Identification of a carbohydrate response element in rat S14 gene

Abstract

To characterize the mechanism by which carbohydrate feeding regulates S14 gene transcription, we created mutations within footprinted sequences of the carbohydrate response region of the S14 gene and either transiently transfected these mutations into rat primary hepatocytes or end-labeled them for gel mobility shift assays. The wild type DNA gave a 2.9 ± 1.0-fold response to raising the media glucose concentration. Mutations within the 3′ footprint eliminated the glucose response and also eliminated hepatic nuclear factor binding. However, mutations within the 5′ footprint did not inhibit the glucose responsiveness and had no effect on hepatic nuclear binding. The gel mobility shift assays were confirmed by appropriate competition gel assays. The 3′ footprint contains a sequence similar to the USF (upstream stimulating factor) binding site (CACGTG) that has been suggested to play a role in carbohydrate regulation. However, the gel shift pattern with purified USF1 is distinctly different from that of rat hepatic nuclear extract. These studies demonstrate that the sequence GGCACCCGTGT is required for the carbohydrate response in the S14 gene.