• Refine Query
  • Source
  • Publication year
  • to
  • Language
  • 11
  • 5
  • 4
  • 1
  • Tagged with
  • 27
  • 6
  • 6
  • 5
  • 5
  • 5
  • 5
  • 4
  • 4
  • 4
  • 4
  • 4
  • 3
  • 3
  • 3
  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Investigating the Function and Therapeutic Potential of the GCN5b Bromodomain in Toxoplasma Gondii

Hanquier, Jocelyne Nicole 06 1900 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / The obligate intracellular protozoan parasite Toxoplasma gondii is a medically relevant pathogen that has infected a third of the world’s population. Toxoplasma is the causative agent of toxoplasmosis, which can have severe repercussions such as encephalitis and even death in immunocompromised patients. Current treatments for toxoplasmosis only target acute infection and can be toxic to patients, resulting in complications including allergy and bone marrow suppression. Thus, the identification of novel drug targets and therapies for toxoplasmosis is vital. Epigenetic modulators of lysine acetylation, including ‘writers,’ ‘erasers,’ and ‘readers,’ have been identified as promising drug targets for protozoan parasites. The lysine acetyltransferase (KAT) GCN5b appears to be an essential gene for Toxoplasma viability. The KAT domain of GCN5b is essential to GCN5b function and is targetable by small molecule inhibitors. While the acetyltransferase activity of this gene is well-characterized, the functionality of its C-terminal bromodomain (BRD) remains to be understood. Bromodomains are readers of lysine acetylation, and recently, bromodomain inhibitors have shown promise in a number of human diseases, as well as in protozoan parasites. We hypothesized that the GCN5b bromodomain is critical for Toxoplasma viability. The data reported herein suggest that the GCN5b bromodomain is important for tachyzoite viability and may serve as a novel therapeutic target in Toxoplasma.
2

Transcriptional regulation and chromatin remodeling mechanisms at PHO5

Carvin, Christopher Dumas 29 August 2005 (has links)
Regulation of gene expression is vital for proper growth and prevention of disease states. In eukaryotes this regulation occurs in the context of chromatin which creates an inherent barrier for the binding of trans-acting factors, such as transcription factors and RNA polymerase. This dissertation focuses on the role of transcriptional activators and chromatin remodeling coactivators in the regulation of the repressible acid phosphatase gene PHO5. Our studies show that histone methylation at lysine 4 of histone H3 is required for the full repression of PHO5and GAL1-10. We show that bromodomains, a domain conserved in chromatin remodeling coactivators, may function to stabilize binding. Finally, we present a strategy using DNA methyltransferases as in vivo probes to detect DNA-protein interactions and examine chromatin structure. We extend this strategy to zinc-finger proteins which can be engineered to bind to any desired DNA sequence as a means of targeting methylation with potential use in epigenetic silencing.
3

Transcriptional regulation and chromatin remodeling mechanisms at PHO5

Carvin, Christopher Dumas 29 August 2005 (has links)
Regulation of gene expression is vital for proper growth and prevention of disease states. In eukaryotes this regulation occurs in the context of chromatin which creates an inherent barrier for the binding of trans-acting factors, such as transcription factors and RNA polymerase. This dissertation focuses on the role of transcriptional activators and chromatin remodeling coactivators in the regulation of the repressible acid phosphatase gene PHO5. Our studies show that histone methylation at lysine 4 of histone H3 is required for the full repression of PHO5and GAL1-10. We show that bromodomains, a domain conserved in chromatin remodeling coactivators, may function to stabilize binding. Finally, we present a strategy using DNA methyltransferases as in vivo probes to detect DNA-protein interactions and examine chromatin structure. We extend this strategy to zinc-finger proteins which can be engineered to bind to any desired DNA sequence as a means of targeting methylation with potential use in epigenetic silencing.
4

Etude fonctionnelle et inhibition de la protéine Bdf1 chez Candida albicans / Functional role and inhibition of Bdf1 protein in Candida albicans

Champleboux, Morgane 04 November 2016 (has links)
Candida albicans représente la première cause d’infection fongique chez l’Homme. Chez les patients immunodéficients, ce pathogène est extrêmement virulent et le nombre de décès suite à une infection systémique par Candida atteint 200 000 chaque année dans le monde. Quatre classes de médicaments antifongiques existent mais les traitements ont un coût élevé et des problèmes récurrents de résistance. Il y a donc un besoin urgent de trouver de nouvelles options thérapeutiques.Notre projet explore une nouvelle cible potentielle de la famille des protéines BET (Bromo and extra-terminal) pour traiter les infections fongiques. Nous étudions la protéine BET de levure, bromodomain factor 1 (Bdf1), impliquée dans la régulation de la transcription. Afin de caractériser son rôle fonctionnel, j’ai combiné différentes approches en biochimie, protéomique et transcriptomique. J’ai démontré que Bdf1 et ses deux bromodomaines, Bd1 et Bd2, sont essentiels pour la croissance et la survie de C. albicans.La seconde partie de ma thèse s'inscrit dans un projet de recherche d'inhibiteurs des bromodomaines de Bdf1 chez C. albicans, inspiré par la découverte de composés anti-cancéreux ciblant les bromodomaines mammifères. Grâce à un criblage haut débit, nous avons identifié des inhibiteurs sélectifs des bromodomaines de Bdf1. Ils agissent comme des molécules antifongiques et inhibent la croissance de C. albicans.Ces découvertes indiquent que les bromodomaines de Bdf1 sont une nouvelle cible pour le développement de nouveaux traitements antifongiques. Leurs inhibitions pourraient représenter une stratégie thérapeutique innovante pour traiter les patients infectés par C. albicans. / C. albicans is the most prevalent human fungal pathogen. In immunocompromised patients, this pathogen is highly virulent and the number of deaths because of systemic infections by C. albicans reaches 200.000 per year worldwide. The limited number, high cost and toxicity of currently available antifungal drugs indicate that new therapeutic agents against C. albicans are urgently needed.We propose to investigate a novel target of BET (Bromo and extra-terminal) proteins family to treat fungal infections. We are interested in the yeast BET protein named bromodomain factor 1 (Bdf1), involved in transcription regulation. To characterize its functional role, I combined several approaches in biochemistry, proteomic and transcriptomic. I discovered that Bdf1 and its two bromodomains Bd1 and Bd2 are essentials for the growth of C. albicans.The second part of my thesis is involved in a research project that aims to identify Bdf1 bromodomains inhibitors in C. albicans, inspired by recently discovered anti-cancer compounds that target mammal bromodomains. High-throughput chemical screens have identified selective Bdf1 bromodomain inhibitors. They act as antifungal compounds and inhibit the growth of C. albicans.Altogether, these discoveries indicate that Bdf1 bromodomains are a valid antifungal target. Hopefully, their inhibition represents a new and innovative therapeutic strategy to treat patients infected with C. albicans
5

Chemical Probes and the Exploration of Bromodomains in Cancer Biology

McKeown, Michael Robert 04 June 2016 (has links)
The post-translational modification of histones and their interaction with transcription factors is essential to gene regulation. Furthermore, these targets would greatly benefit from probe molecules to fully elucidate their biological actions and to potentially lead to therapeutics. However, these protein-protein interactions have been considered difficult to inhibit and few high-quality chemical probes currently exist for the study of epigenetic biological systems in particular.
6

Evaluation of a strategy based on multi-drug targeting of cancer proteins in breast cancer cell lines

Nortje, Evangeline January 2020 (has links)
Therapeutic inefficacy of conventional cancer treatment is a particular dilemma associated with metastatic triple negative breast cancer (TNBC), with patients still facing poor prognosis. The design and development of novel anticancer agents specifically targeted to cancer-associated pathways is of therapeutic interest. The rationale is twofold: firstly, targeted therapy overcomes widespread toxicity and adverse effects of conventional chemotherapy due to the selectivity of the treatment modality. Secondly, synergistic combinations of different classes of highly targeted therapies could hold therapeutic promise to overcome resistance by simultaneously circumventing multiple cancer hallmarks. This study evaluates the in vitro antiproliferative activity of six compounds using breast cancer cell lines as experimental model. Five of these compounds are novel, agents designed in silico to selectively target cancer hallmarks via inhibition of specific cancer-associated proteins. The compounds include an antimitotic (STX1972), three variants of bromodomain 4 (BRD4) inhibitors (Bzt-W41, Bzt-W49 and Bzt-W52), an inhibitor of both sirtuin (SIRT) 1 and 2 (W137) and an inhibitor of janus kinases 1 and 2 (Ruxolitinib). The synergism between paired combinations was also explored. Two breast cancer cell lines, MDA-MB-231 and MCF-7 were used as experimental models. The MDA-MB-231 cell line is oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) negative and is therefore commonly used to model triple negative breast cancer with invasive and metastatic properties. MCF-7 cells are ER and PR positive and represent the hormone-dependent breast cancer model. The endothelial EA.hy926 cell line was used to represent non-cancerous cells. A crystal violet assay was used to determine the half maximal inhibitory concentration (IC50) of the six compounds on the tested cell lines after 48 h exposure. Drug combination studies based on the Chou-Talalay method of paired drug combinations were performed. Effects of treatment on cell morphology was assessed by means of confocal-microscopy. Flow cytometry was used to study the effects on cell cycle progression, apoptosis, autophagy/lysosomal activity, reactive oxygen species (ROS) production, changes in mitochondrial membrane potential (ΔΨm) and the serine 70 phosphorylation status of Bcl-2. Real-time quantitative PCR was used to analyse the effects of the compounds on the mRNA expression levels of p53, c-myc and bcl-2. Quantitative protein expression of c-MYC was analysed by means of enzyme-linked immunosorbent assay. In vitro screening for antiproliferative activity revealed that the compounds showed cancer-selective cytotoxic effects when compared to the EA.hy926 control cell line. The initial screening identified three compounds for further investigative inclusion, namely the antimitotic (STX1972), the BRD4i (Bzt-W41) and the SIRTi (W137). STX1972 was found to inhibit cell growth in the nanomolar concentration range, whilst the rest of the compounds showed growth inhibition in micromolar concentration ranges. Bzt-W41 showed significant preferential selectivity for the TNBC MDA-MB-231 cell line versus the hormone-dependent MCF-7 cell line, while STX1972 and W137 exhibited only slight differential selectivity. Two combinations (STX + Bzt-W41 and Bzt-W41 + W137) exhibited synergism, whilst the STX + W137 combination exhibited antagonistic interaction. Cell cycle and apoptosis analysis revealed that STX1972 and Bzt-W41, alone and in combination, selectively induced cell cycle arrest and apoptosis in cancer cells. However, the W137 +Bzt-W137 combination did not show preferential targeting of breast cancer cell lines, with apoptosis induced equally or even more so in the control EA.hy926 cell line. STX1972 and Bzt-W41, as well as their paired combination, was further probed in aim of deciphering their individual and combined mode of action. STX1972, Bzt-W41 as well as the paired combination proved to selectively inhibit cancer targets resulting in several molecular changes, leading to downstream pathway activation which culminates in both apoptotic and autophagy-related cellular demise. The study contributed towards deducing possible hypotheses regarding the mechanistic behaviours of the individual compounds and elucidated their combined effect during dual treatment. Results warrant future studies to further probe the intricate interaction of pathways involved in the synergistic combination of antimitotics and epigenetic regulators as a novel anticancer therapeutic modality. / Thesis (PhD)--University of Pretoria, 2020. / Physiology / PhD / Unrestricted
7

The Bromodomain Proteins GTE9 and GTE11 Associate with BT2-based E3 Ligase Complex and Mediate Responses to Multiple Signals in Arabidopsis thaliana.

Misra, Anjali 2011 December 1900 (has links)
BT2 is an Arabidopsis thaliana protein with N-terminal BTB, central TAZ and a C-terminal calmodulin binding domain and associates with Cullin3 to form an E3 ubiquitin ligase. We have shown previously that BT2 regulates telomerase activity in mature vegetative organs and controls a variety of hormone, stress and metabolic responses in Arabidopsis thaliana. Loss of BT2 results in plants that are hypersensitive to inhibition of germination by ABA and sugars. Conversely, overexpression of BT2 results in resistance to ABA and sugars, suggesting that BT2 is a negative regulator of ABA and sugar responses. Here, we report the roles of BT2-interacting partners GTE9 and GTE11, bromodomain and extraterminal-domain proteins of Global Transcription Factor Group E, in BT2-mediated responses to sugars and hormones. Loss-of-function mutants gte9-1 and gte11-1 phenocopy the bt2-1-null mutant responses; germination in all three mutants is hypersensitive to inhibition by glucose and ABA. Loss of either GTE9 or GTE11 in a BT2 over-expressing background blocks resistance to sugars and ABA, indicating that both GTE9 and GTE11 are required for BT2 function. Additionally, loss of GTE9 or GTE11, similar to loss of BT2, suppresses transcriptional gene activation mediated by CaMV 35S enhancers in Arabidopsis. The suppressed phenotype was accompanied by decreased transcription and hypermethylation of the 35S enhancers in the activation-tagged lines. We showed that BT2 and GTE9 co-immunoprecipitate and physically interact in vivo to mediate diverse responses to biotic and abiotic signals and 35S enhancer activity. Our working model is that the GTE9 and GTE11 function as chromatin adaptors that localize the BT2-CULLIN3 E3 ubiquitin ligase complex to acetylated chromatin on transcriptionally competent promoters in response to calcium signals detected by BT2’s calmodulin-binding domain.
8

Small hepatitis Delta antigen mimics a histone H3 epitope to facilitate the remodeling of the Hepatitis D virus (HDV) viral ribonucleoprotein / La petite protéine du virus de l’hépatite Delta (HDV) imite un épitope de l’histone H3 pour faciliter le remodelage de la ribonucléoprotéine virale pour la réplication de l’ARN viral

Abeywickrama Samarakoon, Natali 20 October 2016 (has links)
Le virus de l'hépatite Delta (HDV) est un agent infectieux transmissible satellite du virus de l'hépatite B (HBV), induisant des maladies du foie plus sévères que la mono–infection par le HBV. Aucun traitement totalement efficace n'est disponible contre l'HDV et les 15 millions de personnes infectées par le HDV dans le monde sont exposées a un risque élevé de cirrhose et de carcinome hépatocellulaire. HDV est un virus unique qui ne code pas pour une polymérase virale contrairement aux autres virus a ARN. La réplication de l'ARN HDV s'effectue par un double mécanisme de cercle roulant générant des brins d'ARN de longueur génomique ou antigénomiques unitaires. La synthèse de l'ARN génomique est sensible à de faibles concentrations d'alpha–amanitine, ce qui suggère qu'elle soit médiée par l'ARN polymérase II (ARN Pol II) classiquement ADN dépendante. Ce processus repose sur la petite protéine du HDV (S–HDAg), qui doit être acétylée sur l'acide amine K72 pour activer la synthèse de l'ARN génomique. Nous avons récemment identifié la protéine BAZ2B (Bromodomain Associated Zinc finger protein 2B) comme un interactant majeur de S–HDAg par capture par affinité, couplée à la spectrométrie de masse à partir de l'expression de S– HDAg étiqueté par un double motif Strep–TagR dans les cellules HepaRG différentiées. La fonction biologique de BAZ2B est inconnue. Cependant, en comparant avec des protéines apparentées BAZ (BAZ–1A/1B/2A), on postule que BAZ2B représente la sous–unité accessoire d'un nouveau complexe de remodelage de chromatine de type ISWI, qui régule le positionnement des nucléosomes par hydrolyse de l'ATP. Des études récentes ont révélé que le bromodomaine de BAZ2B (BRD) reconnait la signature épigénétique spécifique K14ac–X–X–R sur l'histone H3. Cela pourrait impliquer le mode d'action du complexe de remodelage de la chromatine dont BAZ2B représente l'unité régulatrice reconnaissant des marques spécifiques d'acétylation des histones propagées séquentiellement modifiant la dynamique de la chromatine et favorisant le recrutement de l'ARN Pol II pour activer la transcription. Nous émettons l'hypothèse que l'acétylation, médiée par p300, du motif K72–X–X–R conserve dans les S–HDAg interagissant avec l'ARN antigénomique pseudo double brin, mimerait l'acétylation des histone H3 en K14 permettant de recruter le complexe de remodelage de la chromatine BAZ2B associée et de lancer la réplication HDV. Brièvement, pour confirmer la pertinence fonctionnelle du recrutement BAZ2B pour la réplication HDV, nous avons transfecté des lignées cellulaire Huh–7 exprimant de façon stable, soit la protéine sauvage S–HDAg ou le mutant R75A pour étudier la réplication HDV à partir plasmide pSVLD2m défectif pour l'expression de S–HDAg. Nos résultats indiquent que la synthèse de l'ARN génomique est fortement réduite dans les cellules exprimant le mutant R75A S–HDAg par rapport aux cellules exprimant le type sauvage S–HDAg, alors que la quantité d'ARN antigénomique est restée le même dans les deux cas. Des expériences de co–cristallisation et de siRNA sont actuellement menées afin de mieux caractériser au niveau moléculaire l'association entre BAZ2B BRD et des peptides dérivés de la séquence de S–HDAg et d'étudier les conséquences de l'inhibition par siRNA de BAZ2B. L'implication des BAZ2B dans la réplication de HDV pourra ouvrir des possibilités de développement de médicaments anti–HDV, basées sur l'optimisation des inhibiteurs émergents de BAZ2B–BRD / Hepatitis Delta Virus (HDV) is a satellite of Hepatitis B Virus (HBV), leading to more severe life threatening liver diseases than HBV mono–infection. No efficient therapy is available against HDV and the estimated 15 million HDV infected individuals worldwide are at a high risk of cirrhosis and hepatocellular carcinoma. HDV is a unique RNA virus as it does not encode a viral polymerase. HDV RNA replication occurs via a double rolling circle mechanism generating unit–length genomic or antigenomic RNA strands. The synthesis of the genomic RNA is sensitive to low concentrations of α–amanitin, suggesting that the RNA–dependent RNA synthesis is mediated by DNA–dependent RNA polymerase II (RNA Pol II). This process relies on the HDV encoded Small Hepatitis Delta antigen (S–HDAg), which must be acetylated at K72 to activate the synthesis of the genomic RNA. We recently identified BAZ2B (Bromodomain Associated to Zinc finger protein 2B) as a major interactant of S–HDAg by affinity capture coupled to mass spectrometry in differentiated HepaRG cells. The biological function of BAZ2B is however unknown. In comparison with related BAZ proteins (BAZ–1A/1B/2A), it is postulated that BAZ2B is the accessory subunit of a new chromatin remodeling complex of ISWI–type, which regulates nucleosome positioning through ATP hydrolysis. Recent studies revealed that the BAZ2B bromodomain (BRD) recognizes the distinct epigenetic signature K14ac–X–X–R on histone H3. This suggests that the mode of action of BAZ2B associated chromatin remodeling complex involves recognizing propagated specific histone acetylation marks to subsequently alter the chromatin dynamic and recruit the RNA Pol II for transcriptional activation. We hypothesized that the p300–mediated acetylation of the conserved K72–X–X–R motif in S–HDAg mimics acetylated histones on the pseudo–double stranded antigenomic RNA, to recruit the BAZ2B associated chromatin remodeling complex to initiate RNA Pol II mediated synthesis of HDV genome. To confirm the functional relevance of BAZ2B recruitment for HDV replication, we transfected Huh 7 cells stably expressing either wild–type S–HDAg or R75A mutant S–HDAg with the HDV replication defective plasmid pSVLD2m. Our results indicate that the synthesis of genomic RNA was greatly reduced in cells expressing the R75A mutant S–HDAg in comparison to cells expressing wild–type S–HDAg, whereas the amount of antigenomic RNA remained the same in both cases. Co–crystallization experiments are currently being carried out to better characterize at the molecular level the association between BAZ2B BRD and S–HDAg derived peptides. Furthermore, siRNA experiments directed against the BAZ2B gene are expected to reveal the consequences of BAZ2B inhibition on HDV viral replication. The involvement of BAZ2B in HDV replication may open anti–HDV drug development opportunities, based on the optimization of emerging BAZ2B–BRD inhibitors
9

Impact of BET bromodomain inhibition on KRAS-mutated non-small cell lung cancer

Klingbeil, Olaf 21 December 2016 (has links)
Nicht-kleinzelliger Lungenkrebs (NSCLC) hat bis heute einen hohen medizinischen Bedarf an effektiveren Therapien. Inhibitoren der Bromodomain and extra-terminal domain (BET) Familie wie JQ1 wirken in verschiedenen Krebsarten, einschließlich Lungenkrebs. Während ihre Aktivität auf die Expression von Onkogenen wie c-Myc in vielen Studien untersucht wurde, bleibt der Effekt von BET-Inhibition auf den Apoptose Signalweg weitgehend unbekannt. In dieser Arbeit wurde die Aktivität von BET-Inhibitoren auf den Zellzyklus und auf Komponenten der Apoptose-Antwort der Zelle untersucht. Genomweite Transkriptionsanalysen haben zusammen mit Chromatin Immunpräzipitation und anschließender Sequenzierung geholfen das MYC Gen und dessen assoziierte Super-enhancer als primäres Ziel des BET-Inhibitors JQ1 zu identifizieren. Mittels einer Gruppe von NSCLC Modellen belegt diese Arbeit, dass Zelllinien die auf die BET-Inhibitoren reagieren in Apoptose gehen und eine Reduktion der S-Phasen Population zusammen mit gleichzeitiger de-regulation der c-Myc Expression aufwiesen. Andererseits konnte die ektopische Überexpression von c-Myc der anti-proliferativen Wirkung entgegenwirken. Die Auswirkung von BET-Inhibition auf die Expression von 370 Genen, die in der Apoptose Regulation involviert sind, wurde in sensitiven und resistenten Zellen verglichen und dabei wurde die starke BET-Abhängigkeit der Expression von zwei Schlüsselgenen der Apoptose FLIP und XIAP festgestellt. Die Kombination von JQ1 mit dem tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) oder dem Chemotherapeutikums Cisplatin die verstärke die Induktion von Apoptose in sowohl BET-Inhibitor sensitiven als auch in resistenten Zellen. Des Weiteren zeigte die Kombination einen verbesserten Antitumor-Effekte in A549 tumortragenden Mäusen. Insgesamt zeigen diese Ergebnisse, dass die Identifizierung von BET-abhängigen Genen unterstützend für die Wahl von therapeutischen Kombinationspartnern in der Krebsbehandlung sein kann. / Non-small cell lung cancer (NSCLC) has a high medical need for more effective therapies. Small molecule inhibitors of the bromodomain and extra terminal domain (BET) family such as JQ1 are active in different cancer types, including lung cancer. While their activity on oncogene expression such as c-Myc has been addressed by many studies, the effects of BET inhibition on the apoptotic pathway remain largely unknown. This work evaluates the activity of BET bromodomain inhibitors on cell cycle distribution and on components of the apoptotic response. Genome-wide transcriptional analyses together with chromatin immunoprecipitation followed by sequencing helped to identify the MYC gene and associated super-enhancers as a primary target of JQ1. Using a panel KRAS-mutated NSCLC models, it was found that cell lines responsive to BET inhibitors underwent apoptosis and reduced their S-phase population, concomitant with down-regulation of c-Myc expression. Conversely, ectopic c-Myc overexpression rescued the anti-proliferative effect of JQ1. The effects of BET inhibition on the expression of 370 genes involved in apoptosis were compared in sensitive and resistant cells and the expression of the two key apoptosis regulators FLIP and XIAP was found to be highly BET-dependent. Consistent with this, combination treatment of JQ1 with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the pro-apoptotic chemotherapeutic agent cisplatin enhanced induction of apoptosis in both BET inhibitor sensitive and resistant cells. Furthermore the combination of JQ1 with cisplatin led to significantly improved anti-tumor efficacy in A549 tumor-bearing mice. Altogether these results show that the identification of BET-dependent genes provides guidance for the choice of drug combinations in cancer treatment.
10

Molecular Basis of Erythroid Cell Proliferation and Differentiation / Les bases moléculaires de la prolifération et de la différentiation érythroide

Penglong, Tipparat 20 April 2015 (has links)
Pour assurer la production de milliards de globules rouges, l’érythropoièse doit parfaitement contrôler les processus de prolifération et de différenciation. Ces deux processus sont régulés par l’expression de gènes spécifiques dépendant d’une coordination entre l’activité des facteurs de transcription (FT) et les fonctions épigénétiques portées par exemple par les protéines à bromodomaine. Cette étude se concentre sur les conséquences de l’association ou la dissociation du FT clef de l’érythropoièse GATA-1 avec les FT déterminant pour le cycle cellulaire, pRb et E2F. Dans la première partie de ma thèse, j’ai participé à l’étude du rôle de l’association/dissociation de GATA-1 et FOG-2 avec pRb/E2F dans le contrôle la balance prolifération/différenciation cellulaire. Nos résultats montrent que les souris exprimant une mutation de GATA-1 sur la sérine 310 (GATA-1S310A), qui a la capacité accrue à séquestrer E2F-2, présentent une anémie létale lorsqu’un mécanisme de compensation de production de E2F-2 induit par l’IGF-1 est inhibé. Puis, nous avons trouvé que les propriétés décrites pour GATA-1 sont partagées par le FT FOG-2 et montré que l’abrogation de sa fixation avec pRb induit une perturbation de l’adiposité dans des souris FOG-2pRb-. Dans la deuxième partie, l’expression de c-Myc étant régulé différentiellement par GATA-1 et E2F, j’ai testé si la drogue « JQ1 », premier inhibiteur épigenétique chimique de l’expression de c-Myc, pouvait contrôler l’érythropoièse. Pour cela, j’ai utilisé la ligné érythroleucémique UT7 qui prolifère sans se différencier en présence d’érythropoiétine (stade proérythroblaste). Les résultats montrent que le traitement par JQ1 bloque la prolifération des cellules UT7 et permet de réinitier le programme de différentiation érythroide terminale. J’ai alors recherché les mécanismes moléculaires impliqués dans cette régulation et trouvé que l’inhibition transcriptionnelle de c-Myc par JQ1 est associée à l’inhibition de l’activité transcriptionnelle de STAT5 sans modification de son état de phosphorylation. Enfin, j’ai montré que JQ1 pouvait avoir une activité comparable à celle du TGF-b mais sans implication les voies Smad. Des études in vivo montre que JQ1 augmente la viabilité cellulaire et accélère la maturation des cellules érythroides à la fois chez les souris sauvages et thalassémiques. Cette différence d’action de JQ1 sur l’érythropoièse normale et pathologique implique des modifications épigénétiques différentielles entre ces deux types cellulaires et sont à la base de nouvelles stratégies du traitement du cancer. Le rôle clef de la régulation de l’association/dissociation de GATA-1 ou FOG-2 avec pRb/E2F dans l’érythropoièse et l’adipogénèse, nous a conduit, dans une troisième partie, à déterminer in vivo, les conséquences physiologiques de la séquestration de E2F par pRb. Pour cela nous avons crée une souris transgénique exprimant de façon conditionnelle un peptide contenant la partie N terminale de GATA-1 qui se fixe à pRb (GATA-1Nter). In vitro, ce peptide séquestre E2F dans le complexe GATA-1Nter/pRb et inhibe la prolifération cellulaire de façon irréversible. In vivo, aucune souris transgéniques exprimant le peptide GATA-1Nter n’a pu être sélectionnée et une mortalité au stade embryonnaire est observée. Une expression induite de ce peptide au stade adulte ne produit que des souris chimériques avec une fréquence de recombinaison du transgène GATA-1Nter importante. L’établissement de lignées stables de souris exprimant le peptide GATA-1Nter permettra de déterminer les conséquences physiologiques de la séquestration de E2F dans le complexe GATA-1Nter/pRb. / To ensure the generation of billions of erythrocytes daily, erythropoiesis must be well controlled by proliferation and differentiation processes. These two processes are regulated by expressions of specific genes, coordinated by transcription factors (TFs) and epigenetic factors, such as bromodomain proteins. This study focused on the effects of the binding and dissociation of a key erythroid TF, GATA-1, to the crucial cell cycle TFs, pRb and E2F. In the first part of this thesis, the role of GATA-1 and FOG-2 binding to pRb/E2F in a control balances between cell proliferation and differentiation was studied. Mice bearing a GATA-1 mutation (GATA-1S310A) displayed higher levels of E2F2 sequestration and suffered from fatal anemia when the compensatory pathway of E2F2 production via IGF-1 signaling was also inhibited. The properties described for GATA-1 were found to be common to FOG-2, and the abolition of FOG-2 binding to pRb led to obesity resistance in FOG-2pRb- mice. In the second part of this work, as c-Myc is regulated by GATA-1 and E2F, the first chemical epigenetic inhibitor repressing c-Myc expression to be described, JQ1, was investigated to see if it could control erythropoiesis. The UT7 erythroleukemia cell line, which proliferates without differentiating was used. This cell line stops differentiation at the proerythroblast stage, in response to erythropoietin. JQ1 treatment inhibited UT7 proliferation and restored terminal erythroid differentiation. The molecular mechanism underlying this regulation by JQ1 was shown that the inhibition of c-Myc expression was associated with the inhibition of STAT5 transcription, with no change in the phosphorylation of this protein. It was found that JQ1 had a putative TGF--like activity, which did not involve the Smad pathway. It was shown in the ex vivo studies that JQ1 increased the viability of erythroid cells and accelerated the maturation of these cells in both WT and thalassemic mice. The observed differences between leukemic and normal erythropoiesis involved differential epigenetic modifications that could be at the basis of new strategies regarding cancer treatment.The key role of the association of GATA-1 or FOG-2 had with pRb/E2F, and the dissociation of these factors, in erythropoiesis and adipogenesis, respectively, led us to investigate, in vivo, the physiological consequences of E2F sequestration by pRb. As a result, transgenic mice displaying conditional expression of a peptide containing the N-terminal part of GATA-1 that binds to pRb (GATA-1Nter) were developed. In vitro, this peptide traps E2F in a GATA-1Nter/pRb complex, resulting in the irreversible inhibition of cell proliferation. The yield of transgenic mice expressing the GATA-1Nter peptide in vivo was unsuccessful, as this expression lead to lethality at the embryonic stage. Using an alternative approach, based on the inducible expression of the peptide in adults, chimeric mice with a high frequency of recombination of the GATA-1Nter transgene were obtained for this study. The establishment of a stable mouse line expressing the GATA-1Nter peptide should make it possible to determine the pathophysiological consequences of E2F sequestration in the GATA-1Nter/pRb complex.

Page generated in 0.4438 seconds