DKK1, SFRP1, WIF-1, and Wnt5a encode Wnt pathway genes that are frequently silenced by promoter hypermethylation in colorectal cancer. Despite attractive biological consequences of these events, it is unclear whether they contribute to patient prognostication or may influence tumour cell biology within distinct patient subsets. I sought to determine the prognostic roles of these methylation events in a large cohort of colorectal carcinomas from Ontario and Newfoundland. Methylation was quantified and associated with patient clinicopathlogical features. Methylation was present in cancer tissue. DKK1, Wnt5a, and SFRP1 were strongly and independently associated with tumour subtype in a manner that suggested subtype-specific activity of Wnt signaling. Methylation of DKK1 was a borderline prognosticator of favourable outcome. These results offer intriguing insight into subtype-specific biology and lead to a proposed model whereby methylation-induced Wnt bias may contribute to patient outcome.

High throughput transcriptomic analyses have realized us with the fact that eukaryotic genome encodes thousands of noncoding RNAs (ncRNAs) with unknown function. In my thesis, I sought to address epigenetic regulation of transcription by ncRNA using the Kcnq1 imprinted cluster as a model system. Genomic imprinting is an epigenetic phenomenon whereby one of the parental alleles is silenced by epigenetic mechanism in a parent of origin-specific manner. A long ncRNA Kcnq1ot1 regulates imprinting of nearly 8 protein coding genes in the Kcnq1 imprinted cluster. Expression of Kcnq1ot1 is restricted to the paternal chromosome while that of protein-coding genes to the maternal chromosome. Kcnq1ot1 is a 91kb long, moderately stable, nuclear localized and RNAPII encoded transcript. We demonstrated that Kcnq1ot1 RNA itself mediates lineage specific silencing on the paternal chromosome by interacting with chromatin and recruiting the repressive chromatin modifiers to the imprinted gene promoters. Previously we identified an 890bp silencing domain (SD) at the 5´end of the Kcnq1ot1 RNA which is responsible for gene silencing. Targeted deletion of the 890SD in mouse resulted in specific loss of silencing of ubiquitously imprinted genes. We have further shown that Kcnq1ot1 interacts with Dnmt1 and recruit Dnmt1 at the somatic DMRs flanking some of the ubiquitously imprinted genes. We next addressed the stability of the Kcnq1ot1 mediated epigenetic silencing using transgenic mouse where we have conditionally deleted the Kcnq1ot1 RNA at different developmental stages and we found that Kcnq1ot1 RNA is required to maintain the silencing of the ubiquitously imprinted genes. In addition, DNA methylation, which controls imprinting of the ubiquitous genes require Kcnq1ot1 for its maintenance. To characterize the ncRNAs that mediate gene regulation through chromatin interaction we have isolated chromatin associated RNAs (CARs) from sucrose gradient fractioned chromatin. High-throughput sequencing of the CARs resulted in the identification of the 141 intronic and 74 intergenic regions harboring CARs. We characterized one of the intergenic CARs which regulate the transcription of the two neighboring genes by modulating the chromatin marks. In summary current thesis has uncovered unprecedented role of ncRNAs in gene expression via chromatin level regulation.

DNA methylation is increasingly recognised as a significant epigenetic event that may initiate and drive the process of neoplasia in humans. In the colon, DNA methylation of key genes is common in a subset of colorectal cancers. The extent to which DNA methylation at various genes contributes to initiation of colorectal neoplasms is less clear. This study sought to clarify the biological and clinicopathological significance of methylation of various genes in the development of sporadic and familial colorectal neoplasia. Quantitative methylation-specific PCR (qMSP) assays (capable of detecting down to a measureable proportion of 0.1% of the total input DNA) were developed to determine the presence of CpG methylation at a given gene. Methylation of MLH1-C was found in the apparently normal mucosa samples from seven of 104 (7%) of individuals with sporadic colorectal cancer (CRC) showing microsatellite instability (MSI). No methylation of MLH1-C was found in the biological samples of individuals with microsatellite stable (MSS) counterparts (n=131). MLH1-C methylation may be a field defect that predisposes to the development of sporadic colorectal neoplasia, particularly those demonstrating MSI. Methylation of three of five genes within the 3p22 region including AB002340, MLH1, ITGA9, PLCD1 and DLEC1 (regional 3p22 methylation) was found in 83% of sporadic MSI (n=86) and 12% of MSS cancers demonstrating BRAF V600E mutation (n=42). Regional 3p22 correlated strongly with CpG island methylator phenotype (CIMP), and other clinicopathological characteristics typical of CIMP. Thus, regional 3p22 methylation and CIMP may be overlapping phenomena. Regional 3p22 methylation and the BRAF V600E mutation were found in normal colonic mucosa of four individuals with sporadic MSI CRC, and these cases also had multiple synchronous serrated polyps. These molecular aberrancies may predispose some individuals to the development of metachronous serrated neoplasia. Germline epimutations of APC do not contribute towards the development of FAP, AFAP, or hyperplastic polyposis syndromes. However, APC methylation in normal colonic mucosa of these individuals may represent a field defect in the development of futher neoplasms. In conclusion, different patterns of DNA methylation in normal colonic mucosa may represent a field defect important in the development of different subtypes of colorectal neoplasia.

The Centromere is a vital chromosomal structure that ensures faithful segregation of replicated chromosomes to their respective daughter cells. With such an important structure, one would expect the underlying centromeric DNA sequence would be highly conserved across all species. It turns out that the underlying centromeric DNA sequences between species ranging from the yeast, fly, mouse to humans are in fact highly diverged suggesting a DNA sequence independent or an epigenetic mechanism of centromere formation. / Neocentromeres are centromeres that form de-novo at genomic locations that are devoid of highly repetitive a-satellite DNA sequences of which normal centromeres are usually comprised from. To date, the 10q25 neocentromere is the most well-characterised, fully functional human centromere that has been used previously to characterise the extent of a number of centromeric protein binding domains and characterise the properties of the underlying DNA sequence. Along with other factors, the existence of neocentromeres has given rise to a hypothesis where centromeres are defined by epigenetic or DNA sequence independent mechanisms. / The putative 10q25 neocentromere domain was recently redefined by high resolution mapping of Centromeric protein A (CENP-A) binding through a chromatin immunoprecipitation and array (CIA) analysis. The underlying DNA sequence was investigated to determine and confirm that the formation of the 10q25 neocentromere was through an epigenetic mechanism. Through a high-density restriction fragment length polymorphism (RFLP) analysis using overlapping PCR amplified DNA derived from genomic DNA representing the 10q25 region before and after neocentromere activation. No sequence polymorphisms, large insertions or deletions were detected and confirmed the epigenetic hypothesis of centromere formation. / DNA methylation is one of many epigenetic factors that are important for cellular differentiation, gene regulation and genomic imprinting. As the mechanisms and functions of DNA methylation have been well characterised, its role at the 10q25 neocentromere was investigated to try and identify the candidate epigenetic mechanism involved in the formation of centromeres. DNA methylation across the neocentromere was assessed using sodium bisulfite PCR and sequencing of selected CpG islands located across the 10q25 neocentromere. Overall, the methylation level of the selected CpG islands demonstrated no difference in DNA methylation before and after neocentromere activation. However, significant hypomethylation upon neocentromere formation was detected close to the protein-binding domain boundaries mapped previously suggesting that this may have a role in demarcating protein binding domains at the neocentromere. / Further analysis of DNA methylation investigated non-CpG island methylation at sites defined as CpG islets and CpG orphans. Interestingly, the DNA methylation level measured at selected CpG islets and CpG orphans across the 10q25 neocentromere were not completely hypermethylated as previously thought, but demonstrated variable methylation that became fully hypermethylated upon neocentromere activation in most sites investigated. These results suggested that a role for DNA methylation existed at the 10q25 neocentromere and that it occurred at sites devoid of CpG islands. / This study has found that DNA methylation at non-CpG island sites was variable contrary to popular belief and, was linked with neocentromere formation through the observation of increased DNA methylation at the 10q25 neocentromere. Inhibition of DNA methylation demonstrated increased neocentromere instability and a decrease in methylation of these CpG islets and CpG orphans confirming the importance of DNA methylation at neocentromeres. This study has characterised a new class of sequences that are involved in the maintenance of chromatin structure through DNA methylation at the 10q25 neocentromere.

Thesis (M.Med.Sc.)--University of Western Australia, 2006.

Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2009. / Committee Chair: Dr. Soojin Yi; Committee Member: Dr. Eric Vigoda; Committee Member: Dr. James Thomas; Committee Member: Dr. John McDonald; Committee Member: Dr. Kirill Lobachev; Committee Member: Dr. Michael Goodisman. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 149-172).

DNA methylation (DNAm) is the covalent modification of DNA by addition of a methyl group primarily at the cytosine directly upstream of a guanine. DNAm level plays a central role in transcriptional regulation and is linked to disease. Therefore, understanding genetic and environmental influences on DNAm level in healthy tissue is an important step in the elucidation of trait and disease etiology. However, at present only a minority of easy to access human tissues and ethnicities have been investigated. Therefore, we studied DNAm level measured in five human tissues: cerebellum, frontal cortex, pons, temporal cortex and colon in either North American or South American samples. We applied a novel statistical approach to estimate the heritability attributable to genomic regions (regional heritability, ĥ²/r,g ) for DNAm level at thousands of individual DNAm sites genome-wide. In all five tissues, DNAm level was significantly associated with the local genomic region for more DNAm sites than expected by chance. Moreover, DNAm level could be predicted from the local sequence variants with an accuracy that scaled with the estimated ĥ²/r,g . Our results inform on molecular mechanisms regulating DNAm level and trait etiology in several ways. Firstly, DNAm level at DNAm sites located in genomic risk regions and measured in a tissue relevant to the disease can be influenced by the local genetic variants. Specifically, we found that genetic variation within a region associated with Fluid Intelligence was also associated with local DNAm level at the proline-rich coiled-coil 1 (PRRC1) gene in healthy temporal cortex tissue. Additionally, we replicated the finding of a Colorectal Cancer risk variant (rs4925386) associated with two DNAm sites in healthy colon tissue. More generally, we showed that DNAm sites located within a susceptibility region and measured in a relevant tissue exhibit a similar overall pattern of estimated ĥ²/r,g to DNAm sites outwith a susceptibility region. Secondly, the propensity for DNAm level to be associated with the local sequence variation differs with respect to CpG dinucleotide density and genic location. Most notably, DNAm sites located in CpG dense regions of the genome are less likely to be heritable than DNAm sites located in CpG sparse regions of the genome. Additionally, within both CpG dense and CpG sparse regions of the genome intergenic DNAm sites are more likely to be heritable than intragenic DNAm sites. Overall, our study suggests that variation in DNAm level at some DNAm sites is at least partially controlled by nuclear genetic variation. Moreover, DNAm level in healthy tissue has the potential to act as an intermediary in trait variation and etiology.

The MAPK pathway is important in various biological functions. It is also important in regulating processes associated with gene transcription via different mechanisms such as by phosphorylation of transcription factors, coactivators/corepressors and histone modifier complexes. H3K4 methylation is highly associated with active transcription. Deposition of this mark is catalysed by SET-domain methyltransferases which consists of a WAR complex (WDR5, ASH2L and RBBP5), a catalytic SET-domain protein and other subunits. However, potential links between ERK MAPK signaling and H3K4 methylation in gene expression are not well understood. Thus, the aim of this study was to probe the potential links between these two pathways towards gene-regulatory networks. This study attempted to elucidate their direct functional interaction by studying whether components of the SETD1A complex could be phosphorylated upon ERK activation. Our results showed that the core components of SETD1A complex were not phosphorylated in vivo and in vitro by ERK. Importantly, we reported that at least two splicing variants of RBBP5 exist. ERK-dependent stabilization of exogenous RBBP5 was observed but the mechanism underlying this is unknown. Surprisingly, we found that WAR complex depletion increased the pre-mRNA expression of immediate-early (IE) genes which did not necessarily reflect changes in their mRNA levels. In addition, this occurred in an H3K4me3-independent manner. This regulation is likely to be posttranscriptional that involves pre-mRNA processing events. First, we noticed a decrease of transcription initiation in WAR complex-depleted cells upon ERK activation. Second, depletion of the WAR complex affects the splicing efficiency of FOS and EGR1. Third, RBBP5 occupancy was observed and was significantly reduced upon siRNA-mediated RBBP5 depletion at the coding region and the 3' end of FOS gene. Therefore, we propose that the WAR complex regulates the pre-mRNA processing of IE genes through an interaction between RBBP5 and a splicing factor that has yet to be identified.

Epigenetics refers to heritable changes in phenotype without alterations to the genotype. Epigenetic changes involve two main mechanisms: DNA methylation and histone modification. Methylation of DNA at cytosine bases is the best-studied epigenetic process to date. CpG methylation states are thought to be maintained throughout cell divisions. However, loss of DNA methylation or DNA demethylation has been observed in specific stages of mammalian development. Such prominent examples of developmental DNA demethylation processes occur in developing primordial germ cells and in preimplantation embryos. However, little is known about DNA methylation changes of other tissues in mammalian development. Therefore, the first aim of this PhD study was to investigate changing nuclear distributions and levels of DNA methylation during development in order to discover dynamic variations amongst developing mouse tissues. In addition, a transgenic MBD-GFP mouse was employed to visualise DNA methylation in tissues. Several hypothetical mechanisms for the enzymatic removal of 5mC have been proposed. One of the proposed candidates is Tet-mediated successive oxidation of 5mC to generate 5hmC, 5fC and 5caC. 5hmC has therefore been considered as a transient intermediate in an active cytosine demethylation pathway. Nevertheless, some studies suggest that 5hmC may also function as an epigenetic modification in its own right. Thus, the second aim of this study was to address the research question of how and where 5hmC originates during development. In order to be able to identify tissues undergoing dynamic nuclear changes in DNA methylation and hydroxymethylation states during early mouse development, new working protocols for immunodetection of 5mC and 5hmC on tissue cryosections were required. The protocol optimisation for 5mC immunodetection is discussed in greater detail in Chapter 3. It was found that DNA methylation immunostaining of cryosections required heat-mediated DNA denaturation, which was partly compatible with protein immunostaining. Next, Chapter 4 focuses on identifying tissues undergoing dynamic changes in 5mC and 5hmC patterns during development from E9.5 to E14.5 mouse embryonic stages, using optimised immunohistochemistry protocols. These protocols revealed interesting dynamic observations of 5mC and 5hmC in the developing cerebral neocortex, surface ectoderm, liver, red blood cells, diaphragm and heart. These findings suggested that dynamic changes of 5mC and 5hmC during neocortical and compact myocardial development were in good agreement with a model where the formation of 5hmC may correlate with the loss of old 5mC, but the observations were also consistent with an involvement of de novo methylation in the generation of 5hmC. In other developing tissues, including surface ectoderm, liver, red blood cells, diaphragm and cardiac trabeculae, dynamic changes in 5mC and 5hmC levels were in line with a model where the 5hmC may act as a new epigenetic mark that functions independently. The optimised protocol also confirmed DNA demethylation of the germ cells at E12.5. The presence of three Tet family enzymes (Tet1, Tet2, Tet3) and de novo methyltransferase DNMT3A in mouse E12.5 tissues is reported in the second part of Chapter 4. It was found that Tet1, Tet2, Tet3 and Dnmt3a were present at detectable levels in neocortex, liver, diaphragm and heart. Contrastingly, no apparent signals for Tet1, Tet2, Tet3 and Dnmt3a were observed in red blood cells. This result was expected due to the very low levels of 5hmC staining in E12.5 red blood cells. The third aim of the present study was to investigate the existence of crosstalk between various epigenetic mechanisms. Thus, Chapter 5 focuses on exploring the relationship between 5mC and repressive histone marks, H3K9me3 and H3K27me3. Histone methylation dynamics at H3K9 and H3K27 were observed during mouse fetal development in neocortex and heart. The overall distribution patterns of H3K9me3 and H3K27me3 demonstrated strong association with developmental changes in 5mC, suggesting that these three repressive epigenetic marks work in concert to establish a silenced state of heterochromatin. Chapter 6, on the other hand, focuses on visualising DNA methylation in tissues using mouse transgenic tools. It was found that brain, liver, heart and neural tube expressed high levels of GFP. But no apparent developmental dynamics of GFP was observed. In conclusion, this study will contribute scientific understanding of dynamic DNA methylation and nuclear heterochromatin organisation during mammalian development, and its role in the specification and maintenance of cell lineages forming tissues and organs. This knowledge will provide insight into current barriers to cell fate reprogramming, which will be of benefit to cell regenerative biomedical technologies.