Two CACGTG Motifs with Proper Spacing Dictate the Carbohydrate Regulation of Hepatic Gene Transcription

Regulatory sequences involved in the transcriptional induction of the rat S14 gene in response to increased glucose metabolism in the hepatocyte were investigated and compared with those of the liver-type pyruvate kinase (L-PK) gene. The carbohydrate response element (ChoRE) of the S14 gene was found to consist of two motifs related to the consensus binding site for the c-myc family of transcription factors, CACGTG. These two motifs are separated by five base pairs, a similar arrangement to that found in the L-PK ChoRE. In its natural context, the S14 ChoRE requires a novel accessory factor to support the full response glucose. This factor, as well as the factor hepatic nuclear factor-4, are both capable of binding to the L-PK gene to enhance its carbohydrate regulation. The need for an accessory factor for supporting the glucose response can be overcome in two ways. First, multimers of the ChoREs of either the L-PK or S14 genes can function independently to support the glucose response. Second, mutations in the S14 ChoRE that create a perfect match to the consensus CACGTG motif at each locus no longer require an accessory factor site. The spacing of the two CACGTG motifs, but not the nature of the bases within the spacer, are critical for control. These observations suggest that a carbohydrate responsive factor binds to both motifs in a highly specific spatial orientation to confer the response to increased carbohydrate metabolism.