It is evident that components of the 26S proteasome function beyond protein degradation in the regulation of transcription. Studies in yeast implicate the 26S proteasome, specifically the 19S cap, in the epigenetic regulation of transcription. Saccharomyces cerevisiae 19S ATPases remodel chromatin by facilitating histone acetylation and methylation. However, it is unclear if the 19S ATPases play similar roles in mammalian cells. We previously found that the 19S ATPase Sug1 positively regulates transcription of the critical inflammatory gene MHC-II and that the MHC-II promoter fails to efficiently bind transcription factors upon Sug1 knockdown. MHC-II transcription is regulated by the critical coactivator CIITA. We now find that Sug1 is crucial for regulating histone H3 acetylation at the cytokine inducible MHC-II and CIITA promoters. Histone H3 acetylation is dramatically decreased upon Sug1 knockdown with a preferential loss occurring at lysine 18. Research in yeast indicates that the ortholog of Sug1, Rpt6, acts as a mediator between the activating modifications of histone H2B ubiquitination and H3 methylation. Therefore, we characterized the role the 19S proteasome plays in regulating additional activating modifications. As with acetylation, Sug1 is necessary for proper histone H3K4 and H3R17 methylation at cytokine inducible promoters. In the absence of Sug1, histone H3K4me3 and H3R17me2 are substantially inhibited. Our observation that the loss of Sug1 has no significant effect on H3K36me3 implies that Sug1’s regulation of histone modifications is localized to promoter regions as H3K4me3 but not H3K36me3 is clustered around gene promoters. Here we show that multiple H3K4 histone methyltransferase subunits bind constitutively to the inducible MHC-II and CIITA promoters and that over-expressing one subunit significantly enhances promoter activity. Furthermore, we identified a critical subunit of the H3K4 methyltransferase complex that binds multiple histone modifying enzymes, but fails to bind the CIITA promoter in the absence of Sug1, implicating Sug1 in recruiting multi-enzyme complexes responsible for initiating transcription. Finally, Sug1 knockdown maintains gene silencing as elevated levels of H3K27 trimethylation are observed upon Sug1 knockdown. Together these studies strongly implicate the 19S proteasome in mediating the initial reorganization events to relax the repressive chromatin structure surrounding inducible genes.

Major Histocompatibility Complex (MHC) class II molecules are indispensable arms of the im-mune system that present extracellular antigens to CD4+T cells and initiate the adaptive immune response. MHC class II expression requires recruitment of a master regulator, the class II trans-activator (CIITA). How this master transcriptional regulator is recruited, stabilized and degraded is unknown. The 26S proteasome, a master regulator of protein degradation, is a multi-subunit complex composed of a 20S core particle capped on one or both ends by 19S regulatory particles. Previous findings have linked CIITA and MHC class II transcription to the ubiquitin proteasome system (UPS) as mono-ubiquitination of CIITA increases its transactivity whereas poly-ubiquitination targets CIITA for degradation. Increasing evidence indicates individual ATPase subunits of the 19S regulator play non-proteolytic roles in transcriptional regulation and histone modification. Our initial observations indicate proteasome inhibition decreases CIITA transac-tivity and MHC class II expression without affecting CIITA expression levels. Following cyto-kine stimulation, the 19S ATPase Sug1 associates with CIITA and with the MHC class II enhan-ceosome complex. Absence of Sug1 reduces promoter recruitment of CIITA and proteasome inhibition fails to restore CIITA binding, indicating Sug1 is required for CIITA mediated MHC class II expression. Furthermore, we identify a novel N-terminal 19S ATPase binding domain (ABD) within CIITA. The ABD of CIITA lies within the Proline/Serine/Threonine (P/S/T) re-gion of CIITA and encompasses a majority of the CIITA degron sequence. Absence of the ABD increases CIITA half-life, but blocks MHC class II surface expression, indicating that CIITA requires interaction with the 19S ATPases for both its deployment and destruction. Finally, we identify three degron proximal lysine residues, lysines (K): K315, K330 and K333, and a phosphorylation site, serine (S), S280, located within the CIITA degron, that regulate CIITA ubiquitination, stability and MHC class II expression. These are the first lysine residues identified as sites of CIITA ubiquitination that are essential for MHC class II expression. These observations increase our understanding of the role of the UPS in modulating CIITA mediated MHC class II transcription and will facilitate the development of novel therapies involving manipulation of MHC class II gene expression.

La différentiation entre le « soi » et le « non-soi » est un processus biologique essentiel à la vie. Les peptides endogènes présentés par les complexes majeurs d’histocompatibilité de classe I (CMH I) représentent le fondement du « soi » pour les lymphocytes T CD8+. On donne le nom d’immunopeptidome à l’ensemble des peptides présentés à la surface cellulaire par les molécules du CMH I. Nos connaissances concernant l’origine, la composition et la plasticité de l’immunopeptidome restent très limitées. Dans le cadre de cette thèse, nous avons développé une nouvelle approche par spectrométrie de masse permettant de définir avec précision : la nature et l’abondance relative de l’ensemble des peptides composant l’immunopeptidome. Nous avons trouvé que l’immunopeptidome, et par conséquent la nature du « soi » immun, est surreprésenté en peptides provenant de transcrits fortement abondants en plus de dissimuler une signature tissu-spécifique. Nous avons par la suite démontré que l’immunopeptidome est plastique et modulé par l’activité métabolique de la cellule. Nous avons en effet constaté que les modifications du métabolisme cellulaire par l’inhibition de mTOR (de l’anglais mammalian Target Of Rapamycin) provoquent des changements dynamiques dans la composition de l’immunopeptidome. Nous fournissons également la première preuve dans l’étude des systèmes que l’immunopeptidome communique à la surface cellulaire l’activité de certains réseaux biochimiques ainsi que de multiples événements métaboliques régulés à plusieurs niveaux à l’intérieur de la cellule. Nos découvertes ouvrent de nouveaux horizons dans les domaines de la biologie des systèmes et de l’immunologie. En effet, notre travail de recherche suggère que la composition de l’immunopeptidome est modulée dans l’espace et le temps. Il est par conséquent très important de poursuivre le développement de méthodes quantitatives au niveau des systèmes qui nous permettront de modéliser la plasticité de l’immunopeptidome. La simulation et la prédiction des variations dans l’immunopeptidome en réponse à différents facteurs cellulaires intrinsèques et extrinsèques seraient hautement pertinentes pour la conception de traitements immunothérapeutiques. / Self/non-self discrimination is a fundamental requirement of life. Endogenous peptides presented by major histocompatibility complex class I (MHC I) molecules represent the essence of self for CD8 T lymphocytes. These MHC I peptides (MIPs) are collectively referred to as the immunopeptidome. Very little is known about the origin, composition and plasticity of the immunopeptidome. Here, we developed a novel high-throughput mass spectrometry approach that yields an accurate definition of the nature and relative abundance of peptides presented by MHC I molecules. Surprisingly, we found that the immunopeptidome, and therefore the nature of the immune self, is biased toward peptides derived from highly abundant transcripts and conceals a tissue-specific signature. Then, we showed that the immunopeptidome is plastic and moulded by cellular metabolic activity. In fact, we found that altering cellular metabolism via the inhibition of the mammalian target of rapamycin (mTOR) results in dynamic changes in the cell surface MIPs landscape. Importantly, we provide the first systems-level evidence that the immunopeptidome projects at the cell surface a faithful representation of biochemical networks and metabolic events regulated at multiple levels inside the cell. Our discoveries open up new perspectives in systems biology and immunology. Indeed, our work suggests that the composition of the immunopeptidome is modulated in space and time. Therefore, it is imperative to further develop and exploit systems-level quantitative methods that will enable modelling of the immunopeptidome’s plasticity. Simulating and predicting variations in the immunopeptidome in response to cell-intrinsic and -extrinsic factors could be relevant to numerous contexts, including the rational design of immunotherapeutic interventions.

Understanding the genetic basis of biological processes is a fundamental component of modern ecology and evolutionary biology studies. With the recent advent of next generation sequencing (NGS) technologies, it is now possible to perform large genome and transcriptome projects for ecologically important non-model species. In this thesis, I focused on the development and application of genomic resources of two non-model bird species, the black grouse (Tetrao tetrix) and the great snipe (Gallinago media). Using the chicken genome as a reference, I developed a reference guided NGS pipeline to assemble the complete draft genome of black grouse. The draft genome has a good coverage of the main 29 chromosomes of the chicken genome. The genome was used to develop a vast number of genetic markers. Comparing this genome with that of other species, I identified the genomic regions which were important for the lineage specific evolution of black grouse. I also sequenced and characterised the spleen transcriptome of the black grouse. I identified and validated a large number of gene-based microsatellite markers from the transcriptome and identified and confirmed the expression of immune related genes. Using a similar RNA-Seq approach, I also sequenced the blood transcriptomes of 14 great snipe males with different mating success. I identified genes and single nucleotide polymorphisms (SNPs) which might be related to male mating success in this species, both in terms of gene expression levels and genetic variation structure. For the immunologically important major histocompatibility complex (MHC) gene region of black grouse, I constructed a fosmid library and used it to sequence the complete core MHC region of this species. This resource allowed me to perform a comprehensive comparative genomics analysis of the galliform MHC, by which I found that some genes in this region were affected by selective forces. I was also able to develop a single locus genotyping protocol for the duplicated MHC BLB (class IIB) genes and found that the two black grouse BLB loci followed different evolutionary trajectories. This thesis set an example of developing genomic resources in non-model species and applying them in addressing questions relevant to ecology and evolutionary biology.

La différentiation entre le « soi » et le « non-soi » est un processus biologique essentiel à la vie. Les peptides endogènes présentés par les complexes majeurs d’histocompatibilité de classe I (CMH I) représentent le fondement du « soi » pour les lymphocytes T CD8+. On donne le nom d’immunopeptidome à l’ensemble des peptides présentés à la surface cellulaire par les molécules du CMH I. Nos connaissances concernant l’origine, la composition et la plasticité de l’immunopeptidome restent très limitées. Dans le cadre de cette thèse, nous avons développé une nouvelle approche par spectrométrie de masse permettant de définir avec précision : la nature et l’abondance relative de l’ensemble des peptides composant l’immunopeptidome. Nous avons trouvé que l’immunopeptidome, et par conséquent la nature du « soi » immun, est surreprésenté en peptides provenant de transcrits fortement abondants en plus de dissimuler une signature tissu-spécifique. Nous avons par la suite démontré que l’immunopeptidome est plastique et modulé par l’activité métabolique de la cellule. Nous avons en effet constaté que les modifications du métabolisme cellulaire par l’inhibition de mTOR (de l’anglais mammalian Target Of Rapamycin) provoquent des changements dynamiques dans la composition de l’immunopeptidome. Nous fournissons également la première preuve dans l’étude des systèmes que l’immunopeptidome communique à la surface cellulaire l’activité de certains réseaux biochimiques ainsi que de multiples événements métaboliques régulés à plusieurs niveaux à l’intérieur de la cellule. Nos découvertes ouvrent de nouveaux horizons dans les domaines de la biologie des systèmes et de l’immunologie. En effet, notre travail de recherche suggère que la composition de l’immunopeptidome est modulée dans l’espace et le temps. Il est par conséquent très important de poursuivre le développement de méthodes quantitatives au niveau des systèmes qui nous permettront de modéliser la plasticité de l’immunopeptidome. La simulation et la prédiction des variations dans l’immunopeptidome en réponse à différents facteurs cellulaires intrinsèques et extrinsèques seraient hautement pertinentes pour la conception de traitements immunothérapeutiques. / Self/non-self discrimination is a fundamental requirement of life. Endogenous peptides presented by major histocompatibility complex class I (MHC I) molecules represent the essence of self for CD8 T lymphocytes. These MHC I peptides (MIPs) are collectively referred to as the immunopeptidome. Very little is known about the origin, composition and plasticity of the immunopeptidome. Here, we developed a novel high-throughput mass spectrometry approach that yields an accurate definition of the nature and relative abundance of peptides presented by MHC I molecules. Surprisingly, we found that the immunopeptidome, and therefore the nature of the immune self, is biased toward peptides derived from highly abundant transcripts and conceals a tissue-specific signature. Then, we showed that the immunopeptidome is plastic and moulded by cellular metabolic activity. In fact, we found that altering cellular metabolism via the inhibition of the mammalian target of rapamycin (mTOR) results in dynamic changes in the cell surface MIPs landscape. Importantly, we provide the first systems-level evidence that the immunopeptidome projects at the cell surface a faithful representation of biochemical networks and metabolic events regulated at multiple levels inside the cell. Our discoveries open up new perspectives in systems biology and immunology. Indeed, our work suggests that the composition of the immunopeptidome is modulated in space and time. Therefore, it is imperative to further develop and exploit systems-level quantitative methods that will enable modelling of the immunopeptidome’s plasticity. Simulating and predicting variations in the immunopeptidome in response to cell-intrinsic and -extrinsic factors could be relevant to numerous contexts, including the rational design of immunotherapeutic interventions.

<p>The capacity of marine organisms to adapt to natural and anthropogenic stressors is an integral component of ocean health. Harmful algal blooms (HABs), which are one of many growing threats in coastal marine ecosystems, represent a historically present natural stressor that has recently intensified and expanded in geographic distribution partially due to anthropogenic activities. In the Gulf of Mexico, HABs of <italic>Karenia brevis</italic> occur almost annually and produce neurotoxic brevetoxins that have been associated with large-scale mortality events of many marine species, including the common bottlenose dolphin (<italic>Tursiops truncatus</italic>). The factors resulting in large-scale dolphin mortality associated with HABs are not well understood, particularly in regards to the seemingly different impacts of HABs in geographically disjunct dolphin populations. My dissertation investigates a genetic basis for resistance to HABs in bottlenose dolphins in central-west Florida and the Florida Panhandle. I used both genome-wide and candidate gene approaches to analyze genetic variation in dolphins that died putatively due to brevetoxicosis and live dolphins from the same geographic areas that survived HAB events. Using restriction site-associated DNA sequencing, I identified genetic variation that suggested both a common genetic basis for resistance to HABs in bottlenose dolphins across the Gulf coast of Florida and regionally specific resistance. Many candidate genes involved in the immune, nervous, and detoxification systems were found in close genomic proximity to survival-associated polymorphisms throughout the bottlenose dolphin genome. I further investigated two groups of candidate genes, nine voltage-gated sodium channel genes selected because of their putative role in brevetoxin binding and four major histocompatibility complex (MHC) loci selected because of their genomic proximity to a polymorphism exhibiting a strong association with survival. I found little variation in the sodium channel genes and conclude that bottlenose dolphins have not evolved resistance to HABs via mutations in the toxin binding site. The immunologically relevant MHC loci were highly variable and exhibited patterns of genetic differentiation among geographic regions that differed from neutral loci; however, genetic variation at the MHC also could not fully explain variation in survival of bottlenose dolphins exposed to HABs. In my final chapter, I consider the advantages and drawbacks of the genome-wide approach in comparison to a candidate gene approach and, as laid out in my dissertation, I recommend using both complementary approaches in future investigations of adaptation in genome-enabled non-model organisms.</p> / Dissertation

It is evident that components of the 26S proteasome function beyond protein degradation in the regulation of transcription. Studies in yeast implicate the 26S proteasome, specifically the 19S cap, in the epigenetic regulation of transcription. Saccharomyces cerevisiae 19S ATPases remodel chromatin by facilitating histone acetylation and methylation. However, it is unclear if the 19S ATPases play similar roles in mammalian cells. We previously found that the 19S ATPase Sug1 positively regulates transcription of the critical inflammatory gene MHC-II and that the MHC-II promoter fails to efficiently bind transcription factors upon Sug1 knockdown. MHC-II transcription is regulated by the critical coactivator CIITA. We now find that Sug1 is crucial for regulating histone H3 acetylation at the cytokine inducible MHC-II and CIITA promoters. Histone H3 acetylation is dramatically decreased upon Sug1 knockdown with a preferential loss occurring at lysine 18. Research in yeast indicates that the ortholog of Sug1, Rpt6, acts as a mediator between the activating modifications of histone H2B ubiquitination and H3 methylation. Therefore, we characterized the role the 19S proteasome plays in regulating additional activating modifications. As with acetylation, Sug1 is necessary for proper histone H3K4 and H3R17 methylation at cytokine inducible promoters. In the absence of Sug1, histone H3K4me3 and H3R17me2 are substantially inhibited. Our observation that the loss of Sug1 has no significant effect on H3K36me3 implies that Sug1’s regulation of histone modifications is localized to promoter regions as H3K4me3 but not H3K36me3 is clustered around gene promoters. Here we show that multiple H3K4 histone methyltransferase subunits bind constitutively to the inducible MHC-II and CIITA promoters and that over-expressing one subunit significantly enhances promoter activity. Furthermore, we identified a critical subunit of the H3K4 methyltransferase complex that binds multiple histone modifying enzymes, but fails to bind the CIITA promoter in the absence of Sug1, implicating Sug1 in recruiting multi-enzyme complexes responsible for initiating transcription. Finally, Sug1 knockdown maintains gene silencing as elevated levels of H3K27 trimethylation are observed upon Sug1 knockdown. Together these studies strongly implicate the 19S proteasome in mediating the initial reorganization events to relax the repressive chromatin structure surrounding inducible genes.

[Truncated abstract] Polymorphic Alu insertions (POALINs) are known to contribute to the variation and genetic diversity of the human genome. In this report specific POALINs of the Major Histocompatibility Complex (MHC) were studied. Previous population studies on the MHC POALINs have focused on individuals of African, European and Asian descent. In this study, we expand the research by studying a new and previously uncharacterised population, focusing on the Bedouin from the Middle East. Specifically we report on the individual insertion frequencies of four POALINs within the MHC class I region of this population. POALINs are members of a young Alu subfamily that have only recently been inserted into the human genome. POALINs are either present or absent at particular sites. Individuals that share the inserted (or deleted) polymorphism inherited the insertion (or deletion) from a common ancestor, making Alu alleles identical by decent. In population genetics a comparison of the resulting products from each population can then be done by comparing the lengths of the PCR products in a series of unrelated individuals and may also detect polymorphisms with regard to the presence or absence of the Alu repeats. As a direct result of their abundance and sequence identity, they promote genetic recombination events that are responsible for large-scale deletions, duplication and translocations. The deletions occur mostly in the A-T rich regions and have found to be unlikely to have been created independently of the insertions of the Alu elements (Callinan et al, 2005) The easy genotyping of the POALINs has proven to be very valuable as lineage markers for the study of human population genetics, pedigree and forensics as well as genomic diversity and evolution. POALINs have been used in a range of applications, primarily focusing on anthropological analysis of human populations. As a result of its ease of use and its utility as a marker in human evolutions studies, combining the POALINs along with other markers used in forensics could lead to improved identity testing in forensic science. More specifically, in combination with more traditional markers, race specific genotypes and haplotypes could be used for profiling crime scene samples. ... This is supported by previously reported molecular data using various types of genetic markers. In a study using six separate Alu genes, Antunez-de-Mayolo et al were able to generate a phylogenetic tree, in which the biogeographical groups followed a pattern. The biogeographical groups started with African populations that were found to relate closely to the hypothetical ancestral African population. The African populations were then followed in order by Southwest Asian populations, European populations which include Middle Eastern groups (Antunez-de-Mayolo et al, 2002). This study shows the similarities and differences between the frequencies of the Middle Eastern Bedouin and the rest of the compared populations. Though no clear results were determined, the information from the POALINs along with information provided from other genetic markers can lead to further research on the Bedouin population and the improvement of the forensic population database in order to accurately test individual ethnic background of samples to be analysed.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 90 pages. Includes vita. Includes bibliographical references.

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references (leaves 70-84). Also available online.