Dissertations / Theses on the topic 'MEF2D'

Myocyte enhancer factor 2B (MEF2B) is a transcription factor with somatic mutation hotspots at K4, Y69 and D83 in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma. The recurrence of these mutations indicates that they may drive lymphoma development. However, inferring the mechanisms by which they may drive lymphoma development was complicated by our limited understanding of MEF2B’s normal functions. To expand our understanding of the cellular activities of wildtype and mutant MEF2B, I developed and addressed two hypotheses: (1) identifying genes regulated by wildtype MEF2B will allow identification of cellular phenotypes affected by MEF2B activity and (2) contrasting the DNA binding sites, effects on gene expression and effects on cellular phenotypes of mutant and wildtype MEF2B will indicate mechanisms through which MEF2B mutations may contribute to lymphoma development. To address these hypotheses, I first identified genome-wide MEF2B binding sites and transcriptome-wide gene expression changes mediated by MEF2B. Using these data I identified and validated novel MEF2B target genes. I found that target genes of MEF2B included the cancer genes MYC, TGFB1, CARD11, NDRG1, RHOB, BCL2 and JUN. The identification of target genes led to findings that MEF2B promotes expression of mesenchymal markers, promotes HEK293A cell migration, and inhibits DLBCL cell chemotaxis. I then investigated how K4E, Y69H and D83V mutations change MEF2B’s activity. I found that K4E, Y69H and D83V mutations decreased MEF2B’s capacity to promote gene expression in both HEK293A and DLBCL cells. These mutations also reduced MEF2B’s capacity to alter HEK293A and DLBCL cell movement. Overall, these data support the concept that MEF2B mutations may promote lymphoma development by reducing expression of MEF2B target genes that would otherwise function to help confine germinal centre B-cells to germinal centres. My research demonstrates how observations from genome-scale data can aid in the functional characterization of candidate driver mutations. Moreover, my work provides a unique resource for exploring the role of MEF2B in cell biology. I map for the first time the MEF2B regulome, demonstrating connections between a relatively understudied transcription factor and genes significant to oncogenesis.
Science, Faculty of
Graduate

The MEF2 family of transcription factors (MEF2A, MEF2C, and MEF2D) are crucial during skeletal muscle differentiation. Although the roles of MEF2D isoforms are well established, the roles of MEF2A and MEF2C are not as well understood. This thesis, we investigated the expression, localization, and function of MEF2A and MEF2C, using specific antibodies. While MEF2A is expressed in both proliferating and differentiated myoblasts, protein levels of MEF2C were only detected during differentiation. During early stages of differentiation MEF2A is expressed in both the cytoplasm and the nucleus. However during later stages of differentiation, it is localized predominately in the nucleus. MEF2C appears to be localized differently depending on which isoform is being investigated. Using an affinity purification and mass spectrometry based approach we identified PRMT1 as a unique interacting protein with MEF2A during skeletal muscle differentiation. PRMT1 is a protein arginine methyltransferase which mediates the addition of methyl groups onto various proteins including histone H4 arginine 3 (H3R4) which is associated with gene activation. Both MEF2A and PRMT1 occupy genomic targets of MEF2A. Inhibition of PRMT1 with a specific inhibitor delays C2C12 myoblast differentiation in the early stages of differentiation but no effect was observed during late stage differentiation. The MEF2 family of transcription factors show distinct but overlapping function during skeletal muscle differentiation.

Articular cartilage is a highly specialised tissue composed of a mechanically competent matrix and a single cell population - the chondrocytes. The maintaining of a specialised phenotype requires the integration of intracellular signalling, that in response to appropriate extracellular stimuli, results in expression of cell-specific genes. Previous work in our laboratory has identified hypoxia as one such key stimulus, which through HIF-2α, enhances expression of cartilage master regulator SOX9 and its matrix-encoding target genes (COL2A1, AGC and COL9A1). MEF2C transcription factor is known to be involved in muscle and cardiovascular development; however, recently it has been shown to play a key role in chondrocyte hypertrophy co-ordinately with SOX9. In a previous microarray analysis, we found that MEF2C was upregulated during hypoxia-induced re-dedifferentiation of human articular chondrocytes (HACs); interestingly where its suggested genetic target - COL10A1 - was barely detectable. In this research we therefore investigated a possible new and unknown function of MEF2C transcription factor as a potential genetic regulator of the permanent articular chondrocyte phenotype. In this study, MEF2C protein was detected with a nuclear localisation in chondrocytes in situ in intact healthy human articular cartilage. Experiments in isolated HACs revealed that, at both gene and protein levels, hypoxia enhances MEF2C expression in a HIF-2α and SOX9 dependent fashion. Subsequently, depletion experiments of MEF2C indicated that it is required for SOX9 gene expression both in normoxia and hypoxia. Our results, therefore suggest a mutual positive regulation between MEF2C and SOX9 transcription factors in articular cartilage. Thus, based on our studies a new and critical function for transcription factor MEF2C in HACs has been identified, where it helps promote expression of the differentiated chondrocyte phenotype through mutual regulation with SOX9. These findings give important new insights into our understanding of the transcription factor networks that regulate expression of the articular chondrocyte phenotype.

Muscle differentiation is a complex process involving the transition from undifferentiated mesoderm to a final functional musculature. Mef2 is an essential positive regulator central to the co-ordination of this process. It targets a plethora of key genes both early and late in the differentiation program and its activity must be tightly controlled (Pothoff and Olson, 2007). The aim of my research was to investigate the role of Mef2 in orchestrating Drosophila muscle differentiation. I did this by analysing the formation of the larval somatic musculature under conditions that either increased or decreased Mef2 activity using gain and loss-of -function of either Mef2 itself, Him, a repressor of Mef2 activity (Liotta et al, 2007) or of Zfhl, a potential regulator of Mef2 expression (Postigo et al, 1999). Part of this investigation involved the generation and characterisation of Mef2 dominant negative proteins and isolation of a Him mutant. Detailed analysis revealed a distinct subset of somatic muscles that are missing when Mef2 activity is reduced and another subset of muscles that are duplicated when Mef2 activity is increased. This suggests a role for Mef2 in patterning of the musculature that has not been established previously. In addition, I identified a role for Mef2 in the regulation of Him expression, revealing a mechanism whereby Mef2 could be involved in its own repression. I also investigated the role of mesol8E in muscle differentiation a previously uncharacterised novel gene identified as an early target of Mef2 (Taylor, 2000). I found this to be a Myb-like domain containing protein that is a direct target of Mef2. Over-expression caused a severe disruption to the somatic musculature, revealing a potential role for mesol8E in muscle guidance. Generation of mesol8E mutant alleles by FRT element mediated recombination showed the gene to be essential for development.

The Myocyte Enhancing Factor 2 (MEF2) family of proteins have been implicated in a wide variety of cellular mechanisms including: muscle and neuronal differentiation, inhibition of apoptosis, upregulation of c jun expression, and embryonic and post-natal cardiac development. In the course of my research I have identified a novel MEF2-responsive gene referred to as h&barbelow;uman D&barbelow;NA R&barbelow;eplication Related E&barbelow;lement F&barbelow;actor (hDREF). Three putative MEF2 consensus-binding sites have been found within the two untranslated regions (UTRs) of the hDREF sequence (which lie 5' and 3' to the open reading frame). Using CHromatin I&barbelow;mmunolP&barbelow;recipitation (CHIP) assays I have shown that MEF2 proteins associate with the MEF2 binding site found within the 5'UTR of hDREF under both growth and differentiation conditions. Furthermore, mutation of the MEF2 binding sites results in a failure to repress expression, which indicated that MEF2 acts to negatively regulate hDREF expression during differentiation. Endogenous expression studies indicate that hDREF is highly expressed during growth and downregulated during differentiation. Functional studies suggest that hDREF is directly involved in regulating DNA synthesis, and that overexpression of hDREF leads to aberrant DNA replication in post-mitotic cells leading to polyploidy. Based on my research I propose that hDREF represents a novel MEF2 regulated gene, and that the primary function of hDREF is to ensure DNA synthesis with the cell cycle.

I peptidi sono sempre più impiegati come agenti terapeutici per diverse malattie. Possiedono caratteristiche intermedie tra i farmaci chimici e biologici come dimensioni inferiori, sintetizzabili chimicamente, facilmente modificabili, con bassa tossicità, antigenicità ridotta, affinità di legame e specificità. Queste caratteristiche uniche li rendono una modalità attraente per lo sviluppo di inibitori di interazioni proteina-proteina. Lo scopo del progetto è sviluppare nuovi inibitori a base di peptidi in grado di bloccare l’interazione del fattore di trascrizione “myocyte enhancer 2” (MEF2) con alcuni membri della classe IIa delle istone deacetilasi (HDAC), una sottofamiglia di quattro proteine (HDAC4, HDAC5, HDAC7 e HDAC9) che sono state collegate ad una varietà di tumori solidi ed ematologici. A tal fine sono stati prodotti una serie di peptidi HDAC della classe II impiegando un sistema automatizzato di sintesi peptidica su fase solida, purificati con HPLC su fase inversa e caratterizzati via spettroscopia di massa e dicroismo circolare. L’affinità di legame dei peptidi verso MEF2 è stata testata con saggi di Polarizzazione di Fluorescenza. Infine l’affinità di legame di un peptide è stata incrementata inserendo una struttura sintetica intramolecolare (un braccio), che aiuta a bloccare il peptide in una conformazione specifica, in modo tale da ridurre l’entropia conformazionale.

Le métabolisme cellulaire est la source principale d’énergie et les cellules cancéreuses ont un métabolisme différent des cellules non transformées. La cellule tumorale a tendance à éviter l’activité mitochondriale et ainsi la phosphorylation oxydative, pour lui préférer la voie de la glycolyse pour la production d’énergie (Effet Warburg). Cette altération du métabolisme est si bénéfique pour les cellules en croissance que cela favorise la croissance tumorale et supprime la réponse immunitaire anticancéreuse. La spécificité de ce métabolisme en fait une cible intéressante pour le développement de thérapies anticancéreuses. Mon travail de thèse comporte deux parties. La première partie décrit que lorsque les cellules cancéreuses sont forcées à utiliser la voie mitochondriale comme source d’énergie à travers l’oxydation phosphorylative, elles initient un mécanisme antioxydant pour tolérer les effets délétères des espèces oxygénées réactives (EOR ou ROS pour reactive oxygene species) produites au cours de l’activité mitochondriale. La stimulation mitochondriale entraîne l’activation de la voie de signalisation ERK5-MEF2, et cette dernière engendre un mécanisme antioxydant de deux façons.Initialement, nous avons observé que MEF2 régule positivement l’expression de miR23a, et ce dernier inhibe l’expression de KEAP1. Cette protéine est responsable de la dégradation ubiquitine dépendante de NRF2, un régulateur clé de la réponse antioxydante cellulaire. L’inhibition de KEAP1 empêche la dégradation cytoplasmique de NRF2. Consécutivement à cela la concentration cytoplasmique en NRF2 augmente ce qui engendre sa translocation dans le noyau où il se lie à une séquence élément de réponse antioxydant (ARE) dans la région promotrice de nombreux gènes antioxydants, initiant ainsi leur transcription. Plus tard nous avons observé que l’activation de la voie ERK5-MEF2 induisait directement la synthèse de novo de NRF2, induisant sa translocation nucléaire et un mécanisme antioxydant. L’inhibition de la voie ERK5-MEF2 altère la réponse antioxydante, sensibilisant ainsi les cellules au stress oxydant.La seconde partie de mon travail a exploré les mécanismes à l’origine des effets hypolipémiants du dichloroacétate (DCA). Le DCA est une petite molécule qui inhibe la PDK1 et permet au pyruvate d’entrer dans la mitochondrie. Il a été utilisé en clinique dans le passé pour baisser les taux plasmatiques de cholestérol mais le mécanisme n’était pas clair et nous l’avons décris. Le DCA force les cellules à entrer en oxydation phosphorylative ce qui active la voie ERK5-MEF2. Cette voie augmente directement l’expression du LDLR (Low Density Lipoprotein Receptor ; récepteur aux lipoprotéines de basse densité) qui permet l’endocytose des LDL riches en cholestérol qui sont responsables de la plupart des maladies cardiovasculaires. L’inhibition de cette voie supprime l’afflux de lipides et par conséquent serait une cible intéressante pour de futures recherches puisque de hauts taux de cholestérols sont directement corrélés avec une augmentation du risque d’athérosclérose et de toutes les complications mortelles qu’il entraine.Notre prochain objectif est d’explorer les autres mécanismes cellulaires régulés par la voie ERK5-MEF2. Sur la base de nos résultats préliminaires, nous proposons que cette voie non seulement régule l’expression du LDLR mais aussi celle de nombreux autres gènes qui sont impliqués directement ou indirectement dans le métabolisme des lipides
Cellular metabolism is the main source of energy and cancer cells has different metabolism than non-transformed cells. Tumor cell tends to avoid mitochondrial activity and oxidative phosphorylation (OXPHOS) and prefer glycolysis for energy production (Warburg effect). This alteration in metabolism is beneficial for growing cells in many ways that promote tumor growth and suppress the anti-cancer immune response. This specific metabolism is an auspicious target for the better development of cancers chemotherapies.My thesis work comprises two parts. The first portion describes that when cancer cells are forced to utilize their mitochondria in order to obtain the energy from OXPHOS they initiate an antioxidant mechanism to cope with the deleterious effects of reactive oxygen species (ROS) produced during mitochondrial activity. Mitochondrial stimulation leads to activation of ERK5-MEF2 signaling pathway, which triggers the antioxidant mechanism by at least two ways.Initially we observed that MEF2 up regulates the expression of miR23a, which inhibits KEAP1 expression. This protein is responsible for ubiquitinational degradation of NRF2, a master regulator of the antioxidant response in cells. The inhibition of KEAP1 prevents the NRF2 cytoplasmic degradation. This results in high built up of NRF2 in cytoplasm that translocates to nucleus where it binds to ARE (antioxidant response element) in the upstream promoter region of many antioxidant genes hence initiates their transcription. Latter we observed that activation of ERK5-MEF2 pathway directly results in de novo synthesis of NRF2, resulting in nuclear translocation and triggering of the antioxidative mechanism. Inhibition of ERK5-MEF2 pathway impairs the cellular antioxidant response, thus sensitizing cells towards oxidative stress.The second part of my work explored the mechanism behind the lipid lowering effects of dichloroacetate (DCA). DCA is a small molecule, which inhibits the PDK1 and enables pyruvate to enter the mitochondria. It was used clinically in past to lower the plasma cholesterol level but the underlying mechanism was not clear and we describe it here. DCA forces cells to perform OXPHOS, which activate the ERK5-MEF2 pathway. This pathway directly up-regulates the expression of Low Density Lipoprotein Receptors (LDLR) that are mainly involved in the endocytosis of cholesterol-rich low density lipoproteins, which are responsible for the majority of cardiovascular diseases. Inhibition of this pathway suppresses lipid influx and hence, it would be an interesting target of future investigation since high cholesterol level is the main cause of various life threatening diseases and the development of atherosclerosis.Our next goal is to exploit other possible cellular mechanism regulated by ERK5-MEF2 pathway. Based on our preliminary data, we propose that this pathway not only regulate the LDLR expression but many other genes, which are directly or indirectly involved in lipid metabolism

Development of the central nervous system requires the precise coordination of intrinsic genetic programs to instruct cell fate, synaptic connectivity and function. The MEF2 family of transcription factors (TFs) plays many essential roles in neural development; however, the mechanisms of gene regulation by MEF2 in neurons remain unclear. This dissertation focuses on the molecular mechanisms by which MEF2 binds to the genome, activates enhancers, and regulates gene expression within the developing nervous system. We find that one MEF2 family member in particular, MEF2D, is an essential regulator of the development and function of retinal photoreceptors, the primary sensory neurons responsible for vision. Despite being expressed broadly across many tissues, in the retina MEF2D binds to retina-specific enhancers and regulates photoreceptor-specific transcripts, including critical retinal disease genes. Functional genome-wide analyses demonstrate that MEF2D achieves tissue-specific binding and action through cooperation with a retina-specific TF, CRX. CRX recruits MEF2D away from canonical MEF2 binding sites by promoting MEF2D binding to retina-specific enhancers that lack a strong consensus MEF2 binding sequence. MEF2D and CRX then synergistically co-activate these enhancers to regulate a cohort of genes critical for normal photoreceptor development. These findings demonstrate that MEF2D, a broadly expressed TF, contributes to retina-specific gene expression in photoreceptor development by binding to and activating tissue-specific enhancers cooperatively with CRX, a tissue-specific co-factor. A major unresolved feature of MEF2D function in the retina is that the number of MEF2D binding sites significantly exceeds the number of genes that are dependent on MEF2D for expression. We investigated causes of this discrepancy in an unbiased manner by characterizing the activity of MEF2D-bound enhancers genome-wide. We find that many MEF2D-bound enhancers are inactive. Furthermore, less than half of active MEF2D-bound enhancers require MEF2D for activity, suggesting that significant redundancies exist for TF function within enhancers. These findings demonstrate that observed TF binding significantly overestimates direct TF regulation of gene expression. Taken together, our results suggest that the broadly expressed TF MEF2D achieves tissue specificity through competitive recruitment to enhancers by tissue-specific TFs and activates a small subset of enhancers to regulate genes.

Orientador: Orientador : Kleber Gomes Franchini
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Durante os primeiros dias do desenvolvimento pós-natal, os miócitos cardíacos perdem a capacidade de proliferação, sendo o crescimento adicional do coração decorrente de hipertrofia e não hiperplasia dos miócitos cardíacos. No entanto, em situações de estresse os miócitos cardíacos diferenciados podem apresentar desdiferenciação e reestabelecimento do ciclo celular. Os mecanismos envolvidos nesse fenômeno são ainda pouco compreendidos. No presente estudo, demonstramos que a ativação do fator de transcrição MEF2C (Myocyte Enhancer Factor 2-C) tem papel crítico no processo de desdiferenciação de miócitos cardíacos. Essa conclusão foi obtida por meio de experimentos de ganho de função pela superexpressão de MEF2C em miócitos ventriculares de ratos neonatos em cultura (MVRNs). Demonstramos que a superexpressão de MEF2C em MVRNs induziu a desdiferenciação e a ativação de mecanismos envolvidos na progressão do ciclo celular. Esses resultados foram obtidos por meio de experimentos de microarranjo de DNA, PCR em tempo real, western blotting e análise do fenótipo celular por microscopias de luz, confocal e eletrônica de transmissão. Esses fenômenos foram atenuados pela superexpressão da quinase de adesão focal (FAK), uma proteína que reconhecidamente exerce efeitos pró-hipertróficos em miócitos cardíacos adultos. Experimentos in vivo e in vitro demonstraram a interação direta entre o fator de transcrição MEF2C e a FAK. Estudos com base em ensaios de reação cruzada associada à espectrometria de massas, dinâmica molecular, espalhamento de raios-X a baixos ângulos e mutação sítio dirigida, demonstraram que as hélices 1 e 4 do domínio FAT da FAK interagem diretamente com a domínio de ligação ao DNA do dímero de MEF2C. Estudos de afinidade e de gel shift demonstraram que a porção FAT da FAK desloca a interação MEF2C/DNA in vitro. Ensaios de gene repórter demonstraram que a FAK, mediada pela região C-terminal, diminui a atividade transcricional de MEF2C em células C2C12. O conjunto de dados demonstra que a ativação do fator de transcrição MEF2C em MVRNs induz a desdiferenciação e ativação de mecanismos de progressão do ciclo celular e que a FAK impede esses efeitos através da interação inibitória no domínio de ligação de MEF2C ao DNA
Abstract: During the first days of postnatal development, cardiac myocytes lose their ability to proliferate, and the further growth of the heart is due to hypertrophy and not hyperplasia of cardiac myocytes. However, in response to stress, cardiac myocytes may have dedifferentiation and re-establishment of the cell cycle. The mechanisms involved in this phenomenon are still poorly understood. In the present study, we demonstrated that activation of the transcription factor MEF2C (myocyte enhancer factor 2-C) plays a critical role in the process of dedifferentiation of cardiac myocytes. This conclusion was obtained by gain-of-function experiments through overexpressing MEF2C in neonatal rat ventricular myocytes in culture (NRVMs). We also showed that overexpression of MEF2C in NRVMs induced the dedifferentiation and activation of mechanisms involved on cell cycle progression. These results were obtained by DNA microarray experiments, real time PCR, western blotting and cell phenotype analysis by light microscopy, confocal and electronic transmission. These effects were attenuated by overexpression of focal adhesion kinase (FAK) protein known to exert pro-hypertrophic effects on adult cardiac myocytes. In vivo and in vitro experiments demonstrated the direct interaction between the transcription factor MEF2C and FAK. A model based on crosslinking technology coupled with mass spectrometry, small angle X-ray scattering and the site directed mutation analyses indicated that alpha-helices 1 and 4 of FAK FAT domain interacts directly with the DNA binding domain of MEF2C dimer. Affinity studies and gel shift assay demonstrated that the FAK FAT domain displaces the MEF2C/DNA interaction in vitro. Reporter gene assays demonstrated that FAK, mediated by the C-terminal region, decreases the transcriptional activity of MEF2C in C2C12 cells. The data set shows that the activation of the transcription factor MEF2C in MVRNs induces dedifferentiation and activation of cell cycle progression and that FAK prevents these effects by inhibitory interaction with DNA binding domain of MEF2C
Doutorado
Biologia Estrutural, Celular, Molecular e do Desenvolvimento
Doutor em Ciências

Orientador: Kleber Gomes Franchini
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Os fatores MEF2 (Myocyte Enhancer Factor 2) pertencem à família MADS Box (MCM1-Agamous-Deficiens-Serum response factor) e foram descritos pela primeira vez como fatores de transcrição que se ligam a sequencias de DNA ricas em A/T nos promotores de vários genes músculo específicos. Existem 4 genes da família MEF2 que foram identificados em vertebrados: MEF2A, B, C e D que são expressos de forma distinta durante a embriogênese e nos tecidos adultos. Estudos anteriores do nosso laboratório demonstraram que o fator de transcrição MEF2 é ativado por estiramento mecânico e influencia a expressão de genes relacionados à hipertrofia cardíaca. Utilizando a tecnologia de siRNA para MEF2C (siRNAMEF2C) demonstramos a atenuação da hipertrofia cardíaca induzida por coarctação da aorta nos animais que receberam o siRNAMEF2C. Por outro lado trabalhos demonstraram que animais transgênicos com a superexpressão de MEF2A ou de MEF2C e submetidos à sobrecarga de pressão por coarctação da aorta, não apresentam hipertrofia cardíaca compensatória. Nesses animais a superexpressão de MEF2A ou de MEF2C no coração está associada à deterioração cardíaca funcional e estrutural e o desenvolvimento de cardiomiopatia dilatada. Contudo, a caracterização fenotípica e os mecanismos moleculares envolvidos na superexpressão de MEF2C em miócitos cardíacos ainda são desconhecidos. Da mesma forma não é conhecido o papel do fator de transcrição MEF2C na resposta hipertrófica do miócito cardíaco após coarctação da aorta. No presente trabalho foi demonstrado que a superexpressão de MEF2C em miócitos cardíacos de ratos neonatos (NRMV), com o uso de partículas adenovirais, induziu a desdiferenciação celular e a ativação de mecanismos envolvidos na progressão do ciclo celular. Esses resultados foram obtidos por meio de experimentos de microarranjo de DNA, proteoma, PCR em tempo real e western blotting. A análise do fenótipo celular por microscopias de luz, confocal e eletrônica de transmissão demonstra que NRMV possuem aumento na binucleação e desorganização sarcomérica, alterações coerentes com o quadro de desdiferenciação celular e ativação da progressão do ciclo celular. Por meio da técnica de incorporação de iodeto de propídeo e citometria de fluxo confirmamos o aumento de células em ciclo celular. Para confirmar os achados nos cardiomiócitos neonatos passamos a investigar o efeito da superexpressão de MEF2C em cardiomiócitos de ratos adultos. Para isso padronizamos a técnica de isolamento destas células e tratamos com AdMEF2C. Sendo assim o tratamento com AdMEF2C em miócitos cardíacos de ratos adultos resultou em aumento da expressão de MEF2C após 48 horas de tratamento. O efeito observado foi semelhante ao encontrado em cardiomiócitos neonatos, sendo que os adultos apresentaram aumento da expressão de genes relacionados ao ciclo celular e diminuição dos genes estruturais. O nível ultraestrutural observado por microscopia eletrônica de transmissão no tempo de 48 horas de tratamento não observamos diferenças na estrutura sarcomérica das células tratadas com AdMEF2C. Por fim demonstramos que o silenciamento de MEF2C pela injeção de lentivírus no coração demonstrou ser capaz de impedir o desenvolvimento da hipertrofia cardíaca em camundongos coarctados por 15 dias. A hipertrofia do coração foi avaliada por meio da espessura da parede posterior do ventrículo esquerdo e gravimetria do ventrículo esquerdo e dos pulmões. O conjunto de dados demonstra que a superexpressão de MEF2C leva a alterações estruturais no miócito cardíaco compatíveis com quadro de deterioração e insuficiência cardíaca e que o silenciamento de MEF2C no coração impede o desenvolvimento da hipertrofia cardíaca decorrente da coarctação da aorta
Abstract: The factors MEF2 (myocyte enhancer factor 2) belong to the family MADS box (MCM1-Agamous-deficiens-Serum response factor) and were first described as transcription factors that bind DNA sequences rich in A / T in the promoters of multiple muscle-specific genes. There are four MEF2 family genes that were identified in vertebrates MEF2A, B, C and D are expressed differently during embryogenesis and in adult tissues. Previous studies from our laboratory demonstrated that the transcription factor MEF2 is activated by mechanical stretch and influences the expression of genes related to cardiac hypertrophy. Using siRNA technology to MEF2C (siRNAMEF2C) demonstrated attenuation of cardiac hypertrophy induced by aortic coarctation in animals that received siRNAMEF2C. On the other hand studies have demonstrated that transgenic mice with overexpression of MEF2A or MEF2C and subjected to pressure overload by aortic coarctation show no compensatory cardiac hypertrophy. In these animals the overexpression of MEF2A or MEF2C in the heart is associated with structural and functional cardiac deterioration and development of dilated cardiomyopathy. However, the phenotypic and molecular mechanisms involved in the overexpression of MEF2C in cardiac myocytes are still unknown. Likewise, there is known the role of the transcription factor MEF2C in cardiac myocyte hypertrophic response after aortic coarctation. In the present study it was shown that overexpression of MEF2C in neonatal rat cardiac myocytes (NRMV) with the use of adenoviral particles, and cellular dedifferentiation induced activation mechanisms involved in cell cycle progression. These results were obtained by DNA microarray experiments, proteomics, real time PCR and western blotting. The analysis of cell phenotype by light microscopy, confocal and transmission electron shows that NRMV have increased binucleation and sarcomeric disorganization, changes consistent with the framework of cellular dedifferentiation and activation of cell cycle progression. By means of the propidium iodide incorporation technique and flow cytometry, confirmed increasing cells in the cell cycle. To confirm the findings in neonatal cardiomyocytes we investigate the effect of overexpression of MEF2C in cardiomyocytes of adult rats. For this standardized technique and isolation of these cells treated with AdMEF2C. Thus treatment with AdMEF2C in adult rat cardiac myocytes resulted in increased expression of MEF2C after 48 hours of treatment. The observed effect was similar to that found in cardiomyocytes neonates, adults who showed increased expression of genes related to cell cycle and decreased structural genes. The ultrastructural level observed by transmission electron microscopy in the time of 48 hours of treatment showed no difference in sarcomeric structure of cells treated with AdMEF2C. Finally we show that MEF2C silencing by lentivirus injection in the heart has been shown to prevent the development of cardiac hypertrophy in mice after 15 days of pressure overload. The heart hypertrophy was evaluated by the thickness of the posterior wall of the left ventricle and the left ventricle gravity and lungs. The data set shows that overexpression of MEF2C leads to structural changes in the cardiac myocyte compatible framework of deterioration and failure, and MEF2C silencing of the heart prevents the development of cardiac hypertrophy due to aortic coarctation
Doutorado
Biologia Estrutural, Celular, Molecular e do Desenvolvimento
Doutora em Ciências

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Brain and Cognitive Sciences, February, 2021
Cataloged from the official PDF version of thesis. "February 2021."
Includes bibliographical references (pages 119-126).
Recent increases in human longevity have been accompanied by a rise in the incidence of dementia. While a large proportion of aged individuals display pathological hallmarks of neurodegenerative disease, a small number of these individuals are able to maintain healthy cognitive function even in the presence of extensive brain pathology. The molecular mechanisms that govern this neuro-protected state remain unknown, but individuals that exhibit cognitive resilience (CgR) represent a unique source of insight into potential therapies that could preserve brain function in the face of neurodegenerative disease. Here, we employ a two-pronged approach to dissect the mechanism underlying CgR. First, using multiple integrated repositories of clinical and brain transcriptomic data we identified individuals who maintained normal cognition despite harboring a large burden of Alzheimer's disease (AD) pathology.
We observe significant up-regulation of MEF2 family members in these resilient patients when compared to patients whose cognition declined in the presence of pathology. Second, we utilize the only existing animal model of CgR -- environmental enrichment --
to investigate the molecular mechanisms involved in the induction of resilience. Accessibility of Mef2 binding sites, and expression of Mef2 targets are significantly increased upon enrichment. Additionally, knockdown of Mef2 family members just prior to the initiation of enrichment block its cognitive benefits, demonstrating the necessity of Mef2 activity for achieving the enhanced cognitive potential afforded by enrichment. Neurons lacking Mef2 are hyperexcitable, which is also one of the earliest pathological alterations observed in AD. These results suggest a potential mechanistic link between the Mef2 transcriptional network induced by enrichment and the prevention of disease-associated hyperexcitability. To determine the causal impact of Mef2 on cognition in the context of neurodegeneration, we use a viral approach to manipulate the PS19 mouse model of tauopathy.
Remarkably, in the absence of enrichment, Mef2 overexpression alone is sufficient to improve cognition and reduce hyperexcitability in PS19 mice. Overall, our findings reveal a novel role for MEF2 transcription factors in promoting cognition throughout life, and maintaining cognitive resilience in the context of neurodegenerative disease.
by Scarlett J.V. Barker.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Brain and Cognitive Sciences

Early growth response gene – 1 (Egr1) encodes a protein widely present in mammalian body, such as connective tissue, cardiac tissue, the liver, and the brain. As a transcription factor (TF), it is involved in processes that take place in the endocrine, digestive, immune, musculo-skeletal and central nervous systems, for instance, B cell maturation upon B cell receptor activation, tendon repair upon mechano-stimulation, and long-term spatial memory formation. In mammalian brains, EGR1 controls the responses to environmental stimuli such as chronic stress and physical contact. It also participates in processes such as long-term memory consolidation and synapse re-structuring. It plays a role in enacting responses and qualities of gene transcription cascades upon neuronal stimulation. Inside the epigenetic realm, EGR1 recruits Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) to remove DNA methylation at target loci. Due to its critical functions during brain development and upon neuronal activation, mis-regulation of EGR1 is associated with neuropsychological disorders such as post-traumatic stress disorder (PTSD) and schizophrenia (SCZ) in humans. In this study, we performed bioinformatics analysis with brain methylomes and predicted EGR1 may interact with myocyte enhancer factor 2C (MEF2C), which is known to be involved in many similar processes as EGR1, such as synapse architecture, cell migration, and learning and memory. EGR1 and MEF2C ChIP-seq data derived from mouse frontal cortex suggest these two proteins may regulate a common set of downstream genes. To begin, co-immunoprecipitation experiments were performed with HEK293T cells co-transfected with EGR1-FLAG and MEF2C-HA tagged constructs, allowing for specific interaction identification without endogenous protein expression interference. Furthermore, co-immunoprecipitation experiments performed with brain tissues additionally indicated the two proteins interact with each other endogenously. Altogether, this study provides protein-protein interaction evidence for EGR1 and MEF2C in cultured HEK293 cells and in the cortices of adult male mice. This information provides a foundation for future examinations of how these two TFs interact to initiate cascading events following neuronal stimulation.
Master of Science
Early growth response gene – 1 (EGR1) encodes a protein that can be found in animals such as fruit flies, mice, rats, and humans. In mammals, it is widely expressed in the cardiovascular, endocrine, digestive, immune, musculo-skeletal and central nervous systems (CNS). Within the CNS, EGR1 is known as an essential transcription factor involved in brain development. More specifically, EGR1 plays a role in how the early brain develops in response to environmental stimuli, formation of synapse architecture and certain types of memories. Many gene networks involved in growth and development rely on EGR1 to regulate functions such as synapse reformation after exposure to the environment. EGR1 is known to have numerous partners with whom it interacts to execute its functions. It is also involved in epigenetic regulation, which is a process by which genes are silenced or activated without changing DNA sequences in the genome. EGR1 may directly interact with TET1 to demethylate EGR1 target sites in the genome, and to increase gene transcription. In memory development, EGR1 plays a key role ensuring short-term auditory fear memory can be converted to long-term memory, and also ensures long-term spatial memory. In this study, our computational analyses suggest that EGR1 may interact with MEF2C. This work provides evidence of a protein-protein interaction of EGR1 and MEF2C in cultured cells and in the brain cortical areas of mice. Such an interaction may explain why these two genes regulate overlapped biological processes within the brain and sheds lights on how cascading events are initiated following neuronal stimulation.

Orientador: Kleber Gomes Franchini
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-11T20:15:36Z (GMT). No. of bitstreams: 1 Pereira_AnaHelenaMacedo_M.pdf: 6567293 bytes, checksum: b9a8c070b3b65fe7a4fb095ccfc59d42 (MD5) Previous issue date: 2008
Resumo: Doenças do coração são freqüentemente associadas à hipertrofia miocárdica. Estímulos mecânicos induzem o crescimento hipertrófico e contribuem para a degeneração e morte dos miócitos cardíacos. Dentre os fatores de transcrição envolvidos no processo de hipertrofia miocárdica, estão os da família MEF2 (Myocyte Enhancer Factor-2), que é composto por 4 membros, MEF2A, B, C e D. O MEF2C é descrito como o principal transcrito no miocárdio. Tanto a deleção quanto a hiperexpressão de seu gene causam efeitos deletérios na formação e na função do músculo cardíaco. Estudos anteriores do nosso laboratório demonstraram que o MEF2 é ativado por estiramento de cardiomiócitos e influencia a expressão de genes do programa hipertrófico. O presente estudo tem como objetivo avaliar os efeitos do silenciamento gênico do MEF2C nas alterações estruturais e funcionais do ventrículo esquerdo de camundongos submetidos à sobrecarga pressórica. Para isso, utilizamos a técnica de interferência por RNA para o MEF2C. A padronização constituiu de: 1) avaliação do silenciamento do MEF2C em cultura de células C2C12 e no ventrículo esquerdo de camundongos Swiss; 2) determinação da dose necessária de siRNA para o silenciamento da expressão protéica do MEF2C; 3) determinação do curso temporal do silenciamento; 4) avaliação dos efeitos do tratamento com molécula irrelevante de siRNA direcionada para a proteína exógena GFP; 5) avaliação da especificidade do silenciamento (off-targets) pela análise do RNAm para o MEF2A e das proteínas FAK, GAPDH, JNK1/2 e SHP2; 6) avaliação do silenciamento em outros órgãos, como pulmão e rim; 7) avaliação da efetividade do silenciamento de MEF2C em miócitos cardíacos isolados do ventrículo esquerdo de camundongos. O tratamento com siRNA diminuiu a expressão protéica do MEF2 em 70% das células C2C12. Também verificamos que o tratamento com siRNA silenciou 85% da expressão protéica e do RNAm do MEF2C no ventrículo esquerdo de camundongos em até 4 dias de seguimento. Não foi verificada alteração na expressão de RNAm para o MEF2A e das proteínas FAK, GAPDH, JNK1/2 e SHP2. O silenciamento foi efetivo no pulmão e nos cardiomiócitos isolados do ventrículo esquerdo de camundongos tratado com siRNAMEF2C. Após a padronização do silenciamento, procedeu-se à determinação dos efeitos do silenciamento na estrutura e na função do ventrículo esquerdo de camundongos submetidos à sobrecarga pressórica crônica. Para isso, realizaram-se as análises ecocardiográfica, hemodinâmica, gravimétrica e morfométrica do ventrículo esquerdo de camundongos submetidos à coarctação da aorta com seguimento de 15 dias. Demonstramos que o tratamento com siRNAMEF2C atenuou a hipertrofia cardíaca nos animais coarctados. Esta conclusão foi baseada em dados de ecocardiografia que revelaram menor espessura da parede posterior (30% menor) e por gravimetria que revelou atenuação de aproximadamente 45% da massa do ventrículo esquerdo. Apesar de ter havido aumento do gradiente sistólico nos animais coarctados, a pressão arterial sistêmica não apresentou diferença estatisticamente significativa com o tratamento do siRNAMEF2C. Morfologicamente, o siRNA atenuou a fração de colágeno no ventrículo esquerdo de camundongos coarctado com 15 dias de seguimento. Entretanto, o diâmetro dos miócitos e o infiltrado de células inflamatórias foram comparáveis dentre os grupos. Somente os animais coarctados por 24 horas tiveram maior expressão de ß- MHC, e quando tratados com siRNAMEF2C apresentaram menor razão ATP/ADP. Dessa forma, esses dados sugerem que o MEF2C regula múltiplos aspectos da hipertrofia cardíaca induzida por sobrecarga pressórica tais como a expressão de genes sarcoméricos e genes envolvidos na adaptação metabólica do músculo cardíaco.
Abstract: Heart diseases are frequently associated with myocardial hypertrophy. Mechanical stimuli can trigger hypertrophic growth as well as degeneration and death of the cardiac myocytes. The MEF2C family of transcription factors plays a role in the process of myocardial hypertrophy. It is composed by 4 members, MEF2A, B, C and D, and the MEF2C is the main transcript in the heart. Both the deletion and overexpression of mef2c induce deleterious effects in the formation and function of the heart. Previous studies of the our laboratory has shown that the transcription factor MEF2C is activated by mechanical stretch in cardiomyocytes and regulates the expression of genes related to cardiac hypertrophy. This study was performed to address the effects of MEF2C gene silencing in the structural and functional changes of the left ventricle (LV) induced by pressure overload in mice. To silence MEF2C, it was employed the RNA interference technique, specific siRNA target to MEF2C was administered through the mice jugular vein. To optimize the MEF2C knockdown, it was necessary to 1) analyze the MEF2C silencing in C2C12 cells, 2) determine the dose required to induce significant MEF2C silencing in LV of mice, 3) determine the time course of gene silencing, 4) assess the effects of the treatment with irrelevant siRNA target to the protein GFP, 5) evaluate the specificity of gene silencing by siRNAMEF2C through the expression analysis of the transcription factor MEF2A and other non-related proteins, 6) analyze of the MEF2C knockdown in other organs, 7) determine the effectiveness of the MEF2C silencing in cardiac myocytes harverst from the LV of mice treated systemically with siRNAMEF2C. Treatment with 100ng/mL of siRNAMEF2C induced MEF2C silencing (~70%) in C2C12 cells. Intrajugular delivery of 30µg of siRNAMEF2C in mice induced the reduction in the mRNA and protein levels (~85%) until 4 days after the injection. The treatment with siRNAMEF2C did not affect the expression of MEF2A and other non-related proteins. The MEF2C silencing was effective in lung and in cardiac myocytes harverst from LV of mice treated with siRNAMEF2C. After knockdown optimization, echocardiographic, hemodynamic, gravimetric and morphometric analysis was performed to address the effects of MEF2C silencing in the structure and function of the LV from 15 days aorticbanded mice. Myocardial MEF2C silencing attenuated the load-induced hypertrophy in banded mice, indicated by the reductions of the wall thickness and the mass (~45%) of the LV. An increase in transconstriction gradient was observed in banded mice but the systemic blood pressure did not shown a significant statistically difference with the siRNAMEF2C treatment. The siRNAMEF2C injection reduced the collagen fraction in the LV of 15 days banded mice. On the other hand, the myocytes diameter and inflammatory cells level were comparable between the groups. Only the 24 hours banded mice showed an increase in the â-MHC expression and the treatment with siRNAMEF2C reduced ATP/ADP ratio. This study indicate that MEF2C regulates many aspects of the cardiac hypertrophy induced by pressure overload, like the expression of sarcomeric genes and genes involved in metabolic adaptation of the heart muscle.
Mestrado
Medicina Experimental
Mestre em Fisiopatologia Médica

Thesis (Ph.D.)--Boston University
Skeletal muscle regenerates in response to disease or injury through the activation of quiescent muscle stem cells and their subsequent differentiation into multi-nucleated myotubes. Understanding the molecular mechanisms of regeneration is critical to exploit this pathway for use in tissue repair. Data shown here demonstrate that MEF2A plays an essential role in skeletal muscle regeneration in adult mice. Regenerating muscle from MEF2A knockout mice displays widespread necrosis, reduced myofiber cross-sectional area, and a significant reduction in Pax7-positive progenitors. The existence of activated progenitor cells that co-express MEF2A and Pax7 is also documented in adult regenerative myogenesis. MEF2A controls this process through its direct regulation of the largest known mammalian rnicroRNA (rniRNA) cluster, the Gtl2-Dio3 locus. All miRNAs (>40) within this cluster are coordinately downregulated in MEF2A-deficient regenerating muscle, and a subset of the Gtl2-Dio3 rniRNAs represses secreted Frizzled- related proteins (sFRPs), inhibitors of Wnt signaling. Consistent with downregulation of this rniRNA cluster, expression of sFRPs is upregulated and Wnt signaling is inhibited in MEF2A-deficient regenerating muscle. Furthermore, overexpression of Gtl2-Dio3 miRNAs, miR-433 and miR-410, restores myotube formation in MEF2A-deficient myoblasts. Thus, miRNA-mediated modulation of Wnt signaling by MEF2A is a requisite step for proper muscle regeneration, and represents an attractive pathway for enhancing regeneration of diseased muscle.

A insulina regula a expressão de GLUT4, porém os mecanismos envolvidos nesta regulação não estão definidos. Alguns fatores de transcrição e proteínas cinases estão relacionados com a expressão de GLUT4. Assim, o objetivo desta pesquisa foi investigar a participação dos fatores de transcrição MEF2, HIF-1a e NF-kB, e das proteínas cinases mTOR, PI3K e AKT na regulação da expressão de Slc2a4/GLUT4 induzida pela insulina. Para isso, músculos sóleos de ratos foram incubados por 3 horas em tampão Krebs, tratados ou não com insulina, wortmanina, rapamicina, ML-9 ou TNF-a. Nesses tecidos foram avaliados o conteúdo das proteínas GLUT4 e mTOR (Western), o conteúdo de mRNA de GLUT4, NF-kB1, HIF-1a e MEF2A/C/D (PCR) e a atividade de ligação de proteínas nucleares no sítio de ligação de NF-kB, AT-rich element e E-Box do promotor do gene Slc2a4 (EMSA). O tratamento com insulina aumentou a expressão de Slc2a4/GLUT4 no músculo sóleo, in vitro, ativando os fatores de transcrição MEF2A/D e possivelmente MyoD, através da via da PI3K/AKT e diminuindo a expressão e atividade de NF-kB.
Insulin regulates the GLUT4 expression, but the mechanisms involved in this regulation are not defined. Some transcription factors and protein kinases are related to the expression of GLUT4. Thus, the aim of this research was to investigate the role of the transcription factors MEF2, HIF-1a and NF-kB, and the proteins kinases mTOR, PI3K and AKT, in regulation of Slc2a4 and GLUT4 expression by insulin. For this, rat soleus muscles were incubated for 3 hours in Krebs buffer, treated or not with insulin, wortmanina, rapamycin, ML-9 or TNF-a. In these tissues were evaluated the GLUT4 and mTOR protein content (Western), the content of GLUT4, NF-kB1, HIF-1a and MEF2A/C/D mRNAs (PCR) and the binding activity of protein nuclear in binding site of NF-kB, AT-rich element and E-Box in the promoter of the gene Slc2a4 (EMSA). Insulin treatment increased the expression of Slc2a4/GLUT4 in the soleus muscle in vitro, activating the transcription factors MEF2A/D and possibly MyoD, via PI3K/AKT and decreasing the expression and activity of NF-kB.