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Strawberry plants (Fragaria x ananassa) and Scots pine (Pinus sylvestris) represent species, withinagriculture and forestry respectively, that are traditionally protected by utilization of pesticidesincluding neurotoxic insecticides. More environmentally friendly protection strategies are thereforehighly desirable. Treating plants with specific metabolites naturally occurring in their tissues might alterepigenetic mechanisms, which in turn may strengthen plants self-defense against diseases and weevilattacks. F. x ananassa and P. sylvestris seeds were treated with 2,5 mM nicotinamide and 2,5 mMnicotinic acid in order to investigate possible epigenetical effects by analyzing changes in the level ofthe DNA methylation. The epigenetic changes, for both plants, were analyzed on the global DNA level.Reduction in the DNA methylation level in strawberry leaves as well as the DNA methylation increase inpine needles were observed by means of LUMA-analysis when HpaII restriction enzyme was used in theanalysis. Further investigation is required in order to understand if NIC and NIA may have a significantimpact on pathogen attack in strawberry plants and Scots pine. More research may also unveil ifnicotinamide and nicotinic acid can play a potential role in more sustainable defense strategies ofplants. / Jordgubbsplantor (Fragaria x ananassa) och tallar (Pinus sylvestris) representerar växter inom jord- ochskogsbruk som traditionellt skyddas genom användning av bekämpningsmedel, detta inklusiveneurotoxiska insekticider. Mer miljövänliga skyddsstrategier är därför mycket önskvärda. Behandling avväxter med specifika naturligt förekommande metaboliter genererade av växterna kan påverkaepigenetiska mekanismer. Förändringar på den epigenetiska nivån kan, i sin tur, bidra till förstärkningav växternas eget självförsvar mot sjukdomar och insektsangrepp. Frön av både F. x ananassa och P.sylvestris behandlades med 2,5 mM nikotinamid och 2,5 mM nikotinsyra i syfte att undersökaeventuella epigenetiska effekter. Detta genom att analysera förändringar i graden av DNA metylering ide behandlade plantorna. De epigenetiska förändringarna för jordgubbsplantor och tallar analyseradespå den globala DNA-nivån. Minskad DNA-metylering i jordgubbsblad samt ökad DNA-metylering itallbarr observerades med hjälp av restriktionsenzymet Hpall och LUMA-analys. Ytterligareundersökningar behövs för att kunna förstå om NIC och NIA kan ha en inverkan på patogenangrepp ijordgubbsplantor och tall. Mer forskning kan också avslöja om nikotinamid och nikotinsyra kan ha enbetydande roll inom hållbara försvarsstrategier för växter.

<p>KDM5b acts as a transcriptional repressor through its ability to demethylate tri-methylated lysine (K) 4 on histone H3 (H3K4me3). Demethylation of this histone modification leads to transcriptional repression and downstream biological effects on gene expression. KDM5b is involved in the regulation of differentiation and can exert an oncogenic and a tumour suppressive role depending on cellular context, making it an attractive future target for pharmaceutical intervention. Work from our group has shown that KDM5b expression is linked to differentiation, and that recruitment of the enzyme does not always result in an alteration of H3K4me3. Additionally, work from our group, as well as others, has failed to observe H3K4me3 demethylation by KDM5b in nucleosomal preparations. We therefore hypothesized that KDM5b may exert its demethylase potential on alternative histone targets and that KDM5b requires enzymatic co-factors to demethylate nucleosomes, similar to what is observed for other histone-modifying proteins. In this thesis, we describe KDM5b as having an alternate histone target, di-methylated histone H2B lysine 43 (H2BK43me2). We show that this methyl mark is the primary target for KDM5b, and that the expression level of H2BK43me2 is directly related to the process of differentiation. We additionally present a novel co-factor for KDM5b, the co-repressor TLE4 of the Groucho/TLE family. The presence of TLE4 is required and sufficient to confer nucleosomal demethylase activity to KDM5b, a novel discovery for any of the KDM5 family members. Overall, this work has described both an additional KDM5b target, and detailed requirements for KDM5b nucleosomal demethylation, advancing our understanding of how this enzyme is regulated <em>in vivo</em>. The novel aspects of KDM5b regulation presented within this thesis provide a framework from which future studies can be designed. This work contributes to our overall understanding of epigenetic regulation and will potentially aid in the development of novel anti-cancer therapeutic strategies.</p> / Doctor of Philosophy (PhD)

<p>The generation of hematopoietic progenitors from human pluripotent cell sources for use in personalized medicine is an attainable goal for the ease of clinical intervention using these cells. Furthermore, generated platelets and mature red blood cells are enucleated which allows for the use of induced pluripotent stem cells as a starting source or other sources of genetic manipulation. Generating these cells has proven difficult as the cells appear to be stuck in a primitive state of differentiation and do not mature into an adult phenotype. This thesis shows that inhibition of the hedgehog signaling pathway early in the differentiation of pluripotent stem cells induces a maturation towards definitive hematopoiesis. Generated erythroid cells were shown to express beta globin at the transcript as well as protein level. This maturation effect was confirmed to occur through central hedgehog repressor, Gli3R, through genetic manipulation. Further interrogation of this mechanism showed that globin regulation was not mediated by chromatin methylation by the polycomb repressive complex. Finally, Gli3R was also shown to not act as a transcription factor influencing globin expression directly and is therefore engaging separate regulatory mechanisms. This data provides great strides towards the generation of clinically relevant hematopoietic populations from pluripotent sources, however Gli3R’s direct mechanism of action remains to be determined.</p> / Master of Science (MSc)

Epigenetics was a term first coined to understand how cells with the same genetic make up can differentiate into various cell types. Elegant research over the past 30 years has shown that these mechanisms include heritable marks such as DNA methylation and histone modifications along with stable expression of non- coding RNAs. Within the realm of epigenetics is a phenomenon known as genomic imprinting. Imprints are marks that distinguish the maternal from the paternal chromosomes in the form of methylation. Methylation marks can influence transcript expression, resulting in only one allele being expressed. One imprinted domain is the Kcnq1 domain located on chromosome 11p15.5 in humans and chromosome 7 in the mouse. This domain is thought to be under the control of a paternally expressed long noncoding RNA (ncRNA) Kcnq1ot1. The Kcnq1ot1 ncRNA is expressed on the paternal chromosome due to a differentially methylation region located within its promoter. The promoter is methylated on the maternal allele thus inhibiting ncRNA expression, whereas the promoter is unmethylated on the paternal allele. In the placenta, a most of the genes located within a one mega-basepair region are exclusively expressed from the maternal chromosome, whereas the transcripts on the paternal chromosome are silenced by the ncRNA. The placenta seems to follow the classic idea of an imprinted domain. However, in the embryo and more specifically, in the embryonic heart, this is not the case. In the embryonic heart, only a 400kb region is restricted to maternal expression. In addition, one the genes, Kcnq1, starts out expressed exclusively from the maternal allele in early development but switches to biallelic expression during mid-gestation. The purpose of my research is to determine the underlying complexities that are involved in the regulation of transcripts within the Kcnq1 domain. This involves the Kcnq1 gene itself, which has been shown to transition from mono- to biallelic expression during mid-gestation and the Kcnq1ot1 ncRNA per se. I hypothesize that regulation by the Kcnq1ot1 ncRNA is not occurring in a uniform manner in the embryo; rather, the amount of regulation by the ncRNA is dependent on the developmental stage and specific tissue. In addition, this regulation involves complex interactions between enhancers, insulators and other regulatory elements to control the amount of silencing by the Kcnq1ot1 ncRNA. First, through a series of experiments looking at the Kcnq1 promoter, the mechanism of Kcnq1 paternal expression was determined. It was confirmed that Kcnq1 becomes biallelic during mid-gestation in the heart. Bisulfite mutagenesis and methylation sensitive chromatin immunoprecipitation were used to test the hypothesis that the Kcnq1 promoter was methylated in early development and then lost its methylation mark. However, a lack of methylation disproved this mechanism of paternal Kcnq1 activation. Rather, chromosome conformation capture (3C) determined that the Kcnq1 promoter interacts in a tissue-specific manner with regions within the domain that have enhancer activity. The role of the ncRNA within our system was also investigated. Interestingly, when Kcnq1ot1 allelic expression was profiled throughout development in heart, it transitioned to biallelic expression during heart development but remained monoallelic in the liver and brain. Several possibilities could account for this phenomenon, including loss of promoter methylation and/or an alternative transcript start site. Both of these options were explored using bisulfite mutagenesis and 5' RACE. However, the Kcnq1ot1 promoter region retained its methylation mark even after the maternal transcript was turned on, disproving this idea. Rather, a maternal specific transcript was found in the heart to start downstream of the CpG islands. Lastly, to gain a better understand of the Kcnq1ot1 ncRNA, experiments were carried out on a mutant mouse in which a truncated form of the ncRNA was transmitted paternally; this is dubbed the "Kterm" mouse. Unexpectedly, Kcnq1 still followed the same mono- to biallelic transition as seen in the wild-type, whereas the head and body counterparts from the same stage embryos were biallelic for Kcnq1. Also, the immediate upstream genes, Cdk1nc and Slc22a18, lost their mono-allelic expression in neonatal heart, liver and brain when the Kterm mutation was transmitted. This suggested that Kcnq1ot1 did not function as a silencer for Kcnq1 paternal expression in the heart, but rather had an alternative and previously unknown function. From qRT-PCR, 3C and ChIP assays, it was determined that the Kcnq1ot1 ncRNA plays a role in regulating Kcnq1 gene expression in the heart by limiting its interaction to specific cis-acting enhancers. When the ncRNA was absent, the Kcnq1 promoter interacted with non-native sites along the domain, possibly causing the increase in transcript expression. This phenomenon was specific to the heart and was not seen in other tissues. These findings showed that Kcnq1 paternal expression is the result of strong developmental and tissue specific enhancers. Chromatin interactions in cis put a strong enhancer in contact with the Kcnq1 promoter to increase its expression in later development. In addition, a truncation mutation model identified a key role for the Kcnq1ot1 ncRNA in regulating Kcnq1 expression. Instead of regulating the imprinting status of Kcnq1, the ncRNA regulates the amount of Kcnq1 transcript being produced in the heart by regulating chromatin interactions. Finally, these studies identified a maternally expressed Kcnq1ot1 transcript whose role in heart development is still not fully understood. Taken together, these findings support a model where an inhibitory factor(s) silence the paternal Kcnq1 transcript and maternal Kcnq1ot1 transcript and in later development, this factor is released allowing for expression and chromatin interactions to occur. / Molecular Biology and Genetics

Epstein Barr virus (EBV) is a gammaherpesvirus that infects more than 95% of the human population worldwide. EBV latent infection of B cells is associated with a variety of lymphomas and epithelial cancers and accounts for approximately 1% of all human cancers. The EBV genome persists in infected host cells as a chromatinized episome and is subject to chromatin-mediated regulation. Binding of the host insulator protein CTCF to the EBV genome has an established role in maintaining viral latency type, and in other herpesviruses, loss of CTCF binding at specific regions correlates with viral reactivation. CTCF is post-translationally modified by the host enzyme PARP1, which can affect CTCF’s insulator activity, DNA binding capacity, and ability to form chromatin loops. Both PARP1 and CTCF have been implicated in the regulation of EBV latency and lytic reactivation. Here, we show that PARP activity regulates CTCF in type III EBV latency to maintain latency type-specific gene expression. Further, PARP1 supports chromatin looping between the OriP enhancer and other regions throughout the EBV genome. Further, we show that CTCF is not involved in EBV lytic reactivation, although it is known to restrict reactivation in other herpesviruses. Both PARP activity and PARP1 binding function to restrict EBV lytic reactivation in response to physiological lytic induction. Overall, we show that PARP1 has specific functions throughout the EBV genome, and CTCF function is specifically regulated by PARP activity at specific loci. Taken together, we suggest a model in which PARP1 acts as a stress sensor to determine the fate of the virus in the host cell. These data provide a mechanistic understanding of PARP1 function throughout the EBV genome that suggest potential therapeutic application of PARP inhibitors in EBV-associated treatment strategies. We propose two distinct strategies specific to EBV latency type that could target EBV-infected cancer cells beyond the current chemotherapeutic standard-of-care. / Biomedical Sciences

Quebec platelet disorder (QPD) is a bleeding disorder characterized by a gain of function defect in fibrinolysis. The hallmark feature of QPD is the marked overexpression of urokinase plasminogen activator (uPA) in megakaryocytes (MK) and platelets. The genetic cause of QPD is a tandem duplication of a ~78 kb region that encompasses the uPA gene, PLAU. As the mechanism of PLAU overexpression is unknown, gene regulatory mechanisms specifically epigenetics were evaluated at the PLAU locus in QPD MK and granulocytes, a QPD unaffected lineage. The aims of the thesis were to assess if QPD is associated with 1) genome wide methylation changes of promoter CpG islands, particularly at PLAU and 2) genome wide changes of active histone modifications H3K27Ac, H3K36me3 and H3K4me2, particularly at the region of PLAU duplication. Methylation and active histone enrichment analysis revealed that in QPD and control subjects, PLAU promoter CpG island was characterized by unaltered hypo-methylation and changes in active histone peak enrichments that were within the realm of having one extra copy of PLAU in both MK and granulocytes. The findings imply that the PLAU CNV mutation does not induce altered promoter methylation status and/or significantly alter active histone markers as the reason for the marked PLAU overexpression in QPD MK. Instead, the rearrangement of an active enhancer element, particularly an H3K27Ac enhancer expressed in MK but not granulocytes, that is upstream of the second copy of PLAU might underlie the marked PLAU expression by differentiated QPD MK. The thesis provides novel insights into the epigenetic regulation of PLAU that will be crucial to identifying the mechanism underlying the aberrant PLAU expression in QPD. / Thesis / Master of Science (MSc)

Plants are sessile organisms and have developed varied mechanisms to tolerate stress. One such mechanism is DNA methylation, which plays a vital role within and across generational stress adaptation. To understand the role of DNA methylation in transgenerational stress adaptation, we exposed Arabidopsis thaliana for four generations of sub-lethal doses of glyphosate, trifloxysulfuron, clipping, and shading, which we further classified into the broader categories of stress ecological (shading and clipping) and herbicides (glyphosate and trifloxysulfuron). We analyzed phenotypic and whole-genome bisulfite sequencing data and found that the Arabidopsis phenotype adapts more rapidly to herbicide stress as compared to ecological stresses. DNA methylation changes for glyphosate were minimal after four generations of stress whereas the other three stresses showed dynamic change in the DNA methylation patterns. To understand within generation stress response, Sorghum bicolor was exposed to the same stresses at sub-lethal doses and we analyzed its phenotypic, whole genome bisulfite sequencing, and gene expression responses. Ecological stresses had higher negative impact on S. bicolor plant growth as compared to herbicide stresses. Similarly, we found higher differentially expressed genes for clipping as compared to both herbicides. All four stresses changed the methylome in a unique way; where we found 998 differentially methylated regions (DMR) for trifloxysulfuron, 193 for shading, 141 for clipping and 60 for glyphosate. Out of these DMR's some occurred genic region, which could potentially change gene expression and help plants withstand stress. Overall, DNA methylation can potentially help plants to withstand stress due to their dynamic and specific response to a variety of stresses both transgenerational and within generation. This information to better understand stress adaptation mechanisms in plants and used to develop stress-resilient crops. / Doctor of Philosophy / Environmental and anthropogenic stresses can negatively impact plant growth and development. Plants can have stress memory through epigenetic changes which helps them withstand stress in future generations. Epigenetics is the field of science where changes on the DNA and not sequence, that can be an addition or deletion of a methyl group, modification of histones, or production of small RNAs. We wanted to understand short and long-term effects of common anthropogenic and ecological stresses on how DNA methylation changes can help plants to withstand stress. We used the model plant Arabidopsis thaliana and the non-model crop/weed Sorghum bicolor. We exposed plants to sub-lethal doses of two herbicides, clipping, and shade stress, at levels high enough to cause significant visible injury but still allowed them to recover and reproduce for a single generation for S. bicolor and four generations for A. thaliana. We found that A. thaliana rapidly more adapts to herbicide stress as compared to ecological stresses. DNA methylation changes for glyphosate were minimal after four generations of stress whereas the other three stresses showed dynamic changes in the DNA methylation patterns. Each stressed impacted S. bicolor phenotype, DNA methylation, and gene expression in unique ways. We found ecological stresses greatly affected the phenotype of the S. bicolor plants as compared to herbicide stresses. Overall, our results showed that stress can cause DNA methylation changes and in transgenerational stress DNA methylation can potentially play a role in stress adaptation. This information could be useful for scientists to further understand stress resilience in plants.