Paired MyoD-binding sites regulate myosin light chain gene expression.

The muscle-specific enhancer element located downstream of the myosin light chain (MLC) locus encoding MLC1 and MLC3 contains three binding sites (A, B, and C) for the myogenic determination factor MyoD. A 173-base-pair region of the MLC gene enhancer, including these three sites, retains full enhancer function when transfected into muscle cells. Whereas mutation of either upstream MyoD binding site (A or B) has a mild effect on muscle-specific enhancer activity, mutation of the third MyoD binding site (C) substantially weakens the enhancer, both in muscle cells or in nonmuscle cells cotransfected with a MyoD, myogenin, or myf5 expression vector. Site C is necessary but insufficient, since double mutation of two MyoD binding sites (A plus B) abrogates enhancer activity. Thus, site C requires either site A or B for enhancer function. This study shows a hierarchy of function among the three MyoD binding sites in the MLC enhancer. We propose that a protein-DNA complex is formed with at least two of these sites (A and C or B and C) to effect activation of the locus encoding MLC1/3 during myogenesis.