Harrington R H, Sharma A
Section of Islet Transplantation & Cell Biology, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
J Biol Chem. 2001 Jan 5;276(1):104-13. doi: 10.1074/jbc.M008415200.
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.
与胰岛素增强子区域RIPE3b结合的转录因子介导胰岛素基因的β细胞类型特异性和葡萄糖应答性表达。早期研究表明，存在于β细胞特异性RIPE3b1结合复合物中的激活剂对这些作用至关重要。RIPE3b1激活剂的DNA结合活性在葡萄糖刺激下被诱导，在糖毒性条件下被抑制。RIPE3b区域内的C1元件被认为是RIPE3b1激活剂的结合位点。RIPE3b区域还包含另一个元件A2，它与胰岛素增强子中的A元件具有同源性。与A元件结合的转录因子（PDX-1和HNF-1α）是胰岛素基因表达和/或胰腺发育的关键调节因子。因此，为了了解C1和A2元件在调节胰岛素基因表达中的作用，我们系统地突变了RIPE3b区域，并分析了这些突变对基因表达的影响。我们的结果表明，C1和A2元件共同构成了RIPE3b1激活剂的结合位点。除了C1-A2（RIPE3b）结合复合物外，在胰岛素瘤细胞系的核提取物中还发现了三种特异性识别A2元件的结合复合物；A2.2复合物仅在胰岛素产生细胞系中检测到。此外，A2元件中的两个碱基对对于RIPE3b1和A2.2激活剂的结合至关重要。瞬时转染结果表明，C1-A2和A2特异性结合激活剂协同激活胰岛素基因表达。此外，RIPE3b1和A2特异性激活剂对葡萄糖的反应不同，表明它们重叠的结合特异性和功能合作可能在调节胰岛素基因表达中起重要作用。
