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Generation of a highly sensitive reporter plasmid for the detection of the transcripcional response to hypoxia

Abstract:

Decreased oxygen supply to the tissues at both systemic and local level induces a genic program which allows for adaptation to the hypoxia status. Such program induces the transcriptional activation of different genes such as those encoding for vascular endothelial growth factor (VEGF), erythropoietin (EPO), etc. The transcriptional induction of these genes is mediated by a transcription factor called HIF-1 (hypoxia-inducible factor) for which recognition sequences exist in the DNA of the promoter areas of these hypoxia-activated genes (VEGF, EPO and others). HIF-1 is a heterodimer consisting of two subunits α and β. Hypoxia regulates the levels of subunit α, which dimerizes with subunit β, thus acquiring its transcriptional capacity. This study describes the generation of a reporter plasmid to study the induction of HIF in hypoxia. This construct was generated by tandem coupling of nine copies of the sequence - 985 to -951 of the VEGF promoter, which contains a consensus sequence for recognition of HIF and adjacent sequences. Various experiments have shown this reporter gene to be highly sensitive and specific in response to hypoxia, since its activation: a) required the presence of the β chain of the transcription factor, and it was not induced during hypoxia in β chain-deficient cells); b) responded to exogenous HIF addition; c) could be inhibited in the presence of a negative dominant for HIF; d) responded to chemical agents such as deferoxamine, which chemically mimic hypoxia; and e) was dependent on the presence of p300/CBP co-activators. These data show that a highly sensitive and very specific reporter gene has been generated for studying transcriptional response to hypoxia.