Isolation and characterization of a novel transcription factor that binds to and activates insulin control element-mediated expression.

Abstract

Pancreatic i-cell-type-specific transcription of the insulin gene is principally regulated by a single cis-acting DNA sequence element, termed the insulin control element (ICE), which is found within the 5'-flanking region of the gene. The ICE activator is a heteromeric complex composed of an islet at/-cell-specific factor associated with the ubiquitously distributed E2A-encoded proteins (E12, E47, and E2-5 showed that p-cell-specific transcription was regulated by 5'-flanking insulin DNA sequences (21). These studies, which were conducted with the rat insulin II gene, demonstrated that residues which lie between bp -695 and + 1 relative to the site of initiation are capable of directing 3-cell-type-specific expression. Rodents have two nonallelic insulin genes (I and II), which differ in their number of introns and chromosomal locations (45). The equivalent region of the human insulin gene has also been shown to direct cell-specific expression within transgenic mice (9,17,39).Experiments conducted with insulin-and non-insulin-producing cell lines have demonstrated that the 5'-flanking sequences of the insulin gene that are essential for pancreatic ,3-cell-type-specific expression reside between bp -340 and -91 relative to the transcription start site (for a review, see reference 44). This region exhibits enhancer-like properties and is regulated by both positively and negatively acting transcription factors (12,13,15,31,55). Detailed characterization of the insulin gene enhancer indicates that transcription mediated by these sequences is predominantly regulated by a single element (13, 25), whose core motif, 5'-GCCATCTG-3', is found within the transcription unit of all characterized insulin genes (45). This element, which we refer to as the insulin control element (ICE), is a site of both positive and * Corresponding author. Phone: (615) 322-7236. negative transcriptional control (24,26,55). The ICE is also important in homeostatic control of the insulin gene in glucose-treated a cells (19,40).The ICE activator appears to be restricted to islet ao and L cells (32,38). Experiments conducted with antisera to the expressed E2A gene products, E12, E47, or ITF-1 (or antigenically related proteins), have demonstrated that these ubiquitously distributed proteins are present in the ICE activator complex (11,18,42). (ITF-1 is a generally distributed E47-like factor [23].) Mutagenesis studies have shown that the nucleotides (shown underlined) that define the binding motif for proteins in the basic helix-loop-helix (bHLH) family, CANNTQ, are essential for trans activation (54). In addition, ICE activation is inhibited in ,B cells by a negative regulator of bHLH-mediated activation, Id (11). This factor appears to suppress bHLH activation by sequestering the E2A gene products into transcriptionally nonfunctional complexes (5). These results are all consistent with the proposal that the ICE activator is composed, at least in part, of E2A proteins. The restricted distribution of this ICE activator binding activity sugge...

Chronic Hyperglycemia, Independent of Plasma Lipid Levels, Is Sufficient for the Loss of β-Cell Differentiation and Secretory Function in the db/db Mouse Model of Diabetes

“…Furthermore, parallel increases were observed for a gene capable of inhibiting differentiation, ID-1, as well as other normally suppressed genes. In normal islets, ID-1 expression is low, but it is induced in isolated islets and ␤-cell lines cultured under high glucose conditions, with little effect in other cell types (11), and its forced expression can inhibit insulin promoter activity (36,37). These findings raise the possibility that increased ID-1 plays a role linking hyperglycemia to ␤-cell dedifferentiation in db/db mice.…”

Functional Characterization of the Transactivation Properties of the PDX-1 Homeodomain Protein

“…We first examined whether PDX-1 or XIHbox8 could synergize with E activator factors to stimulate transcription from an insulin gene reporter construct. These experiments were performed with HeLa cells, which lack endogenous PDX-1 (26, 32), ␤2 (33), and INSAF (47).…”

“…We next sought to determine if the N-terminal conserved sequences of PDX-1 are important in functional interactions with the bHLH activators required in insulin E element-mediated transcription. The E element activator is a heteromeric complex composed of the generally distributed proteins (i.e., E2/5, E12, E47 [5,8,15,53], and HEB [42]) and distinct islet-enriched factors (␤2/NeuroD [25,33] and INSAF [47]). Previous studies have indicated that pancreatic ␤-cell-type-specific transcription of the insulin gene resulted from cooperative interactions between PDX-1 and the E activator proteins (16,38,50).…”

“…PDX-1 is the predominant binding activity detected with an insulin A3 element probe in ␤-cell extracts (38,40,41). The activator of E element-directed expression is a heterodimer composed of proteins in the basic helix-loophelix (bHLH) family that are enriched in islets (INSAF [47] or ␤2 [33]) and the more generally distributed E2A (5,8,15,52,53)-and HEB (42) encoded proteins. (The ␤2 homolog has been isolated from Xenopus and termed NeuroD [25].…”

c-jun inhibits insulin control element-mediated transcription by affecting the transactivation potential of the E2A gene products

“…The ICE activator is a heteromeric complex composed of the insulin activator factor (INSAF) proteins (34), which are uniquely distributed to ␣ and ␤ cells, and the generally expressed E2A-encoded basic helix-loop-helix (B-HLH) proteins, E12, E47, and/or E2-5 (9,16,32,35,38). The E2A proteins (E12, E47, and E2-5) are all very similar (21,22,42).…”