• ALTERANTIVE-SPLICING
• MEF2
• MUSCLE
• PHOSPHORYLATION
• REGENERATION

The MEF2 family of transcription factors, composed of four factors (MEF2A-D), regulates many developmental programs, including myogenesis. MEF2 gene transcripts are subjected to alternative splicing, which leads to protein isoforms with different functions. Among other MEF2 proteins, MEF2C has showed to not play a redundant function in muscle maturation.

MEF2C is an important regulator of the myogenic program and it is composed of 13 exons and its transcript is alternatively spliced. The alternative splicing affects alpha, beta and gamma exons – but only alpha exons are mutually exclusive. All of these exons play a different role in MEF2C function, such as, when beta exon is included MEF2C is more transcriptionally active than when is included the gamma exon – which inhibits its transcriptional activity.

The two alpha exons, alpha1 and alpha2 are mutually exclusive and play different roles during myogenesis. It was seen that MEF2Cα2 is required for enhancing MEF2C myogenic activity, on the other hand MEF2Cα1 appears to inhibit the transcription of muscle-related genes, such as MyoD.

These two variants differ also for post-translational modifications, as a matter of fact MEF2Cα1 presents in its amino acidic sequence two relevant phosphoserines residues followed by a proline (SP motif), located respectively at 98 and 110 amino acidic residues. These phosphoserines regulate MEF2Cα1 stability and function during myogenesis and muscle regeneration.

In order to evaluate the role of alpha exons and its phosphorylation status during muscle regeneration, I analysed via H&E and Immunostaining the biological role of these splicing variants and of the MEF2Cα1-2SA – a MEF2Cα1 mutant protein that presents two alanine residues instead of Ser98 and Ser110, so it cannot be phosphorylated. In this way, I was able to understand the different roles played by the two alpha exons and how phosphorylation could affect MEF2Cα1 activity.

These analyses revealed that MEF2Cα1 when phosphorylated is able to promote cell proliferation, as demonstrated by Ki67 Immunostaining analysis – on the other hand the mutant, MEF2Cα1-2SA, activates the IGF-Akt-mTOR pathway, which regulates muscle hypertrophy. These results demonstrated that MEF2Cα1 has a double activity depending on its phosphorylation status.

MEF2Cα2 showed a strong pro-myogenic and pro-differentiation activity compared to MEF2Cα1 isoform – which is slightly traced by MEF2Cα1-2SA. This is probably due to the different ability of these MEF2C isoforms to interact with others transcription factors and their ability to interact with co-activators.

So, MEF2Cα1 depending on its phosphorylation status could regulate cell-fate promoting cell differentiation or inducing cell proliferation. In addition, I have described a new role of MEF2C α-exon, which is able to activate the Akt-Hypertrophic pathway – demonstrating that transcription factors could lead to the expression of different cell programs.

The MEF2 family of transcription factors, composed of four factors (MEF2A-D), regulates many developmental programs, including myogenesis. MEF2 gene transcripts are subjected to alternative splicing, which leads to protein isoforms with different functions. Among other MEF2 proteins, MEF2C has showed to not play a redundant function in muscle maturation. MEF2C is an important regulator of the myogenic program and it is composed of 13 exons and its transcript is alternatively spliced. The alternative splicing affects alpha, beta and gamma exons – but only alpha exons are mutually exclusive. All of these exons play a different role in MEF2C function, such as, when beta exon is included MEF2C is more transcriptionally active than when is included the gamma exon – which inhibits its transcriptional activity. The two alpha exons, alpha1 and alpha2 are mutually exclusive and play different roles during myogenesis. It was seen that MEF2Cα2 is required for enhancing MEF2C myogenic activity, on the other hand MEF2Cα1 appears to inhibit the transcription of muscle-related genes, such as MyoD. These two variants differ also for post-translational modifications, as a matter of fact MEF2Cα1 presents in its amino acidic sequence two relevant phosphoserines residues followed by a proline (SP motif), located respectively at 98 and 110 amino acidic residues. These phosphoserines regulate MEF2Cα1 stability and function during myogenesis and muscle regeneration. In order to evaluate the role of alpha exons and its phosphorylation status during muscle regeneration, I analysed via H&E and Immunostaining the biological role of these splicing variants and of the MEF2Cα1-2SA – a MEF2Cα1 mutant protein that presents two alanine residues instead of Ser98 and Ser110, so it cannot be phosphorylated. In this way, I was able to understand the different roles played by the two alpha exons and how phosphorylation could affect MEF2Cα1 activity. These analyses revealed that MEF2Cα1 when phosphorylated is able to promote cell proliferation, as demonstrated by Ki67 Immunostaining analysis – on the other hand the mutant, MEF2Cα1-2SA, activates the IGF-Akt-mTOR pathway, which regulates muscle hypertrophy. These results demonstrated that MEF2Cα1 has a double activity depending on its phosphorylation status. MEF2Cα2 showed a strong pro-myogenic and pro-differentiation activity compared to MEF2Cα1 isoform – which is slightly traced by MEF2Cα1-2SA. This is probably due to the different ability of these MEF2C isoforms to interact with others transcription factors and their ability to interact with co-activators. So, MEF2Cα1 depending on its phosphorylation status could regulate cell-fate promoting cell differentiation or inducing cell proliferation. In addition, I have described a new role of MEF2C α-exon, which is able to activate the Akt-Hypertrophic pathway – demonstrating that transcription factors could lead to the expression of different cell programs.