A SKELETAL MUSCLE-SPECIFIC ENHANCER REGULATED BY FACTORS BINDING TO EAND CARG BOXES IS PRESENT IN THE PROMOTER OF THE MOUSE MYOSIN LIGHT-CHAIN 1A GENE

The mouse myosin light-chain 1A (MLC1A) gene, expressed in the atria o f the adult heart, is one of the first muscle genes to be activated wh en skeletal as well as cardiac muscles form in the embryo. It is also transcribed in skeletal muscle cell lines at the onset of differentiat ion. Transient transfection assays of mouse skeletal muscle cell lines with DNA constructs containing MLC1A promoter fragments fused to the chloramphenicol acetyltransferase (CAT) gene show that the first 630 b p of the promoter is sufficient to direct expression of the reporter g ene during myotube formation. Two E boxes located at bp -76 and -519 a re necessary for this regulation. MyoD and myogenin proteins bind to t hem as heterodimers with E12 protein and, moreover, transactivate them in cotransfection experiments with the MLC1A promoter in nonmuscle ce lls. Interestingly, the effect of mutating each E box is less striking in primary cultures than in the C2 or Sol8 muscle cell line. A DNA fr agment from bp -36 to -597 confers tissue- and stage-specific activity to the herpes simplex virus thymidine kinase promoter in both orienta tions, showing that the skeletal muscle-specific regulation of the MLC 1A gene is under the control of a muscle-specific enhancer which exten ds into the proximal promoter region, At bp -89 is a diverged CArG box , CC(A/T)(6)AG, which binds the serum response factor (SRF) in myotube nuclear extracts, as does the wild-type sequence, CC(A/T)(6)GG. Both types of CArG box also bind a novel myotube-enriched complex which has contact points with the AT-rich part of the CArG box and adjacent 3' nucleotides. Mutations within the CArG box distinguish between the bin ding of this complex and binding of SRF; only SRF binding is directly involved in the specific regulation of the MLC1A gene in skeletal musc le cell lines.