No description available.

No description available.

No description available.

Owing to the presence of muscle stem cells (MuSC), adult skeletal muscle is capable of regenerating after injury. Quiescent muscle stem cells become activated and proliferate into myoblasts which undergo myogenic differentiation to repair damaged tissue. The transcription factor (TF) Six1 is a known regulator of muscle stem cells which potentially plays a role in the early stages of MuSC activation. When bound to the appropriate cofactor, Six family transcription factors are capable of activating or repressing transcription. Previous work suggests that Six1 establishes the accessibility landscape required for the myogenic regulatory factor (MRF) MyoD to bind to DNA. It was hypothesized that Six1 recruits p300 to acetylate Histone H3 lysine 122 which then renders DNA more accessible and facilitates gene transcription. The objective of this research was to investigate the role of Six1 in regulating the epigenetic and accessibility state of DNA in myoblasts. It was found that Six1 and the histone acetyltransferase p300 coincide at many gene enhancers. In addition, Six1 knock-down is associated with reduced DNA accessibility at a large number of loci in C2C12 myoblasts and with gene downregulation. In this research, we determined that recruitment of p300 by Six1 alters chromatin accessibility and gene expression in proliferating myoblasts, providing evidence of Six1 pioneer factor activity.

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

Epigenetics is a rapidly growing field that has provided insight into the etiology of many physiological mechanisms. Research around post-traumatic stress disorder (PTSD) has evolved immensely since expanding to include an epigenetic lens. Researchers have studied which gene loci are associated with PTSD to understand how genes can become either over or under expressed when exposed to trauma. The three main epigenetic factors that assist with regulating the genome are: DNA methylation, histone modification (including methylation and acetylation), and noncoding RNA. Each factor utilizes a different mechanism to help with either the upregulation or downregulation of a specific gene. Within PTSD research, the impacts of these genome modifications have been studied to understand how they regulate the common physiological symptoms associated with PTSD diagnoses. These symptomologies include decreased basal cortisol levels, decreased cardiovascular health, decreased immune function, and increased mortality. Many epigenetic studies have explored how changes in specific gene loci contribute to these physiological dysregulations. Some genes of interest include nuclear receptor subfamily 3 group C member 1 (NR3C1), FK506 binding protein 5 (FKBP5), and spindle and kinetochore-associated protein 2 (SKA2). Many studies have been conducted examining the DNA methylation activity of each gene in those with PTSD diagnoses and those without. However, research continues to produce mixed results. While some studies show an increase of DNA methylation for a specific gene in subjects with PTSD, other studies evidence a decrease of DNA methylation for the same gene. Examining the reasons for conflicting evidence is valuable to further understand the epigenetic mechanisms that occur. After conducting a literature review, four confounding factors have been identified as contributors to such mixed results. The first factor is the difference in each study’s definition of trauma, as well as the diagnostic tools they use to identify subjects with PTSD. The second factor is the samples used to detect epigenetic changes. Most samples collected in epigenetic studies of PTSD include whole blood samples, salivary samples, and only rarely, brain tissue samples. These different sample types, when cross-compared, can contribute to discrepancies in DNA methylation data. Furthermore, whole blood samples are not only vulnerable to intrinsic factor variabilities, but external factor variabilities. The third factor is a difference in subject population across the literature. Many studies are focused on either combat-veterans (with all male subjects) or child cohorts. These differences in demographics make it difficult to compare groups, as research indicates several epigenetic factors such as DNA methylation activity are sex, ethnicity, and age dependent. Finally, the fourth confounding factor is age at onset of trauma. Many studies show that trauma exposure in childhood leads to more severe symptoms compared to trauma exposure in adulthood. It is important to consider these factors and account for confounding variables when conducting future research. In doing so, more robust and accurate research can be produced. A more refined understanding of the epigenetic etiology of PTSD, as well as its epigenetic biomarkers, will likely yield greater insight into PTSD diagnoses, as well as best treatment practices.

Type 2 Diabetes Mellitus (T2DM), a metabolic disorder denoted by elevated blood glucose levels and insufficient insulin action, is growing in prevalence worldwide . Barriers to improving disease outcome resolve primarily around identifying and intervening during the preliminary stages of insulin resistance, a state clinically referred to as pre-diabetes. Emerging evidence suggests that mitochondrial dysfunction may underlie , and potentially precede, progressive insulin resistance, suggesting that biomarkers indicative of mitochondrial dysfunction could predict disease risk and status. In this study, we examined epigenetic modifications, in the form of DNA methylation, in the promoter region of peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC-1α), a known regulator of mitochondrial biogenesis. Following the initiation of a high fat diet, we observed significant genotypic (DNA methylation) and phenotypic (mitochondrial copy number) alterations in C57/BL6 rodent models. These changes preceded overt disease onset, as classified by clinically utilized indices, which included the homeostatic model assessment for insulin resistance (HOMA-IR), the homeostatic model assessment for β-cell dysfunction (HOMA- β), and the quantitative insulin-sensitivity check index (QUICKI). Our data indicate that methylation analysis may serve as an effective clinical parameter to use in conjunction with physiological criterion for the diagnosis of pre-diabetes and the assessment of T2DM disease risk, and adds to the growing body of work seeking to elucidate the role. / Doctor of Philosophy / High blood glucose, referred to as type 2 diabetes (T2DM), increases the risk for heart and kidney disease, blindness, stroke, and death. Efforts to prevent T2DM have centered primarily around behavioral interventions, which include increased physical activity and decreased caloric intake. Importantly, the interventions are most effective when implemented early on in disease progression. In this study, we sought to examine the effects of a high fat diet on the epigenetic profile of PGC-1α, a gene responsible for maintaining mitochondrial biogenesis. The mitochondria, the powerhouse of the cell, is responsible for maintaining the energy systems in the body. Therefore, we examined how increasing in caloric intake resulted in changes in the epigenetic profile of the PGC-1α promoter, and how these changes impacted mitochondrial number. Further, we sought to examine how hypermethylation of PGC-1α led to changes in gene and protein expression in the mitochondria. Results from our study indicate that DNA methylation changes preceded disease onset, as characterized by the homeostatic model assessment for insulin resistance (HOMA-IR), the homeostatic model assessment for β-cell dysfunction (HOMA- β), and the quantitative insulin-sensitivity check index (QUICKI). Our data indicate that methylation analysis may serve as diagnostic and risk assessment tool for pre-diabetes and T2DM in conjunction with physiological measures.

The developing brain is highly sensitive to environmental influences. Unfavorable nutrition is one kind of stress that can cause acute metabolic disorders during the neonatal period [1,2,3] and severe diseases in later life [4,5]. These early life experiences occurring during heightened periods of brain plasticity help determine the lifelong structural and functional aspects of brain and behavior. In humans, for example, weight gain during the first week of life increased the propensity for developing obesity several decades later [5]. This susceptibility is, if not all, related to the dynamic reversible epigenetic imprints left on the histones [6,7,8], especially during the prenatal and postpartum period [9]. Histones are highly dynamic and responsive towards environmental stress [10,11]. Through covalent modification of the histone tail, histones are able to direct DNA scaffolding and regulate gene expression [10,12]. Thus far, various types of post translational modifications have been identified on various histones tails [12]. Among them, the methylation and acetylation on lysine residue (K) 27 on histone 3 (H3) has been tightly linked to gene repression [13,14] and activation [15], respectively. EZh2 (enhancer of zeste 2) in the polycomb repressive complex 2 (PRC2) is the only methyltransferase that has been linked to catalyze this methylation reaction. In addition, SUZ (suppressor of zeste) and EED (embryonic ectoderm development) are two other key proteins in PRC2 function core that help EZH2. As previous reported, increased H3K27 methylation was monitored after fasting stress during neonatal period in chicks' paraventricular nucleus (PVN). In this study, we investigated the detailed mechanism behind changes in H3K27 methylation following fasting stress. After 24 hours fasting on 3 days-of-age (D3), chicks exhibited elevated mRNA levels of PRC2 key components, including EZH2, SUZ and EED, in the PVN on D4. Western blots confirmed this finding by showing increased global methylation status at the H3K27 site in the PVN on D4. In addition, until 38 days post fasting, SUZ and EZH2 remained inhibited. A newly identified anorexigenic factor, Brain-derived neurotrophic factor (BDNF), was used as an example of multiple hormones expressed in PVN to verify this finding. Both BDNF protein and mRNA exhibited compatible changes to global changes of tri- (me3) and di-methylated (me2) H327. Furthermore, by using chromatin immunoprecipitation assays (ChIP), we were able to monitor the changes of H3K27me2/me3 deposition along the Bdnf gene. Fasting significantly increased H3K27me2/me3 as well as EZH2 at the Bdnf's promoter, transcription start site and 3'-untranslated region. These data show that fasting stress during the early life period could leave epigenetic imprinting in PVN for a long time. Next, we tried to understand the function of this epigenetic imprinting in the chicks' PVN. Thus, we compared naive chicks (never fasted) to chicks that received either a single 24 hour fast on D3 or two 24 hour fast on both D3 and 10 days-of-age (D10). We found that the D3 fasted group significantly increased the level of PRC2 key components and its product H3K27me2/me3 compared to the naive group. However, D3 fasting and D10 fasting together decreased the surges of H3K27me2/me3, SUZ and EED (not EZH2) compared to the naive group. We called this phenomenon "epigenetic memory". The Western blot, qPCR and CHIP assay results from BDNF all confirmed the existence of "epigenetic memory" for PRC2. These data suggested that fasting stress during the early period of brain development could leave long term epigenetic modifications in neurons. These changes could be beneficial to the body, which keeps homeostasis of inner environment and prevent massive response to future same stress. The EZH2 protein was knocked down and the H3K27 methylation status changes were monitored after applying the same treatment. We first confirmed that EZH2 antisense oligonucleotides (5.5 ug), but not EZH2 siRNA and artificial cerebrospinal fluid (ACSF), inhibit EZH2 protein by 86 % in the PVN. Then, on D3, chicks were subjected to a 24 hour fasting stress (D3-fasting) post either EZH2 antisense or ACSF injection. The EZH2 antisense blocked the surge of both EZH2 mRNA and H3K27 methylation after D3-fasting. At the same time, BDNF exhibited elevated expression levels and less methylated H3K27 deposition along the Bdnf gene. In addition, we were also interested in the changes of "epigenetic memory" post EZH2 antisense injection. We found that after EZH2 antisense injection, chicks' PVN no longer exhibited any "epigenetic memory" to repetitive fasting stress. While EZH2 mRNA was constantly inhibited, SUZ, EED and H3K27me2/3 levels were unpredictable. These findings suggested that neurons in the PVN utilized PRC2 as a major H3K27 methylation tool. Knockdown of EZH2 in the PRC2 impaired the proper response in PVN to fasting stress and PVN's ability to acclimate to repetitive fasting stresses. Thus, EZH2 is an important H3K27 methyltransferase inside chicken hypothalamus to maintain homeostasis. In conclusion, fasting stress during the early life period could leave epigenetic markers on chromosomes of neurons in the feeding regulation center. These epigenetic markers will be left on chromosomes for a long period of time and have a beneficial role in keeping homeostasis when individuals face future fasting stress again. H3K27 methylation is one of these epigenetic markers and inhibits expression of various genes inside neurons. EZH2 is so far the only detected methyltransferases for H3K27 that form the PRC2. Thus EZH2 plays a key function in the body's response to fasting. / Ph. D.

The risk of breast cancer transiently increases immediately following pregnancy. Hispanic women have one of the highest rates of postpartum breast cancers of all racial/ethnic minority groups in the US. The biology that underlies this risk window and the effect on the natural history of the disease is unknown. MicroRNAs (miRNAs) are small non-coding RNAs that have been shown to be dysregulated in breast cancer. In this study, we measured the miRNA expression of 56 tumors from a case series of multiparous Hispanic women and assessed the pattern of expression by time since last full-term pregnancy. A data-driven splitting analysis on the pattern of 355 miRNAs separated the case series into two groups: a) an early group representing women diagnosed with breast cancer ≤ 5.2 years postpartum (n=12), and b) a late group representing women diagnosed with breast cancer ≥ 5.3 years postpartum (n=44). We identified 15 miRNAs that are differentially expressed between the early and late postpartum groups; 60% of these miRNAs are encoded on the X chromosome. Ten miRNAs had a two-fold or higher difference in expression; miR-138, miR-660, miR-31, miR-135b, miR-17, miR-454, and miR-934 were overexpressed in the early versus the late group; while miR-892a, miR-199a-5p, and miR-542-5p were under expressed in the early versus the late postpartum group. The DNA methylation of three out of five tested miRNAs (miR-31, miR-135b, and miR-138) was lower in the early versus late postpartum group, and negatively correlated with miRNA expression. Taken together, the results of this study show that miRNAs are differentially expressed and differentially methylated between tumors of the early versus late postpartum, suggesting that potential differences in epigenetic dysfunction may be operative in postpartum breast cancers.

Methyl-CpG binding protein 2 (MeCP2) was discovered as a protein binding to methylated DNA more than 20 years ago. It is very abundant in the brain and was shown to be able to repress transcription. The mutations in MeCP2 cause Rett syndrome, an autism-spectrum neurological disorder affecting girls. Yet, the exact role of MeCP2 in Rett disease, its function and mechanism of action are not fully elucidated. In order to shed some light on its role in the disease the aim of this project was to identify proteins interacting with MeCP2. Affinity purification of MeCP2 from mouse brains and mass spectrometry analysis revealed new interactions between MeCP2 and protein complexes. Detailed analysis confirmed the findings and narrowed down the top interactions to distinct regions of MeCP2. One of the domains interacts with identified NCoR/SMRT co-repressor complex and is mutated in many patients with Rett syndrome. In vitro assays proved that these mutations abolish the putative transcriptional repressor function of MeCP2. We propose a model in which Rett syndrome is caused by two types of mutations: either disrupting the interaction with DNA or affecting the interaction with the identified complex, which has an effect on the global state of chromatin. The presented findings can help to develop new therapies for Rett syndrome in the future.