Identifikation und Analyse genregulatorischer Elemente des hypoxieinduzierbaren Faktors 2 alpha der Maus

The hypoxia inducible factor alpha is a transcription factor mainly expressed in endothelial cells and discussed as important regulator of gene expression of the vascular endothelial growth factor (VEGF) and its receptor KDR/Flk-1. Aim of this work was to identify the regulatory sequences of the murine HIF-2 alpha gene and to find the elements responsible for the endothelial expression of the transcription factor. A cDNA probe complementary to a 5?untranslated region of the murine HIF-2 alpha mRNA was used to isolate a genomic clone containing 4,7 kb of the HIF-2 alpha gene from a genomic phage library. A 3383 bp fragment upstream the start ATG was effective as promoter for a heterologue reporter gene (Luciferase) after transfection in different eucaryotic cultured cells. The highest relative activity of the promoter could be seen in human umbilical endothelial cells. Transcription start could be detected at nucleotide -578 in relation to start ATG via primer extension, RNase protection and PCR as well as a functional TATA box at position -37 in relation to the transcription start. Deletion analysis indicated a 1,1 kb fragment upstream the transcription start to be sufficient to function as promoter. HIFs and Ets transcription factors transactivated HIF-2 alpha promoter reporter constructs and showed strong cooperative action. DNaseI footprint analysis indicated that in the HIF-2 alpha promoter neighbouring potential Ets and HIF binding sites at -935 and -910 are occupied by proteins from endothelial nuclear extracts but not from non endothelial nuclear extracts. Functionality of the HIF binding site could be indicated by additional mutagenesis experiments. In transgene experiments a 2380 bp promoter fragment showed a strong neuronal expression in mouse embryo. In a reporter gene construct containing an additional 1,27 kb intron 1 fragment from the HIF-2 alpha gene the same neuronal expression could be seen. Additional intron fragments lead to disappearance of the neuronal expression but no endothelial expression could be detected. The HIF-2 alpha promoter seems to contain strong positive gene regulatory elements recognised by endothelial transcription factors, HIF-2 alpha activating its own transcription. Elements responsible for endothelial expression of HIF-2 alpha in vivo seem to be situated outside the area analysed in this thesis.