TRANSCRIPTION OF THE HUMAN BETA-ENOLASE GENE (ENO-3) IS REGULATED BY AN INTRONIC MUSCLE-SPECIFIC ENHANCER THAT BINDS MYOCYTE-SPECIFIC ENHANCER FACTOR-2 PROTEINS AND UBIQUITOUS G-RICH-BOX BINDING-FACTORS

To provide evidence far the cis-regulatory DNA sequences and trans-act ing factors involved in the complex pattern of tissue- and stage-speci fic expression of the beta enolase gene, constructs containing fragmen ts of the gene fused to the chloramphenicol acetyltransferase gene wer e used in transient-transfection assays of C2C12 myogenic cells. Delet ion analysis revealed the presence of four major regions: two negative regions in the 5'-flanking sequence, a basal promoter region which di rects expression at low levels in proliferating and differentiated mus cle cells, and a positive region within tbe first intron that confers cell-type-specific and differentiation-induced expression. This positi ve regulatory element is located in the 3'-proximal portion of the fir st intron (nucleotides +504 to +637) and acts as an enhancer irrespect ive of orientation and position from the homologous beta enolase promo ter or the heterologous thymidine kinase promoter, conferring in both cases muscle-specific expression to the linked reporter gene. Deletion of a putative myocyte-specific enhancer factor 1 (MEF-1) binding site , containing a canonical E-box motif, had no effects on muscle-specifi c transcription, indicating that this site is not required for the act ivity of the enhancer. Gel mobility shift assays, competition analysis , DNase I footprinting, and mutagenesis studies indicated that this el ement interacts through an A/T-rich box with a MEF-2 protein(s) and th rough a G-rich box with a novel ubiquitous factor(s). Mutation of eith er the G-rich box or the API-rich box resulted in a significantly redu ced activity of the enhancer in transient-transfection assays. These d ata indicate that MEF-2 and G-rich-box binding factors are each necess ary for tissue-specific expression of the beta enolase gene in skeleta l muscle cells.