Characterization of an intronic hormone response element of the rat liver/skeletal muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene

Sara C. McFarlan, Qing Zhang, Richard J. Miksicek, Alex J. Lange

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

The glucocorticoid response element of the rat liver/skeletal muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene was characterized. The element is composed of two tandem hormone receptor binding sites separated by 12 base pairs. Addition of dexamethasone to HeLa cells transiently transfected with a chloramphenicol acetyl transferase (CAT) reporter plasmid containing the hormone response element and cotransfected with glucocorticoid receptor stimulated transcription 24-fold in an orientation- and position-independent manner. Deletion or mutation of essential G/C pairs of the distal binding site abolished hormone-stimulated CAT activity, whereas deletion or mutation of the proximal binding site decreased the hormone-stimulated response only slightly. Mutation of both distal and proximal binding sites resulted in complete loss of hormone-stimulated CAT activity. Experiments carried out using testosterone and progesterone with their respective receptors revealed qualitatively similar results to those seen with glucocorticoid. Binding of glucocorticoid receptor or androgen receptor DNA binding domains to the hormone response element, visualized by gel mobility shift, was unaffected in the proximal binding site mutant, markedly decreased in the distal binding site mutant, and abolished in the double mutant. In gel mobility shift analysis of separate distal and proximal binding sites, only the native distal site demonstrated high affinity binding to glucocorticoid and androgen receptor DNA binding domains. The results demonstrate that this element is responsible for glucocorticoid, androgen, and progesterone stimulation of transcription of the 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase gene and that the distal receptor binding site is dominant.

Original languageEnglish (US)
Pages (from-to)219-227
Number of pages9
JournalMolecular and Cellular Endocrinology
Volume129
Issue number2
DOIs
StatePublished - 1997

Bibliographical note

Funding Information:
We thank Keith Yamamoto and Roger Miesfeld for providing hormone receptors. This work was supported by grants NIH-R01-DK38354 to AJL (formerly R37-DK38354 to Simon J. Pilkis) and NIH-CA 47384 and CD 479 from the American Cancer Society to RJM.

Keywords

  • 6-phosphofructo-2-kinase/fructose-2-6-biphosphatase
  • DNA-binding
  • Gene regulation
  • Hormone response element
  • Steroid hormones

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