Molecular Imaging Into In Vivo Interaction of HIF-1α and HIF-2α with ARNT

Fluorescence resonance energy transfer (FRET) combined with confocal laser microscopy is a powerful tool to analyze protein-protein interaction in vivo. We have applied this combination to study the assembly of the hypoxia-inducible factor (HIF) complex in living cells under hypoxic conditions. In hypoxia, the basic helix-loop-helix/Period/ARNT/Single-minded (PAS) proteins HIF-1 alpha and HIF-2 alpha accumulate and are translocated into the nucleus. Here, HIF-1 alpha and HIF-2 alpha dimerize with HIF-1 beta, also known as aryl hydrocarbon receptor nuclear translocator (ARNT), to form HIF-1/HIF-2 complexes, which control the expression of specific target genes. Therefore, a new Java-based analyzing program was developed at our institute to calculate the nanometer distance between alpha and beta subunits of the transcriptionally active HIF-1/-2 complex bound to DNA. Fusion proteins of HIF subunits and variants of green fluorescent proteins (cyan and yellow fluorescent proteins) were expressed in living cells and protein-protein interactions were imaged in vivo by means of FRET.