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.

The MEF2 transcription factor is a central regulator of skeletal and cardiac muscle development. Recently, we showed that MEF2A regulates skeletal muscle regeneration through direct regulation of the Gtl2-Dio3 microRNA mega-cluster. In addition to their expression in skeletal muscle, temporal expression analysis of selected Gtl2-Dio3 microRNAs revealed high enrichment in cardiac muscle. Therefore, I investigated the role of selected microRNAs from the Gtl2-Dio3 noncoding RNA locus in the heart. First, I found that Gtl2-Dio3 microRNAs are expressed at higher levels in perinatal hearts compared to adult, suggesting they function in cardiac maturation shortly after birth. I also demonstrated that these microRNAs are dependent on MEF2A in the perinatal heart and in neonatal cardiomyocytes. To determine the specific role in cardiac muscle, I overexpressed selected microRNA mimics in neonatal rat ventricular myocytes (NRVMs). My results showed that miR-410 and miR-495 stimulate cell cycle re-entry and proliferation of terminally differentiated NRVMs. Subsequent target prediction analyses revealed a number of candidate target genes known to function in the cell cycle and/or in cardiac muscle. One of these was Cited2, a cofactor required for proper cardiac development. Subsequently, I showed that Cited2 is a direct target of these miRNAs and that siRNA knockdown of Cited2 in NRVMs resulted in robust cardiomyocyte proliferation. This phenotype was associated with reduced expression of Cdkn1c/p57/Kip2, a cell cycle inhibitor, and increased expression of Vegfa, a growth factor with proliferation-promoting effects. Given the exciting possibility of manipulating the expression of these microRNAs to repair the damaged heart by stimulating cardiomyocyte proliferation, I then investigated whether they were regulated in cardiac disease and function in pathological signaling. Toward this end, I examined expression of miR-410, miR-495, miR-433, as well as the Gtl2 lncRNA in various cardiomyopathies. Interestingly, the microRNAs and lncRNA were dynamically regulated in mouse models of cardiac disease including myocardial infarction and chronic angiotensin II stimulation. Furthermore, I showed for the first time that the Gtl2 lncRNA and miRNAs are differentially regulated in myocardial infarction, indicating that the complex regulation of the Gtl2-Dio3 noncoding RNA locus may be important for response to cardiac injury. Lastly, I showed that inhibiting select Gtl2-Dio3 microRNAs in pathological signaling attenuated cardiomyocyte hypertrophy in vitro. Therefore, differential targeting of the Gtl2-Dio3 noncoding RNAs could provide new therapeutic strategies to control the response of the heart to cardiac diseases with diverse etiologies.

Regulation of striated muscle differentiation and development are complex processes coordinated by an array of transcription factors. MEF2 is a crucial transcription factor required for muscle differentiation, but the roles of the individual MEF2 family members, MEF2A-D, have not been extensively evaluated. Acute ablation of Mef2 expression in skeletal myoblasts revealed a required role for MEF2A activity in myoblast differentiation that was not shared with the other MEF2 factors. We hypothesized that a transcriptomic level analysis of Mef2-deficient skeletal myoblasts would reveal distinct regulatory roles for each MEF2 isoform. Comparative microarray analysis supported our hypothesis and we observed distinct gene programs preferentially-sensitive to individual MEF2 isoforms. While there was no variance in the consensus binding site associated with regulation by individual MEF2 isoforms, we did observe uniquely enriched binding sites for candidate co-regulatory proteins that mediate these complex regulatory patterns. Based on our observations in skeletal myoblasts, we performed a series of acute Mef2 knockdowns in neonatal cardiomyocytes and uncovered a requirement for MEF2A and -D, but not MEF2C in cardiomyocyte survival. Comparative microarray analysis confirmed that, similar to skeletal myoblasts, the MEF2 family regulated distinct but overlapping gene programs in cardiomyocytes. Additionally, this analysis uncovered a previously uncharacterized antagonistic regulation of a subset of cell cycle and sarcomere genes. Interestingly, Mef2a and -d knockdowns caused an upregulation of cell cycle markers and downregulation of sarcomere genes, with the opposite regulatory pattern in Mef2c knockdown. Further investigation of the proximal promoter region of these genes revealed enriched binding sites for transcription factors associated with key signaling pathways in the developing embryo, Hedgehog and Notch. Overexpression of constitutively active components of these signaling pathways revealed that Notch function requires the presence of MEF2A and -D, while Hedgehog does not appear to interact with these two isoforms. We have shown through our studies that MEF2, a core muscle transcription factor, takes part in complex regulatory interactions that are critical for the appropriate development of striated muscle tissues. / 2018-08-09T00:00:00Z

Alcohol use disorder is a global health issue that affects a significant portion of the population, with affects including both negative mental and physical consequences. Currently, there are few treatment options available to those who suffer from alcohol use disorder, alcohol abuse, or alcohol dependence. Identifying candidate genes or environmental influences would therefore improve the means for possible treatments or identification of those people at risk for alcohol use disorder. Previous studies in humans have demonstrated an inverse association between initial sensitivity and risk for alcohol abuse. This connection allows investigators, and our laboratory, to investigate genetic and environmental factors that may influence initial ethanol sedation. Our laboratory utilizes Drosophila melanogaster (flies) as a model organism to identify these such factors influencing acute behavioral responses to alcohol. Our lab has found evidence for both environmental and genetic factors that influence initial alcohol sensitivity in flies. In one study, flies that are fed increased amounts of dietary yeast are resistant to ethanol. We have found that this ethanol resistance is related to the amount of nutrients that is consumed, which then affects alcohol uptake/metabolism, to influence initial alcohol sensitivity. Importantly, we found that serotonergic neuron function is essential for regulating the consumption of high dietary yeast media for the increased nutrient intake to occur. In two separate projects, we identified a role for myocyte enhancer factor 2 (Mef2) and nitric oxide synthase (Nos) in initial alcohol sensitivity. Mef2 was obtained via a GWAS study identifying genes with an association with initial sensitivity in humans. We found that decreasing or altering Mef2 expression, using mutants or Mef2 RNAi, resulted in flies having decreased sensitivity to alcohol. The gene Nos, came out of a previous genetic interaction screen in the laboratory. Multiple reagents to assess Nos’s role in alcohol behavior were obtained and consistent evidence from three piggyBac transposon insertion flies and, importantly, a Nos null fly, demonstrate that decreased Nos expression results in increased ethanol sensitivity. Other preliminary results suggest that Nos expression during adulthood, as well as the mechanism of S-nitrosation, may be important for ethanol sedation in Drosophila.

The Hedgehog (Hh) signalling pathway is one of the key signalling pathways orchestrating intricate organogenesis, including the development of neural tube, heart and skeletal muscle. Yet, insufficient mechanistic understanding of its diverse roles is available. Here, we show the molecular mechanisms regulating the neurogenic, cardiogenic and myogenic properties of Hh signalling, via effector protein Gli2, in embryonic and adult stem cells. In Chapter 2, we show that Gli2 induces neurogenesis, whereas a dominant-negative form of Gli2 delays neurogenesis in P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (ES) cell model. Furthermore, we demonstrate that Gli2 associates with Ascl1/Mash1 gene elements in differentiating P19 cells and activates the Ascl1/Mash1 promoter in vitro. Thus, Gli2 mediates neurogenesis in P19 cells at least in part by directly regulating Ascl1/Mash1 expression. In Chapter 3, we demonstrate that Gli2 and MEF2C bind each other’s regulatory elements and regulate each other’s expression while enhancing cardiomyogenesis in P19 cells. Furthermore, dominant-negative Gli2 and MEF2C proteins downregulate each other’s expression while imparing cardiomyogenesis. Lastly, we show that Gli2 and MEF2C form a protein complex, which synergistically activates cardiac muscle related promoters. In Chapter 4, we illustrate that Gli2 associates with MyoD gene elements while enhancing skeletal myogenesis in P19 cells and activates the MyoD promoter in vitro. Furthermore, inhibition of Hh signalling in muscle satellite cells and in proliferating myoblasts leads to reduction in MyoD and MEF2C expression. Finally, we demonstrate that endogenous Hh signalling is important for MyoD transcriptional activity and that Gli2, MEF2C and MyoD form a protein complex capable of inducing skeletal muscle-specific gene expression. Thus, Gli2, MEF2C and MyoD participate in a regulatory loop and form a protein complex capable of inducing skeletal muscle-specific gene expression. Our results provide a link between the regulation of tissue-restricted factors like Mash1, MEF2C and MyoD, and a general signal-regulated Gli2 transcription factor. We therefore provide novel mechanistic insights into the neurogenic, cardiogenic and myogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo functions. These results may also be important for the development of stem cell therapy strategies.

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.

Η cAMP αποτελεί ένα σημαντικό δεύτερο μήνυμα που ρυθμίζει την ανοσολογική απόκριση. Η αύξηση της ενδοκυττάριας cAMP αυξάνει την παραγωγή της IL-10 από μονοκύτταρα. Σκοπός της μελέτης είναι η αποσαφήνιση της συμμετοχής της cAMP στην παραγωγή της IL-10 από Τ-λεμφοκύτταρα όπου τα δεδομένα είναι ακόμα ασαφή. Ανθρώπινα Τ-λεμφοκύτταρα περιφερικού αίματος διεγέρθηκαν με anti-CD3/anti-CD28, anti-CD3 ή Ionomycin/PMA παρουσία ή απουσία παραγόντων που αυξάνουν την ενδοκυττάρια cAMP (10-6 Μ Forskolin, 10-6 Μ PGE2, 5x10-6 Μ Rolipram και 10-6 Μ 8-Br-cAMP). Το πρωτεϊνικό προϊόν της IL-10 μετρήθηκε με ELISA ενώ η παραγωγή mRNA της IL-10 με Real Time PCR. Η ενεργότητα του υποκινητή της IL-10 ελέγχθηκε με διαμόλυνση των κυττάρων με πλασμίδια που φέρουν τον υποκινητή του γονιδίου (1327 bp) ή τμήματα αυτού (-1010, -500, -310, -235, -135 bp). Η δέσμευση των μεταγραφικών παραγόντων MEF-2 και CREB ελέγχτηκε σε πυρηνικά πρωτεϊνικά εκχυλίσματα με πειράματα EMSA, ενώ η ενεργότητα τους ελέγχθηκε με πειράματα διαμολύνσεως με πλασμίδια που ελέγχουν την ενεργότητα της λουσιφεράσης υπό τον έλεγχο των MEF-2 και CREB. Αύξηση της cAMP ελαττώνει την παραγωγή της IL-10 σε πρωτεϊνικό επίπεδο κατά 50-60% μετά από διέγερση με Ion/PMA, και κατά 80-90% με anti-CD3 ή με anti-CD3/anti-CD28. Η IL-10 παράγεται ακόμα και μετά από διέγερση μόνο με anti-CD3, εύρημα ειδικό για την IL-10, καθώς δεν παρατηρήθηκε αύξηση της παραγωγής άλλων κυτταροκινων (IL-2 & IL-4). Η ελάττωση της παραγωγής της IL-10 αντανακλάται και σε επίπεδο mRNA όπου οι αντίστοιχες μειώσεις είναι κατά 50% με όλους τους τρόπους διέγερσης. Η ενεργότητα του υποκινητή της IL-10 δεν επηρεάζεται από αλλαγές στα επίπεδα της cAMP όταν η διεγερση παρακάμπτει τον Τ κυτταρικό υποδοχέα. Ωστοσο, μειώνεται παρουσία αυξημένων συγκεντρώσεων cAMP όταν τα κύτταρα διεγείρονται μέσω του Τ κυτταρικού υποδοχέα. Το τμήμα του υποκινητή της IL-10 που επηρεάζεται από την ανασταλτική δραση της cAMP (50 % αναστολή) βρίσκεται στις πρώτες 500 bp πριν το TATA box, και περιέχει σημεία πρόσδεσης των μεταγραφικών παραγόντων MEF-2 και CREB, όπως ελέγχθηκε με το πρόγραμμα Consite. Η δέσμευση του MEF-2 σε πυρηνικά εκχυλίσματα διεγερμένων Τ-λεμφοκυττάρων μειώνεται κατά 70% παρουσία αυξημένης cAMP ενώ η ενεργότητα του δεν επηρεάζεται σημαντικά. Αντίθετα, η αύξηση της cAMP αυξάνει τόσο τη δέσμευση (x 2,5) όσο και την ενεργότητα του CREB (x 2). Η δράση της cAMP στην παραγωγή της IL-10 είναι ειδική για τα Τ-λεμφοκύτταρα και εξαρτάται από τον τρόπο διέγερσής τους. Η ρύθμισή της γίνεται τόσο σε μεταγραφικό όσο και σε μετά-μεταγραφικό επίπεδο. Η αύξηση της cAMP μπορεί να επηρεάσει την παραγωγή IL-10 από τα Τ-λεμφοκύτταρα παρεμβαίνοντας στην δέσμευση και την ενεργότητα των παραγόντων μεταγραφής MEF-2 και CREB. Ο τρόπος της αλληλεπίδρασης/συνεργασίας των MEF-2 και CREB παραμένει υπό διερεύνηση. / cAMP is a second messenger playing a crucial role in the signal transduction which controls the immune response, while IL-10 is considered to be an important regulator of this response. Elevation of intracellular concentration of cAMP has been shown to increase IL-10 production by monocytes. The aim of this study was the elucidation of the role of cAMP in IL-10 production by normal T lymphocytes, a mechanism that remains unclear. Fresh Human Τ-lymphocytes derived from PBMC of healthy donors where stimulated with anti-CD3/anti-CD28 or Ionomycin/PMA, in the presence or absence of cAMP elevating agents (10-6 Μ Forskolin, 10-6 Μ PGE2, 5x10-6 Μ Rolipram and 10-6 Μ 8-Br-cAMP). The protein product of IL-10 was measured by ELISA, the production of IL-10 mRNA by Real Time PCR and IL-10 mRNA stability was determined by the use of Actinomycin D (10 μM). The activity of IL-10 promoter was measured by luciferase reporter assay, after transfection of cells with plasmids carrying the wild type promoter (1037bp) or promoter fragments (constructs of -1010, -500, -310, -235, -135bp). PKA role was examined either by cotransfection experiments with a plasmid carrying a constitutively active mutant of the catalytic subunit of PKA-α isoform, or by the use of a specific PKA inhibitor Rp-8- Br-cAMP (10-50 μM). The presence of binding sites of transcription factors in the first 500bp of the IL-10 promoter, was validated using the web-based program CONSITE. Binding of the transcription factors MEF2 and CREB was investigated in nuclear extracts of stimulated human T cells with EMSA experiments. The activity of MEF2 and CREB was investigated independently with transfection experiments using plasmids containing the lusiferase reporter under the control of the transcription factors. Intracellular cAMP elevation, inhibits IL-10 protein production by 50-60%, when T cells are stimulated with Ionomycin/PMA, and by 80-90% after stimulation with anti-CD3 or anti-CD3/anti-CD28, while PKA blocking by Rp-8- Br-cAMP reversed cAMP mediated inhibition.. IL-10 steady state mRNA levels follow the same pattern of inhibition only after anti-CD3/anti-CD28 stimulation. cAMP elevation decreases IL-10 mRNA stability after I/PMA stimulation, whereas in the anti-CD3/anti-CD28 stimulated cells, the mechanism of inhibition is mainly transcriptional. IL-10 promoter activity is reduced up to 60% when cells are stimulated with anti CD3/anti CD28 in the presence of cAMP elevating agents, but is not affected after stimulation with Ionomycin/PMA or cotransfection of the cells with constitutively active PKA mutant. Transfection assays with the different IL-10 promoter fragments revealed that the most responsible part of IL-10 promoter to cAMP mediated inhibition, is the first 500 bp after the TATA box. This part contains binding sites for the transcription factors MEF-2 and CREB, as validated by the web-based program Consite. Increased intracellular cAMP reduces the binding of MEF2 to nuclear extracts of stimulated T cells by 70 %, however its activity is not affected significantly. On the contrary, both the binding and the activity of CREB are increased in the presence of elevated cAMP. cAMP mediated inhibition of IL-10 production is PKA mediated and specific for T lymphocytes, depending on the nature/strength of stimulation. cAMP-dependent regulation of IL-10 production is controlled by transcriptional and/or post-transcriptional mechanisms depending on the nature of stimulus. Transcriptional mechanisms involve the transcription factors MEF2 and CREB, however the exact mechanisms of action of these factors deserves further elucidation. Cell and stimulus specific mechanism of regulation of IL-10 production is necessary for its immunoregulatory function.

The Hedgehog (Hh) signalling pathway is one of the key signalling pathways orchestrating intricate organogenesis, including the development of neural tube, heart and skeletal muscle. Yet, insufficient mechanistic understanding of its diverse roles is available. Here, we show the molecular mechanisms regulating the neurogenic, cardiogenic and myogenic properties of Hh signalling, via effector protein Gli2, in embryonic and adult stem cells. In Chapter 2, we show that Gli2 induces neurogenesis, whereas a dominant-negative form of Gli2 delays neurogenesis in P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (ES) cell model. Furthermore, we demonstrate that Gli2 associates with Ascl1/Mash1 gene elements in differentiating P19 cells and activates the Ascl1/Mash1 promoter in vitro. Thus, Gli2 mediates neurogenesis in P19 cells at least in part by directly regulating Ascl1/Mash1 expression. In Chapter 3, we demonstrate that Gli2 and MEF2C bind each other’s regulatory elements and regulate each other’s expression while enhancing cardiomyogenesis in P19 cells. Furthermore, dominant-negative Gli2 and MEF2C proteins downregulate each other’s expression while imparing cardiomyogenesis. Lastly, we show that Gli2 and MEF2C form a protein complex, which synergistically activates cardiac muscle related promoters. In Chapter 4, we illustrate that Gli2 associates with MyoD gene elements while enhancing skeletal myogenesis in P19 cells and activates the MyoD promoter in vitro. Furthermore, inhibition of Hh signalling in muscle satellite cells and in proliferating myoblasts leads to reduction in MyoD and MEF2C expression. Finally, we demonstrate that endogenous Hh signalling is important for MyoD transcriptional activity and that Gli2, MEF2C and MyoD form a protein complex capable of inducing skeletal muscle-specific gene expression. Thus, Gli2, MEF2C and MyoD participate in a regulatory loop and form a protein complex capable of inducing skeletal muscle-specific gene expression. Our results provide a link between the regulation of tissue-restricted factors like Mash1, MEF2C and MyoD, and a general signal-regulated Gli2 transcription factor. We therefore provide novel mechanistic insights into the neurogenic, cardiogenic and myogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo functions. These results may also be important for the development of stem cell therapy strategies.

The Hedgehog (Hh) signalling pathway is one of the key signalling pathways orchestrating intricate organogenesis, including the development of neural tube, heart and skeletal muscle. Yet, insufficient mechanistic understanding of its diverse roles is available. Here, we show the molecular mechanisms regulating the neurogenic, cardiogenic and myogenic properties of Hh signalling, via effector protein Gli2, in embryonic and adult stem cells. In Chapter 2, we show that Gli2 induces neurogenesis, whereas a dominant-negative form of Gli2 delays neurogenesis in P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (ES) cell model. Furthermore, we demonstrate that Gli2 associates with Ascl1/Mash1 gene elements in differentiating P19 cells and activates the Ascl1/Mash1 promoter in vitro. Thus, Gli2 mediates neurogenesis in P19 cells at least in part by directly regulating Ascl1/Mash1 expression. In Chapter 3, we demonstrate that Gli2 and MEF2C bind each other’s regulatory elements and regulate each other’s expression while enhancing cardiomyogenesis in P19 cells. Furthermore, dominant-negative Gli2 and MEF2C proteins downregulate each other’s expression while imparing cardiomyogenesis. Lastly, we show that Gli2 and MEF2C form a protein complex, which synergistically activates cardiac muscle related promoters. In Chapter 4, we illustrate that Gli2 associates with MyoD gene elements while enhancing skeletal myogenesis in P19 cells and activates the MyoD promoter in vitro. Furthermore, inhibition of Hh signalling in muscle satellite cells and in proliferating myoblasts leads to reduction in MyoD and MEF2C expression. Finally, we demonstrate that endogenous Hh signalling is important for MyoD transcriptional activity and that Gli2, MEF2C and MyoD form a protein complex capable of inducing skeletal muscle-specific gene expression. Thus, Gli2, MEF2C and MyoD participate in a regulatory loop and form a protein complex capable of inducing skeletal muscle-specific gene expression. Our results provide a link between the regulation of tissue-restricted factors like Mash1, MEF2C and MyoD, and a general signal-regulated Gli2 transcription factor. We therefore provide novel mechanistic insights into the neurogenic, cardiogenic and myogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo functions. These results may also be important for the development of stem cell therapy strategies.

Chez le poisson zèbre, le muscle squelettique axial est composé de deux types de fibres musculaires différentes, les fibres lentes et les fibres rapides, organisées le long de l’axe antéro-postérieur et délimités par des frontières somitiques. Les cellules cuboïdes adaxiales, précurseurs des fibres lentes, sont les premières cellules musculaires à se différencier. En cours de somitogenèse elles s’allongent et migrent à partir de la notochorde radialement vers l’extérieur du somite formant une couche monocellulaire de fibres lentes mononuclées. Au sein de ces précurseurs en cours de réarrangement, se déroule l’initiation de la myofibrillogénèse. Ces premières étapes de formation des myofibrilles sont peu connues et nous aimerions comprendre les mécanismes sous-jacents liés à cette initiation. La structure et la composition du sarcomère sont conservées au cours de l’évolution, offrant la possibilité d’utiliser le poisson zèbre comme model afin de mieux comprendre les processus de myofibrillogénèse chez les Vertébrés et potentiellement d’expliquer l’origine des Myopathies Myofibrillaires qui affectent le développement des myofibrilles chez l’Homme. Récemment, nous avons montré que la perte de fonction la protéine Quaking A chez le poisson zèbre perturbait, entre autre, la maturation finale des fibres musculaires lentes. Cette protéine de liaison aux ARN fait partie de la famille des protéines à domaine STAR, elle possède généralement d’autres isoformes chez les Vertébrés. Au cours de ma thèse, j’ai identifié chez le poisson zèbre, par comparaison de séquence in silico, un homologue du gène qkiA que nous avons nommé qkiC. L’expression des gènes qkiA et qkiC est recouvrante sur le territoire des cellules adaxiales. Bien que la perte de fonction de QkiC n’ait aucuns effets sur développement des fibres lentes, la perte de fonction conjointe de QkiA et QkiC induit un phénotype cellulaire autonome sévère et ce, dès les stades précoce de myofibrillogénèse. Ensemble nos données suggèrent une interaction fonctionnelle des deux homologues dans les cellules adaxiales que nous avons cherché à comprendre et à décrire. Un phénotype similaire est induit par la perte de fonction des protéines Mef2C/D, Nous avons montré que ces deux voies agissent en parallèle afin d’initier et d’accompagner le programme de myofibrillogénèse. A 24hpf, une accumulation des protéines de Myosine et une dissection/désolidarisation des filaments épais sont observées dans les fibres lentes, fortement lié à une destruction importante de la bande-Z. Ces phénotypes sont similaires à ceux utilisés par les pathologistes pour décrire les Myopathies Myofibrillaires. Ainsi, notre étude montre un nouveau type de régulation précoce de la myofibrillogénèse et offre un model potentiel pour étudier chez le poisson zèbre les myopathies myofibrillaire. / In zebrafish, myotomes are organized along the antero-posterior axis within repeated units called somites. Contractile fibers are subdivided into two muscle cell types, the slow muscle fibers and the fast muscle fibers. The slow muscle cells are located on the surface of the embryo body while the fast muscle cells are located deeper in the somite, underneath the slow muscle cells. Myogenesis correspond to transitions from unspecified mesodermal cells to mature and functional muscle fibers. These cellular transitions have been extensively studied. However relatively little is known about early developmental mechanisms that are required to form premyofibrils, neither about maturation processes, during which premyofibrils evolved in contractile myofibers. This process called myofibrillogenesis involved a dynamic assembly of the elementary components of the sarcomere that occurred first in adaxial cells, the muscle precursors of slow muscle fibers. Here we show that QkiA and QkiC, two RNA-binding proteins with STAR domain, are required during the early step of myofibrillogenesis where Moysin proteins are not correctly assembled. This early phenotype leads to a strong and specific alteration in the maturation of thick Myosin filaments at 24hpf. The combined QkiA/QkiC loss of function induced a dissection of thick filaments followed by the accumulation of Myosin proteins at the tip of slow muscle cells in a cell autonomous manner. Interestingly, the loss of function of Mef2C/D, two myogenic enhancers from the same family, induced a similar phenotype. However we have shown that Quaking and Mef2 proteins act in parallel ways to control and regulate myofibrillogenesis. Remarkably, we have seen that the accumulation of Myosin, the dissection of thick filaments and the alteration of the Z-disk, induced by QkiA/C loss of function, are the pathologic phenotypes found in Human Myofibrillar Myopathies (MFM). This subgroup of myopathies has been created recently and very few is known about mechanisms involved in those diseases. We propose that QkiA and QkiC is another regulated system that is required to initiated and maintained myofibrillogenesis.