Interaction of the myogenic determination factor myogenin with E12 and a DNA target: mechanism and kinetics

The myogenic determination factors MyoD, myogenin, myf5, and MRF4 are members of the basic helix-loop-helix (bHLH) family of transcription factors and crucial agents of myogenesis. The bHLH regions of these proteins enable them to dimerize with E proteins, another class of the bHLH family, and to bind a specific DNA element known as an E box (CANNTG consensus sequence), which results in the activation of muscle-specific gene expression. As a model for such assembly of the myogenic determination factor/E protein-DNA ternary complex, we have studied the physiologically relevant association of myogenin, E12, and the 3′ E box of the acetylcholine receptor (AChR) α-subunit gene enhancer. Using the technique of electrophoretic mobility shift assay combined with order-of-addition and time-course experiments, we find that heterodimerization of myogenin with E12 occurs prior to DNA-binding. In addition, we deduce the dissociation (Kd) and rate (k) constants for each step in the formation of the myogenin/E12-DNA ternary complex. Kinetic simulations indicate that at 37°C myogenin and E12 heterodimerize with a Kd of 36 μM (kon of 573 M−1 s−1 and koff of 0.0205 s−1), and that subsequently the heterodimer binds the AChR α-subunit gene enhancer 3′ E box with a Kd of 8.8 nM (with possible kon and koff values ranging from 1.0×108 to 14.1×108 M−1 s−1, and 0.875 to 12.3 s−1, respectively).