Gene Expression Profiling of siRNA Knockdown and overexpression of SMYD3 in C2C12

Members of the SMYD family of Histone Lysine Methyltransferases, including SMYD3, were shown to be involved in both cardiac and skeletal myogenesis. Currently, very little is known about their mechanisms of action and their potential target genes. To study the cellular and molecular function of SMYD3 in skeletal muscle differentiation, we used loss and gain of function approaches in C2C12 myoblasts. We used RNA sequencing (RNA-seq) to generate gene expression profiling during myogenic differentiation under SMYD3 transient knockdown by siRNAs or SMYD3 stable overexpression in C2C12. We identified multiple genes whose expression is significantly affected by Smyd3 levels, among them the key myogenic regulatory factor (MRF) Myogenin. Overall design: For knockdown condition, C2C12 myoblasts were transfected with siRNAs against SMYD3 or control 24h before start of differentiation. For overexpression condition, we generated a stable C2C12-derived cell line (SMYD3-CL5) that expresses a HA-Flag-tagged SMYD3, and a control cell line (empty vector). For both conditions, cells were harvested for gene expression profiling when differentiation was induced (0h, confluent cells) and after 24h of differentiation in differentiation medium. For each time point, three replicates were performed per siRNA, and two replicates were performed for the clonal cell lines overexpressing SMYD3.