A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation

Gregg L. Semenza, Guang L. Wang

Research output: Contribution to journalArticle

Abstract

We have identified a 50-nucleotide enhancer from the human erythropoietin gene 3′-flanking sequence which can mediate a sevenfold transcriptional induction in response to hypoxia when cloned 3′ to a simian virus 40 promoter-chloramphenicol acetyltransferase reporter gene and transiently expressed in Hep3B cells. Nucleotides (nt) 1 to 33 of this sequence mediate sevenfold induction of reporter gene expression when present in two tandem copies compared with threefold induction when present in a single copy, suggesting that nt 34 to 50 bind a factor which amplifies the induction signal. DNase I footprinting demonstrated binding of a constitutive nuclear factor to nt 26 to 48. Mutagenesis studies revealed that nt 4 to 12 and 19 to 23 are essential for induction, as substitutions at either site eliminated hypoxia-induced expression. Electrophoretic mobility shift assays identified a nuclear factor which bound to a probe spanning nt 1 to 18 but not to a probe containing a mutation which eliminated enhancer function. Factor binding was induced by hypoxia, and its induction was sensitive to cycloheximide treatment. We have thus defined a functionally tripartite, 50-nt hypoxia-inducible enhancer which binds several nuclear factors, one of which is induced by hypoxia via de novo protein synthesis.

Original languageEnglish (US)
Pages (from-to)5447-5454
Number of pages8
JournalMolecular and cellular biology
Volume12
Issue number12
DOIs
StatePublished - Dec 1992

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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