SNAI1 target genes in myoblasts

SNAI proteins are zinc finger transcription factors that act as transcriptional repressors through a conserved domain (SNAG domain) located in the N-terminus of the protein. These factors bind to a palindromic sequence of the E-box group (CANNTG) in the regulatory regions of their target genes. The role of SNAI11 and SNAI2 is well known in the epithelial mesenchymal transition, where they act as regulators increasing the capacity of tumor cells to metastasize. Less is known about their role as mediators in tissue homeostasis and differentiation. Recent studies have showed SNAI1 and SNAI2 as repressors of muscle differentiation, with the function of maintaining myoblasts in an undifferentiated state during the proliferative phase. In this study, we explored the function of SNAI1 and SNAI2 in myogenesis both in vitro and in vivo. In vitro, we analyzed the expression of SNAI1 and SNAI2 in proliferating murine myoblasts, at various time points after inducing their differentiation. To evaluate their expression during myogenesis in vivo, we induced skeletal muscle regeneration by injecting the myotoxic agent Bupivacaine in the tibialis anterior muscles of wild-type and transgenic mice. We demonstrated that SNAI1 and SNAI2 are upregulated in proliferating myoblasts both in vitro and in vivo. Through the analysis of the transcriptome in C2C12 myoblasts silenced for the expression of SNAI1, we have identified several target genes, among which Fgf21 and Atf3. FGF21 is a growth factor involved in muscle differentiation as well as in glucose and lipid metabolism. In muscle differentiation, FGF21 expression is increased during myogenic differentiation and its knockdown impairs myogenic differentiation in C2C12 cells. ATF3 is a transcription factor that induces endoplasmic reticulum stress (ER-stress), phenomenon behind numerous physiological processes, including muscle differentiation and metabolism regulation. Recent studies have showed that ATF3 is able to regulate chemokine mRNA expression in C2C12 myotubes and it attenuates inflammation of skeletal muscle upon muscle-damaging eccentric exercise. Herein, we analyzed the direct involvement of SNAI1 in the regulation of Fgf21 and Atf3. For this purpose, several Fgf21 and Atf3 promoter deletion mutants, cloned in front of the reporter gene for luciferase, were generated in order to progressively exclude the possible binding sites for SNAI1. We used the Dual-Luciferase Reporter Assay System and ChIP-qPCR analysis to demonstrate that SNAI1 directly binds to the promoter region of Fgf21 and Atf3, leading to the activation of Fgf21 and Atf3 expression in mouse C2C12 myoblasts. Finally, we generated a SNAI1 knockout C2C12 cell line, using the CRISPR-Cas9 genome editing technique and we confirmed that SNAI1 acts as repressor of Fgf21 and Atf3 in proliferating myoblasts.