Academic literature on the topic 'R-methylation'

Background Low socioeconomic status neighborhood exposure to stress and violence may be sources of negative stimuli that poses significant health risks for children, adolescents and throughout the life course of an individual. The study aims to investigate if aberrant epigenetic DNA methylation changes may be a potential mechanism for regulating neighborhood exposures and health outcomes. Methods Exposure to environmental stressors identified in 98 young African American (AA) adults aged 18–25 years old from the Washington D.C., area were used in the study. We correlated the association between stress markers; cortisol, CRP, IgG, IGA, IgM, and self-reported exposure to violence and stress, with quantitative DNA methylation changes in a panel of gene-specific loci using saliva DNA. Results In all participants studied, the exposure to violence was significant and negatively correlated with DNA methylation of MST1R loci (p = 0.032; r = -0.971) and nominally significant with NR3C1 loci (p = 0.053; r = -0.948). In addition, we observed significant and negative correlation of DNA methylation changes of LINE1 (p = 0.044; r = -0.248); NR3C1 (p = 0.017; r = -0.186); MSTR1 (p = 0.022; r = -0.192); and DRD2 (p = 0.056; r = -0.184; albeit nominal significant correlation) with IgA expression. On the other hand, we observed a significant and position correlation of DNA methylation changes in DRD2 (p = 0.037; r = 0.184) with IgG expression. When participants were stratified by sex, we observed in AA young male adults, significant DNA methylation changes of MST1R (p< 0.05) and association with exposure to violence and IgG level. We also observed significant DNA methylation levels of DRD2 (p< 0.05) and association with IgA, IgG, and cortisol level. Furthermore, we observed significant DNA methylation changes of NR3C1 (p< 0.05) with stress, IgA, and IgG in the male participants only. On the other hand, we only observed significant and a positive association of IgG with DNA methylation levels of ESR1 (p = 0.041) in the young AA female participants. Conclusion Our preliminary observation of significant DNA methylation changes in neuronal and immune genes in saliva samples supports our recently published genome-wide DNA methylations changes in blood samples from young AA male adults indicating that saliva offers a non-invasive means for DNA methylation prediction of exposure to environmental stressors in a gender-specific manner.

Abstract RNA 2′-O-methylation is widely distributed and plays important roles in various cellular processes. Mycoplasma genitalium RNase R (MgR), a prokaryotic member of the RNase II/RNB family, is a 3′-5′ exoribonuclease and is particularly sensitive to RNA 2′-O-methylation. However, how RNase R interacts with various RNA species and exhibits remarkable sensitivity to substrate 2′-O-methyl modifications remains elusive. Here we report high-resolution crystal structures of MgR in apo form and in complex with various RNA substrates. The structural data together with extensive biochemical analysis quantitively illustrate MgR’s ribonuclease activity and significant sensitivity to RNA 2′-O-methylation. Comparison to its related homologs reveals an exquisite mechanism for the recognition and degradation of RNA substrates. Through structural and mutagenesis studies, we identified proline 277 to be responsible for the significant sensitivity of MgR to RNA 2′-O-methylation within the RNase II/RNB family. We also generated several MgR variants with modulated activities. Our work provides a mechanistic understanding of MgR activity that can be harnessed as a powerful RNA analytical tool that will open up a new venue for RNA 2′-O-methylations research in biological and clinical samples.

Abstract In paramutation two alleles of a gene interact so that one of the alleles is epigenetically silenced. The silenced state is then genetically transmissible for many generations. The large (220 kbp) multigenic complex R-r is paramutable: its level of expression is changed during paramutation. R-r was found to exhibit increases in its level of cytosine methylation (C-methylation) following paramutation. These C-methylation changes are localized to the 5′ portions of the two genes in the complex that are most sensitive to paramutation. These methylation changes flank a small region called σ that is thought to have been derived from a transposon named doppia. A mutant derivative of R-r that has a deletion of the σ region fails to become methylated under conditions in which R-r is heavily methylated. This suggests that the presence of σ sequences at the locus is required for the methylation changes that are observed following paramutation.

DNA methylation is a part of the regulatory mechanisms of gene expression, including chromatin remodeling and the activity of microRNAs, which are involved in the regulation of T-cell differentiation and function. However, the role of cfDNA methylation in T-cell differentiation is entirely unknown. In patients with endometrial polyps (EPs), we have found an imbalance of T-cell differentiation and an aberrant cfDNA methylation profile, respectively. In this study, we investigated the relationship between cfDNA methylation profiles and T-cell differentiation in 14 people with EPs and 27 healthy controls. We found that several differentially methylated genes (DMGs) were associated with T-cell differentiation in people with EPs (ITGA2-Naïve CD4, r = −0.560, p = 0.037; CST9-EMRA CD4, r = −0.626, p = 0.017; and ZIM2-CM CD8, r = 0.576, p = 0.031), but not in healthy controls (all p > 0.05). When we combined the patients’ characteristics, we found a significant association between ITGA2 methylation and polyp diameter (r = 0.562, p = 0.036), but this effect was lost when adjusting the level of Naïve CD4 T-cells (r = 0.038, p = 0.903). Moreover, the circulating sex hormone levels were associated with T-cell differentiation (estradiol-Naïve CD4, r = −0.589, p = 0.027), and the cfDNA methylation profile (testosterone-ZIM2, r = −0.656, p = 0.011). In conclusion, this study has established a link between cfDNA methylation profiles and T-cell differentiation among people with EPs, which may contribute to the etiology of EPs. Further functional studies are warranted.

A key benefit of long-read nanopore sequencing technology is the ability to detect modified DNA bases, such as 5-methylcytosine. The lack of R/Bioconductor tools for the effective visualization of nanopore methylation profiles between samples from different experimental groups led us to develop the NanoMethViz R package. Our software can handle methylation output generated from a range of different methylation callers and manages large datasets using a compressed data format. To fully explore the methylation patterns in a dataset, NanoMethViz allows plotting of data at various resolutions. At the sample-level, we use dimensionality reduction to look at the relationships between methylation profiles in an unsupervised way. We visualize methylation profiles of classes of features such as genes or CpG islands by scaling them to relative positions and aggregating their profiles. At the finest resolution, we visualize methylation patterns across individual reads along the genome using the spaghetti plot and heatmaps, allowing users to explore particular genes or genomic regions of interest. In summary, our software makes the handling of methylation signal more convenient, expands upon the visualization options for nanopore data and works seamlessly with existing methylation analysis tools available in the Bioconductor project. Our software is available at https://bioconductor.org/packages/NanoMethViz.

Abstract Objectives Gestational diabetes mellitus (GDM) is associated with alterations in DNA methylation in the placenta and offspring tissues. Nutrients participating in the methionine cycle (e.g., choline, betaine, folate, vitamin B12, methionine) influence the supply of methyl groups. The objective of this research was to determine whether maternal intake and status of these nutrients during pregnancy may interact with the GDM status to shape the offspring epigenome. Methods We conducted 3-day dietary recalls and collected blood samples from pregnant women with and without GDM (n = 22/group) to quantify methylation-related nutrient intakes and status. At delivery, we collected cord blood samples and measured global DNA methylation. Results GDM was associated with a 25% increase (P = 0.041) in global DNA methylation in the cord blood. Maternal choline intake (r = −0.602, P = 0.006) as well as cord blood methionine (r = −0.553, P = 0.014) and betaine (r = −0.566, P = 0.011) levels were negatively correlated with cord blood DNA methylation only in non-GDM women, while intakes and maternal blood levels of other methylation-related nutrients were not related to cord blood DNA methylation. Conclusions GDM and methyl nutrient intake/status interact to modify offspring DNA methylation in humans. Funding Sources Egg Nutrition Center.

Interspersed repetitive sequences (IRSs) are a major contributor to genome size and may contribute to cellular functions. IRSs are subdivided according to size and functionally related structures into short interspersed elements, long interspersed elements (LINEs), DNA transposons, and LTR-retrotransposons. Many IRSs may produce RNA and regulate genes by a variety of mechanisms. The majority of DNA methylation occurs in IRSs and is believed to suppress IRS activities. Global hypomethylation, or the loss of genome-wide methylation, is a common epigenetic event not only in senescent cells but also in cancer cells. Loss of LINE-1 methylation has been characterized in many cancers. Here, we evaluated the methylation levels of peripheral blood mononuclear cells of LINE-1, Alu, and human endogenous retrovirus K (HERV-K) in 177 samples obtained from volunteers between 20 and 88 yr of age. Age was negatively associated with methylation levels of Alu (r = −0.452, P < 10−3) and HERV-K (r = −0.326, P < 10−3) but not LINE-1 (r = 0.145, P = 0.055). Loss of methylation of Alu occurred during ages 34–68 yr, and loss of methylation of HERV-K occurred during ages 40–63 yr and again during ages 64–83 yr. Interestingly, methylation of Alu and LINE-1 are directly associated, particularly at ages 49 yr and older (r = 0.49, P < 10−3). Therefore, only some types of IRSs lose methylation at certain ages. Moreover, Alu and HERV-K become hypomethylated differently. Finally, there may be several mechanisms of global methylation. However, not all of these mechanisms are age-dependent. This finding may lead to a better understanding of not only the biological causes and consequences of genome-wide hypomethylation but also the role of IRSs in the aging process.

Arsenic has been associated with significant effects on human health. Exposure to inorganic arsenic has been associated with the changes in gene expression. Promoter of CDKN1A antisense DNA damage activated RNA (PANDAR) expression is induced by p53 protein and DNA damage response. Here, we investigated whether the ability of arsenic metabolism in individuals affected the expression of PANDAR, DNA damage, and DNA methylation. Levels of gene expression and DNA damage were examined by the quantitative polymerase chain reaction and DNA methylation was measured by the methylation-sensitive high-resolution melting curve. In our study, we demonstrated that arsenic exposure increased PANDAR expression and DNA damage among arsenic smelting plant laborers. The PANDAR expression and DNA damage were positively linked to monomethylarsonic acid % ( R = 0.25, p < 0.05 and R = 0.32, p < 0.01) and negatively linked to dimethylarsinic acid % ( R = −0.21, p < 0.05 and R = −0.31, p < 0.01). Subjects with low primary methylation index had increased levels of DNA damage (51.62 ± 2.96 vs. 60.93 ± 3.10, p < 0.05) and methylation (17.14 (15.88–18.51) vs. 15.83 (14.82–18.00), p < 0.05). Subjects with low secondary methylation index had increased levels of PANDAR expression (4.88 ± 0.29 vs. 4.07 ± 0.23, p < 0.01) and DNA damage (17.38 (15.88–19.29) vs. 15.83 (14.82–17.26), p < 0.01). DNA methylation of PANDAR gene was linked to the regulation of its expression in peripheral blood lymphocytes among laborers ( Y = −2.08 × X + 5.64, p < 0.05). These findings suggested arsenic metabolism ability and exposure affected the expression of PANDAR, DNA damage, and DNA methylation.

Objective To investigate the association between DNA methylation and the stable warfarin dose through genome-wide DNA methylation analysis and pyrosequencing assay. Method This study included 161 patients and genome-wide DNA methylation analysis was used to screen potential warfarin dose-associated CpGs through Illumina Infinium HumanMethylation 450 K BeadChip; then, the pyrosequencing assay was used to further validate the association between the stable warfarin dose and alterations in the methylation of the screened CpGs. GenomeStudio Software and R were used to analyze the differentially methylated CpGs. Results The methylation levels of CpGs surrounding the xenobiotic response element (XRE) within the CYP1A1 promoter, differed significantly between the different dose groups (P < 0.05), and these CpGs presented a positive correlation (r> 0, P < 0.05) with an increase in the stable dose of warfarin. At the VKORC1 promoter, two CpGs methylation levels were significantly different between the differential dose groups (P < 0.05), and one CpG (Chr16: 31106793) presented a significant negative correlation (r < 0, P < 0.05) among different dose (low, medium, and high) groups. Conclusion This is a novel report of the methylation levels of six CpGs surrounding the XRE within the CYP1A1 promoter and one differential CpG at the VKORC1 promoter associated with stable warfarin dosage; these methylation levels might be applied as molecular signatures for warfarin.

Restriction Modification (R-M) systems prevent the invasion of foreign genetic material into bacterial cells and are therefore important in maintaining the integrity of the host genome. The spread of antibiotic resistance, which is proposed to occur via the transfer of foreign genes to the bacterial genome, makes the subject of R-M systems extremely relevant. R-M systems are currently classified into four types (I to IV) on the basis of differences in composition, target recognition, cofactors and the manner in which they cleave DNA. Kennaway et al (2012) proposed that there is an evolutionary link between Types I and II. Comparing the structures of examples from two of the subfamilies of Type II systems (IIB and IIG) to those of Type I structures, similarities can be observed. Due to the fact that Type II R-M systems cut DNA at fixed positions, they can be used to obtain genetic material selectively. They have therefore proven to be invaluable in molecular biology. One aspect of this project aims to create a novel R-M system, a pseudo-Type II system, by removing the molecular motors from the restriction subunit of a Type I system and fusing the remaining nuclease domain to a known Type I methyltransferase (MTase). This will not only provide evidence to support the theory that evolution has produced a pared down form of the Type I systems in the Type II systems, but it may also become a useful biological tool. This thesis describes the several attempts at doing this and how the subsequent constructs were expressed, purified and assayed to varying degrees of success. An important characteristic of the Type I systems is their ability to methylate DNA, and it is the mechanism via which host DNA is protected from restriction. This is another subject investigated in this project. As with the nuclease activity of the Type I systems, the site at which DNA is methylated is dictated by the HsdS subunit. It is described here how this subunit can be altered to change the sequence of DNA that is recognised by the system. Again, using Type II system subtypes as a reference, various mutations were made to the HsdS subunit of an MTase from Staphylococcus aureus. This is in an effort to bring about a new mode of action, but also to provide further evidence for an evolutionary link between the two system types. The HsdM and HsdS subunits are expressed from two separate genes at the same locus. There is a frameshift between the genes where the start of the hsdS gene occurs a few base pairs upstream from the stop codon of the hsdM gene. This work shows that removing this frameshift creates an MS fusion product, and in vivo studies show that this product has methylase activity and can form an active restriction complex when the HsdR subunit is added. The product can also be over-expressed and purified, and shows in vitro restriction activity on addition of the HsdR subunit protein. The HsdS subunit is composed of two target recognition domains (TRDs), each dictating one part of the bipartite recognition sequence. These TRDs can be altered, bringing about a change in the sequence of DNA recognised by the enzyme. In this thesis, it is shown that the C-terminal TRD can be removed and that the subsequent “Half S” enzyme possesses both methylase and restriction activity in vivo and that its recognition sequence is different from that of the wild-type enzyme. After the successful creation of both “MS fusion” and “Half S” recombinant proteins of the Sau1, Type I system from a CC398 strain of Staphylococcus aureus, a further construct was produced. This possesses both in vivo and in vitro activity. The novel “M Half S Fusion” enzyme not only links the two aspects of this project but also creates a structure similar to some seen in the Type II systems. This shows that the Type I systems can be manipulated to change their mode of action but also supports the idea that Types I and II are evolutionarily linked. By making the alterations in a step-wise fashion identifies that these structural changes can create viable enzymes, and that they could have occurred through the process of evolution.

Various post-translational modifications (PTMs) have been described to regulate RNA-binding protein (RBP) activity, subcellular localization, and interactions with other proteins or RNAs. Proteome-wide experiments recently carried out in our group have shown that RBPs are the most abundant arginine (R)-methylated proteins. Protein Arginine Methyltransferases (PRMTs) are the enzymes responsible for the deposition of methylation on arginine. Recent evidence has indicated that R-hypomethylation could influence RBP phase-separation and consequent formation of Membrane-Less Organelles (MLOs). In my Ph.D. project, we implemented a quantitative proteomic approach to profile global changes of RBP-RNA interactions upon the modulation of R-methylation, both directly by the use of PRMT inhibitor and indirectly through cisplatin (CDDP)-induced PRMT1 re-localization on chromatin. In particular, by coupling the Orthogonal Organic Phase Separation (OOPS) strategy with mass spectrometry (MS) analysis, we profiled RNA-protein interaction in dependence on R-methylation remodeling. Biochemical and immunofluorescence analysis validated the differential association of a set of RBPs with RNA upon PRMT1 inhibition but also that altered modification is linked to MLOs formation. We then applied our strategy in ovarian cancer in the context of CDDP-induced protein R-methylation rewiring to understand if it may affect RBP-RNA interaction. Preliminary analysis of the significantly regulated RBPs from a new OOPS-MS experiment carried out in these conditions revealed a strong modulation of RBP-RNA interaction and suggested new interesting targets. We are currently analyzing the R-methylation state of the most promising RBPs and in parallel the RNAs which interact differentially with the RBPs in the same conditions.

DNA methylation is a postreplicative covalent modification of the DNA which is catalysed by the DNA methyltransferase enzyme. DNA methylation plays an important role in controlling the gene expression profile of mammalian cells. The hypothesis presented in this thesis is that the expression of the DNA methyltransferase gene is upregulated by cellular oncogenic pathways, and that this induction of MeTase activity results in DNA hypermethylation and plays a causal role in cellular transformation. Novel DNA methyltransferase inhibitors may inhibit the excessive activity of DNA methyltransferase in cancer cells and induce the original cellular genetic program. These inhibitors may also be used to turn on alternative gene expression programs. Therefore specific DNA methyltransferase antagonists might provide us with therapeutics directed at a nodal point in the regulation of genetic information.

One of the main questions of modern biology is how our cells interpret our genetic and epigenetic information. DNA methylation is a covalent modification of the genome that is essential for mammalian development and plays an important role in the control of gene expression, genomic imprinting and X-chromosome inactivation (Bird and Wolffe, 1999; Szyf et al., 2000). Furthermore, changes in DNA methylation and DNA methyltransferase 1 (DNMT1) activity have been widely documented in a number of human cancers (Szyf, 1998a; Szyf et al., 2000).<br>In Escherichia coli, timing and frequency of initiation of DNA replication are controlled by the levels of the bacterial methyltransferase and by the methylation status of its origin of replication (Boye and Lobner-Olesen, 1990; Campbell and Kleckner, 1990). In mammalian cells, however, the importance of methyltransferase activity and of DNA methylation in replication is only now starting to emerge (Araujo et al., 1998; Delgado et al., 1998; DePamphilis, 2000; Knox et al., 2000).<br>The work described in this thesis focuses mainly on understanding the functional relationship between changes in DNA methylation and DNMT1 activity on mammalian DNA replication. In higher eukaryotes, DNA replication initiates from multiple specific sites throughout the genome (Zannis-Hadjopoulos and Price, 1999). The first part of the thesis describes the identification and characterization of novel in vivo initiation sites of DNA replication within the human dnmt1 locus (Araujo et al., 1999). Subsequently, a study of the temporal relationship between DNA replication and the inheritance of the DNA methylation pattern is presented. We also demonstrate that mammalian origins of replication, similarly to promoters, are differentially methylated (Araujo et al., 1998). We then tested the hypothesis that DNMT1 is a necessary component of the replication machinery. The results presented indicate that inhibition of DNMT1 results in inhibition of DNA replication (Knox et al., 2000). Finally, results are presented, demonstrating that the amino terminal region of DNMT1 is responsible for recognizing hemimethylated CGs, DNMT1's enzymatic target. Taken together, the results presented in this thesis demonstrate that DNMT1 is necessary for proper DNA replication and that DNA methylation may modulate origin function.

Cancer cells have aberrant DNA methylation patterns which are characterized by hypomethylation of a large set of promoters and hypermethylation of tumor suppressor genes. The dynamic nature of the epigenome makes it a valuable target for therapeutic interventions. This thesis focuses on understanding the use of various inhibitors towards DNA methylation-related proteins and their respective anti-cancer activities at both global and gene-specific levels. The widely used demethylating agent 5-azacytidine and 5-aza-2'-deoxycytidine (5-azaCdR) are FDA-approved drugs for the treatment of myelodysplastic syndrome. However, these nucleoside analogs which trap the DNA methyltransferases (DNMTs) are non-specific. Studies have shown that 5-azaCdR induced pro-metastatic genes and caused long distance metastasis. This raises serious safety concerns for their clinical use. On the contrary, targeting the DNMTs individually or in combination did not result in dramatic induction of pro-metastatic genes as with 5-azaCdR treatment. In particular, single DNMT1-specific inhibition resulted in maximum growth suppression when compared to inhibition of all three major DNMTs, while not increasing cell invasiveness. DNMT1 has been shown to be important for cancer growth. Our study supports the idea that DNMT1 has a major role in cancer over the other DNMTs and that DNMT1 inhibitors could be effective anti-cancer drugs. 5-azaCdR has nevertheless been proven to be a potent suppressor of cancer growth. We tested the idea of a combinatorial treatment that may minimize its side-effects on cell invasion while maintaining its growth suppressor effects. The methyl-CpG binding protein 2 (MBD2) protein has been shown to demethylate pro-metastatic genes. Its inhibition in concurrent with 5-azaCdR treatment synergistically suppressed cancer growth, while reversed the 5-azaCdR-induced invasion. In order to have a deeper understanding of the impact of the treatments, microarrays studies on the methylome and transcriptome of the treated cells were carried out. Bioinformatics analysis indicated that the combined treatment suppressed gene networks that were involved in cell mobility, while synergistically enhanced gene networks that were involved in cell death. This data indicate that combining 5-azaCdR treatment with MBD2 inhibition results in more potent anti-cancer effects than either treatment alone. In order to explore the currently available drugs that inhibit MBD2, we tested the combination of S-adenosylmethionine (SAM) with 5-azaCdR on the same cancer cell lines. SAM remethylated gene promoters of pro-metastatic genes and repressed 5-azaCdR-induced invasion similarly to MBD2 inhibition. We then investigated the relationship between SAM and MBD2 downregulation and observed hypermethylation on both CpG and non-CpG sites in the MBD2 promoter upon SAM treatment. Interestingly, inhibition of MBD2 using short interference RNA also resulted in hypermethylation of its own promoter. This observation suggested that SAM treatment could directly downregulate MBD2 expression, which is further downregulated through a feedback loop. These results also suggested that SAM treatment could have a direct effect on MBD2 promoter, which in turn affects multiple MBD2 targets that are involved in invasion. Together, the data from this thesis support the idea that targeting the epigenome could be a highly efficacious anti-cancer therapy and that combining drugs that target DNA methylation could increase the potency over individual treatments.<br>Les cellules cancéreuses présentent un profil de méthylation caractérisé par l'hypométhylation d'un grand nombre de promoteurs et l'hyperméthylation de gènes suppresseurs de tumeur. La nature dynamique de l'épigénome en fait une cible de choix pour les interventions thérapeutiques. Cette thèse vise à comprendre l'utilisation de divers inhibiteurs visant des protéines liées à la méthylation de l'ADN et leurs activités anticancéreuses à une échelle génomique globale et au niveau de gènes particuliers. Les agents déméthylants 5-azacytidine et 5-aza-2'-deoxycytidine (5-azaCdR) sont des médicaments pour le traitement du syndrome myélodysplasiqueapprouvés par la FDA. Cependant, ces analogues de nucléosides qui piègent les DNA méthyltransférases (DNMTs) ne sont pas spécifiques. Des études ont montrées que la 5-azaCdR induisait l'expression de gènes pro-métastatiques et l'apparition de métastases. Ceci soulève de sérieuses interrogations quant à leur utilisation en clinique. À l'inverse, le ciblage spécifique des DNMTs ne conduit pas à une induction dramatique des gènes pro-métastatiques. Plus particulièrement, l'inhibition spécifique de DNMT1 résulte en une suppression de la croissance maximale des tumeurs, sans effet sur l'invasion cellulaire, lorsque l'on compare à l'inhibition des trois principales DNMTs. Notre étude supporte l'idée que DNMT1 à un rôle majeur dans le cancer et que le développement d'inhibiteurs de DNMT1 pourraient conduire à des médicaments anti-cancéreux efficaces.Il a néanmoins été montré que la 5-azaCdR était un suppresseur potentiel de la croissance cancéreuse. Nous avons testé l'hypothèse qu'un traitement combiné permettrait de minimiser ses effets secondaires sur l'invasion cellulaire tout en maintenant ses effets suppresseurs de croissance. Il a été montré que la protéine methyl-CpG binding protein 2 (MBD2) participait à la déméthylationde gènes pro-métastatiques. Son inhibition simultanée à un traitement 5-azaCdR abolit de façon synergétique la croissance cancéreuse, tout en inhibant l'invasion induite par la 5-azaCdR. Des analyses du méthylome et du transcriptome ont été réalisées par micropuces à partir de cellules traitées avec un siRNA dirigé contre l'ARNm de MBD2 et la 5-azaCdR afin d'avoir une meilleure compréhension de l'impact de la combinaison des traitements. Les analyses bioinformatiques ont indiqué que le traitement combiné réprimait des réseaux de gènes impliqués dans la mobilité cellulaire tandis que les réseaux de gènes activés étaient impliqués dans la mort cellulaire. Ces données indiquent que le traitement à la 5-azaCdR combiné avec l'inhibition de MBD2 résulte en de plus puissants effets anti-cancéreux que l'un ou l'autre des traitements individuels.Nous avons également testé la combinaison de la S-adenosylmethionine (SAM), un médicament actuellement disponible sur le marché et inhibant l'activité de MBD2, avec la 5-azaCdR sur les lignées cellulaires utilisées précédemment. La SAM, de façon similaire à l'inhibition de MBD2 par un siRNA, permet la méthylation des promoteurs de gènes pro-métastatiques et réprime l'invasion induite par la 5-azaCdR. Nous avons ensuite examiné la relation entre la SAM, la diminution de l'expression de MBD2 et l'hyperméthylation observée à la fois aux sites CpG et non-CpG au niveau du promoteur de MBD2 après traitement avec la SAM. De façon intéressante, l'inhibition de MBD2 par des petits ARN interférant résulte également en une hyperméthylation de son propre promoteur. Cette observation suggère que le traitement avec SAM pourrait directement réduire l'expression de MBD2, qui serait réduite encore plus via une boucle de rétrocontrôle. L'ensemble des données de cette thèse supporte l'idée que le ciblage de l'épigénome pourrait être une thérapie anti-cancéreuse hautement efficace et que la combinaison de médicaments qui ciblent la méthylation de l'ADN pourrait augmenter l'efficacité des traitements individuels.

Arginine methylation is a post-translational modification occurring in higher eukaryotes that results in the addition of one or two methyl group on the nitrogen in the side chain of arginines. The enzymes responsible for protein arginine methylation have been classified in three groups. Type I enzymes promote the formation of both NG-monomethylated and asymmetric o-NG,NG-dimethylated arginines (aDMA). Type II enzymes catalyze the formation of monomethylated and symmetrical o-N G,N'G-dimethylated arginines (sDMA). The type III enzyme found in yeast catalyzes the monomethylation of the delta-guanidino nitrogen atom of the arginine residue. Although some abundant proteins have been described as being substrates for arginine methyltransferases for some time, there are still few known proteins to bear this modification. The primary goal of the work presented in this thesis was to identify new arginine methylated proteins and functionally characterize the roles of arginine methylation in new cellular processes. First, we generated four arginine methyl-specific antibodies: ASYM24 and ASYM25 are specific for aDMA whereas SYM10 and SYM11 recognize sDMA. Cell extracts were used to purify the protein complexes recognized by each of the four antibodies and the proteins were identified by microcapillary reverse-phase liquid chromatography coupled on line with electrospray ionization tandem mass spectrometry (LC/MS/MS). The analysis of 2 tandem mass spectra for each methyl-specific antibody resulted in the identification of 247 proteins, of which 197 are putatively arginine methylated.<br>The DNA repair MRE11/RAD50/NBS1 (MRN) complex was purified using one of the aDMA specific antibody. Since a role of protein arginine methylation in DNA damage checkpoint control and DNA repair had not been previously reported we chose to investigate the consequence of MRE11 methylation in DNA damage. Our results show that the MRE11 checkpoint protein is arginine methylated as determined by mass spectrometry and methylarginine-specific antibodies. The glycine-arginine rich (GAR) domain of MRE11 was specifically methylated by protein arginine methyltransferase 1 (PRMT1). Mutation of the arginines within MRE11 GAR domain severely impaired the exonuclease activity of MRE11. Cells treated with methyltransferase inhibitors displayed a DNA damage-resistant DNA synthesis phenotype and prevented the re-localization of the MRN complex to sites of DNA damage. Downregulation of PRMT1 with small interfering RNAs (siRNA) also yielded a damage-resistant DNA synthesis phenotype that was rescued with the methylated MRE11 complex. Taken together, the work presented in this thesis allowed the identification of many new potentially arginine methylated proteins and demonstrated a novel role for arginine methylation in the regulation of DNA repair enzymes and of the intra-S phase DNA damage checkpoint.

Maintaining appropriate patterns of gene expression in the gametes and during early embryogenesis is essential for normal development. DNA methylation is an epigenetic means of regulating gene expression and is an important molecular mark regulating the sex-specific expression of genes subject to genomic imprinting. Imprinted genes are expressed from only one of two inherited chromosomes and are differentially marked during gametogenesis to allow for their parental allele specific expression. These genes affect embryo growth, placental function, behavior after birth and are implicated in the etiology of a number of human diseases. The primary objective of this thesis was to gain a better understanding of the developmental dynamics and origins of DNA methylation profiles regulating maternally methylated imprinted genes during mouse oocyte development. Studies revealed that maternally methylated imprinted genes acquire methylation within their DMRs during postnatal oocyte growth and that this acquisition occurs in a gene and allele specific manner. It was also observed that maternal methylation imprint acquisition is related to oocyte diameter and that a repetitive parasitic element also acquires methylation during this period. DNA methylation is catalyzed by DNMTs and investigations into the developmental expression profiles of Dnmt3a, Dnmt3b and Dnmt3L indicated that transcript accumulation of these enzymes during oocyte development coincided with the timing of maternal methylation imprint establishment. Moreover, expression analysis in DNMT-depleted oocytes suggested these enzymes to be coordinately regulated. Additional studies aimed at developing another model of oocyte imprinting lead to the identification and characterization of a putative bovine Snrpn DMR. Its DNA methylation profile was found to be conserved with that of mouse and human. Snrpn DNA methylation analysis in bovine IVF and SCNT embryos revealed slight loss of methylatio

One of the elements commonly seen in cancer is the change in methylation status of the genome. These aberrations in methylation appear to be critical for the neoplastic phenotype and manifest as changes to gene expression of oncogenes and tumour suppressors. In addition to epigenetic alterations, the proteins involved in maintaining the plastic methylation status of the genome, DNA methyltransferases and demethylases, also show methylation-independent protein-protein interactions that have effects on cell cycle progression and proliferation. As changes in gene expression and mitotic regulation are seminal elements of cancer, and because several methylated DNA binding proteins show differential expression in a wide variety of cancers, these proteins serve as prime targets for anticancer therapies. This thesis relates to exploring both current and forthcoming possibilities and mechanisms of utilizing the DNA methylation machinery for pharmacological intervention of cancer. Chapter two deals with an antisense drug, currently in clinical trials, targeted to reduction of DNA methyltransferase 1, the maintenance methylation enzyme in mammalian cells. Our data indicate that the existence of a common truncation mutation of the adenomatous polyposis coli gene seen in some forms of sporadic and familial colorectal cancer may lead to downstream upregulation of DNA methyltransferase 1, as reconstitution of the wildtype protein reduces DNA methyltransferase 1 mRNA and protein. Reduction of the transcripts of this methylation enzyme with an antisense oligonucleotide decreases the tumourigenicity of these colorectal cancer cells, and provides a rationale for use of this drug in colorectal cancer patients and prophylactic treatment of adenomatous polyposis coli mutation-bearing individuals. Chapter three describes the rationale, design, and in vitro and in vivo testing of antisense molecules against the methylated DNA binding protein MBD2. These drugs red

Proteins are known to be post-translationally modified. This thesis will discuss arginine methylation, one of the many post-translational modifications that occur within the cell. The enzymes that catalyze this post-translational modification are called arginine methyltransferases. The three main types of methylated arginines include monomethylated arginine (MMA), asymmetric dimethylated arginine (aDMA) and symmetric dimethylated arginines (sDMA). Type I arginine methyltransferases catalyze the formation of MMA and aDMA; Type II enzyme catalyze the formation of MMA and sDMA. Protein arginine methylation has been implicated in the regulation of many different cellular processes, including transcription, cellular localization, protein-protein interaction and signal transduction.<br>The purpose of this work was to further characterize arginine methylation by identifying new members of the arginine methyltransferase enzyme family in Drosophila melanogaster and to study the effects of protein arginine methylation on novel substrates. I identified and characterized nine homologues of arginine methyltransferases in Drosophila that were named DART1 to DART9, for drosophila arginine methyltransferases 1-9. All nine enzymes are expressed at various developmental stages. I discovered that a substrate of mammalian enzyme protein arginine methyltransferase 1 (PRMT1) can also be methylated by PRMT5. I also identified HIV-1 Tat protein as the first substrate of the novel enzyme PRMT6.