MyoD uses overlapping but distinct elements to bind E-box and tetraplex structures of regulatory sequences of muscle-specific genes

Muscle differentiation and expression of muscle-specific proteins are initiated by the binding of heterodimers of the transcription factor MyoD with E2A proteins to E-box motif d(CANNTG) in promoters or enhancers of muscle-specific genes. MyoD homodimers, however, form tighter complexes with tetraplex structures of guanine-rich regulatory sequences of some muscle genes. In this work, we identified elements in MyoD that bind E-box or tetraplex structures of promoter sequences of the muscle-specific genes alpha 7 integrin and sarcomeric Mitochondrial Creatine Kinase (sMtCK). Deletions of large domains of the 315 amino acids long recombinant MyoD indicated that the binding site for both E-box and tetraplex DNA is its basic region (KRK) under bar TTNAD (RRK) under bar AATMRE (RRR) under bar that encompasses the three underlined clusters of basic residues designated R-1, R-2 and R-3. Deletion of a single or pairs of R triads or R111C substitution completely abolished the E-box-binding capacity of MyoD. By contrast, the MyoD deletion mutants Delta 102-114, Delta R-3, Delta R1R3 or Delta R2R3 maintained comparable tetraplex DNA-binding capacity as reflected by the similar dissociation constants of their proteinDNA complexes. Only deletion of all three basic clusters abolished the binding of tetraplex DNA. Implications of the binding of E-box and tetraplex DNA by non-identical MyoD elements are considered.