Cell Fate Specification and the Regulation of RNA-dependent DNA Methylation in the Arabidopsis Root Meristem

The Arabidopsis root apical meristem (RAM) is a complex tissue capable of generating all the cell types that ultimately make up the root. The work presented in this thesis takes advantage of the versatility of high-throughput sequencing to address two independent questions about the root meristem. Although a lot of information is known regarding the cell fate decisions that occur at the RAM, cortex specification and differentiation remain poorly understood. In the first part of this thesis, I used an ethylmethanesulfonate (EMS) mutagenized marker line to perform a forward genetics screen. The goal of this screen was to identify novel genes involved in the specification and differentiation of the cortex tissue. Mapping analysis from the results obtained in this screen revealed a new allele of BRASSINOSTEROID4 with abnormal marker expression in the cortex tissue. Although this allele proved to be non-cortex specific, this project highlights new technology that allows mapping of EMS-generated mutations without the need to map-cross or back-cross. In the second part of this thesis, using fluorescence activated cell sorting (FACS) coupled with high throughput sequencing, my collaborators and I generated single-base resolution whole genome DNA methylomes, mRNA transcriptomes, and smallRNA transcriptomes for six different populations of cell types in the Arabidopsis root meristem. We were able to discover that the columella is hypermethylated in the CHH context within transposable elements. This hypermethylation is accompanied by upregulation of the RNA-dependent DNA methylation pathway (RdDM), including higher levels of 24-nt silencing RNAs (siRNAs). In summary, our studies demonstrate the versatility of high-throughput sequencing as a method for identifying single mutations or to perform complex comparative genomic analyses.

Adiposity, Sex Hormones, and Repetitive Element DNA Methylation in Healthy Postmenopausal Women

INTRODUCTION: Low levels of methylation within repetitive DNA elements, such as long interspersed nuclear element-1 (LINE-1) and Alu repeats, are believed to epigenetically predispose an individual to cancer and other diseases. The extent to which lifestyle factors affect the degree of DNA methylation within these genomic regions has yet to be fully understood. Adiposity and sex hormones are established risk factors for certain types of cancer and other illnesses, particularly amongst postmenopausal women. The aim of the current investigation is to assess the impact of adiposity and sex hormones on LINE-1 and Alu methylation in healthy postmenopausal women. METHODS: A cross-sectional study was conducted using baseline data from an ancillary study of the Alberta Physical Activity and Breast Cancer Prevention (ALPHA) Trial. Current adiposity was measured using a dual-energy x-ray absorptiometry (DXA) scan, computed tomography (CT) scan, and balance beam scale. Historical weights were self-reported in a questionnaire. Current endogenous sex hormone concentrations were measured in fasting blood serum. Estimated lifetime number of menstrual cycles was used as a proxy for cumulative exposure to ovarian sex hormones. Repetitive element methylation was quantified in white blood cells using a pyrosequencing assay. Linear regression was used to model the relations of interest while adjusting for important confounders. RESULTS: Adiposity and serum estrogen concentrations were positively related to LINE-1 methylation but were not associated with Alu methylation. Cumulative ovarian sex hormone exposure had a “U-shaped” relation with LINE-1 regardless of folate intake and a negative relation with Alu methylation amongst low folate consumers. Androgens were not associated with repetitive element DNA methylation in this population. CONCLUSION: Adiposity and estrogens appear to play a role in maintaining high levels of repetitive element DNA methylation in healthy postmenopausal women. LINE-1 methylation may be a mechanism whereby estrogen exposure protects against cardiovascular and neurodegenerative illnesses. These results add to the growing body of literature showing how the epigenome is shaped by our lifestyle choices. Future prospective studies assessing the relation between levels of repetitive element DNA methylation in healthy individuals and subsequent disease risk are needed to better understand the clinical significance of these results.

Epigenetics and the overhealing wound: the role of DNA methylation in fibrosis

Fibrosis is a progressive and potentially fatal process that can occur in numerous organ systems. Characterised by the excessive deposition of extracellular matrix proteins such as collagens and fibronectin, fibrosis affects normal tissue architecture and impedes organ function. Although a considerable amount of research has focused on the mechanisms underlying disease pathogenesis, current therapeutic options do not directly target the pro-fibrotic process. As a result, there is a clear unmet clinical need to develop new agents. Novel findings implicate a role for epigenetic modifications contributing to the progression of fibrosis by alteration of gene expression profiles. This review will focus on DNA methylation; its association with fibroblast differentiation and activation and the consequent buildup of fibrotic scar tissue. The potential use of therapies that modulate this epigenetic pathway for the treatment of fibrosis in several organ systems is also discussed.

Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.

CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.

De novo methylation of tumor suppressor gene p16/INK4a is a frequent finding in multiple myeloma patients at diagnosis.

The p16 gene competes with cyclin D for binding to CDK4/CDK6 and therefore inhibits CDK4/6 complex kinase activity, resulting in dephosphorylation of pRb and related G1 growth arrest. Inactivation of this gene has been involved in a variety of tumors by different mechanisms: homozygous/hemyzygous deletions, point mutations and methylation of a 5' CpG island into exon E1alpha of the p16 gene. Homozygous deletions have been rarely found in multiple myeloma (MM) and no point mutations have been reported. Two recent studies have reported a high prevalence of methylation in the exon E1alpha of the p16 gene, but included only a small number of cases. We have analyzed the methylation pattern of exon E1alpha of the p16 gene in 101 untreated MM and five primary plasma cell leukemias (PCL). A PCR assay, relying on the inability of some restriction enzymes to digest methylated sequences, was used to analyze the methylation status. Southern blot analysis was used to confirm these results. Forty-one of 101 MM patients (40.5%) as well as four of the five (80%) primary PCL patients had shown methylation of the exon E1alpha. Our study confirms that hypermethylation of the p16 gene is a frequent event in MM. Leukemia (2000) 14, 183-187.

HNF1B variants associate with promoter methylation and regulate gene networks activated in prostate and ovarian cancer

Two independent regions within HNF1B are consistently identified in prostate and ovarian cancer genome-wide association studies (GWAS); their functional roles are unclear. We link prostate cancer (PC) risk SNPs rs11649743 and rs3760511 with elevated HNF1B gene expression and allele-specific epigenetic silencing, and outline a mechanism by which common risk variants could effect functional changes that increase disease risk: functional assays suggest that HNF1B is a pro-differentiation factor that suppresses epithelial-to-mesenchymal transition (EMT) in unmethylated, healthy tissues. This tumor-suppressor activity is lost when HNF1B is silenced by promoter methylation in the progression to PC. Epigenetic inactivation of HNF1B in ovarian cancer also associates with known risk SNPs, with a similar impact on EMT. This represents one of the first comprehensive studies into the pleiotropic role of a GWAS-associated transcription factor across distinct cancer types, and is the first to describe a conserved role for a multi-cancer genetic risk factor.

Development and application of sensitive genome-wide platforms to study the genetic and epigenetic (DNA methylation) makeup of gametes and early bovine embryos

Pour ce projet, nous avons développé une plateforme pour l’analyse pangénomique de la méthylation de l’ADN chez le bovin qui est compatible avec des échantillons de petites tailles. Cet outil est utilisé pour étudier les caractéristiques génétiques et épigénétiques (méthylation de l’ADN) des gamètes soumis aux procédures de procréation médicalement assisitée et des embryons précoces. Dans un premier temps, une plateforme d’analyse de biopuces spécifiques pour l’étude de la méthylation de l’ADN chez l’espèce bovine a été développée. Cette plateforme a ensuite été optimisée pour produire des analyses pangénomiques de méthylation de l’ADN fiables et reproductibles à partir d’échantillons de très petites tailles telle que les embryons précoces (≥ 10 ng d’ADN a été utilisé, ce qui correspond à 10 blastocystes en expansion). En outre, cet outil a permis d’évaluer de façon simultanée la méthylation de l’ADN et le transcriptome dans le même échantillon, fournissant ainsi une image complète des profils génétiques et épigénétiques (méthylation de l’ADN). Comme preuve de concept, les profils comparatifs de méthylation de l’ADN spermatique et de blastocystes bovins ont été analysés au niveau de l’ensemble du génome. Dans un deuxième temps, grâce à cette plateforme, les profils globaux de méthylation de l’ADN de taureaux jumeaux monozygotes (MZ) ont été analysés. Malgré qu’ils sont génétiquement identiques, les taureaux jumeaux MZ ont des descendants avec des performances différentes. Par conséquent, l’hypothèse que le profil de méthylation de l’ADN spermatique de taureaux jumeaux MZ est différent a été émise. Dans notre étude, des différences significatives entre les jumeaux MZ au niveau des caractéristiques de la semence ainsi que de la méthylation de l’ADN ont été trouvées, chacune pouvant contribuer à l’obtention de performances divergentes incongrues des filles engendrées par ces jumeaux MZ. Dans la troisième partie de ce projet, la même plateforme a été utilisée pour découvrir les impacts d’une supplémentation à forte concentration en donneur de méthyle universel sur les embryons précoces bovins. La supplémentation avec de grandes quantités d’acide folique (AF) a été largement utilisée et recommandée chez les femmes enceintes pour sa capacité bien établie à prévenir les malformations du tube neural chez les enfants. Cependant, plus récemment, plusieurs études ont rapporté des effets indésirables de l’AF utilisé à des concentrations élevées, non seulement sur le développement de l’embryon, mais aussi chez les adultes. Au niveau cellulaire, l’AF entre dans le métabolisme monocarboné, la seule voie de production de S-adénosyl méthionine (SAM), un donneur universel de groupements méthyles pour une grande variété de biomolécules, y compris l’ADN. Par conséquent, pour résoudre cette controverse, une forte dose de SAM a été utilisée pour traiter des embryons produits in vitro chez le bovin. Ceci a non seulement permis d’influencer le phénotype des embryons précoces, mais aussi d’avoir un impact sur le transcriptome et le méthylome de l’ADN. En somme, le projet en cours a permis le développement d’une plateforme d’analyse de la méthylation de l’ADN à l’échelle du génome entier chez le bovin à coût raisonnable et facile à utiliser qui est compatible avec les embryons précoces. De plus, puisque c’est l’une des premières études de ce genre en biologie de la reproduction bovine, ce projet avait trois objectifs qui a donné plusieurs nouveaux résultats, incluant les profils comparatifs de méthylation de l’ADN au niveau : i) blastocystes versus spermatozoïdes ; ii) semence de taureaux jumeaux MZ et iii) embryons précoces traités à de fortes doses de SAM versus des embryons précoces non traités.

Reprogramming of DNA Methylation in Pollen Guides Epigenetic Inheritance via Small RNA

Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA.

The metabolic and epigenetic effects of the isocitrate dehydrogenase-1 mutation in glioma

Cancer cells have been noted to have an altered metabolic phenotype for over ninety years. In the presence of oxygen, differentiated cells predominately utilise the tricarboxylic acid (TCA) cycle and oxidative phosphorylation to efficiently produce energy and the metabolites necessary for protein and lipid synthesis. However, in hypoxia, this process is altered and cells switch to a higher rate of glycolysis and lactate production to maintain their energy and metabolic needs. In cancer cells, glycolysis is maintained at a high rate, even in the presence of oxygen; a term described as “aerobic glycolysis”. Tumour cells are rapidly dividing and have a much greater need for anabolism compared to normal differentiated cells. Rapid glucose metabolism enables faster ATP production as well as a greater redistribution of carbons to nucleotide, protein, and fatty acid synthesis, thus maximising cell growth. Recently, other metabolic changes, driven by mutations in genes related to the TCA cycle, indicate an alternative role for metabolism in cancer, the “oncometabolite”. This is where a particular metabolite builds up within the cell and contributes to the tumorigenic process. One of these genes is isocitrate dehydrogenase (IDH) IDH is an enzyme that forms part of the tricarboxylic acid (TCA) cycle and converts isocitrate to α-ketoglutarate (α-KG). It exists in three isoforms; IDH1, IDH2 and IDH3 with the former present in the cytoplasm and the latter two in the mitochondria. Point mutations have been identified in the IDH1 and IDH2 genes in glioma which result in a gain of function by converting α-KG to 2-hydroxyglutarate (2HG), an oncometabolite. 2HG acts as a competitive inhibitor of the α-KG dependent dioxygenases, a superfamily of enzymes that are involved in numerous cellular processes such as DNA and histone demethylation. It was hypothesised that the IDH1 mutation would result in other metabolic changes in the cell other than 2HG production, and could potentially identify pathways which could be targeted for therapeutic treatment. In addition, 2HG can act as a potential competitive inhibitor of α-KG dependent dioxygenases, so it was hypothesised that there would be an effect on histone methylation. This may alter gene expression and provide a mechanism for tumourogenesis and potentially identify further therapeutic targets. Metabolic analysis of clinical tumour samples identified changes associated with the IDH1 mutation, which included a reduction in α-KG and an increase in GABA, in addition to the increase in 2HG. This was replicated in several cell models, where 13C labelled metabolomics was also used to identify a possible increase in metabolic flux from glutamate to GABA, as well as from α-KG to 2HG. This may provide a mechanism whereby the cell can bypass the IDH1 mutation as GABA can be metabolised to succinate in the mitochondria by GABA transaminase via the GABA shunt. JMJ histone demethylases are a subset of the α-KG dependent dioxygenases, and are involved in removing methyl groups from histone tails. Changes in histone methylation are associated with changes in gene expression depending on the site and extent of chemical modification. To identify whether the increase in 2HG and fall in α-KG was associated with inhibition of histone demethylases a histone methylation screen was used. The IDH1 mutation was associated with an increase in methylation of H3K4, which is associated with gene activation. ChiP and RNA sequencing identified an increase in H3K4me3 at the transcription start site of the GABRB3 subunit, resulting in an increase in gene expression. The GABRB3 subunit forms part of the GABA-A receptor, a chloride channel, which on activation can reduce cell proliferation. The IDH1 mutation was associated with an increase in GABA and GABRB3 subunit of the GABA-A receptor. This raises the possibility of GABA transaminase as a potential therapeutic target. Inhibition of this enzyme could reduce GABA metabolism, potentially reducing any beneficial effect of the GABA shunt in IDH1 mutant tumours, and increasing activation of the GABA-A receptor by increasing the concentration of GABA in the brain. This in turn may reduce cell proliferation, and could be achieved by using Vigabatrin, a GABA transaminase inhibitor licensed for use in epilepsy.

Analiza proteomiczna kompleksów tworzonych przez białko hDPY30 w komórkach ludzkich

Wydział Biologii

RARβ gene methylation is a candidate for primary glioblastoma treatment planning.

Background: We screened RARβ methylation in primary glioblastoma multiforme (GBM) and the results were evaluated based on the clinical data and treatment type. Objective: The objective of this study was to find new areas for the usage of MS-HRM applications in the determination of methylation levels in primary GBM samples and it shows the association of RARβ methylation with the clinical outcome. Methods: In our study, tumor samples were collected during surgical resection by the Department of Neurosurgery. The clinical and radiologic data was carefully reviewed, compared, and evaluated with the histological results. The methylation status of RARβ was determined by using MS-HRM. Results: RARβ gene methylation was detected in 24 out of 40 cases (60%), with different quantitative methylation levels. The mean survival time was 19 months form ethylated cases and 15 months for the non-methylated cases. The survival time of the patients who received treatment was 25 months and the survival time of the patients who received radiotherapy alone or where no treatment protocol applied was 15-20 months. Therefore, a significant difference in survival rates has been observed (P<0.05). This study indicates a potential prognostic value for GBM treatment planning. Conclusion: Our study is the first study to investigate RARβ methylation in primary GBMs. We conclude that the RARβ gene could be a new prognostic and predictive candidate marker to designate the treatment protocol for primary GBMs. Keywords:

Geminivirus Rep Protein Interferes with the Plant DNA Methylation Machinery and Modifies the Host Epigenome

The apparent simplicity of viruses hides the complexity of their interactions with their hosts. Viruses are masters at circumventing host defenses and manipulating the cellular environment for their own benefit. The replication of the largest known family of single-stranded DNA viruses, Geminiviridae, is impaired by DNA methylation and Arabidopsis mutants affected in cytosine methylation are hypersusceptible to geminivirus infection. This implies that plants might use methylation as a defense against geminiviruses and that the viral genome is a target for plant DNA methyltransferases. We have found a novel counter-defense strategy used by geminiviruses, that reduces the expression of the plant maintenance DNA methyltransferases, MET1 and CMT3, in both, locally and systemically infected tissues. Furthermore, we demonstrated that the virus-mediated repression of these two maintenance DNA methyltransferases is widely spread among different geminivirus species. Additionally, we identified Rep as the geminiviral protein responsible for the repression of MET1 and CMT3, and another viral protein, C4, as an ancillary player in MET1 downregulation. The presence of Rep, suppresses TGS of an Arabidopsis transgene and of host loci whose expression is strongly controlled by CG methylation. Bisulfite sequencing analyses showed that the expression of Rep caused a substantial reduction in the levels of DNA methylation at CG sites. Our findings suggest that Rep, the only viral protein essential for geminiviral replication, displays TGS suppressor activity through a mechanism distinct from the one thus far described for geminiviruses.

Reprogramming of DNA Methylation in Pollen Guides Epigenetic Inheritance via Small RNA

Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA.