|Tipo di tesi||Tesi di dottorato di ricerca|
|Titolo||Splicing alternativo, fosforilazione e interazione proteina-proteina conferiscono a MEF2C un nuovo ruolo pro-proliferativo in cellule staminali muscolari adulte|
|Titolo in inglese||Alternative splicing, phosphorylation and protein-protein interaction confer to MEF2C a novel pro-proliferative role in adult muscle stem cells|
|Settore scientifico disciplinare||BIO/10 - BIOCHIMICA|
|Corso di studi||Scuola di D.R. in MEDICINA MOLECOLARE E RIGENERATIVA|
|Data inizio appello||2014-04-09|
|Disponibilità||Accessibile via web (tutti i file della tesi sono accessibili)|
Il muscolo scheletrico è caratterizzato da una notevole capacità rigenerativa che deriva principalmente dalle cellule satellite (SCs), cellule staminali adulte, localizzate in una nicchia tra la lamina basale e il sarcolemma delle fibre muscolari. In condizioni normali le SCs sono mitoticamente quiescenti e caratterizzate dall’espressione del fattore trascrizionale Pax7, tuttavia in seguito a danno muscolare possono attivarsi e generare colonie di mioblasti che esprimono il determinante miogenico MyoD.
Skeletal muscle possesses remarkable regenerative capacity to repair muscle damage and the major contribution to skeletal muscle regeneration derives from satellite cells (SCs), adult stem cells located in a niche between the basal lamina and the sarcolemma of skeletal muscle fibers. In normal conditions SCs are mitotically quiescent and are characterized by the expression of the paired-box transcription factor Pax7, but upon muscle injury they can undergo rapid expansion, generating colonies of myoblasts that express MyoD, a member of the myogenic regulatory factors (MRFs) family. Later, satellite cell-derived myoblasts down-regulate Pax7, maintain MyoD and induce the MRFs Myogenin and MRF4 to promote differentiation and, finally, they fuse with each other or to existing myofibers to repair damage. Some of the activated SCs do not proliferate or differentiate, but self-renew and return to the quiescent state to replenish the SC pool. Efficient activation of the differentiation program depends also on the presence of proteins belonging to the Myocyte Enhancer Factor 2 (MEF2) family, in particular it has been shown that MEF2C and MEF2A are expressed in SCs and contribute to regulate MyoD expression and muscle regeneration. It is well known that MEF2C activity is finely modulated at several levels but some aspects of this regulation still remains uncharacterized; for example, MEF2C is already expressed in proliferating myoblasts, but it is transcriptionally silent unless the cells are stimulated to withdraw the cell cycle and differentiate. Several studies demonstrated that phosphorylation of MEF2 factors at so-called Ser/Thr-Pro amminoacidic motifs can modulate protein function through the induction of conformational changes catalyzed by the peptidyl–prolyl cis/trans isomerase Pin1. In the past we identified two novel critical phosphorylation sites in MEF2C, Ser98 and Ser110, located in the alternative spliced exon α1 and essential for the binding with the negative regulator Pin1. In the present work I investigate a new regulatory mechanism of MEF2C that involves MEF2C phosphorylation and its interaction with Pin1, which might play an important role in allowing the expansion of SCs pool, preventing their premature differentiation. I provide evidence that the phosphorylation of MEF2C on the Pin1 binding sites negatively affects MEF2C myogenic function and plays a positive role in promoting cell proliferation. I analyzed the dynamics of MEF2C phosphorylation on Ser98 and Ser110 together with the alternative splicing pattern of mef2c transcripts during myogenic progression of SCs. I showed that, although MEF2C and Pin1 are expressed in quiescent, proliferating and differentiating SCs, the conditions required for the interaction between MEF2C and Pin1 are satisfied exclusively in proliferating SCs, where Pin1 is nuclear localized and the MEF2C isoform with exon α1 phosphorylated on the Ser98 and Ser110 Pin1 binding sites specifically appears. I confirmed the physical interaction between Pin1 and MEF2Cα1 in MyoD+ SCs through a bimolecular complementation assay (BiFC). I observed that the constitutive over-expression of MEF2Cα1 or Pin1 increases the proliferation rate of SCs and that the infection with lentiviral vectors encoding for MEF2C protein mutated on the Pin1 binding sites accelerates myogenic differentiation. Taken together these results lead us to speculate that phosphorylation of MEF2Cα1 isoform on Ser98 and Ser110 and the consequent interaction with Pin1 plays an important role in proliferating SCs, contributing to keep silent the MEF2C-dependent transcription of muscle specific genes and promoting the expansion of the SCs pool to prevent a precocious differentiation and to guarantee a proper regeneration.