Doctor of Philosophy

dissertationOur work focussed on how germ cell DNA is packaged and if it is poised by distinctive chromatin to influence embryo development. Finally, is misregulation of that poising a common theme observed in infertility and cancer? We profiled the epigenome in mature human sperm and found that packaging in mature sperm revealed the presence of two programs - a future program, involved in guiding embryo development and a past program, involved in spermatogenesis (Chapter 2). Next, the clearest place a chromatin problem can manifest is in infertility. We asked if the DNA methylation status of seven imprinted regions can serve as a diagnostic to inform two groups of infertile patients about the risk of their offspring developing a disorder. Although our results did not provide a causal link for the trans-generational inheritance of DNA methylation defects leading to imprinting disease, it showed a strong correlation between infertility in males and aberrations of DNA methylation at select imprinted loci (Chapter 3). Taken together, our data suggests that germ cell chromatin plays a significant role in early embryonic development and infertility. Finally, we investigated how defects in a metabolic enzyme, succinate dehydrogenase (SDH) can have an impact on chromatin packaging and transcriptome of paragangliomas. We also queried the epigenetic status of paragangliomas lacking mutations in SDH. We compared our two PGL subclasses to a progenitor cell type, neural crest cells (NCCs). Strikingly, we found that both subclasses of PGLs are phenotypically very similar. Furthermore, they share the majority of regions that gain and lose DNA methylation compared to neural crest cells. Whole exome sequencing of both PGL subclasses shows iv mutations in many epigenetic modifier genes and hence we speculate that in PGLs lacking SDH mutations, epigenetic enzymes may harbor mutations that could phenocopy the misregulation in SDH deficient tumors (Chapter 4). Together, we hope that by querying the epigenetic status in a normal system and comparing these findings to perturbed systems, we have gained more insight into the role of epigenetic misregulation in infertility and cancer

A practical view of fine-mapping and gene prioritization in the post-genome-wide association era

Over the past 15 years, genome-wide association studies (GWASs) have enabled the systematic identification of genetic loci associated with traits and diseases. However, due to resolution issues and methodological limitations, the true causal variants and genes associated with traits remain difficult to identify. In this post-GWAS era, many biological and computational fine-mapping approaches now aim to solve these issues. Here, we review fine-mapping and gene prioritization approaches that, when combined, will improve the understanding of the underlying mechanisms of complex traits and diseases. Fine-mapping of genetic variants has become increasingly sophisticated: initially, variants were simply overlapped with functional elements, but now the impact of variants on regulatory activity and direct variant-gene 3D interactions can be identified. Moreover, gene manipulation by CRISPR/Cas9, the identification of expression quantitative trait loci and the use of co-expression networks have all increased our understanding of the genes and pathways affected by GWAS loci. However, despite this progress, limitations including the lack of cell-type- and disease-specific data and the ever-increasing complexity of polygenic models of traits pose serious challenges. Indeed, the combination of fine-mapping and gene prioritization by statistical, functional and population-based strategies will be necessary to truly understand how GWAS loci contribute to complex traits and diseases

PASD1: a promising target for the immunotherapy of haematological malignancies

In general, there is a lack of good immunotherapy targets within the spectrum of haematological malignancies. However haematopoietic stem cell transplants and continuing antigen discovery have allowed further insight into how further improvements in outcomes for patients might be achieved. Most patients with haematological malignancies can be treated with conventional therapies such as radio- and chemotherapy and will attain first remission. However the removal of residual diseased cells is essential to prevent relapse and its associated high mortality. PASD1 is one of the most tissue restricted cancer-testis (CT) antigens with expression limited to primary spermatagonia in healthy tissue. However, characterisation of PASD1 expression in cancers has been predominantly focussed on haematological malignancies where the inappropriate expression of PASD1 was first identified. PASD1 has one of the highest frequencies of expression of all CT antigens in acute myeloid leukaemia, with some suggestion of its role as a biomarker in diffuse large B-cell lymphoma. Here we describe the characterisation of the function and expression patterns of PASD1 in cell lines and primary tissues. Development of DNA vaccines targeting PASD1 epitopes demonstrate effective ex vivo T-cell responses in terms of IFNγ secretion and tumour cell killing. Of particular note these vaccines have led to the destruction of cells which process and present endogenous PASD1 indicating that effectively primed CTLs could kill PASD1-positive tumour cells

Widespread Epigenetic Reprogramming in Aging Human Hematopoietic Stem Cells

At the root of the hematopoietic hierarchy reside the Janus-faced hematopoietic stem cells (HSC), capable of both self-renewal and differentiation. Aging impairs HSC function, leading to increased self-renewal, reduced homing ability and a myeloid differentiation bias. In addition, hematopoietic cells acquire somatic mutations as they age, frequently affecting epigenetic modifier genes. In this dissertation work, I provide a comprehensive characterization of epigenomic changes during normal human HSC aging and demonstrate that aged HSCs undergo widespread reduction in H3K27ac, H3K4me1 and H3K4me3, with little change in H3K27me3. Age-associated loss of enrichment of the activating histone marks H3K27ac and H3K4me3 was particularly prominent at active enhancers and bivalent promoters, respectively. Functional annotation of enhancers lost with age suggests that enhancer deregulation may contribute to HSC myeloid bias and the immune impairments observed in older individuals. Focal changes in DNA methylation were also observed with age, affecting WNT and cadherin associated pathways, and at regions that may predispose to leukemogenesis. DNA 5-hydroxymethylation displayed age-related gains, targeting GATA and KLF binding sites. Concurrent with these epigenetic changes were transcriptional downregulation and mis-splicing of epigenetic modifiers, spliceosome components, transcription factors, including many in the KLF family, and LMNA, which is mutated in Hutchinson-Gilford progeria syndrome. Together, these results establish that multiple levels of epigenetic deregulation with age converge on key hematopoietic regulatory genes and pathways contributing to aged HSC dysfunction.PHDMolecular & Cellular PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/145985/1/adelmane_1.pd

The Effects of Maternal and Postnatal Dietary Methyl Nutrients on Epigenetic Changes that Lead to Non-Communicable Diseases in Adulthood

The risk for non-communicable diseases in adulthood can be programmed by early nutrition. This programming is mediated by changes in expression of key genes in various metabolic pathways during development, which persist into adulthood. These developmental modifications of genes are due to epigenetic alterations in DNA methylation patterns. Recent studies have demonstrated that DNA methylation can be affected by maternal or early postnatal diets. Because methyl groups for methylation reactions come from methionine cycle nutrients (i.e., methionine, choline, betaine, folate), deficiency or supplementation of these methyl nutrients can directly change epigenetic regulation of genes permanently. Although many studies have described the early programming of adult diseases by maternal and infant nutrition, this review discusses studies that have associated early dietary methyl nutrient manipulation with direct effects on epigenetic patterns that could lead to chronic diseases in adulthood. The maternal supply of methyl nutrients during gestation and lactation can alter epigenetics, but programming effects vary depending on the timing of dietary intervention, the type of methyl nutrient manipulated, and the tissue responsible for the phenotype. Moreover, the postnatal manipulation of methyl nutrients can program epigenetics, but more research is needed on whether this approach can rescue maternally programmed offspring

Computational Integrative Models for Cellular Conversion: Application to Cellular Reprogramming and Disease Modeling

The groundbreaking identification of only four transcription factors that are able to induce pluripotency in any somatic cell upon perturbation stimulated the discovery of copious amounts of instructive factors triggering different cellular conversions. Such conversions are highly significant to regenerative medicine with its ultimate goal of replacing or regenerating damaged and lost cells. Precise directed conversion of damaged cells into healthy cells offers the tantalizing prospect of promoting regeneration in situ. In the advent of high-throughput sequencing technologies, the distinct transcriptional and accessible chromatin landscapes of several cell types have been characterized. This characterization provided clear evidences for the existence of cell type specific gene regulatory networks determined by their distinct epigenetic landscapes that control cellular phenotypes. Further, these networks are known to dynamically change during the ectopic expression of genes initiating cellular conversions and stabilize again to represent the desired phenotype. Over the years, several computational approaches have been developed to leverage the large amounts of high-throughput datasets for a systematic prediction of instructive factors that can potentially induce desired cellular conversions. To date, the most promising approaches rely on the reconstruction of gene regulatory networks for a panel of well-studied cell types relying predominantly on transcriptional data alone. Though useful, these methods are not designed for newly identified cell types as their frameworks are restricted only to the panel of cell types originally incorporated. More importantly, these approaches rely majorly on gene expression data and cannot account for the cell type specific regulations modulated by the interplay of the transcriptional and epigenetic landscape. In this thesis, a computational method for reconstructing cell type specific gene regulatory networks is proposed that aims at addressing the aforementioned limitations of current approaches. This method integrates transcriptomics, chromatin accessibility assays and available prior knowledge about gene regulatory interactions for predicting instructive factors that can potentially induce desired cellular conversions. Its application to the prioritization of drugs for reverting pathologic phenotypes and the identification of instructive factors for inducing the cellular conversion of adipocytes into osteoblasts underlines the potential to assist in the discovery of novel therapeutic interventions

Computational Integrative Models for Cellular Conversion: Application to Cellular Reprogramming and Disease Modeling

The groundbreaking identification of only four transcription factors that are able to induce pluripotency in any somatic cell upon perturbation stimulated the discovery of copious amounts of instructive factors triggering different cellular conversions. Such conversions are highly significant to regenerative medicine with its ultimate goal of replacing or regenerating damaged and lost cells. Precise directed conversion of damaged cells into healthy cells offers the tantalizing prospect of promoting regeneration in situ. In the advent of high-throughput sequencing technologies, the distinct transcriptional and accessible chromatin landscapes of several cell types have been characterized. This characterization provided clear evidences for the existence of cell type specific gene regulatory networks determined by their distinct epigenetic landscapes that control cellular phenotypes. Further, these networks are known to dynamically change during the ectopic expression of genes initiating cellular conversions and stabilize again to represent the desired phenotype. Over the years, several computational approaches have been developed to leverage the large amounts of high-throughput datasets for a systematic prediction of instructive factors that can potentially induce desired cellular conversions. To date, the most promising approaches rely on the reconstruction of gene regulatory networks for a panel of well-studied cell types relying predominantly on transcriptional data alone. Though useful, these methods are not designed for newly identified cell types as their frameworks are restricted only to the panel of cell types originally incorporated. More importantly, these approaches rely majorly on gene expression data and cannot account for the cell type specific regulations modulated by the interplay of the transcriptional and epigenetic landscape. In this thesis, a computational method for reconstructing cell type specific gene regulatory networks is proposed that aims at addressing the aforementioned limitations of current approaches. This method integrates transcriptomics, chromatin accessibility assays and available prior knowledge about gene regulatory interactions for predicting instructive factors that can potentially induce desired cellular conversions. Its application to the prioritization of drugs for reverting pathologic phenotypes and the identification of instructive factors for inducing the cellular conversion of adipocytes into osteoblasts underlines the potential to assist in the discovery of novel therapeutic interventions