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Otx2 ChIP-seq reveals unique and redundant functions in the mature mouse retina.

Samuel A, Housset M, Fant B, Lamonerie T - PLoS ONE (2014)

Bottom Line: Surprisingly, photoreceptor survival does not require Otx2 expression in the neural retina, where the related Crx homeobox gene, a major regulator of photoreceptor development, is also expressed.To assess the potential redundancy of Otx2 and Crx, we compared our data with Crx ChIP-seq data.Overall, this study provides a better understanding of gene-regulatory networks controlling photoreceptor homeostasis and disease.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Valrose, University of Nice Sophia Antipolis, CNRS UMR7277, Inserm U1091, Nice, France.

ABSTRACT
During mouse retinal development and into adulthood, the transcription factor Otx2 is expressed in pigment epithelium, photoreceptors and bipolar cells. In the mature retina, Otx2 ablation causes photoreceptor degeneration through a non-cell-autonomous mechanism involving Otx2 function in the supporting RPE. Surprisingly, photoreceptor survival does not require Otx2 expression in the neural retina, where the related Crx homeobox gene, a major regulator of photoreceptor development, is also expressed. To get a deeper view of mouse Otx2 activities in the neural retina, we performed chromatin-immunoprecipitation followed by massively parallel sequencing (ChIP-seq) on Otx2. Using two independent ChIP-seq assays, we identified consistent sets of Otx2-bound cis-regulatory elements. Comparison with our previous RPE-specific Otx2 ChIP-seq data shows that Otx2 occupies different functional domains of the genome in RPE cells and in neural retina cells and regulates mostly different sets of genes. To assess the potential redundancy of Otx2 and Crx, we compared our data with Crx ChIP-seq data. While Crx genome occupancy markedly differs from Otx2 genome occupancy in the RPE, it largely overlaps that of Otx2 in the neural retina. Thus, in accordance with its essential role in the RPE and its non-essential role in the neural retina, Otx2 regulates different gene sets in the RPE and the neural retina, and shares an important part of its repertoire with Crx in the neural retina. Overall, this study provides a better understanding of gene-regulatory networks controlling photoreceptor homeostasis and disease.

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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.
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pone-0089110-g002: 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.

Mentions: We next examined to which extent the double antibody approach enhanced the reliability of OBR identification, by comparing the pairs of datasets obtained in the NR and the RPE (Fig. 2A). In the NR, 69.5% of the OBRs detected in the GFP assay overlapped with OBRs detected in the WT experiment, forming a core set of 4167 peaks. We analysed the gene ontology of the corresponding 3308 closest genes using the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool (Table 1). We found an enrichment for the following ontology terms: visual perception (p = 9.9×10−15, Fisher Exact P-value) sensory perception of light stimulus (p = 1.6×10−14), vision (p = 2.6×10−11), detection of light stimulus (p = 1.1×10−6), response to light stimulus (p = 8.2×10−6). By contrast, such enrichment was absent in the 5903 closest genes corresponding to the non-core set of peaks. This indicates that the intersection of GFP and WT datasets in the NR corresponds to a core set of high confidence Otx2 bound regions, with strong relevance to NR function. Similarly, in the RPE, 55.6% of the OBRs detected in the WT assay were common with the OBRs detected in the GFP assay. A total core set of 1638 peaks was deduced. DAVID analysis indicated that the 1374 closest genes were enriched in specific ontology terms (Table 2): cell adhesion (p = 2.3×10−5), cell junction (p = 1.0×10−5), metal-ion binding (p = 3.5×10−4), eye development (p = 2.2×10−3), melanocyte differentiation (p = 2.4×10−3) whereas these enrichments were absent or strongly reduced (p>8.8×10−3) in the 2151 non-core specific closest genes. Therefore, the intersection of GFP and WT datasets in the RPE also represents a core set of high confidence OBRs with relevance to specialized function of the RPE.


Otx2 ChIP-seq reveals unique and redundant functions in the mature mouse retina.

Samuel A, Housset M, Fant B, Lamonerie T - PLoS ONE (2014)

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.
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pone-0089110-g002: 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.
Mentions: We next examined to which extent the double antibody approach enhanced the reliability of OBR identification, by comparing the pairs of datasets obtained in the NR and the RPE (Fig. 2A). In the NR, 69.5% of the OBRs detected in the GFP assay overlapped with OBRs detected in the WT experiment, forming a core set of 4167 peaks. We analysed the gene ontology of the corresponding 3308 closest genes using the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool (Table 1). We found an enrichment for the following ontology terms: visual perception (p = 9.9×10−15, Fisher Exact P-value) sensory perception of light stimulus (p = 1.6×10−14), vision (p = 2.6×10−11), detection of light stimulus (p = 1.1×10−6), response to light stimulus (p = 8.2×10−6). By contrast, such enrichment was absent in the 5903 closest genes corresponding to the non-core set of peaks. This indicates that the intersection of GFP and WT datasets in the NR corresponds to a core set of high confidence Otx2 bound regions, with strong relevance to NR function. Similarly, in the RPE, 55.6% of the OBRs detected in the WT assay were common with the OBRs detected in the GFP assay. A total core set of 1638 peaks was deduced. DAVID analysis indicated that the 1374 closest genes were enriched in specific ontology terms (Table 2): cell adhesion (p = 2.3×10−5), cell junction (p = 1.0×10−5), metal-ion binding (p = 3.5×10−4), eye development (p = 2.2×10−3), melanocyte differentiation (p = 2.4×10−3) whereas these enrichments were absent or strongly reduced (p>8.8×10−3) in the 2151 non-core specific closest genes. Therefore, the intersection of GFP and WT datasets in the RPE also represents a core set of high confidence OBRs with relevance to specialized function of the RPE.

Bottom Line: Surprisingly, photoreceptor survival does not require Otx2 expression in the neural retina, where the related Crx homeobox gene, a major regulator of photoreceptor development, is also expressed.To assess the potential redundancy of Otx2 and Crx, we compared our data with Crx ChIP-seq data.Overall, this study provides a better understanding of gene-regulatory networks controlling photoreceptor homeostasis and disease.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Valrose, University of Nice Sophia Antipolis, CNRS UMR7277, Inserm U1091, Nice, France.

ABSTRACT
During mouse retinal development and into adulthood, the transcription factor Otx2 is expressed in pigment epithelium, photoreceptors and bipolar cells. In the mature retina, Otx2 ablation causes photoreceptor degeneration through a non-cell-autonomous mechanism involving Otx2 function in the supporting RPE. Surprisingly, photoreceptor survival does not require Otx2 expression in the neural retina, where the related Crx homeobox gene, a major regulator of photoreceptor development, is also expressed. To get a deeper view of mouse Otx2 activities in the neural retina, we performed chromatin-immunoprecipitation followed by massively parallel sequencing (ChIP-seq) on Otx2. Using two independent ChIP-seq assays, we identified consistent sets of Otx2-bound cis-regulatory elements. Comparison with our previous RPE-specific Otx2 ChIP-seq data shows that Otx2 occupies different functional domains of the genome in RPE cells and in neural retina cells and regulates mostly different sets of genes. To assess the potential redundancy of Otx2 and Crx, we compared our data with Crx ChIP-seq data. While Crx genome occupancy markedly differs from Otx2 genome occupancy in the RPE, it largely overlaps that of Otx2 in the neural retina. Thus, in accordance with its essential role in the RPE and its non-essential role in the neural retina, Otx2 regulates different gene sets in the RPE and the neural retina, and shares an important part of its repertoire with Crx in the neural retina. Overall, this study provides a better understanding of gene-regulatory networks controlling photoreceptor homeostasis and disease.

Show MeSH
Related in: MedlinePlus