Methylation of histone 3 (H3) at lysine 36 (K36) has been implicated in several biological processes, such as DNA replication, DNA repair, and transcription. To date, at least eight distinct mammalian enzymes have been described to methylate H3K36 in vitro and/or in vivo. In this work, Set2, Nsd1, and Nsd3 Venus tagged proteins were successfully expressed in mouse embryonic stem cells and, then, analyzed by confocal microscopy, mass spectrometry (MS), and chromatin immunoprecipitation sequencing (ChIP-seq). MS analysis revealed that Setd2, Nsd1, and Nsd3 do not associate in protein complexes with each other. Setd2 was associated with RNA polymerase II subunits and two transcription elongation factors (Supt5 and Supt6), whereas Nsd1 associated with the transcription factor Zfx. In contrast, Nsd3 interacted with multiple protein complexes including Kdm1b and Brd4 complexes. Interestingly, Nsd1 and Zfx seem to be bound to chromatin during cell division. ChIP-seq analysis of the H3K36 methyltransferases showed different binding profiles at transcribed genes: Nsd1 binds near the transcription start site (TSS), Setd2 loading starts near the TSS and spreads along the gene body, while, Nsd3 is preferentially enriched at the 5’ and 3’ gene regions. Sequential deletion of PWWP and zinger-finger like domains was achieved to study any possible changes in Nsd1 and Nsd3 function. Deletion of either PHD1-4 or PHD5/C5HCH domains decreased Nsd1 recruitment to chromatin. Particularly, the PHD5/C5HCH were identified as the protein-protein interface for Zfx interaction. In agreement, Zfx knockdown also decreased Nsd1 deposition at the Oct4 and Tcl1 promoter regions. Furthermore, Nsd1 depletion reduced bulk histone H3K36me2 and histone H3K36me3 loading at the coding regions of Oct4, Rif1, Brd2, and Ccnd1. In addition, Nsd1 knockdown led to an increased Zfx deposition at promoters. Our findings suggest Zfx recruits Nsd1 to its target loci, whereas Nsd1 regulates Zfx chromatin release and further contributes to transcription regulation through its H3K36 dimethylase activity. On the other hand, loss of Nsd3’s PHD5/C5HCH or PWWP domains decreased Nsd3 binding to DNA. In addition, we demonstrate that Nsd3 is recruited to target genes in a Brd4-dependent manner. Herein, we provided further insights on how H3K36 methyltransferases are regulated, and how they contribute to changes in the epigenetic landscape in mouse embryonic stem cells.fi

Bipolar disorder is a psychiatric illness characterized by recurrent fluctuations in mood and increased risk of suicide. Twin and family studies have identified the highly heritable nature of the disorder, but the limitations of the current DNA-centric paradigm underscore the need for a new perspective to gain a clearer understanding of its basis. This project investigates various facets of bipolar disorder from an epigenetic standpoint. We used mass spectrometry-based mapping of individual DNA modification differences of the brain-derived neurotrophic factor gene. Moreover, the epigenetic basis of suicidal behaviour in bipolar disorder was investigated using DNA methylation microarrays. We also used a newly-developed enrichment technique, mTAG, to interrogate chromosome-wide DNA modification profiles using tiling microarrays in post-mortem brains of bipolar disease patients and controls. Findings from these experiments highlight observable features of epigenomes of patients affected with mood disorders, and may further the understanding of the molecular origin of psychiatric diseases.

Transcription elongation by RNA polymerase II is regulated by an array of protein complexes. Among various elongation factors, Spt5 is conserved in the three kingdoms of life. I investigated functional interactions of its C-terminal repeats (CTR) domain with several elongation protein complexes in Saccharomyces cerevisiae. By using genetics and molecular biology methods, I established two major pathways in this thesis. The first describes how BUR kinase-mediated phosphorylation of CTR domain leads to co-transcriptional recruitment of the PAF complex to regulate histone modifications on active genes. The second describes how CTR phosphorylation facilitates recruitment of capping enzymes to enhance gene splicing. Finally, several Spt5-associated protein complexes were studied, and potential molecular mechanisms underlying these observations are proposed and discussed.

Bipolar disorder is a psychiatric illness characterized by recurrent fluctuations in mood and increased risk of suicide. Twin and family studies have identified the highly heritable nature of the disorder, but the limitations of the current DNA-centric paradigm underscore the need for a new perspective to gain a clearer understanding of its basis. This project investigates various facets of bipolar disorder from an epigenetic standpoint. We used mass spectrometry-based mapping of individual DNA modification differences of the brain-derived neurotrophic factor gene. Moreover, the epigenetic basis of suicidal behaviour in bipolar disorder was investigated using DNA methylation microarrays. We also used a newly-developed enrichment technique, mTAG, to interrogate chromosome-wide DNA modification profiles using tiling microarrays in post-mortem brains of bipolar disease patients and controls. Findings from these experiments highlight observable features of epigenomes of patients affected with mood disorders, and may further the understanding of the molecular origin of psychiatric diseases.

Transcription elongation by RNA polymerase II is regulated by an array of protein complexes. Among various elongation factors, Spt5 is conserved in the three kingdoms of life. I investigated functional interactions of its C-terminal repeats (CTR) domain with several elongation protein complexes in Saccharomyces cerevisiae. By using genetics and molecular biology methods, I established two major pathways in this thesis. The first describes how BUR kinase-mediated phosphorylation of CTR domain leads to co-transcriptional recruitment of the PAF complex to regulate histone modifications on active genes. The second describes how CTR phosphorylation facilitates recruitment of capping enzymes to enhance gene splicing. Finally, several Spt5-associated protein complexes were studied, and potential molecular mechanisms underlying these observations are proposed and discussed.

Inhibitor of growth (ING) proteins have been classified as type II tumour suppressor proteins due to their ability to facilitate cellular events such as chromatin remodelling, apoptosis, angiogenesis, DNA replication, DNA repair, cell cycle progression, cell senescence and hormone response regulation. These processes are all associated with combating oncogenesis; conversely, recent evidence suggesting that ING proteins also function as oncogenes in certain cancers has spurred the investigation of ING proteins as potential anticancer targets. In order to better understand the complex role ING proteins play in the cell, the mechanisms that direct ING proteins to the chromatin template require extensive study. This dissertation investigates the role the chromatin environment plays in recruiting ING proteins by characterizing the interaction between ING proteins and chromatin. ING proteins have been shown to interact with the histone H3 lysine 4 trimethylated (H3K4me3) epigenetic mark through binding studies between peptides comprising the ING plant homeodomain (PHD) finger and the H3 N-terminal tail. However, these studies do not take into account the effect of organizing H3 into a nucleosome or the effect of the remaining ING protein structural domains. In order to address these elements, this dissertation describes binding studies between the PHD finger of Yng1 (Yng1PHD) and H3K4me3 in the context of a nucleosome, and between full-length Xenopus laevis ING1 (xING1) and H3K4me3 in the context of a nucleosome. A 6XHis tagged xING1 protein was purified, Yng1PHD was obtained from Dr. Leanne Howe, and an analog of H3K4me3 (H3KC4me3) was installed into recombinant H3 protein and used to reconstitute nucleosomes. Affinity-tag based anti-Yng1PHD and anti-xING1 pull-down assays were then used to display an in vitro H3K4 methylation-dependent interaction between Yng1PHD / xING1 and H3KC4me3 containing nucleosomes. In addition, analytical ultracentrifuge (AUC) analysis of the xING1 protein displayed the presence of 3 species containing sedimentation coefficients consistent with those that would be expected from monomeric, dimeric and tetrameric forms of xING1. Several studies have focused on the interaction between ING proteins and DNA binding proteins such as transcription factors and hormone receptors which recruit ING proteins to specific genes. However, little knowledge is available regarding the role chromatin plays in recruiting ING proteins with the exception of the interaction between the ING PHD fingers and H3K4me3. This dissertation addresses this gap in knowledge by investigating the nature of chromatin bound by the human ING1b (hING1b) protein. For this purpose, HEK293 cells were transfected with a Flag-hING1b construct. Upon fractionation of the HEK293 chromatin, Flag-hING1b was found to localize exclusively to the “Pellet” fraction. ChIP analysis of the HEK293 chromatin showed that Flag-hING1b bound nucleosomes were deprived of H3K9me3, H3K27me3 and H3S10P, contained no enrichment for H3K4me3 and H3K36me3, and were significantly enriched for H2A.Z. Lastly, a hING1b-GFP construct was transiently transfected into SKN-SH human neuroblastoma cells and found to be evenly distributed throughout the nucleus with moderate enrichment on chromatin and within the nucleolus. / Graduate

The genetic information required for the existence of a living cell of any kind is encoded in the sequence information scripted in the double helix DNA. A modern trend in biology struggles to come to grip with the amazing fact that there are so many different cell types in our body and that they are directed from the same genomic blueprint. It is clear, that the key to this feature is provided by epigenetic information that dictates how, where and when genes should be expressed. Epigenetic states “dress up” the genome by packaging it in chromatin conformations that differentially regulate accessibility for key nuclear factors and in coordination with differential localizations within the nucleus will dictate the ultimate task, expression. In the imprinted Igf2/H19 domain, this feature is determined by the interaction between the chromatin insulator protein CTCF and the unmethylated H19 imprinting control region. Here I show that CTCF interacts with many sites genome-wide and that these sites are generally protected from DNA methylation, suggesting that CTCF function has been recruited to manifest novel imprinted states during mammalian development. This thesis also describes the discovery of an epigenetically regulated network of intra and interchromosomal complexes, identified by the invented 4C method. Importantly, the disruption of CTCF binding sites at the H19 imprinting control region not only disconnects this network, but also leads to significant changes in expression patterns in the interacting partners. Interestingly, CTCF plays an important role in the regulation of the replication timing not only of the Igf2 gene, but also of all other sequences binding this factor potentially by a cell cycle-specific relocation of CTCF-DNA complexes to subnuclear compartments. Finally, I show that epigenetic marks signifying active or inactive states can be gained and lost, respectively, upon exposure to stress. As many genes belonging to the apoptotic pathway are upregulated we propose that stress-induced epigenetic lesions represent a surveillance system marking the affected cells for death to the benefit of the individual. This important observation opens our minds to the view of new intrinsic mechanisms that the cell has in order to maintain proper gene expression, and in the case of misleads there are several check points that direct the cell to towards important survival decisions.

Pediatric acute lymphoblastic leukemia (ALL) is the most common malignancy in children, which results from the malignant transformation of progenitor cells in the bone marrow into leukemic cells. The precise mechanisms for this transformation are not well defined, however recent studies suggest that aberrant regulation of gene expression or DNA methylation may play an important role. Hence, the aim of this thesis was to use novel methods to investigate genome-wide gene expression and DNA methylation patterns in a large collection of primary ALL cells from pediatric patients. With these studies, we aimed to increase the understanding of factors that regulate gene expression and DNA methylation in ALL. In the first study of the thesis we found that data obtained from genome-wide digital gene expression analysis enabled excellent cytogenetic subtype-specific classification of ALL cells and revealed new features of gene expression within the disease, such as prevalent antisense transcription and alternative polyadenylation. In the second study we used technology developed for large-scale single nucleotide polymorphism (SNP) genotyping for quantitative analysis of allele-specific gene expression (ASE), revealing widespread ASE in ALL cells. Analysis of DNA methylation in promoter regions of the genes displaying ASE using DNA-microarrays revealed frequent regulation of gene expression by DNA methylation. In the third study, using the same DNA methylation array, we identified differences in the DNA methylation patterns in ALL cells at diagnosis compared to healthy mononuclear cells from the bone marrow of the same children at remission. In the fourth study we measured the DNA methylation of >450,000 CpG sites across the genome in a large collection of ALL samples and non-leukemic control cells. We found that ALL cells displayed highly divergent DNA methylation patterns depending on their cytogenetic subtype and widespread regions of differential methylation were enriched for repressive histone marks. DNA methylation levels at distinct regions in the genome were substantially increased at relapse compared to matched cells from diagnosis. Collectively, the results presented in this thesis provide new insights into the patterns of gene expression and epigenetic changes in ALL and further increase our understanding of the development and progression of the disease, which will hopefully lead to better treatment options in the future.

Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly occurring in association with gene silencing. However, understanding the dynamics of epigenetic changes is often hindered due to the absence of adequate in vitro model systems that accurately reflect events occurring in vivo. Human mammary epithelial cells (HMECs) grown under standard culture conditions enter a growth arrest termed selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection cells, have many of the hallmarks seen in the earliest lesions of breast cancer, including transcriptional silencing and hypermethylation of the p16INK4A tumour suppressor gene. The overall aim of my thesis was to use post-selection HMECs as model system to identify and dissect the mechanism involved in early epigenetic aberrations. Firstly, using a microarray approach, I found that multiple members of the TGF-β signalling pathway were concordantly suppressed in post-selection cells, and this was associated with functional disruption of the TGF-β pathway. Interestingly, concordant gene suppression was not associated with aberrant DNA methylation, but with repressive chromatin remodelling. Secondly, to further understand the mechanism underpinning epigenetic silencing, I demonstrated using laser capture technology, that p16INK4A silencing is a precursor to DNA methylation and histone remodelling. Thirdly, I found that individual post-selection HMEC strains during the early passages shared a common 'wave' pattern of regional-specific methylation within the p16INK4A CpG island. Interestingly, the 'wave' pattern of early de novo methylation correlated with the apparent footprint of nucleosomes within the p16INK4A CpG island. Lastly, to further characterise the properties of the HMEC culture system, I demonstrated that post-selection cells do not possess a natural tumour-inducing property when transplanted into the mammary fat pad of immunocompromised mice. However, post-selection HMECs were associated with high expression of a variety of stem/progenitor markers, as well as stem/progenitor associated polycomb genes, thus demonstrating that these cells share some common features of stem/progenitor cells. The research presented in this thesis demonstrate that epigenetic changes occur early in the growth of post-selection HMECs and many of these changes are common in breast cancer.

The p53 protein is a primary mediator of apoptosis and growth arrest after exposure to DNA-damaging agents. Previous work has categorised a wild type p53 gene in the majority of childhood acute lymphoblastic leukaemia (ALL) cases, in which instance the p53 protein functions as a modulator of chemotherapy-induced cell death. In contrast, certain p53-induced proteins, such as p21WAF1, can act in an anti-apoptotic manner, and bestow resistance to chemotherapy. Previous studies of the p53 pathway in ALL have utilised cell lines and primary material. In this study a model of ALL was utilised that had previously been developed from a heterogeneous panel of patient biopsies established as xenografts in immune-deficient mice, and are adaptable for short term in vitro culture. A wild-type p53 protein response to etoposide and nutlin-3 exposure was a feature of the whole ALL xenograft panel, irrespective of clinical characteristics and disease biology. While a range of p53 target genes were induced in B-cell precursor (BCP)-ALL and T-ALL xenografts after etoposide exposure, there was negligible induction of p21WAF1 in T- ALL samples. Further work with the histone deacetylase inhibitor vorinostat facilitated p53-independent induction of p21WAF1 in BCP-ALL samples, yet failed to induce p21WAF1 in T- ALL. An association was observed between reduced p21WAF1 expression in the T-ALL samples and decreased histone H3 acetylation in the p21WAF1 promoter together with increased cytosine methylation in the first exon/intron of the p21WAF1 gene. These results suggest that p21WAF1 in T-ALL cells is subject to epigenetic modifications that cause transcriptional silencing. Defective induction of p21WAF1 in T-ALL xenografts was associated with increased sensitivity to the death-inducing effects of drugs, phosphatidylserine (PS) externalisation and caspase-3/-7 activity after drug exposure, indicating that p21WAF1 may exert an anti-apoptotic activity. As proof of principle, p21WAF1 was silenced in Nalm-6 cells by micro-RNA transduction and these cells exhibited increased sensitivity and rapid PS externalisation after drug exposure. A combination of a p21WAF1 inhibitory agent and vorinostat gave some pharmacological evidence to suggest that p21WAF1 inhibition could enhance drug efficacy. Overall, these investigations provide insight into the epigenetic regulation of p21WAF1 and demonstrate an anti-apoptotic role for p21WAF1 in childhood ALL cells.