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

  • ️Thu Jan 01 2015

. 2015 May 26;43(10):4833-54.

doi: 10.1093/nar/gkv370. Epub 2015 Apr 20.

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

Amandine Chatagnon et al. Nucleic Acids Res. 2015.

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.

© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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Figures

Figure 1.
Figure 1.

RAR/RXR heterodimers binding regions. (A) Time course analysis of RAR (green) and RXR (red) binding profiles in the Hoxa locus after RA stimulation of F9 EC cells. A screenshot (

http://genome.ucsc.edu

) of the RAR and RXR binding signal at the different time points after RA stimulation, is shown. (B) Characterization of RAR/RXR binding region relative to specific genomics features. Open and blue bars show the proportion of the indicated genomics features in the genome and ChIP regions, respectively. (C) Distribution of the distance to the first annotated transcription start site (TSS) (blue bars: absolute distance < 20 kb, black bars: absolute distance > 20 kb). (D) Multiple species alignment scores (Phastcons) for RAR/RXR binding regions (blue curve) and randomly selected genome regions (brown curve).

Figure 2.
Figure 2.

RAR and RXR binding dynamics in differentiating F9 EC cells. (A) The number of RAR- and RXR-binding sites detected by ChIP-seq during PrE differentiation. Results are shown as Venn diagrams representing the number of binding sites during differentiation for RAR only (green), RXR only (red) and shared RAR/RXR (brown). Circle sizes are representative of the number of binding sites at the indicated time point. (B) Intersection of RAR, RXR and RAR/RXR binding regions occupation throughout the PrE differentiation process. Results are shown as four-way Venn diagrams representing the proportion (%) of binding sites assigned to specific temporal behavior in each sub-category. (C) Distribution of the occupancy level over time for binding sites exhibiting no binding of RAR (green) and significant binding of RXR (red) in untreated condition. Occupancy levels are expressed in read per region and per million mapped reads. (D) K-means clustering of RAR/RXR dynamic regions based on their changes in RAR occupancy during PRE differentiation. Results are shown as heat map representing the normalized RAR binding region coverage intensity over time. (E) RAR (green) and RXR (red) binding profiles variation in the Cyp26a1 locus after RA stimulation of F9 EC cells. Upstream and downstream binding region are highlighted by gray box. (F) qPCR quantification of RAR binding intensity at the Cyp26a1 upstream (open bars) and downstream (blue bars) binding sites. RAR binding intensity was normalized on binding level in untreated cells for each binding region. The data shown represent mean ± SD of triplicate experiments. Student's t-test was applied to assess statistical difference of the mean (*: P < 0.05; **: P < 0.01). (G) qPCR quantification for temporal binding pattern of RAR at Cyp26a1 upstream (open bars) and downstream (blue bars) binding sites in wild-type (WT) and Rarb -/- F9 cells. Statistical analysis similar to (F). (H) qPCR quantification of temporal open chromatin enrichment at Cyp26a1 upstream (open bars) and downstream (blue bars) RAR/RXR binding sites. Results show open chromatin enrichment in RA-stimulated relative to untreated F9 EC cells. Statistical analysis similar to (F).

Figure 3.
Figure 3.

Analysis of Hormone Response Elements (HRE) in RAR/RXR target regions. (A) Position weight matrix (PWM) of core half-site motif. Matrices predicted by Balmer and Blomhoff (upper panel) and by the de novo motif-discovery algorithm DREME (middle and lower panel). (B) ROC curves. Enrichment in the various types of HRE was assessed by comparing the frequency of discovery of the consensus in the RAR/RXR bound regions (y-axis) and in a set of randomly generated sequences (same size, same GC content, x-axis). Results show compound motif (DR0–1–2–5) (red curve) and DR0 (green curve), DR1 (yellow curve), DR2 (purple curve) and DR5 (blue curve) motifs enrichment. (C) Areas under the curve measuring enrichment of RAR/RXR binding region in Direct Repeat (DR), Inverted Repeat (IR) and Everted Repeat (ER) with spacer from 0 to 9 nucleotides are shown as heat map. (D) Prevalence of the different motifs (DR-IR-ER with spacer 0–9) in the RAR/RXR binding region set. (E) Number of occurrences per motif in the RAR/RXR binding region set. Grey bars represent the number of predicted isolated motifs in each category. (F) The number of identified regions encompassing at least one DR0, DR1, DR2 or DR5 motif. Results are shown as four-way Venn diagrams representing the number of region in each subcategory. In D-F panels, motifs are predicted based on their alignment score with the corresponding PWM. Score threshold was defined as the score giving 10% of positive matches in control sequences. (G) Motif association analysis. Results show the percentage of predicted HRE (DR0, DR1, DR2 or DR5) that share one of the half core with another predicted motif. Blue link represent the percentage of embedded motifs and red link represent the percentage of motifs sharing the central half core.

Figure 4.
Figure 4.

RAR/RXR targeted region coincide with pluripotency- and differentiation-associated transcription factors binding sites. (A) Correlation between RAR/RXR and the indicated factors binding sites. Data sets used for the correlation study were obtained in mouse ES cells (open bars) or mouse F9 EC cells (blue bars). (B) Distance distribution of the orphan nuclear receptors ESRRB (upper panel) and NR5A2 (lower panel) binding regions identified in mouse ES cells that overlap with RAR/RXR binding regions identified in F9 EC cells relative to RAR/RXR region center. (C) Distance distribution of the pluripotency-associated transcription factors binding regions identified in mouse ES cells that overlap with RAR/RXR binding regions identified in F9 EC cells relative to RAR/RXR region center. (D) Distance distribution of POU5F1 and SOX17 binding regions that overlap with RAR/RXR binding regions identified in F9 EC cells relative to RAR/RXR region center. The number between brackets represents the absolute number of RAR/RXR binding regions (peak summit ± 250 bp, blue area) that overlap with the indicated transcription factor.

Figure 5.
Figure 5.

RAR/RXR binding dynamics and region-associated features. (A) Each bar represents the log odds ratio from Fisher's exact test between a cluster assignment and the presence of a feature within 0.5kb of the RAR/RXR peak summit. A red (resp. green) bar indicates that a feature is significantly more frequent (resp. less frequent) in the cluster than in other RAR/RXR binding regions. Error bars indicate 95% confidence intervals. (B) Effect of Esrrb KD on RAR binding intensity. Upper panels show a screenshot (

http://genome.ucsc.edu

) of RAR and ESRRB binding signal on the genomic loci analyzed in wild-type F9 cells and ES cells respectively. Predicted RARE motifs present under the peak are indicated. Lower panels show ChIP-qPCR quantification of RAR binding in Control and Esrrb KD F9 cells. RAR binding intensity is expressed relative to input amount. The data shown represent mean ± SD of replicate experiments. (C) Effect of Sox17 KD on RAR binding intensity. Upper panels show a screenshot (

http://genome.ucsc.edu

) of RAR and SOX17 binding signal on the genomic loci analyzed in untreated and RA treated wild-type F9 cells. Predicted RARE motifs present under the peak are indicated. Lower panels show ChIP-qPCR quantification of RAR binding in untreated and RA treated Control and Sox17 KD F9 cells. RAR binding intensity is expressed relative to input amount. The data shown represent mean ± SD of replicate experiments.

Figure 6.
Figure 6.

Gene expression profiles during RA-induced PrE differentiation of mouse F9 EC cells. (A) Identification of differentially expressed genes at different time points after RA stimulation relative to their untreated expression level. Results are shown as a volcano plot representing Benjamini-Hochberg adjusted P-value versus log2 fold change on the y- and x-axes, respectively. Up- and down-regulated genes are shown in red and green, respectively. (B) Proportion of genes in the indicated set that exhibit an RAR/RXR target regions within the indicated distance range from their closest annotated TSS. Blue bars correspond to the percentage of genes exhibiting an RAR/RXR target region at less than 20 kb upstream or downstream of their annotated TSS. (C) Same as (B) panel for gene up- or down-regulated at the indicated time point after RA stimulation. (D) Clustering of differentially expressed gene expression profiles. Gene associated to Primitive Endoderm differentiation or pluripotency maintenance are indicated in the corresponding annotated boxes. (E) Boxplot representing the distance distribution of the closest RAR/RXR target region relative to annotated TSS of differentially expressed genes for the previously defined (D) expression clusters.

Figure 7.
Figure 7.

Gene expression profiles and associated regulatory region features. The results are shown as the association score between specific gene expression cluster assignment and presence of the indicated feature within a 40 kb window centered on the annotated TSS. Each bar represents the enrichment of the indicated feature in each cluster with respect to all differentially expressed genes and represents the log odds ratio and 95% confidence interval as computed by Fisher's exact test. The red and green bars correspond to the significantly enriched and depleted features, respectively.

Figure 8.
Figure 8.

Integrated model of RAR/RXR binding dynamics during RA-induced PrE differentiation of F9 EC cells.

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