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. 1998 Aug 1;333(Pt 3):705–712. doi: 10.1042/bj3330705

Identification of upstream stimulatory factor as transcriptional activator of the liver promoter of the glucokinase gene.

P B Iynedjian 1
PMCID: PMC1219635  PMID: 9677331

Abstract

A functionally important cis-acting element termed P2 was identified in the liver promoter of the glucokinase gene. Element P2 was delineated by footprinting in vitro with nuclear proteins from rat liver and spleen. Its core sequence in the rat gene is a canonical CACGTG E-box. In the electrophoretic mobility-shift assay with nuclear proteins from rat liver, hepatocytes and hepatoma cells, an oligonucleotide with P2 in the context of the glucokinase promoter sequence gave rise to a DNA-protein complex shown to contain the upstream stimulatory factor (USF) by specific competition experiments and by reactivity with anti-USF antibodies. Transient transfection of hepatoma HepG2 cells, combined with site-directed mutagenesis, demonstrated that the P2 element was important for liver glucokinase promoter activity. Co-transfection of an expression plasmid coding for USF1 activated reporter gene expression in a manner dependent on an intact P2 element, whereas an expression plasmid for c-Myc was ineffective. Expression of a truncated form of USF1 lacking the transcription activation domain and the basic region decreased reporter activity by a dominant-negative effect. The functional significance of the P2 element was also demonstrated in transient transfection of primary hepatocytes.

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Selected References

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