Identification of a functional hypoxia-responsive element that regulates the expression of the egl nine homologue 3 (egln3/phd3) gene.
Low oxygen levels induce an adaptive response in cells through the activation of HIFs (hypoxia-inducible factors). These transcription factors are mainly regulated by a group of proline hydroxylases that, in the presence of oxygen, target HIF for degradation. The expression of two such enzymes, EGLN1 [EGL nine homologous protein 1, where EGL stands for egg laying defective (Caenorhabditis elegans gene)] and EGLN3, is induced by hypoxia through a negative feedback loop, and we have demonstrated recently that hypoxic induction of EGLN expression is HIF-dependent. In the present study, we have identified an HRE (hypoxia response element) in the region of the EGLN3 gene using a combination of bioinformatics and biological approaches. Initially, we isolated a number of HRE consensus sequences in a region of 40 kb around the human EGLN3 gene and studied their evolutionary conservation. Subsequently, we examined the functionality of the conserved HRE sequences in reporter and chromatin precipitation assays. One of the HREs, located within a conserved region of the first intron of the EGLN3 gene 12 kb downstream of the transcription initiation site, bound HIF in vivo. Furthermore, this sequence was able to drive reporter gene expression under conditions of hypoxia in an HRE-dependent manner. Indeed, we were able to demonstrate that HIF was necessary and sufficient to induce gene expression from this enhancer sequence.
Pescador
Nuria
N
Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo s/n, 28029 Madrid, Spain.
Cuevas
Yolanda
Y
Naranjo
Salvador
S
Alcaide
Marisa
M
Villar
Diego
D
Landázuri
Manuel O
MO
Del Peso
Luis
L
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D049308:Dioxygenases; D004790:Enzyme Induction; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D008969:Molecular Sequence Data; D010100:Oxygen / Q000502:physiology*; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D020218:Response Elements / Q000502:physiology*; D012689:Sequence Homology, Nucleic Acid
D049308:Dioxygenases; D011393:Procollagen-Proline Dioxygenase; C488894:EGLN3 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D010100:Oxygen