DNA methylation is a common epigenetic mark that affects gene regulation, genomic stability and chromatin structure. In mammals, methylation is mainly found in the CpG dinucleotides. The CpG methylation signals can be recognised by the Methyl-CpG-Binding Protein (MBP) family which includes MeCP2, MBD1, MBD2, MBD3, MBD4 and Kiaso. MeCP2 and MBD1-4 (except mammalian MBD3) recognise methyl-CpG via their MBD domain whereas Kaiso interprets methylation through its Zn finger DNA binding domain. The TRD domains of MeCP2, MBD1 and MBD2 have been reported to recruit transcriptional co-repressors to the methylated DNA. A thymine DNA glycosylase domain is located at the C-terminal region of MBD4. This study concerns the molecular details of the methyl-CpG recognition by the MBD domain of MeCP2. To achieve this, the MeCP2 MBD domain has been expressed, purified and co-crystallised with a 20 bp DNA fragment from the BDNF promoter. The DNA-protein cocrystal diffracted X-rays to a maximum resolution of 2.5Å using synchrotron sources. It belongs to space group C2 with unit cell dimensions: a = 79.71Å, b = 53.60Å, c = 65.73Å, and β = 132.1°. The X-ray structure of the MeCP2 MBD-DNA complex was solved using the SAD method. Structural analyses of the refined X-ray structure reveal that the methyl groups of the DNA make contact with a predominantly hydrophilic surface that includes tightly bound water molecules. From a structure of the MBD domain in MBD1, established by NMR, the binding specificity of the MBD domain had been thought to depend on hydrophobic interactions between the cytosine methyl groups and a hydrophobic patch within the MBD domain. The findings of this study suggest that MeCP2 recognises the hydration pattern of the major groove of methylated DNA rather than cytosine methylation per se. The X-ray structure also identifies a unique role of T158 and R106, the sites of the two most frequent Rett missense mutations. Both residues stabilise the tandem Asx-ST motif at the C-terminal region of MBD domain. Disruption of this tandem motif destabilises the DNA-protein interaction. The BDNF sequence in this study contains an AT run which displays unique properties of AT tract DNA. Previously, mutation of the AT run has been reported to decrease MeCP2 binding specificity. This study however demonstrated that a significant reduction can only be observed when both AT runs close to the methyl-CpG have been mutated. The X-ray structure of the MeCP2 MBD-DNA complex in this study rationalises the effects of the most common Rett mutations and provides a general model for methylated DNA binding that is dependent on structured water molecules.

In mammals, the correct spatio-temporal patterns of gene expression are coordinated by transcription factor networks in combination with epigenetic signalling pathways. CpG methylation is an epigenetic modification of DNA involved in the heritable transmission of gene silencing patterns. Increasing evidence suggest a primary role for CpG methylation in the direct regulation of gene expression, at least for a subset of promoters. An example of this direct regulation is represented by the ectopic expression of genes involved in genome defence pathways upon global loss of methylation. However, the mechanistic relationship between CpG methylation and transcriptional regulation is not well understood. To explore this, we have applied Cap Analysis of Gene Expression (CAGE), to cells deficient in CpG methylation (Mouse Embryonic Fibroblasts with hypomorphic mutation of Dnmt1 ) and matched controls. This provides a quantitative, single nucleotide resolution, genome wide map of methylation responsive transcription initiation. Integrating this with RNA-seq, genome wide measures of CpG methylation and ChIP-seq for histone modifications in the same system, provides a detailed view of how reduced CpG methylation alters the chromatin and transcriptional landscape of the genome. Our results show dramatic shifts in the cellular RNA pool, with the pronounced up-regulation or de-repression of promoters in a specific sub-family of transposable elements. Tens of other genic and non-coding RNA promoters similarly show dramatic inductions. Contrary to a prior hypothesis, we found no evidence for increased rates of transcriptional initiation from anonymous genomic sites not previously implicated in promoter activity. Transcription initiation at CpG island promoters is generally unaffected by hypomethylation, however a set of TSSs located in CpG island shores and a class to transposable element overlapping TSSs do appear to be sensitive to methylation and are significantly up-regulated upon hypomethylation.

Graduation date: 1995

Glioblastoma (GBM) is the most common and malignant of the glial tumors. These tumors may develop from lower-grade astrocytomas (diffuse astrocytoma; grade II or anaplastic astrocytoma; grade III) through a progressive pathway, but, more frequently, they manifest de novo without any evidence of a pre-malignant lesion. The treatment of GBM includes surgery, radiotherapy, and chemotherapy with temozolomide. Despite improvements in treatment protocols, the median survival of GBM patients remains very low at 12-15 months. The cause of glioma (either development or progression) can be genetic and epigenetic modification driven changes. In contrast to genetic modifications, where DNA sequence is changed, epigenetic modifications are those gene expression regulatory mechanisms which do not involve the change in the DNA sequence. It includes DNA methylation, chromatin modifications and miRNA mediated changes in gene expression. Aberrant DNA methylation is one of the common molecular lesions occurring in the cancer cell. The 5th position of cytosine (CpG) is the most preferred site of DNA methylation in mammalian cells. The methylated cytosines are prone to undergo oxidative deamination, and get mutated to thymine in DNA. Consequently, this led to decrease in CpG abundance in the genome. In normal conditions, promoters of majority of the genes escape methylation, because of which CpG of these regions remain same. This phenomenon led to the restriction of CpGs in the promoter regions of most of the genes. These CpG rich regions of the promoters are known as CpG islands, and the methylation status of these islands have a major role in regulating gene expression. The cancer genome is shown to undergo genome-wide hypomethylation whereas CpG islands undergo hypermethylation compared to normal tissue, resulting in net loss of total methylation, as the CpGs from non-island areas far exceed in number compared to the CpGs from islands. The most studied change of DNA methylation in neoplasms is the silencing of the tumor suppressor genes by CpG island promoter hypermethylation. Apart from few studies, the role of DNA methylation in glioma development and progression is poorly known. With this background, we have focused our study on DNA methylation changes in GBM. To identify GBM specific DNA methylation alterations, we have performed the genome wide methylation profile of 44 GBM and 8 normal samples using Infinium methylation array. Beta value, which is a measure of methylation, was calculated for all the CpG probes. Beta value ranges between 0-1 (from no methylation to complete methylation). We sought to understand the clinical importance, with particular importance to patient survival, of the DNA methylation pattern observed. We also undertook steps to understand the contribution of the differential DNA methylation and the associated gene expression changes in GBM development. This work has been divided into three parts: Part I –Identification of GBM specific methylome and development of a DNA methylation prognostic signature for GBM To identify the differentially methylated genes in GBM, we compared the methylation levels of 27,578 CpGs between GBM and normal control samples using statistical methods. We then compared the list of differentially methylated genes with the expression data generated by The Caner Genome Atlas (TCGA) to find out genes whose expression oppositely correlates with the DNA methylation status. This resulted in the identification of 62 genes hypermethylated and down regulated, while 65 genes hypomethylated and up regulated. We believe that this set of differentially methylated genes may play important role in glioma development. Next, to identify GBM specific DNA methylation survival signature, we correlated the survival data of 44 GBM patients with beta values of all the 27,578 probes. Using Cox regression method, we identified a set of 9 genes, whose methylation predicted the survival in GBM patients. A risk score was then calculated using methylation values and regression co-efficient of each of the genes. The methylation risk score was found to be an independent predictor of survival in a multivariate analysis in TCGA data set and the Bent et al data set (independent validation sets). Using methylation risk score, we were able to divide the patients into low and high risk groups with significant difference in survival. To discover the biology behind the difference in the survival of low and high risk groups, we performed network analysis, using differentially expressed genes between low and high risk patients, which revealed an activated NFkB pathway association with poor prognosis. The inhibition of NFkB pathway sensitized the glioma cells for chemotherapeutic drugs only in NFkB activated cell lines, suggesting a pivotal role for NFkB pathway imparting chemoresistance in poor surviving group. Part II -NPTX2, a methylation silenced gene, inhibits NFkB through a p53-PTEN-PI3K-AKT signaling pathway To understand the mechanism behind the prediction of survival by methylation of 9 genes, we took NPTX2 as a candidate gene for further investigation. NPTX2, a risky methylated gene, is highly methylated in high risk group with poor survival, which suggests that it may have a growth inhibitory activity in GBM. Bisulphite sequencing confirmed the hypermethylation status of NPTX2 promoter in GBM samples and glioma cell lines compared to normal brain tissue. As expected, NPTX2 transcript level was significantly down regulated in GBMs and glioma cell lines compared to normal samples, and could be re-expressed upon methylation inhibitor treatment in glioma cells. Exogenous over expression of NPTX2 inhibited proliferation, colony formation and sensitized glioma cells to chemotherapeutic drugs. Moreover, NPTX2 also inhibited soft agar colony formation in vitro, which confirms its growth inhibitory function in GBM. As NPTX2 was methylated and silenced in the high risk group, which has high activation of NFkB pathway, we then checked if NPTX2 could inhibit NFkB activity. Indeed, we observed that NPTX2 overexpression inhibited expression from NFkB dependent luciferase reporter, sequence-specific DNA-binding of NFkB, nuclear translocation of NFkB sub unit (p65) and it also significantly repressed key NFkB target genes. We also show that NPTX2 mediated inhibition of NFkB could be abrogated by co-expression of constitutively active forms of PI3 kinase, AKT and IKKα, suggesting an involvement of PI3K-AKT-IKKα axis in NPTX2 mediated NFkB inhibition. Further, we found that NPTX2 repressed NFkB activity by inhibiting AKT through an ATM-p53-PTEN-PI3K dependent pathway. Thus, these results explain the need for hypermethylation and down regulation of NPTX2 in high risk GBM wherein the NFkB pathway is activated. Part III -Methylation silencing of ULK2, an autophagy gene, is important for astrocyte transformation and cell growth Among the differentially methylated genes (see part I), ULK2 was one of the most hypermethylated and down regulated genes. ULK2 is a known initiator protein in autophagy pathway, which is a type II cell death mechanism. There are many contradictory reports with respect to the role of autophagy in GBM development. For example, it has been shown that autophagy has a tumor suppressor activity and is essential for temozolomide mediated cell toxicity in GBM cells, whereas others studies implicate its involvement in tumor growth and progression. Hence, we carried out experiments to understand the role of ULK2 in GBM development. Using bisulphite sequencing, we validated ULK2 promoter hypermethylation status in GBM and glioma cell lines. In good correlation, ULK2 was found to be down regulated in GBMs and glioma cell lines, which was reexpressed by methylase inhibitor treatment in glioma cell lines. The over expression of ULK2 was found to inhibit colony formation, proliferation and soft agar colony formation of glioma cells. As expected, ULK2 overexpressing cells showed higher autophagy, compared to control cells. Interestingly, we also found increased apoptosis in ULK2 overexpressing cells. The cell death caused by ULK2 overexpression was compromised when cells were treated with 3-MA (an autophagy inhibitor) or Z-VAD-FMK (a pan caspase inhibitor). However, ULK2 failed to inhibit cell growth in autophagy deficient cells (ATG5-/-), thereby suggesting the importance of autophagy in ULK2 induced cell death. Further, ULK2 overexpression, increased catalase degradation and Reactive Oxygen Species (ROS) generation, which suggests that increase in ROS may play a role in ULK2 dependent cell death. In good correlation, N-Acetyl Cysteine, a ROS inhibitor, treatment rescued the cells from ULK2 mediated cell death, confirming the role of ROS in ULK2 induced cell death. Kinase deficient ULK2 overexpression failed to induce cell growth inhibition, autophagy and apoptosis, suggesting kinase activity of ULK2 is important for ULK2 function. Co-transfection of ULK2 inhibited Ras mediated transformation of immortalized normal human astrocytes. Taken together, we have identified and validated ULK2 as one of the DNA methylation silenced genes in GBM. ULK2 was found to be growth inhibitory in GBM cells by increasing autophagy dependent apoptosis. ULK2 inhibited Ras mediated transformation, suggesting essentiality of DNA methylation mediated ULK2 down regulation in GBM. In conclusion, the present work sheds light on the importance of methylation of genes in GBM progression. As observed, two of the genes, NPTX2 and ULK2 play as critical growth inhibitors in GBM. Also, we have identified a robust, independent and a highly sensitive 9 gene methylation signature, for GBM patient’s survival prediction.

BACKGROUND: The study of genetic variants alone is not enough to explain a complex disease like cancer. Alterations in DNA methylation patterns have been associated with different types of tumor. In order to detect markers of susceptibility for the development of cutaneous melanoma and breast cancer in the Uruguayan population, we integrated genetic and epigenetic information of patients and controls. METHODS: We performed two case-control studies that included 49 individuals with sporadic cutaneous melanoma and 73 unaffected controls, and 179 women with sporadic breast cancer and 209 women controls. We determined the level of global leukocyte DNA methylation using relative quantification of 5mdC by HPLC, and we compared methylation levels between cases and controls with nonparametric statistical tests. Since the Uruguayan population is admixed and both melanoma and breast cancer have very high incidences in Uruguay compared to other populations, we examined whether individual ancestry influences global leucocyte DNA methylation status. We carried out a correlation analysis between the percentage of African, European and Native American individual ancestries, determined using 59 ancestry informative markers, and global DNA methylation in all participants. RESULTS: We detected global DNA hypomethylation in leukocytes of melanoma and breast cancer patients compared with healthy controls (p < 0.001). Additionally, we found a negative correlation between African ancestry and global DNA methylation in cancer patients (p <0.005). CONCLUSIONS: These results support the potential use of global DNA methylation as a biomarker for cancer risk. In addition, our findings suggest that the ancestral genome structure generated by the admixture process influences DNA methylation patterns, and underscore the importance of considering genetic ancestry as a modifying factor in epigenetic association studies in admixed populations such as Latino ones.

In mammals, epigenetic modifications and trans-acting effectors coordinate gene expression during development and impose transcriptional memories that define specific cell lineages and cell-types. Methylation at CpG dinucleotides is an epigenetic mechanism through which transcriptional silencing is established and heritably maintained through development. Functionally, DNA methylation regulates key biological processes such as X-chromosome inactivation, transposon repression and genomic imprinting. However, the extent to which DNA methylation is the primary regulator of single-copy gene expression and the precise mechanism of methylation-dependent silencing remain undetermined. Here, I identify a novel set of germline-specific candidate genes putatively regulated by DNA methylation. Analysis of one candidate gene, Tex19, demonstrates that promoter CpG methylation is the primary and exclusive mechanism for regulating developmental silencing in somatic lineages. Genetic or pharmacological removal of CpG methylation triggers robust de-repression of Tex19 and loss of transcriptional memory. Moreover, Tex19 critically relies on de novo methylation, mediated by Dnmt3b, to impose silencing in differentiating ES cells and somatic cells in vivo from embryonic day (E)7.5. Reporter gene and ChIP analysis demonstrate that Tex19 is strongly activated by general transcription factors and is not marked by repressive histone modifications in somatic lineages, consistent with differential DNA methylation per se being the primary mechanism of regulating expression. Full transcriptional silencing of Tex19 is critically dependent on the methyl-binding protein (MBP) Kaiso, which is only recruited to methylated Tex19 promoter. The reliance on DNA methylation and Kaiso for silencing in somatic cells establishes an epigenetic memory responsible for maintaining expression in germline and pluripotent cell types through successive developmental cycles. This thesis represents the first causal report of lineagespecific promoter DNA methylation directing silencing of an in vivo gene through recruitment of an MBP.

Chromatin structure and epigenetic mechanisms play an important role in initiating and maintaining the intricate patterns of gene expression required for embryonic development. One such mechanism, DNA methylation (5mC), involves the chemical modification of cytosine bases in DNA and is implicated in maintaining patterns of transcription. However, many fundamental aspects of DNA methylation are not fully understood, including the mechanisms by which it influences transcriptional states. Recent data suggest functional links between DNA methylation and a second epigenetic mechanism that has important roles in transcriptional repression, the polycomb group (PcG) repressor system. Here, I suggest that an intact DNA methylation system is required for the repression of many PcG target genes by influencing the genomic targeting of the polycomb repressor 2 complex (PRC2) and its signature histone modification, H3K27me3 (K27me3). I demonstrate differential genomic localisation of K27me3 at gene promoter regions in hypomethylated mouse embryonic fibroblast (MEF) cells deficient for the major maintenance DNA methyltransferase, Dnmt1. Globally, Dnmt1-/- MEFs have a higher level of the K27me3 mark than controls, as assessed by western blot and immunofluorescence. I observe increased K27me3 at a relatively small number of gene promoters in Dnmt1-/- MEFs that often are associated with high levels of DNA methylation in wildtype MEFs, consistent with the notion that DNA methylation is capable of antagonising PRC2 binding at certain loci. Conversely, I show that a large number of developmentally important genes that are normally repressed and highly bound by K27me3, including classic polycomb targets, the Hox genes, display dramatically reduced association with K27me3 in Dnmt1-/- MEFs. Many of these genes, but not all, show reciprocal increases in promoter H3K4me3 modification and are transcriptionally de-repressed in Dnmt1-/- MEFs. I suggest that these genes are mostly associated with CpG-rich promoters with low levels of DNA methylation in wildtype cells, implying that their silencing is not dependent on the canonical role of DNA methylation. Consistent with the findings of recently published work, I suggest a working model where PRC2 binding in wildtype cells is restricted by CpG methylation. According to this model, the differential genomic location of K27me3 in hypomethylated Dnmt1-/- MEFs is explained by a redistribution of PRC2 to normally DNA methylated, unbound loci, resulting in a titration effect and coincident loss of K27me3 from normal targets. It was also apparent that certain PRC2-target genes, including the developmentally important Hox gene clusters, are strongly affected in Dnmt1-/- MEFs, displaying striking loss of K27me3. As intergenic transcription has been implicated in relief from polycomb silencing and abundant intergenic transcription has been reported within Hox clusters, I measured RNA expression at Hox clusters and a small number of other PcG target genes in Dnmt1-/- MEFs using highdensity tiling arrays. In Dnmt1-deficient MEFs, widespread increases in intergenic transcription were observed within Hox clusters. In addition, mapping of the elongatingpolymerase- associated H3K36me3 histone modification showed widespread increases in this mark at intergenic and promoter regions in Dnmt1-/- MEFs. Increased local intergenic RNA and H3K36me3 were found to correlate with K27me3 loss for this cohort of genes. I suggest a working model where increased intergenic transcription and H3K36me3 in Dnmt1-/- MEFs leads to accelerated loss of K27me3 at certain loci, including Hox clusters. Taken together with recently published data, this work suggests that a major role of DNA methylation is in shaping the PRC2/K27me3 landscape. The potential implications of this putative role for DNA methylation are widespread, including our knowledge of how DNA methylation influences transcriptional regulation, and the consequence of rearranged DNA methylation patterns that are observed in many diseases including cancers.

Virtually all multicellular organisms are capable of developing differently in response to environmental variation. At the molecular level, such developmental plasticity requires interpretation and perpetuation of environmental signals without changing the underlying genotype. Such non-genetic, heritable information is known as epigenetic information. This dissertation examines epigenetic information among social insects, and how differences in such information relate to phenotypic caste differences. The studies included herein primarily focus on one form of epigenetic information: DNA methylation. In particular, these studies explore DNA methylation as it relates to and impacts (i) alternative phenotype and particular gene expression differences in two social insect species, (ii) histone modifications, another important form of epigenetic information, in insect genomes, and (iii) molecular evolutionary rate of underlying actively transcribed gene sequences. We find that DNA methylation exhibits marked epigenetic and evolutionary associations, and is associated with alternative phenotype in multiple insect species. Thus, DNA methylation is emerging as one important epigenetic mediator of phenotypic plasticity in social insects.

One of the most frequently reported deficits seen in individuals with Fetal Alcohol Spectrum Disorder (FASD) is impairments in learning and memory, which is likely attributed to the teratogenic effects of ethanol on the developing hippocampus. TGF-β (transforming growth factor-β), hedgehog and Wnt signaling pathways have been identified as high probability candidate pathways associated with brain deficits seen in FASD. Increasing evidence indicates that ethanol may induce changes in DNA methylation that could alter transcription regulating factors within signaling pathways critical in brain development. The purpose of this study was to test the hypotheses that prenatal ethanol exposure during i) the first trimester-equivalent period, or ii) throughout the entire gestational period induces changes in DNA methylation and alters the transcription/translation of TGF-β1, SHH (sonic hedgehog) and Wnt3a within the developing hippocampus. Pregnant Dunkin-Hartley-strain guinea pigs were assigned to one of three groups: ethanol (4 g/kg maternal body weight), isocaloric-sucrose/pair-feeding, or no treatment. Embryonic telencephalon tissue (which gives rise to the hippocampus) and fetal hippocampus were collected at gestational day (GD) 23 or GD 65, respectively. GD 23 ethanol-exposed and nutritional control embryos exhibited decreased crown-rump and head lengths. GD 65 ethanol-exposed fetuses exhibited decreased body and brain weights compared with the control groups. Ethanol exposure during the first trimester-equivalent period, but not during the entire gestational period, resulted in an increase in global DNA methylation. First trimester-equivalent ethanol exposure did not alter TGF-β1, SHH and Wnt3a gene expression within the GD 23 telencephalon. However, ethanol exposure throughout the entire pregnancy led to an increase in the expression of all three genes within the GD 65 hippocampus. No change in TGF-β1 protein was seen in the hippocampus of ethanol-treated fetuses. Post-translationally modified (ptm) SHH, but not unmodified SHH protein, was decreased in the hippocampus of ethanol-exposed fetuses. A decrease in unmodified, but not ptm Wnt3a protein, was observed in both ethanol-exposed and nutritional control hippocampus. These results suggest that prenatal ethanol exposure may affect hippocampal development through alterations in i) DNA methylation as shown at early gestation and ii) the expression of transcription regulating factors, especially SHH, as shown at term. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2012-12-03 12:36:33.035

Although the many cells within a mammal share the same DNA sequence, their gene expression programmes are highly heterogeneous, and their functions correspondingly diverse. This heterogeneity within an isogenic population of cells arises in part from the ability of each cell to respond to its immediate surroundings via a network of signalling pathways. However, this is not sufficient to explain many of the transcriptional and functional differences between cells, particularly those that are more stable, or, indeed, differences in expression between parental alleles within the same cell. This conundrum lead to the emergence of the field of epigenetics - the study of heritable changes in gene expression independent of DNA sequence. Such changes are dependent on “epigenetic modifications”, of which DNA methylation is one of the best characterised, and is associated with gene silencing. The establishment of correct DNA methylation patterns is particularly important during early development, leading to cell type specific and parental allele specific gene regulation. Besides DNA methyltransferases, various other proteins have recently been implicated in DNA methylation. The absence of these proteins leads to defects in DNA methylation and development that can be even more severe than those in DNA methyltransferase knockouts themselves. In this study I focus on three such accessory proteins: LSH (a putative chromatin remodelling ATPase), G9a (a histone lysine methyltransferase) and SmcHD1 (a structural maintenance of chromosomes protein). To compare DNA methylation between WT cells and cells knocked out for each of these proteins, I used whole genome methylated DNA affinity purification and subsequent hybridization to promoter microarrays. This enabled me to compare the requirement for each protein in DNA methylation at specific genomic regions. The absence of LSH in mouse embryonic fibroblasts (MEFs) resulted in the loss of DNA methylation at 20% of usually methylated promoters, and the misregulation of associated protein coding genes. This revealed a requirement for LSH in the establishment of DNA methylation at promoters normally methylated during pre-implantation as well as post-implantation development. Secondly, I identified hypomethylation at 26% of normally methylated promoters in G9a-/- compared to WT ES cells. Strikingly, this revealed that G9a is required for maintenance of DNA methylation at maternal as well as paternal imprinting control regions (ICRs). This is accompanied by expression defects of imprinted genes regulated by these ICRs. Finally, in collaboration with the Brockdorff lab at the University of Oxford I identified a role for SmcHD1 in establishing DNA methylation at promoters on the X chromosome normally methylated slowly during X chromosome inactivation. Interestingly, SmcHD1 was also required for DNA methylation at autosomal gene promoters, contrary to previous reports that it is mainly involved in X chromosome methylation. I conclude that different accessory proteins are required to facilitate correct DNA methylation and gene repression at distinct regions of the genome, as well as at different times during development. This function of accessory proteins may be in part dependent on the prior establishment of specific chromatin signatures and developmental signals, together comprising a precisely regulated system to establish and maintain appropriate DNA methylation throughout development.