VISUALIZATION OF PIT-1 TRANSCRIPTION FACTOR INTERACTIONS IN THE LIVING CELL-NUCLEUS BY FLUORESCENCE RESONANCE ENERGY-TRANSFER MICROSCOPY

The pituitary-specific transcription factor Pit-1 forms dimers when in teracting with specific DNA elements and has been shown to associate w ith several other nuclear proteins. Recently, techniques have become a vailable that allow visualization of protein-protein interactions as t hey occur in single living cells. In this study, the technique of fluo rescence resonance energy transfer (FRET) microscopy was used to visua lize the physical interactions of Pit-1 proteins fused to spectral var iants of the jellyfish green fluorescent protein (GFP) that emit green or blue light [blue fluorescent protein (BFP)]. An optimized imaging system was used to discriminate fluorescence signals from single cells coexpressing the BFP- and GFP-fusion proteins, and the contribution o f spectral overlap to background fluorescence detected in the FRET ima ges was established. Energy transfer signals from living cells express ing a fusion protein in which GFP was tethered to BFP by short protein linker was used to demonstrate acquisition of FRET signals. Genetic v ectors encoding GFP- and BFP-Pit-1 proteins were prepared, and biologi cal function of the fusion proteins was confirmed. FRET microscopy of HeLa cells coexpressing the GFP- and EFP-Pit-1 demonstrated energy tra nsfer, which required the two fluorophores to be separated by less tha n 100 A. Biochemical studies previously demonstrated that Pit-1 physic ally interacts with both c-Ets-1 and the estrogen receptor. FRET imagi ng of cells coexpressing BFP-Pit-1 and GFP-Ets-1 demonstrated energy t ransfer between these fusion proteins, a result consistent with their association in the nucleus of these living cells. In contrast, there w as no evidence for energy transfer between the BFP-Pit-1 and an estrog en receptor-GFP fusion proteins. It is likely that the FRET imaging ap proach described here can be applied to many different protein-partner pairs in a variety of cellular contexts.