In vertebrates, the embryonic hindbrain is transiently subdivided along its anterior-posterior (A-P) axis into 8 well defined segments termed rhombomeres (r1-8). Each rhombomere represents a true cellular compartment in transcriptional profile, lineage restriction and neuronal organization. Thus, the vertebrate hindbrain provides a beautiful model for studying mechanisms of anterior-posterior patterning, signal transduction and interpretation, initiation and maintenance of transcriptional profiles, cell sorting and border formation. The Kreisler/MafB gene, which encodes a basic leucine zipper (bZIP) transcription factor that regulates some Hox genes, is one of the first genes to be expressed segmentally in the hindbrain, and is subject to a dynamic and complex regulatory process. However, unlike the Hox genes, Kreisler/MafB is not located within a large cluster of genes and therefore provides a simple system for dissecting the molecular mechanisms involved in hindbrain compartmentalization. In dissecting the mechanisms that govern Kreisler/MafB regulation, we have identified the S5 regulatory element that directs early MafB expression in the future r5-r6 domain. We have found a binding site within S5 that is specific for the Variant Hepatocyte Nuclear Factor 1 (vHNF1) to be essential, but not sufficient for early induction of r5-r6-specific expression. Thus, early inductive events that initiate MafB expression are clearly distinct from later acting ones that modulate its expression levels. Using mouse mutants, we have shown that MafB is dependent on the M33 polycomb protein and other mechanisms of chromatin remodeling. We then utilized transgenic flies and mice as well as binding assays to identify and validate a PcG/trxG response element (PRE), PRE1 which acts to reorganize the surrounding chromatin, regulating S5-dependent expression. To our knowledge, PRE1 is the first validated vertebrate PcG/trxG response element. Thus, PRE1 provides a springboard for further exploration of the mechanisms governing chromatin remodeling.

In vertebrates, the embryonic hindbrain is transiently subdivided along its anterior-posterior (A-P) axis into 8 well defined segments termed rhombomeres (r1-8). Each rhombomere represents a true cellular compartment in transcriptional profile, lineage restriction and neuronal organization. Thus, the vertebrate hindbrain provides a beautiful model for studying mechanisms of anterior-posterior patterning, signal transduction and interpretation, initiation and maintenance of transcriptional profiles, cell sorting and border formation. The Kreisler/MafB gene, which encodes a basic leucine zipper (bZIP) transcription factor that regulates some Hox genes, is one of the first genes to be expressed segmentally in the hindbrain, and is subject to a dynamic and complex regulatory process. However, unlike the Hox genes, Kreisler/MafB is not located within a large cluster of genes and therefore provides a simple system for dissecting the molecular mechanisms involved in hindbrain compartmentalization. In dissecting the mechanisms that govern Kreisler/MafB regulation, we have identified the S5 regulatory element that directs early MafB expression in the future r5-r6 domain. We have found a binding site within S5 that is specific for the Variant Hepatocyte Nuclear Factor 1 (vHNF1) to be essential, but not sufficient for early induction of r5-r6-specific expression. Thus, early inductive events that initiate MafB expression are clearly distinct from later acting ones that modulate its expression levels. Using mouse mutants, we have shown that MafB is dependent on the M33 polycomb protein and other mechanisms of chromatin remodeling. We then utilized transgenic flies and mice as well as binding assays to identify and validate a PcG/trxG response element (PRE), PRE1 which acts to reorganize the surrounding chromatin, regulating S5-dependent expression. To our knowledge, PRE1 is the first validated vertebrate PcG/trxG response element. Thus, PRE1 provides a springboard for further exploration of the mechanisms governing chromatin remodeling.

Orientadores: Nilson Ivo Tonin Zanchin, Beatriz Gomes Guimaraes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T09:39:45Z (GMT). No. of bitstreams: 1 Scapin_SandraMaraNaressi_D.pdf: 11335048 bytes, checksum: 153f9eea9142fb7f3cb17de59a608da6 (MD5) Previous issue date: 2007 / Resumo: As GTPases da família Rab regulam o transporte intracelular de vesículas em eucariotos. Cada Rab atua em uma via de transporte específica e seu mecanismo de ação se dá através da realização de um ciclo de ligação e hidrólise de GTP. Neste trabalho, foi determinada a estrutura cristalográfica das formas inativa (ligada a GDP) e ativa (ligada a GppNHp) da GTPase Rab11b, um membro da subfamília Rab11 que está envolvida na reciclagem de proteínas dos endossomos para a membrana plasmática, no tráfego de vesículas da rede trans-Golgi para a membrana plasmática e na fagocitose. Os resultados foram confrontados com os dados estruturais da Rab11a descritos anteriormente. A Rab11b inativa cristalizou como um monômero, o que gera conflitos a respeito da formação de dímeros funcionais pela Rab11a. A Rab11b e a Rab11a ativas divergiram em relação à posição e à interação da serina 20, que é importante na hidrólise de GTP, mas apresentaram taxas hidrolíticas semelhantes in vitro. Visando uma investigação mais ampla da família Rab, a GTPase Rab21 também foi cristalizada, mas os cristais difrataram até 2.90 Å de resolução. Ensaios de desnaturação térmica revelaram que a Rab21 é estruturalmente mais instável do que a Rab11, talvez pela presença de cisteínas que estão susceptíveis à oxidação, contribuindo para a agregação e precipitação da proteína. A Rab11 é bastante estável, e possivelmente forma estruturas do tipo beta-amilóide em altas temperaturas. Este trabalho envolveu também o estudo funcional da interação entre a proteína TIP41 humana (TIPRL) e o fator de transcrição MafB. A TIPRL é uma proteína conservada que foi identificada como uma ativadora de MAP quinases enquanto sua homóloga em levedura foi caracterizada como um antagonista da via de sinalização da quinase TOR que regula o crescimento celular. A MafB está envolvida no controle transcricional em diversos processos de desenvolvimento, mas seus reguladores ainda não estão bem estabelecidos. A interação direta entre a TIPRL e a MafB inteira, ou seu domínio bZIP isolado, foi confirmada através de ensaios de ligação in vitro. As proteínas co-localizaram no núcleo de células HEK293 e nossos resultados preliminares mostram que a TIPRL inibe a atividade transcricional da MafB in vivo, embora apenas interfira na ligação in vitro do domínio bZIP da MafB ao seu DNA-alvo mediante a estabilização do complexo TIPRL-bZIP. A TIPRL pode, portanto, constituir um novo regulador da atividade de MafB / Abstract: GTPases of the Rab family are responsible for the intracellular transport of vesicles. Each family member acts on a specific transport pathway and their function is regulated by GTP binding and hydrolysis, cycling between inactive (GDP-bound) and active (GTP-bound) forms. In this work, we describe the crystal structure of inactive and active forms of the GTPase Rab11b, a member of the Rab11 subfamily which is involved in recycling of proteins from endosomes to the plasma membrane, in polarized transport in epithelial cells, in the transport of molecules of the trans-Golgi network to the plasma membrane and in phagocytosis. The Rab11b structure showed several differences from the Rab11a isoform previously described. Inactive Rab11b crystallized as a monomer, contradicting the hypothesis about functional dimers formed by Rab11a. Active Rab11b differ from Rab11a relative to the position of the serine 20 sidechain, which is involved in GTP hydrolysis, although both GTPases show similar GTP hydrolysis rates in vitro. In order to obtain structural information on Rab GTPases, Rab21 was also crystallized, but the crystals diffracted to a relatively low resolution (2.90 Å). Rab21 is a cysteine rich protein, showing a higher instability relative to Rab11b. Thermal unfolding followed by circular dicroism confirmed this hypothesis. Both Rab11b and Rab11a show a relatively high thermal stability and circular dicroism analysis indicate that they undergo conversion to structures rich in beta-strands upon thermal denaturation. This work includes also studies on the function of TIPRL in regard to its interaction with the transcription factor MafB. TIPRL is a conserved human protein identified as an activator of MAP kinases whereas its yeast counterpart Tip41 functions as an antagonist of the TOR kinase pathway. MafB is a large member of the Maf family of bZIP transcription factors controlling developmental processes in vertebrates. Regulation of MafB is critical, for example, during erythroid differentiation. A direct interaction between TIPRL and full length MafB and the bZIP domain of MafB was confirmed by in vitro interaction assays. TIPRL is localized throughout the whole cell and overlaps with MafB in the nucleus of HEK293 cells. Preliminary assays showed that TIPRL inhibits transcriptional activation mediated by MafB in HEK293 cells, although MafB shows a higher binding affinity to its target DNA relative to TIPRL in vitro. This evidence indicates that TIPRL may control MafB activity in vivo / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Les macrophages ont une contribution essentielle dans la bonne santé et la maladie. Comment les macrophages sont capables d'auto-renouvellement reste une question sans réponse. Au sein du laboratoire il a était démontré que les macrophages déficients pour MafB et c-Maf (Maf-DKO) ont la capacité de s'autorenouveller indéfiniment in vitro et ceci sans perdre leur identité de macrophages ni devenir cancéreux (Aziz et al. 2009). En utilisant les macrophages Maf-DKO comme outil d'étude de l'auto-renouvellement, nous avons pu identifier un réseau de genes qui permet l'auto-renouvellement des macrophages en absence de MafB. De plus nous montrons que des macrophages génétiquement non modifiés sont capables d'exprimer des genes du réseau d'auto-renouvellement des cellules souches embryoniques. Ce réseau d'auto-renouvellement est inhibé par MafB, qui peut-être sous exprimé in vivo. Les macrophages alvéolaires (MA) expriment constitutivement de faibles niveaux de MafB et c-Maf comme montré par Gautier et al. 2013. Les MA montrent une importante capacité d'auto-renouvellement, ils peuvent être amplifiés ex vivo. La surpression de MafB dans les MA in vitro et in vivo réduit la capacité d'auto-renouvellement de ces derniers. Nous avons finalement identifié GSK3 comme une cible pharmacologique pour l'inhibition de MafB dans les macrophages. Il a était montré que GSK3 tait nécessaire pour l'activation de MafB par phosphorylation directe. Nous avons montré que par inhibition de GSK3, les macrophages étaient capables s'auto-renouveler même s'ils exprimés de façon endogène/exogène MafB et c-Maf. / Macrophages contribute to essential functions in health and disease. Some macrophages are short lived but some macrophages are able to self-renew. However, in which manner macrophages are able to self-renew remains an open question. In our lab, we have demonstrated that macrophages deficient in MafB and c-Maf (Maf-DKO macrophages) can self-renew indefinitely in vitro, without neither loosing their macrophage identity nor becoming cancerous (Aziz et. al 2009).Using Maf-DKOs as a tool to study molecular mechanisms of self-renewal of macrophages, we have now been able to identify a network of genes, which allows macrophage self-renewal in the absence of MafB. We identified 25 genes, which affected only self-renewal. Additionally, we show that genetically unmodified macrophages are able to express self-renewal gene network. This self-renewal network is inhibited by MafB, which can be downregulated in vivo after mitogenic stimuli. Recently, Gautier et al., showed that Alveolar macrophages (AMs) constitutively express very low levels of MafB and c-Maf. We were able to demonstrate that AMs are able to self-renew in vitro and in vivo. Overexpression of MafB in AM in vitro and in vivo reduced the ability of AMs to self-renew. Additionally, we identified GSK3 as a pharmaceutical target for MafB regulation in macrophages. GSK3 has been shown to be required for Maf activation through direct phosphorylation. We showed that by inhibiting GSK3, macrophages were able to self-renew even if they were expressing endogenous or exogenous MafB and c-Maf.

La démonstration du rôle de la CDT (« Cytolethal Distending Toxin ») de Helicobacterhepaticus dans le développement de l’hépatocarcinome murin fait de cette toxine un candidatpertinent dans l'activation de processus pro-cancéreux. Comme la toxine CagA de Helicobacterpylori, la sous-unité active CdtB de la CDT pourrait être une oncoprotéine. Nous avons étudié lerôle de la CdtB des Hélicobacters dans l’inflammation et la carcinogenèse digestive via unestratégie lentivirale d’expression constitutive ou conditionnelle de la CdtB ou de son mutant pourl’activité DNase. Nous avons réalisé une étude du transcriptome et montré que la CdtB deH. hepaticus induisait une réponse inflammatoire en surexprimant des cytokines, chimiokines,peptides antimicrobiens et en activant la voie du NF-κB des cellules épithéliales. La CdtB réguleégalement l’expression et la localisation nucléaire du facteur de transcription et oncogène MafB.Ces résultats ont été confirmés pour la CdtB de Helicobacter pullorum. Des expériencesd'infection des cellules avec des souches sauvages et mutées pour la CDT (deH. hepaticus & H. pullorum) ont permis de valider les résultats obtenus et de les attribuer à laCdtB et notamment à son activité DNase. Nous avons aussi développé un nouveau modèle dexénogreffes de cellules épithéliales inductibles pour l’expression de la CdtB de H. hepaticus.Dans ce modèle, la CdtB, en plus de ses effets déjà connus, retarde la croissance tumorale,induit l’apoptose, la sénescence et la surexpression du marqueur nucléaire de prolifération,Ki-67, suggérant la survie cellulaire. L’ensemble de ces résultats fournit de nouveaux argumentsen faveur du potentiel oncogénique de la CDT. / The demonstration of the role of the Cytolethal Distending Toxin (CDT) of Helicobacter hepaticusin the development of hepatocarcinoma in mice, makes this toxin a relevant candidate in theactivation of precancerous processes. As in the case of the CagA toxin of Helicobacter pylori, theCdtB active subunit of CDT could be an oncoprotein. We studied the role of Helicobacter CdtB ininflammation and digestive carcinogenesis using a lentiviral strategy for constitutive or conditionalexpression of the CdtB subunit or its corresponding DNase mutant. We conducted a study of thetranscriptome and showed that CdtB induced an inflammatory response by overexpressingcytokines, chemokines, antimicrobial peptides and activating the NF-kB pathway in epithelialcells. The CdtB also regulated the expression and nuclear localization of the transcription factorand oncogene MafB. These results were confirmed for the CdtB of Helicobacter pullorum.Infection of cells with wild type strains and the corresponding CDT-mutant strains (of H. hepaticus& H. pullorum) were used to validate the results and to attribute the effects to the CdtB and, inparticular, to its DNase activity. We also developed a novel epithelial cell xenograft model toevaluate the inducible expression of H. hepaticus CdtB. In this model, the CdtB, in addition to itspreviously well-known effects, delayed tumor growth, induced apoptosis, senescence and theoverexpression of nuclear proliferation marker, Ki-67, suggesting cell survival. All of these resultsprovide new arguments in favor of the oncogenic potential of the CDT.

PIWI Proteine sind die zentralen Darsteller eines RNA-basierten Mechanismus, der die Mobilisierung transponierbarer Elemente im Genom unterdrückt, um genetische Stabilität zu gewährleisten. Demzufolge sind PIWI-Proteine für die langfristige Erhaltung verschiedener Stamzellpopulationen notwendig. Beispiele dafür sind verschiedene adulte somatische Stammzellen in Drosophila und die Stammzellen der Keimbahn aller bisher untersuchten Tierarten. Bei Säugetieren sind die beschriebenen Funktionen von PIWI Proteinen strikt auf die männliche Keimbahn beschränkt. Trotz Andeutungen auf eine Rolle von PIWI-Proteinen in somatischen Zellen von Säugetieren, wurde eine Funktion bisher nicht beschrieben. Ähnlich wie Stammzellen, können sich Makrophagen in verschiedenen Geweben selbst-erneuern, um ihre Populationen zu erhalten. Diese Selbsterneuerung beruht auf der geringen Expression der Transkriptionsfaktoren MafB und cMaf, was die Aktivierung eines stammzell-ähnliches Gen-Netzwerk, das die Proliferation vorantreibt. Makrophagen mit einer genetischen Deletion von MafB und cMaf (MafDKO-Makrophagen) oder Makrophagen mit natürlich niedriger Expression von MafB oder cMaf, wie z.B. alveoläre Makrophagen, weisen dementsprechend eine erweiterte Kapazität zur Selbsterneuerung auf. Wie haben festgestellt, dass eine kurze Isoform des Maus- Gens Piwil2, die wir ‚Piwito’ genannt haben, in MafDKO und alveolären Makrophagen exprimiert wird. Die Expression von Piwito ist für die normale Selbsterneuerung der untersuchten Makrophagen notwendig, wie die in vitro und in vivo Untersuchungen darlegen. Eine Abwesenheit von Piwito in alveolären Makrophagen führt zu einer Verkürzung derer Lebenspanne in Kultur. Außerdem beweisen wir, dass Piwito von MafB in nicht-proliferierenden Makrophagen gebunden und unterdrückt wird. Diese Studie ist somit der erste Bericht über eine somatische Funktion von PIWI-Proteinen in nicht transformierten Zellen von Säugetieren. / PIWI proteins are the main players of an RNA-based gene regulatory machinery that represses transposable elements in the genome to prevent their mobilization and ensure genetic stability. PIWI proteins have thus highly conserved stem-cell functions. They are indispensable for the long-term maintenance of the somatic stem cells that drive regeneration in invertebrates, of various adult somatic stem cells in Drosophila and, most prominently, of the germline of all species studied so far. In mammals, their described functions are strictly restricted to the male germline. Despite suggestive observations for a role of PIWI proteins in the mammalian soma, robust evidence remains absent. Similar to stem cells, tissue macrophages can locally self-renew to maintain their populations. Mechanistically, their self-renewal relies on low expression of the macrophage transcription factors MafB and cMaf, since it allows the induction of a stem cell-like network of genes that drives proliferation. Macrophages with a genetic deletion of MafB and cMaf (MafDKO macrophages) acquire therefore the capacity to self-renew, defined by an indefinite growth in culture that does not comprise their identity and does not involve cancerogenic transformation. Similarly, macrophages with naturally low levels of MafB or cMaf, such as alveolar macrophages, display an extended self-renewal capacity in vivo and in vitro. We have found that a short isoform of the murine Piwil2 gene, that we named ‘Piwito’, is expressed in MafDKO and alveolar macrophages. Piwito expression is necessary for the unaltered self-renewal of macrophages, as shown by in vitro and in vivo assays. To highlight is the fact that Piwito deficiency limits the extended lifespan of alveolar macrophages in culture. Additionally, we show that Piwito is bound and repressed by MafB in quiescent macrophages. This study thus represents the first report of a somatic function for mammalian PIWI proteins in non-transformed cells.

The present COVID-19 pandemic has revealed that several characteristics render patients especially prone to developing severe COVID-19 disease, i.e., the male sex, obesity, and old age. An explanation for the observed pattern of vulnerability has been proposed which is based on the concept of low sensitivity of the TLR7-signaling pathway at the time of infection as a common denominator of vulnerable patient groups. We will discuss whether the concept of established TLR-tolerance in macrophages and dendritic cells of the obese and elderly prior to infection can explain not only the vulnerability of these two demographic groups towards development of a severe infection with SARS-CoV-2, but also the observed cytokine response in these vulnerable patients, which is skewed towards pro-inflammatory cytokines with a missing interferon signature.

The inner ear, the sensory organ responsible for hearing and balance, contains specialized sensory and non-sensory epithelia arranged in a highly complex threedimensional structure. To achieve this complexity, a tight coordination between morphogenesis and cell fate specification is essential during otic development. Tisúes surrounding the otic primordium, and more particularly the adjacent segmented hindbrain, have been implicated in specifying structures along the anteroposterior and dorsoventral axes of the inner ear. In this work we have first characterized the generation and axial specification of the otic neurogenic domain, and second, we have investigated the effects of the mutation of kreisler/MafB -a gene transiently expressed in the rhombomeres 5 and 6 of the developing hindbrain- in early otic patterning and cell specification. We show that kr/kr embryos display an expansion of the otic neurogenic domain, due to defects in otic patterning. Although many reports have pointed to the role of FGF3 in otic regionalization, we provide evidence that FGF3 is not sufficient to govern this process. Neither Krox20 nor Fgf3 null mutant embryos, in which Fgf3 is either downregulated or absent in r5 and r6, present ectopic otic neuroblasts in the otic primordium. However, Fgf3-/-Fgf10-/- double mutants show a phenotype very similar to kr/kr embryos: they present ectopic neuroblasts along the AP and DV otic axes. Finally, and remarkably, partial rescue of the kr/kr phenotype is obtained when Fgf3 or Fgf10 are ectopically expressed in the hindbrain of kr/kr embryos. These results highlight a compensatory mechanism between FGFs, and the importance of hindbrain-derived signals in instructing otic patterning and the establishment of the neurogenic domain.