In contrast to protein structure and to metabolic networks, an organism's transcriptional regulatory network (TRN) is highly plastic and has been reported to be the target of numerous mutations affecting the phenotype. The ascomycetes fungi are an ideal phylogenetic branch to look at TRN evolution by assessing the conservation of transcription factors (TF) and TF binding sites. In this thesis I 1) describe new tools to assess protein function in Candida albicans, 2) report a novel mode of TRN evolution that we named TF substitution that occurred in the evolution of the ribosomal protein (RP) regulon and 3) explore the large-scale transcription network reorganization of two RP TRNs with distinct wirings and TF substitutions. This work leads to a broader understanding of the span of TRN evolution at the organismal level in ascomycetes. These findings illustrate the extent of TRN plasticity in the evolution of fungi. They also raise questions regarding the rate and importance of TRN changes in the evolution of eukaryotes. / Contrairement à la structure des domaines protéiques et aux réseaux métaboliques qui sont difficilement interchangeables, le réseau de régulation transcriptionnelle (TRN) d'un organisme se doit d'être extrêmement plastique et peut être sujet à plusieurs types de mutations influant sur le phénotype. Les levures ascomycètes constituent un groupe phylétique idéal pour évaluer la conservation des facteurs de transcription (TFs) et de leurs sites de liaison sur l'ADN. Dans cette thèse, 1) je décris de nouveaux outils permettant l'analyse fonctionnelle des protéines de C. albicans, 2) Je rapporte le premier exemple d'un changement dans le TRN baptisé substitution de TF qui s'est produit dans l'évolution du régulon des protéines ribosomales (RP) et 3) j'explore les modalités de réorganisation du réseau transcriptionnel dans un système recâblé suite à une substitution de TF. Ces travaux pavent la voie à une étude élargie de l'évolution du TRN chez les ascomycètes. Ils illustrent aussi l'extrême plasticité du TRN ribosomal dans l'évolution des ascomycètes et soulèvent d'intéressantes questions quant à l'importance des changements du réseau régulatoire dans l'évolution des eucaryotes.

Cellular differentiation is accompanied by the modulation of gene expression. I have compared the expression of various genes, using the slot-blot technique, in two different systems. First, the level of expression of a wide variety of genes was analyzed in the hypertrophied heart of transgenic mice expressing the polyomavirus large T-antigen gene, and compared to normal control heart. I have shown that most changes in gene expression occurred mainly during early stages of heart hypertrophy. These genes code for proteins known to play a role in signal transduction, and transcriptional and growth control. The latter stages of cardiac hypertrophy are accompanied by changes in the expression of genes that are mostly involved in stress responses. Second, we analyzed the expression of various genes in three mouse myogenic cell lines undergoing differentiation in several culture conditions. The adult (C2C12) and fetal-derived (G7 and G8) myoblast cell lines were exposed to either retinoic acid, dimethyl sulfoxide, or transforming growth factor $ beta$. These three molecules are known to have profound effects on cellular growth and differentiation. I have shown that these treatments result in significant changes in expression of a wide variety of genes. Interestingly, all three cell lines differed considerably in their pattern of gene expression. Results from the analysis of these two systems demonstrate that differentially induced morphological changes of muscle cells, result in cell type specific changes in expression of a variety of genes.

A novel S1 nuclease mapping method was devised to measure the efficiency and sites of transcription termination in vivo by RNA polymerase II on polyomavirus DNA. To determine termination efficiency, pulse-labeled nuclear RNA was hybridized with a DNA fragment which spans the transcription initiation region. Since polyomavirus DNA is circular, any RNA polymerases which do not terminate transcription will traverse the entire genome and pass through the initiation region again. Hybrids were treated with ribonuclease, bound to nitrocellullose filters, eluted from the filters by degrading single-stranded DNA with S1 nuclease, and analyzed by gel electrophoresis. The results show that about 70% of polymerases terminate transcription per genome traverse at 16 hours post infection; only 50% terminate at 28 hours. At 16 hours, the major transcription initiation site on the L DNA strand is at nucleotide 5050; but at 28 hours, the major initiation site is shifted upstream to nucleotide 5128. RNA polymerase terminates and/or pauses at many discrete sites which lie mostly downstream of the polyadenylation site. Some pausing and/or termination sites are also observed upstream of the polyadenylation site and are probably created by polymerases that traverse the genome more than once. Our experiments also show that the 3$ sp prime$ end at nucleotide 1230 found by Triesman and Kamen (1981) in steady state nuclear RNA is created by a post-transcriptional RNA processsing event and not by termination of transcription.

Knowing the tissues, cells, and sub-cellular compartments where a protein is expressed can provide important insights about its biological and physiological roles. CDP/Cux is a transcription factor that is proteolytic processed as cells progress from G1 to S phase of the cell cycle. With the aim of investigating CDP/Cux expression in tissues and in tissue culture cells, I characterized a panel of polyclonal and monoclonal antibodies raised against various regions of the protein. I established an immunohistochemistry assay and demonstrated that CDP/Cux is expressed in certain epithelial cells in several tissues. Using the technique of immunofluorescence I studied the localization of CDP/Cux proteins throughout the cell cycle. I obtained results suggesting that CDP/Cux isoforms may localize to different compartments during S phase. Finally, using 3 different antibodies, I showed that cathepsin L, the potential protease that cleaves CDP/Cux, may translocate to the nucleus during progression through the cell cycle.

RARbeta2 is a tumour suppressor activated by retinoic acid (RA). It controls gene expression by heterodimerizing with RXRs on RA response elements. We hypothesised that Erk1/2 inhibits RARbeta2 transcriptional activity in RARbeta2-expressing and RA-resistant cell lines by phosphorylating RXRalpha. Treatment of RA-resistant, RARbeta2-expressing cell lines (A549 and Hs578t) and a RA sensitive cell line (H157) with PD98059 synergized with RA in inhibiting growth. Cloning an active mutant of MEK1 (MEK1DD) into H157 made these cells around 40% less sensitive to ATRA. EGF-mediated Erk1/2 activation inhibited the ATRA-induced transcription of both a synthetic reporter and an endogenous (RARbeta) gene. In both cases, PD98059 reversed this inhibition. Finally, MEK1DD inhibited to approximately 90% transcription from the reporter gene. These results indicate that Erk1/2 reduces RA-mediated transcription by all RARs, possibly by phosphorylating RXRalpha and that RA may be effective clinically when used in combination with drugs acting upstream of MEK1/2 such as Iressa and Herceptin.

Inactivating mutations of the TGF-beta signaling pathway, including SMAD4, have been identified in a wide variety of human tumors and can lead to resistance to TGF-beta induced growth arrest. SMAD4 itself has been firmly established as a tumor suppressor while despite the massive information gathered on TGF-beta signaling the contribution of SMAD4 inactivation in tumor progression remains elusive. It is largely unknown which particular genes involved in tumor progression require SMAD4 for their transcriptional regulation. Smad4-/- murine fibroblasts were shown to display normal growth inhibition in response to TGF-beta and we hypothesized that in Smad4-/- tumors there are as yet unidentified physiological targets of Smad4 involved in tumor progression. A cDNA microarray was performed on Smad4+/+ and -/- murine fibroblasts with and without TGF-beta stimulation in order to identify genes that were deregulated in the absence of Smad4. Several potential novel targets were identified and confirmed by RT-PCR and Northern analysis. Consistent with the role of Smad4 as a tumor suppressor, when Smad4 is inactivated, fibroblasts appear to present with an increase in factors that enhance angiogenesis and a decrease in factors that inhibit the degradation of the extracellular matrix. This could potentially lead to an increased vascularization of tumors and an augmentation of metastatic ability. Further, in the absence of Smad4, TGF-beta stimulation appears to amplify the deregulation of target genes, a mechanism which could potentially enhance tumor progression in Smad4-/- tumors.

Cathepsin K is a cysteine protease of the papain family. It is expressed predominantly in the osteoclast and is believed to be the main degradative agent of the bone organic matrix. / Collagen types I and II were treated with recombinant human cathepsin K in the presence or absence of chondroitin-4 sulfate. The reaction was stopped at 0, 1, 2, 4, 8, and 24 h by the addition of a specific cysteine protease inhibitor E-64. The effect of chondroitinase ABC pre-treatment, to remove possible chondroitin sulfate contaminant in the collagen preparation, was investigated. In order to obtain a clear picture of the degradative potential of this enzyme and to investigate whether the accessibility of the proteoglycans in the extracellular matrix affects their susceptibility to proteolysis, the action of recombinant cathepsin K on the components of cartilage as isolated molecules, was compared with their cleavage in the intact tissue. The isolated molecules were obtained by extracting bovine nasal cartilage with guanidinium chloride (Gdn-HCl). Cartilage tissue was cut into 1 mm3 pieces and treated with cathepsin K. The tissue was then extracted in Gdn-HCl. All experiments were performed at pH 5.5. Proteoglycans and collagen were analyzed by SDS/PAGE and western blotting. / All proteoglycans studied were substrates for cathepsin K. However, a marked difference in their accessibility to this enzyme in cartilage was found. Aggrecan had the same relative accessibility in both extracts and in intact cartilage, whereas the small proteoglycans were resistant to cleavage indicating protection by association with other cartilage macromolecules. The present study suggest that cathepsin K would be an effective cartilage degrading enzyme and that cartilage may "extend" the life of certain components through interactions with others.

In mammals, Nck, represented by two genes, is a 47kDa protein lacking intrinsic enzymatic function. It is composed solely of three N-terminal Src-homology 3 (SH3) domains and a single C-terminal Src-homology 2 (SH2) domain. Nck is classified as an adapter molecule that links cell surface receptors, via its SH2 domain, to downstream effectors, through its SH3 domains. Two cDNAs coding for the carboxy-terminal region of the beta subunit of the eukaryotic initiation factor 2 (eIF2beta) were isolated from a two-hybrid screen to identify new effector molecules interacting with the SH3 domains of Nck. eIF2beta is a component of the molecular complex eIF2 responsible for one of the early steps in the initiation of protein synthesis. In this thesis, I determined in vivo that the first and the third SH3 domains of Nck were both required for its interaction with eIF2beta. Furthermore, Nck and eIF2beta colocalized in an enriched ribosomal fraction. The localization of Nck in this compartment is enhanced upon insulin stimulation. I also established that Nck-1 overexpression is concomitant with an increase in protein translation. Interestingly, the effect of Nck on translation was dependent on its first and third SH3 domains originally found to mediate its interaction with eIF2beta. To elucidate how Nck modulates protein translation, I examined whether Nck maintains its positive effects under ER stress conditions where protein synthesis is normally inhibited. Interestingly, my data revealed that Nck-1 overexpression antagonized ER stress-induced inhibition of translation. Furthermore, I demonstrated that overexpression of Nck-1 prevented PERK activation, eIF2alpha phosphorylation, BiP induction and cell survival in response to ER stress treatments. This integrates Nck in the regulation of the ER unfolded protein response (UPR). I also provided evidence that the effects of Nck on the UPR involve its second SH3 domain and a phosphatase-dependent mechanis

Premature labour and subsequent premature birth is a leading cause of neonatal morbidity and mortality. We studied marine labour at term and preterm with models of intrauterine infection and ovariectomy using Affymetrix microarray U74Av2 (containing 12,488 probe sets) and real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) to identify novel candidate genes involved in normal and preterm labour. Strict statistical analysis revealed 320, 188, and 74 genes to be significantly induced or suppressed during normal, infection-induced, and ovariectomy-induced labour, respectively. Novel genes identified include: associated with normal labour: alpha fetoprotein, apolipoprotein A1, fibrinogen polypeptides, cytochrome P45011a, Purkinje cell protein 4 (PCP4), and chloride channel calcium activated 3; associated with infection: numerous inflammatory mediators, alpha fetoprotein, apolipoprotein Al, and fibrinogen polypeptides. Small proline-rich protein 2 family genes were induced by ovariectomy and infection. PCP4 gene was induced after ovariectomy, but suppressed at normal labour. Only seven genes were significantly regulated (each induced) at labour in all models implying that unique gene networks are involved in normal and preterm labour induced by various stimuli. These included genes for plasminogen activator inhibitor 1 and for contraction associated proteins (CAPS) required for uterine activation and uterotonin stimulation of contractions. / The oxytocin receptor (OTR) gene encodes one such CAP. Northern blot and real-time RT-PCR demonstrated its up-regulation prior to labour in each model, preferentially in normal labour. Uterine contraction promotes increased central and peripheral oxytocin release and synaptic plasticity. To further examine the role of the OTR, we developed an OTR-lacZ reporter mouse. We mapped, by X-gal histochemistry, the distribution of OTR gene expression in the early postparturient mouse brain and identified novel regions of expression. These included the piriform cortex, entorhinal cortices, and parasubiculum, which support memory function. Dorsal tegmental, vestibular, and lateral reticular nuclei expression suggests the transmission of locomotor inputs. Hypoglossal, facial, and spinal trigeminal nuclei support maternal behaviours. We also more accurately demarcated OTR gene expression in the solitary tract nucleus responsible for relaying contraction stimulation of oxytocin release. / These studies provide a more accurate knowledge base for the development of successful therapies to decrease the incidence of premature labour.

Maintaining appropriate patterns of gene expression in the gametes and during early embryogenesis is essential for normal development. DNA methylation is an epigenetic means of regulating gene expression and is an important molecular mark regulating the sex-specific expression of genes subject to genomic imprinting. Imprinted genes are expressed from only one of two inherited chromosomes and are differentially marked during gametogenesis to allow for their parental allele specific expression. These genes affect embryo growth, placental function, behavior after birth and are implicated in the etiology of a number of human diseases. The primary objective of this thesis was to gain a better understanding of the developmental dynamics and origins of DNA methylation profiles regulating maternally methylated imprinted genes during mouse oocyte development. Studies revealed that maternally methylated imprinted genes acquire methylation within their DMRs during postnatal oocyte growth and that this acquisition occurs in a gene and allele specific manner. It was also observed that maternal methylation imprint acquisition is related to oocyte diameter and that a repetitive parasitic element also acquires methylation during this period. DNA methylation is catalyzed by DNMTs and investigations into the developmental expression profiles of Dnmt3a, Dnmt3b and Dnmt3L indicated that transcript accumulation of these enzymes during oocyte development coincided with the timing of maternal methylation imprint establishment. Moreover, expression analysis in DNMT-depleted oocytes suggested these enzymes to be coordinately regulated. Additional studies aimed at developing another model of oocyte imprinting lead to the identification and characterization of a putative bovine Snrpn DMR. Its DNA methylation profile was found to be conserved with that of mouse and human. Snrpn DNA methylation analysis in bovine IVF and SCNT embryos revealed slight loss of methylatio