While metabolic enzymes inside the cell nucleus were initially considered “contaminants”, recent evidence has shown that these fulfill essential functions in epigenetic regulation. Indeed a model is emerging in which local metabolite pools influence various nuclear processes. In this model, the subcellular distribution and organization of metabolic factors have a crucial role in the complex logic and regulation of nuclear functions. Cancer cells exploit nuclear metabolic enzymes to alter the synthesis and utilization of metabolites that sustain their transcriptional programs allowing their abnormal proliferation. Understanding the precise molecular mechanisms that modulate the distribution of nuclear metabolic enzymes and their related biological functions has the potential to uncover novel therapeutic vulnerabilities of malignant cells. Here, we describe an unexpected subcellular distribution of acetyl-CoA carboxylase alpha (ACC1), the rate-limiting enzyme of de novo fatty acid synthesis. We found that in cancer cells, ACC1 is not restricted to the cytoplasm. Instead, at mitosis and after the nuclear envelope breakdown, it transiently redistributes into filament-like structures that contact condensed chromosomes. Simultaneous profiling of protein-protein and -DNA interactions defined ACC1 association with different factors associated with the cellular machinery that modulates chromosome segregation, including the centromere, the kinetochore, and the fibrous corona. Inducible depletion of ACC1 resulted in altered mitotic progression and accumulation of chromosome segregation defects – effects that are abolished only with the reconstituted expression of catalytically active mutants of ACC1 but not its inactive counterparts. We further found that the abundance of malonyl-CoA – the main product of ACC1 enzymatic activity – gradually increases towards the onset of mitosis, being a significant determinant for histone malonylation. Overall we uncovered a previously unknown function of ACC1 in modulating mitotic progression and chromosome segregation. Our findings support a model where local niches of malonyl-CoA might act as signal molecules for faithful chromosome segregation.

Systematic characterization of microbes in several tumors including colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) has revealed the presence of multiple species of intracellular bacteria within tumors. However, there is limited knowledge on how these bacteria colonize tumors, how they survive inside host cells, how they modulate host cell phenotypes, and if their elimination should complement cancer therapy. This is, in part, due to the lack of representative animal models, challenges in co-culture of host epithelial cells and bacteria, and limited resolution of available analytical techniques to study host-microbial interactions. I have addressed these challenges by harnessing multiple technologies from microbiology, genetic engineering, tissue engineering, and microfluidics, in order to investigate the role of an emerging oncomicrobe, Fusobacterium nucleatum, in the tumor microenvironment (TME). F. nucleatum is a Gram-negative, anaerobic bacterium that is normally found within the oral cavity. However, its selective enrichment in CRC and PDAC tumors is correlated with poor clinical outcomes. My work along with collaborators in the Verbridge, Slade, and Lu labs at Virginia Tech has revealed a multifactorial impact of F. nucleatum in influencing cancer progression. First, in CRC, we discovered that F. nucleatum infection of host cancer cells induced robust secretion of select cytokines that increased cancer cell migration, impacted cell seeding, and enhanced immune cell recruitment. In PDAC, we uncovered additional cytokines that were secreted from both normal and cancerous pancreatic cell lines upon infection with F. nucleatum that increased cancer cell proliferation and migration via paracrine and autocrine signaling, notably in the absence of immune cell participation. In order to examine the contribution of a hypoxic TME on infection dynamics, we used a multi-omics approach that combined RNA-seq and ChIP-seq of H3K27ac to determine epigenomic and transcriptomic alterations sustained within hypoxic CRC cells upon infection with F. nucleatum. Our findings revealed that F. nucleatum can subvert host cell recognition in hypoxia and can modulate the expression of multiple cancer-related genes to drive malignant transformation. Insights gained from this research will pave the way for future studies on the impact of the tumor microbiome in cancer and will identify novel targets for therapy and clinical intervention to control bacteria-induced exacerbation of cancer. / Doctor of Philosophy / Colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) are the second and third leading causes of cancer death in the United States, respectively. Recent systematic characterization of various tumor types revealed the presence of distinct bacteria within tumors. However, there is limited knowledge on how these bacteria colonize tumors, how they survive inside host cells, how they modulate host cell phenotypes, and if their elimination should complement cancer therapy. This is, in part, due to the lack of representative animal models, challenges in developing host cell-microbe co-culture models, and limited resolution of available analytical techniques to study host-microbial interactions. I have addressed these challenges by harnessing multiple technologies from microbiology, genetic engineering, tissue engineering, and microfluidics, in order to investigate the role of an emerging cancer-associated microbe, Fusobacterium nucleatum, in the tumor microenvironment (TME). F. nucleatum is a microbe commonly found within the oral cavity. However, clinical studies revealed that selective enrichment of F. nucleatum in CRC and PDAC tumors significantly correlated with poor prognosis. My work along with collaborators in the Verbridge, Slade, and Lu labs at Virginia Tech has revealed a multifactorial impact of F. nucleatum in influencing cancer progression. First, in CRC, we discovered that F. nucleatum invasion of host cancer cells induced the secretion of select proteins called cytokines that cells use to signal and communicate with each other. These cytokines directly stimulated the cell migration of host cancer cells which is usually associated with increased cancer aggressiveness. In PDAC, F. nucleatum infection induced the secretion of additional cytokines from both cancer cells and normal cells that, in addition to cell migration, impacted the proliferation of cancer cells, another feature of aggressive cancers. F. nucleatum usually thrives in a low oxygen environment that is prevalent in cancer tissue and hence, we examined how a low oxygen environment can influence infection dynamics using sequencing technologies that probe the genomic constitution within cells. Our findings revealed that F. nucleatum can escape recognition in low oxygen environments and can modulate the expression of multiple cancer-related programs within the cell to drive cancer progression. Insights gained from this research will pave the way for future studies on the impact of the tumor-associated microbes in cancer and will identify novel targets for therapy and clinical intervention to control bacteria-induced exacerbation of cancer.

Epigenetic alterations are observed in virtually all cancer types, yet there is limited understanding of their role in tumorigenesis and evolution. The role of DNA methylation has been particularly elusive in this context. While this epigenetic mark has been extensively profiled in healthy and cancerous samples, our ability to understand its relationship with underlying biological processes is still limited. Moreover, recent advancements in the profiling of cell-free DNA in circulation have sparked renowned attention toward tissue-specific and cancer-specific DNA methylation patterns. In this thesis, I present results to improve and refine the computational characterization of DNA methylation in cancer, focusing on metastatic castration-resistant prostate cancer. The first contribution is the development and performance assessment of Rockermeth, a computational methodology to leverage large-scale DNA methylation profiling data to nominate robust differentially methylated regions (DMRs). Rocker-meth can retrieve biologically relevant DNA methylation changes, as demonstrated by extensive integrative analyses with gene expression, chromatin states, and genomic annotations. The second contribution is the generation of a map of DNA methylation changes across prostate cancer progression. The application of Rockermeth and other tailored methodologies can be used to trace the critical evolutionary steps of this disease, from the healthy tissue to the most lethal metastatic AR-independent counterpart. The main result is the evidence of the ability of DNA methylation to capture a snapshot of the active transcription factors in each state of the disease, offering orthogonal information compared to standard genomic sequencing. The third contribution is the design and development of NEMO, a tailored liquid biopsy sequencing panel approach to allow non-invasive neuroendocrine castration-resistant prostate cancer detection in patients with metastatic disease. Based on previous results and the comprehensive analysis of multiple datasets, I designed a set of informative genomic regions to estimate disease burden and evidence of neuroendocrine transdifferentiation. The actual implementation of the NEMO panel produced a scalable and cost-effective strategy, which has been extensively benchmarked using both in silico and in vitro approaches. The application of NEMO to patient-derived cfDNA samples demonstrated accurate tumor content estimation and robust detection of neuroendocrine disease, making it a promising instance of liquid biopsy for CRPC.

Prenatal exposure to mixture N1, a chemical mixture consisting of four phthalate diesters,three pesticides and Bisphenol A, has been associated with behavioural changes as well as changes in gene expression in mice. In this study it was investigated whether the changes in gene expression could be explained by changes in DNA methylation. Mixture N1 was found to significantly change DNA methylation in three different genes (Nr3c1, Nr3c2 and Crhr1) on totally eight different Cytosine Guanine dinucleotides (CpG) positions. To further investigate whether these changes could be induced already during differentiation of cells, a hippocampal cell model HT22 was exposed to mixture N1 during differentiation. In this mode, mixture N1 induced a statistically significant change in the promoter region of Nr3c1. Unfortunately, this change could however not be validated, and the experiments would need to be repeated. In conclusion, this study showed that exposure to mixture N1 can result in changes in DNA methylation.

No description available.

The aim of this thesis is to bridge the gap between the state-of-the-art bioinformatic tools and resources, currently at the forefront of epigenetic analysis, and their emerging applications to non-model species in the context of plant ecology. New, high-resolution research tools are presented; first in a specific sense, by providing new genomic resources for a selected non-model plant species, and also in a broader sense, by developing new software pipelines to streamline the analysis of bisulfite sequencing data, in a manner which is applicable to a wide range of non-model plant species. The selected species is the annual field pennycress, Thlaspi arvense, which belongs in the same lineage of the Brassicaceae as the closely-related model species, Arabidopsis thaliana, and yet does not benefit from such extensive genomic resources. It is one of three key species in a Europe-wide initiative to understand how epigenetic mechanisms contribute to natural variation, stress responses and long-term adaptation of plants. To this end, this thesis provides a high-quality, chromosome-level assembly for T. arvense, alongside a rich complement of feature annotations of particular relevance to the study of epigenetics. The genome assembly encompasses a hybrid approach, involving both PacBio continuous long reads and circular consensus sequences, alongside Hi-C sequencing, PCR-free Illumina sequencing and genetic maps. The result is a significant improvement in contiguity over the existing draft state from earlier studies. Much of the basis for building an understanding of epigenetic mechanisms in non-model species centres around the study of DNA methylation, and in particular the analysis of bisulfite sequencing data to bring methylation patterns into nucleotide-level resolution. In order to maintain a broad level of comparison between T. arvense and the other selected species under the same initiative, a suite of software pipelines which include mapping, the quantification of methylation values, differential methylation between groups, and epigenome-wide association studies, have also been developed. Furthermore, presented herein is a novel algorithm which can facilitate accurate variant calling from bisulfite sequencing data using conventional approaches, such as FreeBayes or Genome Analysis ToolKit (GATK), which until now was feasible only with specifically-adapted software. This enables researchers to obtain high-quality genetic variants, often essential for contextualising the results of epigenetic experiments, without the need for additional sequencing libraries alongside. Each of these aspects are thoroughly benchmarked, integrated to a robust workflow management system, and adhere to the principles of FAIR (Findability, Accessibility, Interoperability and Reusability). Finally, further consideration is given to the unique difficulties presented by population-scale data, and a number of concepts and ideas are explored in order to improve the feasibility of such analyses. In summary, this thesis introduces new high-resolution tools to facilitate the analysis of epigenetic mechanisms, specifically relating to DNA methylation, in non-model plant data. In addition, thorough benchmarking standards are applied, showcasing the range of technical considerations which are of principal importance when developing new pipelines and tools for the analysis of bisulfite sequencing data. The complete “Epidiverse Toolkit” is available at https://github.com/EpiDiverse and will continue to be updated and improved in the future.:ABSTRACT ACKNOWLEDGEMENTS 1 INTRODUCTION 1.1 ABOUT THIS WORK 1.2 BIOLOGICAL BACKGROUND 1.2.1 Epigenetics in plant ecology 1.2.2 DNA methylation 1.2.3 Maintenance of 5mC patterns in plants 1.2.4 Distribution of 5mC patterns in plants 1.3 TECHNICAL BACKGROUND 1.3.1 DNA sequencing 1.3.2 The case for a high-quality genome assembly 1.3.3 Sequence alignment for NGS 1.3.4 Variant calling approaches 2 BUILDING A SUITABLE REFERENCE GENOME 2.1 INTRODUCTION 2.2 MATERIALS AND METHODS 2.2.1 Seeds for the reference genome development 2.2.2 Sample collection, library preparation, and DNA sequencing 2.2.3 Contig assembly and initial scaffolding 2.2.4 Re-scaffolding 2.2.5 Comparative genomics 2.3 RESULTS 2.3.1 An improved reference genome sequence 2.3.2 Comparative genomics 2.4 DISCUSSION 3 FEATURE ANNOTATION FOR EPIGENOMICS 3.1 INTRODUCTION 3.2 MATERIALS AND METHODS 3.2.1 Tissue preparation for RNA sequencing 3.2.2 RNA extraction and sequencing 3.2.3 Transcriptome assembly 3.2.4 Genome annotation 3.2.5 Transposable element annotations 3.2.6 Small RNA annotations 3.2.7 Expression atlas 3.2.8 DNA methylation 3.3 RESULTS 3.3.1 Transcriptome assembly 3.3.2 Protein-coding genes 3.3.3 Non-coding loci 3.3.4 Transposable elements 3.3.5 Small RNA 3.3.6 Pseudogenes 3.3.7 Gene expression atlas 3.3.8 DNA Methylation 3.4 DISCUSSION 4 BISULFITE SEQUENCING METHODS 4.1 INTRODUCTION 4.2 PRINCIPLES OF BISULFITE SEQUENCING 4.3 EXPERIMENTAL DESIGN 4.4 LIBRARY PREPARATION 4.4.1 Whole Genome Bisulfite Sequencing (WGBS) 4.4.2 Reduced Representation Bisulfite Sequencing (RRBS) 4.4.3 Target capture bisulfite sequencing 4.5 BIOINFORMATIC ANALYSIS OF BISULFITE DATA 4.5.1 Quality Control 4.5.2 Read Alignment 4.5.3 Methylation Calling 4.6 ALTERNATIVE METHODS 5 FROM READ ALIGNMENT TO DNA METHYLATION ANALYSIS 5.1 INTRODUCTION 5.2 MATERIALS AND METHODS 5.2.1 Reference species 5.2.2 Natural accessions 5.2.3 Read simulation 5.2.4 Read alignment 5.2.5 Mapping rates 5.2.6 Precision-recall 5.2.7 Coverage deviation 5.2.8 DNA methylation analysis 5.3 RESULTS 5.4 DISCUSSION 5.5 A PIPELINE FOR WGBS ANALYSIS 6 THERE AND BACK AGAIN: INFERRING GENOMIC INFORMATION 6.1 INTRODUCTION 6.1.1 Implementing a new approach 6.2 MATERIALS AND METHODS 6.2.1 Validation datasets 6.2.2 Read processing and alignment 6.2.3 Variant calling 6.2.4 Benchmarking 6.3 RESULTS 6.4 DISCUSSION 6.5 A PIPELINE FOR SNP VARIANT ANALYSIS 7 POPULATION-LEVEL EPIGENOMICS 7.1 INTRODUCTION 7.2 CHALLENGES IN POPULATION-LEVEL EPIGENOMICS 7.3 DIFFERENTIAL METHYLATION 7.3.1 A pipeline for case/control DMRs 7.3.2 A pipeline for population-level DMRs 7.4 EPIGENOME-WIDE ASSOCIATION STUDIES (EWAS) 7.4.1 A pipeline for EWAS analysis 7.5 GENOTYPING-BY-SEQUENCING (EPIGBS) 7.5.1 Extending the epiGBS pipeline 7.6 POPULATION-LEVEL HAPLOTYPES 7.6.1 Extending the EpiDiverse/SNP pipeline 8 CONCLUSION APPENDICES A. SUPPLEMENT: BUILDING A SUITABLE REFERENCE GENOME B. SUPPLEMENT: FEATURE ANNOTATION FOR EPIGENOMICS C. SUPPLEMENT: FROM READ ALIGNMENT TO DNA METHYLATION ANALYSIS D. SUPPLEMENT: INFERRING GENOMIC INFORMATION BIBLIOGRAPHY

Cardiovascular disease (CVD) is the leading cause of death due to its prevalence in tandem with the propensity of atherosclerosis to worsen and cause myocardial infarction and stroke. The greatest risk factor for CVD development is age. The multifactorial etiology of atherosclerosis has made CVD difficult to model and consequently little is known about CVD onset and progression. Hutchinson-Gilford Progeria Syndrome (HGPS) is a severe human premature aging disorder caused by a mutation in Lamin A that leads to the accumulation of an aberrant Lamin A protein termed progerin. Patients who harbour this mutation develop atherosclerosis and die from myocardial infarction or stroke at an average age of 13 years old. Autopsies reveal deterioration of vascular smooth muscle cells (VSMCs) in HGPS patients, underlining a strong connection between VSMC loss and predisposition to CVD development. The major aim of this thesis was to model normative vascular aging and disease using HGPS induced pluripotent stem cell (iPSC)-derived VSMCs and monitor the onset of defective epigenetic inheritance in vitro. My results indicate reprogramming of patient fibroblasts to restores a normal nuclear phenotype. Patient derived iPSC lines generated from fibroblasts are nearly indistinguishable from healthy controls in terms of pluripotency, nuclear membrane integrity, as well as transcriptional and epigenetic profiles. However, differentiation of HGPS iPSCs to generate HGPS VSMCs recapitulates many aspects of normative vascular aging exemplified by increased ROS, DNA damage and transcriptomic aberrations. Furthermore, using a multi-omic approach including RNA-sequencing, and accelerated native isolation of protein on nascent DNA, HGPS VSMCs demonstrate loss of histone acetylation due to defective MOF abundance that contributed to impaired engagement with DNA damage repair pathway. This dissertation provides insights on the mechanisms that drive the epigenetic and transcriptomic changes in HGPS vasculature, illuminating druggable pathways that may also drive CVD in the general population.

The azanucleosides decitabine and azacytidine are used widely in the treatment of myeloid neoplasia and increasingly in the context of combination therapies. Although they were long regarded as being largely interchangeable in their function as hypomethylating agents, the azanucleosides actually have different mechanisms of action; decitabine interferes primarily with the methylation of DNA and azacytidine with that of RNA. Here, we examine the role of DNA methylation in the lineage commitment of stem cells during normal hematopoiesis and consider how mutations in epigenetic regulators such as DNMT3A and TET2 can lead to clonal expansion and subsequent neoplastic progression. We also consider why the efficacy of azanucleoside treatment is not limited to neoplasias carrying mutations in epigenetic regulators. Finally, we summarise recent data describing a role for azacytidine-sensitive RNA methylation in lineage commitment and in the cellular response to stress. By summarising and interpreting evidence for azanucleoside involvement in a range of cellular processes, our review is intended to illustrate the need to consider multiple modes of action in the design and stratification of future combination therapies.

Infertility is an issue for approximately 12% of couples attempting to have a child. Of this group, 50% of the cases are due to male factor infertility. There are many reasons for decreased fecundity in men, but there remains 10% to 15% of infertile men that are diagnosed with the most severe form of infertility, non-obstructive azoospermia (NOA). A diagnosis of NOA implies the lack of sperm cells in the ejaculate with no physiological reason. The current diagnostic test and treatment consist of microscopic examination of seminal fluid and a biopsy to extract any viable sperm from the testis. This treatment is known to be problematic because of the destructive nature of surgery as well as expense. A non-invasive diagnostic test that could identify the presence of sperm in the testis at the beginning of fertility treatment would inform the patient and the physician about the functionality of the testis and thus lead to more informed decisions about treatment and potentially a decrease in cost. The ability to identify the tissue source of DNA present in the reproductive tract could facilitate a fertility diagnostic tool. Tissue specific epigenetic mechanisms are known to play a role in an organism's development. The identification of an epigenetic signature unique to sperm DNA would allow for the identification of sperm DNA in a heterologous mixture. Our lab has been able to identify a methylation signature that can consistently differentiate between sperm DNA and somatic DNA. We compared the sperm DNA signature with that of blood and testicular tissue and found that there was no overlap in epigenetic markers. To create an assay that could evaluate the presence of sperm DNA we used an Oxford Nanopore next-generation sequencing platform. Sequencing bisulfite converted DNA; we were able to retrieve the methylation status at locations of interest. A bioinformatic tool was created to analyze the thousands of reads obtained and analyze the individual methylation points within single molecules of DNA. To create a more accessible fertility test, we used the sperm DNA analysis tool to evaluate seminal cell-free DNA (cfDNA). The presence of sperm cfDNA in a patient's seminal fluid may indicate that there is sperm somewhere in the male reproductive tract even if the cells are not intact. A clinician could use this information to better advise the patient about treatment and potentially decrease cost of care.

Ovarian cancer (OVCA) is the most lethal gynecologic cancer. Chemoresistance remains a major hurdle to successful therapy and patient survival. The secreted isoform of the actin-associated protein, gelsolin (plasma gelsolin; pGSN), is highly expressed in chemoresistant than chemosensitive OVCA cells, although the mechanism underlying the differential expression is not known. Also, its overexpression significantly correlates with shortened survival of OVCA patients. DNA methylation plays a key role in the regulation of genes expression and contributing to cancer development and chemoresistance with the help of DNA methyltransferases (DNMTs) or Ten eleven translocation (TETs) enzymes. TET1 is the most studied isoform of TETs family and primarily responsible for 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) oxidation to initiate demethylation and increase in the expression of methylated genes. Whether pGSN expression in OVCA cells is regulated by DNA methylation and TET1 regulates the differential pGSN expression between chemosensitive and resistant OVCA cells is not known. In this study, we hypothesized pGSN overexpression in chemoresistant OVCA cells is due to the hypomethylation at its promoter region by TET1. Our objective was to investigate whether DNA methylation and specifically TET1 plays a role in the regulation of differential pGSN expression and chemosensitivity in OVCA. Chemosensitive and resistant OVCA cell lines of different histological subtypes were used in this study to measure pGSN and TET1 mRNA abundance and protein contents by qPCR and Western blotting respectively. Cisplatin-induced chemoresponsiveness was morphologically assessed by Hoechst staining (apoptosis). Infinium HumanMethylation450 BeadChip assay was used for global methylation analysis of twelve (12) different OVCA cells and to investigate the role of DNA methylation specifically in pGSN regulation and pGSN-induced chemoresistance. DNMTs and TETs were pharmacologically inhibited in sensitive and resistant OVCA cell using specific inhibitors. Gain-and-loss-of-function assays were carried to identify the relationship between TET1 and pGSN in OVCA chemoresponsiveness. Differential protein and mRNA expressions of pGSN and TET1 were observed between sensitive and resistant OVCA cells and cisplatin reduced their expression in sensitive but not in resistant cells. Global methylation analysis revealed hypomethylation in resistant cells compared to sensitive cells. Pharmacological inhibition of DNMTs increased pGSN protein levels in sensitive OVCA cells and decreases their responsiveness to cisplatin, however we did not observe any difference in methylation level at pGSN promoter region. TETs inhibition resulted in hypermethylation at multiple CpG sites and decreased pGSN protein level in resistant OVCA cells which was also associated with enhanced response to cisplatin, findings that suggested the methylation role of TETs in the regulation of pGSN expression in OVCA cells. Further, we found that TET1 is inversely related to pGSN and positively related to chemoresponsiveness of OVCA cells. This project does not only broaden our knowledge about the mechanistic insights into the epigenetic regulation of pGSN in OVCA chemoresistance, but it also reveals a new potential target to re-sensitize chemotherapy resistant OVCA cells. This may provide a future strategy to improve overall OVCA patient survival.