Transcription factors binding the insulin enhancer region, RIPE3b, mediate ␤-cell type-specific and glucose-responsive expression of the insulin gene. Earlier studies demonstrate that activator present in the ␤-cellspecific RIPE3b1-binding complex is critical for these actions. The DNA binding activity of the RIPE3b1 activator is induced in response to glucose stimulation and is inhibited under glucotoxic conditions. The C1 element within the RIPE3b region has been implicated as the binding site for RIPE3b1 activator. The RIPE3b region also contains an additional element, A2, which shares homology with the A elements in the insulin enhancer. Transcription factors (PDX-1 and HNF-1␣) binding to A elements are critical regulators of insulin gene expression and/or pancreatic development. Hence, to understand the roles of C1 and A2 elements in regulating insulin gene expression, we have systematically mutated the RIPE3b region and analyzed the effect of these mutations on gene expression. Our results demonstrate that both C1 and A2 elements together constitute the binding site for the RIPE3b1 activator. In addition to C1-A2 (RIPE3b) binding complexes, three binding complexes that specifically recognize A2 elements are found in nuclear extracts from insulinoma cell lines; the A2.2 complex is detected only in insulin-producing cell lines. Furthermore, two base pairs in the A2 element were critical for binding of both RIPE3b1 and A2.2 activators. Transient transfection results indicate that both C1-A2 and A2-specific binding activators cooperatively activate insulin gene expression. In addition, RIPE3b1-and A2-specific activators respond differently to glucose, suggesting that their overlapping binding specificity and functional cooperation may play an important role in regulating insulin gene expression.In adult mammals, the insulin gene is expressed only in the pancreatic ␤-cells in the islets of Langerhans. In studies using transgenic animals and transient transfection analysis, the proximal 5Ј-flanking region of the insulin promoter was shown sufficient for directing ␤-cell-specific expression of the insulin gene (1-7). Further, mutational analysis of the promoter proximal region identified several cis-acting enhancer elements that are important for insulin expression. The insulin enhancer elements, with the exception of E-box elements, have been classified based on the nucleotide sequence of the element (8). Most of these enhancer elements are well conserved in various species, suggesting the presence of a common regulatory mechanism(s) controlling insulin expression.Three conserved insulin enhancer elements, A3 (Ϫ201 to Ϫ196 bp) 1 (9 -13), RIPE3b/C1-A2 (Ϫ126 to Ϫ101 bp) (14), and E1 (Ϫ100 to Ϫ91 bp) (4, 15, 16), play an important role in regulating cell-specific expression of the insulin gene. Factors binding these sites have a very limited cellular distribution. Transcription factors that bind and activate expression from two of the three conserved insulin enhancer elements (A3 and E1) have been cloned. PDX-1, a ...

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