Cupriavidus metallidurans CH34 est une béta-protéobactérie, résistante aux métaux lourds, isolée des sédiments d'une usine de métallurgie non-ferreuse en Belgique. <p>Le génome de cette bactérie contient un chromosome (3.6 Mb), un mégaplasmide (2.6 Mb) et deux plasmides pMOL28 (171 kb) et pMOL30 (234 kb) déjà connus pour porter des gènes de résistance aux métaux lourds. <p>Nous avons d'abord fait le catalogue des gènes impliqués dans la résistance aux métaux lourds et, ensuite, cherché à mesurer leur expression par deux approches transcriptomiques :RT-PCR et puces à ADN.<p> L'analyse du génome montre au moins 170 gènes relatifs à la résistance aux ions métalliques localisés sur les 4 réplicons, principalement sur les deux plasmides. Ces gènes codent essentiellement pour des systèmes d'efflux tel que les HME-RND (transport chimioosmotique avec flux de protons à contresens), les ATPases de type P ou encore pour le système de résistance aux ions Cu(II). Dans le génome de C. metallidurans, nous avons identifié 13 opérons qui codent pour des systèmes HME-RND, seuls trois, localisés sur les plasmides, sont surexprimés en présence de métaux lourds. Huit gènes codent pour des ATPases de type P, dont deux appartiennent à une classe dont les substrats ne sont pas métalliques. Deux ATPases appartiennent à une famille spécialisée pour l'efflux du Cu(II) et les quatre autres à une autre grande famille impliquée dans l'efflux des ions Cd(II), Pb(II) et Zn(II). Les analyses transcriptomiques montrent la surexpression des deux premières classes d'ATPases P en présence des métaux lourds. La mutagenèse du gène zntA (mégaplasmide), codant pour l'une des ATPases, provoque une diminution de la viabilité en présence de Zn(II), Cd(II) et dans une moindre mesure de Pb(II), Tl(I) et Bi(III). <p>Sur pMOL30, la résistance au cuivre implique un groupe de 19 gènes cop codant pour la résistance au cuivre au niveau du périplasme et du cytoplasme, et vraisemblablement pour une forme de stockage du cuivre essentiel. Ces 19 gènes sont surexprimés en présence de cuivre, mais une quinzaine de gènes proches semblent aussi requis pour une expression optimale de la résistance au cuivre. <p>L'annotation des plasmides a mis en évidence la parenté du plasmide pMOL28 avec le plasmide pHG1 (hydrogénotrophie, fixation du CO2) de C. eutrophus H16 et le plasmide pSym (fixation de l'azote) de C. taiwanensis, et chez pMOL30, la présence de deux îlots génomiques concentrant la plupart des résistances aux métaux lourds. Les puces montrent la surexpression de 83 sur 164 gènes dans pMOL28, et de 143 sur 250 gènes dans pMOL30. Elles montrent aussi que les gènes présents sur les deux plasmides sont davantage surexprimés que ceux localisés sur les deux mégaréplicons. Parmi les gènes surexprimés les plus intéressants du plasmide pMOL30, il faut mentionner des transposases tronquées et des gènes impliqués dans la synthèse des membranes (glycosyltransférases). L'analyse de l'expression des gènes plasmidiens de résistance aux métaux lourds montre la surexpression en présence de plusieurs ions métalliques ajoutés indépendamment et pas seulement par les substrats métalliques de ces opérons, ce qui suggère l'intervention de deux types de régulation dont les gènes correspondants sont aussi localisés sur le chromosome et le mégaplasmide.<p>Ce travail met en évidence la spécialisation de la bactérie dans la réponse à un grand spectre de concentrations de métaux lourds, jusqu'à la limite majeure de la toxicité observée pour les bactéries mésophiles hétérotrophes. Cette spécialisation correspond bien aux biotopes industriels de divers continents dans lesquels on l'a trouvée. <p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

La grande famille des facteurs de transcription Ets est caractérisée par un domaine de liaison à l’ADN, le domaine ETS, qui présente une structure de type hélice-tour-hélice ailé et qui reconnaît la séquence nucléotidique GGAA/T. Ces facteurs sont des protéines modulaires, dont les domaines sont structurellement conservés, régulent la transcription de leurs gènes cibles. L’action régulatrice de ces facteurs de transcription, ainsi que leur spécificité, dépendent de leurs sites d’expression, du taux auquel ils sont exprimés ainsi que des modifications post-traductionnelles qui les touchent. Au sein de la famille Ets, les trois membres du groupe PEA3 - Erm, Pea3 et Er81 - sont impliqués dans divers processus tant physiologiques tels que le développement des neurones sensitifs et moteurs que pathologiques tels que la croissance et l’invasion tumorale ou l’apparition de métastases, au niveau mammaire notamment.<p><p>Notre travail a eu pour ambition de mieux comprendre les relations structure/fonction des membres du groupe PEA3, et plus particulièrement de Erm. <p><p>\ / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

Les protéines de la Famille Ets sont des facteurs de transcription qui reconnaissent par l'intermédiaire d'un domaine de liaison à l'ADN, appelé le domaine ETS, une séquence nucléotidique centrée sur un core consensus 5'-GGAA/T-3'. Le gène fev, un membre de cette famille, a été isolé chez l'humain après avoir été identifié dans une tumeur de Ewing chez l'enfant comme le résultat d'une translocation chromosomique. Une étude préliminaire de 1997 indiquait que chez l'Homme, Fev possède une expression tissulaire restreinte au petit intestin et à la prostate "adulte". Dans la première partie de ce travail, nous avons observé une surexpression de l'ARNm de ce facteur de transcription dans une large série d'adénocarcinomes prostatiques de différenciation variable en comparaison au profil d'expression observé dans un groupe témoin de prostates hyperplasiées. En recherchant des lignées cellulaires qui expriment ce facteur, nous avons mis en évidence la présence de ce messager dans la lignée humaine d'origine prostatique LNCaP. De même, les lignées humaines d'origine hématopoïétiques Dami/HEL92.1.7, K-562, KU-812 F et U-937 expriment ce facteur. Parallèlement, nous avons réalisé l'étude de l'expression de Fev dans le cerveau humain. En effet, chez le rat et chez la souris, l'homologue de Fev (mPet-1/Pet-1) est exprimé spécifiquement dans les neurones sérotoninergiques. Nous avons ainsi montré sur le cerveau humain que l'ARNm de Fev est exclusivement exprimé dans les noyaux du raphé qui contiennent les neurones sérotoninergiques. Il est co-exprimé avec deux marqueurs de ces neurones, la SERT/5-HTT et la TPH2.<p>Parallèlement, nos travaux de caractérisation fonctionnelle de la protéine Fev ont permis de définir ce facteur comme un répresseur transcriptionnel. En effet, ce facteur possède un domaine carboxy-terminal riche en résidus alanines qui lui confère, en partie, sa fonction de répresseur de la transcription (répression active); la délétion de cette région conduisant à une réduction drastique de l'effet répresseur. Parallèlement, nous avons montré que le domaine ETS de Fev est responsable d'une activité répressive passive par l'occupation des sites de liaison aux facteurs Ets. Néanmoins, nous ne connaissons pas, à l'heure actuelle, les gènes-cibles spécifiques qui sont directement réprimés par Fev dans les cellules dans lesquelles le gène est normalement exprimé. Nous avons tenté de développer, par établissement de clones stables, des modèles cellulaires où le gène d'intérêt est surexprimé, mais ces tentatives furent infructueuses. Cet effet drastique de Fev est conféré par la partie carboxy-terminale. Il est ainsi probable que la surexpression de ce répresseur transcriptionnel spécifique de certains gènes cibles de la famille Ets provoque des effets sur la survie des cellules en régulant des gènes indispensables à la croissance cellulaire, et ainsi empêchant la prolifération de clones cellulaires.<p> / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

Indiana University-Purdue University Indianapolis (IUPUI) / My thesis comprises of two individual projects: 1) we have developed a database for operon prediction using high-throughput sequencing datasets for bacterial genomes. 2) Genomics and mechanistic insights of convergent transcription in bacterial genomes. In the first project we developed a database for the prediction of operons for bacterial genomes using RNA-seq datasets, we predicted operons for bacterial genomes. RNA-seq datasets with different condition for each bacterial genome were taken into account and predicted operons using Rockhopper. We took RNA-seq datasets from NCBI with distinct experimental conditions for each bacterial genome into account and analyzed using tool for operon prediction. Currently our database contains 9 bacterial organisms for which we predicted operons. User interface is simple and easy to use, in terms of visualization, downloading and querying of data. In our database user can browse through reference genome, genes present in that genome and operons predicted from different RNA-seq datasets. Further in the second project, we studied the genomic and mechanistic insights of convergent transcription in bacterial genomes. We know that convergent gene pairs with overlapping head-to-head configuration are widely spread across both eukaryotic and prokaryotic genomes. They are believed to contribute to the regulation of genes at both transcriptional and post-transcriptional levels, although factors contributing to their abundance across genomes and mechanistic basis for their prevalence are poorly understood. In this study, we explore the role of various factors contributing to convergent overlapping transcription in bacterial genomes. Our analysis shows that the proportion of convergent overlapping gene pairs (COGPs) in a genome is affected due to endospore formation, bacterial habitat, oxygen requirement, GC content and the temperature range. In particular, we show that bacterial genomes thriving in specialized habitats, such as thermophiles, exhibit a high proportion of COGPs. Our results also conclude that the density distribution of COGPs across the genomes is high for shorter overlaps with increased conservation of distances for decreasing overlaps. Our study further reveals that COGPs frequently contain stop codon overlaps with the middle base position exhibiting mismatches between complementary strands. Further, for the functional analysis using cluster of orthologous groups (COGs) annotations suggested that cell motility, cell metabolism, storage and cell signaling are enriched among COGPs, suggesting their role in processes beyond regulation. Our analysis provides genomic insights into this unappreciated regulatory phenomenon, allowing a refined understanding of their contribution to bacterial phenotypes.

Indiana University-Purdue University Indianapolis (IUPUI) / Disruptions of the endoplasmic reticulum (ER) cause perturbations in protein folding and result in a cellular condition known as ER stress. ER stress and the accumulation of unfolded protein activate the unfolded protein response (UPR) which is a cellular attempt to remedy the toxic accumulation of unfolded proteins. The UPR is implemented through three ER stress sensors PERK, ATF6, and IRE1. Phosphorylation of the α-subunit of eIF2 by PERK during ER stress represses protein synthesis and also induces preferential translation of ATF4, a transcriptional activator of stress response genes. Early UPR signaling involves translational and transcriptional changes in gene expression that is geared toward stress remedy. However, prolonged ER stress that is not alleviated can trigger apoptosis. This dual signaling nature of the UPR is proposed to mimic a 'binary switch' and the regulation of this switch is a key topic of this thesis. Adaptive gene expression aimed at balancing protein homeostasis encompasses the first phase of the UPR. In this study we show that the PERK/eIF2~P/ATF4 pathway facilitates both the synthesis of ATF6 and trafficking of ATF6 from the ER to the Golgi where ATF6 is activated. Liver-specific depletion of PERK significantly lowers expression of survival genes, leading to reduced expression of protein chaperones. As a consequence, loss of PERK in the liver sensitizes cells to stress which ultimately leads to apoptosis. Despite important roles in survival, PERK signaling is often extended to the vii activation of other downstream transcription factors such as CHOP, a direct target of ATF4-mediated transcription. Accumulation of CHOP is a hallmark of the second phase in the binary switch model where CHOP is shown to be required for full activation of apoptosis. Here the transcription factor ATF5 is found to be induced by CHOP and that loss of ATF5 improves the survival of cells following changes in protein homeostasis. Taken together this study highlights the importance of UPR signaling in determining the balance between cell survival and cell death. A topic that is important for understanding the more complex pathological conditions of diseases such as diabetes, cancer, and neurodegeneration.

Indiana University-Purdue University Indianapolis (IUPUI) / The class IV alcohol dehydrogenase (ADH7, µ-ADH, σ-ADH) efficiently metabolizes ethanol and retinol. ADH7 is expressed mainly in the upper gastrointestinal tract with no expression in the liver unlike the other ADHs, and is implicated in various diseases including alcoholism, cancer and fetal alcohol syndrome. Genome wide studies have identified significant associations between ADH7 variants and alcoholism and cancer, but the causative variants have not been identified. Due to its association with two important metabolic pathways and various diseases, this dissertation is focused on studying ADH7 regulation and the effects of variants on this regulation using cell systems that replicate endogenous ADH7 expression. We identified elements regulating ADH7 transcription and observed differences in the effects of variants on gene expression. A7P-G and A7P-A, two promoter haplotypes differing in a single nucleotide at rs2851028, had different transcriptional activities and interacted with variants further upstream. A sequence located 12.5 kb upstream (7P10) can function as an enhancer. These complex interactions indicate that the effects of variants in the ADH7 regulatory elements depend on both sequence and cellular context, and should be considered in interpretation of the association of variants with alcoholism and cancer. The mechanisms governing the tissue-specific expression of ADH7 remain unexplained however. We identified an intergenic region (iA1C), located between ADH7 and ADH1C, having enhancer blocking activity in liver-derived HepG2 cells. This enhancer blocking function was cell- and position- dependent with no activity seen in CP-A esophageal cells. iA1C had a similar effect on the ectopic SV40 enhancer. The CCCTC-binding factor (CTCF) bound iA1C in HepG2 cells but not in CP-A cells. Our results suggest that in liver-derived cells, iA1C blocks the effects of downstream ADH enhancers and thereby contributes to the cell specificity of ADH7 expression. Thus, while genetic factors determine level of ADH7 transcriptional activity, iA1C helps determine the cell specificity of transcription.

Colorectal cancer (CRC) is the third leading cause of cancer related deaths in the United States. The nuclear factor κB (NF-κB) is an important family of transcription factors whose aberrant activation has been found in many types of cancer, including CRC. Therefore, understanding the regulation of NF-κB is of ultimate importance for cancer therapy. Using a novel validation-based insertional mutagenesis (VBIM) strategy, our lab has identified the novel adenomatous polyposis coli (APC) like protein (ALP) gene as a negative regulator of NF-κB. Preliminary studies from our lab demonstrated that overexpression of ALP led to decreased NF-κB activity by κB reporter assay and electrophoresis mobility gel shift assay (EMSA). The current project aims to further evaluate the role of ALP in the regulation of NF-κB signaling in CRC cells. We found that overexpression of ALP in human CRC HT29 cells greatly reduced both the number and the size of colonies that were formed in a soft agar assay. ALP overexpression also decreased the cell growth rate and cell migration ability, while shRNA mediated knockdown of ALP showed opposite effects, confirming that ALP is a tumor suppressor in CRC HT29 cells. Overexpression of ALP led to decreased NF-κB activity by κB reporter assay and condition media assay in CRC HT29 cells. Furthermore, immunohistochemical analysis with human colon vii tissues revealed that there is a gradual loss of ALP protein with tumor progression. We also found that ALP predominantly localizes in the cytoplasm, and binds to the p65 subunit of NF-κB, and might be functioning downstream of IκB kinase (IKK). In summary, in this study, we provide evidence regarding the tumor suppressor role of ALP in CRC by functioning as novel negative regulator of NF-κB. This discovery could lead to the establishment of ALP as a potential biomarker and therapeutic target in CRC.

Indiana University-Purdue University Indianapolis (IUPUI) / RNA-binding proteins (RBPs) are vital post-transcriptional regulatory molecules in transcriptome of mammalian species. It necessitates studying their expression dynamics to extract how post-transcriptional networks work in various mammalian tissues. RNA binding proteins (RBPs) play important roles in controlling the post-transcriptional fate of RNA molecules, yet their evolutionary dynamics remains largely unknown. As expression profiles of genes encoding for RBPs can yield insights about their evolutionary trajectories on the post-transcriptional regulatory networks across species, we performed a comparative analyses of RBP expression profiles across 8 tissues (brain, cerebellum, heart, lung, liver, lung, skeletal muscle, testis) in 11 mammals (human, chimpanzee, gorilla, orangutan, macaque, rat, mouse, platypus, opossum, cow) and chicken & frog (evolutionary outgroups). Noticeably, orthologous gene expression profiles suggest a significantly higher expression level for RBPs than their non-RBP gene counterparts, which include other protein-coding and non-coding genes, across all the mammalian tissues studied here. This trend is significant irrespective of the tissue and species being compared, though RBP gene expression distribution patterns were found to be generally diverse in nature. Our analysis also shows that RBPs are expressed at a significantly lower level in human and mouse tissues compared to their expression levels in equivalent tissues in other mammals: chimpanzee, orangutan, rat, etc., which are all likely exposed to diverse natural habitats and ecological settings compared to more stable ecological environment humans and mice might have been exposed, thus reducing the need for complex and extensive post-transcriptional control. Further analysis of the similarity of orthologous RBP expression profiles between all pairs of tissue-mammal combinations clearly showed the grouping of RBP expression profiles across tissues in a given mammal, in contrast to the clustering of expression profiles for non-RBPs, which frequently grouped equivalent tissues across diverse mammalian species together, suggesting a significant evolution of RBPs expression after speciation events. Calculation of species specificity indices (SSIs) for RBPs across various tissues, to identify those that exhibited restricted expression to few mammals, revealed that about 30% of the RBPs are species-specific in at least one tissue studied here, with lung, liver, kidney & testis exhibiting a significantly higher proportion of species specifically expressed RBPs. We conducted a differential expression analysis of RBPs in human, mouse and chicken tissues to study the evolution of expression levels in recently evolved species (i.e., humans and mice) than evolutionarily-distant species (i.e., chickens). We identified more than 50% of the orthologous RBPs to be differentially expressed in at least one tissue, compared between human and mouse, but not so between human and an outgroup chicken, in which RBP expression levels are relatively conserved. Among the studied tissues (brain, liver and kidney) showed a higher fraction of differentially expressed RBPs, which may suggest hyper- regulatory activities by RBPs in these tissues with species evolution. Overall, this study forms a foundation for understanding the evolution of expression levels of RBPs in mammals, facilitating a snapshot of the wiring patterns of post-transcriptional regulatory networks in mammalian genomes. In our second study, we focused on elucidating novel features of post-transcriptional regulatory molecules called as circRNA from LongPolyA RNA-sequence data. The debate over presence of nonlinear exon splicing such as exon-shuffling or formation of circularized forms has finally come to an end as numerous repertoires have shown of their occurrence and presence through transcriptomic analyses. It is evident from previous studies that along with consensus-site splicing non-consensus site splicing is robustly occurring in the cell. Also, in spite of applying different high-throughput approaches (both computational and experimental) to determine their abundance, the signal is consistent and strongly conforming the plausible circularization mechanisms. Earlier studies hypothesized and hence focused on the ribo-minus non-polyA RNA-sequence data to identify circular RNA structures in cell and compared their abundance levels with their linear counterparts. Thus far, the studies show their conserved nature across tissues and species also that they are not translated and preferentially are without poly (A) tail, with one to five exons long. Much of this initial work has been performed using non-polyA sequencing thus probably underestimates the abundance of circular RNAs originating from long poly (A) RNA isoforms. Our hypothesis is if the circular RNA events are not the artifact of random events, but has a structured and defined mechanism for their formation, then there would not be biases on preferential selection / leaving of polyA tails, while forming the circularized isoforms. We have applied an existing computational pipeline from earlier studies by Memczack et. al., on ENCODE cell-lines long poly (A) RNA-sequence data. With the same pipeline, we achieve a significant number of circular RNA isoforms in the data, some of which are overlapping with known circular RNA isoforms from the literature. We identified an approach and worked upon to identify the precise structure of circular RNA, which is not plausible from the existing computational approaches. We aim to study their expression profiles in normal and cancer cell-lines, and see if there exists any pattern and functional significance based on their abundance levels in the cell.

Indiana University-Purdue University Indianapolis (IUPUI) / Although rare, classification of over 200 hereditary cancer susceptibility syndromes accounting for ~5-10% of cancer incidence has enabled the discovery and understanding of cancer predisposition genes that are also frequently mutated in sporadic cancers. The need to prevent or delay invasive cancer can partly be addressed by characterization of cells derived from healthy individuals predisposed to cancer due to inherited "single-hits" in genes in order to develop patient-derived samples as preclinical models for mechanistic in vitro studies. Here, we present microarray-based transcriptome profiling of Li-Fraumeni syndrome (LFS) patient-derived unaffected breast epithelial cells and their phenotypic characterization as in vitro three-dimensional (3D) models to test pharmacological agents. In this study, the epithelial cells derived from the unaffected breast tissue of a LFS patient were cultured and progressed from non-neoplastic to a malignant stage by successive immortalization and transformation steps followed by growth in athymic mice. These cell lines exhibited distinct transcriptomic profiles and were readily distinguishable based upon their gene expression patterns, growth characteristics in monolayer and in vitro 3D cultures. Transcriptional changes in the epithelial-to-mesenchymal transition gene signature contributed to the unique phenotypes observed in 3D culture for each cell line of the progression series; the fully transformed LFS cells exhibited invasive processes in 3D culture with disorganized morphologies due to cell-cell miscommunication, as seen in breast cancer. Bioinformatics analysis of the deregulated genes and pathways showed inherent differences between these cell lines and targets for pharmacological agents. After treatment with small molecule APR-246 that restores normal function to mutant p53, we observed that the neoplastic LFS cells had reduced malignant invasive structure formation from 73% to 9%, as well as an observance of an increase in formation of well-organized structures in 3D culture (from 27% to 91%) by stereomicroscopy and confocal microscopy. Therefore, the use of well-characterized and physiologically relevant preclinical models in conjunction with transcriptomic profiling of high-risk patient derived samples as a renewable laboratory resource can potentially guide the development of safer and more effective chemopreventive approaches.

Indiana University-Purdue University Indianapolis (IUPUI) / Intestinal smooth muscle development involves complex transcriptional regulation leading to cell differentiation of the circular, longitudinal and muscularis mucosae layers. Differentiated intestinal smooth muscle cells express high levels of smooth muscle-specific contractile and regulatory proteins, including telokin. Telokin is regulatory protein that is highly expressed in visceral smooth muscle. Analysis of cis-elements required for transcriptional regulation of the telokin promoter by using hypoxanthine-guanine phosphoribosyltransferase (Hprt)-targeted reporter transgenes revealed that a 10 base pair large CC(AT)₆GG ciselement, called CArG box is required for promoter activity in all tissues. We also determined that an additional 100 base pair region is necessary for transgene activity in intestinal smooth muscle cells. To examine how transcriptional regulation of intestinal smooth muscle may be altered under pathological conditions we examined the effects of diabetes on colonic smooth muscle. Approximately 76% of diabetic patients develop gastrointestinal (GI) symptoms such as constipation due to intestinal dysmotility. Mice were treated with low-dose streptozotocin to induce a type 1 diabetes-like hyperglycemia. CT scans revealed decreased overall GI tract motility after 7 weeks of hyperglycemia. Acute (1 week) and chronic (7 weeks) diabetic mice also had decreased potassium chloride (KCl)-induced colon smooth muscle contractility. We hypothesized that decreased smooth muscle contractility at least in part, was due to alteration of contractile protein gene expression. However, diabetic mice showed no changes in mRNA or protein levels of smooth muscle contractile proteins. We determined that the decreased colonic contractility was associated with an attenuated intracellular calcium increase, as measured by ratio-metric imaging of Fura-2 fluorescence in isolated colonic smooth muscle strips. This attenuated calcium increase resulted in decreased myosin light chain phosphorylation, thus explaining the decreased contractility of the colon. Chronic diabetes was also associated with increased basal calcium levels. Western blotting and quantitative real time polymerase chain reaction (qRT-PCR) analysis revealed significant changes in calcium handling proteins in chronic diabetes that were not seen in the acute state.These changes most likely reflect compensatory mechanisms activated by the initial impaired calcium response. Overall my results suggest that type 1 diabetes in mice leads to decreased colon motility in part due to altered calcium handling without altering contractile protein expression.