Abstract
Transcription factors binding the insulin enhancer region, RIPE3b, mediate beta-cell type-specific and glucose-responsive expression of the insulin gene. Earlier studies demonstrate that activator present in the beta-cell-specific 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 alpha) 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.
Highlights
In adult mammals, the insulin gene is expressed only in the pancreatic -cells in the islets of Langerhans
We demonstrate that the presence of both C1 and A2 elements are essential for binding of the RIPE3b1 activator
The RIPE3b1 complex is detected in insulin-producing cells, whereas the RIPE3b2 complex is detected in nuclear extracts from all cell lines examined to date [14, 27,28,29,30,31]
Summary
Tissue Culture—The HIT T-15 cell line was obtained from American Type Culture Collection (Manassas, VA), and all experiments were conducted with cells between passage numbers 68 and 80. TC-3 and TC1⁄7TET cell lines have been described earlier [55, 56] and were provided by Dr Shimon Efrat. For the preparation of medium with different glucose concentration, glucosefree Dulbecco’s modified Eagle’s medium and dialyzed fetal bovine serum (Life Technolgies Inc.) were used. Other insulin-producing and non-insulin-producing cell lines were cultured for 4 – 6 days, and nuclear extracts were prepared as described [58]. To prepare extracts from HIT T-15 cells grown in the presence of different concentrations of glucose, cells were cultured for 4 days in Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium. Binding reaction conditions for the RIPE3b, Ϫ139 to Ϫ113 and thyroid transcription factor-1 probes were identical (10 mM Tris, pH 7.8, 150 mM NaCl, 2 mM EDTA, 8 mM dithiothreitol, 0.4 mg of poly(dI-dC), 5% glycerol, 50 –100 fmol of probe, and 3–5 g of nuclear extracts). Competition experiments were performed by simultaneous addition of radiolabeled probe and excess unlabeled competitors to the binding reaction. After completion of the run, gels were dried and scanned on a Molecular Dynam-
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