozygotes have a range of hematopoietic abnormalities including splenomegaly, anaemia and reticulocytosis. Homozygotes die at birth and appear pale. The increased GFP in the peripheral blood appears to be the consequence of an increase in reticulocytes. The mutation is linked to a 1.5 Mb interval on Chromosome 7. MommeD7 mice appear to have hematopoietic abnormalities that affect the expression of the erythroid-specific GFP reporter transgene. MommeD7 mice serve as a reminder that, as well as discovering bona fide modifiers of epigenetic reprogramming, the ENU screen can also identify hematopoietic mutants.

Gene transfer techniques such as retroviral transduction have many applications such as cell marking, cell reprogramming, and therapeutics. Transgene expression, however, is often variable and maintaining long-term expression is problematic in progenitor cell types. To better control transgene expression, research has focused on the optimized use of cis-regulatory elements, such as promoters, enhancers and insulators. In addition to controlling gene expression, these regulatory elements modulate the nuclear organization of the transgene. The integration site also exerts significant effects on steady state and temporal transgene expression via the neighbouring chromatin environment. The first part of this thesis describes the co-operation of modified β-globin intronic elements in providing high-level expression and favorable nuclear localization. I demonstrate that these elements are compatible with efficient lentivirus transduction for globin gene therapy purposes. In the second chapter, I examine high-expressing EGFP retroviral transgenes and show that such steady state expression may exhibit rapid transcriptional fluctuations, which is modulated by different transcriptional dynamics at different integration sites. Finally, in the last chapter, I evaluate the use of a 3’D4Z4 insulator element in maintaining long-term EGFP transgene expression in ES cells, and discover integration-site specific temporal dynamics in retroviral vector expression. Overall, my results demonstrate that using multiple regulatory elements and insulating these elements from different types of genomic loci optimize transgene expression and dynamics in progenitor cells.

Gene transfer techniques such as retroviral transduction have many applications such as cell marking, cell reprogramming, and therapeutics. Transgene expression, however, is often variable and maintaining long-term expression is problematic in progenitor cell types. To better control transgene expression, research has focused on the optimized use of cis-regulatory elements, such as promoters, enhancers and insulators. In addition to controlling gene expression, these regulatory elements modulate the nuclear organization of the transgene. The integration site also exerts significant effects on steady state and temporal transgene expression via the neighbouring chromatin environment. The first part of this thesis describes the co-operation of modified β-globin intronic elements in providing high-level expression and favorable nuclear localization. I demonstrate that these elements are compatible with efficient lentivirus transduction for globin gene therapy purposes. In the second chapter, I examine high-expressing EGFP retroviral transgenes and show that such steady state expression may exhibit rapid transcriptional fluctuations, which is modulated by different transcriptional dynamics at different integration sites. Finally, in the last chapter, I evaluate the use of a 3’D4Z4 insulator element in maintaining long-term EGFP transgene expression in ES cells, and discover integration-site specific temporal dynamics in retroviral vector expression. Overall, my results demonstrate that using multiple regulatory elements and insulating these elements from different types of genomic loci optimize transgene expression and dynamics in progenitor cells.

RNA interference (RNAi) is an important mechanism regulating gene expression. In plants, RNAi is triggered by double-stranded RNA (dsRNA) which is processed into small RNAs (sRNAs), usually 21-24 nt long. The sRNAs are loaded into Argonaut (AGO) protein and recognize the target based on sequence complementarity. When the target is mRNA, they can slice it or block translation leading to posttranscriptional gene silencing (PTGS). When the target is DNA, they can induce DNA methylation and chromatin changes, which when present in the promoter can lead to transcriptional gene silencing (TGS). The individual components of RNAi are well described, but less is known about the impact of different types of dsRNA precursors on the dynamics of RNAi. To study these aspects of RNAi, we used tobacco BY-2 cell line expressing GFP reporter and inducible silencers. The silencers used different ways of triggering the dsRNA formation by transcripts from antisense (AS), unterminated sense (UT) and inverted repeat (IR) GFP sequence to initiate PTGS. Additionally, one IR silencer based on the CaMV 35S promoter initiated TGS. This allowed us to study RNAi from the beginning throughout the steady state level and till the recovery phase, all in the highly homogeneous system. Using this system, we described several features...

Le glioblastome (GBM) représente environ 50 % des gliomes, le type de tumeur cérébrale le plus répandu. Sa forte agressivité se traduit par un faible et inquiétant taux de survie médian de 8 mois. La résistance intensive aux traitements actuels du GBM souligne la nécessité d'approches innovantes. Malgré l'essor récent d’immunothérapies ces dernières années, le GBM présente plusieurs attributs qui entravent l’efficacité de ces thérapies. Les cellules Natural Killer (NK) possèdent d’intéressantes propriétés anti-tumorales qui encouragent leur utilisation en tant que source prometteuse de thérapie cellulaire adoptive pour relever les défis posés par le GBM. Ce projet est une collaboration avec Scott McComb (National Research Center, Ottawa), dont l'équipe a développé des vecteurs lentiviraux de récepteurs antigéniques chimériques (CARs) ciblant EGFRvIII et HER2, respectivement exprimés dans 30 % et surexprimés dans 80 % des cas de GBM. Nous proposons d’adapter ces constructions aux domaines activateurs transmembranaires et intracellulaires optimisés pour la signalisation des cellules NK. L’objectif est d'optimiser la stabilité de l'expression CAR dans les cellules NK. Cela permettra d'augmenter durablement l'efficacité et la spécificité de la cytotoxicité cellulaire de cellules cancéreuses EGFRvIII+ et HER2+. Des vecteurs lentiviraux codant pour le rapporteur eGFP sous 5 promoteurs différents ont été produits. Des cellules NK primaires activées ont été modifiées par transduction lentivirale et triées en fonction de l'expression du transgène afin d'enrichir la population de cellules modifiées. Nos données suggèrent que la séquence régulatrice minimale UCOE pourrait conférer à elle seule une expression stable et robuste du transgène dans les cellules NK pendant 12 semaines. Ce promoteur a ensuite été incorporé dans les constructions CAR-NK pour optimiser la stabilité d’expression du CAR. L’efficience des CAR anti-EGFRvIII et des CAR anti-HER2 ont été évaluées in vitro en utilisant la lignée cellulaire U87 dérivée du GBM dans des tests de cytotoxicité. Parmi les constructions sélectionnées, CAR-F269 et sdCAR-HER2-6 ont montré une cytotoxicité significativement améliorée par rapport aux cellules NK non modifiées contre des cibles spécifiques de l'antigène. L'amélioration de la cytotoxicité observée avec sdCAR-HER2-6 était positivement corrélée à l'intensité de l'expression de HER2 dans les cellules cibles. Ce projet représente une preuve de principe qui suggère le potentiel de la thérapie CAR-NK comme une voie prometteuse dans la lutte contre le GBM et d'autres tumeurs solides. / Glioblastoma (GBM) accounts for approximately 50% of gliomas, the most prevalent type of brain tumor. Its high aggressiveness results in a worrying poor median survival rate of 8 months. Intensive resistance to current treatments for GBM highlights the need for innovative approaches. Despite recent growth in immunotherapies, GBM exhibits several hallmarks that hinder effectiveness of such therapies. Natural killer (NK) cells display interesting anti-tumoral properties that encourage their use as a promising adoptive cell therapy source to address the GBM challenges. This project is a collaboration with Scott McComb (National Research Center, Ottawa), whose team developed lentiviral vectors coding for chimeric antigen receptors (CARs) targeting EGFRvIII and HER2, which are respectively expressed in 30% and overexpressed in 80% of GBM cases. We propose to adapt these constructs to transmembrane and intracellular activator domains which are optimized for NK cell signaling. The objective is to optimize the stability of CAR expression in NK cells. This will allow for a sustained increase in killing efficacy and specificity of EGFRvIII+ and HER2+ cells. First, lentiviral vectors encoding the eGFP reporter under 5 different promoters were produced. Activated primary NK cells were modified by lentiviral transduction and sorted for transgene expression to enrich the population of modified cells. Our data suggest that the minimal regulatory sequence UCOE may confer alone a stable and robust transgene expression in NK cells for 12 weeks. The UCOE promoter was then incorporated into CAR-NK constructs to optimize the stability of CAR expression. The efficiency of anti-EGFRvIII CARs and anti-HER2 CARs were evaluated in vitro using the GBM-derived U87 cell line in cytotoxicity assays. Among the screened constructs, CAR-F269 and sdCAR-HER2-6 exhibited significantly enhanced cytotoxicity against antigen-specific targets, in comparison to unmodified NK cells. The enhancement of cytotoxicity observed with sdCAR-HER2-6 was found to positively correlate with the intensity of HER2 expression in the target cells. This project represents a proof of principle that suggests the potential of CAR-NK therapy as a promising avenue of in the fight against GBM.

The RNA interference is a mechanism, which allows cells to regulate their genes functions, to establish and maintain heterochromatin and to defend them against invasive nucleic acids. In plants, RNA interference is initiated by double-stranded RNA, which is processed by Dicer into small RNAs, usually 20-24nt long. These small RNAs form a complex with Argonaut protein that participates in different processes based on sequence complementarity. This complex can guide mRNA cleavage, translation blocking and chromatin modifications, resulting either into posttranscriptional silencing (by preventing translation of already existing mRNA, PTGS) or transcriptional silencing (by preventing transcription of mRNA, TGS). The first step of this thesis was to establish different ways of triggering PTGS and to evaluate their functionality and efficiency. The next step was a preparation of a system which would allow to study the transition from posttrancriptional to transcriptional silencing. These so called "indicator lines" should allow to observe the timing and dynamics of this process by utilizing fluorescent proteins. This system is also going to enable to evaluate, how different factors are involved in this process - one of the factors is RNA-dependent RNA polymerase 6 (RDR6) which plays an essential role in...