I have investigated epigenetic mechanisms of acquired endocrine-resistance in breast cancer using an in vitro model system based on estrogen-dependent MCF7 cells and their derivatives, LCC1 and LCC9. LCC1 cells, derived from MCF7 after passage in ovariectomised mice and routinely cultured in vitro in the absence of estrogen, exhibit estrogen-independent growth. They retain sensitivity to tamoxifen and fulvestrant. LCC9 cells, derived from LCC1 cells by growing them in increasing concentrations of fulvestrant, are completely estrogen-independent and are resistant to fulvestrant and cross-resistant to tamoxifen. When compared to MCF7 cells, LCC1 cells have marked up-regulation of the estrogen receptor α (ERα) protein that is not concomitant with increased estrogen receptor 1 (ESR1) transcription, suggesting a role for estrogen in controlling the proteasomal degradation of ERα. However, despite being grown in the same estrogen-deprived conditions, LCC9 cells do not have up-regulated ERα levels. As LCC1 cells retain sensitivity to tamoxifen and fulvestrant, these data suggest that LCC1 have developed estrogen-independence through ERα uncoupled from its ligand. However, LCC9 cells appear to have developed an alternative mechanism which is not dependent on ERα, presumably explaining their resistance to fulvestrant. I have studied global gene expression changes in the presence and absence of estrogen in these cell lines, using oligonucleotide microarrays, and correlated these data with global DNA methylation data derived from methylation arrays, which interrogate the methylation status of approximately 27,000 CpG dinucleotides in the genome. The analysis led to the discovery of more than 5,000 genes that were potentially either up-regulated or down-regulated by estrogen in MCF7 cells, either directly or indirectly. The transcriptional response to estrogen was generally muted in LCC1 and LCC9 compared with MCF7, but was not completely absent. I used various methods based on differential gene expression to parse the data, including gene ontology analysis, aiming to select genes for further mechanistic study. However, none of these methods led to the conclusive identification of a specific gene (or set of genes) that might have accounted for the physiological differences between the cell lines. In one strategy, I reasoned that, as the endocrine-resistant cells had lost their estrogen-dependence, genes involved might be regulated in an estrogen-dependent manner in MCF7 cells, without exhibiting misregulation in LCC9. This led to the identification of DUSP1 as a candidate gene, which was taken forward for mechanistic study because of its potential role in regulating ERα expression. However, when over-expressing DUSP1 in LCC9 cells, I could not demonstrate any effect on ERα levels. The final approach taken was to identify genes that might have been epigenetically deregulated, being both estrogen-regulated and deregulated in association with aberrant DNA methylation in the estrogen-independent cell lines. Surprisingly, given the phenotypic differences between the cell lines, only a very few genes were significantly methylated between cell lines. Of those that were differentially methylated between MCF7 cells and LCC1/9, only three exhibited the expected inverse correlation between methylation and expression. Of these, the gene CYBA was selected for further investigation. CYBA is a critical component of the NAPDH oxidase complex which is involved in generating oxygen free-radicals. My work suggests CYBA expression is estrogen-dependent, and that chronic estrogen deprivation leads to the epigenetic inactivation of CYBA in breast cancer cells. I speculate that the epigenetic suppression of CYBA may protect cells from the oxidant damage that results from estrogen deprivation and may be part of the mechanism that leads to acquired endocrine-resistance in previously sensitive cells.

Asthma is a heterogeneous group of conditions that typically begin in early life and result in recurrent, reversible bronchial obstruction. The role played by epigenetic mechanisms in the pathogenesis of childhood asthma is understood only in part. Here we discuss asthma epigenetics within a developmental perspective based on our recent demonstration that the epigenetic trajectory to childhood asthma begins at birth. We next discuss how this trajectory may be affected by prenatal environmental exposures. Finally, we examine in vitro studies that model the impact of asthma-associated exposures on the epigenome. All of these studies specifically surveyed human DNA methylation and involved a genome-wide component. In combination, their results broaden our understanding of asthma pathogenesis and the role the methylome plays in this process.

In patients with multiple sclerosis (MS), microglia become activated due to the autoimmune inflammatory response which is directed against the central nervous system (CNS). Following the first disease relapse, microglia remain activated and do not return to a resting state during remissions. Chronically-activated microglia release inflammatory mediators that cause CNS tissue damage, and as such, MS progression has been associated with widespread, chronic microglial activation that correlates with neurodegeneration. To date, only one histone demethylase, Jmjd3, has been described to have a role in inflammation. In agreement with this, up-regulation of Jmjd3 expression was observed following microglial treatment with several pro-inflammatory stimuli, including a range of toll-like receptors ligands and cytokines, suggesting a universal role of Jmjd3 during microglial activation. Subsequent ChIP-qPCR assays revealed that Jmjd3 was recruited to the promoters of Il6, Ccl3, Ccl5 and Nos2 following activation, which, in turn, presented a decrease in their H3K27me3 levels. Using an experimental autoimmune encephalomyelitis (EAE) mouse model of MS, Jmjd3 expression was shown to be increased in activated microglia from mice in the acute and late phases of disease. Immunization with complete Freud’s adjuvant (CFA) alone, also caused microglial activation with Jmjd3 induction, indicating a CFA-mediated TLR2 and TLR4 stimulation of microglia. Further investigation, in which primary microglia were isolated from mice deficient in Jmjd3 (Jmjd3-/-), however demonstrated that the absence of Jmjd3 alone had no resultant effect on the expression of a subset of immune response and inflammation related genes, including the Jmjd3 target genes Il6, Ccl3, Ccl5 and Nos2, before or after activation. This suggested that Jmjd3 acts in concert with a repertoire of other demethylases to facilitate microglia activation, and as such was rendered redundant in this setting. Deciphering the epigenetic profile of microglia in MS and determining whether it is involved in the maintenance of chronic microglial activation in the progressive phase of the disease remains an important line of investigation, and through a clearer understanding of its role in MS pathophysiology, could lead to the development of novel therapeutic interventions in the future.

Thesis advisor: Hugh P. Cam / Meiotic homologous recombination (HR) is not uniform across eukaryotic genomes, creating regions of strong recombination activity dubbed recombination hotspots, and regions of low recombination activity dubbed coldspots. Considerable attention has led to discoveries of a host of factors controlling the formation of hotspots. However, the determinants of coldspots are not as clearly defined. I have previously shown that CENP-B homologs of the fission yeast Schizosaccharomyces pombe have a genome surveillance role in regulating the nuclear organization and expression of Tf2 retrotransposons. Here, I reveal an additional role for CENP-Bs in suppressing meiotic recombination of Tf2s. I describe the development of a random sporulation assay to rapidly screen thousands of meiotic progeny for recombination across a locus in a variety of genetic backgrounds. Loss of any CENP-B family members (Abp1, Cbh1, Cbh2), results in increased HR at Tf2s. I show that Abp1, which acts as the primary determinant of HR suppression at Tf2s, is required to maintain proper recombination exchange of homologous alleles flanking a Tf2. In addition, Abp1-mediated suppression of HR at Tf2s requires all three of its domains with distinct functions in transcriptional repression and higher-order genome organization. I show that this suppression is likely mediated by Abp1 binding to specific motifs near the 3’end of flanking LTRs. I demonstrate that HR suppression of Tf2s can be robustly maintained despite disruption to chromatin factors essential for transcriptional repression and nuclear organization of Tf2s. Intriguingly, I uncover a surprising cooperation between the histone methyltransferase Set1 responsible for histone H3 lysine 4 methylation and the non-homologous end joining pathway in ensuring the suppression of HR at Tf2s. Furthermore, I identify a role for the architectural protein condensin involved in 3D chromatin organization and chromosome condensation in restricting HR at Tf2s. My study identifies a molecular pathway involving functional cooperation between a transcription factor with epigenetic regulators, DNA repair pathway, and chromosome organizers to regulate meiotic recombination at interspersed repeats. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

DNA methylation plays a crucial role in human health, especially cancer. Traditional DNA methylation analysis aims to identify CpGs/genes with differential methylation (DM) between experimental groups. Differential variability (DV) was recently observed that contributes to cancer heterogeneity and was also shown to be essential in detecting early DNA methylation alterations, notably epigenetic field defects. Moreover, studies have demonstrated that environmental factors may modify the effect of DNA methylation on health outcomes, or vice versa. Therefore, this dissertation seeks to develop new statistical methods for epigenetic data focusing on DV and interactions when efficient analytical tools are lacking. First, as neighboring CpG sites are usually highly correlated, we introduced a new method to detect differentially methylated regions (DMRs) that uses combined DM and DV signals between diseased and non-diseased groups. Next, using both DM and DV signals, we considered the problem of identifying epigenetic field defects, when CpG-site-level DM and DV signals are minimal and hard to be detected by existing methods. We proposed a weighted epigenetic distance-based method that accumulates CpG-site-level DM and DV signals in a gene. Here DV signals were captured by a pseudo-data matrix constructed using centered quadratic methylation measures. CpG-site-level association signal annotations were introduced as weights in distance calculations to up-weight signal CpGs and down-weight noise CpGs to further boost the study power. Lastly, we extended the weighted epigenetic distance-based method to incorporate DNA methylation by environment interactions in the detection of overall association between DNA methylation and health outcomes. A pseudo-data matrix was constructed with cross-product terms between DNA methylation and environmental factors that is able to capture their interactions. The superior performance of the proposed methods were shown through intensive simulation studies and real data applications to multiple DNA methylation data.

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disorder caused by a homozygous GAA repeat expansion mutation in intron 1 of the frataxin gene (FXN), which instigates transcriptional issues. As a consequence, reduced levels of frataxin protein lead to mitochondrial iron accumulation, oxidative stress and ultimately cell death; particularly in dorsal root ganglia (DRG) sensory neurons and the dentate nucleus of the cerebellum. In addition to neurological disability, FRDA is associated with cardiomyopathy, diabetes mellitus and skeletal deformities. Currently there is no effective treatment for FRDA and patients die prematurely. Recent findings suggest that abnormal GAA expansion plays a role in histone modification, subjecting the FXN gene to heterochromatin silencing. Therefore, as an epigenetic-based therapy, I investigated the efficacy and tolerability of two histone methyltransferase (HMTase) inhibitor compounds, BIX0194 (G9a-inhibitor) and GSK126 (EZH2-inhibitor), to specifically target and reduce H3K9me2/3 and H3K27me3 levels, respectively, in FRDA human and mouse primary fibroblasts. We show that a combination treatment of BIX0194 and GSK126, significantly increased FXN gene expression levels and reduced the repressive histone marks. However, no increase in frataxin expression was seen. Nevertheless, our results are still promising and may encourage to investigate HMTase inhibitors with other synergistic epigenetic-based therapies for further preliminary studies. Additionally, it has been reported that ubiquitin-proteasome pathway (UPP) controls frataxin stability, thus leading to the development of new therapeutic approaches aimed at preventing the degradation of frataxin. Here we investigated the efficacy of various proteasome inhibitors (MG132, Bortezomib, Salinosporamide A and Ixazomib) using human primary fibroblasts. Only treatments using ixazomib indicated a small increase in frataxin protein; II however, an increase in the cell cycle stress modulator, p27Kip1, was also observed. Therefore, at this stage the use of proteasome inhibitor compounds cannot be advocated for FRDA therapy. Moreover, a study has proposed that increased degradation of D-serine by D-amino acid oxidase (DAO), may lead to low NMDA functioning and impair neural signalling, causing ataxia. Therefore, we investigated a DAO inhibitor, TAK-831, on the YG8sR FRDA mouse model, and detected a significant improvement in ataxia motor coordination deficits. TAK 831 is now proposed for further studies and is currently undergoing randomized Phase 2 clinical trials for FRDA in USA.

Transcriptional repression is maintained by many molecular processes, including DNA methylation and polycomb repression. These two systems are both associated with chromatin modification at the promoters of silent genes, and are both essential for mammalian development. Previous work has shown that DNMT proteins are required for correct targeting of polycomb repressive complexes (PRCs). In this thesis, I investigate whether targeting of DNA modification has a reciprocal dependence on the polycomb machinery by mapping DNA modification in wild-type and PRC-mutant ES cells (Ring1B null, EED null, and Ring1B/EED duble null). I find that the loss of PRCs results in increased DNA modification at sites normally targeted by de novo DNA methyltransferase which lose H3K4 methylation upon PRC removal. This increased DNA modificaiton is associated with increased gene expression when found at CpG island shores of genes marked by the PRC-mediated histone modifications H3K27me3 and H2AK119ub, but not genes lacking these marks. Gene misregulation may be further linked to DNA modification changes by increased DNA modification at enhancers. While loss of either Ring1B or EED led primarily to increases in DNA modification at regions dependant on DNMT3A/DNMT3B, the combined loss of Ring1B and EED results in widespread loss of DNA modification at sites more dependent on DNMT1 activity. This thesis suggests an interplay between PRCs and DNA modification placement which is relevant to the cntrol of gene expression.

Plant viruses can cause many plant diseases, which result in substantial damage to crop production. To overcome viral infections, plants evolved RNA silencing which can recognise viral RNAs during their replications and slice them into small RNA (sRNA) using antiviral nucleases called DICER or Dicer-like (DCL). The resulting virus-derived small interfering RNA (vsiRNA, 21-24 nucleotides) then guides effector nucleases, namely ARGONAUTE (AGO), to cleave viral RNAs in the cytoplasm in a nucleotide-specific manner. However, the activity of vsiRNA is not restricted to the control of viral RNA accumulation. Virus-derived sRNAs can regulate host gene expression if host mRNAs share sequence complementarity with vsiRNAs. Interestingly, vsiRNAs are also able to target and methylate homologous DNA sequences in the nucleus indicating that vsiRNAs have potential to regulate endogenous genes at transcriptional level by modifying the epigenetic status of gene promoter sequences. This mechanism is referred to as transcriptional gene silencing (TGS). Thus, RNA silencing opens up new strategies to stably and heritably alter gene expression in plants. However, the mechanisms and efficacy of plant virus-induced TGS are largely unknown. The aim of my PhD was to investigate the molecular and environmental factors that are involved in virus-induced epigenetic modifications in the infected plants and in their progeny. First, I examined the required sequence complementary between sRNAs and their nuclear target sequence. I demonstrated for the first time that nuclear-imported vsiRNAs can induce RNA-directed DNA methylation (RdDM) and subsequently heritable virus-induced transcriptional gene silencing (ViTGS) even when they do not share 100% nucleotide sequence complementarity with the target DNA. This finding reveals a more dynamic interaction between viral RNAs and the host epigenome than previously thought. Secondly, I explored how environmental stimuli such as light and temperature can affect the efficacy of ViTGS. I found that ViTGS is greatly inhibited at high temperature. Using RNA-seq, I established that inefficient ViTGS at high temperature is due to the limited production of secondary sRNAs that may limit the initiation, amplification and spreading of virus-induced DNA methylation to neighbouring cells and down generations. Lastly, I studied the link between the viral suppressors of RNA silencing (VSRs): viral proteins that can interfere with plant RNA silencing and ViTGS. I established that VSRs of certain viruses can impair TGS in infected tissues, suggesting that viruses may alter the epigenome and consequently plant gene expression in the infected plants and their progeny. Collectively, my work reveals how viruses can re-program the epigenome of infected plants, and deepens our knowledge of how we can harness pathogens to modify the epigenome for plant breeding.

Cancer has been thought of as a mostly genetic phenomenon, however recent research into epigenetic causes of cancer emphasizes that these causes of cancer are also important. RIZ1 is a tumor suppressor which is silenced in many human leukemias, such as human Acute Myeloid Leukemia and Chronic Myelogenous Leukemia. It was the goal of this thesis to re-express RIZ1 using three epigenetic drugs: decitabine, a DNA methylation inhibitor, Trichostatin A, a histone deacetylase inhibitor and chaetocin, an inhibitor of SUV39h1. Cells were treated with these drugs and analyzed for toxicity, methylation status, and RIZ1 expression levels. The synergy between the drugs was also determined. It was found that cells treated with decitabine and chaetocin had an induction of RIZ1 expression. Chaetocin induced RIZ1 expression without affecting the methylation status of the cell. Also, cells which were treated with decitabine paired with either Trichostatin A or chaetocin showed the highest amount of RIZ1 expression. Cells treated with all three drugs together had a higher amount of RIZ1 expression than cells treated with either drug alone, however had less expression than cells which had been treated with decitabine paired with either Trichostatin A or chaetocin. Using these data a model was developed in which H3K9 methylation is the dominant epigenetic event in transcriptional silencing.

Cancer has been thought of as a mostly genetic phenomenon, however recent research into epigenetic causes of cancer emphasizes that these causes of cancer are also important. RIZ1 is a tumor suppressor which is silenced in many human leukemias, such as human Acute Myeloid Leukemia and Chronic Myelogenous Leukemia. It was the goal of this thesis to re-express RIZ1 using three epigenetic drugs: decitabine, a DNA methylation inhibitor, Trichostatin A, a histone deacetylase inhibitor and chaetocin, an inhibitor of SUV39h1. Cells were treated with these drugs and analyzed for toxicity, methylation status, and RIZ1 expression levels. The synergy between the drugs was also determined. It was found that cells treated with decitabine and chaetocin had an induction of RIZ1 expression. Chaetocin induced RIZ1 expression without affecting the methylation status of the cell. Also, cells which were treated with decitabine paired with either Trichostatin A or chaetocin showed the highest amount of RIZ1 expression. Cells treated with all three drugs together had a higher amount of RIZ1 expression than cells treated with either drug alone, however had less expression than cells which had been treated with decitabine paired with either Trichostatin A or chaetocin. Using these data a model was developed in which H3K9 methylation is the dominant epigenetic event in transcriptional silencing.