Article (Scientific journals)
Site-directed mutagenesis studies of the hypoxia-inducible factor-1alpha DNA-binding domain.
Michel, Gaetan; Minet, Emmanuel; Mottet, Denis et al.
2002In Biochimica et Biophysica Acta, 1578 (1-3), p. 73-83
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Keywords :
Amino Acid Sequence; Cell Hypoxia; DNA/metabolism; DNA-Binding Proteins/chemistry/genetics/metabolism; Electrophoretic Mobility Shift Assay; Genes, Reporter; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Molecular Sequence Data; Mutagenesis, Site-Directed; Nuclear Proteins/chemistry/genetics/metabolism; Response Elements; Transcription Factors; Transfection; Tumor Cells, Cultured
Abstract :
[en] Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor, is activated when cells are exposed to hypoxia. It is composed of two subunits, HIF-1alpha and ARNT. When activated, it binds to the hypoxia-responsive element (HRE) and up-regulates the expression of several genes (vascular endothelial growth factor (VEGF), erythropoietin (EPO), enolase, em leader ). By molecular modeling, a 3-D model for the complex between the DNA-binding domain of HIF-1 (bHLH domain) and its consensus DNA sequence has been developed. Specific interactions between three amino acids (Ser22, Ala25, Arg30) of the HIF-1alpha subunit and DNA bases were identified. In order to further investigate the role of these amino acids, we generated four mutants of the HIF-1alpha subunit using site-directed mutagenesis. The activity of each mutant was tested using a reporter gene containing either 6 HRE sequences or the authentic human VEGF promoter. The results show that three mutants, Ala25Ser, Ala26Glu and Arg30Ala, were no longer active in the reporter gene assay. On the other hand, the Ser22Ala mutant increased the reporter gene expression, in normoxia as well as in hypoxia. These results correlate with the ones obtained when the DNA-binding capability of the mutants was assayed by electrophoretic mobility shift assays (EMSA): Arg30Ala and Ala26Glu mutants bind very weakly to HRE while the Ser22Ala mutant has the same binding capacity as the wild-type HIF-1alpha. These results bring new insights on the specificity of the protein/DNA interactions for HIF-1 towards HRE.
Disciplines :
Biochemistry, biophysics & molecular biology
Oncology
Author, co-author :
Michel, Gaetan
Minet, Emmanuel
Mottet, Denis ;  Université de Liège - ULiège > Département des sciences biomédicales et précliniques > GIGA-R : Labo de recherche sur les métastases
Remacle, Jose
Michiels, Carine
Language :
English
Title :
Site-directed mutagenesis studies of the hypoxia-inducible factor-1alpha DNA-binding domain.
Publication date :
2002
Journal title :
Biochimica et Biophysica Acta
ISSN :
0006-3002
eISSN :
1878-2434
Publisher :
Elsevier, Amsterdam, Netherlands
Volume :
1578
Issue :
1-3
Pages :
73-83
Peer reviewed :
Peer Reviewed verified by ORBi
Funders :
F.R.S.-FNRS - Fonds de la Recherche Scientifique [BE]
FRIA - Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture [BE]
BELSPO - SPP Politique scientifique - Service Public Fédéral de Programmation Politique scientifique
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