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Acute myeloid leukemia (AML) is a highly lethal malignancy characterized by unchecked expansion of immature myeloid blasts. While certain genetic and cytogenetic aberrations have been associated with chemotherapy response and disease risk, clinical outcomes remain heterogeneous. AML harbors relatively few somatic mutations compared to other cancers, however, it shows marked enrichment for epigenetic regulator alterations, and has been shown to harbor DNA methylation defects. My focus has been to dissect these epigenetic defects using high-throughput DNA methylation data. I first characterized two genome-wide hypermethylation signatures in AML: AML-CpG island methylator phenotype (A-CIMP+), and IDH-associated CIMP (I-CIMP+). While I-CIMP+ leukemias showed significant enrichments for mutations in IDH1 or IDH2, A-CIMP+ cases were mutation independent, and were best defined by their epigenetic defects, and associated transcriptomic changes. Importantly, A-CIMP+ leukemias had relatively favorable clinical outcomes, while I-CIMP+ patients did not. I next sought to characterize epigenetic defects involving demethylation of normally methylated genomic regions. I identified two distinct demethylator phenotypes (DMPs): DMP.1+ and DMP.2+. DMP.1+ AML was largely defined by mutations in DNMT3A, FLT3, and NPM1, while DMP.2+ leukemias harbored favorable-risk genomic rearrangements and a distinct gene expression profile. Both DMPs also carried prognostic information in AML; DMP.1+ cases had poor outcomes, while DMP.2+ patients tended to have favorable survival. Using both CIMP and DMP signatures, I then built an integrated epigenetic model for AML prognosis I termed MethylScore. The MethylScore algorithm was prognostic independent of age and cytogenetic risk in multivariate Cox regression models, suggesting that DNA methylation defects may augment existing clinical tools for risk stratification, and/or treatment selection. Finally, I explored whether DNA methylation signatures and genetic mutations could serve as biomarkers of response to epigenetic therapy, and found that DNA hypermethylation correlated with poor overall survival, and a gene mutation profile was associated with lack of complete remission after treatment with a DNA methylation inhibitor. These data provide evidence of distinct epigenetic signatures in AML that define transcriptionally, genetically, and clinically distinct populations that should be evaluated in future translational/clinical studies. / Biomedical Sciences

Background: Infertility has become a growing concern across the world as cases continue to increase each year. Research has now shifted to identifying novel biomarkers to predict male fertility. While mtDNAcn has recently been found to show promising results as potential biomarker, its regulation remains unclear. Method: Triplex probe-based PCR was used to quantify mtDNA levels, while 850K Array was used to measure methylation levels. A-clustering algorithm followed by generalized estimating equations (GEE) lead to clustering of individual CpG sites, containing a minimum of 2 CpGs within 1000 base pairs of each other. These clusters were used for analysis of the association between mtDNAcn and DNA methylation within sperm. Metascape1 was used to annotate gene ontology terms. Result: Generalized estimating equation model analysis produced 6,038 FDR significant (q Conclusion: Thus, we show that sperm mtDNAcn is strongly associated with sperm DNA methylation and the associated implicates mtDNAcn as an influence on infertility.

Obesity is a major health condition in which little is known about the molecular mechanisms that drive it. The hypothalamus is the primary control center for controlling both food intake and energy expenditure in order to maintain the body's energy balance and dysregulation of molecular processes in this region have been implicated in the development and progression of obesity. Recently, several studies have shown altered DNA methylation of critical appetite genes, including the satiety gene Pomc, in the hypothalamus of rodents fed a high fat obesogenic diet. However, it has not previously been studied whether diet-induced changes in DNA methylation of critical appetite genes in the hypothalamus contributes to the development and persistence of the obesity phenotype. Further, DNA 5-hydroxymethylation (5-hmC) is one type of DNA methylation that is 10 times more abundant in the brain than peripheral tissues. However, to date, no study has been conducted examining whether DNA 5-hmC becomes altered in the brain following weight gain and/or contributes to the obesity phenotype. Additionally, there is also evidence to support that exposure to a high fat diet dysregulates the activity of the ubiquitin-proteasome system, the master regulator of protein degradation in cells, in the hypothalamus of male rodents. Despite this, whether this can occur in both sexes and directly contributes to abnormal weight gain has not been investigated. Here, we used a rodent diet-induced obesity model in combination with quantitative molecular assays and CRISPR-dCas9 manipulations to test the role of hypothalamic 1) DNA 5-hmC levels, 2) Pomc methylation, and 3) dysregulated ubiquitin-proteasome signaling in abnormal weight gain following exposure to obesogenic diets. We found that males, but not females, have decreased levels of DNA 5-hmC in the hypothalamus following exposure to a high fat diet, which tracked body weight. Short-term exposure to a high fat diet, which does not result in significant weight gain, resulted in decreased hypothalamic DNA 5-hmC levels, suggesting these changes occur prior to obesity development. Moreover, decreases in DNA 5-hmC persist even after the high fat diet is removed. Importantly, CRISPR-dCas9 mediated upregulation of DNA 5-hmC enzymes in the male, but not female, hypothalamus significantly reduced the percentage of weight gained on the high fat diet relative to controls. Next, we used the CRISPR-dCas9-TET1 and dCas9-DNMT3a systems to test the role of Pomc DNA methylation in the hypothalamus in abnormal weight gain following acute exposure to a high fat diet in male rats. We found that exposure to a high fat diet increases Pomc DNA methylation and reduces gene expression in the hypothalamus. Despite this, we found that CRISPR-dCas9-TET1-mediated demethylation of Pomc was not sufficient to prevent abnormal weight gain following exposure to a high fat diet. Moreover, CRISPR-dCas9-DNMT3a-mediated methylation of Pomc did not alter weight gain following exposure to standard or high fat diets. Finally, we found that both males and females showed dynamic downregulation of proteasome activity, decreases in proteasome subunit expression and an accumulation of degradation-specific K48 polyubiquitinated proteins in the hypothalamus. However, while the CRISPR-dCas9 system was able to selectively increase some forms of proteasome activity, it was unable to prevent diet-induced proteasome downregulation or abnormal weight gain. Collectively, this data reveals novel, sex-specific differences in the engagement of the ubiquitin proteasome system and role of DNA 5-hydroxymethylation in the hypothalamus during the development of the obesity phenotype. / Doctor of Philosophy / Obesity affects 34% of the American population at an annual cost of more than $340 billion in healthcare and is a risk factor for the development of diabetes and certain cancers. Genetic and environmental factors have also been shown to influence the expression of genes that play a role in the development of obesity. The hypothalamus coordinates many integral activities such as hormone regulation and feed intake and numerous studies have observed altered hypothalamic gene regulation in obesity models. Recently, several studies have shown altered DNA methylation of critical appetite genes, including the satiety gene Pomc, in the hypothalamus of rodents fed a high fat obesogenic diet. However, it has not previously been studied whether diet-induced changes in DNA methylation of critical appetite genes in the hypothalamus contributes to the development and persistence of the obesity phenotype. Further, DNA 5-hydroxymethylation (5-hmC) is one type of DNA methylation that is 10 times more abundant in the brain than peripheral tissues. However, to date, no study has been conducted examining whether DNA 5-hmC becomes altered in the brain following weight gain and/or contributes to the obesity phenotype. Additionally, there is also evidence to support that exposure to a high fat diet dysregulates the activity of the ubiquitin-proteasome system, the master regulator of protein degradation in cells, in the hypothalamus of male rodents. Despite this, whether this can occur in both sexes and directly contributes to abnormal weight gain has not been investigated. In this document, I outline a series of experiments designed to elucidate novel, sex-specific differences in the role of the ubiquitin proteasome system and DNA 5-hydroxymethylation in the hypothalamus during the development of the obesity phenotype.