Figure 1.
Tissue-specific Otx2 genome binding in the retina.
A. Experimental design: four independent ChIP experiments were performed. RPE and neural retine (NR) nuclei of Otx2+/Otx2−GFP mice were subjected to the GFP assays using a GFP antibody, and RPE and NR nuclei of Otx2+/+ mice were subjected to the WT assays using an Otx2 antibody. B. Genome distribution of Otx2 bound regions (OBRs). Upper panel: colour-coded pie chart showing peak distribution of each ChIP-seq assay compared to global genome distribution. Below, number of OBRs and peak height distribution of each assay are shown. Lower panel: percentage of peaks in defined functional domains of the genome for each assay. Colour-code is as in pie charts.
Figure 2.
Dual ChIP-seq assays identify relevant neural retina and RPE OBR core sets.
A. Venn diagrams showing the overlap of peaks identified in both ChIP-seq assays in each tissue. Intersections of GFP (green) and WT (red in NR, blue in RPE) assays represent two core sets of 4167 and 1638 binding sites in the NR and in the RPE, respectively. B. Motif enrichment analysis on the core datasets. Shown is the highest enriched TAATCC Otx2 binding consensus motif. C. Distribution of the TAATCC motif in 1 kb of genomic sequence around the centre of the core set of OBRs. D. GC content 1 kb around the centre of Otx2 bound regions in NR (red) and RPE (blue) compared to a random selection of 1000 regions in the genome (grey). E. RPE specific microarray confirmed genes with a called peak in their vicinity.
Table 1.
Ontology term enrichment in core and non-core set of the neuroretina.
Table 2.
Ontology term enrichment in core and non-core set of the RPE.
Figure 3.
Evolutionary conservation marks OBR relevance.
A. Principle of the relevance assay method: OBRs are sorted according to a given criterion and the mean rank of OBRs close to Otx2 target genes (red) is compared to the average mean rank of all called OBRs according to this criterion. A similar rank indicates a neutral criterion and a lower rank indicates a relevant criterion. B. Shown is the ratio of all OBRs mean rank to the mean rank of OBRs close to microarray confirmed Otx2 target genes according to evolutionary conservation, GC content, DNase hypersensibility (DHS) DNase sensibility and sensibility peaks (DSP), histone H3 lysine 4-mono- and tri-methylation (H3K4Me1, H3K4Me3), CpG islands (CPG), Consensus Coding sequences (CCDS) and known transcription factor bound regions (TFBS). The dashed line at the value of 1 represents the neutrality of all criteria. C. Application of the conservation criterion to the NR: expected and observed percentage of OBRs close to genes relevant to neural retina Gene Ontology terms among the 2/3 and 1/2 most conserved OBRs.
Figure 4.
Contrasted Otx2 genome occupancy in neuroretina and RPE.
A. Venn diagram showing the overlap of OBR core sets in NR (red) and RPE (blue). B. Venn diagram showing the overlap of the closest genes to core set OBRs. C. Representative examples of OBR localization in RPE-specific (Ttr), NR-specific (Atf3) and common (Elmo2) genes. Shown are browser captures for the 4 ChIP-seq assays and control. The gene is indicated in blue at the bottom. D. Distribution of distance from the closest transcription start site (TSS) in NR and RPE for all OBRs lying within 10 kb from a known TSS. E. Distribution of Sp1 and Nkx2-5 motif occurrence 1 kb around the centre of OBRs in the NR (red) and the RPE (blue).
Figure 5.
Otx2 and Crx redundancy in the neuroretina.
A. Heatmap and dendrogram representation of Diffbind clustering of the indicated ChIP-seq experiments. B. Venn diagrams showing overlapping Crx bound regions (CBRs) and OBRs in the NR and in the RPE. CBRs represented the intersection of both Crx ChIP-seq replicates. C. Venn diagram showing the overlap between the above 415 common OBRs/CBRs in the RPE (grey) and the 426 OBRs common to RPE and NR (purple) shown in Fig. 4A. D. Otx2 and Crx transactivation of the RBP3 promoter.