Abstract
Nuclear receptors are members of ligand-inducible factors that regulate the transcription of many genes. Nuclear receptor RevErbα constitutively inhibits the transcription of target genes via the recruitment of the corepressor complex NCor-HDAC3 (Nuclear CoRepressor and Histone DeAcetylase 3). This complex plays an important role in circadian clock and glucogenesis via regulation of the transcription of the G6P (Glucose-6-Phosphate) and PEPCK (PhosphoEnol Pyruvate Carboxy Kinase) genes, both coding for proteins involved in glucogenesis, which is central to type 2 diabetes. Here we have investigated the interaction of the nuclear receptor RevErbα with two corepressors: NCor and SMRT (Silencing Mediator for Retinoid and Thyroid Receptors). We choose to study the interaction of the RN with SMRT because of sequences similarity between the two corepressors and also because they are most of the time interacting with the same nuclear receptor. On the other hands, several studies reveled that SMRT and RevErbα are not interacting in vivo. But peptides from this proteins associate in vitro. To study these interactions, we use two complementary fluorescence techniques: Number & Brightness and fluorescence anisotropy to investigate the effect of several ligands on this interaction. Three ligands will be tested: heme which is reported to be the natural ligand of RevErbα and two synthetics and non-naturals ligands of RevErbα (SGN and SD7). By fluorescence anisotropy (in vitro) we confirmed an interaction between RevErbα and an NCor peptide and reveal the effect of the three ligands on this interaction. We also study the interaction between RevErbα and several peptides from NCor corresponding to the necessary domains for it binding to RevErbα. We succeed to determine affinity constants for these interactions. We demonstrate, as well the destabilizing effect of heme binding to RevErbα on it interaction with NCor in vitro. We also confirm an interaction between RevErbα and a SMRT peptide corepressor. In order to confirm the functional relevance of these results, we used 2 photons 2 colors Cross Number and Brightness (N&B), an imaged-based fluorescence fluctuation technique (Digman et al, 2008) to study specific interactions of RevErbα with NCor and SMRT full length in cellulo as well as the effect of several ligands above mentioned. We show for the first time a specific interaction between RevErbα and SMRT corepressor full length in cellulo. The ultimate goal of those studies is to identify ligands that enhance the recruitment of the corepressor complex NCor/HDAC3 by RevErbα, thus leading to a decrease expression of the target gene. Such a ligand could be of interest in the quest to decrease blood glucose levels in type 2 diabetes.