Abstract
Retinoic acid (RA) is a key signal for the specification of the pancreas. Still, the gene regulatory cascade triggered by RA in the endoderm remains poorly characterized. In this study, we investigated this regulatory network in zebrafish by combining RNA-seq, RAR ChIP-seq and ATAC-seq assays. By analysing the effect of RA and of the RA receptor (RAR) inverse-agonist BMS493 on the transcriptome and on the chromatin accessibility of endodermal cells, we identified a large set of genes and regulatory regions regulated by RA signalling. RAR ChIP-seq further defined the direct RAR target genes in zebrafish, including hox genes as well as several pancreatic regulators like mnx1, insm1b, hnf1ba and gata6. Comparison of zebrafish and murine RAR ChIP-seq data highlighted the conserved direct target genes and revealed that some RAR sites are under strong evolutionary constraints. Among them, a novel highly conserved RAR-induced enhancer was identified downstream of the HoxB locus and driving expression in the nervous system and in the gut in a RA-dependent manner. Finally, ATAC-seq data unveiled the role of the RAR-direct targets Hnf1ba and Gata6 in opening chromatin at many regulatory loci upon RA treatment.
Highlights
Retinoic acid (RA) is a key signal for the specification of the pancreas
Principal component analysis of all RNA-seq data (Fig. 1A) shows (i) a tight clustering of all triplicate samples confirming a high reproducibility in the experiment, (ii) a strong difference between the transcriptome of endodermal and non-endodermal (NE) cells, (iii) relatively similar transcriptomes of cells isolated at 3-S and 8-S stages, and (iv) a clustering of BMS493 samples near DMSO samples indicating a much weaker effect of BMS493 treatments compared to the RA treatments
We found a large overlap among the sets of genes having an endodermal-enriched expression at 3-S and at 8-S stages (Fig. S1A; list of genes given in Table S1) and these sets include all known endodermal markers including sox[17], gata5/6 and foxa1/2/3, validating the accurate sorting of endodermal cells
Summary
Retinoic acid (RA) is a key signal for the specification of the pancreas. Still, the gene regulatory cascade triggered by RA in the endoderm remains poorly characterized. Genome-wide identification of RAR binding sites has been achieved by ChIP-seq experiments mostly using murine or human cell lines[19, 30,31,32] Such studies confirmed that the RAR/RXR heterodimers bind to direct repeats of the RGKTCA motif (R = A/G, K = G/T) usually separated by 5 bases (DR5), and to repeats of this motif with other spacing and orientations. These data uncovered several thousand RAR/RXR binding sites in the murine and human genomes, some located near RA-induced genes like the Hox, Cyp26a, or Rar genes. RAR sites with essential function can be identified in vertebrate genomes
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