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.

The synaptic remodeling of neural circuits is thought to underly memory formation. Both long-term memory formation and remote memory formation are thought to involve a process restructuring of synapses in specific areas of the brain. The transcription factor myocyte enhancer factor 2 (MEF2) has been shown to restrict spine growth in both in vivo and in vitro. It has been suggested that MEF2 is a critical molecule involved in memory formation, however, MEF2‘s role in adult memory formation it is largely unexplored. Thus, we have sought to characterize MEF2’s involvement in the formation of long-term and remote memory formation. We first showed that acute overexpression of MEF2 in the hippocampus blocks long-term spatial memory formation and activity-dependent spine formation. We next found that acute overexpression of MEF2 in the lateral amygdala likewise blocks long-term fear memory formation, suggesting that MEF2 is critical protein involved in synaptic plasticity necessary for long-term memory formation. We next demonstrated the bi-directionality of MEF2 by decreasing MEF2 function in the hippocampus and amygdala and showing a facilitation in memory formation. Together, these results suggest that MEF2 is a critical protein, which regulates spine formation important for the formation of long-term memories. We next investigated whether similar synaptic plasticity mechanisms are involved in the systems consolidation. We acutely overexpressed MEF2 in the anterior cingulate cortex at different time points following contextual fear conditioning. We noted that there was a critical window, where MEF2 blocks spine density increases in the ACC and remote memory formation. Results from this study, suggest that cortical synapses undergo a process of strengthening and remodeling and that MEF2 is a critical regulator involved. Our results demonstrate that MEF2 is involved in the synaptic consolidation of long-term and remote memories.

The synaptic remodeling of neural circuits is thought to underly memory formation. Both long-term memory formation and remote memory formation are thought to involve a process restructuring of synapses in specific areas of the brain. The transcription factor myocyte enhancer factor 2 (MEF2) has been shown to restrict spine growth in both in vivo and in vitro. It has been suggested that MEF2 is a critical molecule involved in memory formation, however, MEF2‘s role in adult memory formation it is largely unexplored. Thus, we have sought to characterize MEF2’s involvement in the formation of long-term and remote memory formation. We first showed that acute overexpression of MEF2 in the hippocampus blocks long-term spatial memory formation and activity-dependent spine formation. We next found that acute overexpression of MEF2 in the lateral amygdala likewise blocks long-term fear memory formation, suggesting that MEF2 is critical protein involved in synaptic plasticity necessary for long-term memory formation. We next demonstrated the bi-directionality of MEF2 by decreasing MEF2 function in the hippocampus and amygdala and showing a facilitation in memory formation. Together, these results suggest that MEF2 is a critical protein, which regulates spine formation important for the formation of long-term memories. We next investigated whether similar synaptic plasticity mechanisms are involved in the systems consolidation. We acutely overexpressed MEF2 in the anterior cingulate cortex at different time points following contextual fear conditioning. We noted that there was a critical window, where MEF2 blocks spine density increases in the ACC and remote memory formation. Results from this study, suggest that cortical synapses undergo a process of strengthening and remodeling and that MEF2 is a critical regulator involved. Our results demonstrate that MEF2 is involved in the synaptic consolidation of long-term and remote memories.

Members of the Mef2 transcription factor family are extensively studied within the muscle field for their ability to cooperate with the myogenic regulatory factors MyoD and myogenin during muscle differentiation. Although it is known that Mef2 pre-mRNAs undergo alternative splicing, the different splice forms have not been functionally annotated. In this thesis, my studies aimed to characterize three Mef2D splice forms: MEF2Dα'β, MEF2Dαβ, MEF2Dαø. Our results show that MEF2D splice forms can be differentially phosphorylated by p38 MAPK and PKA in vitro. Gene expression analysis using cell lines over-expressing each Mef2D splice form suggests that they can differentially activate desmin, myosin heavy chain and myogenin expression. Mass spectrometry analyses from our pull-down assays reveal known and novel MEF2D binding partners. Our work suggests that Mef2D splice forms have overlapping but distinct roles and provides new insight into the importance of Mef2D alternative splicing during skeletal myogenesis.

Rhabdomyosarcoma (RMS) is a highly malignant pediatric cancer that is the most common form of soft tissue tumors in children. RMS cells have many features of skeletal muscle cells, yet do not differentiate. Thus, our studies have focused on the molecular defects present in these cells that block myogenesis. We have found MEF2D is absent in RMS cell lines representing both major subtypes of RMS and primary cells derived from an embryonal RMS mice model. We have shown that the down regulation of MEF2D is a major cause for the failure of RMS cells to differentiate. We find MEF2D cannot bind to muscle specific gene promoters. Exogenous expression of MEF2D activates muscle specific luciferase constructs, upregulates p21 expression and increases muscle specific gene expression including the expression of myosin heavy chain, a marker for skeletal muscle differentiation. Restoring expression of MEF2D also inhibits proliferation, cell motility, anchorage independent growth in vitro, and tumor growth in vivo by xenograft assay. We also have found MEF2C is deregulated in rhabdomyosarcoma with the aberrant alternative splicing. We have shown that exon α in MEF2C is aberrantly alternatively spliced in RMS cells, with the ratio of α2/α1 being highly downregulated in RMS cells compared with normal myoblasts. We find that MEF2Cα1 is the ubiquitously expressed isoform which exhibits no myogenic activity and that MEF2Cα2, the muscle specific MEF2C isoform, is required for efficient differentiation. Compared with MEF2Cα2, MEF2Cα1 more strongly interacts with and recruits HDAC5 to myogenic gene promoters to repress muscle specific genes. Overexpression of the MEF2Cα2 isoform in RMS cells increases myogenic activity and promotes differentiation in RMS cells. We have also identified a serine protein kinase, SRPK3, which is downregulated in RMS cells and found that expression of SRPK3 promoted the splicing of the MEF2Cα2 isoform and induced differentiation. Restoration of either MEF2Cα2 or SPRK3 inhibited both proliferation and anchorage independent growth of RMS cells. The NAC complex performs many diverse biological functions, and the deregulation of its subunits has been correlated with many cancers. We sought to understand the function of the NAC complex in normal myogenesis and tumor progression in rhabdomyosarcoma cells. We found that the muscle specific subunit of the NAC complex, skNAC, which is the alternatively spliced isoform of NACα, was induced in normal cells and downregulated in RMS cells, while BTF3, also known as NACβ, was induced in normal cells and severely downregulated in RMS cells. We also showed that skNAC associated with muscle specific promoters together with BTF3 in differentiated normal cells, and this association was dependent on the expression of BTF3. We further investigated the involvement of skNAC in RMS progression. We found that the muscle specific expressed methyltransferase Smyd1 was nuclear localized in RMS cells and its interaction partner skNAC was switched with corepressors (HDAC1 and TBX2). We also confirmed the expression of skNAC was regulated by the splicing factor kinase SRPK3 and overexpression of SPRK3 induced skNAC expression and muscle differentiation in RMS cells. We also confirmed the overexpression of BTF3 in patient RMS tumors and depletion of BTF3 induced apoptosis in RMS cells and decrease RMS cell survival. BTF3 depletion also sensitized TRAIL induced cell apoptosis in RMS cells. However, BTF3 played a different role in normal cells. Deletion of BTF3 in C2C12 cells does not induce cell apoptosis, which suggests BTF3 functions as an anti-apoptosis factor in RMS cells and could be used as a cancer specific therapeutic target in RMS cells.

Members of the Mef2 transcription factor family are extensively studied within the muscle field for their ability to cooperate with the myogenic regulatory factors MyoD and myogenin during muscle differentiation. Although it is known that Mef2 pre-mRNAs undergo alternative splicing, the different splice forms have not been functionally annotated. In this thesis, my studies aimed to characterize three Mef2D splice forms: MEF2Dα'β, MEF2Dαβ, MEF2Dαø. Our results show that MEF2D splice forms can be differentially phosphorylated by p38 MAPK and PKA in vitro. Gene expression analysis using cell lines over-expressing each Mef2D splice form suggests that they can differentially activate desmin, myosin heavy chain and myogenin expression. Mass spectrometry analyses from our pull-down assays reveal known and novel MEF2D binding partners. Our work suggests that Mef2D splice forms have overlapping but distinct roles and provides new insight into the importance of Mef2D alternative splicing during skeletal myogenesis.

The Myocyte Enhancer Factor-2, or MEF2, transcription factor family is necessary for the differentiation and regeneration of both skeletal and cardiac muscle tissue. The transcription factors in this family are responsible for the activation of many muscle specific growth factor-induced and differentiation genes. There are four individual isoforms of MEF2; MEF2A, -B, -C, and –D, and the roles of these individual transcription factors are not completely understood. Knockdowns of these individual isoforms revealed that a MEF2A knockdown mouse model displays severe myofibrillar defects in cardiac muscle. This knockdown also has shown that MEF2A is required for myogenesis in vitro, where the other 3 isoforms, -B, -C, and –D, are not necessary for this process. One method of knocking down MEF2A to study its roles further is through the use of short hairpin RNAs (shRNA). The purpose of my research was two-fold. First, in order to test the specificity of this shRNA method, an shRNA-resistant MEF2A over expression construct in an adenoviral vector was created to perform rescue experiments. Second, to compare individual MEF2 isoform knockouts to a complete knockout of the entire MEF2 family, a dominant negative construct was created in an adenoviral vector. In both cases, a pShuttle-CMV adenoviral vector was used. The results of this experiment can be used to further investigate the roles of MEF2A in both regeneration and differentiation of skeletal and cardiac muscle tissue.

Chez l’homme, les cellules de Leydig sont les principales productrices d’hormones stéroïdiennes dans le testicule. Ces hormones, dont font partie la testostérone, la dihydroprogestérone (DHP) et la dihydrotestostérone (DHT), sont indispensables à la spermatogenèse, au développement des caractéristiques sexuelles primaires et secondaires ainsi qu'au maintien de la fertilité masculine. Les niveaux d’hormones stéroïdiennes produits par ces cellules doivent être étroitement régulés au cours du développement. En outre, la stéroïdogenèse est source de formation d'espèces réactives de l'oxygène (ERO). La présence d'ERO en excès dans les cellules de Leydig, inhibe la stéroïdogenèse. Notre laboratoire a récemment identifié un nouveau facteur de transcription, myocyte enhancer factor 2 (MEF2), présent dans le testicule, tout au long de la vie. Ce facteur, premièrement identifié dans le cœur et le cerveau, est essentiel à l'organogenèse ainsi qu'à la différenciation cellulaire. Dans un premier temps, mon travail de doctorat met en évidence le rôle clé du facteur MEF2 dans la régulation de l’expression génique dans la lignée de cellules de Leydig MA-10. Dans un second temps, ce travail caractérise le rôle de MEF2 dans la régulation de l’expression des gènes impliqués dans les mécanismes de détoxification cellulaire, qui permettent l’élimination des ERO produites par la stéroïdogenèse. Le facteur MEF2 régule, seul ou en coopération avec la Ca2+/calmoduline-dependent protein kinase I (CAMKI), l’expression du gène Gsta1 qui code pour une enzyme antioxydante, la glutathion-S transférase A1. De plus, MEF2 n’est pas le seul facteur de transcription présent dans les cellules de Leydig et la coopération entre différents facteurs de transcription permet la régulation de l’expression des gènes. Ainsi, dans un troisième temps, mon travail de doctorat met en évidence une nouvelle coopération entre les facteurs de transcription MEF2 et COUP-TFII dans les cellules de Leydig MA-10. MEF2 et COUP-TFII régulent l’expression du gène Akr1c14 codant pour une 3α-hydroxystéroïde déshydrogénase, permettant la régulation des niveaux de DHP et DHT qui sont des stéroïdes métaboliquement très actifs. En conclusion, mes travaux mettent en évidence le rôle du facteur MEF2 sur l’expression des gènes impliqués dans le maintien et la régulation des fonctions stéroïdogéniques des cellules de Leydig. / In male, the Leydig cells are the main producer of steroid hormones in the testis. These steroids, including testosterone, DHT and DHP, are essential for spermatogenesis, for the development of primary and secondary male sexual characteristics and for the maintenance of male fertility. The steroid levels produced by these cells must be tightly regulated throughout fetal and adult life. In addition to synthesizing steroids, steroidogenesis produces a significant amount of reactive oxygen species (ROS), which in turn disrupt steroid production. Our lab has recently identified the presence of a novel transcription factor, myocyte enhancer factor 2 (MEF2), in the mouse testis, throughout fetal and adult life. MEF2 factor is an important regulator of organogenesis and cell differentiation in various tissues and was first identified in the heart and the brain. Initially, my Ph.D. work highlights the key role of MEF2 factor in the regulation of gene expression in the MA-10 Leydig cell line. Secondly, my work characterized the role of MEF2 in the regulation of genes involved in cellular detoxification mechanisms, which seek the elimination of ROS produced by steroidogenesis. The transcription factor MEF2 regulates, alone or in cooperation with the Ca2+/calmodulin-dependent protein kinase I (CAMKI), the expression of Gsta1 gene that encodes for an antioxidant enzyme, glutathione S-transferase A1. Furthermore, MEF2 is not the sole transcription factor present in Leydig cells and the cooperation between different transcription factors allows for proper regulation of steroidogenic gene expression. Thereby, the third part of my Ph.D. work highlighted a new cooperation between two transcription factors, MEF2 and COUP-TFII in MA-10 Leydig cells. In these cells, MEF2 and COUP-TFII cooperate to regulate Akr1c14 gene expression. This gene encode for a 3α-hydroxysteroid dehydrogenase that regulates the bioavailabilities of DHP and DHT, which are potent steroids. In conclusion, my work identifies novel important roles for MEF2 factor in the expression of genes involved in the maintenance and regulation of Leydig cell functions.

Les cellules de Leydig sont les principales cellules stéroïdogéniques dans le testicule. La stéroïdogenèse est un mécanisme crucial pour la masculinisation pendant l’embryogenèse et la puberté ainsi que pour le maintien des caractéristiques mâles durant l’âge adulte. Elle est donc finement régulée par l’axe hypothalamo-hypophysaire mais également directement dans la cellule de Leydig. Un des mécanismes majeurs, ayant lieu dans la cellule de Leydig, est la régulation de l’expression des gènes stéroïdogéniques par des facteurs de transcription. Pendant ma thèse, j’ai caractérisé la présence des facteurs de transcription MEF2 dans les cellules de Leydig. Ces facteurs font partie de la famille des facteurs de transcription MEF2 qui compte quatre membres : MEF2A, 2B, 2C et 2D. J’ai mis en évidence que les facteurs MEF2, et plus précisément les facteurs MEF2A et MEF2D, sont présents dans la lignée cellulaire de Leydig MA-10 et qu’ils activent l’expression du gène Nr4a1 ainsi que du gène Star. NR4A1 est connu comme un régulateur de l’expression de gènes stéroïdogéniques et STAR comme réalisant une étape limitante de la stéroïdogenèse. De plus, MEF2 régule l’expression de ces gènes en coopération avec la CAMKI, la forskoline ou l’AMPc. Ces molécules sont connues pour mimer l’activation par la LH ou faire partie d’une des voies activées par la LH. De plus, MEF2 est exprimé dans le testicule tout au long du développement embryonnaire et de la vie adulte mais à aucun de ces stades dans l’ovaire. Ceci suggère un/des rôle(s) bien particulier(s) de MEF2 dans le développement et la fonction de la gonade mâle. Afin de mieux comprendre son (ses) rôle(s), des expériences de micropuces ont été réalisées à partir de cellules de Leydig dans lesquelles l’expression de MEF2 a été diminuée par des petits ARN interférents. Ces expériences ont permis d’identifier des nouveaux gènes cibles de MEF2 dans les cellules de Leydig. Ma thèse a donc permis d’identifier un nouveau facteur de transcription dans les cellules de Leydig et de commencer à décrypter son rôle dans ces cellules. / Leydig cells are the main steroidogenic cells in the testis. Steroidogenesis is an essential mechanism for the development of male characteristics during embryogenesis and puberty and their maintenance throughout adulthood. Therefore, steroidogenesis is tightly regulated by the hypothalamo-pituitary axis, but also directly within the Leydig cell. One mechanism that occurs in Leydig cells is the regulation of steroidogenic gene expression by transcription factors. During my PhD, I have identified MEF2 as new transcription factors present in Leydig cells. These factors are member of the MEF2 family of transcription factors which contains four members: MEF2A, 2B, 2C and 2D. MEF2 factors and more specifically MEF2A and MEF2D factors are present in the MA-10 Leydig cell line and they activate Nr4a1 and Star gene expression. NR4A1 is known as a key regulator of several steroidogenic genes and STAR is essential for the rate-limiting step in steroidogenesis. Furthermore, MEF2 was found to regulate expression of these genes in cooperation with CAMKI, cAMP or forskolin. These molecules are known to mimic the LH activation pathways. Moreover, MEF2 is present in the testis throughout embryonic development and into adulthood whereas MEF2 expression was not detected at any stage in the ovary. This suggests broad roles for MEF2 factors in male gonadal formation and function. To better understand the role(s) of MEF2, microarray experiments were performed using Leydig cells in which MEF2 expression was downregulated by siRNA. These experiments lead to the identification of several new MEF2 target genes in Leydig cells. In conclusion, during my doctoral work, I was able to identify a novel transcription factor in Leydig cells and to characterize its role in these cells.

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-1<font face=\"Symbol\">a e NF-<font face=\"Symbol\">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-<font face=\"Symbol\">a. Nesses tecidos foram avaliados o conteúdo das proteínas GLUT4 e mTOR (Western), o conteúdo de mRNA de GLUT4, NF-<font face=\"Symbol\">kB1, HIF-1<font face=\"Symbol\">a e MEF2A/C/D (PCR) e a atividade de ligação de proteínas nucleares no sítio de ligação de NF-<font face=\"Symbol\">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-<font face=\"Symbol\">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-1<font face=\"symbol\">a and NF-<font face=\"Symbol\">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-<font face=\"Symbol\">a. In these tissues were evaluated the GLUT4 and mTOR protein content (Western), the content of GLUT4, NF-<font face=\"Symbol\">kB1, HIF-1<font face=\"Symbol\">a and MEF2A/C/D mRNAs (PCR) and the binding activity of protein nuclear in binding site of NF-<font face=\"Symbol\">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-<font face=\"Symbol\">kB.