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Background  Nuclear receptors (NR) regulate transcription of genes involved in many biological processes such as development, cell proliferation, differentiation and cell death. Amongst them, PPARG2 and THR control tissue glucose and lipid homeostasis which are deregulated in severe pathophysiological conditions such as metabolic syndromes.    Methodology/Principal Findings  Here, we describe a real time BRET approach to monitor heterodimerization between RXR and PPARG2 or THR  in vitro  or in living cells. The presence of a specific DNA target was required to induce  in vitro  a BRET shift reflecting heterodimerization of RXR/PPARG2 or RXR/THR. As in electrophoretic mobility shift assay (EMSA), the stringency and specificity of the BRET shift assay depended upon assay condition optimization including MgCl 2  concentration. For the nuclear receptors, we found by mutagenesis analysis that each heterodimer partner must harbor an intact DNA binding domain to induce BRET and heterodimerization on a DNA target. Moreover the interaction between the PPARG2 ligand binding domain and the RXR DNA binding domain stabilized the heterodimer on its DNA target. BRET microscopy in living cells highlighted the heterodimerization of RXR/PPARG2 within the nucleus clustered in discrete foci that may represent active target gene transcription regulation regions. BRET imaging also suggested that heterodimerization between RXR and PPARG2 required the DNA binding of PPARG2.    Conclusions/Significance  The BRET approach described here allowed us to study the dynamic interactions which exist between NR  in vitro  or in living cells and can provide important information on heterodimerization modes, affinity with a given RE and subcellular localization of the heterodimers. This method could be used to study real time changes of NR heterodimers occurring on DNA depending upon cell activation, chromatin state and help to define the mechanisms of ligands or drug action designed to target NRs.

Analysis of RXR/THR and RXR/PPARG2 Heterodimerization by Bioluminescence Resonance Energy Transfer (BRET)