DEFINITION OF THE CARBOHYDRATE RESPONSE ELEMENT OF THE RAT S-14 GENE - CONTEXT OF THE CACGTG MOTIF DETERMINES THE SPECIFICITY OF CARBOHYDRATE REGULATION

Transcription of the S-14 gene in primary hepatocytes is stimulated in response to increased carbohydrate metabolism. We have demonstrated p reviously that a 30-base pair (bp) segment of the S-14 gene from -1457 to -1428 is a carbohydrate response element (ChoRE). This element con tains a (5')CACGTG motif that is essential for control. DNase I footpr inting experiments with liver nuclear extract revealed two factors bin ding within the S-14 ChoRE. In transient transfection experiments, mut ation of the upstream site between -1457 and -1450 did not affect the response to elevated glucose, whereas the downstream 21-bp site betwee n -1448 and -1428 was sufficient to mediate the glucose induction. Ele ctrophoretic mobility shift assays indicated that the hepatic factor b inding to this site in vitro is closely related or identical to the ma jor late transcription factor (MLTF). However, replacement of the 21-b p S-14 ChoRE with the authentic MLTF binding site from the adenovirus major late promoter failed to elicit the glucose response. By systemat ically exchanging bases between the functional S-14 and nonresponsive adenovirus sites, the sequence (5')CACGTGNNNGCC was found to be essent ial for carbohydrate regulation. A segment containing this specific mo tif from the rat fatty acid synthase gene, another carbohydrate-respon sive gene in hepatocytes, conferred a carbohydrate response when linke d to the S-14 promoter. Thus, the context of the CACGTG motif provides the specificity for regulation by carbohydrate metabolism.