This study examined the DNA methylation profile of endothelial nitric oxide synthase (eNOS) in the placental villous trophoblast in first, second trimester and healthy term placentas. Syncytiotrophoblast DNA revealed a heterogeneous methylation pattern in the first trimester eNOS proximal promoter and transitioned to a densely methylated pattern at term. Healthy, term syncytiotrophoblast and cytotrophoblast obtained through cytotrophoblast isolation technique provided purified cell samples for RNA and DNA extraction. Real-time PCR (rt-PCR) verified the presence and quantity of eNOS mRNA. In summary, the main findings of this thesis are heterogeneous methylation in first trimester compared to hypermethylation at term; greater eNOS expression and variable methylation in term syncytiotrophoblast compared to cytotrophoblast. A heterogeneous methylation pattern in eNOS has been identified in this study and recent stem cell studies in our lab, and we propose it represents plasticity in gene expression, particularly in early development.

Eukaryotic organisms utilize post-translational modifications of highly conserved histone proteins to control gene expression programs. Methylation of lysine 4 on histone H3 (H3K4me) in particular, is thought to be associated with actively transcribed DNA. Paradoxically, recent evidence has suggested that H3K4me has a repressive function as well. Pho23, a member of the highly conserved ING family of tumour suppressor proteins, binds H3K4me and is a component of the gene repressive complex, Rpd3L. My genetic analysis suggests that Pho23 controls transcriptional repression via H3K4me and that Pho23 is itself regulated by the sequence-specific DNA-binding protein Ume6. Moreover, this Ume6-regulated function appears to be governed by Ume6 phosphorylation by Mck1, an evolutionarily conserved kinase. Finally, while Ume6/Pho23 are known to function together with the histone deacteylase Rpd3, my findings suggest the existence of an Rpd3-independent function for Pho23.

This study examined DNA methylation patterns at promoters of endothelial cell (EC)-enriched genes during differentiation of mouse ES cells towards the EC. We have previously shown that eNOS, CD31, VE-cadherin and vWF, which have an EC-enriched pattern of gene expression are differentially methylated between EC and vascular smooth muscle cells. Given that differential promoter DNA methylation is functionally important we asked when these distinct patterns are established. Using the hanging drop method to differentiate ES cells, followed by FACS, we isolated early (EB-day4 VEGFR2-positive) and late (EB-day7 CD31-positive) endothelial progenitor cells. Though current paradigms suggest that lineage-restricted genes are methylated in ES cells, we show heterogeneous promoter DNA methylation. We show DNA demethylation at the CD31 promoter in EB-day 7 CD31-positive cells. In contrast, the eNOS promoter is still heavily methylated in EB-day 7 CD31 positive cells compared with murine EC where there is no DNA methylation.

This study examined the DNA methylation profile of endothelial nitric oxide synthase (eNOS) in the placental villous trophoblast in first, second trimester and healthy term placentas. Syncytiotrophoblast DNA revealed a heterogeneous methylation pattern in the first trimester eNOS proximal promoter and transitioned to a densely methylated pattern at term. Healthy, term syncytiotrophoblast and cytotrophoblast obtained through cytotrophoblast isolation technique provided purified cell samples for RNA and DNA extraction. Real-time PCR (rt-PCR) verified the presence and quantity of eNOS mRNA. In summary, the main findings of this thesis are heterogeneous methylation in first trimester compared to hypermethylation at term; greater eNOS expression and variable methylation in term syncytiotrophoblast compared to cytotrophoblast. A heterogeneous methylation pattern in eNOS has been identified in this study and recent stem cell studies in our lab, and we propose it represents plasticity in gene expression, particularly in early development.

During early mammalian embryogenesis, the embryonic genome undergoes critical reprogramming events that include changes in both DNA methylation and histone modifications necessary to control chromatin structure and thus, gene expression. Improper reprogramming of the epigenome during this window of development can lead to a vast number of imprinting anomalies, which are increased in children and livestock conceived in vitro. In the bovine, which closely resembles human preimplantation development, epigenetic changes occur from fertilization through the blastocyst stages. In particular, and concurrent with embryonic genome activation (EGA), de novo DNA methylation begins at the 8-cell stage. In order to explore the roles of histone-modifying enzymes during this crucial period of development, we characterized the transcript expression of several enzymes key enzymes across in vitro bovine preimplantation development using quantitative real-time PCR. Two of the 7 genes analyzed (Suz12 and Lsh) exhibited notable increases at the 8-16 cell stages, with basal levels observed both before and after this. These increases coincided with both EGA and de novo DNA methylation. We further explored their roles in bovine preimplantation embryos by knocking down expression via the use of gene-specific targeting siRNAs. Independent suppression of either Suz12 or Lsh via cytoplasmic microinjection of targeting siRNAs resulted in lower development rates (p < 0.0001), and poorer embryo quality of the morulas and blastocysts that survived. In addition, Suz12 suppression led to reductions in both H3K27 (p < 0.0001) and H3K9 (p = 0.07) trimethylation, and an increase in DNA methylation levels (p < 0.0001), as compared to the null-injected controls. Lsh suppression did not change H3K27, but led to a reduction in H3K9 trimethylation (p = 0.006) and an increase in DNA methylation (p < 0.0001). Clearly our data demonstrate that these epigenetic modifiers play a critical role in formation of the embryonic epigenome, but further research would be necessary in order to fully characterize gene activities during this developmental window.

Although structurally unrelated, the human killer cell immunoglobulin-like (KIR) and the rodent lectin-like Ly49 receptors serve similar functional roles in natural killer (NK) cells. Moreover, both gene families display variegated and mostly mono-allelic expression patterns established at the transcriptional level. DNA methylation, but not histone modifications, has recently been shown to play an important role in maintenance of the expression patterns of KIR genes but the potential role of DNA methylation in the expression of Ly49 genes was unknown. My thesis focuses on the role of epigenetic modifications, especially DNA methylation, in the maintenance of mouse Ly49 gene expression. I show that hypomethylation of the region encompassing the main promoter of Ly49a and Ly49c in primary C57BL/6 (B6) mouse NK cells correlates with expression of these genes. Using B6 x BALB/c Fl hybrid mice, I demonstrate that the expressed allele of Ly49a is hypomethylated while the non-expressed allele is heavily methylated, indicating a role for epigenetics in maintaining mono-allelic Ly49 gene expression. Furthermore, the Ly49a promoter region is heavily methylated in fetal NK cells but variably methylated in non-lymphoid tissues. In apparent contrast to the KIR genes, I show that histone acetylation state of the promoter region strictly correlate with Ly49A and Ly49G expression status. Also, the instability of Ly49G expression on some lymphoid cell lines is at least in part due to changes in the level of histone acetylation of the promoter region. As for the activating Ly49 receptors, it seems that although DNA methylation levels of the promoter regions do correlate with the state of expression of these receptors, the pattern of DNA methylation is different from that of the inhibitory Ly49a and c genes. In conclusion, my results support a role for epigenetic mechanisms in the maintenance of Ly49 expression. Moreover, these epigenetic mechanisms appear to vary among the Ly49 genes and also differ from those governing KIR expression.

<p>Genomic imprinting is the parent-of-origin dependent monoallelic expression of select developmentally important genes that are regulated by epigenetic mechanisms. It is believed to have co-evolved with placentation in the Therian lineage, but it is unclear whether this phenomenon arose in a convergent or divergent manner in the Metatherians (those with a rudimentary placenta) and Eutherians (true placental mammals). Moreover, the precise epigenetic mechanisms involved in establishing genomic imprinting (DNA methylation or histone modifications) are still poorly defined. Thus, I studied Metatherian orthologues of Eutherian imprinted loci using Monodelphis domestica as a model organism. L3MBTL and HTR2A were monoallelically expressed; PEG1/MEST had one imprinted and one non-imprinted transcript, while IMPACT, COPG2 and PLAGL1 were not imprinted, thus revealing that this phenomenon is conserved at some, but not all loci between the two groups of Therians. Moreover, differential methylation patterns and the associated regulatory non-coding RNA are also not conserved amongst them, exemplified by the novel DMR identified within IGF2R which had no associated anti-sense transcript. However, histone modifications, specifically the activating H3 Lysine 4 dimethylation mark at the active allele's promoter seems to be important in both lineages and probably serves as the primordial imprint mark. Although the evidence does not resolve the issue of convergence or divergence, it raises the intriguing possibility that both forms of evolution occurred during establishment of imprinting in these mammals.</p> <p>The imposition of functional haploidy in the genome by such epigenetic mechanisms necessarily makes imprinted genes more susceptible to deleterious mutations and regulatory perturbations. Thus, imprinting is implicated in a number of developmental disorders, but its role in the etiology of complex human diseases and neurological disorders, like autism and schizophrenia, remains to be determined. I chose to investigate the imprint status of the duplicated locus DGCR6/DGCR6L lying within the 22q11.2 microdeletion causative of DiGeorge Syndrome (DGS), because our lab previously predicted genes at this genomic location to be imprinted. My studies revealed that both genes DGCR6 and DGCR6L are monoallelically expressed in the primate lineage, but not in a parent-of-origin dependent manner. Interestingly, DGCR6L is not present in the mouse, and Dgcr6 is expressed from both parental alleles.</p> <p>Although DGS primarily manifests as facial, limb and heart abnormalities in children, a number of these patients also ultimately present with variable neurocognitive defects. Thus, I focused my studies on determining the effect of the microdeletion at this chromosomal region on DGCR6 and DGCR6L expression because of their potential role in neural crest cell migration. This revealed that DGS subjects have a highly dysregulated pattern of DGCR6 and DGCR6L expression as compared to that in controls. Moreover, increased expression of these genes correlated significantly with decreased performance in sustained-attention tests. This provides the first evidence that disruption of the normal monoallelic expression pattern of DGCR6 and DGCR6L by hemizygous deletion is involved in the variability in neurocognitive symptoms associated with DiGeorge Syndrome. The results of my studies highlight the importance of searching for novel imprinted domains to better understand not only their evolution, but also the potential role of such epigenetically labile regions in modulating complex human diseases and neurological disorders.</p> / Dissertation

The advent of somatic cell nuclear transfer in cattle has provided the opportunity for researchers to generate genetically identical animals as well as animals that possess precise genetic modifications for agriculture and biomedical purposes. However, in spite of the revolutionary impact this technology presents, problems remain which hinder the production of healthy animals on a consistent basis. Research on cloned mice implicates improper reprogramming of epigenetic modifications and genomic imprinting for the low pregnancy rates and high incidence of abnormalities that are manifested in cloned animals; however, a systematic and comprehensive analysis of nuclear reprogramming in cloned cattle remains undone. The purpose of this research is to assess and characterize the patterns of genomic imprinting in normal and nuclear transfer derived bovine fetuses. To facilitate the identification of imprinted genes in the bovine, a Bos gaurus/Bos taurus interspecies model has been incorporated to maximize the genetic heterozygosity that exists between the alleles of putative imprinted genes for allelic discrimination and parental inheritance. The sequence of twenty-six genes, previously reported as imprinted in mice and humans, was analyzed in Bos gaurus (Gaur) and Bos taurus (Angus) cattle for the presence of single nucleotide polymorphisms (SNP). SNPs were detected in the Gene trap locus 2 (GTL2), Insulin like growth factor 2 (IGF2), Wilms tumor 1 (WT1) and the X chromosome inactivation specific transcript (XIST). Allelic expression analysis in interspecies hybrids indicated maternal genomic imprinting at the IGF2 and XIST loci, paternal genomic imprinting at the GTL2 locus and no imprinting at the WT1 locus. Analysis in cloned hybrids indicated fidelity of allelic expression at the IGF2 and GTL2 loci, however disruption of imprinting was observed at the XIST locus in the placenta of clones. These results are the largest identification of imprinted genes in the bovine and the first identification of the disruption of an imprinted gene in an animal derived from somatic cell nuclear transfer.

In the red-eared slider turtle, Trachemys scripta, gonadal sex is determined by the incubation temperature during the mid-trimester of development; temperature effects can be overridden by exogenous ligands if they are administered during the temperature-sensitive period of development. How the physical signal of temperature is transduced into a biological signal that ultimately results in determining gonad sex is not known. My thesis research focuses on five candidate sex determining genes: cyp19a1 (aromatase), Forkhead box protein L2, R-spondin1, Doublesex mab3-related transcription factor 1, and Sex-determining Region on Y chromosome-box 9. The first three genes are markers of ovarian differentiation while the latter two genes are markers of testicular differentiation. Both in ovo (egg) and in vitro (gonadal explants) studies were conducted. Chapters 1 and 2 examine how exogenous steroid ligands interact with candidate genes as the gonads differentiate into testes or ovaries. Topical application of testosterone with aromatase inhibitor to eggs incubating at the female-producing temperature (31 ºC; FPT) suppresses expression of ovarian markers while increasing expression of testicular markers. Administration of 17β-estradiol (E2) to eggs incubating at a male-producing temperature (26 ºC; MPT) increases expression of ovarian markers while testicular markers are suppressed. This suggests that exogenous ligands modify gonadal trajectory by re-directing (suppression and activation) the expression of candidate genes. Chapter 3 identifies the gonad-specific promoter and the temperature-dependent DNA methylation signatures of the aromatase gene during gonadal differentiation. DNA methylation of the aromatase promoter is lowest at FPT relative to MPT. Exogenous E2 and certain polychlorinated biphenyls retain typical methylation patterns observed at MPT (Chapter 4). This suggests that despite the ability of exogenous ligands to alter the transcriptional profiles and gonad phenotypes, the MPT set the temperature typical epigenetic marks first at the beginning of TSP. Recruitment of modified histone proteins, H3K4me3 and H3K27me3, at the aromatase promoter is FPT-specific during gonad determination. Temperature shift experiments suggest a lack of histone enrichment is due to MPT cue, but is not reversible by FPT. Preliminary analysis of modified histones by Next-generation sequencing shows high duplication levels across samples, leaving room for technical improvement in future study.

It has become increasingly apparent that the misregulation of histone modification actively contributes to cancer. The histone H3 lysine 79 (H3K79) methyltransferase Dot1l has been implicated in the development of leukemias bearing translocations of the Mixed Lineage Leukemia (MLL) gene. We studied the global epigenetic profile for H3K79 dimethylation and found abnormal H3K79 dimethylation profiles exist not only in leukemias driven by MLL-fusion proteins with nuclear partners like AF9, but also in leukemia with MLL-fusions containing cytoplasmic partners like AF6. Genetic inactivation of Dot1l led to downregulation of fusion target genes and impaired both in vitro bone marrow transformation and in vivo leukemia development by MLL-AF10, CALM-AF10 as well as MLL-AF6, suggesting that aberrant H3K79 methylation by DOT1L sustains fusion-target gene expression in MLL rearranged leukemias and CALM-AF10 rearranged leukemias. Pharmacological inhibition of DOT1L selectively killed MLL-AF10 and MLL-AF6 transformed cells but not Hox9/Meis1 transformed cells, pointing to DOT1L as a potential therapeutic target in MLL-rearranged leukemia. We further characterized the DOT1L complex under physiological conditions from human leukemia cells and identified AF10 as a key DOT1L complex component. Given the importance of H3K79 methylation in MLL-rearranged leukemia, we sought to study the role of DOT1L complex component AF10 in H3K79 methylation and MLL leukemia. We generated conditional knockout mice in which the Dot1l-interacting octapeptide-motif leucine-zipper (OM-LZ) domain of Af10 was flanked by LoxP sites. Cre induced deletion of \(Af10^{OM-LZ}\) is predicted to abrogate the Af10-Dot1l interaction. Our histone mass spectrometry data demonstrated that deletion of the endogenous \(Af10^{OM-LZ}\) domain abrogated global H3K79 dimethylation but retained H3K79 monomethylation. Interestingly, bone marrow transformation by MLLAF6 and MLL-AF9 is abrogated by induced deletion of endogenous \(Af10^{OM-LZ}\), while bone marrow transformation by MLL-AF10 and CALM-AF10 is not affected by deletion of endogenous \(Af10^{OM-LZ}\), confirming the importance of Af10-Dot1l interaction in MLL- or CALM fusion-leukemias. Moreover, we showed \(Af10^{OM-LZ}\) deletion prolonged survival of MLL-AF9 leukemia in vivo and led to chromotin compaction and downregulation of MLL fusion targets in MLL-AF9 leukemia. Therefore our results demonstrate a role for Af10 in the conversion of H3K79 monomethylation to dimethylation and reveal the AF10-DOT1L interaction as an attractive therapeutic target in MLL-rearranged leukemias.