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

Abstract

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 α7 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 KRKTTNADRRKAATMRERRR that encompasses the three underlined clusters of basic residues designated R1, R2 and R3. 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 Δ102-114, Δ3, ΔR1R3 or ΔR2R3 maintained comparable tetraplex DNA-binding capacity as reflected by the similar dissociation constants of their protein-DNA 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. © 2007 The Author(s).