I initially set out to identify the cap structure on the spliced leader RNA of Ciona intestinalis. During this investigation, I discovered a previously unobserved 53 nt transcript containing the spliced leader sequence. This transcript contained the usual metazoan spliced leader RNA cap, trimethylguanosine, while the canonical spliced leader RNA lacked this moiety, but likely contained a mP7PG cap. The cap structure of the trans-spliced troponin I mRNA matched that of the canonical spliced leader RNA, indicating that the canonical spliced leader RNA, not the 53 nt transcript, is the donor for troponin I. Further immunoprecipitation studies showed that both the canonical spliced leader RNA and the novel 53 nt transcript exist in association with Sm proteins. I cloned a genomic DNA segment containing four tandem repeats of the spliced leader RNA gene, and this was used as a probe in an in situ hybridization study that showed the vast majority of the spliced leader RNA genes reside on chromosome 8. Finally, I performed some preliminary work showing that outrons, the 5'-segments of pre-mRNAs removed by trans-splicing, may exist in sufficient quantities as to be detected by PCR amplification. / Au d'épart, j'ai entrepris d'identifier la structure coiffe de l'ARN spliced leader de Ciona intestinalis. Durant cette recherche, j'ai découvert un nouveau transcript d'RN de 53 nt qui contient la séquence spliced leader. Ce transcrit contient la coiffe usuele des ARNs spliced leader des métazoaires, le trimethylguanosine, tandis que l'ARN spliced leader canonique ne possède pas cette modification, mais contient, fort probablement, une coiffe mP7PG. La structure de la coiffe de l'ARNm de la troponine I trans-épissé correspond à celle de l'ARN spliced leader canonique, indiquant que l'ARN spliced leader est le donneur pour la troponine I, et non l'ARN de 53 nt. Des études d'immunoprécipitation supplémentaires ont montré que l'ARN spliced leader canonique et le nouvel ARN de 53 nt existent en association avec des protéines Sm.J'ai cloné une sequence d'ADN génomique contenant quatre repetitions en tandem du gène l'ARN spliced leader. Ce clone a été utilisé comme sonde lors d'une experience d'hybridation in situ qui a montré que la grande majorité des gènes l'ARN spliced leader réside sur le chromosome 8.Finalement, j'ai effectué une étude préliminaire montrant que les outrons, les segments 5' des ARNs pré-messagers enlevés par le trans-épissage, existent en quantité suffisante et peuvent être détecté par l'amplification PCR.

The objective of this thesis is to research at the molecular level the mechanisms involved in the initiation of mammalian DNA replication. Human WI-38 cells were synchronized to the G1/S border by serum starvation and aphidicolin block. Cells were relased from arrest and followed as they progressed into S phase by microfluorometric analysis. Early replicating DNA was extruded from replication bubbles, purified from the high molecular weight parental DNA and cloned into the NruI site of pBR322. The recombinant plasmids were surveyed for properties previously associated with origins of replication. In a random sample of 10 human origin enriched sequences (hors) that were analyzed, 5 were capable of autonomous replication in a transient BrdU substitution assay. Two clones contained DNA fragments that migrate anomalously on acrylamide gels suggestive of bent DNA. One clone contained a weak DNA unwinding element as judged by sensitivity to the single strand specific enzyme mung bean nuclease. Primary sequence analyses of five of the hors clones (hors 98, 106, 112, 129 and 133) revealed that they were enriched for the AP2-A, NF-1 related and iron response consensus sequences. The replicating clones also contained potential cruciform structures within 50 bp of an A/T rich region. A DNA binding activity was identified in HeLa nuclear extracts that binds to a sub-fragment of one of the replicating clones (hors106) by bandshift assays and it was partially purified by DEAE and PC-11 column chromatography.

The fragment 2 domain (F2) of human prothrombin is known to interact with both thrombin and factor Va contributing to enhanced prothrombinase activity in the absence of phospholipid membranes. Using P.Pastoris, we have over-expressed recombinant F2 and achieved uniform labeling of F2 with both 15N and 15N/13C isotopes. The 1H-15N HSQC NMR spectra of F2 and other backbone (13Calpha and some 13C beta) resonances have been assigned by a combination of proton homonuclear TOCSY/NOESY and triple-resonance NMR experiments. The 13C alpha conformational shifts indicate that human F2 assumes secondary structure elements similar to those found in the crystal structure of human F2 in complex with thrombin. Less than stoichiometric amounts of thrombin and factor Va were found to perturb subsets of the HSQC peaks of F2, indicating specific and reversible binding of human F2 with both thrombin and factor Va. The binding sites on F2 for thrombin and factor Va are located at distinct surface areas, as shown through an analysis of residue-specific shifts in the assigned HSQC spectra of F2. Most of the binding residues on F2 identified by NMR correlate well with those identified through functional studies and/or in the crystal structure of the human F2-thrombin complex. More interestingly, our NMR studies demonstrate for the first time direct involvement of the C-terminal region of F2, or the junction residues between kringle 2 and thrombin in prothrombin, for thrombin binding. Therefore, using recombinant F2 we have been able to characterize the thrombin- and Va-binding domains on F2 and establish that F2 binds to these proteins via different binding sites.

Hereditary nonpolyposis colorectal carcinoma (HNPCC) is linked to inherited defects in human genes (hMLH1, hMSH2, hMSH6, hPMS1, and hPMS2) that are involved in the repair of mismatched bases that may occur during DNA replication. Germline missense mutations in human MLH1 (hMLH1) are frequently detected and their functional characterization is critical to the development of genetic testing for HNPCC. We used several functional assays to characterize two hMLH1 mutations: T117M and R182G. Saccharomyces cerevisiae were transformed with hMLH1 cDNA expression vectors containing either mutation. The presence of functional hMLH1 produces an accumulation of mutations in mismatch repair (MSM)-proficient yeast due to MLH1 protein homology and interference with normal MMR. The transformants were tested for increased mutation rates using three assays: mutation of the gene for canavanine resistance (CAN1 ), reversion of the hom 3--10 allele, and insertion-deletions at a dinucleotide repeat regulating expression of beta-galactosidase. Based on the results of these assays T117M is likely a functional mutation while R182G may be a polymorphism. We conclude that this assay may be applicable in genetic testing for HNPCC.

Trans-activation response (TAR) RNA-binding protein (TRBP), is a cellular protein that binds to the human immunodeficiency virus type 1 (HIV-1) TAR RNA and activates viral expression. TRBP also binds the interferon induced, double-stranded RNA-activated protein kinase, PKR, that exerts antiviral activities. Activated PKR inhibits translational initiation by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIf-2alpha). TRBP blocks the inhibitory effects of PKR, whereas PKR activating protein (PACT), another cellular protein, heterodimerizes with and activates PKR in vitro and in vivo in the absence of viral infection. TRBP and PACT are highly homologous but have opposite effects on PKR. / We studied the interplay between TRBP and PACT on PKR activation and on the regulation of HIV expression. By the yeast two-hybrid method, we showed an interaction between TRBP and PACT and we characterized domains implicated in this interaction. We confirmed this interaction in the human cellular context by coimmunoprecipitation. Immunofluorescence studies showed that TRBP and PACT colocalize in the cytoplasm. In the context of HIV-1 expression, PKR mediates an inhibition of LTR (long terminal repeat) expression, but no further decrease was obtained with PACT. Interestingly, in coimmunoprecipitation studies, we found that the concentration of PACT protein is decreased when PACT and TRBP were cotransfected, whereas PACT mRNA level remains unchanged. This result suggests that TRBP either blocks PACT translation or induces its degradation, which open new ways of PKR and viral regulation.

Autosomal dominant polycystic kidney disease (PKD) is caused by mutations of the PKD1 or PKD2 genes. Cyst cells exhibit sustained expression of fetal genes such as PAX2. Normally, PAX2 is involved in kidney development and is rapidly downregulated after birth. Overactivity of the canonical beta-catenin signaling pathway has also been linked to the formation of renal cysts. To determine whether beta-catenin activity is linked to the level of PAX2 expression in vivo, we created a transgenic mouse overexpressing PAX2 in mature proximal tubules of the kidney. Here we report that the canonical beta-catenin signaling activity is increased in mice bearing the targeted PAX2 transgene. / There is also evidence that non-canonical Writ signaling may be involved in the development of renal cysts but this pathway is uncharacterized. We therefore studied the ontogeny of a downstream marker of this pathway (NFAT) and its localization in the developing kidney. Here we report that NFAT activity is high in early stages of kidney development and is rapidly downregulated at birth. The NFAT signal is diffuse and is expressed in both mesenchymal and epithelial cells of the developing kidney.

HOX proteins are evolutionarily conserved homeodomain (HD)-containing transcription factors that mediate environmental signal cues and specify regional identities. PBC (including EXD in Drosophila and PBX in vertebrates) and MEIS/PREP proteins are two families of transcription factors within the broader three-amino-acid-loop-extension (TALE) class of homeoproteins. They form cooperative DNA-binding complexes with HOX proteins and have been shown to assist HOX functional specificity and participate in HOX-mediated transcriptional regulation. / The function of HOX cofactors is regulated at multiple levels. The availability of PBC proteins is controlled via differential subcellular localization which is partially directed by MEIS. However, cytoplasmic anchoring factors could also contribute to PBX subcellular localization. Part of this thesis project is devoted to identifying such factors. A cytoplasmic retention factor, non-muscle myosin II heavy chain B (NMHCB) has been uncovered as a PBX-interacting protein via a yeast two-hybrid screen. The NMHCB fragment induces cytoplasmic retention of PBX and EXD in both mammalian cells and Drosophila S2 cells. In addition, the subcellular distribution of EXD is deregulated in Drosophila bearing a mutated zipper gene, a homolog of mammalian NMHCB. These results reveal a conserved mechanism that promotes the cytoplasmic localization of PBX and EXD. / Nuclear PBX and MEIS form cooperative complexes with subsets of HOX proteins. In this project, we further study the transcriptional activity of MEIS-PBX-HOX heterotrimers and try to dissect the role of the third partner in the complex. We show that MEIS1 is recruited to the Hoxb1 auto-regulatory element (ARE), a known PBX-HOX target, in conjugation with RA-induced Hoxb1 transcriptional activation in P19 cells. We map TSA- and protein kinase A (PKA)-responsive transactivation domains to the C-termini of MEIS1 proteins. We propose that cell signaling modulates the transcriptional activity of MEIS and MEIS-PBX-HOX complexes through this region. In addition, the DNA binding of MEIS and protein-protein interaction between MEIS and PBX both contribute to the activity. Moreover, the C-terminal regions appear to confer functional differences among MEIS/PREP family proteins.

Ligand-dependent corepressor LCoR was discovered almost ten years ago in the laboratory of Dr. John White through a yeast two-hybrid screen in which the ligand binding domain (LBD) of the estrogen receptor alpha (ERalpha) was used as bait. LCoR represses gene transcription by recruitment of the corepressor C-terminal binding proteins (CtBPs) and histone deacetylases (HDACs) through distinct domains. The action of HDACs strengthens the interactions of histones with negatively charged DNA resulting in stable nucleosomal and chromatin structures and repression of transcription. CtBPs interact with many transcriptional repressors through a common motif P/VLDLS/TXK/R, of which there are two in LCoR. We hypothesize that LCoR is a regulator of endogenous nuclear receptor target gene expression. Initial data characterized the function of LCoR as a nuclear receptor (NR) corepressor by using a series of in vitro and over expression assays. LCoR was also shown to have many protein-interacting domains that might contribute in its corepressor function. The goal of this thesis was to determine the role of LCoR in the control of endogenous estrogen and progesterone target gene expression. Through reporter gene assays with overexpression of LCoR and truncated forms of LCoR lacking the interacting domains, we have shown that both HDAC6 and CtBP1 contribute to transcriptional corepression observed with LCoR. To find out whether these cofactors are recruited to regulatory regions of endogenous ERalpha and PR target genes, ChIP and reChIP assays techniques were applied. These have shown a ligand-depenedent and orderly corecruitment of LCoR, HDAC6, CtBP1 and corresponding NR on estrogen and progesterone target genes. SiRNA-mediated gene silencing of LCoR, HDAC6 and CtBP1 in combination with reporter gene assay showed a transcriptional derepression in ERalpha- and PR-mediated gene transcription, confirming the transcriptional corepressor function of these coregulators. However, the in / Il y a dix ans, le laboratoire de Dr. John White a découvert un corépresseur nucléaire surnommer LCoR. La fonction du corépresseur est dépendante de la présence d'agoniste. LCoR a été découvert grâce au système du double hybride de levure pour lequel le domaine de liaison du ligand (LBD) du récepteur estrogen alpha (ERalpha) a été utilisé comme appât. LCoR inhibe la transcription d'un gene en s'associant avec le corépresseur C-terminal binding proteins (CtBPs) et les histones déacétylases (HDACs). L'association de LCoR avec ces deux familles protéiques ce fait à travers deux domaines distincts. L'action enzymatique des HDACs stabilisent les structures du nucléosome et des histones en renforçant les liens entres ces derniers et l'ADN. Cette stabilité structurale a pour but d'inhiber la transcription génique. Les CtBPs intéragissent avec un grand nombre de répresseurs transcriptionnel en s'associant au motif d'intéraction P/VLDLS/TXK/R, dont LCoR en possède deux. Nous émettons l'hypothèse que LCoR est un régulateur transcriptionnel des gènes cibles de récepteurs nucléaires in vivo. Les données initiales (basées sur des techniques in vitro) ont caractérisé LCoR en tant que répresseur de transcription médiée par les récepteurs nucléaires. Ces données ont également démontrée la contribution des cofacteurs CtBP1 and HDAC6 à l'activité trancriptionnelle répressive de LCoR. Le but de cette thèse est de déterminer le rôle de LCoR dans le contrôle transcriptionnel des gènes cibles des récepteurs nucléaires ERalpha et progestérone (PR) in vivo. Les immunoprécipitations chromatiques des cofacteurs LCoR, CtBP1, HDAC6, ERalpha et PR ont démontrée une association de ceux-ci aux éléments de réponses hormonales de manière ordonnées et séquentielles. L'ablation d'expression de LCoR, HDAC6 and CtBP1 par rapproche RNA inhibiteur a démontrée une perte de répression transcriptionnelle médiée par les expressions

Now that the genome has successfully been sequenced, the next aim will be to characterize and annotate features. With the development of high resolution tools, such as Chromosome Conformation Capture (3C) technology, the 3-dimensional architecture of large linear spans of DNA can be determined. We have established that the mouse HoxA gene cluster is structured in such a way as to contain four distinct looping regions with a central interacting rosette core in its resting pluripotent state. This central core feature might directly affect the spatiotemporal regulation of the Hox genes during normal development as well as differentiation via retinoic acid administration in the P19 embryonal carcinoma cell line. The DNA looping might be mediated by cis-regulatory elements, which could function in a cooperative manner to regulate HoxA gene expression. Regulatory elements might include the previously identified retinoic acid response elements (RAREs) known to mediate specific developmental cues. Interestingly, Hox genes expressed earlier and more anteriorly are within loops containing higher amounts of identified RAREs as well as other conserved non-coding sequences that also appear to play a potential role in orchestrating the collinear mechanism of action. Loops containing the more 5' located genes have fewer regulatory elements and therefore may respond later to developmental cues. / Maintenant que le génome été séquencé avec succès, le but suivant sera de caractériser et annoter les caractéristiques regulataire. Avec le développement d'instruments de haute résolution, comme la technologie du Capture de Conformation Chromatin (3C), l'architecture 3- dimensionnelle de grandes durées linéaires d'ADN peut être déterminée. Nous avons établi que le groupe de gène de HoxA dans la souris est structurée d'une telle façon pour contenir quatre régions boucles distinctes avec un centre de rosette réagissant réciproquement central dans son état de reposant pluripotent. On croit que cette caractéristique centrale de base directement affecte le règlement spatiotemporel des gènes Hox pendant le développement normal aussi bien que la différentiation via l'administration acide rétinoïque dans le P19 ligne de cellule de carcinome. Les éléments cis-de-contrôle responsables de définir ces boucles ont l'air de fonctionner dans une manière coopérative dans laquelle le pluripotent HoxA l'état est plein d'assurance de réagir à l'administration acide rétinoïque dans une manière colinéaire contrôlée d'expression de gène. Auparavant identifié les éléments de réponse acide rétinoïque (ERARs) sont organisés pour tenir compte de la réorganisation dynamique du groupe sur la réception de signaux spécifiques du développement. Les gènes de Hox ont exprimé tôt et sont plus antérieurement dans les boucles qui contiennent de plus hautes quantités de ERARs identifié aussi bien que d'autres ordres de non-codification conservés qui ont aussi l'air de jouer un rôle potentiel dans le fait d'orchestrer le mécanisme colinéaire d'action.

Protein methylation is a post-translational modification which can take place on both lysine and arginine residues. Classically, the characterization of these modifications on histones has shaped the field of epigenetics. These modifications are regulated by enzymes which specifically catalyze the transfer of a methyl group from a methyl donor to the amino groups of lysine or arginine residues. Methylation of non-histone proteins has also been proven to be crucial in many cellular processes. Understanding the role that protein methylation plays in DNA damage has elucidated the function of many proteins involved in the complex signalling cascade occurring at double-stranded DNA breaks (DSBs). Examining protein methylation has allowed us to identify new proteins implicated in DNA damage and to better clarify the function of existing proteins involved in the DNA damage response. The work presented in this thesis aims to develop new ways to identify methylated proteins involved in DNA damage as well as to verify any functional relevance of newly discovered proteins. Tudor domains traditionally have been shown to bind methylated residues. P53-binding protein 1 (53BP1) is a prominent mediator of DNA damage response and is recruited to DSBs via its tandem Tudor domain by binding methylated lysine on H4 histone protein. A proteomic screen of 53BP1's Tudor domain showed that it could bind brahma-related gene 1 (BRG1), a critical part of the human chromatin remodelling complex. One of the goals of the work presented in this thesis was to verify any in vivo significance of this interaction and if the chromatin remodelling complex plays a significant role in DNA damage response. The existence of an endogenous interaction was verified by a co-immunoprecipitation of BRG1 with 53BP1. In vivo methylation assay showed that BRG1 does in fact contain methylated residues. I hypothesised that 53BP1 could recruit BRG1 to DNA damage sites via this interaction, however, extensive analy / La méthylation de protéines est une modification post-translationelle qui a lieu sur des résidus de lysine et d'arginine. Classiquement, la caractérisation de ces modifications sur les histones a formé le domaine de l'étude épigénétique en expliquant le règlement de l'expression des gènes. Ces modifications sont réglées par les enzymes qui catalysent spécifiquement le transfert d'un groupe méthylique à partir d'un donateur méthylique aux groupes aminés des résidus de lysine ou d'arginine. On s'est également avéré que la méthylation des protéines non-histone est cruciale dans plusieurs des processus cellulaires. Un meilleur arrangement du rôle que la méthylation de protéines joue dans le dommage d'ADN a élucidé la fonction de nombreux protéines impliquées dans une cascade de signalisation complexe se produisant aux cassures double-brin (CDB). L'étude de la méthylation de protéines nous a permise d'identifier des nouvelles protéines impliquées dans le dommage d'ADN et de clarifier la fonction des protéines existantes impliquées dans la réponse au dommage d'ADN. Cette thèse a comme objectif de développer des nouvelles méthodes pour identifier les protéines méthylées impliquées dans le dommage d'ADN et de vérifier la pertinence fonctionnelle des protéines nouvellement découvertes. Les domaines de Tudor traditionnellement ont été démontrés de lier les résidus méthylés. La protéine 53BP1 est une médiatrice de la réponse au dommage d'ADN et est recrutée aux CDBs par l'intermédiaire de son domaine tandem de Tudor en liant la lysine méthylée aux protéines de l'histone H4. Un écran protéomique des domaines de Tudor 53BP1 a prouvé qu'il pourrait lier le gène BRG1, une partie critique de la chromatine humaine transformant le complexe. Un des buts du travail présenté dans cette thèse est de vérifier la signification in vivo de cette interaction et de déterminer si la chromatine transformant le complexe j