Winter wheat has several advantages over spring varieties, higher (25 % more) yield, efficient use of spring moisture, reduction of soil erosion by providing ground cover during the fall and early spring, rapid initial spring growth to out - compete weeds and circumvent the peak of Fusarium head blight infections by flowering early. Winter wheat is planted in early autumn when it germinates and developing seedlings acclimate to cold. The crown survives under snow cover and in spring rapidly grows into a vigorously growing plant for grain to be harvested in summer. However, the harsh Canadian prairie winters require that winter wheat has increased cold hardiness and improved winter survival to reduce losses from sudden cold snaps during winter and spring. Low temperature (LT) tolerance is one of the major components of cold hardiness. Genetic mapping studies have revealed a major quantitative trait locus (Fr-A2) at wheat chromosome 5A which can explain at least 50 % of LT tolerance in wheat. Physical mapping of 5A LT QTL in a hardy winter wheat cv Norstar revealed a cluster of at least 23 C - repeat binding factors (CBF) coinciding with peak of Fr-A2 QTL. The objective of this study is biochemical, and molecular characterization of CBF co - located at Fr-A2 to identify key CBF participating in conferring LT tolerance in winter wheat. A comparative analysis of CBF gene cluster at the Fr-A2 collinear region among Poaceae members showed an expansion in the number of CBF genes with increased LT tolerance. Rice, a cold sensitive member, had only three CBF genes, whereas cold hardy winter wheat cv Norstar has 23 CBF genes. Amino acid sequence - based cluster analysis of complete CBF genes, or their major functional components such as the AP2 - DNA binding domain and C - terminal trans - activation domain, divide Norstar CBF into Pooideae specific clades. However, analyses of Norstar CBF amino acid sequences of different functional groups revealed a shift in clade members. These results suggest divergence of CBF functions which could lead to possible differences / similarity in the regulon activated by a CBF in a specific group. The 15 CBF genes from winter wheat cv Norstar were expressed in E. coli to produce recombinant TrxHisS - CBF fusion proteins in adequate quantities for structural and functional assays. All CBF fusion proteins could be recovered in the E. coli soluble phase of cell extract, except that the CBF17.0 fusion protein could only be recovered with 6 M urea extraction. Eleven of the 15 CBF fusion proteins were very stable in heat (98 oC), 10 % SDS and 6 M urea treatment. The five other CBF members were very labile under native conditions, but were stable in E. coli cell extracts or when extracted under denaturing conditions. Most of the CBF recombinant proteins in denaturing gel electrophoresis migrated slower than expected from their predicted molecular mass, based on amino acid sequence. The slow migration could be associated to their elongated protein structure as determined by dynamic light scattering (DLS). CBF 12.2 and CBF 17.0 were highly resistant to denaturation and retained their secondary structure in these conditions as determined by circular dichroism (CD) spectra. The high stability of these two CBF proteins may be important for cold acclimation or maintenance of cold hardiness in wheat. CBF proteins are transcription factors that bind to the dehydration-responsive element / C-repeat element (DRE / CRT) motif (CCGAC). Ten of the 15 Norstar recombinant CBFs whether purified under native or denaturing conditions showed in vitro binding to the CRT motif. Within hours of cold exposure (4 oC) the native CBF increased their affinity to CRT interaction which could be due to changes in the CBF secondary structures. Some of the CBF for binding preferred the core GGCCGAC motif while others preferred TGCCGAC. Similarly binding assays with truncated CBF revealed that for some CBF proteins, the second signature motif (DSAWR) and remaining C - terminal were not needed, while for others a considerable portion of the C -terminal region was needed for binding. Norstar CBF 12.1 has a memory of cold experience, and upon exposure to cold, has a high and immediate affinity to CRT elements. A homolog CBF12.2 in less cold - hardy winter wheat cv Cappelle - Desprez had a non - functional protein due to a R → Q substitution in a highly conserved residue within the AP2 domain. Several of the cv Norstar CBFs showed increased activity under LT and denaturing conditions, which may be the reason for the greater cold hardiness in Norstar. In conclusion, detailed and extensive analyses of CBF in this study characterized their structure and function relationships, which are important for understanding and improving LT tolerance in plants. The identification of specific CRT binding motifs and two CBFs which were very stable under adverse conditions may be prime candidates for further study to improve LT tolerance in plants.

Computational systems biology is an emerging area of research that focuses on understanding the holistic view of complex biological systems with the help of statistical, mathematical and computational techniques. The regulation of gene expression in gene regulatory network is a fundamental task performed by all known forms of life. In this subsystem, modelling the behaviour of the components and their interactions can provide useful biological insights. Statistical approaches for understanding biological phenomena such as gene regulation are proving to be useful for understanding the biological processes that are otherwise not comprehensible due to multitude of information and experimental difficulties. A combination of both the experimental and computational biology can potentially lead to system level understanding of biological systems. This thesis focuses on the problem of inferring the dynamics of gene regulation from the observed output of gene expression. Understanding of the dynamics of regulatory proteins in regulating the gene expression is a fundamental task in elucidating the hidden regulatory mechanisms. For this task, an initial fixed structure of the network is obtained using experimental biology techniques. Given this network structure, the proposed inference algorithms make use of the expression data to predict the latent dynamics of transcription factor proteins. The thesis starts with an introductory chapter that familiarises the reader with the physical entities in biological systems; then we present the basic framework for inference in transcriptional regulation and highlight the main features of our approach. Then we introduce the methods and techniques that we use for inference in biological networks in chapter 2; it sets the foundation for the remaining chapters of the thesis. Chapter 3 describes four well-known methods for inference in transcriptional regulation with pros and cons of each method. Main contributions of the thesis are presented in the following three chapters. Chapter 4 describes a model for inference in transcriptional regulation using state space models. We extend this method to cope with the expression data obtained from multiple independent experiments where time dynamics are not present. We believe that the time has arrived to package methods like these into customised software packages tailored for biologists for analysing the expression data. So, we developed an open-sources, platform independent implementation of this method (TFInfer) that can process expression measurements with biological replicates to predict the activities of proteins and their influence on gene expression in gene regulatory network. The proteins in the regulatory network are known to interact with one another in regulating the expression of their downstream target genes. To take this into account, we propose a novel method to infer combinatorial effect of the proteins on gene expression using a variant of factorial hidden Markov model. We describe the inference mechanism in combinatorial factorial hidden model (cFHMM) using an efficient variational Bayesian expectation maximisation algorithm. We study the performance of the proposed model using simulated data analysis and identify its limitation in different noise conditions; then we use three real expression datasets to find the extent of combinatorial transcriptional regulation present in these datasets. This constitutes chapter 5 of the thesis. In chapter 6, we focus on problem of inferring the groups of proteins that are under the influence of same external signals and thus have similar effects on their downstream targets. Main objectives for this work are two fold: firstly, identifying the clusters of proteins with similar dynamics indicate their role is specific biological mechanisms and therefore potentially useful for novel biological insights; secondly, clustering naturally leads to better estimation of the transition rates of activity profiles of the regulatory proteins. The method we propose uses Dirichlet process mixtures to cluster the latent activity profiles of regulatory proteins that are modelled as latent Markov chain of a factorial hidden Markov model; we refer to this method as DPM-FHMM. We extensively test our methods using simulated and real datasets and show that our model shows better results for inference in transcriptional regulation compared to a standard factorial hidden Markov model. In the last chapter, we present conclusions about the work presented in this thesis and propose future directions for extending this work.

In the adult brain, the thalamocortical tract conveys sensory information from the external environment to the cortex. The cortex analyzes and integrates this information and sends neural responses back to the thalamus through the corticothalamic tract. To reach their final target both thalamocortical and corticothalamic axons have to cover long distances during embryogenesis, changing direction several times and passing through different brain territories. The ventral telencephalon plays a major role in the early development of these tracts. At least three main axon guidance mechanisms act in the ventral telencephalon. First, two different populations of pioneer neurons in the lateral ganglionic eminence (LGE) (LGE pioneer neurons) and medial ganglionic eminence (MGE) (MGE pioneer neurons) provide scaffolds which allow growing corticothalamic and thalamocortical axons to cross the pallium sub pallium boundary (PSPB) and the diencephalic telencephalic boundary (DTB), respectively. Second, the ventral telencephalon forms a permissive corridor for thalamic axons by tangential migration of Isl1 and Ebf1 expressing cells from the LGE into the MGE. Finally, thalamortical and corticothalamic axons guide each other once they have met in the ventral telencephalon (“handshake hypothesis”). The Gli3 transcription factor has been shown to be essential for normal early embryonic regionalization of the mammalian forebrain, although roles of Gli3 in later aspects of forebrain development, like the formation of axonal connections, have not been investigated previously. Here, I present the analysis of axonal tract development in the forebrain of the Gli3 hypomorphic mutant mouse Polydactyly Nagoja (Pdn). These animals lack the major axonal commissures of the forebrain: the corpus callosum, the hippocampal commissure, the anterior commissure and the fimbria. In addition, DiI injections and neurofilament (NF) staining showed defects in the formation of the corticothalamic and thalamocortical tracts. Although the Pdn/Pdn cortex forms early coticofugal neurons and their axons, these axons do not penetrate the LGE and instead run along the PSPB. Later in development, although a thick bundle of Pdn/Pdn cortical axons is still observed to project along the PSPB, some Pdn/Pdn cortical axons eventually enter the ventral telencephalon navigating along several abnormal routes until they reach thalamic regions. In contrast, Pdn/Pdn thalamic axons penetrate into the ventral telencephalon at early stages of thalamic tract development. However, rostrally they deviate from their normal trajectory, leaving the internal capsule prematurely and only few of them reach the developing cortex. Caudally, an ectopic Pdn/Pdn dorsal thalamic axon tract projects ventrally in the ventral telencephalon not entering the internal capsule at all. These defects are still observed in newborn Pdn/Pdn mutant mice. Next, I investigated the developmental mechanisms causing these pathfindings defects. No obvious defects are present in Pdn/Pdn cortical laminae formation and in the patterning of the Pdn/Pdn dorsal thalamus. In addition, Pdn/Pdn thalamocortical axons are able to respond to ventral telencephalic guidance cues when transplanted into wild type brain sections. However, these axonal pathfinding defects correlate with patterning defects of the Pdn/Pdn LGE. This region is partially ventralized and displays a reduction in the number of postmitotic neurons in the mantle zone due to an elongated cell cycle length of LGE progenitor cells. Finally, Pdn/Pdn mutant display an upregulation of Shh expression and Shh signalling in the ventral telencephalon. Interestingly, these patterning defects lead to the absence of DiI back-labelled LGE pioneer neurons, which correlates with the failure of corticothalamic axons to penetrate the ventral telencephalon. In addition, ventral telencephalic thalamocortical guidance mistakes happen at the same time of abnormal formation of the corridor cells. Taken together these data reveal a novel role for Gli3 in the formation of ventral telencephalic intermediate cues important for the development of the thalamocortical and corticothalamic connections. Indeed, Pdn animals are the first known mutants with defective development of the LGE pioneer neurons, and their study provides a link between early patterning defects and axon pathfinding in the developing telencephalon.

<p>Transcription factors (TFs) control the temporal and spatial expression of target genes by interacting with DNA in a sequence-specific manner. Recent advances in high throughput experiments that measure TF-DNA interactions in vitro and in vivo have facilitated the identification of DNA binding sites for thousands of TFs. However, it remains unclear how each individual TF achieves its specificity, especially in the case of paralogous TFs that recognize distinct target genomic sites despite sharing very similar DNA binding motifs. In my work, I used a combination of high throughput in vitro protein-DNA binding assays and machine-learning algorithms to characterize and model the binding specificity of 11 paralogous TFs from 4 distinct structural families. My work proves that even very closely related paralogous TFs, with indistinguishable DNA binding motifs, oftentimes exhibit differential binding specificity for their genomic target sites, especially for sites with moderate binding affinity. Importantly, the differences I identify in vitro and through computational modeling help explain, at least in part, the differential in vivo genomic targeting by paralogous TFs. Future work will focus on in vivo factors that might also be important for specificity differences between paralogous TFs, such as DNA methylation, interactions with protein cofactors, or the chromatin environment. In this larger context, my work emphasizes the importance of intrinsic DNA binding specificity in targeting of paralogous TFs to the genome.</p> / Dissertation

Chez le riz, la céréale modèle, le système racinaire est principalement constitué de racines issues de la tige, nommées racines coronaires (RC). Peu de gènes contrôlant le développement des RC sont connus, parmi eux CROWN ROOTLESS1 (CRL1) code une protéine à domaine AS2/LOB (ASL/LBD), qui est probablement un facteur de transcription. Le gène CRL1 est nécessaire à l'initiation des primordia de RC, il est directement activé par l'auxine et est situé en amont du réseau de gènes contrôlant le programme de différentiation des RC. Afin de mieux connaître les processus génétiques impliqués dans l'initiation des RC, l!objectif principal de cette thèse est de comprendre la fonction moléculaire de la protéine CRL1 en validant sa fonction de facteur de transcription et en identifiant ses gènes cibles. L'interaction de la protéine CRL1 avec l!ADN a été montrée in vitro et une expérience de SELEX a permis d'identifier sa séquence de fixation à l!ADN : CACA(A/C)C (CRL1-box). Des expériences en levure ont permis de montrer que CRL1 est un activateur de la transcription. Une comparaison entre le sauvage et le mutant crl1, ainsi que l'élaboration d!un système inductible à la dexaméthasone permettant d'activer l'expression de CRL1 dans le fond génétique mutant crl1, ont été utilisés pour identifier des gènes cibles précoces de CRL1 grâce à des analyse de transcriptome. 277 gènes sont activés dès quatre heures après induction de CRL1, les deux tiers contiennent au moins une CRL1-box dans leur promoteur et peuvent donc être des cibles directes de CRL1. CRL1 induit l'expression d!un ensemble de gènes permettant la mise en place des processus de régulation de l'information génétique, de division, de croissance et de différenciation cellulaires nécessaires à la création d'un méristème de racine coronaire organisé et fonctionnel. Parmi eux, QHB code un facteur de transcription clé nécessaire au maintien des cellules souches des méristèmes racinaires. Ce résultat établit pour la première fois un lien moléculaire entre la signalisation de l!auxine et des gènes impliqués dans la mise en place ou le maintien des cellules souches lors de la formation d!un nouveau méristème racinaire au cours du développement post-embryonnaire. Par ailleurs, une étude histologique a permis de révéler que les RC sont issues d!une couche de péricycle dans la tige, un tissu équivalent en termes de localisation et de potentiel rhizogène au péricycle de la racine à partir duquel sont initiées les racines latérales. Les données acquises suggèrent de fortes similarités dans les processus cellulaires et génétiques de la différentiation des méristèmes racinaires au cours du développement post-embryonnaire chez les monocotylédones et les dicotylédones. La découverte de gènes spécifiques au développement de racines issues de la tige ouvre une voie importante vers la compréhension du déterminisme génétique de l!architecture du système racinaire chez les céréales et offre un nouveau potentiel de ressources génétiques pour l!amélioration variétale. / In rice, the model cereal, the root system is mainly composed of stem-derived roots, named crown roots (CR). Very few genes that control the root system development are known, among them CROWN ROOTLESS1 (CRL1) encodes an AS2/LOB-domain protein that is a putative transcription factor (TF). CRL1 is necessary for CR primordium initiation, it is directly activated by auxin and is situated upstream of the gene regulatory network that control the CR differentiation programme. To better known the genetic processes involved in CR initiation, the main objective of this thesis is to understand the molecular function of the CRL1 protein by validating its function of TF and by identifying its target genes. The interaction of CRL1 with DNA was shown in vitro and a consensus CRL1 DNA-binding motif was identified with a SELEX method : CACA(A/C)C named CRL1-box. A yeast assay showed that CRL1 is a transcriptional activator. A comparison between wild type and c rl1 mutant, and the development of a dexamethasone inducible system to ectopically express CRL1 in the crl1 background, were used to identify CRL1 early target genes by transcript profiling. 277 genes were induced from four hours following CRL1 activation, the two-thirds possess at least one CRL1-box and may be CRL1 direct target genes. CRL1 activates the expression of a broad range of genes that allow to orientate genome expression and to initiate cell division, growth and differentiation mechanisms required for the building of an organized and functional CR meristem. Among these genes, QHB encodes a key TF required for the maintenance of root meristem stem cells. This result evidences for the first time a molecular link between auxin signalling and major genes involved in stem cell patterning and maintenance in the formation of a new root meristem during post-embryonic development. Otherwise, an histological study showed that CR are derived from a shoot pericycle, a tissue equivalent to the root pericycle, from which lateral roots develop, in terms of location and rhizogenic potential. All these data suggest strong similarities between monocots and dicots in cellular and genetic mechanisms that control root meristem differentiation during post-embryonic development. The identification of genes specifically involved in stem-derived root development pave the way towards the understanding of the genetic control of root system architecture in cereals and offer a new potential of genetic resources for plant breeding.

La bilharziose reste un problème de santé majeur. L'équipe du Pr Dessein a montré que les infections élevées étaient déterminées par un locus majeur en 5q31 et que des polymorphismes dans un gène à ce locus,IL13, aggravent l'infection. Notre premier objectif était d'évaluer si des variants d'autres gènes de la voie de l'IL13 intervenaient dans le contrôle de l'infection. Nous avons observé une association entre le SNP rs324013, dans le promoteur de STAT6,et les niveaux d'infection à S. haematobium. Ce polymorphisme a un effet additif avec le polymorphisme IL13rs1800925. Ce SN modifie la fixation de facteurs nucléaires au niveau du promoteur de STAT6. L'équipe du Pr Dessein avait également montré que les fibres hépatiques avancées et sévères étaient déterminées par un autre locus majeur localisé en 6q23. Notre deuxième objectif fut d'évaluer dans le laboratoire du Pr Dessein et en étroite collaboration avec le laboratoire du Pr Li(Yueyang Institute of Parasitic disease)deux gènes candidats(IFNGR1 et CTGF) situés dans cette région chromosomique. Nous avons observé une association entre les deux polyporphismes(rs17066192 er rs673156)localisés dans le promoteur du gène. Nous avons observé une association entre les deux polymorphismes(rs17066192 et rs673156)localisés dans le promoteur du gène IFNGR1 et la fibrose hépatique: le génotype rs673156A/A et rs17066192C/C sont associés à un risque 7.3 fois et 1.5 fois plus élevé, respectivement, de fibrose avancée. Nous avons également montré que les variants rs9402373 et rs12526196 du gène CTGF sont indépendamment associés à la fibrose chez les fermiers et pêcheurs chinois infectés par S.japonicum. Sur la population chinoise d'étude, les risques relatifs associés aux polymorphismes rs9402373 et rs12526196 sont de 2.8 et 3 / Schistosomiasis remains one of the world’s most prevalent diseases. It comprises a group of chronic diseases caused by helminths of the Schistosoma genus. Schistosoma haematobium causes obstructive nephropathy that can be aggravated by urinary bacterial infections. S.japonicum and S.mansoni cause hepatic fibrosis associated with portal blood hypertension, which can be lethal. In previous studies, our laboratory had shown that worm burden in S.haematobium infections were aggravated by IL13 variants and that severe hepatic fibrosis (HF) was controlled by gene(s) located on 6q23. The present study is to further evaluate other IL-13 pathway genes (STAT6) in the control of infection in Malian farmers and to test candidate genes in the 6q23 region in hepatic fibrosis (HF) in S.japonicum infected Chinese fishermen and farmers. First we have developped an improved FTA® technology technique to perform SNP genotyping. This technique allows us to use saliva samples for genotyping SNPs. Subsequently, this improved FTA® technology was used in our study on HF.Our work on a Malian sample infected with S. haematobium indicated that a polymorphism (rs324013) in the promoter of STAT6 gene was associated with the control of S. haematobium infection levels and has an additive effect with IL13rs1800925, a polymorphism previously associated with infection in this same population. Both SNPs modify the binding of nuclear factors to the promoter regions of their respective genes. Thus, both SNPs may play a crucial role in controlling S. haematobium infection levels. In order to study HF in S.japonicum infections, we have participated actively in the study that recruited of a large sample of Chinese fishermen and farmers who had been exposed to the infection for most of their life. HF was evaluated by ultrasound and covariates that could affect HF were evaluated by interviews. Then, we tested two genes (IFNGR1, CTGF) of the 6q23 region that were good candidates for the control of HF on these samples. Both genes encode molecules that were shown in animal and human studies to have strong effect on extracellular matrix proteins deposition and turnover. We found that two polymorphisms (rs17066192 and rs673156) in IFNGR1 promoter were associated with HF: the rs673156A/A genotype was associated with a 7.3-fold increased risk of advanced HF; and rs17066192C/C genotype with a 1.5-fold increased risk of HF. These results must now be confirmed in another population sample. We also found that variants of CTGF rs9402373 and rs12526196 were independently associated with HF in Chinese fishermen and farmers, in Sudanese, and in Brazilians infected with either S. japonicum or S. mansoni. Our results provide additional evidence for a protective role of IL-13 in schistosome infections, and they also demonstrate that TGFβ / CTGF pathway plays a key role in HF and should be targeted by chemotherapy. Ongoing studies evaluate whether CTGF variants could be used in the prognosis of the HF caused by schistosomes and also by other infectious agents.

Neurodegenerative disorders (NDD) are an ever-increasing burden on healthcare; consequently, elucidating the mechanisms underlying neurodegeneration (ND) is critical for the development of effective treatments for these diseases. In order to unravel the molecular pathways underlying movement disorders and identify new genes involved in ND, two ataxic mouse mutants characterised by cell death in the cerebellum were studied in detail using a combination of in vitro and in vivo techniques. The robotic mouse demonstrated the key role of a transcription factor, Af4, in Purkinje cell (PC) survival and how only small changes in the levels of a single transcriptional cofactor could deleteriously affect normal cerebellum function. Expression array studies of the robotic PCs revealed the first confirmed targets of Af4-mediated transcription, including insulin-like growth factor 1 (Igf-1). It was demonstrated that Igf-1 is critical for PC survival, highlighting the role of the IGF-1 signalling pathway as a potential therapeutic target for the treatment of cerebellar ataxia in humans. Detailed analysis of the bella mutant demonstrated that ataxia and apoptotic cerebellar degeneration is caused by loss of the oxidative resistance 1 (Oxr1) gene. In vitro modelling experiments went on to show that the levels of this previously uncharacterised gene are critical for controlling the sensitivity of neuronal cells to oxidative stress (OS). Moreover, this study showed that Oxr1 was up-regulated both in human and pre-symptomatic mouse models of amyotrophic lateral sclerosis (ALS), demonstrating that Oxr1 was an early marker of ROS defence, prior to pathology, and potentially a novel neuroprotective factor in NDD. Preliminary interaction studies show that Oxr1 is likely to be a multi-functional protein that forms complexes with proteins known to be mutated in NDD. Thus, the study of both the robotic and the bella mouse has demonstrated the value of the phenotype-driven approach to investigate novel neurodegenerative pathways.

Legionella pneumophila, an intracellular pathogen of protozoa, is well known for its dimorphic life cycle that alternates between the vegetative replicative form (RF) and highly infectious cyst-like form (CLF). To this date several virulence factors including LpRpoS, LpIHF, and the Dot/Icm secretion system have been found to be required for the survival of L. pneumophila in macrophage and protozoa. Here we have identified and characterised Lpg1967, an orthologue of Pseudomonas PsrA in L. pneumophila. PsrA (Lpg1967) was found to regulate the expression of previously known virulence factors such non-coding RNAs, RsmY/Z, RpoS, LpIHF, flagella and Dot/Icm Type IV secretion system. In addition, the ΔpsrA mutant strain was unable to establish Legionella-containing vacuole and thus displayed a severe growth defect in the U937 derived macrophage cell line. Thus, PsrA was found to play an important role in controlling the regulatory cascade governing virulence in L. pneumophila. / October 2014

Introduction: Despite many advances in therapeutic and surgical techniques for glioblastoma multiforme (GBM), this form of brain cancer still remains incurable. A hallmark feature of GBM is the ability of the glioma cells to infiltrate surrounding brain tissue. The invasive nature of glioma cells is a key challenge in considering treatment for patients with GBM. Certain members of the matrix metalloproteinase (MMP) family play a role in tumor cell invasion and metastasis (Coussens, et al., 2002). A functional SNP resulting from an additional guanine at position -1607 in the MMP-1 promoter creates an erythroblastosis twenty six transcription factor protein (ETS) DNA consensus binding site, which results in significantly higher transcriptional activity of MMP-1 (Rutter et al., 1998). Several published studies show the incidence of this 2G allele is significantly higher in aggressive and metastatic tumors. Binding of an adjacent transcription factor DNA consensus site, activator protein -1 (AP1) site at -1607 has been shown to cooperate with ETS binding to activate transcription of the MMP-1 gene. We have reported a significant increase in the 2G/2G MMP-1 genotype in glioblastomas (pPurpose: To determine if a novel SNP decoy can inhibit the 2G genotype-dependent increase in MMP-1 transcriptional activity, three specific aims were tested: one, to verify specificity of binding of a transcription factor decoy designed to mimic the -1607 SNP site within the MMP-1 promoter; two, to determine the effect of transcription factor decoy ODN on transcriptional activity of an MMP-1 promoter containing the 2G SNP at -1607; and three, to assess the effect of the transcription factor decoy ODN on MMP-1 mRNA and protein expression in treated glioma cells. Methods: Modified and unmodified decoys were designed to mimic position -1607 to -1593 of the MMP-1 promoter. The SNP decoy contains both ETS and AP1 DNA consensus sites and MMP-1 flanking sequences. We first determined optimal binding conditions with electromobility shift assays (EMSAs). The EMSA assays were used to determine the presence of Ets-1 and AP1 DNA binding activity within the glioma cell lines, T98 and U87. EMSAs were also used to determine if these transcription factors could bind to the MMP-1 promoters with and without the SNP. Lastly, EMSAs were done to determine the binding characteristics of the two modified SNP decoys (LNA-locked nucleic acid, and a PS-phosphothioate modification). The effect of the decoy on MMP-1 transcriptional activity was assessed using a Dual-Luciferase Reporter Assay. The effect of the SNP decoys on mRNA was assessed using quantitative RT-PCR, and on protein expression using a sandwich enzyme-linked immunoassay (ELISAs). Statistical analysis was done using a two-way ANOVA to evaluate the effect of the decoy on MMP-1 transcriptional activity, and protein expression. Results: EMSA results indicate that Ets-1 and AP1 probes, and MMP-1 promoter probes effectively bind proteins from glioma cell nuclear extracts. Addition of excess decoy was able to inhibit protein interactions with the 2G MMP-1 promoter probe and to a lesser extent the 1G promoter probe. The scrambled decoy had no effect. Promoter studies showed a significant increase in transcriptional activity of the 2G promoter and addition of 5 mm PS-SNP decoy could effectively prevent the increase in activity (pConclusions: U87 and T98 cell lines contain DNA binding activity of the transcription factors of interest, namely ETS-1 and AP1. The candidate transcription factors can bind to the MMP-1 promoter in the presence or absence of the 2G. Both the LNA and PS-SNP modified decoys can inhibit nuclear proteins from binding to the MMP-1 2G promoter. The PS-SNP decoy was able to inhibit MMP-1 (2G) gene transcription in a dose dependent manner, whereas the control decoy showed a consistent non-specific effect. The PS-SNP decoy inhibited MMP-1 mRNA and protein expression in glioma cells containing the 2G genotype, and to lesser extent in glioma cells containing the 1G genotype. The results presented here support the conclusion that the chimeric SNP decoy can selectively inhibit the MMP-1 promoter containing the 2G genotype.

Le facteur de transcription Growth factor independent 1b (GFI1b) est impliqué à différents stades dans la régulation de l'hématopoïèse. Il est notamment fortement exprimé dans les cellules souches hématopoïétiques et au cours de la différenciation des cellules des lignées érythroïdes et mégacaryocytaires. Grâce à un modèle de délétion conditionnelle chez la souris par le système CreLox, nous avons montré que l'absence de GFI1b entraîne une prolifération de cellules mégacaryocytaires incapables de produire des plaquettes. Notre étude ne permet pas de confirmer formellement la prolifération des cellules souches dans la moelle osseuse précédemment observée par notre équipe dans un autre modèle murin. En revanche, les souris GFI1b "knockout" présentent une augmentation des cellules souches circulantes dans le sang périphérique. Une analyse moléculaire préliminaire montre que GFI1b pourrait influer sur la régulation par le cycle circadien de la mobilisation de ces cellules dans le sang. Finalement, notre étude des effets biologiques de la délétion de GFI1b dans le compartiment hématopoïétique des souris adultes nous a permis de définir de façon plus précise le rôle de GFI1b dans l'hématopoïèse et de confirmer son rôle majeur dans la régulation de la mégacaryopoïèse et la production de plaquettes matures. / Growth factor independent 1b (GFI1b) is a transcription factor implicated in the regulation of hematopoiesis. It is highly expressed in hematopoietic stem cells (HSCs) and throughout the differentiation of erythroid and megakaryocytic lineages. Using a CreLox system, we have generated mice in which GFI1b is conditionally deleted in megakaryocytic lineages. These mice exhibit a strong proliferation of immature megakaryocytes, which are unable to produce platelets. We were unable to confirm the results of a previously published study from our group, which reported an increase of hematopoietic stem cells in the bone marrow using a different mouse model. However, in agreement with this publication, an increase in the circulating HSC pool can be detected in our GFI1b knockout mice. A preliminary analysis of the molecular role of GFI1b suggests that this transcription factor might be implicated in the circadian regulation of HSC mobilization in the blood. Finally, our experiments on the biological effects of the deletion of GFI1b in the adult murine hematopoietic compartment allowed us to better understand the role of GFI1b in hematopoiesis, and to confirm the major contribution of GFI1b to the development of megakaryocytes and the production of mature platelets.