Orientador: Lucia Elvira Alvares / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T08:30:05Z (GMT). No. of bitstreams: 1 Mantovani_CarolinaStefano_M.pdf: 7530088 bytes, checksum: 5c8cf5e001032d6962337caaccfcbcd8 (MD5) Previous issue date: 2015 / Resumo: A Miostatina (MSTN) é uma proteína que regula negativamente a formação de musculatura esquelética, e sua estrutura e função são conservadas em diversas espécies, incluindo humanos. O nocaute de seu gene causa hiperplasia e hipertrofia das fibras musculares, o que despertou grande interesse médico e agropecuário desde a descoberta deste gene. Trabalhos anteriores descrevem a função da proteína, mas ainda faltam dados sobre a regulação da sua expressão gênica. Recentemente, o promotor do gene da Mstn foi identificado e caracterizado pelo nosso grupo de pesquisa, a partir de uma abordagem filogenética. Entretanto, além do promotor, também existem outros elementos cis-reguladores que podem atuar como estimuladores ou silenciadores do processo de transcrição gênica. Em conjunto com o promotor gênico, esses elementos controlam a taxa de transcrição e conferem especificidade espacial e temporal para sua expressão. Sendo assim, a proposta deste trabalho foi identificar e caracterizar possíveis elementos cis-reguladores da Mstn a fim de ampliar o conhecimento sobre a sua regulação transcricional. Para tal, análises de genômica comparativa, semelhantes àquelas empregadas na identificação do promotor gênico da Mstn, foram realizadas para localizar regiões evolutivamente conservadas (ECRs) adjacentes ao lócus da Mstn de humano, camundongo e galinha, bem como para identificar potenciais sítios de ligação para fatores transcricionais conservados nessas regiões. Além disso, foi realizada uma análise da evolução desses possíveis elementos cis-reguladores, a fim de identificar polimorfismos que tenham o potencial de afetar a atividade transcricional da Mstn. Nossos resultados revelaram a existência de um arcabouço regulatório ancestral composto por sítios de ligação conservados em várias das ECRs analisadas, o qual tem sido mantido entre 310 e 430 milhões de anos. Além disto, foram identificados polimorfismos, bem como sítios de ligação grupo-específicos, os quais podem estar envolvidos na regulação diferencial da atividade da Mstn e, portanto, na geração de diferentes fenótipos musculares entre os animais. Dentre as oito ECRs estudadas, duas possuem maior potencial de estarem envolvidas na miogênese, uma vez que nelas foram identificados sítios de ligação para importantes fatores relacionados a este processo. Análises funcionais das ECRs 2 e 5/6 em células C2C12 em diferenciação confirmaram o potencial dessas ECRs de humano e camundongo de atuarem como elementos cis-reguladores, uma vez que mostraram que eles são capazes de modificar a expressão do gene repórter EGFP em comparação ao controle. As ECRs de galinha, por outro lado, não geraram resultados significativos, reforçando a hipótese de que as diferenças nos TFBS encontradas no grupo das aves geram alterações funcionais nas ECRs. O desenrolar de novos estudos a partir dos resultados obtidos neste trabalho permitirão estabelecer o papel específico das ECRs identificadas, bem como determinar a importância das variações de sequências destes elementos reguladores em diferentes animais. No longo prazo, nossas pesquisas poderão subsidiar o desenvolvimento de estratégias que possibilitem a modulação da atividade da Mstn em patologias humanas que afetam a musculatura esquelética / Abstract: The Myostatin protein (MSTN) is an important regulator of skeletal muscle deposition in vertebrates, and its structure and function are conserved in several species, including humans. The knockout of this gene causes hyperplasia and hypertrophy of muscle fibers, which has aroused great medical and agricultural interest since its discovery. Previous studies have described the function of this protein, but data on the regulation of Mstn gene expression are still scarce. Recently, the Mstn gene promoter has been identified and characterized by our research group from a phylogenetic approach. However, in addition to the promoter, there are also other cis-regulatory elements that can act as enhancers or silencers of gene transcription process. Along with the gene promoter, these elements control the transcription rate and provide spatial and temporal specificity for its expression. Thus, the purpose of this study was to identify and characterize potential Mstn cis-regulatory elements in order to increase knowledge of the transcriptional regulation of this gene. For this purpose, comparative genomic analysis similar to those employed in the identification of the Mstn promoter were performed to locate evolutionary conserved regions (ECRs) adjacent to the locus of the Mstn gene in human, mouse and chicken, as well as to identify potential transcription factors binding sites (TFBS) conserved in these regions. Furthermore, an evolutionary analysis of the putative cis-regulatory elements has been performed in order to identify polymorphisms that have the potential to affect the transcriptional activity of Mstn. Our results indicate the existence of an ancestral regulatory framework that comprises conserved TFBS in almost all of the ECRs analyzed, which has been maintained between 310 and 430 million years. Furthermore, polymorphisms were identified, as well as group-specific binding sites, which may be involved in the differential regulation of Mstn activity and, therefore, in the generation of different muscle phenotypes in animals. Among the eight ECRs studied, two of them have great potential to be involved in myogenesis, given that binding sites for important factors related to this processes were identified. Functional analyses of ECRs 2 and 5/6 in differentiating C2C12 cells confirmed the potential of the human and mouse ECRs as cis-regulatory elements, once they have shown that they are able to enhance the expression of the EGFP reporter gene, when compared to the control. The chicken ECRs, on the other hand, did not generate significant results, supporting the hypothesis that the differences found in the TFBS pattern in birds generate functional modifications in the ECRs. The development of new studies from the results obtained here may help to establish the specific role of the ECRs identified, as well as determine the importance of the variation in the sequence of these regulatory elements in different animals. In the long run, our research may lay the foundation to the development of strategies that allow the modulation of Mstn activity in human pathologies affecting skeletal muscle / Mestrado / Biologia Tecidual / Mestra em Biologia Celular e Estrutural

Même si les acteurs majeurs du développement musculaire ont été identifiés et les voies de transductions décrites, d’autres régulateurs restent encore à découvrir. Un crible ARNi pratiqué sur un modèle cellulaire couramment utilisé, la lignée myoblastique C2C12, a identifié 20 nouveaux gènes potentiellement impliqués dans la myogenèse in vitro. Au cours de ma thèse, deux de ces gènes ont été invalidés sur modèle souris en utilisant la technologie CRISPR/Cas9 pour valider in vivo leur implication. Pour l’un d’entre eux, seuls les animaux hétérozygotes ont pu être étudiés puisqu’une létalité précoce a été observée chez les homozygotes mutés. Aucune anomalie du développement musculaire n’a été mise en évidence. Une étude plus fine dans les premières phases du développement embryonnaire nous a permis de montrer le rôle indispensable de cette protéine précocement. L’étude du second gène – dont les analyses se poursuivent – semble confirmer in vivo le rôle de ce gène au cours de la myogenèse. Pour éviter la survenue de létalité embryonnaire et observer rapidement les effets de l’invalidation d’autres gènes, une technique de transgenèse somatique s’appuyant sur l’ARN interférence a été mis en place via l’injection de lentivirus contenant une cassette d’expression de shRNA directement dans le tibialis antérieur des souris. La validation de cette approche a été faite sur le gène de la myostatine, régulateur négatif du développement musculaire, et a montré une diminution de l’expression du gène associée à une augmentation de l’aire des fibres musculaires. La même approche appliquée à trois autres gènes renforce l’hypothèse de l’implication d’un des gènes dans le développement musculaire. Cette approche permet donc un crible rapide « in vivo » de gènes identifiés in vitro. Cependant, certaines améliorations doivent être apportées au protocole au regard des résultats obtenus. / Even if the major actors and transduction pathways of muscle development have been identified, there are still unknown regulatory factors. An in vitro RNAi screening performed on C2C12 myoblastic cells has permitted to identify 20 novel genes potentially implicated in myogenesis. During my thesis, two of these genes were invalidated on mouse model using CRISPR/Cas9 technology in order to confirm their implication in vivo. For the first gene, due to an early lethality occurring in homozygous mutated animals, only heterozygous animals were studied and there was no muscular development anomaly detected. A refined study of earlier stages of embryonic development permitted to show the essential role of the protein in these phases. The study of the second gene, still in progress, seems to confirm in vivo the implication of the gene on the myogenesis. In order to avoid embryonic lethality due to germline invalidation and to observe more rapidly the effects of gene invalidation in muscle, we developed a technique of somatic transgenesis based on RNA interference. Lentivirus containing a shRNA expression cassette was injected directly into the tibialis anterior of mice. We validated this approach on Myostatin gene, a well-known negative regulator of muscle development, showing that the decrease of Myostatin gene expression was associated to an increase of muscle fibers area. The same approach was used with three genes and support the hypothesis of the implication of one of them in muscle development. Thus, this approach allows a rapid “in vivo” screening of in vitro identified genes. Nonetheless, some improvements should be brought on the protocol according to the first results.

Skeletal muscle differentiation requires synergy between tissue-specific transcription factors, chromatin remodeling enzymes and the general transcription machinery. Here we demonstrate that two distinct protein arginine methyltransferases are required to complete the differentiation program. Prmt5 is a type II methyltransferase, symmetrically dimethylates histones H3 and H4 and has been shown to play a role in transcriptional repression. An additional member of the Prmt family, Carm1 is a type I methyltransferase, and asymmetrically methylates histone H3 and its substrate proteins. MyoD regulates the activation of the early class of skeletal muscle genes, which includes myogenin. Prmt5 was bound to and dimethylates H3R8 at the myogenin promoter in a differentiation-dependent fashion. When proteins levels of Prmt5 were reduced by antisense, disappearance of H3R8 dimethylation and Prmt5 binding was observed. Furthermore, binding of Brg1 to regulatory sequences of the myogenin promoter was abolished. All subsequent events relying on Brg1 function, such as chromatin remodeling and stable binding by muscle specific transcription factors such as MyoD, were eliminated. Robust association of Prmt5 and dimethylation of H3R8 at myogenin promoter sequences was observed in mouse satellite cells, the precursors of mature myofibers. Prmt5 binding and histone modification were observed to a lesser degree in mature myofibers. Therefore, these results indicate that Prmt5 is required for dimethylating histone at the myogenin locus during skeletal muscle differentiation in order to facilitate the binding of Brg1, the ATPase subunit of the chromatin remodeling complex SWI/SNF. Further exploration of the role of Prmt5 during the activation of the late class of muscle genes revealed that though Prmt5 is associated with and dimethylates histones at the regulatory elements of late muscle genes in tissue and in culture, it was dispensable for late gene activation. Previous reports had indicated that Carm1 was involved during late gene activation. We observed that Carm1 was bound to and responsible for dimethylating histones at late muscle gene promoters in tissue and in culture. In contrast to Prmt5, a complete knockout of Carm1 resulted in abrogation of late muscle gene activation. Furthermore, loss of Carm1 binding and dimethylated histones resulted in a disappearance of Brg1 binding and chromatin remodeling at late muscle gene loci. Time course chromatin immunoprecipitations revealed that Carm1 binding and histone dimethylation occurred concurrently with the onset of late gene activation. In vitro binding assays revealed that an interaction between Carm1, myogenin and Mef2D exists. These results demonstrate that Carm1 is recruited to the regulatory sequences of late muscle genes via its interaction with either myogenin or Mef2D and is responsible for dimethylates histones in order to facilitate the binding of Brg1. Therefore, these results indicate that during skeletal muscle differentiation, distinct roles exist for these Prmts such that Prmt5 is required for activation of early genes while Carm1 is essential for late gene induction.

The fast-skeletal-muscle-fiber-specific expression of the troponin I(fast) (TnIfast) gene is driven by an Intronic Regulatory Element (IRE) located within the first intron of the gene. The IRE is a 148 bp transcriptional enhancer that contains several known and suspected cis-regulatory elements. These include the E-box, the closely-spaced MEF2 site and CACT box, the CACC site, and the CAGG element. Previous loss-of-function studies performed using the quail TnIfast IRE suggest that its activity depended on the MEF2 and CACT elements. The goal of my thesis research was to determine whether the MEF2 and CACT sites were not only necessary, but also sufficient, to support IRE activity. I prepared head-to-tail multimers of a 27-bp IRE segment that consisted largely of the near-adjacent MEF2 and CACT elements and did not contain any other known/suspected elements. These multimers were cloned upstream of a reporter gene consisting of the minimal promoter of the quail TnIfast gene linked to sequences encoding human placental alkaline phosphatase. The transcriptional capabilities of the constructs were assessed by gene transfer into the mouse soleus muscle in vivo by intramuscular injection/electroporation, and histochemical analysis of reporter enzyme plap expression including quantitative microdensitometry. I found that expression of these constructs was readily detectable and that it was markedly reduced by prior mutation of the CACT and, especially, of the MEF2 sites. These data indicate that the short DNA segment containing MEF2 and CACT elements is sufficient to drive expression in skeletal muscle and confirms the functional importance of these specific elements. / Although constructs containing the wild-type IRE 27-bp region were expressed, there was little preferential expression in fast fibers, in contrast to expression driven by the complete 148-bp IRE. Thus my results indicate that the MEF2 and CACT elements are not sufficient to drive fast fiber-type-specific expression, and suggest that additional elements outside of the 27-bp region tested are also necessary for fiber-type-specificity.

No description available.

No description available.

No description available.

No description available.

No description available.

Skeletal muscle is a complicated and heterogeneous striated muscle tissue that serves critical mechanical and metabolic functions in the organism. The process of generating skeletal muscle, myogenesis, is elaborately coordinated by members of the protein kinase family, which transmit diverse signals initiated by extracellular stimuli to myogenic transcriptional hierarchy in muscle cells. Mitogen-activated protein kinases (MAPKs) including p38 MAPK, c-Jun N terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) are components of serine/threonine protein kinase cascades that play important roles in skeletal muscle differentiation. The exploration of MAPK upstream kinases identified mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4), a serine/threonine protein kinase that modulates p38 MAPK, JNK and ERK activities in multiple cell lines. Our lab further discovered that Map4k4 regulates peroxisome proliferator-activated receptor γ (PPARγ) translation in cultured adipocytes through inactivating mammalian target of rapamycin (mTOR), which controls skeletal muscle differentiation and hypotrophy in kinase-dependent and -independent manners. These findings suggest potential involvement of Map4k4 in skeletal myogenesis. Therefore, for the first part of my thesis, I characterize the role of Map4k4 in skeletal muscle differentiation in cultured muscle cells. Here I show that Map4k4 functions as a myogenic suppressor mainly at the early stage of skeletal myogenesis with a moderate effect on myoblast fusion during late-stage muscle differentiation. In agreement, Map4k4 expression and protein kinase activity are declined with myogenic differentiation. The inhibitory effect of Map4k4 on skeletal myogenesis requires its kinase activity. Surprisingly, none of the identified Map4k4 downstream effectors including p38 MAPK, JNK and ERK is involved in the Map4k4-mediated myogenic differentiation. Instead, expression of myogenic regulatory factor Myf5, a positive mediator of skeletal muscle differentiation is transiently regulated by Map4k4 to partially control skeletal myogenesis. Mechanisms by which Map4k4 modulates Myf5 amount have yet to be determined. In the second part of my thesis, I assess the relationship between Map4k4 and IGF-mediated signaling pathways. Although siRNA-mediated silencing of Map4k4 results in markedly enhanced myotube formation that is identical to the IGF-induced muscle hypertrophic phenotype, and Map4k4 regulates IGF/Akt signaling downstream effector mTOR in cultured adipocytes, Map4k4 appears not to be involved in the IGF-mediated ERK1/2 signaling axis and the IGF-mediated Akt signaling axis in C2C12 myoblasts. Furthermore, Map4k4 does not affect endogenous Akt signaling or mTOR activity during C2C12 myogenic differentiation. The results presented here not only identify Map4k4 as a novel suppressor of skeletal muscle differentiation, but also add to our knowledge of Map4k4 action on multiple signaling pathways in muscle cells during skeletal myogenesis. The effects that Map4k4 exerts on myoblast differentiation, fusion and Myf5 expression implicate Map4k4 as a potential drug target for muscle mass growth, skeletal muscle regeneration and muscular dystrophy.