Les sites de fixation des facteurs de transcription ou éléments régulateurs sont impliqués dans la régulation de l'expression des gènes. Une meilleure connaissance de l'architecture des promoteurs est aujourd'hui accessible via l’annotation des génomes et les données transcriptomiques. Certains éléments régulateurs sont conservés à une position préférentielle dans les promoteurs. Chez A. thaliana, nous avons mis au point une approche pour caractériser de tels motifs. Ce travail a permis de proposer une cartographie des promoteurs en identifiant 5105 motifs caractérisés par une sur-représentation locale dans les promoteurs proximaux. L’étude du promoteur central où est observée la boîte TATA, élément régulateur conservé entre eucaryotes, a été approfondie. Une liste de 15 variants fonctionnels de la boîte TATA a été identifiée, ainsi qu’une nouvelle classe d’éléments régulateurs qui sont caractérisés par des mêmes contraintes topologiques que la boîte TATA : les motifs-TC. Ils sont conservés chez A. thaliana et O. sativa, mais absents chez les mammifères. Les 18% de gènes d’A. thaliana contenant un motif-TC ont tendance à être exprimés dans des conditions expérimentales spécifiques. Ces éléments pourraient participer à la régulation de l’expression des gènes. L’étude de l’élément initiateur YR chez A. thaliana a mis en évidence une extension de ces 4 dinucléotides dans l’UTR 5’. Des associations entre ces éléments régulateurs peuvent montrer une collaboration fonctionnelle. La recherche de caractéristiques fonctionnelles communes aux gènes possédant une même organisation d'éléments régulateurs pourra permettre de contribuer à l’annotation fonctionnelle de ces éléments. / Transcription factor binding sites are regulatory elements involved in gene expression regulation. The knowledge of promoter architecture is now possible due to genome annotation and transcriptomic data. Some regulatory elements are conserved at a precise location in promoters. We developed an approach to characterize such motifs in A. thaliana. This work led to the promoter cartography by the identification of 5105 over-represented motifs in proximal promoters. The TATA-box is a regulatory element conserved within eukaryotes. The core-promoter where this element is expected has been thoroughly analysed. We identified a list of 15 functional variants of the TATA-box and a new class of regulatory elements that shares the TATA-box topological constraints: the TC-motifs. They are conserved in both A. thaliana and O. sativa and have not been observed in mammalian genomes. The A. thaliana genes containing a TC-motif are 18%. They are mainly expressed in specific experimental conditions. The TC-motifs might be involved in gene expression regulation. We observed that the 4 dinucleotides of the initiator element YR in A. thaliana are extended in 5’ UTR. Associations between these regulatory elements may highlight a functional collaboration. The study of the functional characteristics of genes with a same regulatory elements organization might help in these elements functional annotation.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24524号 / 医博第4966号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 萩原 正敏, 教授 山崎 渉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

<p>Gene expression and regulation are major determinants of phenotypic traits displayed across species. Although the DNA sequence elements that control gene expression play a crucial role in determining species morphology, predicting cis-regulatory elements through sequence analysis alone remains a difficult task. A few regulatory elements, such as the TATA-box and Initiator sequence, have been known to exhibit overrepresentation at specific locations within the proximal promoter. However, the extent to which this occurs among cis-regulatory elements is not well understood. Here, we take a genome-wide approach towards detecting such functional sequence elements, using location-specific overrepresentation as a criterion for regulatory function. We provide evidence that a surprisingly large number of regulatory elements exhibit locational overrepresentation with respect to the transcription start site. We then utilize this characteristic to predict novel cis-regulatory elements overrepresented at particular locations within the proximal promoter.</p><p>Transcriptional regulation is most often controlled not by single protein factors acting in isolation, but instead multiple transcription factors acting together within multi-protein complexes. As protein-protein interactions are largely determined through protein structure, we would expect to see patterns of spatial preference between motif-pairs binding interacting factors. However, in the absence of methods to predict such spatial preferences between motifs, comprehensive assessments of such inter-relationships have not been previously conducted. As our model provides a general tool for detecting positional specificities of a motif relative to a given reference point, we expanded our model to measure distance preferences between pairs of motifs on a genome-wide scale. We show that there often exist patterns of spatial dependencies between pairs of sequence elements that bind interacting protein factors. We find that regulatory motifs binding interacting proteins often have multiple inter-motif distances at which they preferentially occur, and we show that the intervals between preferred distances are highly consistent across motif-pairs. This distance preference `phasing' was empirically found to occur at consistent intervals around ~8-10 bp, corresponding to approximately the number of nucleotides within a single turn of the DNA double-helix. This finding suggests a tendency for protein factor-pairs to interact in a specific orientation with respect to the turn of the DNA molecule, and offers a convenient method by which to determine motif-pairs binding interacting transcription factors de novo. </p><p>While little is known about the mechanisms by which individual cis-regulatory elements ultimately control gene expression, even less is known about how such elements evolve over time. A single transcription factor can potentially target hundreds of genes across the genome, and thus modifications in the binding affinities of such proteins must induce conversions at a multitude of functional sites in order to preserve the set of target genes that the trans-factor regulates. It is therefore commonly assumed that such changes occur rarely and at a slow rate over the course of evolution. Despite this widespread assumption, we find that a surprisingly large number of cis-regulatory elements have been subject to significant changes in consensus sequence in a lineage-specific manner. Here, we demonstrate that the genomic landscape is highly adaptable, rapidly adjusting to global changes in preferred regulatory consensus sequences. Focusing upon regulatory elements exhibiting location-specific overrepresentation, we find that a substantial fraction of regulatory elements have been subject to evolutionary modifications, even between closely related eutherians. These findings have broad implications regarding evolving phenotypes observed across species.</p> / Dissertation

<p> Cellular cholesterol homeostasis is a fundamental and highly regulated process. Transcription factors known as sterol regulatory element binding proteins (SREBP) are responsible for the expression of many genes involved in the uptake and biosynthesis of cholesterol. SREBP activation and lipid dysregulation has been associated with cellular endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR). Our lab has previously reported a relationship between ER stress and SREBP activation causing lipid dysregulation and hepatic steatosis. This project was designed to elucidate the mechanism of ER stress-induced SREBP activation and determine its relationship with cellular pathologies associated with ER stress and lipid accumulation. My research has examined the mechanism by which ER stress activates SREBP-2 in various cell lines, including epithelial and macrophage cells. This research revealed that (1) ER stress-induced SREBP-2 activation is not dependent on caspases and occurs through the conventional sterol-mediated proteolytic pathway; (2) the mechanism of ER stress-induced SREBP-2 activation is sensitive to changes in ER calcium; (3) ER stress is associated with SREBP-2 activation and lipid dysregulation in a model of renal injury; and ( 4) ER stress-induced SREBP activation in vitro is not associated with lipid accumulation in macrophage foam cells. </P> <p> This project has also offered me the opportunity to further enhance our understanding of the mechanism by which ER stress causes SREBP activation in a sterolindependent manner. </P> / Thesis / Doctor of Philosophy (PhD)

No description available.

No description available.

The goal of the Encyclopedia of DNA Elements (ENCODE) project has been to characterize all the functional elements of the human genome. These elements include expressed transcripts and genomic regions bound by transcription factors (TFs), occupied by nucleosomes, occupied by nucleosomes with modified histones, or hypersensitive to DNase I cleavage, etc. Chromatin Immunoprecipitation (ChIP-seq) is an experimental technique for detecting TF binding in living cells, and the genomic regions bound by TFs are called ChIP-seq peaks. ENCODE has performed and compiled results from tens of thousands of experiments, including ChIP-seq, DNase, RNA-seq and Hi-C. These efforts have culminated in two web-based resources from our lab—Factorbook and SCREEN—for the exploration of epigenomic data for both human and mouse. Factorbook is a peak-centric resource presenting data such as motif enrichment and histone modification profiles for transcription factor binding sites computed from ENCODE ChIP-seq data. SCREEN provides an encyclopedia of ~2 million regulatory elements, including promoters and enhancers, identified using ENCODE ChIP-seq and DNase data, with an extensive UI for searching and visualization. While we have successfully utilized the thousands of available ENCODE ChIP-seq experiments to build the Encyclopedia and visualizers, we have also struggled with the practical and theoretical inability to assay every possible experiment on every possible biosample under every conceivable biological scenario. We have used machine learning techniques to predict TF binding sites and enhancers location, and demonstrate machine learning is critical to help decipher functional regions of the genome.

Thesis (Ph.D. in Human Medical Genetics) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 117-126). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

The mechanisms causing luteolysis of the primate corpus luteum are unknown. There is an increase in expression of liver x receptor (LXR) target genes and reduced low density lipoprotein receptor (LDLR) during spontaneous luteolysis in primates. The LXRs belong to the nuclear receptor superfamily and increase cholesterol efflux by inducing transcription of their target genes. Uptake of cholesterol into primate luteal cells occurs primarily via LDL, and LDLR transcription is regulated by sterol regulatory element binding transcription factor 2 (SREBF2). Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) maintain luteal function by binding to the LH/CG receptor (LHCGR), which stimulates progesterone (P4) synthesis via protein kinase A (PKA). It has also been previously reported that there is an increase in 27-hydroxycholesterol (27OH) concentrations during spontaneous luteolysis in primates. Pregnenolone and P4 inhibit the enzyme activity of CYP27A1 (cytochrome p450, family 27, subfamily A, polypeptide 1), which converts cholesterol into 27OH, an oxysterol that is a natural LXR agonist and SREBF2 inhibitor. Therefore, the overall hypothesis is that LXR-induced cholesterol efflux and reduced LDL uptake via inhibition of SREBF2 activity mediate luteolysis of the human CL. The objective of study 1 is to determine the effects of LXR activation and SREBF2 inhibition on P4 production, cholesterol metabolism and gene expression; and how hCG signaling via PKA regulates these effects in human luteinized granulosa cells. Basal and hCG-stimulated P4 secretion were significantly decreased by the combined actions of the LXR agonist T0901317 (T09) and the SREBF2 inhibitor fatostatin, which was associated with alterations in cholesterol metabolism leading to reduced intracellular cholesterol storage. Expression of LXR target genes in the presence of T09 was significantly reduced by hCG, while hCG significantly increased LDLR expression. These effects of hCG were reversed by a specific PKA inhibitor. Chronic hCG exposure had similar effects on LXR target gene and LDLR expression without an exogenous LXR agonist. The objective of study 2 is to determine the effects of 27OH on P4 production and cholesterol metabolism; and to determine if inhibiting the conversion of cholesterol into pregnenolone increases LXR and decreases SREBF2 target gene expression via CYP27A1 in human luteinized granulosa cells. During luteolysis in primates and sheep, CYP27A1 expression significantly increased. 27OH significantly decreased hCG-stimulated P4 secretion and enhanced cholesterol efflux. Aminoglutethimide, which inhibits the conversion of cholesterol to pregnenolone, significantly increased ABCA1 and decreased LDLR. Knock-down of CYP27A1 resulted in a significant increase in P4 secretion, but did not prevent aminoglutethimide-induced effects on ABCA1 and LDLR. Knock-down of steroidogenic acute regulatory protein (STAR), which controls cholesterol transport into the mitochondria where CYP27A1 resides, significantly decreased LDLR transcription. Collectively, the data from study 1 support the hypothesis that LXR-induced cholesterol efflux and reduced LDL uptake via inhibition of SREBF2 activity mediates luteolysis in primates, which is reversed by hCG. Data from study 2 indicates that 27OH produced via CYP27A1 may contribute to reductions in P4 synthesis during luteolysis, partially by serving as a dual LXR agonist and SREBF2 inhibitor, although other oxysterols are also likely involved.