Cell-specific and ubiquitous factors are responsible for the enhancer activity of the rat insulin II gene.

Pancreatic beta-cell-specific expression of the insulin gene is mediated, at least in part, by an enhancer element termed the rat insulin promoter element 3 (RIPES) found within the rat insulin II gene between positions -126 and -86. Here we identify three distinct factors interacting with RIPE3, namely 3a1, 3a2, and 3b1, which bind to the sequences between -100 to -90, -108 to -99, and -115 to -107, respectively. Factors 3a1 and 3b1 are beta-cell specific whereas 3a2 is ubiquitously distributed. The 3a1 site contains the consensus binding sequence (CANNTG) for a group of DNA-binding proteins called basic-helix-loop-helix proteins. We showed in this study that the 3a1 binding activity contains E12/E47, a member of the basic-helix-loop-helix protein family, or an E12/E47-like protein. Sequence comparison of the 3a2 and 3b1 binding sites suggest that they are unique and may bind to novel transcription factors. Mutation analysis of each individual binding site in transient expression experiments indicates that all of the three binding sites contribute to the enhancer activity of the RIPE3 in beta-cells. Mutation in any one of the three binding sites not only disrupts binding of the corresponding factor but decreases RIPE3 enhancer activity by 4-7-fold. The results suggest that interactions among the 3a1, 3a2, and 3b1 factors are required for maximum enhancer activity of the RIPE3 in insulin-producing cells.