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DNA methylation-dependent gene silencing, catalyzed by DNA methyltransferases (DNMTs) and mediated by methyl binding domain proteins (MBDs) and histone deacetylases (HDACs), is essential for mammalian development, with the nervous system demonstrating particular sensitivity to perturbations. Little is known, however, about the role of DNA methylation in the stage-specific differentiation of neurons. In the olfactory epithelium (OE), where neurogenesis is continuous and the cells demonstrate a laminar organization with a developmental hierarchy, we identified sequential, transitional stages of differentiation likely mediated by different DNMT, MBD and HDAC family members. Biochemically, HDAC1 and HDAC2 associate with repressor complexes recruited by both MBD2 and MeCP2. HDAC1 and HDAC2, however, are divergently expressed in the OE, a pattern that is recapitulated in the brain. Rather than simultaneous inclusion in a complex, therefore, the individual association of HDAC1 or HDAC2 may provide specificity to a repressor complex in different cell types. Furthermore, distinct transitional stages of differentiation are perturbed in the absence of MBD2 or MeCP2. MeCP2 is expressed in the most apical immature olfactory receptor neurons (ORNs), and is up-regulated with neuronal maturation. In the MeCP2 null OE there is a transient delay in ORN maturation and an increase in neurons of an intermediate developmental stage. Two protein variants of MBD2 are expressed in the OE, with MBD2b expressed in cycling progenitor cells and MBD2a in the maturing ORNs. MBD2 null ORNs undergo increased apoptotic cell death. There is also a significant increase in proliferating progenitors in the MBD2 null OE, likely due, at least in part, to feedback from the dying ORNs, acting to up-regulate neurogenesis. Increased cell cycling in the MBD2 null is also observed post-lesion, however, in the absence of feedback back from the ORNs, a phenotype that is recapitulated by an acute inhibition of HDACs with valproic acid. Therefore, disruptions at both transitional stages of ORN differentiation are likely in the MBD2 null mouse. Together, these results provide the first evidence for a sequential recruitment of different MBD proteins and repressor complexes at distinct transitional stages of neuronal differentiation. / Medicine, Faculty of / Graduate

Atherosclerosis is a chronic inflammatory and lipid disorder caused by the buildup of cholesterol-loaded cells of monocyte and muscle cell origin in the arterial intima. While the relationship between excess cholesterol and macrophage behavior is well observed, the molecular mechanisms linking the two remain unclear. Therefore, characterizing the pathways from changes in intracellular cholesterol to the resulting inflammatory output is key to understanding the behavioral changes observed in human macrophages in vitro. We identified that THP-1 macrophages acutely depleted of cholesterol increase the expression of JMJD3, an H3K27me3 demethylase. By using IL-10 as a marker for immune-modulating genes and TNF-α as a marker for pro-inflammatory genes, cholesterol-depleted THP-1 macrophages responded inconsistently to LPS and echinomycin, an inhibitor of HIF-1α, as determined by RT-qPCR and ELISA. Further studies investigating other regulators and outputs of macrophage behavior linked to cellular cholesterol modification are required.

Schizophrenia is a neurological disorder found in approximately 1% of the population. It is estimated that as many as 88% of individuals diagnosed which schizophrenia smoke tobacco, a rate which is greatly increased compared to the general population. While increased use of nicotine-containing products such as cigarettes may be detrimental to the long-term health of individuals with schizophrenia, it has been hypothesized that nicotine use is a form of self-medication for these individuals who suffer from serious neurological and psychological symptoms such as hallucinations, delusions, anhedonia, and cognitive impairments. Understanding the biomolecular mechanisms which result in a higher propensity for smoking may lead to an overall better understanding of the disease and new treatment options. This study investigated the effects of nicotine in an epigenetic transmission model of schizophrenia. Rats were treated neonatally with the dopamine D2 agonist quinpirole or saline from P1 to P21 and then raised into adulthood. This paradigm has been previously shown to produce rats with symptoms similar to schizophrenia, including an enhanced response to nicotine. These neonatally treated rats were then bred to produce pups which were not neonatally treated to investigate whether schizophrenia-like symptoms would be transmitted to the untreated offspring of the quinpirole treated parents in the context of nicotine administration, similar to that of their parents. To examine the effects of nicotine, the rats that were the offspring of animals that were neonatally treated quinpirole were behaviorally tested on either a behavioral sensitization or conditioned place preference (CPP) paradigm, and animals received either nicotine or saline. Following behavioral testing, brain tissue was collected, and an ELISA for brain-derived neurotrophic factor (BDNF) was performed. Results revealed that these offspring demonstrated a heightened behavioral response to nicotine as well as increased expression of BDNF following nicotine administration if at least one parent rat was neonatally treated with quinpirole. This indicates that there may be epigenetic information passed from parents neonatally treated with quinpirole to the offspring.

The multiple physiological effects of the indoleamine hormone melatonin, are mediated primarily by its two G protein-coupled MT1 and MT2 receptors. Our group has shown an upregulation of melatonin receptors following treatment with histone deacetylase (HDAC) inhibitors, including valproic acid (VPA) and Trichostatin A, in cultured cells and/or in the rat brain. VPA increases histone H3 acetylation at the MT1 gene promoter region in rat C6 glioma cells, indicating that this epigenetic mechanism underlies its upregulation of MT1 expression. Since HDAC inhibitors can also alter DNA methylation, the possible involvement of this second major epigenetic mechanism in the regulation of MT1 expression, was examined. C6 cells were treated with the DNA demethylating agent, azacytidine (AZA, 1 - 25 µM), for 24 or 48 hours. Treatment of C6 cells with AZA caused a significant upregulation of MT1 mRNA expression, as compared with controls (DMSO 0.05%). Moreover, treatment with AZA (10 or 20 µM) for 24 or 48 hours, suppressed or abolished DNMT1 protein expression, and inhibited DNMT1 mRNA expression, which indicates inhibition of the DNMT1 enzyme activity. A combination of VPA and AZA caused a trend toward additive upregulation of the MT1 receptor. These results show that DNA demethylation plays a role in the regulation of the MT1 receptor, consistent with the well-known effects of this epigenetic mechanism on gene transcription. Epigenetic regulation of melatonin receptor expression could provide a novel strategy for modulating the therapeutic effects of this hormone and its clinically relevant agonists, such as agomelatine, and could also provide avenues for enhancing the antioxidant, neuroprotective, oncostatic and other benefits of this hormone and its agonists. / Thesis / Master of Science (MSc) / The hormone, melatonin, is involved in maintenance of the sleep cycle, and has many neuroprotective effects, initiated by its binding to specific proteins called receptors. Epigenetic or reversible chemical modifications which alter DNA, without changing its sequence, can alter the levels of these receptors. This process can be modulated by drugs, which can increase levels of the melatonin receptor. In this study, the drug 5-Azacytidine (AZA) was used to cause specific chemical changes to DNA, termed demethylation. This thesis shows for the first time, that AZA causes an increase in melatonin receptors. AZA’s ability to cause demethylation was confirmed by observing decreased levels of the protein responsible for DNA methylation, DNA methyltransferase. Melatonin receptors in the brain exhibit changes in disorders such as Alzheimer’s and Parkinson’s disease. Understanding the mechanisms underlying the regulation of these receptors could provide avenues for enhancing the neuroprotective benefits of melatonin and related drugs.

This work contains a summary of the current genetic, epigenetic and metagenomic knowledge of obesity, as well as an extensive overview of current genetic approaches in mouse models of obesity. Lastly, this work presents a candidate gene approach based on mouse models, which finds new variants associated with multiple obesity phenotypes in a multi-ethnic cohort. / The prevalence of obesity has been mainly be attributed to the rise in an obesogenic environment, in which individuals are more prone to high-dense energy foods and live a sedimentary lifestyle. Familial aggregation of obesity however, has led to numerous studies focused on understanding the genetic basis of this complex disorder. To this effect, this thesis summarizes the current knowledge of obesity genetics, including the monogenic, polygenic and epigenetics field. Given the tremendous contribution of animal models, especially mouse models, to our current knowledge of obesity genetics, this thesis summarizes the methodology of genetic studies in mice, and focuses on how the synergy between human and mouse studies has led to not only the discovery of obesity causal genes, but also their biological contribution to obesity. Lastly, this thesis summarizes a candidate gene approach based on the information from mouse models that have led to identification of a novel variant associated with body mass index (BMI), hip circumference and body adiposity index (BAI) in a multi-ethnic cohort. / Thesis / Master of Science (MSc) / Obesity is a heritable disease, and the genetic basis of obesity could result in better prevention and treatment options for this disorder. Obesity patterns are not uniform worldwide, and some ethnic groups are more prone to obesity than others, therefore having a multi-ethnic approach in studying obesity could yield to causal variants that could be generalized to all.

Fear memory formation requires a coordination of molecular events, including protein synthesis, protein degradation, and epigenetic regulation of gene expression, throughout a circuit of brain regions. One mechanism highly studied for its involvement in protein degradation during fear memory is the ubiquitin-proteasome system (UPS), which utilizes the small protein ubiquitin to label proteins. Ubiquitin contains eight linkage sites that each lead to a unique outcome for the protein being labeled and a protein can gain one (monoubiquitination) or multiple (polyubiquitination) ubiquitins. The 26S proteasome is the catalytic component of the UPS and is comprised of a 20S catalytic core surrounded by two 19S regulatory caps. Phosphorylation of 19S cap regulatory subunit RPT6 at serine 120 (pRPT6-S120) has been widely implicated in controlling activity-dependent increases in proteasome activity. Interestingly, sex differences have been observed in proteasome-mediated protein degradation in the amygdala and hippocampus during fear memory formation. However, female subjects have only recently been regularly included in rodent behavioral studies so the majority of data on mechanisms of fear memory apply solely to the male brain. Considering post-traumatic stress disorder (PTSD) is two to three times more prevalent in females compared to males, understanding the mechanisms involved in fear memory in both sexes is important for understanding sex-specific development of fear-based disorders, such as PTSD. Importantly, the UPS also has non-proteolytic functions independent of proteasome-mediated protein degradation. For example, monoubiquitination and some forms of polyubiquitination do not lead to protein degradation. Additionally, 19S cap regulatory subunit RPT6 has been found to function independently of its role in the proteasome, where it has a transcription-like role in the hippocampus of male rats during fear memory formation. Unfortunately, proteasome-independent functions of the UPS have not been extensively studied in terms of different forms of ubiquitination. Additionally, it is unclear whether phosphorylation of RPT6 is necessary for its non-proteolytic roles in memory formation and the role of proteasome-independent RPT6 in general has not been investigated in female subjects. Here, we address these gaps in knowledge by 1) investigating sex-differences in the role of lysine 63 (K63-) polyubiquitination, a proteasome-independent ubiquitin linkage, in the amygdala during fear memory formation, 2) studying the role of proteasome-independent RPT6 in the hippocampus of female rats during fear memory formation, and 3) identifying proteasome-independent RPT6 target genes as well as the role of phosphorylation status of RPT6 at Serine-120 for its transcriptional activity during memory formation in the hippocampus of male rats. We first found that levels of K63-polyubiquitination targeting in the amygdala were increased in female, but not male, rats during fear memory formation. Interestingly, K63-polyubiquitination targeted proteins involved in ATP synthesis and proteasome functions in the amygdala of females and genetic manipulation of the K63 codon in the ubiquitin coding gene led to decreased ATP levels and proteasome activity. Additionally, this manipulation only led to impaired fear memory in females, suggesting that K63-polyubiquitination has a sex-selective role in the amygdala, where it regulates fear memory in females, but not males. We then investigated the role of proteasome-independent RPT6 in the hippocampus of females and males during fear memory formation. In females, we found RPT6 did not bind to DNA regions in the c-fos gene, a previously identified RPT6 target gene in males. However, RPT6 did bind to monoubiquitination of histone H2B at lysine-120 (H2BubiK120), an epigenetic modification identified as an RPT6 binding partner in males, suggesting a potential role for proteasome-independent RPT6 in transcriptional regulation in the hippocampus of female rats. In males, we identified RPT6 targets genes during memory formation, found that RPT6 DNA binding alone altered gene expression, and lastly observed that pRPT6-S120 was necessary for RPT6 to bind DNA and regulate transcription during memory formation. Collectively, these data reveal sex-differences in proteasome-independent roles of the UPS through ubiquitination and proteasomal subunits in both the amygdala and hippocampus during fear memory formation. Considering males and females have differences in PTSD prevalence, understanding proteasome-independent roles of the UPS in both sexes may lead to a better understanding of PTSD development as well as potential therapeutic targets in each sex. / Doctor of Philosophy / In order to remember a fear-provoking memory, or any memory, it must be stored in the brain after the event. To store a fear memory, cells in specific areas of the brain have to destroy some proteins and activate or shut off certain genes using epigenetic mechanisms. Although the DNA itself never changes, epigenetic mechanisms recruit proteins to sit on the DNA to make it more (activate) or less (shut off) accessible. For protein destruction, it has been shown that brain cells use a mechanism called the ubiquitin-proteasome system (UPS) during fear memory storage. The UPS uses a small protein called ubiquitin to label proteins in the cell. Ubiquitin is versatile in its ability to label proteins due to it having eight different binding sites that can be used as a label. Some proteins only gain one ubiquitin (monoubiquitination), while other proteins can gain multiple ubiquitin proteins (polyubiquitination) and both the number of ubiquitins and the label used determines what happens to the protein. When a protein is labeled to be destroyed, the UPS uses a large complex of proteins called the 26S proteasome, which contains a section called the 20S catalytic core and two 19S regulatory caps that sit above and below the 20S core. It has been shown that when one of the proteins in the 19S regulatory cap called RPT6 gains a phosphate molecule at the 120th amino acid, which is a serine amino acid, it increases the number of proteins destroyed by the 26S proteasome. Interestingly, the UPS does not destroy proteins in the same way in male and female brains during storage of a fear memory. This is important because females are 2-3 times more likely to develop post-traumatic stress disorder (PTSD) than males, but it is unclear why, making the study of mechanisms involved in fear memory storage in both males and females important. The UPS also functions in ways that do not involve destroying proteins. For example, proteins with a monoubiquitination label are often not destroyed. Additionally, it has been observed that RPT6, a protein in the 19S regulatory cap, can work outside of the 19S regulatory cap of the 26S proteasome to activate genes in brain cells of male rats. Sadly, the ubiquitin labels that do not cause protein destruction have not been well studied. It is also unclear whether RPT6 must gain a phosphate group to activate genes and if it activates genes in both male and female brains during fear memory storage. In the present study, we investigated the role of a common ubiquitin linkage that does not cause protein destruction called lysine 63- (K63-) polyubiquitination during fear memory storage in the amygdala, the emotional control center of the brain, in male and female rats. We found that K63-polyubiquitination is increased in female, but not male, rats during fear memory storage. In females, K63-polyubiquitination was involved in making new ATP as well as controlling the number of proteins destroyed by the 26S proteasome. Lastly, we found that female, but not male, rats required K63-polyubiquitination in the amygdala for fear memory storage, suggesting a female-specific use of this ubiquitin label. In this study, we also studied the role of RPT6 in the hippocampus, another key area of the brain for memory storage, of male and female rats. In females, we found RPT6 did not activate the same gene we previously identified in male rats, but it did bind with a monoubiquitination label on a protein that DNA is wrapped around, known as a histone. Due to this finding, it appears that RPT6 may act freely of the 19S regulatory cap to alter accessibility of genes in the hippocampus of females. On the other hand, in males we found that RPT6 activates some genes and shuts off other genes and can do so by sitting on DNA by itself. We also found that the addition of phosphate to RPT6 was necessary for it to sit on a gene and activate it during fear memory storage. These results show differences in UPS mechanisms during fear memory storage between males and females, which will be important for understanding why females are more likely to develop PTSD than males and for identifying potential treatment options.

Indiana University-Purdue University Indianapolis (IUPUI) / A subpopulation of tumor cells known as ovarian cancer initiating cells (OCICs) have been shown to be the cells that propagate the tumor phenotype in ovarian cancer. Studies have showed that a very small population (100) of these cells is sufficient to induce a tumor phenotype; while a large quantity of tumor cells (5 X 105) are required to induce such a phenotype. In this study we studied the functional changes in genes expressed in the OCIC phenotype which were important for such efficient propagation of cancers. To enable this analysis, we generated mRNA expression and DNA methylation profiles of OCICs and compared them with those of tumor and normal ovarian surface epithelial cells. We identified four pathways which regulated most of the observed changes and were predicted to be important factors in distinguishing the OCICs from tumors and normal cells. The gene signatures for these pathways were analyzed by unsupervised clustering in order to determine the similarities of OCICs with respect to tumor and normal samples. We further believed that the OCICs can be used as indicators towards the genesis and progression of early events in the ovarian cancers. In light of this, we considered two hypotheses which are currently addressing the genesis of ovarian cancer. The first hypothesis proposed ovarian surface epithelial cells to be cells of origin of the ovarian cancer while the other proposed the fallopian tube cells to be contributing the cell of origin for these cancers. It is also believed that these two cells can be reciprocal cells of origin for the cancer phenotype. In order to test these hypotheses, we integrated the in-house dataset with a public domain fallopian tube gene expression data. The integration of the results obtained from these analyses provided better understanding of the early events in ovarian carcinogenesis.

A large variety of post-translational modifications can exist on the N-terminal tails of histone proteins H2A, H2B, H3 and H4. These have been of great interest as they have increasingly been shown to influence fundamental biological processes and human disease. Studying these modifications provides insight into their physiological functions and enables the search for potent small molecule inhibitors. In this thesis, new fluorescence-based supramolecular tools were developed and used to a) measure the binding of covalently modified peptide tails to a collection of synthetic receptors in neutral aqueous solution and b) monitor an enzyme that installs a post-translational modification (PTM) in real-time. Two different approaches were used to detect binding in these systems. The first was the optimization of a competitive dye-displacement method that relies on the ability of the cationic dye lucigenin. The second was the synthesis of novel conjugates that consist of calixarenes covalently appended with multiple different fluorescent dyes. / Graduate / 0487 / 0490 / 0491

Vernalization is defined as the response to prolonged cold exposure required for acquiring the molecular competence necessary to undergo floral transition. FLOWERING LOCUS C (FLC), a potent floral repressor in Arabidopsis, is highly expressed before vernalizing cold treatment but is repressed during prolonged vernalization. VERNALIZATION INSENSITIVE 3 (VIN3) is a Plant HomeoDomein (PHD)- containing protein that is required for establishing vernalization-mediated repression of FLC. The induction of VIN3 is one of the earliest molecular events in vernalization response and its expression is intimately linked to prolonged cold exposure. However, mechanisms underlying VIN3 induction remain poorly understood. The constitutive repression of VIN3 in the absence of cold is due to multiple repressive components, including a transposable element-derived sequence, LIKE-HETEROCHROMA TIN PROTEIN 1 (LHP1), and POLYCOMB REPRESSION COMPLEX 2 (PRC2). Furthermore, the full extent of VIN3 induction by vernalization requires activating complex components, including EARLY FLOWERING 7 (ELF7) and EARLY FLOWERING IN SHORT DAYS (EFS). Dynamic changes in the histone modifications present at VIN3 chromatin during vernalization were also observed, indicating that chromatin changes play a critical role in regulating VIN3 induction. However, VIN3 induction by vernalization still occurs in the absence of activation complexes and de- repression of VIN3 in the absence of the repressive complexes is not sufficient for achieving complete induction. Thus, unknown cold-influenced regulators responsible for achieving maximum VIN3 induction during vernalization must exist. Therefore, forward genetic screening was undertaken to elucidate upstream regulators of VIN3. Molecular characterization of T-DNA mutant populations elucidated two interesting mutants: a mutant that ectopically expressed VIN3 before cold (ectopic VIN3 induction, evi1) and mutants that failed to induce VIN3 during vernalization (defects in VIN3 induction, dvi1). FLC is over-expressed in dvi1 despite its failure to induce VIN3 expression during vernalization, suggesting that this mutant may regulate both VIN3 and FLC. In evi1, FLC is hyper-repressed after 40 days of vernalization, leading to an acceleration of flowering time. These results indicate that regulators of VIN3 in the vernalization pathway exist and that these regulators may use different mechanisms in order to influence VIN3 expression. / text