Project description:In mouse embryonic cells, a retinoic acid (RA) stimulation triggers a massive change of gene expression leading the pluripotent, proliferating cells to a lineage-specific differentiation process. The retinoic acid receptor (RAR) plays a key role in this response by inhibiting pluripotency-maintaining genes and simultaneously activating some major actors of cell differentiation. To investigate the mechanism underlying this dual regulation, we performed joint RAR/RXR ChIP-seq and mRNA-seq time series during the first 48 hours of the RA-induced Primitive Endoderm differentiation process in F9 embryonic carcinoma cells. We detected significantly more RAR/RXR binding regions than previous studies and identified among them a handful of typical binding intensity patterns during differentiation. We demonstrate that these patterns are correlated with the coincidental binding of essential transcription factors (TFs) for pluripotency maintenance or PrE differentiation of embryonic stem (ES) cells, as well as the presence of variants of RAR binding motifs. Most importantly, early-bound regions coincide with pluripotency-associated transcription factor binding in ES (like Pou5f1, Sox2, Esrrb and Nr5a2) and display an increased frequency of the DR0 type RAR binding motifs; late-bound sites are associated to the PrE marker Sox17 and are enriched in the canonical DR5 binding motif. Our data offer an unprecedently detailed view on the action of RA in triggering pluripotent cell differentiation. Altogether, this work sheds light on the relocation of RAR/RXR binding sites throughout differentiation, and shows how RAR/RXR progressively shift from DR0 enriched regions, which were specifically identified in undifferentiated models, to canonical RAR binding sites containing loci. Time course (0, 2, 6, 12, 24, 48h) after stimulation of F9 cultured cells by retinoic acid: PanRAR and PanRXR ChIP-seq at 0, 2, 24 and 48h (no replicate); WCE-seq at 0h (no replicate); mRNA-seq at 0, 6, 12, 24, 48h (2 replicates except for time 0h, 4 replicates). Additionally, a control time course (culture in DMSO) sampled at 24 and 48h (no replicates).

Project description:Haffez2017 - RAR interaction with synthetic analogues This model is described in the article: The molecular basis of the interactions between synthetic retinoic acid analogues and the retinoic acid receptors Hesham Haffez, David R. Chisholm, Roy Valentine, Ehmke Pohl, Christopher Redfern and Andrew Whiting MedChemComm Abstract: All-trans-retinoic acid (ATRA) and its synthetic analogues EC23 and EC19 direct cellular differentiation by interacting as ligands for the retinoic acid receptor (RARÎ±, Î² and Î³) family of nuclear receptor proteins. To date, a number of crystal structures of natural and synthetic ligands complexed to their target proteins have been solved, providing molecular level snap-shots of ligand binding. However, a deeper understanding of receptor and ligand flexibility and conformational freedom is required to develop stable and effective ATRA analogues for clinical use. Therefore, we have used molecular modelling techniques to define RAR interactions with ATRA and two synthetic analogues, EC19 and EC23, and compared their predicted biochemical activities to experimental measurements of relative ligand affinity and recruitment of coactivator proteins. A comprehensive molecular docking approach that explored the conformational space of the ligands indicated that ATRA is able to bind the three RAR proteins in a number of conformations with one extended structure being favoured. In contrast the biologically-distinct isomer, 9-cis-retinoic acid (9CRA), showed significantly less conformational flexibility in the RAR binding pockets. These findings were used to inform docking studies of the synthetic retinoids EC23 and EC19, and their respective methyl esters. EC23 was found to be an excellent mimic for ATRA, and occupied similar binding modes to ATRA in all three target RAR proteins. In comparison, EC19 exhibited an alternative binding mode which reduces the strength of key polar interactions in RARÎ±/Î³ but is well-suited to the larger RARÎ² binding pocket. In contrast, docking of the corresponding esters revealed the loss of key polar interactions which may explain the much reduced biological activity. Our computational results were complemented using an in vitro binding assay based on FRET measurements, which showed that EC23 was a strongly binding, pan-agonist of the RARs, while EC19 exhibited specificity for RARÎ², as predicted by the docking studies. These findings can account for the distinct behaviour of EC23 and EC19 in cellular differentiation assays, and additionally, the methods described herein can be further applied to the understanding of the molecular basis for the selectivity of different retinoids to RARÎ±, Î² and Î³. This model is hosted on BioModels Database and identified by: BIOMD0000000629. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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Mus musculus

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