Changes in the patterns of electrical activity in neurons and muscle can regulate the function of the nervous system, both during development and adult life. Myogenin is a member of the MyoD family of bHLH transcription factors, known as muscle determination factors, due to their ability to convert non-muscle cells into muscle. These factors bind to a target DNA sequence (CANNTG) known as an E-box and stimulate transcription. An E-box in the proximal promoter of the AChR delta subunit gene is essential for electrical activity-dependent gene expression, since transgenes containing a mutation in this E-box, unlike wild-type transgenes, are not induced following denervation. These results suggest that electrical activity decreases the level and/or activity of transcriptional activators that bind this E-box.
Myogenin is expressed in developing muscle but Myogenin protein and RNA are rapidly down-regulated following innervation. Although other members of the MyoD family are also down-regulated by innervation, Myogenin is far more responsive to electrical activity than other MyoD family members, and Myogenin is the only family member to be down-regulated in all species studied. Because Myogenin can promote expression of other genes known to be regulated by electrical activity, such as subunits of the AChR, and metabolic enzymes, and because response elements for electrical activity-dependent regulation have been mapped to E-boxes in AChR genes, Myogenin is a good candidate for a transcriptional mediator of electrical activity-dependent gene expression.
Phosphorylation of Myogenin can regulate its ability to bind DNA and activate transcription. Importantly, a threonine residue, T87, in the basic region of Myogenin can be phosphorylated by PKC, preventing Myogenin from binding DNA. Electrical activity has been reported to increase PKC activity in skeletal muscle, and the general phosphorylation state of Myogenin in cultured muscle cells has been shown to change with alterations in both electrical and PKC activity, suggesting that electrical activity could stimulate phosphorylation of Myogenin-T87, preventing DNA-binding and transcription of downstream targets. We generated antibodies that are specific for a 13-amino acid phosphopeptide, centered on T87 in Myogenin and found that Myogenin is phosphorylated at T87 in electrically active muscle (Blagden et al., 2004). To determine whether T87 phosphorylation is necessary in vivo to inactivate myogenin and AChR expression, we replaced myogenin with myogenin T87N by homologous recombination. We found that phosphorylation of T87 has a minor role in regulating expression of myogenin and Myg-target genes, indicating that electrical activity can inactivate myogenin expression independent from T87 phosphorylation (Blagden et al., 2004). To elucidate how electrical activity inactivates myogenin expression, we generated transgenic mice carrying gene fusions between the myogenin 5’ regulatory region and a reporter gene, and we found that the MEF2- and MEF3-binding sites are critical cis-acting sensors for electrical activity-dependent expression. These data indicate that electrical activity regulates myogenin expression by controlling the complex of proteins that are bound to the MEF2- and MEF3-binding sites. Current studies are designed to identify the complex of proteins that are loaded onto these MEF2- and MEF3-binding sites in active and inactive muscle and to detemine how the formation of these complexes are regulated by electrical activity and how they activate and repress transcription.
