The HIFs (hypoxia-inducible factors) are a family of heterodimeric transcription factors essential for the adaptation of cells to reduced oxygen supply. Three human PHDs (prolyl hydroxylase domain proteins, PHD1–PHD3) initiate oxygen-dependent degradation of HIF-α-subunits in normoxia. RNA interference directed against PHD2, but not PHD1 or PHD3, is sufficient to stabilize HIF-1α in normoxia. Therefore PHD2 is regarded as the main cellular oxygen sensor. PHD2 itself is up-regulated by hypoxia and may thus limit hypoxic signalling. By sequence analysis, we predicted a promoter approx. 3.5 kb 5′ of the translation start codon and a second promoter located in a CpG island immediately upstream of the coding sequence. A consensus HIF-1-binding site that is conserved in the murine phd2 gene was detected in the CpG island. By electrophoretic mobility-shift assay, we demonstrated binding of HIF-1 to the putative HIF-1-binding site. In luciferase reporter vectors, the isolated upstream promoter was inactive in all cell lines tested unless 200 bp were deleted at the 3′-end. The downstream promoter was active and induced by hypoxia. In reporter vectors containing both promoter sequences, luciferase activity was equal to vectors containing only the downstream promoter. In cells transfected with a vector containing both promoters, a single luciferase transcript was detectable. This transcript had the same length as transcripts from a vector containing the downstream promoter only. We conclude that the phd2 gene is transcribed exclusively from the downstream promoter that contains a functional hypoxia-responsive, cis-regulatory element. Our results establish that PHD2 is a direct HIF target gene.
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Research Article|
April 26 2005
Regulation of the prolyl hydroxylase domain protein 2 (phd2/egln-1) gene: identification of a functional hypoxia-responsive element
Eric METZEN;
1Institute of Physiology, University of Luebeck, Ratzeburger Allee 160, D23538 Luebeck, Germany
2To whom correspondence should be addressed (email metzen@physio.uni-luebeck.de).
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Daniel P. STIEHL;
Daniel P. STIEHL
1
1Institute of Physiology, University of Luebeck, Ratzeburger Allee 160, D23538 Luebeck, Germany
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Kathrin DOEGE;
Kathrin DOEGE
1Institute of Physiology, University of Luebeck, Ratzeburger Allee 160, D23538 Luebeck, Germany
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Jan H. MARXSEN;
Jan H. MARXSEN
1Institute of Physiology, University of Luebeck, Ratzeburger Allee 160, D23538 Luebeck, Germany
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Thomas HELLWIG-BÜRGEL;
Thomas HELLWIG-BÜRGEL
1Institute of Physiology, University of Luebeck, Ratzeburger Allee 160, D23538 Luebeck, Germany
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Wolfgang JELKMANN
Wolfgang JELKMANN
1Institute of Physiology, University of Luebeck, Ratzeburger Allee 160, D23538 Luebeck, Germany
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Publisher: Portland Press Ltd
Received:
October 13 2004
Revision Received:
November 18 2004
Accepted:
November 24 2004
Accepted Manuscript online:
November 24 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 387 (3): 711–717.
Article history
Received:
October 13 2004
Revision Received:
November 18 2004
Accepted:
November 24 2004
Accepted Manuscript online:
November 24 2004
Citation
Eric METZEN, Daniel P. STIEHL, Kathrin DOEGE, Jan H. MARXSEN, Thomas HELLWIG-BÜRGEL, Wolfgang JELKMANN; Regulation of the prolyl hydroxylase domain protein 2 (phd2/egln-1) gene: identification of a functional hypoxia-responsive element. Biochem J 1 May 2005; 387 (3): 711–717. doi: https://doi.org/10.1042/BJ20041736
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