Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Although skeletal myogenesis is largely controlled by myogenic regulatory factors, epigenetic modifications have recently emerged as an essential regulatory mechanism of gene expression. Molecular regulation of stem cell differentiation is exerted through both genetic and epigenetic factors over distal enhancer regions. Understanding the mechanistic action of active or poised enhancers is therefore, imperative for the control of stem cell differentiation. Based on the genome-wide co-occurrence of different epigenetic marks in proliferating myoblasts, we have generated a chromatin state model to profile differentiation- and rexinoid-responsive histone acetylation in early myoblast differentiation. Here, we delineate the functional mode of transcription regulators during early myogenic differentiation using genome-wide chromatin state association. We define a role of transcriptional coactivator p300, when recruited by muscle master regulator MyoD, in the establishment and regulation of myogenic loci at the onset of myoblast differentiation. In addition, we reveal an enrichment of loci-specific histone acetylation at p300 associated active or poised enhancers, mainly when enlisted by MyoD. We have previously established that bexarotene, a clinically approved agonist of retinoid X receptor (RXR), promotes the specification and differentiation of skeletal muscle lineage. Hence, we investigated the genome-wide impact of rexinoids on myogenic differentiation and uncovered a new mechanism of rexinoid action, which is mediated by the nuclear receptor and largely reconciled through direct regulation of MyoD gene expression. In addition, we determined rexinoid-responsive residue-specific histone acetylation at a distinct chromatin state associated with MyoD and myogenin. Finally, through ChIP-seq and RNA-seq analyses, we have identified dystroglycan (Dag1) as a differentiation-dependent and a rexinoid-responsive model target, and we revealed a possible co-regulation of Dag1 by p300 and MyoD accompanied by enrichment of loci-specific histone acetylation. Taken together, we provide novel molecular insights into the regulation of myogenic enhancers by p300 in concert with MyoD. Furthermore, we provide novel mechanistic perceptions into the interplay between RXR signaling and chromatin states pertinent to myogenic programs in early myoblast differentiation. Our studies present a valuable insight for driving condition-specific chromatin state or enhancers pharmacologically to treat muscle-related diseases and for the identification of additional myogenic targets and molecular interactions for therapeutic development.

Cystic Fibrosis (CF) is an autosomal recessive disease that arises from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The deletion of phenylalanine-508 (ΔF508-CFTR) is the most prevalent CF mutation and results in a misfolded protein that fails to exit the endoplasmic reticulum (ER). Previous studies demonstrated that mutation of a di-arginine based ER retention motif (R553AR555) in the first nucleotide binding domain (NBD1) rescues the trafficking defect of ΔF508-CFTR. We hypothesized that if the R553AR555 motif mediates retention of the ΔF508-CFTR protein, peptides that mimic this motif should antagonize mistrafficking mediated by aberrant exposure of the endogenous R553AR555 motif. We generated a peptide bearing the R553AR555 motif (CF-RXR) and conjugated it to the cell penetrating peptide Tat (CPP-CF-RXR) to facilitate intracellular delivery and investigated its efficacy in rescuing the mistrafficking and function of ΔF508-CFTR. Using a variety of biochemical and functional assays we demonstrate that the CPP-CF-RXR peptide is effective at increasing surface expression of ΔF508-CFTR in baby hamster kidney (BHK) and human embryonic kidney (HEK) cell lines. Furthermore, the increased surface expression is accompanied by an increase in its functional expression as a chloride channel. Using Ussing chamber assays, we demonstrate that the CPP-CF-RXR peptide improved ΔF508-CFTR channel function in respiratory epithelial tissues obtained from CF patients. Additionally, we investigated the effects of small molecules on mediating biosynthetic rescue of a ΔF508-CFTR construct bearing the additional mutations R553K and R555K (ΔFRK-CFTR) to inactivate the R553AR555 motif. Interestingly, mutation of the R553AR555 motif exerts an additive effect with correctors VRT-325 and Corrector 4a. Taken together, our data suggests that abnormal accessibility of the RXR motif present in NBD1 is a key determinant of the mistrafficking of the major CF causing mutant.

No description available.

Long-term treatment with all trans-retinoic acid (RA) induces neuronal differentiation and apoptosis. However, the effect of short-term RA treatment on cell proliferation, migration and invasion of neuroblastoma cell lines (SH-SY5Y and IMR-32) remains unclear. RA induces expression of tissue-transglutaminase (TGase) and promotes migration and invasion after 24 h of treatment in SH-SY5Y cells, but not in IMR-32 cells. RA receptor (RAR) agonist (4-(E-2-[5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid) and RAR/retinoid X receptor (RXR) agonist (9-cis-RA) promote expression of TGase, migration and invasion of SH-SY5Y cells, while RXR agonist has no significant effect. RAR antagonist blocks RA effect on migration and invasion, indicating that RAR receptors are required. Retinoid receptors are expressed and activated by RA in both cell lines. However, only transient activation of RAR is observed in IMR-32 cells. These findings suggest that different responses observed in SH-SY5Y and IMR-32 cells could be due to differential activation of retinoid receptors. Overexpression of TGase has no effect on migration or invasion, while overexpression of antisense TGase blocks RA-induced migration and invasion, indicating that other molecules along with TGase mediate RA effects. In addition to the long-term effects of RA that are coupled with cell differentiation, short-term effects involve migration and invasion of neuroblastoma SH-SY5Y cells.

No description available.

La présentation antigénique par les molécules de classe II du complexe majeur d’histocompatibilité (CMH II) est un mécanisme essentiel au contrôle des pathogènes par le système immunitaire. Le CMH II humain existe en trois isotypes, HLA-DP, DQ et DR, tous des hétérodimères composés d’une chaîne α et d’une chaîne β. Le CMH II est entre autres exprimé à la surface des cellules présentatrices d’antigènes (APCs) et des cellules épithéliales activées et a pour fonction de présenter des peptides d’origine exogène aux lymphocytes T CD4+. L’oligomérisation et le trafic intracellulaire du CMH II sont largement facilités par une chaperone, la chaîne invariante (Ii). Il s’agit d’une protéine non-polymorphique de type II. Après sa biosynthèse dans le réticulum endoplasmique (ER), Ii hétéro- ou homotrimérise, puis interagit via sa région CLIP avec le CMH II pour former un complexe αβIi. Le complexe sort du ER pour entamer son chemin vers différents compartiments et la surface cellulaire. Chez l’homme, quatre isoformes d’Ii sont répertoriées : p33, p35, p41 et p43. Les deux isoformes exprimées de manière prédominante, Iip33 et p35, diffèrent par une extension N-terminale de 16 acides aminés portée par Iip35. Cette extension présente un motif de rétention au réticulum endoplasmique (ERM) composé des résidus RXR. Ce motif doit être masqué par la chaîne β du CMH II pour permettre au complexe de quitter le ER. Notre groupe s’est intéressé au mécanisme du masquage et au mode de sortie du ER des complexes αβIi. Nous montrons ici que l’interaction directe, ou en cis, entre la chaîne β du CMH II et Iip35 dans une structure αβIi est essentielle pour sa sortie du ER, promouvant la formation de structures de haut niveau de complexité. Par ailleurs, nous démontrons que NleA, un facteur de virulence bactérien, permet d’altérer le trafic de complexes αβIi comportant Iip35. Ce phénotype est médié par l’interaction entre p35 et les sous-unités de COPII. Bref, Iip35 joue un rôle central dans la formation des complexes αβIi et leur transport hors du ER. Ceci fait d’Iip35 un régulateur clef de la présentation antigénique par le CMH II. / Antigen presentation by the major histocompatibility complex class II molecules (MHCII) is a pathway essential to the immune system control over pathogens. There are three MHCII isotypes in humans, which are HLA-DP, DQ and DR. These are all heterodimers made of an α and a β chain. MHCII is expressed at the cell surface of antigen presenting cells (APCs) and activated epithelial cells and its role is mainly to present peptides of exogenous origin to CD4+ T lymphocytes. MHCII oligomerization and trafficking are largely favored by its chaperone, the invariant chain (Ii). Ii is a non-polymorphic type II protein. It hetero- or homotrimerizes right after biosynthesis in the endoplasmic reticulum (ER), and then its CLIP region interacts with MCHII to form a αβIi structure. This multimer exits the ER and starts its journey towards numerous compartments and the cell surface. Humans have four Ii isoforms: p33, p35, p41 et p43. The two most predominantly expressed are Ii p33 and p35. They differ in a N-terminal extension long of 16 amino acids that is displayed by Iip35. This extension bears an ER retention motif (ERM) made of the RXR residues. The RXR motif must be masked by the MHCII β chain in order for the αβIi multimer to exit the ER. Our group investigated the masking and ER exit mechanisms of the αβIi structures. Here we show that a direct (cis) interaction between the MHCII β chain and Iip35 is essential for ER exit, thus promoting the formation of high order αβIi structures. Moreover, we demonstrate that the bacterial virulence factor NleA impairs the trafficking of Iip35-containing αβIi multimers. This observation is mediated by an interaction between COPII subunits and Iip35. Altogether, Iip35 plays a crucial role in the αβIi multimer formation and ER exit. Iip35 is therefore a key regulator for MHCII antigen presentation.

Un régime alimentaire déséquilibré et/ou la présence de composés contaminantsexogènes peuvent modifier la signalisation endocrine et l’homéostasie des lipides et induirel’obésité. Les travaux réalisés dans le cadre de cette thèse ont permis, dans un premier temps,de développer une méthode simple et rapide, dénommée "zebrafish obesogenic (ZO) test",pour identifier in vivo, par utilisation de la larve de poisson zèbre, des facteurs qui peuventaugmenter ou diminuer la taille de l’adipocyte blanc et ainsi moduler le niveau de l’adiposité(Tingaud-Sequeira, Ouadah, Babin, J. Lipid Res. 52, 1765-1772, 2011). Ce test permetd’identifier des composés et des mélanges de molécules obésogènes et anti-obésogènes etfournit des informations pertinentes pour l'évaluation des risques liés leur présence maiségalement pour élucider les mécanismes impliqués. Les travaux ont, dans un second temps,permis d’apporter des réponses quant aux modalités d’action d’un obésogène puissant, lechlorure de tributylétain, contaminant retrouvé très largement dans notre environnement.Cette molécule agit sur l’adipocyte blanc à une concentration de l’ordre du nano molaire viales récepteurs nucléaires RXR et LXR, et non pas via les isoformes PPARgamma/delta(Ouadah et Babin, manuscrit en préparation). / An unbalanced diet and / or the presence of exogenous compounds contaminants mayalter endocrine signaling and lipid homeostasis and induce obesity. The work done in thisthesis have, at first, developed a simple and rapid method, called "zebrafish obesogenic (ZO)test" to identify in vivo by using the zebrafish larva, the factors that may increase or decreasethe size of the white adipocyte and therefore modulate the level of adiposity (Tingaud-Sequeira, Ouadah, Babin, J. Lipid Res. 52, 1765-1772, 2011). This test helps to identifycompounds and mixtures of obesogenic and anti-obesogenic molecules and providesinformation relevant to the risk assessment of their presence but also to elucidate themechanisms involved. Work in a second time allowed to answer as to how the action oftributyltin chloride, a powerful obesogenic contaminant found widely in the environment.This molecule acts in vivo on white adipocytes in a concentration of the order of nano molarvia nuclear receptors LXR and RXR, and not via the PPARgamma isoforms / delta (Ouadahand Babin, manuscript in preparation).

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Molecular switch systems that activate gene expression by a small molecule are effective technologies that are widely used in applied biological research. Previously, two orthogonal ligand receptor pairs (OLRP) were developed as potential molecular switch systems by modifying nuclear receptors, ligand-activated transcription factors, to bind and activate gene expression with the synthetic ligand LG335 and not with the natural ligand 9-cis retinoic acid (9cRA). The two OLRP previously discovered were RXR variant 130 (I268A, I310A, F313A, and L436F) (also known as GR130) and the RXR variant QCIMFI (Q275C, I310M, and F313I) and (also known as GRQCIMFI). The OLRP were further developed into molecular switches to provide controlled gene expression and potentially benefit gene therapy applications by replacing the DNA binding domain (DBD) with a Gal4 DBD, a yeast transcription factor. Both molecular switches are able to bind Gal4 RE in response to LG335 and activate expression of a luciferase or GFP reporter gene in either a two- or one-component system. When characterizing the GR130 variant in the two-component system, no activation was observed with the natural ligand 9cRA, and the variant displayed a 19±5-fold activation and a 50 nM EC50 value in the presence of LG335. When the GRQCIMFI variant was evaluated in the two-component system, activation was observed in the presence of LG335 with a 10 nM EC50 value and a 6±2-fold induction, and 9cRA induced activation only at the highest concentration. The GRQCIMFI variant was also characterized with the one-component system containing the reporter gene GFP in a transient transfection as well as through retroviral transduction, displaying green fluorescence in 30% of the cells in the presence of 10 µM LG335. Several attempts were made to improve the molecular switch system. The VP16 activation domain was fused to GRQCIMFI in an effort to increase the fold induction; however, the addition of the VP16 created a constitutively active protein. Another approach to improve the molecular switch incorporated error-prone PCR to discover a new variant, Q275C, I310M, F313I, L455M (QCIMFILM), which displayed a 10-fold increase in sensitivity towards LG335 with a 5 nM EC50 value. Examination of the L455 position in the crystal structure of RXR revealed this residue is located outside of the ligand binding pocket on helix 12 (H12), but is able to significantly enhance receptor function. In fact, the single variant, L455M, was able to enhance receptor activation, compensate for a nonfunctional variant, as well as influence coactivator association. The long-term goal of this research is to develop a gene regulation system that would be used in human gene therapy trials. In the process of creating this system a deeper assessment of the nuclear receptor structure and function is made, which can be used for the enhancement and development of transcriptional regulation mechanisms.