Identification of Downstream Effectors of Retinoic Acid Specifying the Zebrafish Pancreas by Integrative Genomics.

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) antagonist BMS439 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-dependant 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.

Rewiring of the epigenome and chromatin architecture by retinoic acid signaling during zebrafish embryonic development

“…In contrast, previous studies using higher concentrations of atRA (1 µM) showed the opposite effect, i.e. body axis truncation (33), suggesting that the effect of RA signaling is highly dose-dependent. Indeed, lower concentrations of atRA require more exposure time to produce morphological effects (57).…”

“…In this sense, a recent report used dissected embryonic trunks from Aldh1a2 knockout mice and identified CREs switching epigenomic state in the absence of RA near well-known RA target genes in the trunk (32). More recently, another study used endodermal cells from zebrafish embryos treated with RA or the inverse agonist BMS493, and found alterations in chromatin accessibility associated with genes involved in pancreatic development (33). Here, we have taken advantage of a zebrafish-specific antibody to profile the dynamic chromatin binding of the RA receptor RARaa by ChIP-seq through early development, covering from gastrulation to the phylotypic stage.…”

“…Previous studies showed changes in histone modifications and chromatin accessibility upon alteration of RA levels (32,33). Thus, we wondered whether changes in gene expression induced by atRA treatment in zebrafish whole embryos could also occur together with alterations in CRE function.…”

“…Despite the large amount of evidence establishing the function of RA signaling and RARs in the regulation of target gene expression, very little is known about their global effect on the chromatin landscape. In this sense, recent findings in mouse and zebrafish pancreas suggest that RA signaling may rewire the chromatin landscape leading to cis-regulatory element (CRE) activation (32,33). Previous studies indicate that active enhancers are engaged in chromatin 3D interactions with target promoters, being established either before or concomitant with gene activation, depending on the context (34,35).…”