Med12 is Essential for Skeletal Muscle Development

Abstract

Tight regulation of tissue‐ and cell‐specific gene expression is essential for healthy physiological development. Mediator is a multisubunit protein complex that links basal transcriptional machinery, transcription factors, and epigenetic modifiers to gene targets to coordinate transcriptional regulation during development. One subunit, MED12, controls tissue‐ and cell‐specific gene expression. Some mutations in MED12 cause neurological developmental disorders, while others are associated with uterine leiomyomas. In addition to these diseases, human MED12 mutations have been linked to muscle disorders such as skeletal muscle hypotonia and congenital heart defects, but the function of MED12 in muscle and muscle cell development has not been elucidated. The objective in this study was to discover a molecular role of MED12 in transcriptional control of muscle development. We hypothesized that MED12 is required for healthy muscle development, and that MED12 helps recruit myogenic transcription factors (TFs) to Mediator at the Pre‐Initiation Complex (PIC) to achieve transcriptional control. Our lab generated a conditional skeletal muscle‐specific Med12 knockout mouse model (Med12mKO) driven by a Myogenin‐Cre transgene that is expressed during embryonic muscle development. Med12mKOmice have a runted phenotype, smaller muscles, disorganized muscle architecture, and suffer premature death, compared to littermate controls. Gene expression analysis by RNA‐sequencing (RNA‐seq) of muscle from 1‐day old Med12mKO and control mice revealed that Med12 deletion dysregulates metabolic and structural genes, including several myosin genes. MED12 is a transcriptional regulator, but does not directly bind DNA. To identify putative candidate TFs that regulate muscle gene expression in a MED12‐dependent manner we performed Upstream Regulator Analysis (URA) of dysregulated genes in Med12mKO muscle. Multiple TFs that regulate muscle gene expression were predicted to do so via MED12, including SRF, which is known to regulate expression of many myosin genes. Using differentiating C2C12 mouse myoblasts as a cell model of myogenesis, knockdown of Med12by shRNA resulted in diminished myotube size and reduced expression of myosin genes, recapitulating the gene expression and muscle phenotype observed in the Med12mKOmodel. However, no interaction was detected between SRF and MED12 in C2C12 myoblasts by Co‐Immunoprecipitation (Co‐IP), which did not support our hypothesis of muscle gene expression regulation through a MED12‐SRF mechanism. To further investigate how loss of MED12 dysregulates muscle gene expression, we used an unbiased approach to identify TFs that interact with MED12. We immunoprecipitated MED12 from C2C12 myoblasts and identified interacting TFs using mass spectrometry. We are currently investigating other TFs through which MED12 could be regulating gene expression during muscle development. We conclude that Med12 is required for normal skeletal muscle development, including normal gene expression of structural and metabolic genes during myogenesis.