Rar chip-seq

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. Combination of 12 ATAC-seq samples. Samples 12 Less Identification of downstream effectors of retinoic acid specifying the zebrafish pancreas by integrative genomics. SOFT formatted family file s. MINiML formatted family file s. As for the zebrafish ndrg1a gene, it is induced by RA in zebrafish embryos but does not harbour any RAR site in its genomic loci revealing an indirect regulation by RA signalling in zebrafish.

The treatment of embryos with exogenous RA leads to a similar number of up- and down-regulated genes; in contrast the treatment with the pan-RAR inverse-agonist BMS mostly causes down-regulation of genes. In contrast, RA causes mainly a release of co-repressors and the recruitment of co-activators. Most RA-downregulated genes detected in the present study likely represent indirect RAR targets such as genes expressed in anterior endodermal cells whose fate is modified upon RA treatment.

This could be the case for the nkx2. We also observed that the number of genes regulated by BMS was much lower compared to the number of RA-regulated genes. The action of BMS as inverse-agonist should repress all direct target genes. Such difference in the number of BMS versus RA- regulated genes can be explained by considering that a large proportion of RAR direct target genes are still in a repressed state in the DMSO-treated control embryos.

This could be due to the low proportion of endodermal cells receiving the endogenous RA signal. In addition, a down-regulation is more difficult to detect for the genes having a low basal expression level or for genes expressed in a few endodermal cells.

Another study comparing gene expression upon agonist RA versus inverse-agonist AGN also revealed less genes affected by the inverse-agonist 23 , likely for the same reasons. In our study, we also determined the RAR cistrome at the end of zebrafish gastrulation to identify RAR direct targets.

However, the majority of the zebrafish and murine RAR binding sequences could not be aligned. For example, the two RAR sites located near the zebrafish hnf1ba and the murine Hnf1b genes are both located in fish and mammalian CNEs, respectively; these two RAR sites have also similar location in the murine and zebrafish genes i.

We can nevertheless reasonably assume that these RAR sites have a common origin and function. Interestingly, 24 RAR sites were found in extremely conserved non-coding regions and sequences could be aligned from zebrafish to human. This is the case for RAR sites located in some hox clusters 17 , near meis2 25 , nrip1a , ncoa3 and in the skap1 gene this study.

Such high conservation suggests that the level of expression of these RA target genes must be precisely controlled. We show in the present study that the highly conserved RAR site in the 4th intron of the zebrafish skap1 gene drives expression in the neural tube and gut in a pattern reminiscent to hoxb1b expression, suggesting that this RARE controls the expression of the neighbouring hoxbb cluster.

This is further supported by recent chromosome conformation capture Hi-C data obtained from adult zebrafish muscle tissue 40 ; mining these data show that the 4th intron skap1 CNE interacts with several hoxbb genes not shown. This observation also reveals that the assignment of RAR sites to the closest neighbour could result in some incorrect links between RAR sites and their putative target genes.

Still, we think that our strategy enabled to accurately define the majority of RAR-gene assignments as revealed by the correlation found between RA gene induction and the number of nearby RAR sites. Our study also highlights that RA modifies the accessibility of thousands of genomic regions in zebrafish endodermal cells. The modest effect of BMS on chromatin is probably due to the small proportion of endodermal cells under the influence of endogenous RA, as already discussed above for the RNA-seq data.

Still, many RAR sites display the same chromatin accessibility despite strong activation of nearby genes by RA treatment. Thus, these two factors seem to act as important effectors of RA signalling by opening chromatin at numerous regulatory regions. Gata6 and Hnf1b have previously been shown to be involved in pancreas development 42 — 45 and Gata factors have also been shown to act as pioneering factors 41 , Whether Hnf1b has an intrinsic ability to bind nucleosomal DNA and to open chromatin or whether this requires the involvement of other co-factors remain to be determined.

By determining the transcriptomic landscape and genome-wide chromatin accessibility of endodermal and non-endodermal cells at the end of gastrulation, the present data also provide the lists of genes and regulatory cis-elements specifically active in the endoderm. For example, the RAR site located downstream from the mnx1 gene is located in a region which is nucleosome-free only in the endoderm. Accordingly, our transgenic reporter assay shows that this region drives expression in the posterior endoderm and in the dorsal pancreas.

Other endoderm-specific ATAC-seq peaks showed similar endoderm-specific activity data not shown. This strategy can also help to assign a regulatory region to its target gene by correlating expression and chromatin structure; the accuracy of such analyses can be increased by including more data from different cell types or from different developmental stages.

In conclusion, the present study provides interesting resources not only to decipher the regulatory network triggered by RA but also to unveil regulatory regions involved in endoderm formation and patterning in zebrafish. Our data confirm the reassignment of anterior endoderm into midtrunk endoderm by RA treatment, as revealed by the downregulation of anterior markers such as nkx2.

Our data reveal the regulation of many transcription factors, in addition to the hox and meis genes, which are potentially involved in the AP patterning of endoderm; among these many belong to the nuclear receptor superfamily such as nr2f1a , nr2f2 , nr2f5 , nr6a1b genes.

All these genes are direct targets of RAR as revealed by the detection of RARa sites in their genomic loci data not shown. Further analyses are required to determine whether some of these genes are involved in AP endoderm patterning. In contrast, the zebrafish posterior endoderm seems not affected by RA signalling as revealed by the unchanged levels of foxa3 , cdx4 or cdx1a. Our study underlings the central role of the RA in the specification of the hepatopancreatic region through the direct activation of Hnf1ba and Gata6 factors.

Fish were maintained in accordance with the national guidelines and all animal experiments described herein were approved by the ethical committee of the University of Liege protocol number No ectopic pancreatic cells were observed at 25 or 50 nM RA, concentrations usually used in cell culture.

The requirement of high RA concentration in zebrafish is probably due to the degradation of exogenous RA by the endogenous Cyp26 enzyme whose expression is induced by RA 27 , Endodermal and non-endodermal cells were obtained using the transgenic line Tg sox17 :GFP Embryos were used either directly at 11 hpf 3-somite stage or either washed in embryos medium, raised until 13 hpf 8-somite stage in embryos medium.

Seventy five bp single-end sequences were obtained using the NextSeq Illumina Sequencer with coverage of about 20 million reads per library.

Raw reads were aligned to the zebrafish genome Zv9, Ensembl genome version 79, ensembl. Normalization and differential expression analysis were performed using DESeq2 Validation of Myc-tagged RARaa protein expression was done by western-blotting on cytosolic and nuclear lysate fractions from injected embryos using a ChIP-grade anti-myc antibody ab, Abcam.

About 0. Around injected embryos were used for chromatin immunoprecipitation essentially as previously described 51 and fixed with 1. Diagenode Bioruptor sonicator was used for chromatin shearing. Dynal Protein A magnetic beads Diagenode and ChIP-graded anti-myc antibody ab, Abcam were used for the immunoprecipitation, an aliquot of chromatin being taken before the immunoprecipitation step and used as negative control. Forty two bp pair-end sequences were obtained using the NextSeq Illumina Sequencer with coverage of 60 million reads per library.

Raw reads were mapped to the zebrafish genome Zv9 using bowtie2 52 using the settings "--threads 3 --very- sensitive". Enriched peaks were called using MACS2 53 with the following settings : callpeak --gsize 1. Annotation of peaks was done using ChIPseeker Libraries were sequenced 42 bp paired end using the NextSeq Illumina Sequencer with coverage of 40 million reads per library.

Three replicates were prepared for each condition. Raw reads were mapped to the zebrafish genome Zv9 using bowtie2 52 with the following settings : --threads 3 --very-sensitive --maxins --no-discordant. ChIPpeakAnno 58 was used to create the density and heatmap plots.

Briefly, the density of mapped reads was calculated for each of the , regions obtained by merging the peaks obtained for each of the 12 individual samples and differential analysis was performed in these regions to identify the regions with differentially opened chromatin in each condition. Annotation of peaks was done using the ChIPseeker For both transgenes, several lines were analyzed and showed similar expression pattern indicating that expression results from the inserted RAR fragment and not from the transgene insertion sites.

GFP expression from the stable transgenic embryos were analyzed with a Leica fluorescence stereomicroscope. We are indebted to Virginie Vonberg for technical help in transgene constructions and for zebrafish egg injections. All authors read, edited and approved the final manuscript. Publisher's note. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

The online version contains supplementary material available at National Center for Biotechnology Information , U. Sci Rep. Published online Nov Ana R. Wardle , 4 Isabelle Manfroid , 1 Marianne L. Voz , 1 and Bernard Peers 1.

Piotr J. Fiona C. Marianne L. Author information Article notes Copyright and License information Disclaimer. Bernard Peers, Email: eb. Corresponding author. Received Jun 24; Accepted Oct The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

Supplementary Information. Abstract Retinoic acid RA is a key signal for the specification of the pancreas. Subject terms: Computational biology and bioinformatics, Developmental biology, Evolution, Molecular biology. Introduction Retinoic acid RA is essential for the development of vertebrate embryos. Results Retinoic acid affects the transcriptome of zebrafish endodermal cells To identify genes regulated by RA in zebrafish endodermal cells, we used the transgenic Tg soxGFP line which drives GFP expression in endodermal cells and allows their selection by fluorescence activated cell sorting FACS.

Open in a separate window. Figure 1. Identification of RAR binding sites in the zebrafish genome To further identify the genes directly regulated by RAR and determine if some pancreatic regulatory genes are direct targets of RA signalling , we performed ChIP-seq experiments at the end of gastrulation. Figure 2. Figure 3. Conservation of RAR binding sites from fish to mammals Functionally important regulatory regions are expected to be conserved during evolution.

Figure 4. Figure 5. RA affects chromatin accessibility in endodermal cells RAR—RXR complexes control gene expression through the recruitment of corepressors e. Figure 6. Figure 7. Discussion In this study, we have used an integrative genomic approach to identify the genes regulated directly and indirectly by RA signalling in the endoderm at the end of gastrulation and notably involved in the specification of pancreatic progenitors.

Materials and methods Zebrafish strains, pharmacological treatments and FACS sample preparation Fish were maintained in accordance with the national guidelines and all animal experiments described herein were approved by the ethical committee of the University of Liege protocol number ChIP-seq library preparation and data analysis About 0.

Supplementary Information Supplementary Table S1. Supplementary Table S2. Supplementary Table S3. Supplementary Table S4. Supplementary Table S5. Supplementary Table S6. Supplementary Table S7. Top 6 enriched motifs were shown. Also labeled are representative genes found at the leading edge. The fold enrichment of ChIPed samples vs input was plotted in the y-axis. It implicates that some of activated targets, such as GFI1 , are dedicated to transcriptional programs needed to maintain the promyelocytic stage for APL maintained consistently or normal promyelocytes transiently.

Although fetal bovine serum used for cell culture might contain traces of retinoids, the contribution of ATRA might be below the physiological level supplemental Figure There is considerable evidence to support our findings.

Firstly, histone deacetylases HDACs may help prevent inappropriate reinitiation of transcription on regulatory regions simultaneously bound by histone acetyltransferases and HDACs. This provides the structural basis for the efficient transcriptional activation. We note some limitations to our work, which was largely limited by available techniques. We anticipate that our work can be further extended with future technological innovations.

Finally, it is worth mentioning that small-molecule compounds selectively targeting super-enhancers have shown tremendous therapeutic potential in inhibiting cancer cell growth.

The publication costs of this article were defrayed in part by page charge payment. Contribution: K. Sign In or Create an Account. Sign In. Skip Nav Destination Content Menu. Close Key Points. Article Navigation. This Site. Google Scholar. Yi Zhang , Yi Zhang. Rongsheng Zhang , Rongsheng Zhang. Shufen Li , Shufen Li. Wen Jin , Wen Jin. Saijuan Chen , Saijuan Chen. Hai Fang , Hai Fang. Zhu Chen , Zhu Chen. Kankan Wang Kankan Wang.

Blood 11 : — Article history Submitted:. Connected Content. Cite Icon Cite. Visual Abstract View large Download slide. View large Download slide. Bioinformatic and statistical analysis are detailed in supplemental Methods. Figure 1. View large Download PPT. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. The online version of this article contains a data supplement.

Conflict-of-interest disclosure: The authors declare no competing financial interests. Search ADS. Acute promyelocytic leukaemia: novel insights into the mechanisms of cure.

Chromatin modifiers and remodellers: regulators of cellular differentiation. Gfi1b:green fluorescent protein knock-in mice reveal a dynamic expression pattern of Gfi1b during hematopoiesis that is largely complementary to Gfi1. Reduced expression but not deficiency of GFI1 causes a fatal myeloproliferative disease in mice.

Inflammatory reactions and severe neutropenia in mice lacking the transcriptional repressor Gfi1.