Imaging Erg and Jun transcription factor interaction in living cells using fluorescence resonance energy transfer analyses.
Physical interactions between transcription factors play important roles in modulating gene expression. Previous in vitro studies have shown a transcriptional synergy between Erg protein, an Ets family member, and Jun/Fos heterodimer, members of the bZip family, which requires direct Erg-Jun protein interactions. Visualization of protein interactions in living cells is a new challenge in biology. For this purpose, we generated fusion proteins of Erg, Fos, and Jun with yellow and cyan fluorescent proteins, YFP and CFP, respectively. After transient expression in HeLa cells, interactions of the resulting fusion proteins were explored by fluorescence resonance energy transfer microscopy (FRET) in fixed and living cells. FRET between YFP-Erg and CFP-Jun was monitored by using photobleaching FRET and fluorescence lifetime imaging microscopy. Both techniques revealed the occurrence of intermolecular FRET between YFP-Erg and CFP-Jun. This is stressed by loss of FRET with an YFP-Erg version carrying a point mutation in its ETS domain. These results provide evidence for the interaction of Erg and Jun proteins in living cells as a critical prerequisite of their transcriptional synergy, but also for the essential role of the Y371 residue, conserved in most Ets proteins, in this interaction.
Camuzeaux
Barbara
B
UMR 8526CNRS/Institut Pasteur de Lille/Université de Lille2, Institut de Biologie de Lille, BP 447, 1 rue Calmette, 59021 Lille cedex, France.
Spriet
Corentin
C
Héliot
Laurent
L
Coll
Jean
J
Duterque-Coquillaud
Martine
M
D001426:Bacterial Proteins / Q000378:metabolism; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D000071230:Transcriptional Regulator ERG; D014162:Transfection
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D004268:DNA-Binding Proteins; C072312:ERG protein, human; D008164:Luminescent Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D008156:Luciferases