Strict distance requirement for transcriptional activation by two regulatory elements of the glucagon gene

The glucagon gene is specifically expressed in A cells of the pancreatic islets. We have previously identified three functional DNA control elements within the 5'-flanking sequence of the glucagon gene: an upstream promoter element (G1), responsible for the A-cell-specific expression, and two enhancer-like elements (G2 and G3). Mutations within G1 completely abolished the enhancer activities of G2 and G3. We show here that the loss of transcriptional activity is not due to an absence of binding of nuclear proteins to G2 or G3, but rather to an essential role of G1 to enable G2 and G3 to exert their effects at a distance. When juxtaposed to the TATA box, both enhancers are capable of inducing transcription as efficiently as when an intact G1 is present. However, the tandem arrangement of G2 plus G3 does not result in additional transcriptional activity compared with a single element. We conclude that G2 and G3 are capable of directly activating transcription but in a highly distance-dependent fashion and without cooperatively interacting. G1 may thus serve only as a tissue-specific relay, bringing together factors that bind to enhancers and the TATA box.