Transcription Factors Recognizing Overlapping C1-A2 Binding Sites Positively Regulate Insulin Gene Expression

Abstract

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 ...

Inhibition of MafA transcriptional activity and human insulin gene transcription by interleukin-1β and mitogen-activated protein kinase kinase kinase in pancreatic islet beta cells

“…Identification of MEKK1/IL-1β response elements within the INS promoter The INS promoter contains several enhancer-like elements among them the CRE bound by CREB, the A1, A3 and A5 elements bound by PDX1, the E1 element bound by BETA2/NEUROD, and the C1 element bound by MafA [6,9,16,18,19]. To identify a region within the INS promoter that confers MEKK1 responsiveness, a 5′-and 3′-deletion analysis was undertaken.…”

“…Effect of MEKK1 and IL-1β on the RIPE3b-binding transcriptional activator MafA The insulin C1 element within RIPE3b [18] has been shown to recruit the Fig. 5 Effect of MEKK1 on 5′-or 3′-deleted INS promoter fragments.…”

“…The overexpression of Beta2 in the hamster insulinoma cell line In1024 enhanced human insulin gene transcription [10], whereas mice deficient in BETA2 showed a reduction of insulin-producing beta cells and failed to develop mature islets [15]. MafA was identified as a glucose-regulated transcription factor binding to the rat insulin promoter element 3b (RIPE3b) within the insulin gene promoter [16,17], containing the C1 and A2 elements [18]. This transcription factor appears to be responsible for the betacell-specific expression of insulin [19].…”

Identification of β-cell-specific insulin gene transcription factor RIPE3b1 as mammalian MafA

“…Wild-type and the mutant rat insulin II oligonucleotides used in the study have been described before (24). The RIPE3b1 and RIPE3b2 factors can bind rat insulin II oligonucleotides RIPE3b, Ϫ139 to Ϫ101 bp, Ϫ114.113m, Ϫ125.124m, but not the oligonucleotides Ϫ139 to Ϫ101 bp, Ϫ110.109m, and Ϫ125.124m (24). Other oligonucleotides used in the EMSA are wild type (5Ј-ACGTAGCATTCCAGCTGCTGACGGTGCAGCCTCT-CCCCCGAG-3Ј), or mutant ␣A-crystallin (5Ј-ACGTAGCAT-TCCAGCTGCTGCCGGTGCAGCCTCTCCCCCGAG-3Ј) (25).…”

Pancreas duodenum homeobox-1 regulates pancreas development during embryogenesis and islet cell function in adulthood

“…Both C1-and A2-elements together constitute the binding site for RIPE3b1. In addition to C1 -A2 binding complexes, three binding complexes that specifically recognize A2-elements have been identified in nuclear extracts from insulinoma cell lines (38). Transient transfection of those domains indicates that both C1 -A2-and A2-specific binding activators are necessary to cooperatively activate the expression of the insulin gene.…”