Evidence suggests that chromatin-modifying complexes contribute to the uncontrolled cell proliferation of cancer cells through dysregulation of the epigenome. Histone deacetylases (HDACs) are a type of chromatin modifying complex that are responsible for removing acetyl groups from modified histone tails. Their dysregulation is known to play a role in oncogenesis of many cancer types by contributing to an aberrant epigenome and transcription program. Recent clinical trials have begun to test the efficacy of using HDACiâs to treat certain breast cancer, but their application to an aggressive type of breast cancer (Triple Negative Breast Cancer, TNBC) has largely been overlooked. My thesis project aims to test the hypothesis that increased chromatin acetylation associated with Panobinostat treatment leads to increased TSG expression and growth inhibition. Here I analyze the phenotypic effect of Panobinostat (an HDACi) in TNBC preclinical models, and found that Panobinostat is a potent anti-cancer agent for a broad panel of TNBC subtypes. I also observed histone acetylation increases in TNBC cells in a dose-dependent manner in response to Panobinostat. After examining bulk histone acetylation levels from nuclear extracts, I examined genome-wide Panobinostat-dependent H3K27Ac changes to identify differentially affected enhancers and promoters. Expression profiling corroborated the observed Pan-dependent epigenetic changes and gene-ontology analysis revealed deregulated tumor suppressor genes and perturbed signaling pathways. In summary, my thesis provides new insights in how Panobinostat leads to impaired TNBC cell growth, presents evidence for H3K27ac associated activity, and provides a rationale for the design of combinatorial drug therapies for TNBC.

Pdx1 is a transcription factor of fundamental importance to pancreas formation and adult islet ?-cell function. However, little is known regarding its role in mature b-cells or the positive- and negative-acting coregulators recruited to mediate transcriptional control. Using a conditional deletion and lineage tracing strategy we revealed a bifunctional role of Pdx1 in maintenance of mature ?-cell identity as Pdx1-deleted cells rapidly lost b-cell features and simultaneously acquired a global transcriptional profile similar to ?-cells. To better understand how Pdx1 mediates its actions in mature b-cells, a coimmunoprecipitation/mass spectrometry approach was undertaken. Numerous Pdx1-interacting factors possessing a wide range of cellular functions linked with this protein, including, but not limited to, coregulators associated with transcriptional activation and repression, DNA damage response, and DNA replication were isolated. Because chromatin remodeling activities are essential to developmental lineage decisions and adult cell function, our analysis focused on investigating the influence of the Swi/Snf chromatin remodeler on Pdx1 action.

The central melanocortin receptors have classically been implicated in the maintenance of energy homeostasis, though their expression in hypothalamus and brainstem. Two melanocortin receptors are predominantly expressed in the central nervous system, namely melanocortin -3 and -4 receptors (MC3R and MC4R). While the MC4R has been extensively studied by our lab and others, the function of the MC3R is less well understood. Through the utilization of a mouse model with the MC3R promoter driving green fluorescent protein expression (MC3R-GFP) and another with genetic knockout of the MC3R (MC3R KO), a more extensive study of the function of the MC3R was performed. High expression of the MC3R-GFP was noted in brain regions including the cortex, thalamus, hypothalamus, and ventral midbrain. Following up expression of the MC3R in the ventral tegmental area, MC3R deficiency was shown to alter dopamine homeostasis and sucrose preference in a female specific manner. The alteration in midbrain dopamine content in the MC3R KO female was normalized by ovariectomy. In addition, the response to ovariectomy in the MC3R KO uncovered a significant increase in body weight and body fat in response to surgery in MC3R KO animals. Further studies examining gender-specific control of energy homeostasis were conducted during pregnancy, when ovarian hormone signaling fluctuates. MC3R KO dams showed a deficiency in pregnancy induced food intake and a defect in the accumulation of fat stores in preparation for lactation. However, MC3R KO dams displayed normal body weight gain and food intake response to lactation. These findings demonstrate that the MC3R exerts multiple effects on the regulation of food intake and energy homeostasis that are specific to female animals.

Caveolin-1 (CAV1) is an essential protein for the formation of caveolae, invaginations found at the plasma membrane. Caveolae are abundant in many cell types and numerous functions have been identified, including modulating cell signaling and buffering cells from mechanical stress. However, exactly how newly identified mutant forms of CAV1 impact caveolae formation and function remains to be elucidated. Previous work has highlighted the importance of the C-terminus of CAV1 in the assembly of CAV1 complexes, and trafficking to the plasma membrane. Investigation of the frameshift mutant CAV1-P158 and the truncation mutant CAV1-F160X revealed that alteration of the amino acid sequence, or truncation of the C-terminus of CAV1 had little effect on caveolae assembly. Interestingly, incorporation of these mutant proteins into hybrid caveolae with wild type CAV1 led to the formation of caveolae with altered biochemical and molecular properties, indicating that an intact C-terminus may also be important for normal caveolae function. Thus, the incorporation of CAV1 proteins with C-terminal mutations into caveolae is a critical factor associated with human diseases.

The enhancer DNA-binding transfactor Repressor activator protein 1 (Rap1) performs several essential cellular functions in the budding yeast Saccharomyces cerevisiae. These functions include regulation of telomere length, transcription repression of both telomere-proximal genes and the silent mating type loci, and transcriptional activation of hundreds of protein-encoding genes, including the ribosomal protein- and glycolytic enzyme-encoding genes. Rap1 was discovered over 30 years ago, and studies of Rap1-dependent transcription repression and telomere length stabilization have produced significant mechanistic insights. By comparison, the mechanism of Rap1 transcription activation remains poorly understood for two reasons. First, Rap1 is encoded by a single copy essential gene and its involvement in many disparate cellular functions prevents easy interpretation of direct Rap1 dissection studies. Second, the existence of conflicting reports of the ability of Rap1-heterolgous DNA-binding domain fusion proteins to serve as chimeric transcriptional activators challenges the use of this conventional approach to study Rap1. In the accompanying dissertation, I address these challenges and uncover Rap1 contributions to transcription activation through the generation of an altered DNA-binding specificity variant of Rap1 (Rap1AS). Rap1AS possesses true altered DNA-binding specificity in that the mutant enhancer recognition it has gained is accompanied by a loss in recognition of the wild-type enhancer. Rap1AS is thus an ideal tool for dissecting Rap1 structure-function relationships. Using Rap1AS, I mapped and characterized a 41-amino acid activation domain (AD) within The Rap1 C-terminus. I found that this AD is required for transcription of both chimeric reporter genes and authentic chromosomal Rap1 enhancer-containing target genes. Finally, as predicted for a bona fide AD, mutation of this newly identified AD reduced the efficiency of Rap1 binding to a known transcriptional coactivator TFIID-binding target, Taf5. Rap1 AD mapping and identification of Taf5 as a coactivator target represent important advances in our understanding of how Rap1 contributes to gene-specific activation and will enable future dissection of Rap1-dependent transcription activation mechanisms.

The giant myofilament protein titin is indispensable for the structural integrity and function of the sarcomere, however, little is known regarding the turnover of titin within the complex macromolecular sarcomere structure in contracting cardiomyocytes. This dissertation presents novel and unique insight into the homeostatic control of titin in the cardiac sarcomere; nevertheless, the projects described in this dissertation served to elucidate key components in our understanding of the titin life cycle. The experiments described in the following chapters have addressed 3 major findings. 1) Titin is post-transcriptionally regulated at its 5â-UTR via cis-regulatory uORFs. 2) Titin is post-transcriptionally regulated by miR-26a/b at its 3â-UTR. 3) Titin is not a dynamic and mobile protein that is undergoing continuous exchange between myofibrils.

The evolutionarily conserved 14-subunit RNA Polymerase II Transcription Factor D (TFIID) complex is composed of TATA-box Binding Protein (TBP) and 13 TBP-associated factors (Tafs). In Saccharomyces cerevisiae, TFIID is essential for life as are each of its subunits; gene deletions and certain mutations result in cell inviability or temperature sensitive (Ts) growth phenotypes. Despite decades of study, the mechanisms by which many Taf subunits contribute to the essential function of TFIID are only poorly understood. In the accompanying dissertation, I address this gap in knowledge for two yeast TFIID subunits, Taf2 and Taf14. To understand the function of Taf2 in vivo, I performed a molecular genetic dissection of the TFIID subunit Taf2. Through systematic site-directed mutagenesis, I discovered a family of taf2ts alleles, two of which display growth defects that can be strongly suppressed by overexpression of the yeast-specific TFIID subunit TAF14 but not by overexpression of any other TFIID subunit. In S. cerevisiae, Taf14 is also a constituent of six other transcription related complexes making interpretation of its role in any of these complexes difficult. While Taf14 is not conserved as a TFIID subunit in metazoans, it is conserved through its chromatin binding YEATS domain. Based on my identification of the Taf2-Taf14 genetic interaction, I demonstrate that Taf2 and Taf14 directly interact and mapped the Taf2-Taf14 interaction domains. I used this information to identify a Taf2 separation-of-function variant (Taf2-ÎC). While Taf2-ÎC no longer interacts with Taf14 in vivo or in vitro, it stably incorporates into the TFIID complex. In addition, purified Taf2-ÎC mutant TFIID is devoid of Taf14 making this variant a powerful reagent for determining the role of Taf14 in TFIID function. Additionally, I developed methodologies for reconstitution of the TFIID complex using inducible overexpression in S. cerevisiae. This methodology could lead to important insights into the molecular architecture and assembly pathway of the TFIID complex. Successful reconstitution of TFIID using recombinant expression technologies could pave the way for in-depth genetic and biochemical dissection of the essential functions TFIID plays in transcriptional regulation.

Diabetic retinopathy (DR) is the leading cause of blindness in working age Americans. The early stages of DR pathogenesis include chronic inflammation, which can eventually lead to the development of later stages of the disease characterized by pathologic pre-retinal neovascularization. The first goal of this dissertation was to determine diabetes-relevant culture conditions for which to test the effect of a novel class of anti-inflammatory lipids, the epoxygenated fatty acids. Epoxygenated fatty acids of the epoxyeicosatrienoic acid (EET) family and epoxydocosapentaenoic acid (EDP) family are generated by cytochrome P450 epoxygenase enzymes. EET and EDP have been shown to be anti-inflammatory, but their levels are limited by the hydrolysis to diol products by the soluble epoxide hydrolase enzyme (sEH). Thus, the second goal of this dissertation was to determine whether restoration of epoxide levels through exogenous addition and inhibition of their metabolism by sEH was able to inhibit retinal vascular inflammation. However, EETs have been shown to exert pro-angiogenic activities, which would be contraindicated for the late stages of DR. Therefore, the final goal was to determine whether EETs were involved in hypoxia-induced retinal neovascularization.

The pleiotropic cytokine interleukin 2 (IL2) disrupts the blood-brain barrier and alters brain microcirculation, underlying vascular leak syndrome that complicates cancer immunotherapy with IL2. The microvascular effects of IL2 also play a role in the development of multiple sclerosis and other chronic neurological disorders. The mechanism of IL2-induced disruption of brain microcirculation has not been previously determined. Signaling through the IL2 receptor complex identified in brain microvascular endothelial cells leads to activation of the transcription factor, nuclear factor kappa B, resulting in expression of proinflammatory cytokines/chemokines. In parallel, IL2 induced disruption of adherens junctions, concomitant with cytoskeletal reorganization, ultimately leading to increased endothelial cell monolayer permeability. IL2-induced phosphorylation of vascular endothelial cadherin, a constituent of adherens junctions, leads to dissociation of its stabilizing adaptor partners. These results unravel the mechanism of deleterious effects induced by IL2 on brain microvascular endothelial cells and may inform the development of new measures to improve IL2 cancer immunotherapy, as well as treatments for autoimmune diseases affecting the central nervous system.

Misregulated hormone secretion from the islets of Langerhans is central to the pathophysiology of Diabetes. While insulin plays a key role in glucose regulation and the modulation of secretion has immediate clinical implications, the importance of glucagon is increasingly acknowledged. However, the mechanisms that regulate glucagon secretion from alpha cells are still unclear. We utilized pseudo-islets reconstituted from dispersed islet cells to study alpha cells with and without various indirect effects from other islet cells. When cultured, both murine and human islet cells reassociated into pseudo-islets, which recovered normal glucose-regulated hormone secretion. We generated small (~40 µm) pseudo-islets using all of the islet cells, or only some of the cell types, which allowed us to characterize novel aspects of regulated hormone secretion. Our data suggest that the recovery of regulated glucagon secretion from alpha cells in small pseudo-islets depends upon the combined action of paracrine factors, like insulin and somatostatin, and juxtacrine signals between EphA4/7 on alpha cells and ephrins on beta cells. While these signals modulate different pathways, both appear to be required for proper inhibition of glucagon secretion in response to glucose. The improved understanding of the modulation of hormone secretion can provide novel therapeutic routes for the treatment of Diabetic individuals.