Two different gene elements are required for glucose regulation of S14 transcription

Carbohydrate feeding increases the transcriptional activity of the hepatic S14 gene. The region of the S14 promoter between -1384/-1275 contributes to the transcriptional regulation by carbohydrate. A previously identified el ement (-1303/-1289) within this region is required but is not sufficient fo r the carbohydrate effect. Therefore, we ligated -1384/-1275 to a heterolog ous promoter and created mutants in this region to identify other potential responsive sequences. We found that mutation within -1365/-1350 eliminated the response to high glucose (27.5 mM). However, three copies of this elem ent ligated to a mouse mammary tumor virus-luciferase vector did not respon d to glucose indicating the -1365/-1350 element is insufficient to confer a glucose response in isolation. Nevertheless, mutating the -1365/-1350 elem ent in the native promoter led to a loss of response to glucose, proving th is element is necessary. Electrophoretic mobility shift assays (EMSA) using three copies of the element showed significant binding to rat hepatic nucl ear extracts, but no difference between the dietary states. Competition EMS A studies showed that the previously identified element at -1303/-1289 was unable to compete for proteins that bind to the -1365/-1350 element. Theref ore, we have demonstrated two separate elements within the -1384/-1275 regi on of the S14 gene that bind different proteins and interact to elicit the carbohydrate effect. (C) 1999 Elsevier Science Ireland Ltd. All rights rese rved.