2 CACGTG MOTIFS WITH PROPER SPACING DICTATE THE CARBOHYDRATE REGULATION OF HEPATIC GENE-TRANSCRIPTION

Regulatory sequences involved in the transcriptional induction of the rat S-14 gene in response to increased glucose metabolism in the hepat ocyte were investigated and compared with those of the liver-type pyru vate kinase (L-PK) gene. The carbohydrate response element (ChoRE) of the S-14 gene was found to consist of two motifs related to the consen sus binding site for the c-myc family of transcription factors, CACGTG . These two motifs are separated by five base pairs, a similar arrange ment to that found in the L-PK ChoRE. In its natural context, the S-14 ChoRE requires a novel accessory factor to support the full response to 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 carbohy drate 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 S-14 genes can function independently to support the glucose response. Second, mutations in the S-14 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 CACGT G motifs, but not the nature of the bases within the spacer, are criti cal for control. These observations suggest that a carbohydrate respon sive factor binds to both motifs in a highly specific spatial orientat ion to confer the response to increased carbohydrate metabolism.