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Splicing alternativo, fosforilazione e interazione proteina-proteina conferiscono a MEF2C un nuovo ruolo pro-proliferativo in cellule staminali muscolari adulte
No full textIl 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.
Successivamente le SCs proliferanti spengono l’espressione di Pax7, mantenendo elevati i livelli di MyoD e attivando i fattori miogenici Miogenina e MRF4 per promuovere il differenziamento muscolare; infine le SCs si fondono fra loro o con le fibre danneggiate per generare nuove miofibre o per riparare quelle esistenti. Alcune SCs attivate non vanno incontro a differenziamento, ma tornano allo stato di quiescenza per ricostituire il pool di cellule staminali muscolari.
La corretta attivazione del programma miogenico dipende anche dalla presenza di fattori trascrizionali appartenenti alla famiglia MEF2 (Myocyte Enhancer Factor 2), in particolare è stato dimostrato che le proteine MEF2C e MEF2A contribuiscono a regolare l'espressione MyoD e al processo di rigenerazione muscolare.
L’attività del fattore MEF2C è finemente modulata a vari livelli, ma alcuni aspetti di questa regolazione rimangono ancora non ben caratterizzati, per esempio MEF2C è già espresso in mioblasti proliferanti, dove però è trascrizionalmente silente finchè le cellule non vengono stimolate a uscire dal ciclo cellulare per differenziare.
La fosforilazione delle proteine MEF2 nei cosiddetti motivi amminoacidici Ser/Thr-Pro può modulare la funzione delle proteine attraverso l'induzione di modificazioni conformazionali catalizzate dalla peptidyl prolyl cis/trans isomerasi Pin1.
In passato nel nostro laboratorio sono stati individuati due siti fosfoaccettori di MEF2C, Ser98 e Ser110, situati nell’esone alternativo α1, essenziali per il legame con il repressore Pin1.
Nel presente lavoro viene indagato un nuovo meccanismo di regolazione che coinvolge la fosforilazione MEF2C e l'interazione con Pin1 e che potrebbe rivestire un ruolo importante per consentire l'espansione del pool di SCs dopo l'attivazione, evitando un differenziamento prematuro.
Ho dimostrato che la fosforilazione di MEF2C sui siti di legame per Pin1 influenza negativamente la funzione pro-miogenica di MEF2C e promuove la proliferazione.
Ho inoltre combinato l'analisi della dinamica di fosforilazione MEF2C sulla Ser98 e Ser110 con lo studio del pattern di splicing alternativo del trascritto di mef2c durante la progressione miogenica delle SCS.
In particolare ho notato che, nonostante MEF2C e Pin1 siano espressi in SCs quiescenti, proliferanti e differenziate, i requisiti necessari per l'interazione tra MEF2C e Pin1 sono soddisfatti esclusivamente nelle SC proliferanti, dove Pin1 è localizzato nel nucleo e l’isoforma MEF2Cα1 fosforilata su Ser98 e Ser110 inizia ad essere espressa; mediante saggio di complementazione bimolecolare (BiFC) ho dato prova dell'interazione fisica tra Pin1 e MEF2Cα1 in SC MyoD+.
Inoltre l’espressione costitutiva di MEF2Cα1 o di Pin1 aumenta il tasso di proliferazione delle SCs, mentre con il mutante di fosforilazione di MEF2C il differenziamento miogenico viene accelerato.
Nel complesso tali risultati mi permettono di ipotizzare che la fosforilazione dell’isoforma α1 di MEF2C sulla Ser98 e Ser110 e la conseguente interazione specifica con Pin1 gioca un ruolo importante nella regolazione delle SCs proliferanti, contribuendo a mantenere silente la trascrizione MEF2C-dipendente di geni muscolo-specifici per promuovere l'espansione delle SCs ed evitare un differenziamento precoce, garantendo così una corretta rigenerazione.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
Role of the CCAAT-transcription factor NF-Y and its splice variants in myogenic differentiation and muscle regeneration
No full textThe expression of NF-YA, the DNA-binding subunit of the CCAAT-binding transcription factor NF-Y, is down-regulated in adult skeletal muscle to allow a complete terminal differentiation. The NF-YA gene encodes for two alternative splice variants, NF-YAs and NF-YAl: in human haematopoietic and mouse embryonic stem cells, the expression of NF-YAs or NF-YAl is crucial to promote proliferation or differentiation, respectively. The role of NF-YA isoforms in skeletal myogenesis and satellite cells fate has never been investigated. Here we show that both NF-YA isoforms are induced in mouse regenerating muscle and are expressed in satellite cells (SCs).
The abrogation of NF-Y activity impairs both proliferation and differentiation of SCs. Forced expression of NF-YAs stimulates SCs proliferation, while NF-YAl enhances their differentiation. The same effects are observed in mouse C2C12 myoblasts, in which the two isoforms activate opposite transcriptional programs.
NF-YAs upregulates genes annotated in growth factor binding, cell adhesion and extra cellular matrix Gene Onthology terms, while NF-YAl increases the transcription of genes belonging to sarcomere, muscle cell differentiation, development and muscle contraction categories. NF-YAl boosts myoblasts differentiation by up-regulating the transcription of novel NF-Y target genes, among which Mef2D, Six4 and Cdkn1C, which are known to be involved in the differentiation program. Overall, our results highlight the functional difference between NF-YA isoforms, whose modulation could be useful to improve stem cell based therapies to treat muscular dystrophy
Regulation of Mef2c activity by alternative splicing in zebrafish muscle development
No full textTeleost fish possess two Mef2c gene paralogues: mef2ca and mef2cb. In zebrafish, Danio rerio, the Mef2cs proteins function to promote cardiomyogenic differentiation and myofibrilogenesis in nascent skeletal muscle fibers. In mouse and human, an array of MEF2C proteins are generated by alternative splicing (AS), yet specific functions have not been ascribed to each isoform in vivo. We found that the transcripts of zebrafish mef2ca and mef2cb are alternatively spliced, the resulting splice variants have differing biological activity. Among the two mef2c paralogues, mef2ca is more abundantly expressed in developing skeletal muscle and the splicing pattern of its transcript is tuned through zebrafish development. At 24 hours post fertilization (hpf), a stage when mef2ca activity is required for proper myogenesis, we found the expression of a highly active full length protein. At earlier stages mef2ca activity is attenuated by AS, indeed a high proportion of the mef2ca transcripts lack part of the transcriptional activation domain. We present evidence that the developmentally regulated AS of mef2ca transcripts is important for a correct development of the zebrafish embryos
Phosphorylation-dependent degradation of MEF2C contributes to regulate G2/M transition
No full textThe Myocyte Enhancer Factor 2C (MEF2C) transcription factor plays a critical role in skeletal muscle differentiation, promoting muscle-specific gene transcription. Here we report that in proliferating cells MEF2C is degraded in mitosis by the Anaphase Promoting Complex/Cyclosome (APC/C) and that this downregulation is necessary for an efficient progression of the cell cycle. We show that this mechanism of degradation requires the presence on MEF2C of a D-box (R-X-X-L) and 2 phospho-motifs, pSer98 and pSer110. Both the D-box and pSer110 motifs are encoded by the ubiquitous alternate α1 exon. These two domains mediate the interaction between MEF2C and CDC20, a co-activator of APC/C. We further report that in myoblasts, MEF2C regulates the expression of G2/M checkpoint genes (14-3-3γ, Gadd45b and p21) and the sub-cellular localization of CYCLIN B1. The importance of controlling MEF2C levels during the cell cycle is reinforced by the observation that modulation of its expression affects the proliferation rate of colon cancer cells. Our findings show that beside the well-established role as pro-myogenic transcription factor, MEF2C can also function as a regulator of cell proliferation
A POTENTIAL ROLE OF MEF2C IN THE MYOGENIC PROGRESSION BESIDE TERMINAL DIFFERENTIATION
No full textMEF2C belongs to the family of Myocyte Enhancer Factor 2 transcription factors, which activate the muscle-specific gene expression program. Its 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. Phosphorylation of MEF2 factors at so-called Ser/Thr-Pro motifs can modulate protein function through the induction of conformational changes by the peptidyl–prolyl cis/trans isomerase Pin1. This regulatory mechanism is based on the physical interaction between Pin1 and the Ser98 and Ser110 phosphoacceptor sites located in the alternative spliced exon α1 of MEF2C. The MEF2C/Pin1 interaction results in the repression of MEF2C stability and transcriptional activity, inhibiting muscle differentiation.
We investigated the function of this regulatory mechanism in primary myogenic stem cells (SCs) combining the analysis of the dynamics of MEF2C phosphorylation with the study of the alternative splicing pattern.
We demonstrated that the conditions necessary for the interaction between MEF2C and Pin1 are satisfied exclusively in proliferating SCs, where:
Pin1 expression is upregulated
MEF2C isoform containing the exon α1 specifically appears in response to activation signals
MEF2C phosphorylation on the Pin1-binding sites occurs specifically in proliferating SCs
Indeed we showed that MEF2C and Pin1 can interact in the nuclei of C2C7 and SCs-derived myoblasts. Overall we provide evidence that this interaction not only would serve as a failsafe mechanism to keep silent the MEF2C-dependent transcription of muscle specific genes in proliferating SCs but we hypothesize that the expression of the MEF2Cα1 isoform phosphorylated on Ser98 and Ser110 and the interaction with Pin1 might actively contribute to promote SCs proliferation, allowing the expansion of the activated SCs pool and avoiding their premature differentiation
NF-Y splice variants differentially affect skeletal myogenesis
No full textThe mechanisms that regulate skeletal muscle development involve the coordinated activity of transcription factors (TFs) and the precise timing of gene expression patterns. NF-Y is a heterotrimeric TF with a pioneer role in the transcriptional and epigenetic regulation of promoters containing the CCAAT-box. NF-Y activates the expression of various genes related to the cell cycle, particularly genes of the G2/M phase.
NF-YA, the regulatory DNA-binding subunit of the complex, is expressed in proliferating myoblasts and down-regulated during terminal differentiation. The NF-YA gene encodes for two alternatively spliced isoforms, namely NF-YAs and NF-YAl, which are not functionally identical.
Using mouse C2C12 cells, we provide evidence of a different role for NF-YA variants in the myogenic program. While NF-YAs enhances myoblasts proliferation, NF-YAl boosts their differentiation by up-regulating the transcription of novel target genes, among which Mef2D, Sixs and Cdkn1C, which are known to be involved in the differentiation program.
We further demonstrate that NF-YA is expressed in resident stem cells (SCs) and the two isoforms are transcribed at different levels during SCs activation and differentiation. The inhibition of NF-Y activity impairs both proliferation and differentiation of SCs and the overexpression of the two NF-YA isoforms differentially affects their fate
IL FATTORE DI TRASCRIZIONE MEF2C AI CROCEVIA TRA PROLIFERAZIONE, SVILUPPO E DIFFERENZIAMENTO MUSCOLARE
No full textIl principale interesse del laboratorio è lo studio della funzione della proteina MEF2C, un fattore di trascrizione abbondante nel tessuto muscolare scheletrico dove dirige il differenziamento terminale dei precursori miogenici (mioblasti). In specifico, ci occupiamo di definire i meccanismi che regolano la funzione di MEF2C nella progressione miogenica. Abbiamo identificato un meccanismo di repressione dell'attività di MEF2C nei mioblasti proliferanti che coinvolge la fosforilazione della proteina e la sua successiva interazione fosfo-specifica con l'enzima Pin1, una peptidil-prolil cis trans isomerasi che diminuisce la stabilità di MEF2C. Questo meccanismo repressivo è importante per inibire un differenziamento prematuro dei precursori miogenici che in tal modo possono amplificarsi. La repressione Pin1-dipendente di MEF2C è attiva sia nella linea cellulare miogenica C2C12 che in cellule satelliti, vale a dire mioblasti primari di muscolo adulto, responsabili dei meccanismi di rigenerazione in caso di danno muscolare. Ci proponiamo di valutare se questo meccanismo è alterato nella distrofia di Duchenne, dove è stata osservata una diminuita capacità proliferativa delle cellule satelliti. L'attività di MEF2C è anche regolata da meccanismi di splicing alternativo del corrispondente trascritto. Nel nostro laboratorio stiamo definendo le funzioni specifiche di due isoforme di splicing di MEF2C che si differenziano per la presenza di due esoni mutualmente esclusivi: esone alpha1 e alpha 2. I nostri dati preliminari indicano che le due varianti di splicing svolgono funzioni opposte nel corso della progressione miogenica, in particolare la variante alpha1 sembra essere importante nel controllare la progressione nel ciclo delle cellule muscolari mentre la variante alpha 2 sembra essenziale per la trascrizione muscolo-specifica. Infine abbiamo osservato che in Zebrafish, Danio rerio, Mef2c svolge un ruolo importante nel regolare lo sviluppo embrionale, specificamente nella determinazione dorso-ventrale dell'embrione, inoltre la sua attività viene modulata attraverso meccanismi di splicing anche in questo organismo
A specific role for the splice variants of the transcription factor NF-Y in modulating the transcriptional activity of the myogenic program
No full textCell proliferation and differentiation programs are highly regulated transcriptional processes essential for myogenesis. The transcription factor NF-Y has been long considered a fundamental player of cell growth by supporting the basal transcription of various cell cycle genes. It is composed by the NF-YB/NF-YC heterodimer and NF-YA, which interacts with the other two subunits and confers the strict sequence specificity to the complex. The NF-YA gene encodes two alternatively splice transcripts (NF-YAs and NF-YAl), which differently regulate cell proliferation and differentiation, as shown in haematopoietic and mouse embryonic stem cells.
NF-YAl expression is down-regulated in terminally differentiated muscle cells and in skeletal and cardiac muscle tissues. Its forced expression in muscle cells committed to differentiate impairs their exit from the cell cycle and indirectly interferes with the differentiation program. Here we show that the two NF-YA isoforms play a different role in the transcriptional activity of the myogenic program and may regulate the activity of muscle satellite cells
Proline Isomerase Pin1 represses terminal differentiation and Myocyte Enhancer Factor 2C function in Skeletal Muscle Cells
No full textMEF2 (myocyte enhancer factor 2) transcription factors (MEF2A-D) are highly expressed in skeletal muscle cells, they bind to a conserved AT rich DNA sequence through their N-ter MADS and MEF2 domains and activate transcription via their C-ter transcriptional activation domains (TAD), the functional domains of MEF2C are indicated in Figure 1. MEF2 proteins interact with members of the MyoD family of basic helix–loop–helix (bHLH) proteins to establish a unique transcriptional code for skeletal muscle gene activation. Recent studies have revealed multiple signaling systems that stimulate and inhibit myogenesis by altering MEF2 phosphorylation and its association with other transcriptional cofactors. We show that the Pin1 isomerase, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds, interacts with phosphorylated MEF2C in muscle cells. This interaction requires two novel phospho-Ser-Pro motifs in MEF2C: Ser(98) and Ser(110), which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenesis. Furthermore Pin1 modulates the skeletal muscle differentiation program because down-regulation of Pin1 markedly promotes myogenic differentiation. We suggest that Pin1 is a novel regulator of MEF2C function and muscle differentiation, it is expressed in muscle cells and a significant proportion of Pin1 in myotubes but not in myoblasts is excluded from the nucleus. We observed a reduction of phosphorylation of the Ser(98) and Ser(110) Pin1 binding sites in differentiated myocytes. Based on these results we propose a model in which, in proliferating myoblasts, Pin1, upon binding to phosphorylated nuclear MEF2C, leads to decreased levels and transcriptional activity of MEF2C. Upon induction of terminal differentiation, to establish a full activity of MEF2 proteins, a reduced Pin1-MEF2C association is required, possibly due to the relegation of Pin1 to the cytoplasm and to a reduced level of phosphorylation of Ser98 and Ser110
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