EXPRESSION OF MYOGENIN DURING EMBRYOGENESIS IS CONTROLLED BY SIX SINEOCULIS HOMEOPROTEINS THROUGH A CONSERVED MEF3 BINDING-SITE/

Myogenin, one of the MyoD family of proteins, is expressed early durin g somitogenesis and is required for myoblast fusion ill vivo. Previous studies in transgenic mice have shown that a 184-bp myogenin promoter fragment is sufficient to correctly drive expression of a beta-galact osidase transgene during embryogenesis. We show here that mutation of one of the DNA motifs present in this region, the MEF3 motif, abolishe d correct expression of this beta-galactosidase transgene. We have fou nd that the proteins that bind to the MEF3 site are homeoproteins of t he Six/sine oculis family. Antibodies directed specifically against Si x1 or Six4 proteins reveal that each of these proteins is present in t he embryo when myogenin is activated and constitutes a muscle-specific MEF3-binding activity in adult muscle nuclear extracts. Both of these proteins accumulate in the nucleus of C2C12 myogenic cells, and trans ient transfection experiments confirm that Six1 and Six4 are able to t ransactivate a reporter gene containing MEF3 sites. Altogether these r esults establish Six homeoproteins as a family of transcription factor s controlling muscle formation through activation of one of its key re gulators, myogenin.