Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb. 12, 2009). Includes bibliographical references.

>Magister Scientiae - MSc / The human arylamine N-acetyltransferase 1 (NAT1) is critical in determining the duration of action and pharmacokinetics of amine-containing drugs such as para-aminosalicylic acid and para-aminobenzoyl glutamate used in clinical therapy of tuberculosis (TB), as well as influencing the balance between detoxification and metabolic activation of these drugs. SNPs in this enzyme are continuously being detected and indicate inter-ethnic and inter-individual variation in the enzyme function. The effect of nsSNPs on the structure and function of proteins are routinely analyzed using SIFT and POLYPHEN-2 prediction algorithms. The false-negative rate of these two algorithms results in as much as 25% of nsSNPs. This study aimed to explore the use of homology modeling including residue interactions, Gibbs free energy change and solvent accessibility as additional evidence for predicting nsSNP effects on enzyme function.This study evaluated the functional effects of 14 nsSNPs identified in a South African mixed ancestry population of which 3 nsSNPs were previously identified in Caucasians. The SNPs were evaluated using structural analysis that included homology modeling, residue interactions, relative solvent accessibility,Gibbs free energy change and sequence conservation in addition to the routinely used nsSNP function prediction algorithms, SIFT and POLYPHEN-2. The structural analysis implemented in this study showed a loss of hydrogen bonds for S259R thereby affecting protein function which contradicts predictions obtained from SIFT and POLYPHEN-2 algorithms. The variant N245I was shown to be neutral but contradicted the predictions from SIFT and POLYPHEN-2. Structural analysis predicted that variant R242M would affect protein stability and therefore NAT1 function in agreement with POLYPHEN-2 predictions but contradicting predictions from SIFT. No structural changes were expected for variant E264K in agreement with predictions obtained from POLYPHEN-2 but contradicting results from SIFT. The functions of the remaining 10 nsSNPs were consistent with those predicted by SIFT and POLYPHEN-2 namely that four variants R117T, E167Q, T193S and T240S do not affect the NAT1 function whereas R166T, F202V, Q210P, D229H, V231G and V235A could affect the enzyme function.This study provided the first evaluation of the functional effects of 11 newly characterized nsSNPs on the NAT1 tuberculosis drug-metabolizing enzyme. The six functionally important nsSNPs predicted by all three methods and the four SNPs with contradictory results will be tested experimentally by creating a SNP construct that will be cloned into an expression vector. These combined computational and experimental studies will advance our understanding of NAT1 structure-function relationships and allow us to interpret the NAT1 genetic polymorphisms in individuals who are slow or fast acetylators. The results, albeit a small dataset demonstrate that the routinely used algorithms are not without flaws and that improvements in functional prediction of nsSNPs can be obtained by close scrutiny of the molecular interactions of wild type and variant amino acids.

The compaction of the eukaryotic genome into a complex, highly folded chromatin structure necessitates cellular mechanisms for allowing access of regulatory proteins to the DNA template. Recent advances have led to the identification of two distinct families of chromatin remodeling enzymes--multi-subunit complexes that harbor a SWI2/SNF2 ATPase family member, and the nuclear acetyltransferases. The Saccharomyces cerevisiae SWI/SNF complex, the prototype for the ATP-dependent chromatin remodeling machines, is required for expression of a subset of genes in yeast. This 2MDa multimeric assembly is believed to facilitate transcriptional enhancement by antagonizing chromatin-mediated transcriptional repression through disruption of histone-DNA contacts. In an attempt to identify components or regulators of the SWI/SNF complex, we have cloned three previously identified genes, ADA2, ADA3, and GCN5, that encode subunits of a complex distinct from SWI/SNF. During the course of this thesis work, one of these gene products, GCN5, was identified as the first catalytic nuclear histone acetyltransferase. The goal of this thesis work was to determine the role of the ADA/GCN5 complex in transcriptional activation in Saccharomyces cerevisiae. Using in vivo functional and genetic analysis, we have found that mutations in ADA2, ADA3, and GCN5 cause phenotypes strikingly similar to those of swi/snf mutants. ADA2, ADA3, and GCN5 are required for full expression of all SWI/SNF-dependent genes tested, including HO, SUC2, INO1, and Ty elements. Furthermore, mutations in the SIN1 gene, which encodes a non-histone chromatin component, or mutations in histones H3 or H4, alleviate the transcriptional defects caused by ada/gcn5 or swi/snf mutations. We have also found that ada2 swi1, ada3 swi1, and gcn5 swi1 double mutants are inviable and that mutations in SIN1 allow viability of these double mutants. To determine the biochemical activities of the native GCN5-containing complex in yeast, we have partially purified three chromatographically distinct GCN5-dependent acetyltransferase activities. We have found that these three acetyltransferase complexes demonstrate unique substrate specificities for free histones and histones assembled into nucleosomal arrays. Additionally, we found that these enzymes not only acetylate histones, but also purified yeast Sin1 protein, a non-histone chromatin component that resembles HMG1. We have also established a functional relationship between GCN5-dependent histone acetylation and polyamine-dependent chromatin condensation. We have found that depletion of cellular polyamines alleviates transcriptional defects caused by inactivation of the GCN5 histone acetyltransferase. In contrast, polyamine depletion does not alter the transcriptional requirements for the SWI/SNF chromatin remodeling complex. We have also found that polyamines facilitate oligomerization of nucleosomal arrays in vitro. Furthermore, this polyamine-mediated condensation reaction requires intact N-terminal domains of the core histones, and is inhibited by hyperacetylation of these domains. The results presented throughout this thesis support roles for the ADA/GCN5 products in antagonizing chromatin. In vivo analysis suggests a functional relationship between the ADA/GCN5 acetyltransferase complex (or complexes) and the SWI/SNF complex. These comp1exes may operate in concert at nucleosomes within specific promoters to facilitate activated transcription. Furthermore, our studies suggest that polyamines are repressors of transcription in vivo, and that an additional role of histone hyperacetylation is to antagonize the ability of polyamines to stabilize highly condensed states of chromosomal fibers.

Indiana University-Purdue University Indianapolis (IUPUI) / Infection with the single-celled parasite Toxoplasma gondii (phylum Apicomplexa) is usually benign in normal healthy individuals, but can cause congenital birth defects, ocular disease, and also life-threatening infection in immunocompromised patients. Acute infection caused by tachyzoites is controlled by a healthy immune response, but the parasite differentiates into a latent cyst form (bradyzoite) leading to permanent infection and chronic disease. Current therapies are effective only against tachyzoites, are highly toxic to the patient, and do not eradicate the encysted bradyzoites, thus highlighting the need for novel therapeutics. Inhibitors of histone deacetylases have been shown to reduce parasite viability in vitro demonstrating that chromatin remodeling enzymes, key mediators in epigenetic regulation, might serve as potential drug targets. Furthermore, epigenetic regulation has been shown to contribute to gene expression and differentiation in Toxoplasma. This dissertation focused on investigating the physiological role of a Toxoplasma GCN5-family histone acetyltransferase (HAT), termed TgGCN5-B. It was hypothesized that TgGCN5-B is an essential HAT that resides within a unique, multi-subunit complex in the parasite nucleus. Studies of TgGCN5-B have revealed that this HAT possesses a unique nuclear localization signal (311RPAENKKRGR320) that is both necessary and sufficient to translocate the protein to the parasite nucleus. Although no other protein motifs have been identified in the N-terminal extension of TgGCN5-B, it is likely that this extension plays a role in protein-protein interactions. All GCN5 homologues function within large multi-subunit complexes, many being conserved among species, but bioinformatic analysis of the Toxoplasma genome revealed a lack of many of these conserved components. Biochemical studies identified several potential TgGCN5-B associating proteins, including several novel apicomplexan transcription factors. Preliminary evidence suggested that TgGCN5-B was essential for tachyzoites; therefore, a dominant-negative approach was utilized to examine the role of TgGCN5-B in the physiology of Toxoplasma. When catalytically inactive TgGCN5-B protein was over-expressed in the parasites, there was a significant decrease in tachyzoite growth and viability, with initial observations suggesting defects in nuclear division and daughter cell budding. These results demonstrate that TgGCN5-B is important for tachyzoite development and indicate that therapeutic targeting of this HAT could be a novel approach to treat toxoplasmosis.

L’acétylation des histones est associée à une activation transcriptionnelle. Cette acétylation est mise en place par des histone acétyltransférases (HATs) qui sont le plus souvent les sous-unités catalytiques de complexes multiprotéiques. Mon travail concerne plus particulièrement deux complexes contenant l’acétyltransférase Mof, MSL et NSL, ainsi que le complexe HAT Tip60-p400 dans les cellules souches embryonnaires de souris (mESCs). Nos analyses de localistaion sur l’ensemble du génome par ChIP-seq indiquent que MSL, NSL et Tip60-p400 se lient aux gènes activement transcrits et agissent comme des co-activateurs transcriptionnels majeurs dans les mESCs. MSL, NSL et Tip60-p400 ont des rôles à la fois chevauchants mais aussi distincts dans la régulation transcriptionnelle dans les mESCs. Chaque complexe présent un profil distinct de liaison à la chromatine. NSL lie principalement des gènes de ménage. MSL et Tip60-p400 sont également présent les gènes impliqués dans le développement. MSL est directement impliqué dans l’augmentation de l’expression de ces gènes au cours de la différenciation des mESCs. / Histone acetylation is involved in transcriptional activation of genes and is carried out by histone acetyltransferases (HATs), which are part of molecular protein complexes. This study focuses on the genome-wide role of Mof-containing MSL and NSL complexes and the Tip60-p400 complex in mouse embryonic stem cells (mESCs). I have analysed these complexes by ChIP-seq, shRNA knockdown and biochemical approaches. The genome-wide binding studies show that NSL, MSL and Tip60-p400 have a global overlap at promoters, but also bind to specific gene sets. There distinct binding profiles propose distinct roles in transcriptional regulation. MSL is the main H4K16 acetylase in mESCs.NSL binds mainly to housekeeping genes, whereas MSL and Tip60 are also present at developmental genes. Importantly, these developmental genes are directly regulated by MSL during cellular differentiation.

The polyamines spermine and spermidine play important roles in many cellular processes, and unusual levels of these polyamines have been associated with numerous human diseases. Spermidine/spermine N¹-acetyltransferase (SSAT) is an enzyme involved in polyamine regulation, where acetylation of polyamines by SSAT ultimately leads to their degradation or export from the cell. In this dissertation, x-ray crystallography and nuclear magnetic resonance (NMR) are used to provide insights into the structure and function of this important enzyme. X-ray crystallography provided two distinct views of SSAT: one of the enzyme in complex with coenzyme A (CoA), and another of the enzyme in complex with CoA and the polyamine spermine. Together, the two structures reveal structural plasticity in the active site of the enzyme. The complex with spermine provides a direct view of polyamine binding by SSAT, and shows that the enzyme relies heavily on associated water molecules to bind spermine; these water molecules also appear to form a "proton relay" between the primary amine of spermine and the side-chain of a conserved glutamate residue. Guided by the structural results, NMR methods were used to test hypotheses regarding the enzyme mechanism of SSAT. The activity of the enzyme over a range of solution conditions, and towards different polyamine substrates, was determined; the effects of mutating single amino acids in the enzyme were also evaluated. The enzyme appeared to be most active between pH 8.5 and 9.5, and mutation of the aforementioned glutamate significantly altered this behavior. This suggests the glutamate is directly involved in the acetyltransfer reaction, where it likely functions as a catalytic base though the proton relay in the enzyme active site. These studies advance our general understanding of how polyamines are regulated in mammalian cells, and have the potential to assist in developing new therapeutic options for human diseases involving polyamines. / text

Aescin is a mixture of several acidic triterpenoid saponin glycosides found in the extracts of the horse chestnut tree. Horse chestnut, Aesculus Hipoocastanum, is one of the 25 domestic species of Aesculus that are mostly large, ornamental shade trees. Although known to be poisonous, the nuts of the horse chestnut are used by Amerindians, after detoxification. Horse chestnuts are said to have several traditional medicinal usages including even cancer. In this study the biochemical, genotoxic, and cytotoxic effects of aescin was studied using isolated lymphocytes, whole blood lymphocytes and HL-60 promyeloid leukemia cell lines. Cytotoxicity of aescin was examined by trypan blue viability staining of the cells in culture treated with varying aescin concentrations. It was observed that aescin was cytotoxic at all concentrations, for all cell types studied, except whole blood lymphocytes, where it was not cytotoxic at 10-9 and 10-10 M concentrations. Genotoxicity of aescin was examined by sister chromatid exchange and micronucleus. The genotoxic effect of Aescin was observed to be more significant over isolated lymphocytes compared to other cell lines. On the otherhand, aescin at 10-8 M and lower concentrations were observed to be non-genotoxic over whole blood lymphocytes whereas this concentration was considerably toxic for isolated lymphocytes and for HL-60 cell lines. Apoptotic properties of aescin were determined by DNA fragmentation, cytochrome c release and negative NAPO staining. All the Aescin concentrations tested resulted in apoptosis over HL-60 cell lines, whereas necrosis was not observed. However, isolated lymphocytes showed both apoptosis and necrosis upon treatment with 10-6 M to 10-8 M aescin, exhibiting apoptosis only at 10-9 M and 10-10 M. Biochemical effects of aescin were investigated by following GST and NAT enzyme activities. An increase in GST enzyme activity was observed over all cell lines treated with increasing aescin concentrations for 72 hours. Whereas NAT activity was decreased upon treatment with aescin in similar manner.

The following work examines the mechanisms by which Tip60-p400 chromatin remodeling complex regulates gene expression in embryonic stem cells (ESCs). Tip60-p400 complex has distinct functions in undifferentiated and differentiated cells. While Tip60-p400 is often associated with gene activation in differentiated cells, its most prominent function in ESCs is to repress differentiation-related genes. I show that Tip60-p400 interacts with Hdac6 and other proteins to form a unique form of the complex in ESCs. Tip60-Hdac6 interaction is stem cell specific and is necessary for Tip60-p400 mediated gene regulation, indicating that Tip60- p400 function is controlled in part through the regulation of Hdac6 during development. Furthermore, I find that Hdac6 is required for the binding of Tip60- p400 to many of its target genes, indicating Hdac6 is necessary for the unique function of Tip60-p400 in ESCs. In addition to accessory proteins like Hdac6, Tip60-p400 also interacts with thousands of coding and noncoding RNAs in ESCs. I show that R-loops, DNA-RNA hybrids formed during transcription of many genes, are important for regulation of chromatin binding by at least two chromatin regulators (Tip60-p400 and PRC2). This finding suggests that transcripts produced by many genes in ESC may serve as a signal to modulate binding of chromatin regulators. However, R-loops might also function to regulate chromatin architecture in differentiated cells as well. Future studies based on this work will be necessary to understand the full repertoire of cell types and chromatin regulators regulated by these structures.

Les histones sont des protéines nucléaires hautement conservées chez les cellules des eucaryotes. Elles permettent d’organiser et de compacter l’ADN sous la forme de nucléosomes, ceux-ci representant les sous unités de base de la chromatine. Les histones peuvent être modifiées par de nombreuses modifications post-traductionnelles (PTMs) telles que l’acétylation, la méthylation et la phosphorylation. Ces modifications jouent un rôle essentiel dans la réplication de l’ADN, la transcription et l’assemblage de la chromatine. L’abondance de ces modifications peut varier de facon significative lors du developpement des maladies incluant plusieurs types de cancer. Par exemple, la perte totale de la triméthylation sur H4K20 ainsi que l’acétylation sur H4K16 sont des marqueurs tumoraux spécifiques a certains types de cancer chez l’humain. Par conséquent, l’étude de ces modifications et des événements determinant la dynamique des leurs changements d’abondance sont des atouts importants pour mieux comprendre les fonctions cellulaires et moléculaires lors du développement de la maladie. De manière générale, les modifications des histones sont étudiées par des approches biochimiques telles que les immuno-buvardage de type Western ou les méthodes d’immunoprécipitation de la chromatine (ChIP). Cependant, ces approches présentent plusieurs inconvénients telles que le manque de spécificité ou la disponibilité des anticorps, leur coût ou encore la difficulté de les produire et de les valider. Au cours des dernières décennies, la spectrométrie de masse (MS) s’est avérée être une méthode performante pour la caractérisation et la quantification des modifications d’histones. La MS offre de nombreux avantages par rapport aux techniques traditionnelles. Entre autre, elle permet d’effectuer des analyses reproductibles, spécifiques et facilite l’etude d’un large spectre de PTMs en une seule analyse. Dans cette thèse, nous présenterons le développement et l’application de nouveaux outils analytiques pour l’identification et à la quantification des PTMs modifiant les histones. Dans un premier temps, une méthode a été développée pour mesurer les changements d’acétylation spécifiques à certains sites des histones. Cette méthode combine l’analyse des histones intactes et les méthodes de séquençage peptidique afin de déterminer les changements d’acétylation suite à la réaction in vitro par l’histone acétyltransférase (HAT) de levure Rtt109 en présence de ses chaperonnes (Asf1 ou Vps75). Dans un second temps, nous avons développé une méthode d’analyse des peptides isomériques des histones. Cette méthode combine la LC-MS/MS à haute résolution et un nouvel outil informatique appelé Iso-PeptidAce qui permet de déconvoluer les spectres mixtes de peptides isomériques. Nous avons évalué Iso-PeptidAce avec un mélange de peptides synthétiques isomériques. Nous avons également validé les performances de cette approche avec des histones isolées de cellules humaines érythroleucémiques (K562) traitées avec des inhibiteurs d’histones désacétylases (HDACi) utilisés en clinique, et des histones de Saccharomyces cerevisiae liées au facteur d’assemblage de la chromatine (CAF-1) purifiées par chromatographie d’affinité. Enfin, en utilisant la méthode présentée précédemment, nous avons fait une analyse approfondie de la spécificité de plusieurs HATs et HDACs chez Schizosaccharomyces pombe. Nous avons donc déterminé les niveaux d’acétylation d’histones purifiées à partir de cellules contrôles ou de souches mutantes auxquelles il manque une HAT ou HDAC. Notre analyse nous a permis de valider plusieurs cibles connues des HATs et HDACs et d’en identifier de nouvelles. Nos données ont également permis de définir le rôle des différentes HATs et HDACs dans le maintien de l’équilibre d’acétylation des histones. Dans l’ensemble, nous anticipons que les méthodes décrites dans cette thèse permettront de résoudre certains défis rencontrés dans l’étude de la chromatine. De plus, ces données apportent de nouvelles connaissances pour l’élaboration d’études génétiques et biochimiques utilisant S. pombe. / Histones are highly conserved, basic proteins found in eukaryotic cell nuclei. They organize and package DNA strands into nucleosome core particles (NCPs), the fundamental repeating units of eukaryotic chromatin. The histones are subject to a wide variety of posttranslational modifications (PTMs) including acetylation, methylation and phosphorylation. These PTMs play an essential role in DNA-replication, transcription, and chromatin assembly. Alterations in histone PTM abundances have been implicated in several types of cancer. For example, the global loss of trimethylation at H4K20 and acetylation at H4K16 is a hallmark of human cancers. Thus, characterization of histone PTMs and their dynamics is extremely useful for elucidating normal cellular functions and molecular pathways that lead to diseases. Traditionally, histone PTMs are analyzed using antibody-based approaches such as western blot and chromatin immunoprecipitation (ChIP) assays. These methods, however, suffer from several limitations including antibody cross-reactivity, epitope occlusion, and the cost and difficulty in producing and validating antibodies. Over the last decade, mass spectrometry (MS) has emerged as a powerful technique for the characterization and quantification of histone PTMs. MS offers several advantages over the traditional approaches including reproducibility, specificity, and ability to rapidly analyze numerous PTMs in a single experiment. In this thesis, the development and applications of novel analytical tools for the identification and quantification of histone PTMs are presented. First, a method useful for measuring the global and site specific changes in histone acetylation is described. This method combines intact mass analysis and peptide sequencing approaches to study the global and site specific changes in histone acetylation during in vitro assays with yeast Rtt109 and its chaperone (Asf1 or Vps75). Second, a method for analysis of isomeric histone peptides is presented. This method combines a high resolution LC-MS/MS with a novel bioinformatics tool called Iso-PeptidAce to deconvolute mixed spectra of co-eluting isomeric peptides. We benchmarked Iso-PeptidAce using mixtures of synthetic isomeric peptides. We demonstrated its capability in histones isolated from human erythroleukemic (K562) cells treated with clinically relevant histone deacetylase inhibitors (HDACi) and in affinity-purified S. cerevisiae histones bound to chromatin assembly factor-1 (CAF-1). Third, by employing the above methods, an in-depth quantitative analysis of the substrate specificities of several fission yeast HATs and HDACs was assessed. We determined the acetylation site occupancy of multiple lysines in histones isolated from a control or mutant strains lacking specific HAT or HDAC activities. Our analysis identified several known and novel HAT and HDAC target sites. Our data also defined the division of labor between the different HATs and HDACs in maintaining the steady-state level of histone acetylation. Overall, we anticipate that the methods described in this thesis will address some of the existing challenges facing the chromatin field. Moreover, the data presented will provide valuable insights for future genetic and biochemical studies involving the fission yeast.