Methyl-CpG Binding Proteins (MCBPs) are thought to function as the interpreters of epigenetic information encoded in cytosine methylation. Their ability to translate DNA methylation into local transcriptional repression has sparked interest in the role of Methyl-Binding Domain Proteins (MBDs) in cancer, where repatterning of CpG methylation is common. In this dissertation I summarize and discuss observations made in the Ginder Lab linking MCBPs to the progression of neoplastic disease. It is clear from our work that the Methyl Binding Domain Protein 2 (MBD2) is necessary for the persistent repression of critical tumor suppressor genes in breast cancer. We show that stable knockdown of MBD2 also leads to growth suppression in cultured human mammary epithelial cancer lines (MCF-7, 49% suppression; MDA-MB-231, 77%; MDA-MB-435, 94%; SK-BR-3, 92%) with the peak cytotoxicity and anti-proliferative effect occurring as late as 2-3 weeks after knockdown. MBD2 knockdown also led to a decrease in viable tumor cells at equivalent doses of the histone deacetylase inhibitor, SAHA (Vorinostat™), and chemotherapeutic agents Doxorubicin, and Paclitaxel. Stable MBD2 knockdown in MCF7 cells led to an increased proportion of normal epithelial structures in 3D culture (70%, [CI=0.55-0.83]) when compared to untransfected (46%, [CI=0.39-0.53], p≤0.038) or scrambled shRNA transfected (37%, [CI=0.29-0.45], p≤0.012) controls. In vivo xenograft studies show tumor growth in BALB/C nu/nu mice was significantly impaired when mice were implanted with human breast cancer cells harboring MBD2 targeted shRNA. Following MBD2 knockdown, tumor suppressor promoter methylation remained unchanged despite sustained increases in gene expression, arguing against the convention that passive demethylation occurs with increased transcription. Our data suggest that uncoupling CpG methylation from histone modifications or other repressor functions by removing MBD2 is sufficient to initiate and maintain anti-tumor gene transcription in the absence of secondary changes in DNA methylation. In this dissertation I present evidence for the pathologic role of MBD2 in breast cancer and provide mechanistic support for the prospect of targeting MBDs in neoplastic disease..

Cytosine methylation of mitochondrial DNA (mtDNA) was first described several decades ago, but neither the mechanism generating this modification nor its functional significance was known. Because mitochondrial dysfunction is a hallmark characteristic of numerous human diseases, including neurological and cardiovascular disease, aging and cancer, this dissertation addressed whether epigenetic modification of mtDNA regulates mitochondrial function. We show that mtDNA contains not only 5-methylcytosine (5mC), but also 5-hydroxymethylcytosine (5hmC), suggesting that previous reports likely underestimated the degree of epigenetic modification within the mitochondrial genome. We questioned how these modifications were generated by looking for mitochondrial isoforms of the nuclear-encoded DNA methyltransferases. We found that an isoform of the most abundant mammalian methyltransferase, DNA methyltransferase 1 (DNMT1) translocates to mitochondria, driven by an in-frame mitochondrial targeting sequence (MTS) located upstream of the nuclear DNMT1 translational start site. This MTS is highly conserved across mammalian species, and directs a heterologous protein to the mitochondria. To investigate the function of mitochondrial DNMT1 (mtDNMT1), we created a cell line that carries a tandem-affinity purification (TAP) tag at the C-terminus of a single endogenous human DNMT1 allele. Using the DNMT1-TAP cell line, we showed that mtDNMT1 specifically binds mtDNA in a manner that is proportional to CpG density, proving its presence in the mitochondrial matrix. mtDNMT1 exhibits CpG-specific methyltransferase activity in vitro that is resistant to trypsin-treatment of intact mitochondria, but moderately susceptible to pharmacologic inhibition by the nucleoside analog 5-aza-2’-deoxycytidine (5-aza-dC). NRF1 and PGC1α, transcription factors that activate nuclear-encoded mitochondrial proteins in response to oxidative stress, were observed to up-regulate expression of mtDNMT1. Loss of p53, a tumor suppressor gene known to help control mitochondrial metabolism, also results in a striking increase in mtDNMT1 expression, and this up-regulation of mtDNMT1 appears to modify mitochondrial transcription in a gene-specific fashion. Our data suggests roles for mtDNMT1 in both the establishment and maintenance of cytosine methylation (from which 5hmC is presumably derived) and in the regulation of mitochondrial transcription. We propose that the enzymes responsible for epigenetic modification of mtDNA have potential as therapeutic targets, with relevance to a broad spectrum of human disorders.

Identification of therapeutically relevant molecules is necessary for the advancement of non-viral reprogramming of human cells for regenerative medicine. We have developed a novel non-viral model system that transforms primary human dermal fibroblasts into cells with induced regeneration competence (iRC). Low oxygen-mediated effects of fibroblast growth factor FGF2 lead to an increased cellular lifespan with a two fold increase in population doublings before senescence, remaining non-tumorigenic when injected into SCID mice while maintaining regeneration competence. This system allows us to study molecules that participate in increased cellular lifespan in a non-tumorigenic system. Analysis of chromatin modification enzymes by hybridization array, RT-PCR, and Western blots revealed upregulation of the arginine methyltransferase PRMT8 in iRC cells, challenging the paradigm that PRMT8 is solely expressed in brain tissue at the plasma membrane. Possibly leading to the erroneous conclusions that PRMT8 is brain specific at the plasma membrane is the fact that PRMT8 has several mRNA variants and protein isoforms. Here, I report expression of a novel PRMT8 variant in human dermal fibroblasts. Essential participation of PRMT8 in cellular proliferation was identified as a novel function for this enzyme through siRNA-mediated knockdown in both non-tumorigenic and tumorigenic cell lines. While other members of the PRMT family have known roles in cell cycle progression, I show for the first time that PRMT8 expression is reduced in both natural senescence and by premature induction of replicative senescence using sub-cytotoxic levels of hydrogen peroxide, implicating a correlation between PRMT8 expression and cell cycle progression. However, PRMT8 overexpression causes no significant change in the number of population doublings or the amount of time spent in culture prior to senescence, and does not alter the expression of key cell cycle regulatory genes. These results suggest that maintenance of PRMT8 expression is critical for cellular proliferation, but overexpression of PRMT8 alone is not sufficient to increase cellular lifespan. I determined that oxygen is the primary mediator of PRMT8 upregulation in the iRC system and therefore investigate histone occupancy of the PRMT8 promoter at hypoxia response elements. Through this analysis, I found bivalent occupancy regardless of culture conditions, indicating that PRMT8 maintains a state of poised readiness for transcriptional accessibility. The mechanism by which PRMT8 participates in cellular proliferation was investigated through binding partner identification. A binding partner of endogenous PRMT8 is identified here for the first time as FGF2 using co-IP and mass spectrometry. As iRC cells demonstrate a unique phenotype that uncouples the mechanisms of increased lifespan from tumorigenesis, I investigated the feasibility of PRMT8 as a cancer biomarker by mining publically available data in light of our own. I showed that PRMT8 is not only expressed in a variety of cancers, but that its expression is amplified. Moreover, PRMT8 expression significantly correlates to patient survival in specific cancers, strengthening the feasibility of this molecule as a biomarker. Aberrant expression of most PRMT family members has been described in various cancers, and specific PRMT variants are currently being used as prognostic markers. As such, I analyzed variant-specific PRMT8 expression in primary cancer cell lines and show that tumorigenic glioblastomas express PRMT8 mRNA variant 2. These data suggest that PRMT8 is a viable candidate for further study as a prognostic cancer biomarker, specifically for brain cancer.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree in Master of Science (Medicine) in the Division of Human Genetics / Foetal Alcohol Syndrome (FAS) is characterised by growth retardation, craniofacial dysmorphology and neurodevelopmental deficits. Whilst, not all alcohol exposed offspring display alcohol-related developmental anomalies, the percentage of affected offspring is greatly underestimated. Common behavioural disorders, such as ADHD and anxiety, are likely to be linked to the transgenerational effects of in utero alcohol exposure. Epigenetics has been highlighted as a potential mechanism in the aetiology of alcohol teratogenesis due to alcohol’s disruptive effects on the folate pathway, and subsequently DNA methylation. The imprinted H19/Igf2 domain is critical in foetal growth and development. The locus is regulated by the methylation-sensitive CTCF binding protein which binds to the H19 imprinting control region (ICR) upstream of the H19 locus. CTCF binding allows for the reciprocal expression of H19 and Igf2 in an allele-specific parent of origin manner. Due to the monoallelic expression of imprinted genes, DNA methylation changes within their control regions can lead to altered gene expression and possibly disease. Furthermore, if these alterations occur in the germline, disease states or susceptibility to disease may be transmittable to future generations. A mouse model was used to investigate the potential transgenerational effects of F0 chronic maternal ethanol exposure on parturition, growth, locomotor activity and anxiety. Furthermore, the transgenerational inheritance of H19 ICR DNA methylation was investigated and its possible contribution to the aforementioned phenotypes was determined. Phenotypic analysis revealed significantly reduced F1 fertility following alcohol exposure (P = 0.003) but no other significant perturbations in parturition. Although not significant at all generations, alcohol’s effects on growth and behaviour were apparent. DNA was extracted from tail biopsies, bisulfite modified and the CTCF1 and CTCF2 regions of the H19 ICR amplified. DNA methylation quantification via Pyrosequencing revealed significantly reduced mean methylation profiles at CTCF1 and CTCF2 within the F1 EtOH exposed group (P = 0.021), with CpG sites 1, 2, 4 and 6 of CTCF1 and CpG sites 1, 2, 3 (P = 0.021) and 5 (P = 0.043) of CTCF2 displaying statistically significant differences. In contrast, the EtOH group of the F2 generation showed an increase in CTCF1 mean methylation that trended towards significance (P = 0.083) suggesting a potential recovery or compensatory mechanism within the epigenetic machinery. The F3 generation EtOH exposed group displayed decreased CTCF1 mean methylation levels (P = 0.083). The F2 and F3 generations showed no significant difference in CTCF2 methylation levels between treatment groups. The significant change in CTCF1 methylation at the F1 generation and the trend towards significance in the F2 and F3 generations indicated potential transgenerational inheritance of altered H19 ICR DNA methylation. Correlations between DNA methylation at the H19 CTCF1 and CTCF2 binding sites with growth rate and behaviour measures revealed no significant relationships. This dissertation supports the involvement of epigenetic mechanisms in alcohol teratogenesis. In addition it contributes to the growing field of transgenerational epigenetic inheritance, with implications for the treatment of those with Foetal Alcohol Syndrome and/or Foetal Alcohol Spectrum Disorders and their progeny.

by Kwok-cheong Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1983. / Bibliography: leaves 194-221.

The N-terminal tails of histones are subject to many chemical modifications that are involved in a variety of biological functions. Histone methylation is a major epigenetic modification found in both single and multicellular organisms and is directly involved in the regulation of gene expression. Methylation of lysine 9 of histone 3 (H3K9) has been shown to have diverse functions depending on the number of methyl groups added; H3K9me1 marks the active promoters, while H3K9me2 and H3K9me3 are present within inactive gene promoters and pericentric heterochromatin. G9a, also known as euchromatic histone-lysine N-methyltransferase 2 (Ehmt2), is a histone methylase that catalyses addition of mono- and dimethyl groups to H3K9 in euchromatic regions of the genome to silence genes. Therefore, it is a vital component of the gene expression regulation machinery. In mouse embryonic stem (ES) cells, G9a forms a stable heterodimer with the G9a-like protein (GLP or Ehmt1), which is further stabilised by the C2H2-type zinc finger protein, widely interspaced zinc finger protein (WIZ). These three proteins form the core G9a complex, which is essential for mouse development. Lack of any G9a complex member leads to embryonic lethality at E9.5 with severe growth defects. The ankyrin repeat domain of G9a/GLP can bind to H3K9me1/2 with high affinity in vitro (Collins et al. 2008). This enables the self-recruitment of the G9a complex to sites with H3K9me1/2 and maintenance of the mark. However, the initial recruitment of the G9a complex to sites lacking H3K9me1/2 mark during differentiation is poorly understood. Neither G9a nor GLP has a DNA/RNA binding domain, so recruitment of the G9a complex to specific sites must be mediated by other binding partners of the G9a complex. Using mass spectrometry, I was able to identify a number of zinc finger proteins as binding partners of G9a. Among these, WIZ was identified in stoichiometric amounts to G9a and GLP, and is a potential DNA binding protein similar to other C2H2-type zinc fingers. The aim of this study was to determine the role of WIZ in the recruitment of the G9a complex to specific sites. I showed that knockdown of WIZ had no significant effect on the chromatin binding of G9a in undifferentiated mouse ES cells, which indicates WIZ is dispensable in the maintenance of H3K9me2. However, I observed a 30% decrease in the G9a levels upon WIZ knockdown, which shows that WIZ might have a role in stabilising G9a. Using recombinant WIZ zinc finger pairs, I was able to show that the 3rd and 4th zinc finger of WIZ bind DNA in vitro. Furthermore, using the systematic evolution of ligands exponential enrichment (SELEX) approach I demonstrated that the zinc fingers of WIZ preferentially bind to G-rich double-stranded DNA sequences. Binding site analysis with synthetic DNA indicated that WIZ ZF3-4 require two binding sites that are a certain distance apart from each other for efficient binding. In addition, ZF3-4 binds ssDNA with higher affinity than dsDNA, and binding to ssDNA is sequence-independent. This study shows for the first time that mouse WIZ zinc finger pairs can bind DNA and RNA in vitro. Therefore, sequence-specific recruitment of G9a might be mediated by WIZ during differentiation. Furthermore, DNA binding preference of WIZ might suggest that WIZ-mediated recruitment of G9a to establish H3K9me2 could occur at the R-loops where G-rich DNA forms a hybrid with newly transcribed RNA or at the G-rich repetitive sequences.

Gene expression in eukaryotes is dependent on the N-7 methylguanosine cap, located at the 5’ end of RNA pol II transcripts, which marks pre-mRNA for processing, stabilisation and translation initiation. The enzymes that catalyse the formation of the N-7 methylguanosine cap are recruited to RNA pol II at the initial stages of transcription. The final step in this process, N-7 methylation of the guanosine cap, is catalysed by the RNA guanine-7 methyltransferase, RNMT. RNA guanine-7 methylation is an essential process for cell viability and its up-regulation has been associated with cell transformation. However, the mechanistic details of RNMT function in mammalian cells remain elusive. In order to gain better understanding of the molecular mechanisms associated with RNA guanine-7 methylation, cellular RNMT complexes were purified from human cells and constituent proteins were identified using mass spectrometry. A novel component of the RNA guanine-7 methyltransferase complex was identified and designated as RAM (RNMT activating mini-protein). The vast majority of RNMT is found in a complex with RAM and vice versa.RAM is an RNA-binding protein, promoting recruitment of RNA to RNMT. RAM increases recombinant and cellular RNMT cap methyltransferase activity and is required for cap methylation in vivo. We therefore, describe RAM as an “obligate activator” of the human cap methyltransferase. As expected of a protein essential for cap methylation, RAM is required for gene expression, and RAM depletion results in loss of cell viability. Current studies are being focused on determining RAM/RNMT crystal structure as well as determining how the RNA guanine-7 methyltransferase complex is regulated within cells.

The phenotypic variability in human populations is partly the result of gene polymorphisms and differential gene expression. Studying the variability of gene expression across human populations is essential to understanding the molecular basis for diversity. However, key issues remain unanswered with respect to human expression variability. For example, the role of gene methylation in expression variability is uncertain, nor is it clear what role tissue-specific factors may have. Moreover, the contribution that expression variability has in aging and development is unknown. Here we classified human genes based on their expression variability in normal human breast and brain samples and identified functional aspects associated with high and low expression variability. Interestingly, both high variability and low variability gene sets are enriched for developmentally essential genes. There is limited overlap between the variably expressed genes of different tissues, indicating that tissue-specific rather than individual-specific factors are at work. We also find that methylation likely has a key role in controlling expression variability insofar as genes with low expression variability are likely to be non-methylated. Importantly, we find that genes with high population expression variability are likely to have age-, but not sex-dependent expression. Taken together, our work indicates that gene expression variability is tissue-specific, methylation-dependent, and is an important component of the natural aging process.

Cytosine methylation of CpG dinucleotides is an epigenetic modification that cells use to regulate gene expression, largely to promote transcriptional silencing. Focal hypermethylation of tumor suppressor genes (TSGs) accompanied by genomic hypomethylation are epigenetic hallmarks of malignancy. DNA methyltransferase 1 (DNMT1) is the principle vertebrate enzyme responsible for maintenance of DNA methylation and its dysregulation has been found to lead to aberrant methylation in cancer. In addition, recent findings demonstrated that the ten-eleven translocation 1 (TET1) protein functions as a 5-methylcytosine dioxygenase that converts 5-methylcytosine (5mC) bases to 5-hydroxymethylcytosine (5hmC) to mediate active DNA demethylation. Emerging evidence suggests that TET1 might function as a TSG. To understand the dynamic regulation of DNA methylation during cellular transformation, my work focused on intrinsic regulation of DNMT1 and how TET1 regulates DNA demethylation in generating a cancer methylome. The replication foci targeting sequence (RFTS) is an N-terminal domain of DNMT1 that inhibits DNA-binding and catalytic activity, suggesting that RFTS deletion would result in gain of DNMT1 function. However, other data suggested that RFTS may be a positively acting domain. To test biochemical and structural predictions that the RFTS domain of DNMT1 is inhibitory, we established cellular systems to evaluate the function of DNMT1 alleles. The data indicate that deletion of RFTS is necessary and sufficient to promote cellular transformation, focal hypermethylation of specific TSGs, and global hypomethylation. These data and human mutation data suggest that RFTS domain is a target of tumor-specific dysregulation. RAS mutations are frequently in multiple malignancies. Methylation-associated silencing of TSGs is a hallmark of RAS-driven-tumorigenesis. I discovered that suppression of TET1 by the ERK signaling cascade is responsible for promoter hypermethylation and the malignant phenotype in KRAS-transformed cells. Restoration of TET1 expression reactivates silenced TSGs and reduces colony formation. Moreover, TET1 knockdown in a cell depleted for KRAS is sufficient to rescue the inhibition of colony formation by KRAS knockdown. My findings suggest that dysregulated TET1-mediated DNA demethylation is a target responsible for epigenetic changes in cancers with KRAS activation.

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.