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

Chatagnon A, Veber P, Morin V, Bedo J, Triqueneaux G, Sémon M, Laudet V, d'Alché-Buc F, Benoit G - Nucleic Acids Res. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Université de Lyon, Université Claude Bernard Lyon1, CGphiMC UMR CNRS 5534, 69622 Villeurbanne, France.

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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.
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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.

Mentions: We next investigated whether the RAR/RXR occupancy profiles identified by clustering (Figure 2D) were specifically associated with molecular features such as the presence of RARE motifs or transcription factor binding sites experimentally identified in F9 and ES cells. To this end, we performed Fisher's exact tests to detect features whose prevalence varied significantly in one cluster compared to all other RAR/RXR binding regions. The results are presented in Figure 5A and Supplementary Figure S8, where red (resp. green) bars indicate features that are significantly enriched (resp. depleted) in the cluster. First we observe that each binding pattern is characterized by a unique combination of molecular features. For instance, the regions assigned to clusters exhibiting high average occupancy in untreated F9 cells are characterized by their association with ESRRB and POU5F1 binding events (clusters B, D and F in Figure 2D and Supplementary Figure S3 panel A). Also, all clusters exhibiting a maximum occupancy in the early phase of the RA-induced PrE differentiation process, namely clusters A–D, are associated with a higher prevalence of DR0 type motif. Among these four clusters, subtle binding pattern variations appear coupled with specific core pluripotency factor binding sites layouts and are independent or negatively associated with PrE differentiation-associated transcription factor like SOX17. Cluster A, which exhibits only a transient occupancy at 2 h after RA stimulation, is only associated with DR0 enrichment while clusters B, C and D are also associated with NR5A2 and SMAD1 binding but differ in their association with SOX2, NANOG, POU5F1, TCF3 and PRDM14 binding events and the presence of DR2 motif.


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

Chatagnon A, Veber P, Morin V, Bedo J, Triqueneaux G, Sémon M, Laudet V, d'Alché-Buc F, Benoit G - Nucleic Acids Res. (2015)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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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.
Mentions: We next investigated whether the RAR/RXR occupancy profiles identified by clustering (Figure 2D) were specifically associated with molecular features such as the presence of RARE motifs or transcription factor binding sites experimentally identified in F9 and ES cells. To this end, we performed Fisher's exact tests to detect features whose prevalence varied significantly in one cluster compared to all other RAR/RXR binding regions. The results are presented in Figure 5A and Supplementary Figure S8, where red (resp. green) bars indicate features that are significantly enriched (resp. depleted) in the cluster. First we observe that each binding pattern is characterized by a unique combination of molecular features. For instance, the regions assigned to clusters exhibiting high average occupancy in untreated F9 cells are characterized by their association with ESRRB and POU5F1 binding events (clusters B, D and F in Figure 2D and Supplementary Figure S3 panel A). Also, all clusters exhibiting a maximum occupancy in the early phase of the RA-induced PrE differentiation process, namely clusters A–D, are associated with a higher prevalence of DR0 type motif. Among these four clusters, subtle binding pattern variations appear coupled with specific core pluripotency factor binding sites layouts and are independent or negatively associated with PrE differentiation-associated transcription factor like SOX17. Cluster A, which exhibits only a transient occupancy at 2 h after RA stimulation, is only associated with DR0 enrichment while clusters B, C and D are also associated with NR5A2 and SMAD1 binding but differ in their association with SOX2, NANOG, POU5F1, TCF3 and PRDM14 binding events and the presence of DR2 motif.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Université de Lyon, Université Claude Bernard Lyon1, CGphiMC UMR CNRS 5534, 69622 Villeurbanne, France.

Show MeSH
Related in: MedlinePlus