Gain of function NOTCH1 mutations are common in both patients with T-ALL and in mouse models of the disease. Inhibiting the Notch pathway in T-ALL cell lines results in growth arrest and/or apoptosis in vitro, suggesting a requirement for Notch signaling in T-ALL. Therefore, we sought to examine the role of Notch1 signaling in both premalignancy and in the maintenance of leukemic growth. Using a murine model of T-ALL, in which expression of the Tal1 and Lmo2 oncogenes arrests thymocyte development, our preleukemic studies reveal that Notch1 mutations are early events that contribute to the clonal expansion of DN3 and DN4 progenitors. We also demonstrate that progenitors are maintained within the tumor and are enriched in leukemia-initiating cell (L-IC) activity, suggesting Notch1 may contribute to L-IC self-renewal. By studying the effects of Notch signaling in murine T-ALL cell lines, we also demonstrate that Notch1 promotes the proliferation and survival of leukemic blasts through regulation of Lef1 and the Akt/mTOR pathways. Given that T-ALL cell lines are dependent on Notch signaling in vitro, we investigated the effects of Notch inhibition in vivo. We provide evidence that Notch1 can be successfully targeted in vivo and that Notch inhibition, with γ-secretase inhibitors (GSIs), significantly extends the survival of leukemic mice. We also demonstrate that administration of GSIs in combination with rapamycin inhibits human T-ALL growth and extends survival in a mouse xenograft model. Given that NOTCH1 may be required to maintain both L-IC and bulk leukemic growth, targeting NOTCH1 may prove to be an efficacious targeted therapy for T-ALL patients with aberrant NOTCH1 activation.

In the mammalian genome, 5‟-CpG-3‟ dinucleotides are frequently methylated, correlating with transcriptional silencing. Genome-wide waves of demethylation are thought to occur only twice during development, in primordial germ cells and in the pre-implantation embryo. They are followed by de novo methylation, setting up a pattern that is inherited throughout development. No global methylation changes are thought to occur during further somatic development, although methylation does alter at gene-specific loci, contributing to tissue-specific patterns of gene expression. Here we studied DNA methylation in differentiating mouse erythroblasts in vivo using several approaches including genomic-scale, reduced representation bisulfite sequencing (RRBS). Surprisingly, demethylation at the erythroid-specific β-globin locus was coincident with a wave of global DNA demethylation at most genomic elements, including repetitive elements and genes silenced in erythropoiesis. Over 30% of total methylation is irreversibly lost during erythroid differentiation. Demethylation occurred through a passive mechanism, requiring the rapid DNA replication triggered with the onset of erythroid terminal differentiation. Global loss of DNA methylation was not associated with a global increase in transcription, as determined by GeneChip analysis. We propose that global demethylation is a consequence of cellular mechanisms required for the rapid demethylation and induction of β-globin and other erythroid genes. Our findings demonstrate that, contrary to previously held dogma, DNA demethylation can occur globally during somatic cell differentiation, providing a new experimental model for the study of global demethylation in development and disease.

Different cell types in multi-cellular organisms heritably maintain different gene expression patterns despite carrying the same genome; a phenomenon termed epigenetics. It is widely believed that the packaging state of the genome, known as chromatin structure, carries epigenetic information. How chromatin states are inherited and how chromatin structure changes during development, moreover how different epigenomes, such as chromatin and DNA modifications communicate with each other during these processes are important questions. Accordingly, understanding the mechanisms that govern pluripotency and differentiation requires details of chromatin dynamics. The major goal of my doctoral thesis was to understand the genome wide view of chromatin dynamics in embryonic stem cells. My studies centered on two aspects of chromatin dynamics in mouse embryonic stem cells—localization and function of two antagonistic chromatin regulators and genome-wide histone variant dynamics. In the first part, we examined the roles of several chromatin regulators whose loss affects the pluripotent state of ES cells. We found that two such regulators, Mbd3 and Brg1, control a large number of genes in ES cells via antagonistic effects on promoter nucleosome occupancy. Moreover, we found that both Mbd3 and Brg1 play key roles in the biology of 5-hydroxymethylcytosine (5hmC), a newly identified DNA modification. Mbd3, which was named by homology to known cytosine methyl binding domains, yet does not bind methylcytosine in vitro, co-localized in ES cells with 5hmC. Furthermore, Mbd3 localization was lost in knockdown cells lacking the major 5mC hydroxylase, Tet1. Our results suggest, contrary to current dogma, that 5hmC is more than just an intermediate in cytosine demethylation pathways, that it may regulate genes via the Mbd3/NuRD complex. Finally, we showed that both Mbd3 and Brg1 are themselves required for normal levels of 5hmC in vivo, identifying a feedback loop between 5hmC and Mbd3. Together, our results identified a possible effector for 5hmC, thereby suggesting a functional role for this DNA modification. Moreover, Brg1 and Mbd3 can now be added to the growing list of regulators with opposite effects on ES cell gene expression, suggesting that pairs of antagonistic chromatin binding proteins may be a common phenomenon in ES cell transcription regulation (Yildirim et al., Cell 2011). The second part of my dissertation concerns the dynamics of several histone variants. Seminal studies in the Henikoff lab showed that certain histone variants are replaced throughout the cell cycle, in contrast to the predominant replication-coupled mode of histone assembly. Work in yeast and flies showed that rapid histone turnover occurs at epigenetically-regulated genomic regions, such as chromatin boundary elements or Polycomb/Trithorax binding sites. Notably, promoter regions of actively transcribed genes exhibit rapid turnover, suggesting that histone turnover may have an important role in gene regulation, as higher histone turnover rate would provide higher probability of DNA element exposure and faster erasure of chromatin marks of the replaced histones. In order to extend such studies to a model for pluripotency and differentiation, we developed a system for measuring histone replacement in mouse ES cells. To be able to carry out turnover experiments in ES cells, we generated stable ES cell lines that can be induced to express epitope-tagged histone variants. Our results confirmed that histone turnover patterns are conserved from yeast to mammals and that turnover profiles are histone variant specific. Murine H3.3 turnover is similar to H3.3 turnover in flies, with peaks at the promoters of highly transcribed genes. MacroH2A2, a variant generally linked to gene repression, had a more complex turnover profile. Surprisingly, we found rapid exchange of macroH2A2 occurring around transcription start sites of a number of highly expressed genes. At poorly expressed genes, on the other hand, macroH2A2 localizes upstream or downstream of transcription start sites and is incorporated slowly, either via slow turnover or via replication-coupled incorporation. Finally, we have used those inducible ES cell lines to generate mice, which will enable future studies on tissue-specific histone replacement in vivo. In summary, my thesis work not only significantly extends our understanding of chromatin regulation in general but also provides a more detailed landscape of chromatin structure and regulation in ES cells. Extending these analyses to differentiating cells and in vivo tissue specific dynamics should provide us with a better understanding not only of cell type specific chromatin organization but also improve our ability to program and re-program genomic landscapes in vitro.

Electronic cigarette usage is becoming increasingly prevalent among school age children and young adults. A known bactericidal agent, propylene glycol, is often used as a carrier for nicotine, flavoring, and additional constituents of electronic cigarette juice. This study examined the relationship between propylene glycol and virulence gene expression in Streptococcus pyogenes, a respiratory tract pathogen commonly found in school-age individuals. A variety of virulence genes controlled by the three stand alone regulators mga, RofA, and Rgg/RopB were sampled in an effort to understand the pathway by which virulence is affected. The genes chosen encode C5a peptidase, fibronectin binding protein, hyaluronate lyase, NAD glycohydrolase, Streptococcal pyrogenic exotoxin A and B, streptodornase, streptokinase, Streptolysin O, and Streptolysin S. No significant change in gene expression was observed, but a novel method to test the effects of aerosols on cells was developed. This method can be used in the future to observe the effect of aerosols, including commercial electronic cigarette juice, on both bacterial and mammalian cells.

The purpose of this thesis is to examine familial hypercholesterolemia (FH) and emerging pharmacogenomics therapies that propose to lower serum low density lipid (LDL) levels. The search of various data bases resulted in nine research articles being selected for review. Syntheses of the articles suggest emerging phamacogenomic drug therapy can improve treatment outcomes for individuals with a diagnosis of FH. The Human Genome Project (HGP) has had far reaching applications for genomic technologies and pharmacagenomic interventions, tailored to human conditions associated with select genomic traits. Synthesis of nine research articles demonstrate that little is known on the topic and reveals extensive gaps in the evidence. This thesis concludes with implications for nursing education, practice, policy and research along with limitations are noted.

A sequential series of mutational events is necessary for the development of leukemia. The misexpression of TAL1, a basic helix-loop-helix (bHLH) transcription factor, is the most common mutation in T cell acute lymphoblastic leukemia (T-ALL). Tal1 transgenic mice develop leukemia with a long latency and incomplete penetrance indicating additional mutations are necessary to develop disease. To investigate additional mutational events that potentially contribute to TAL1-expressing T-ALL patients, we sought to identify cooperating mutations in Tal1 transgenic mice. Clinical studies implicated the loss of the INK4a/ARF locus, which encodes two tumor suppressors, p16INK4a and p14ARF, in the majority of T-ALL patients. We demonstrated disease acceleration in tal1/ink4a/arf+/-, tal1/pl6ink4a+/- and tal1/p19arf+/- mice, thereby providing genetic evidence that Tal1 cooperates with loss of either p16Ink4a or p19Arf in leukemogenesis. The cooperation of Tal1 with the loss of or p16Ink4a or p19Arf, is consistent with our observation that Tal1 alters cell cycle regulation in leukemia by promoting S phase induction and apoptosis in vivo. An additional mutational event common in tal1 tumors is activation of the Notch1 signaling pathway. We provide evidence that the majority of tal1 tumors express increased levels of Notch1, and exhibit activating notch1 mutations. Additionally, tal1 tumors display sensitivity to the pharmacologic inhibition of γ-secretase activity in vitro, indicating that γ-secretase inhibitors may prove an efficacious treatment for TAL1-expressing T-ALL patients. Furthermore, we developed a doxycycline-regulated NotchIC T-ALL cell line, which will allow the identification of important Notch1IC target genes in leukemogenesis.

Nuclear hormone receptors (NHR) constitute a superfamily of ligand inducible transcriptional activators that enable an organism to regulate development and homeostasis through switching on or off target genes in response to stimuli reflecting changes in environment as well as endocrine. NHRs include classical steroid hormone receptors (GR, AR, ER and MR) and retinoid, thyroid hormone receptors. One long-term goal of our lab is to understand the molecular mechanisms through which the transcriptional activity of NHRs is regulated. Extensive studies in the past few years have revealed that in addition to the dependence on ligand availability, the transcriptional activity of NHRs is also regulated by two types of proteins: co activators and corepressors. In the absence of ligand, many NHRs, including TR and RAR can actively repress target gene transcription with the help of corepressors, proteins that physically interact with both NHRs and histone deacetylases (HDACs). Functional interactions between NHRs and corepressors therefore lead to tightly compact and transcriptionally non-permissive chromatin structures after the removal of obstructive acetyl groups from histone tails by HDACs. On the other hand, ligand binding stabilizes NHRs in a conformation that favors interaction with proteins other than corepressors; many of these proteins are able to potentiate the transcriptional activity of NHRs through various mechanisms, such as histone acetylation, chromatin remodeling and recruitment of basal transcription machinery and are collectively termed coactivators. Two highly related corepressors, SMRT (silencing mediator of retinoid and thyroid hormone receptors) and N-CoR (nuclear receptor corepressor), have been cloned. This research in corepressor SMRT started by a systematic study of its subcellular localization. We found that SMRT predominantly forms a specific nuclear punctuate structure that does not appear to overlap with any other well-known subnuclear domains/speckles. Although our searching for specific sequence signals that may determine the specific speckle localization of SMRT did not yield conclusive results, we discovered the colocalization of unliganded RAR and certain HDACs, including HDAC1, 3,4 and 5, in the SMRT nuclear speckles. Moreover, SMRT is likely to be the organizer of such speckles since it appears to be able to recruit other proteins into these speckles. The presence of HDAC1 in the SMRT speckles suggests a direct association between these two proteins, which has not been detected by previous biochemical analyses. Interestingly, HDAC1 point mutants that are completely defective in deacetylase activity failed to locate to SMRT nuclear speckles, while another partially active mutant maintained the colocalization. These discoveries may indicate SMRT nuclear speckles as novel nuclear domains involved in transcriptional repression. More physiologically relevant support for this hypothesis arises from study of HDAC4 and 5. HDAC4 and 5 are potent inhibitors of transcriptional activator MEF2C. Nuclear presence of HDAC4/5 can block the activation of MEF2C, which is required during muscle differentiation. Normally, HDAC4 is predominantly located in cytoplasm. However, we found that in the presence of SMRT overexpression, HDAC4 was found mostly in SMRT nuclear speckles. This accumulation enhanced HDAC4 mediated inhibition on MEF2C transcriptional activity in a transient transfection assay. SMRT overexpression also resulted in accumulation of HDAC5 in the SMRT nuclear speckles compared to the nuclear diffuse distribution in the absence of SMRT. Again, this accumulation of HDAC5 in nuclear speckles correlated with enhanced inhibition of MEF2C. Taken together, our study suggested that instead of being merely a corepressor for NHRs, SMRT might function as an organizer of a nuclear repression domain, which may be involved in a broad array of cellular processes. In contrast to the limited number of corepressors, numerous co activators have been identified; the SRC (or p160) family is relatively well studied. This family includes three highly related members, SRC-1, TIF2/GRIP1, RAC3/AIB1/ACTR/p/CIP. Similar domain structures are shared among these factors, with the most highly conserved region, the bHLH-PAS domain found within the N terminal ~400 amino acid residues. This study of RAC3 aims to identify the function of the highly conserved N terminal bHLH-PAS domain by isolating interacting proteins through yeast two-hybrid screening. One candidate gene isolated encodes the C terminal fragment of the human homologue of the yeast protein MMS19. Functional studies of this small fragment revealed that it specifically interacted with human estrogen receptors (ERs) and inhibited ligand induced transcriptional activity of ERs in the transient transfection assay. Then we cloned the full-length human MMS19 cDNA and characterized the hMMS19 as a weak coactivator for estrogen receptors in the transient transfection assay. Furthermore, when tested on separate AF-1 or AF-2 of ERs, hMMS19 specifically enhanced AF-1 but had no effect on AF-2. These results identified hMMS19 as a specific coactivator for ER AF-1.

Expansion of visceral adipose tissue correlates with the metabolic syndrome and increased cardiovascular risk. Hypertrophied visceral fat becomes inflamed, causing increased lipolysis, decreased triglyceride storage, and lipotoxicity in skeletal muscle and liver resulting in insulin resistance. Perivascular adipose tissue is a normal component of the adventitia of arteries in humans and animals. Whether or not perivascular adipose also becomes inflamed in obesity is an important question, as this may be an additional, direct mechanism by which obesity causes vascular inflammation and disease. Thus, for the first part of my thesis, we asked the question: does perivascular adipose in mice become inflamed with high fat feeding? In contrast to visceral adipose, macrophage gene expression was not increased in perivascular adipose in response to high fat diet, and this correlated with reduced F480 antigen positive cells as seen by immunohistochemistry and flow cytometry. Interestingly, perivascular adipose surrounding the thoracic aorta was similar to brown adipose tissue, a highly thermogenic fat depot, as shown by histology and DNA microarrays. Moreover, inter-scapular brown adipose was also resistant to diet induced inflammation in comparison to visceral adipose. These findings suggest that brown adipose in the perivascular niche may serve to protect the vasculature from diet induced inflammation, or from cold exposure, or both; whether or not brown perivascular adipose tissue exists in humans has yet to be determined. In the second part of my thesis, we evaluated the role of perivascular adipose tissue in the apolipoprotein E knockout mouse, which exhibits severe hyperlipidemia and atherosclerosis, but is resistant to diet induced obesity and glucose intolerance. We tested the hypothesis that in this model of severe atherosclerosis, inflammation of perivascular adipose does occur. However, we were surprised to find that macrophage specific gene expression, as determined by either microarray analysis or quantitative polymerase chain reaction, was not increased in either the perivascular or the visceral adipose of high fat diet fed apolipoprotein E knockout mice. While the visceral adipose of wild type mice had extensive alterations in gene expression in response to high fat diet, in particular, enrichment of inflammatory gene expression and broad down regulation of peroxisome proliferator activated receptor gamma target genes, apolipoprotein E knockout visceral adipose did not. Importantly, the apolipoprotein E knockout visceral adipose instead showed increased expression of genes encoding enzymes in fatty acid oxidation pathways. High fat diet fed apolipoprotein E knockout visceral adipose was also characterized by smaller adipocyte size. We conclude that, 1) inflammation in thoracic perivascular adipose does not occur in conjunction with diet induced obesity in normal animals nor with atherosclerosis in apolipoprotein E knockout mice, 2) thoracic perivascular adipose tissue is essentially identical to brown adipose tissue in mice, thus potentially protecting the vasculature from the cold, and 3) apolipoprotein E knockout mice remain lean on a high fat diet, despite hyperlipidemia and atherosclerosis, and the decreased adiposity correlates with decreased adipocyte size and adipose inflammation but increased oxidation of fatty acids. Consistent with previous work showing apolipoprotein E controls adipocyte uptake and deposition of triglyceride, its absence prevents adipocyte hypertrophy and resultant inflammation of visceral adipose tissue. Thus limiting adipocyte acquisition of fatty acids may be advantageous, provided that compensatory mechanisms to prevent sustained hyperlipidemia and peripheral organ lipotoxicity can be activated.

Very long chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD deficient mice and patients’ clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD deficient mice were treated systemically with 1x10 12 vector genomes of rAAV9-VLCAD. Expression was detected in the liver, heart and muscle. Also substantial expression of VLCAD was noted in the brain, where it was expressed across different sections of the brain and in different cell types with different morphologies. Biochemical correction was observed in vector-treated mice beginning two weeks post-injection, as characterized by a significant drop in long chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks post injection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD-/- mice dropped below 20°C and the mice became lethargic, requiring euthanasia. In contrast all rAAV9-treated VLCAD-/- mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD-/- mice maintained euglycemia, whereas untreated VLCAD-/- mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans.

The Snail protein functions as a transcriptional regulator to establish early mesodermal cell fate in Drosophila. Later, in germ band-extended embryos, Snail is considered a pan-neural protein based on its extensive expression in neuroblasts. The evidence presented in thesis links snail expression and function in CNS. Cloning and functional characterization of a novel snail homologue, in Drosophila, are also described here. Cloning of this gene, worniu (Chinese for snail), revealed that the neural function of snail is masked by this and another closely related gene escargot. Both Escargot and Worniu contain zinc finger domains that are highly homologous to that of Snail. These three members of Snail protein family are redundantly required for CNS development. Although not affecting formation of neuroblasts, the loss of expression of these three members correlates with disruption of Nb asymmetry and division. Downstream targets of Snail protein family, in these processes, are inscuteable and string. In mutant embryos, which have the three genes deleted, the RNA expression of inscuteable and string is significantly lowered. Consistent with the gene expression defects, the mutant embryos have loss of asymmetric localization of prospero RNA in neuroblasts and nuclear localization of Prospero protein in ganglion mother cells. Transgenic expression of inscuteable and string together, in the snail family deletion mutant, efficiently restores the Prospero expression in GMC, demonstrating that the two genes are key targets of Snail in Nbs. Like in the mesoderm, in CNS Snail function depends on interaction with dCtBP co-repressor. These results suggest that Sna [Snail] family of proteins control both asymmetry and cell division of neuroblasts by activating, perhaps indirectly, the expression of inscuteable and string.