Abstract
BackgroundNuclear 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 FindingsHere, 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 MgCl2 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/SignificanceThe 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.
Highlights
Nuclear receptors (NR) are members of a superfamily of ligandactivated transcription factors acting as transcriptional switches involved in the regulation of development, reproduction, and metabolism of lipids, drugs and energy
PPARG agonists have been used in the treatment of dyslipidemia and hyperglycemia [3,4] and many insulin sensitizing drugs targetting PPARG are designed in the treatment of diabetes as a way to lower serum glucose without increasing pancreatic insulin secretion [5]
To develop a Bioluminescence Resonance Energy Transfer (BRET) interaction assay between RXR and PPARG2 or RXR and thyroid hormone receptor (THR), we cloned their cDNAs in fusion with Renilla luciferase (Rluc8) and enhanced yellow fluorescent protein (EYFP) to obtain RXR-Luc, PPARG2-EYFP and THR-EYFP
Summary
Nuclear receptors (NR) are members of a superfamily of ligandactivated transcription factors acting as transcriptional switches involved in the regulation of development, reproduction, and metabolism of lipids, drugs and energy. Genetic studies in humans and rodents support the notion that NRs control a wide variety of metabolic processes by regulating the expression of genes encoding key enzymes, transporters and other proteins involved in metabolic homeostasis [1]. The importance of this family of proteins in metabolic diseases is well supported by the use of NR ligands for the treatment of diabetes mellitus, dyslipidemia, hypercholesterolemia, or other metabolic disorders [2]. PPARG2 and THR control tissue glucose and lipid homeostasis which are deregulated in severe pathophysiological conditions such as metabolic syndromes
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