Mastitis represents a major issue within the dairy industry responsible for economic loss via decreased animal productivity and associated veterinary costs. Currently, there is a push to identify a phenotypic innate immune response that will yield dairy cows with an enhanced resistance to mastitis. Bovine dermal fibroblasts were used as a cell model to measure the response of individuals to Gram-negative bacterial stimuli through the TLR4 signaling pathway. Fibroblast cultures were isolated from 15 dairy heifers at 5, 11, and 16 months of age in order to determine the variability in responsiveness to LPS as well as to monitor the development of the innate immune response in calves. These individuals displayed a large range in response to LPS as measured by IL-8 production. In addition, response within individuals increased dramatically with age. To determine the cause behind this, DNA methylation was investigated as a potential player in the variation in response described both within an individual over time as well as across individuals. Fibroblast exposure to 5-aza-2'-deoxycytidine, a DNA demethylating agent, increased the cellular response to LPS, but more so in cultures that had previously displayed low responding phenotypes. This suggested that DNA methylation acted as an inhibitor of the innate immune response, and may be responsible for some degree of the variation seen in the LPS response. To determine the effect of epigenetic factors on this response, microarray analysis was conducted on RNA isolated from cells either having been epigenetically modified (DNA demethylation and histone hyperacetylation) or without undergoing any epigenetic treatment. This analysis identified 1,758 genes with altered expression due to epigenetic modification. To focus on DNA methylation's role, methylated CpG island recovery assay (MIRA-Seq) libraries were created from fibroblasts to investigate differential methylation from a group of the same individuals sampled at 5 and 16 months of age. In addition, transcriptomic data were generated by RNA-Seq from fibroblasts collected from the young and older samples and exposed to LPS for 0, 2, and 8 hours to characterize age-associated changes in the innate immune response. Cultures from older animals were much more responsive to LPS as indicated by greater expression of IL-8, IL-6, TNF-α, and CCL20 at various times in response to LPS. TLR4 and CD14 were more highly expressed in older cultures, suggesting these fibroblasts are more able to detect the presence of LPS. Analysis of the bovine fibroblast methylome revealed methylation with remarkable stability except for 20 regions along the genome undergoing major shifts due to age. Similar data were collected from fibroblasts isolated from different individuals displaying either a low or high responding phenotype resulting in 843 regions with differential methylation between groups. This suggests that DNA methylation may be playing a role in both the age-dependent and between animal differential responses to LPS, and also gives the first in depth look at the bovine fibroblast methylome and its stability over time.

Appendices data can be found at: A: http://dx.doi.org/10.15142/T35P49 B: http://dx.doi.org/10.15142/T3201B C: http://dx.doi.org/10.15142/T3X59V D: http://dx.doi.org/10.15142/T3SG6K F: http://dx.doi.org/10.15142/T3NP4N

O momento das primeiras divisões celulares pode predizer o potencial de desenvolvimento de um embrião, incluindo sua capacidade de estabelecer prenhez. Além de diferenças relacionadas ao metabolismo, estresse e sobrevivência, embriões com diferentes velocidades de desenvolvimento apresentam padrões transcricionais distintos, principalmente relacionados ao metabolismo energético e lipídico. Como o padrão transcricional é regulado por fatores epigenéticos este estudo visou caracterizar os mecanismos epigenéticos envolvidos neste fenótipo. Para isto, embriões bovinos foram produzidos in vitro utilizando sêmen sexado (fêmeas) e as 40 horas pós inseminação (hpi) foram classificados como Rápidos (4 ou mais células) ou Lentos (2 ou 3 células), permanecendo em cultivo até o estágio de blastocisto (168 hpi) (Capítulo 1) ou sendo avaliados com 40 hpi, 96 hpi ou 168 hpi (Capítulo 2). No capítulo 1, a análise da metilação global do genoma pelo sistema EmbryoGENE Methylation DNA Array (EDMA) identificou 11.584 regiões diferencialmente metiladas (DMRs) (7.976 regiões hipermetiladas em blastocistos derivados do grupo de clivagem rápida FBL - e 3.608 regiões hipermetiladas em blastocistos derivados de embriões de clivagem lenta - SBL). Os FBL apresentaram mais regiões classificadas como hipermetiladas, distribuídas ao longo do genoma, como nos íntrons, exons, promotores e elementos de repetição, enquanto em SBL, as regiões hipermetiladas estavam mais presentes nas ilhas CpG. As DMRs foram agrupadas em relação aos processos biológicos a que estavam envolvidas, e algumas das vias mais afetadas foram relacionadas à sobrevivência/diferenciação celular e metabolismo energético/lipídico. Os perfis dos transcritos dos genes diferencialmente metilados (DMs) relacionados com estas vias também foram avaliados, e a maior parte revelou mudanças na quantificação relativa. No capítulo 2, foi avaliada ao longo do desenvolvimento a presença de metilações e hidroximetilações do DNA e modificações pós-traducionais de histonas (acetilação e trimetilação da lisina 9 da histona 3 e trimetilação da lisina 27 da histona 3 H3K9ac, H3K9me3 e H3K27me3, respectivamente). A cinética das primeiras clivagens influenciou a maioria das modificações epigenéticas estudadas desde os estágios iniciais até o blastocisto (exceto pela hidroximetilação do DNA e H3K27me3). A análise dos transcritos relacionados a inclusão ou remoção dessas modificações corroborou o padrão de modificações encontrado. É possível concluir que a cinética das primeiras clivagens apresenta relação com modificações epigenéticas nos embriões e que se manterão até o final do desenvolvimento pré-implantacional, resultando em blastocistos de padrão transcricional e metabólico distintos, podendo influenciar na viabilidade embrionária. / The timing of the first cell divisions may predict the developmental potential of an embryo, including its ability to establish pregnancy. Besides differences related to metabolism, stress, and survival, embryos with different speeds of development present distinct patterns of gene expression, mainly related to energy and lipid metabolism. As gene expression is regulated by epigenetic factors, and that includes DNA methylation patterns, in this study we compared the global DNA methylation profile of embryos with different kinetics of development in order to identify general pathways and regions that are most influenced by this phenotype. For this, bovine embryos were produced in vitro using sexed semen (females) and the 40 hours post insemination (hpi) were classified as Fast (4 or more cells) or Slow (2 or 3 cells), remaining in culture until the blastocyst stage (168 hpi) (Chapter 1) or evaluated at 40 hpi, 96 hpi or 168 hpi (Chapter 2). In Chapter 1, the analysis of global DNA methylation by EmbryoGENE DNA Methylation Array (EDMA) identified 11,584 differentially methylated regions (DMRs) (7,976 regions hypermethylated in blastocysts derived from the fast cleavage group -FBL- and 3,608 hypermethylated regions in blastocyst derived from the slow cleavage group -SBL). FBL presented more regions classified as hypermethylated, distributed throughout the genome, as in introns, exons, promoters and repeating elements, whereas in SBL, hypermethylated regions were more present in the CpG islands. DMRs were grouped in relation to the biological processes to which they were involved, and some of the most affected pathways were related to cell survival/differentiation and energy/lipid metabolism. Profiles of transcripts of differentially methylated (DMs) genes related to these pathways were also evaluated, and most presented changes in relative quantification. In Chapter 2, the presence of DNA methylations and hydroxymethylations and post-translational histone modifications (histone 3 lysine 9 acetylation and trimethylation and histone 3 lysine 27 trimethylation - H3K9ac, H3K9me3 and H3K27me3, respectively). The kinetics of the first cleavages influenced most of the epigenetic modifications studied from the initial stages to the blastocyst (except DNA hydroxymethylations and H3K27me3). The analysis of the transcripts related to insertion or removal of these modifications corroborated the pattern of modifications. It is possible to conclude that the kinetics of the first cleavages is related with epigenetic modifications in embryos that will be maintained through the pre-implantation development, resulting in blastocysts with different transcriptional and metabolic patterns, which might influence the embryonic viability.

Epigenetic regulation is now known to play an important role in determining stem cell fate during normal tissue development and disease pathogenesis. In this study, we report loss of 5-hydroxymethylcytosine (5-hmC) mediated by ten-eleven translocation (TET) methylcytosine dioxygenases in keratinocyte stem cells (KSCs) and in their progenitor transit-amplifying (TA) cells of psoriatic lesions. We establish the DNA hydroxymethylation profile in both human psoriasis as well as in the imiquimod (IMQ)-induced mouse psoriasis model. Genome-wide mapping of 5-hmC in IMQ-treated mice epithelium revealed a loci-specific reduction of 5-hmC in genes associated with maintaining stem cell homeostasis including those involved in the RAR and Wnt/β-catenin signaling pathways. Restoration of TET expression in human KSC cultures via vitamin C treatment increased 5-hmC levels and induced more normal KSC differentiation. We found that by modulating 5-hmC levels in vitro, we could alter downstream expression of genes important in regulating stem cell homeostasis like nestin as well as IL-17R known to promote the psoriatic phenotype. Our findings demonstrate that loss of 5-hmC is a critical epigenomic phenomenon in KSCs and TA cells during psoriasis pathogenesis. / 2019-12-17T00:00:00Z

The sperm head contains highly compacted genome. This compaction is mediated by protamines. Sperm protamines are replaced by cytoplasmic histones after the sperm entry into the oocyte. Beside the proteins replacement, also some epigenetic remodeling occurs. One of the most studied epigenetic remodeling in early zygotes is DNA demethylation. This phenomenon was observed in some mammals (mouse, rat, monkey) but not in some other mammals (sheep) and what is more, in some of them quite inconsistent data were published (pig, human, goat, rabbit). In our work we were mostly concentrated on porcine zygotes and attempted to explain the reasons of inconsistency in observed data. Three factors were evaluated in our work - the technique of embryo production, sperm factors, and the oocyte quality. In the first part of the study (the technique of embryo production) we compared the zygotes produced by conventional in vitro fertilization and zygotes produced by intracytoplasmic sperm injection. The epigenetic remodeling was evaluated by immunolabeling. There were no differences between zygotes produced by the two mentioned techniques. The paternal genome was not demethylated in any of zygotes. The labeling with anti-H3/K9- me2 (anti dimethyl group on lysine 9 of histone 3) showed the positive labeling of both...

La complexe protéique Sir (Silent Information Regulator) de la levure bourgeonnante est l’acteur principal dans la formation de l’hétérochromatine, qui provoque l’atténuation de l’expression génique par un mécanisme épigénétique. Le complexe Sir lié à la chromatine maternelle est démonté lors de la réplication génomique et puis réformé sur les deux brins nouvellement répliqué. La dynamique de maintien de Sir sur la chromatine pendant le cycle cellulaire et dans de variables conditions de croissance n’est pas bien comprise. Pour comprendre comment la structure chromatinienne telle que l’hétérochromatine peut être héritée et par conséquent comment les structures épigénétiques sont transmises d’une génération cellulaire à l’autre, nous avons besoin de mesurer la vitesse d’échange des sous-unités du complexe Sir au cours du cycle cellulaire dans différentes conditions de croissance. Nous avons donc utilisé le système RITE qui permet d’échanger deux épitopes attachés à Sir3 (une des sous-unités de Sir) et par la suite mesurer la cinétique de remplacement de Sir3. Nous avons constaté que la Sir3 maternelle est complètement remplacée par la Sir3 nouvellement synthétisées dans les régions télomériques durant le premier cycle cellulaire après la sortie de la phase stationnaire. Nous proposons que cette reprogrammation du complexe hétérochromatique est un mécanisme d’adaptation qui assure l’activation des gènes de réponse au stress par la déstabilisation transitoire de la structure hétérochromatinienne. / The budding yeast SIR complex (Silent Information Regulator) is the principal actor in heterochromatin formation, which causes epigenetically regulated gene silencing phenotypes. The maternal chromatin bound SIR complex is disassembled during replication and then, if heterochromatin is to be restored on both daughter strands, the SIR complex has to be reformed on both strands to pre-replication levels. The dynamics of SIR complex maintenance and re-formation during the cell-cycle and in different growth conditions are however not clear. Understanding exchange rates of SIR subunits during the cell cycle and their distribution pattern to daughter chromatids after replication has important implications for how heterochromatic states may be inherited and therefore how epigenetic states are maintained from one cellular generation to the next. We therefore used the tag switch RITE system to measure genome wide turnover rates of the SIR subunit Sir3 before and after exit from stationary phase and show that maternal Sir3 subunits are completely replaced with newly synthesized Sir3 at subtelomeric regions during the first cell cycle after release from stationary phase. We propose that the observed “reset” of the heterochromatic complex is an adaptive mechanism that ensures the activation of subtelomeric stress response genes by transiently destabilizing heterochromatin structure.

Thesis advisor: Marc A.T. Muskavitch / The Notch pathway is an evolutionarily conserved mechanism of intercellular signaling that plays a central role in the development of metazoans. Here I summarize two genetic screens that utilize a rough eye phenotype created by Delta overexpression in the Drosophila eye to identify modulators of Notch pathway signaling activity. Among the many "hits" obtained from both screens, I have mapped to the Not1 gene a single complementation group that exhibits strong genetic interactions with Notch pathway mutants. NOT1 is a component of the CCR4-NOT complex, a global regulator of gene expression that exerts its effects through a variety of mechanisms, including mRNA deadenylation and direct transcriptional repression. I have conducted a series of genetic and molecular experiments in an effort to obtain more insight into the relationship between the CCR4-NOT complex and the Notch pathway. Both Not1 EMS mutations and RNAi-mediated knockdown of NOT1 expression produce phenotypes that mimic those of Notch loss-of-function pathway mutants. Knockdown of NOT1 in the developing bristle organ disrupts Notch-mediated inhibition of neuronal specification, resulting in supernumerary neurons and aberrant sheath cell specification. Knockdown of NOT1 within the developing wing margin disrupts expression of the Notch target genes Cut and Wingless, as well as the Notch ligand Delta. Phenotypic rescue experiments imply that Not1 functions downstream of Notch signal activation and acts directly on Notch target gene expression. These results suggest that NOT1 is required for Notch signal transmission in certain developmental contexts and implicate the CCR4-NOT complex as a positive regulator of the Notch pathway. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis (MSc (Biomedical Sciences))--Cape Peninsula University of Technology, 2018. / Background: Early detection of individuals who are at risk of developing Glucose Intolerance would decrease the morbidity and mortality associated with this disease. MicroRNA is one of the most widely studied biomolecules involved in epigenetic mechanisms, hence it offers unique opportunities in this regard. Circulating microRNAs are associated with disease pathogenesis during the asymptomatic stage of disease. This has therefore attracted a lot of attention as a potential biomarker for identifying individuals who have an increased risk of developing Glucose Intolerance. The identification of high risk biomarkers for Glucose Intolerance will go a long way to eliminate the possible complications that arise due to late diagnosis and treatment of Glucose Intolerance. This could ultimately lead to better ways to prevent, manage and control the Glucose Intolerance epidemic that is rampant worldwide. The aim of the study is to investigate expression of circulating microRNA’s in blood of mixed ancestry subjects with glucose intolerance. Methods: A quantitative cross-sectional study design involving 36 individuals [who were age, gender and BMI (Body Mass Index) matched] from a total population of 1989 participants of mixed ancestry descent, residing in Bellville South, South Africa was used. Participants were classified as controls (normoglycemic), pre-diabetic (preDM) and diabetic (DM) (screen detected diabetic) according to WHO criteria of 1998. MicroRNAs were extracted from serum using the Qiagen miRNeasy Serum/Plasma Kit (ThermoFisher). The purified micro RNAs were reverse-transcribed to cDNA (complementary deoxyribonucleic acid) using the Qiagen RT2 First Strand Kit. Then, using Qiagen miScript SYBR Green PCR kit and miScript miRNA PCR arrays (ThermoFisher), the real time polymerase chain reaction was done to determine the expression profile the circulating micro RNAs present in the serum of the participants. Results: The 36 participants were evenly divided into 3 groups of 12 participants each as mentioned earlier. There were significant differences between groups in the waist (cm) (p=0.0415) and waist/hip ratio (p=0.0011) with highest values in the DM group and lowest in the normal group. Clinical parameters varied significantly according to glycemic status. As expected, the FBG (mmol/L) (p<0.0001), 2 HRs Post Glucose (mmol/L) (p<0.0001), HbA1c (%) (p=0.0009), Fasting Insulin (mIU/L) (p=0.0039), were all highest in the DM and lowest in the control group. In contrast, the 2 HRs Post Insulin (mIU/L) (p = 0.0027) was highest in the preDM group and lowest in the normal group, while the Glucose/Insulin ratio (p=0.0477) was highest in the normal group and lowest in the preDM group. Triglycerides (mmol/L) (p=0.0043) and Total Chol (mmol/L) (p=0.0429) were significantly increased through the three groups, with highest values in the DM group and lowest in the normal group. Furthermore, 12 of the 84 miRNAs studied were expressed through all the 3 groups and they exhibited both inverse and positive correlations between the clinical parameters, especially the glucose parameters (Fasting blood glucose, 2 hours post glucose, Fasting blood insulin, 2 hours post insulin and Glycated Hemoglobin).

Les protéines du groupe Polybomb (PcG) sont conservées de la drosophile jusqu’à l’homme et assurent la « mémoire cellulaire » d’un état transcriptionnel réprimé au cours du développement. Un modèle a été proposé pour expliquer leur fonctionnement, qui propose que les deux principaux complexes, PRC1 et PRC2 (Polycomb Repressive Complexes 1 & 2), sont recrutés ensemble au niveau de séquences spécifiques appelés PREs (Polycomb Responsive Elements) où ils collaborent pour maintenir la chromatine dans un été réprimé.Au cours de ma thèse, j’ai voulu tester ce modèle en utilisant les disques imaginaux d’œil-antenne de drosophile, qui sont des structures larvaires préfigurant l’œil adulte. Étonnamment, alors que les mutants PRC1 et PRC2 présentent des phénotypes similaires dans l’embryon, seuls les clones mutants PRC1 présentent une transformation néoplasique et une surcroissance dans l’œil. Pour comprendre les mécanismes moléculaires qui sous-tendent ce découplage fonctionnel, nous avons réalisé des ChIP-Seq contre plusieurs marques d’histones actives et répressives, ainsi que contre des protéines du PcG. La comparaison de ces ChIP-Seq avec les profils embryonnaires a d’abord révélé un redéploiement majeur du PRC1 au stade larvaire, sur environ 1000 promoteurs actifs. Cette nouvelle classe de cibles, que nous avons appelée « Neo-PRC1 », se trouve au niveau de gènes actifs où la marque H3K27me3 normalement déposée par le PRC2 est remplacée par la marque active H3K27Ac. Ces gènes sont impliqués dans la régulation de la polarité, la prolifération ou encore la signalisation cellulaires, et un nombre substantiel d’entre eux est surexprimé dans les mutants PRC1, mais pas PRC2. Ces résultats suggèrent que l’activité suppresseur de tumeurs du PRC1 au stade larvaire découle de la régulation précise de gènes classiquement dérégulés dans les cancers, et ce en l’absence du PRC2En plus des sites situés sur des promoteurs actifs, nous avons détecté des sites PRC1 sans PRC2 au niveau de régions enrichies pour des marques de séquences amplificatrices (« Enhancers » en anglais) actives. Ces sites correspondent à des séquences amplificatrices spécifiquement actives au stade larvaire, et sont localisés à proximité de gènes codant pour des facteurs de transcription cruciaux pour le développement de l’œil, tels que les gènes du réseau de détermination de la rétine (RDGN). Pour mieux comprendre l’action du PRC1 sur ces cibles, j’ai réalisé des expériences de Hi-C (High-throughput Chromosome Conformation Capture) dans l’œil et l’embryon, révélant ainsi que ces séquences amplificatrices contactent les promoteurs proches spécifiquement au stade larvaire. De plus, la fréquence des contacts est positivement corrélée au niveau de PRC1 fixé. Étonnamment, ces gènes cibles sont sous-exprimés dans les mutants PRC1 mais pas dans les mutants PRC2, ce qui suggère que les contacts PRC1-dépendants entre ces séquences amplificatrices et leurs promoteurs cibles promeuvent la transcription. Pour vérifier cette hypothèse, j’ai étudié l’impact de la délétion via CRISPR de deux sites PRC1 impliqués dans une boucle régulatrice ; l’un situé au promoteur d’un gène du RDGN appelé dac et l’autre sur une séquence amplificatrice putative située en aval du gène. Des expériences de 3D-FISH révèlent que leur délétion entraîne la diminution des contacts entre la séquence amplificatrice et le promoteur, avec pour effet la sous-expression de dac. Ces résultats suggèrent que le PRC1 est impliqué dans la formation de boucles entre les séquences amplificatrices et leurs promoteurs cibles, et que cette topologie est nécessaire pour l’activation de ces gènes au cours du développement.Ma thèse a donc contribué à la découverte de nouvelles fonctions pour le PRC1, qui acquiert de nouvelles cibles au cours du développement et régule la transcription de gènes impliqués dans le cancer ou le développement indépendamment du PRC2, via des mécanismes dédiés. / Polycomb Group (PcG) are a set of highly conserved proteins implicated in cellular memory of transcriptional gene silencing throughout development. A classical model of PcG mode of action proposes that the two main Polycomb Repressive Complexes (PRC), PRC1 and PRC2, are co-recruited at specific DNA sequences called PREs (Polycomb Responsive Elements) where they collaborate to stably maintain a repressed chromatin state.My PhD work has challenged this collaborative model, by using as an experimental system the Drosophila larval Eye-Antennal imaginal Disc (EAD) that prefigures the adult eye. Surprisingly, while PRC1 and PRC2 mutants exhibit similar phenotypes in embryos, only PRC1 mutant clones show neoplastic transformation and massive overgrowth in EAD, while PRC2 mutant clones do not. To understand the molecular basis of this functional uncoupling, we generated ChIP-Seq directed against a large set of repressive and active Histone Marks (HTMs) as well as against core PcG proteins in EAD. A comparative analysis with Chip-Seq embryonic profiles firstly identified a massive de novo redeployment of PRC1 proteins at mostly 1000 active promoters that occurs only at larval stage. This new class of transcriptionally active PcG target genes, that we named “Neo-PRC1”, is devoid of the H3K27me3 epigenetic mark normally deposited by PRC2 and carry instead the active H3K27Ac mark. Moreover, this Neo-PRC1 category of PcG targets is enriched in ontologies linked to cell polarity, proliferation or signalling. A substantial subset of neo-PRC1 targets is up-regulated in PRC1 but not in PRC2 mutants, suggesting that the tumour-suppressor activity of PRC1 during Drosophila development might be exerted by fine-tuning the expression of cancer-related genes independently of PRC2.In addition to neo-PRC1 sites located at promoters, we next detected an enrichment of PRC1, but not PRC2, at regions enriched for active enhancer marks. These neo-sites which correspond to larval stage-specific enhancers are found in the vicinity of genes encoding for transcription factors playing a key role in EAD development, like genes implicated in the Retinal Determination Gene Network (RDGN). To understand the function of PRC1 at these enhancers, we performed comparative Hi-C (High-throughput Chromosome Conformation Capture) experiments between embryos and EADs, and discovered differential chromatin contacts occurring between the stage-specific neo-PRC1 enhancers and their closest promoters. The intensity of these 3D contacts is positively correlated with the PRC1-binding levels. Unexpectedly, in PRC1, but not in PRC2 mutants, these genes are down-regulated, suggesting that PRC1-dependent enhancer-promoter loops promote transcription. To study if larval 3D chromatin loops are PcG-dependent and functionally relevant, we analyzed the topological and transcriptional impact of two CRISPR-generated deletions affecting two PRC1 binding sites known to form a regulatory loop. These two PREs are respectively located close to the promoter and a putative 3’ enhancer of the dac locus encoding for a crucial member of the RDGN. 3D FISH experiments demonstrate that the removal of the dac endogenous PRC1 binding sites is sufficient to significantly decrease dac enhancer-promoter contacts as well as to trigger down-regulation of dac expression. Altogether, these results suggest that PRC1 might contribute to enhancer-promoter contacts at crucial developmental genes in EAD and that these PRC1-dependent long-range interactions could be necessary to allow a proper transcriptional induction during development.To summarize, my PhD project contributed in opening a new perspective, namely that in addition to conveying cellular memory, a main function of PcG correlates with a second wave of PRC1 recruitment during larval stage to subtly regulate and coordinate the expression of cancer-related and developmental genes through non-canonical molecular mechanisms.

Embryonic stem cells are derived from the inner cell mass of a blastocyst-stage embryo and are characterized by the ability to self-renew and differentiate into all cell types of an adult organism, as demonstrated by their transplantation into embryos in the mouse. Isolation of cells with similar properties from human embryos has permitted the study of human cell differentiation in vitro as might occur during development. As such, human ES cells may be useful to assess and predict the developmental toxicity of environmental compounds capable of epigenetic alterations of the genome and its expression. The first objective of my research was to validate the functional significance to maintenance of an undifferentiated human ES cell state of expressed genes whose epigenetic modification is conserved across diverse lines and/or likely to be deterministic of an embryo stem cell associated epigenetic state. The second goal was to determine the sensitivity and relationship of the expression of these genes to environmental factors known to perturb the epigenome, specifically subcytotoxic exposure to diverse organic and metallic compounds and the availability of atmospheric oxygen. siRNA-mediated knockdown of genes previously identified on the basis of the conserved methylation status of gene associated Cytosine-Guanine islands (i.e. GLIS2, HMGA1, PFDN5, TET1 and JMJD2C) and two related family members (TET2 & 3) resulted in induction of cell differentiation in two independent human ES cell lines (RH1 and H9). Differentiation was reflected by morphological changes, reduction or loss of pluripotency associated markers, qualitative and quantitative reduction in genomic 5-hmC and upregulation of diverse germinal lineage markers. Subcytotoxic exposure of the same human ES cell lines to diverse compounds known to alter the epigenome (i.e. 5-azacytidine, sodium arsenite, cadmium chloride and valproic acid) generally induced downregulation of the aforementioned genes, loss of genomic hydroxymethylation and differentiation when applied under normoxia (20% O2), the exception being valproic acid. The same treatment applied under hypoxia (0.5% O2), did not induce differentiation, with the exception of cadmium chloride. Hypoxia is a general feature of developing embryos prior to the establishment of a maternal/fetal placental interface and fetal cardiovasculature. The protective effect of hypoxia was associated with elevation of ROS, expression of the dioxygenases TET1 and JMJD2C, and genomic hydroxymethylation. This research has demonstrated that genes identified on the basis of a conserved pattern of epigenetic modification function in the maintenance of an undifferentiated human ES cell phenotype. Furthermore, a human ES cell-based toxicology test system has been developed with which one can assess the subcytotoxic effects of compounds known to disrupt the epigenome and affect development by assessing their impact on maintenance of an undifferentiated human ES cell state. This is reflected by alterations in pluripotency markers, epigenetically-defined biomarkers and changes in global 5-hmC levels and the expression of genes responsible for this epigenetic modification (TET1-3). The epigenetically-defined biomarkers of pluripotent human ES cell identity (GLIS2, HMGA1, PFDN5, JMJD2C and TET1) could serve as biomarkers for screenings of compounds at an epigenetic level as their expression has been shown to be altered upon compound exposure along with monitoring the expression of 5-hmC.