<p>A controlled and efficient coordination of gene expression is the key for normal development of an organism. In mammals, a subset of autosomal genes is expressed monoallelically depending on the sex of the transmitting parent, a phenomenon known as genomic imprinting.</p><p>The imprinted state of the <i>H19</i> and <i>Igf2</i> genes is controlled by a short stretch of sequences upstream of <i>H19</i> known as the imprinting control region (ICR). This region is differentially methylated and is responsible for the repression of the maternally inherited <i>Igf2</i> allele. It harbors hypersensitive sites on the unmethylated maternal allele and functions as an insulator that binds a chromatin insulator protein CTCF. Hence the <i>H19</i> ICR, which plays an important role in maintaining the imprinting status of <i>H19</i> and <i>Igf2</i>, was shown to lose the insulator property upon CpG methylation.</p><p>Another ICR in the <i>Kcnq1</i> locus regulates long-range repression of <i>p57Kip2</i> and <i>Kcnq1</i> on the paternal allele, and is located on the neighboring subdomain of the imprinted gene cluster containing <i>H19</i> and <i>Igf2</i>, on the distal end of mouse chromosome 7. Similarly to the <i>H19</i> ICR, the <i>Kcnq1</i> ICR appears to possess a unidirectional and methylation-sensitive chromatin insulator property in two different somatic cell types. Hence, methylation dependent insulator activity emerges as a common feature of imprinting control regions.</p><p>The protein CTCF is required for the interpretation and propagation of the differentially methylated status of the <i>H19</i> ICR. Work in this thesis shows that this feature applies genomewide. The mapping of CTCF target sites demonstrated not only a strong link between CTCF, formation of insulator complexes and maintaining methylation-free domains, but also a network of target sites that are involved in pivotal functions. The pattern of CTCF <i>in vivo</i> occupancy varies in a lineage-specific manner, although a small group of target sites show constitutive binding. </p><p>In conclusion, the work of this thesis shows that epigenetic marks play an important role in regulating the insulator property. The studies also confirm the importance of CTCF in maintaining methylation-free domains and its role in insulator function. Our study unravels a new range of target sites for CTCF involved in divergent functions and their developmental control.</p>

Advancements in prognostication have improved the subdivision of chronic lymphocytic leukaemia (CLL) into diverse prognostic subgroups. In CLL, IGHV unmutated and IGHV3-21 genes are associated with a poor-prognosis, conversely, IGHV mutated genes with a favourable outcome. The finding of multiple CLL subsets expressing ‘stereotyped’ B-cell receptors (BCRs) has suggested a role for antigen(s) in leukemogenesis. Patients belonging to certain stereotyped subsets share clinical and biological characteristics, yet limited knowledge exists regarding the genetic and epigenetic events that may influence their clinical behaviour. This thesis aimed to, further investigate Swedish IGHV3-21-utilising patients, screen for genetic and DNA-methylation events in CLL subgroups/subsets and study DNA methylation over time and within different CLL compartments. In paper I, IGHV gene sequencing of 337 CLL patients from a Swedish population-based cohort revealed a lower (6.5%) IGHV3-21 frequency relative to previous Swedish hospital-based studies (10.1-12.7%). Interestingly, this frequency remained higher compared to other Western CLL (2.6-4.1%) hospital-based cohorts. Furthermore, we confirmed the poor-outcome for IGHV3-21 patients to be independent of mutational and stereotypy status. In paper II, genomic events in stereotyped IGHV3-21-subset #2, IGHV4-34-subset #4 and subset #16 and their non-stereotyped counterparts were investigated via SNP arrays (n=101). Subset #2 and non-subset #2 carried a higher frequency of events compared to subset #4. A high frequency of del(11q) was evident in IGHV3-21 patients particularly subset #2 cases, which may partially explain their poor-prognosis. In contrast, the lower prevalence of aberrations and absence of poor-prognostic alterations may reflect the inherent low-proliferative disease seen in subset #4 cases. In papers III and IV, differential methylation profiles in IGHV mutated and IGHV unmutated patients were identified using DNA-methylation microarrays. CLL prognostic genes (CLLU1, LPL), tumor-suppressor genes (TSGs) (ABI3, WISP3) and genes belonging to TGF-ß and NF-kB/TNFR1 pathways were differentially methylated between the subgroups. Additionally, the re-expression of methylated TSGs by use of methyl and deacetyl inhibitors was demonstrated. Interestingly, analysis of patient-paired diagnostic/follow-up samples and patient-matched lymph node (LN) and peripheral blood (PB) cases revealed global DNA methylation to be relatively stable over time and remarkably similar within the different compartments. Altogether, this thesis provides insight into the aberrant genomic and DNA methylation events in divergent CLL subgroups. Moreover this thesis helps distinguish the extent to which DNA methylation changes with respect to time and microenvironment in CLL.

Genomic imprinting marks a subset of autosomal loci expressed in parent of origin-dependent monoallelic expression in a non-Mendelian fashion. To restore totipotency and to reset the imprint according to the sex of the individual, the mark must be erased during germline development. The imprinted Igf2-H19 loci located distally on chromosome 7 in mouse and 11p15.5 in human, share common regulatory elements that regulate differential expression. Where the H19 is silenced when paternally inherited, the Igf2 is silenced when maternally inherited. The differentially methylated 5'-flank of H19 gene, termed imprinting control region (ICR), shown to display a unique chromatin organisation harbours hypersensitive sites in linker regions flanked by positioned nucleosomes on the maternal allele. This unique chromatin conformation functions as a methylation-sensitive and unidirectional chromatin insulator, which later was found to depend on the chromatin insulator protein CTCF. The H19 ICR exhibits default-silencing functions in promoter-proximal positions. The maximal distance between the H19 ICR and the promoter of the reporter gene required for this effect was 1.2 ± 0.3kb which can be compared to the 1.9 kb distance between the endogenous H19 ICR and H19 promoter. Results suggest that the H19 ICR adopts a chromatin conformation that must be separated by a minimal distance from pivotal cis-regulatory elements to avoid adverse effects on neighbouring promoters. Poly(ADP-ribosy)lation represents a novel post-translational epigenetic mark that segregates with exclusively the maternal derived H19 ICR and associated with factors that interact with the CTCF target sites. CTCF is itself poly(ADP-ribosy)lated and the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide relieves the insulator function of the H19 ICR. Designed zinc finger proteins were applied to examine if epigenetic marks provided an obstacle for targeted activation and silencing. The zinc finger protein ZFP809 with activator/repressor domain able to efficiently activate/silence the IGF2 target. Murine hybrid cell lines of human chromosome 11, demonstrated that the ZFP809 overcame the epigenetic marks that repressed maternal IGF2 and paternal H19 allele, respectively. Results suggested that imprinted genes are not normally exposed to strong cis-regulatory elements and that the designed ZFPs can be exploited to develop a therapeutic method for rectifying epigenetic lesions.

Genomic imprinting is an epigenetic phenomenon that causes a subset of mammalian genes to be expressed from only one allele in a parent-of-origin manner. The defects in the imprinting regulation result in disorders that affect development, growth and metabolism. We have used the Kcnq1 imprinted cluster as a model to understand the mechanism of imprinted gene regulation. The imprinting at the Kcnq1 locus is regulated by a long noncoding RNA, Kcnq1ot1, whose transcription on the paternal chromosome is associated with the silencing of at least eight neighboring genes. By destabilizing Kcnq1ot1 in an episomal system, we have conclusively shown that it is the RNA and not the process of transcription that is required for the gene silencing in cis. Kcnq1ot1 RNA interacts with the chromatin modifying enzymes such as G9a and Ezh2 and recruits them to imprinted genes to establish repressive chromatin compartment and gene silencing. Using the episomal system, we have identified an 890 bp silencing domain (SD) at the 5’ end of Kcnq1ot1 RNA, which is required for silencing of neighboring reporter genes. The deletion of the SD in the mouse resulted in the relaxation of imprinting of ubiquitously imprinted genes (Cdkn1c, Kcnq1, Slc22a18, and Phlda2) as well as reduced DNA methylation over the somatic DMRs associated with the ubiquitously imprinted genes. Moreover, Kcnq1ot1 RNA interacts with Dnmt1 and recruits to the somatic DMRs and this recruitment was significantly affected in the SD mutant mice. By using a transgenic mouse, we have conditionally deleted Kcnq1ot1 promoter at different developmental stages and demonstrated that Kcnq1ot1 maintains imprinting of the ubiquitously imprinted genes by regulating DNA methylation over the somatic DMRs. Kcnq1ot1 is dispensable for the maintenance of repressive histone marks and the imprinting of placental-specific imprinted genes (Tssc4 and Osbpl5). In conclusion, we have described the mechanisms by which Kcnq1ot1 RNA establishes and maintains expression of multiple imprinted genes in cis.

Genomic imprinting refers to the parent-of-origin specific monoallelic expression of a gene. Imprinted genes are often clustered in the genome and their expression is regulated by an imprinting centre (IC). ICs are regions of DNA that propagate the parental specific regulation of gene expression, which are usually characterized by differential DNA methylation, histone marks and the presence of non-coding RNAs. Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome associated with the dysregulation of imprinted gene expression on human chromosome band 11p15.5. The 11p15.5 imprinted region has two imprinting centres, IC1 and IC2. IC1 is telomeric and regulates the imprinted expression of the genes H19 and IGF2. IC2 is ~700kb centromeric and is associated with a cluster of nine imprinted genes including CDKN1C, KCNQ1 and an imprinted non-coding RNA associated with IC2, KCNQ1OT1. Loss of differential DNA methylation at IC2 is seen in 50% of patients with BWS with loss of imprint of the non-coding RNA KCNQ1OT1 and associated with a decreased expression of the putative tumour suppressor CDKN1C. Patients with BWS also have a thousand-fold increased risk of pediatric cancer. The focus of this thesis involves investigation of dysregulation of imprinting in three groups of BWS patients. Firstly, I show that BWS patients with alveolar rhabdomyosarcoma have constitutional loss of methylation at IC2 and biallelic expression of KCNQ1OT1. Secondly, loss of methylation at IC2 has been previously associated with female monozygotic twins discordant for BWS. In male monozygotic twins with BWS, however, the molecular lesions reflect the molecular heterogeneity seen in BWS singletons. Thirdly, BWS patients associated with translocations and inversions that have breakpoints within the KCNQ1 gene near IC2 show regional gain of DNA methylation around the breakpoint and decreased expression of CDKN1C. Therefore, using a rare collection of BWS patients, I have attempted to determine the various roles of the imprinting centres IC1 and IC2 and their involvement in tumourigenesis, monozygotic twinning and structural chromosomal rearrangements causing BWS.

Colorectal cancer (CRC) is a major source of morbidity and mortality in the Western world. Approximately 15% of all CRCs develop via the mutator pathway, which results from a deficiency of mismatch repair (MMR) system and leads to genome-wide microsatellite instability (MSI). MLH1 promoter hypermethylation accounts for the majority of MSI CRCs. Numerous single nucleotide polymorphisms have been identified in MMR genes, however their functional roles in affecting MMR system, and therefore susceptibility to MSI CRCs, are unknown. This study uses a multidisciplinary approach combining molecular genetics, epigenetics, and epidemiology to examine the contribution of MMR gene polymorphisms in CRC. Among a panel of MMR SNPs examined, the MLH1 (-93G>A) promoter polymorphism (rs1800734) was shown to be associated with increased risk of MSI CRCs in two Canadian populations, Ontario and Newfoundland. Functional studies of the MLH1-93G>A polymorphism indicate that it has weak effects on the core promoter activity, although it dramatically reduces activity of the shorter promoter constructs in a panel of cell lines. Furthermore, MLH1 gene shares a bi-directional promoter with EMP2AIP1 gene, and the MLH1-93G>A polymorphism increases the activity of the reverse, EPM2AIP1 promoter. Examination of alternative role of the MLH1-93G>A polymorphism in MSI-H CRCs led to evaluation of a 500-kilobase pair chromosome 3 region around the MLH1 gene and identification of two additional SNPs, rs749072 and rs13098279, which are in strong linkage disequilibrium with rs1800734. All three SNPs showed strong associations with MLH1 promoter methylation, loss of MLH1 protein expression, and MSI-H CRCs in three populations, Ontario, Newfoundland, and Seattle. Such findings potentially implicate genetic susceptibility to DNA methylation. Logistic regression models for MSI-H versus non-MSI-H CRCs demonstrate that models including MLH1 IHC status and MLH1 promoter methylation status fit the data most parsimoniously in all three populations combined, however, when rs1800734/rs749072/rs13098279 was added to this model, polymorphisms no longer remained significant indicating that the observed associations of these polymorphisms with the MSI-H CRCs occur through their effect on DNA methylation. This study identified a novel mechanism in which common missense alterations may contribute to complex disease.

Aberrant promoter methylation is known to silence tumor-suppressor genes in prostate cancer. Using a quantitative real-time PCR assay(MethyLight), I determined promoter methylation levels of APC, RASSF1A, CYP26A1 and TBX15 in 219 radical prostatectomies diagnosed between 1998-2001, examined their correlation with clinicopathological follow-up data including Gleason Pattern(GP), Gleason Score(GS) and pathological stage, and explored their potential in predicting biochemical recurrence(BR) using univariate and multivariate analyses. I demonstrated that methylation status of all four genes could accurately differentiate normal from cancerous tissues. Quantitative methylation levels of APC and TBX15 correlated strongly with GP, GS, and pathological stage. Both APC and TBX15 methylation levels could significantly predict BR in univariate analysis(p-value=0.028 and 0.003, respectively). The methylation profiles of APC and TBX15 combined could discriminate patients into high, intermediate, and low risk groups of BR(p-value=0.005). My project demonstrated that quantitative increase in promoter methylation levels of APC and TBX15 were associated with PCa progression.

Aberrant promoter methylation is known to silence tumor-suppressor genes in prostate cancer. Using a quantitative real-time PCR assay(MethyLight), I determined promoter methylation levels of APC, RASSF1A, CYP26A1 and TBX15 in 219 radical prostatectomies diagnosed between 1998-2001, examined their correlation with clinicopathological follow-up data including Gleason Pattern(GP), Gleason Score(GS) and pathological stage, and explored their potential in predicting biochemical recurrence(BR) using univariate and multivariate analyses. I demonstrated that methylation status of all four genes could accurately differentiate normal from cancerous tissues. Quantitative methylation levels of APC and TBX15 correlated strongly with GP, GS, and pathological stage. Both APC and TBX15 methylation levels could significantly predict BR in univariate analysis(p-value=0.028 and 0.003, respectively). The methylation profiles of APC and TBX15 combined could discriminate patients into high, intermediate, and low risk groups of BR(p-value=0.005). My project demonstrated that quantitative increase in promoter methylation levels of APC and TBX15 were associated with PCa progression.

Genomic imprinting refers to the parent-of-origin specific monoallelic expression of a gene. Imprinted genes are often clustered in the genome and their expression is regulated by an imprinting centre (IC). ICs are regions of DNA that propagate the parental specific regulation of gene expression, which are usually characterized by differential DNA methylation, histone marks and the presence of non-coding RNAs. Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome associated with the dysregulation of imprinted gene expression on human chromosome band 11p15.5. The 11p15.5 imprinted region has two imprinting centres, IC1 and IC2. IC1 is telomeric and regulates the imprinted expression of the genes H19 and IGF2. IC2 is ~700kb centromeric and is associated with a cluster of nine imprinted genes including CDKN1C, KCNQ1 and an imprinted non-coding RNA associated with IC2, KCNQ1OT1. Loss of differential DNA methylation at IC2 is seen in 50% of patients with BWS with loss of imprint of the non-coding RNA KCNQ1OT1 and associated with a decreased expression of the putative tumour suppressor CDKN1C. Patients with BWS also have a thousand-fold increased risk of pediatric cancer. The focus of this thesis involves investigation of dysregulation of imprinting in three groups of BWS patients. Firstly, I show that BWS patients with alveolar rhabdomyosarcoma have constitutional loss of methylation at IC2 and biallelic expression of KCNQ1OT1. Secondly, loss of methylation at IC2 has been previously associated with female monozygotic twins discordant for BWS. In male monozygotic twins with BWS, however, the molecular lesions reflect the molecular heterogeneity seen in BWS singletons. Thirdly, BWS patients associated with translocations and inversions that have breakpoints within the KCNQ1 gene near IC2 show regional gain of DNA methylation around the breakpoint and decreased expression of CDKN1C. Therefore, using a rare collection of BWS patients, I have attempted to determine the various roles of the imprinting centres IC1 and IC2 and their involvement in tumourigenesis, monozygotic twinning and structural chromosomal rearrangements causing BWS.

Colorectal cancer (CRC) is a major source of morbidity and mortality in the Western world. Approximately 15% of all CRCs develop via the mutator pathway, which results from a deficiency of mismatch repair (MMR) system and leads to genome-wide microsatellite instability (MSI). MLH1 promoter hypermethylation accounts for the majority of MSI CRCs. Numerous single nucleotide polymorphisms have been identified in MMR genes, however their functional roles in affecting MMR system, and therefore susceptibility to MSI CRCs, are unknown. This study uses a multidisciplinary approach combining molecular genetics, epigenetics, and epidemiology to examine the contribution of MMR gene polymorphisms in CRC. Among a panel of MMR SNPs examined, the MLH1 (-93G>A) promoter polymorphism (rs1800734) was shown to be associated with increased risk of MSI CRCs in two Canadian populations, Ontario and Newfoundland. Functional studies of the MLH1-93G>A polymorphism indicate that it has weak effects on the core promoter activity, although it dramatically reduces activity of the shorter promoter constructs in a panel of cell lines. Furthermore, MLH1 gene shares a bi-directional promoter with EMP2AIP1 gene, and the MLH1-93G>A polymorphism increases the activity of the reverse, EPM2AIP1 promoter. Examination of alternative role of the MLH1-93G>A polymorphism in MSI-H CRCs led to evaluation of a 500-kilobase pair chromosome 3 region around the MLH1 gene and identification of two additional SNPs, rs749072 and rs13098279, which are in strong linkage disequilibrium with rs1800734. All three SNPs showed strong associations with MLH1 promoter methylation, loss of MLH1 protein expression, and MSI-H CRCs in three populations, Ontario, Newfoundland, and Seattle. Such findings potentially implicate genetic susceptibility to DNA methylation. Logistic regression models for MSI-H versus non-MSI-H CRCs demonstrate that models including MLH1 IHC status and MLH1 promoter methylation status fit the data most parsimoniously in all three populations combined, however, when rs1800734/rs749072/rs13098279 was added to this model, polymorphisms no longer remained significant indicating that the observed associations of these polymorphisms with the MSI-H CRCs occur through their effect on DNA methylation. This study identified a novel mechanism in which common missense alterations may contribute to complex disease.