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RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements.

ABSTRACT: In mouse embryonic cells, ligand-activated retinoic acid receptors (RARs) play a key role in inhibiting pluripotency-maintaining genes and 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 h of the RA-induced Primitive Endoderm (PrE) differentiation process in F9 embryonal carcinoma (EC) cells. We show here that this dual regulation is associated with RAR/RXR genomic redistribution during the differentiation process. In-depth analysis of RAR/RXR binding sites occupancy dynamics and composition show that in undifferentiated cells, RAR/RXR interact with genomic regions characterized by binding of pluripotency-associated factors and high prevalence of the non-canonical DR0-containing RA response element. By contrast, in differentiated cells, RAR/RXR bound regions are enriched in functional Sox17 binding sites and are characterized with a higher frequency of the canonical DR5 motif. Our data offer an unprecedentedly detailed view on the action of RA in triggering pluripotent cell differentiation and demonstrate that RAR/RXR action is mediated via two different sets of regulatory regions tightly associated with cell differentiation status.

SUBMITTER: Chatagnon A

PROVIDER: S-EPMC4446430 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements.

Chatagnon Amandine A Veber Philippe P Morin Valérie V Bedo Justin J Triqueneaux Gérard G Sémon Marie M Laudet Vincent V d'Alché-Buc Florence F Benoit Gérard G

Nucleic acids research 20150420 10

In mouse embryonic cells, ligand-activated retinoic acid receptors (RARs) play a key role in inhibiting pluripotency-maintaining genes and 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 h of the RA-induced Primitive Endoderm (PrE) differentiation process in F9 embryonal carcinoma (EC) cells. We show here that this dual regulation is associated with ...[more]

PMID: 25897113

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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:Differentiation of embryonic and adult myogenic progenitors undergoes a complex series of cell rearrangements and specification events which are controlled by distinct gene regulatory networks. Delineation of the molecular mechanisms that regulate skeletal muscle specification and formation should be important for understanding congenital myopathies and muscular degenerative diseases. Retinoic acid (RA) signaling plays an important role in development. However, the role of RA signaling in adult myogenic progenitors is poorly understood. Here, we investigate the role of RA signaling in regulating myogenic differentiation of myoblastic progenitor cells. Using the mouse myoblast progenitor C2C12 line as a model, we have found that the endogenous expression of most RAR and RXR isotypes is readily detected. While the nuclear receptor co-repressors are highly expressed, two of the three nuclear receptor co-activators and the enzymes involved in RA synthesis are expressed at low level or undetectable, suggesting that the RA signaling pathway may be repressed in myogenic progenitors. Using the alpha-myosin heavy chain promoter-driven reporter (MyHC-GLuc), we have demonstrated that either ATRA or 9CRA is able to effectively induce myogenic differentiation, which can be synergistically enhanced when both ATRA and 9CRA are used. Upon ATRA and 9CRA treatment of C2C12 cells the expression of late myogenic markers significantly increases. We have further shown that adenovirus-mediated exogenous expression of RARalpha and/or RXRalpha is able to effectively induce myogenic differentiation in a ligand-independent fashion. Morphologically, ATRA- and 9CRA-treated C2C12 cells exhibit elongated cell body and become multi-nucleated myoblasts, and even form myoblast fusion. Ultrastructural analysis under transmission electron microscope reveals that RA-treated myogenic progenitor cells exhibit an abundant presence of muscle fibers. Therefore, our results strongly suggest that RA signaling may play an important role in regulating myogenic differentiation.

Dataset Information

RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements.

Publications

RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements.

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