Basic helix-loop-helix factors recruit nuclear factor I to enhance expression of the Na_V 1.4 Na⁺ channel gene

We have previously shown that the basic helix-loop-helix (bHLH) transcription factors coordinate Na_V 1.4 Na⁺ channel gene expression in skeletal muscle, but the identity of the co-factors they direct is unknown. Using C2C12 muscle cells as a model system, we test the hypothesis that the bHLH factors counteract negative regulation exerted through a repressor E box (- 90/- 85) by recruiting positive-acting transcription factors to the nucleotides (- 135/- 57) surrounding the repressor E box. We used electrophoretic mobility shift assays to identify candidate factors that bound the repressor E box or these adjacent regions. Repressor E box-binding factors included the known transcription factor, ZEB/AREB6, and a novel repressor E box-binding factor designated REB. Mutations of the repressor E box that interfere with the binding of these factors prevented repression. The transcription factor, nuclear factor I (NFI), bound immediately upstream and downstream of the repressor E box. Mutation of the NFI-binding sites diminished the ability of myogenin and MRF4 to counteract repression. Based on these observations we suggest that bHLH factors recruit NFI to enhance skeletal muscle Na⁺ channel expression.