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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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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.
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pone-0089110-g001: 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.

Mentions: To gain insight into the respective function of Otx2 in RPE and neural retina (NR), we set out to compare the genomic targets of Otx2 in each compartment. We dissected eyes from 4–5 weeks old mice and carried out Otx2-ChIP-seq separately on RPE and NR nuclei. In order to enhance the reliability for identified Otx2-bound sites, we performed two parallel experiments using two different antibodies: first, Otx2Otx2−GFP/+ knock-in mice expressing an Otx2-GFP fusion protein [13] together with an anti-GFP antibody, second, wild type mice together with an anti-Otx2 antibody (Fig. 1A). Two pairs of independent sets of data were generated, thereafter referred to as the GFP and the WT assays (Fig. 1A). A preliminary description of RPE Otx2-ChIP-seq was previously published [12]. These RPE data were subjected to in-depth analysis and comparison with neural retina ChIP-seq datasets. A total of 4-16×106 sequence reads were mapped to the genome for each condition. MACS algorithm with a <1% FDR threshold was used to identify peaks, henceforth referred to as Otx2-bound regions (OBRs). NR-WT and NR-GFP assays respectively yielded 15448 and 5997 OBRs, while RPE-WT and RPE-GFP assays respectively yielded 2941 and 3766 OBRs (Fig. 1B). Control experiments with GFP antibody on wild-type mice and non-relevant Lamin-B1 antibody on wild-type mice failed to show significant sequence enrichment above background across the genome, indicating that Otx2 and GFP antibodies were both specific.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928427&req=5

pone-0089110-g001: 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.
Mentions: To gain insight into the respective function of Otx2 in RPE and neural retina (NR), we set out to compare the genomic targets of Otx2 in each compartment. We dissected eyes from 4–5 weeks old mice and carried out Otx2-ChIP-seq separately on RPE and NR nuclei. In order to enhance the reliability for identified Otx2-bound sites, we performed two parallel experiments using two different antibodies: first, Otx2Otx2−GFP/+ knock-in mice expressing an Otx2-GFP fusion protein [13] together with an anti-GFP antibody, second, wild type mice together with an anti-Otx2 antibody (Fig. 1A). Two pairs of independent sets of data were generated, thereafter referred to as the GFP and the WT assays (Fig. 1A). A preliminary description of RPE Otx2-ChIP-seq was previously published [12]. These RPE data were subjected to in-depth analysis and comparison with neural retina ChIP-seq datasets. A total of 4-16×106 sequence reads were mapped to the genome for each condition. MACS algorithm with a <1% FDR threshold was used to identify peaks, henceforth referred to as Otx2-bound regions (OBRs). NR-WT and NR-GFP assays respectively yielded 15448 and 5997 OBRs, while RPE-WT and RPE-GFP assays respectively yielded 2941 and 3766 OBRs (Fig. 1B). Control experiments with GFP antibody on wild-type mice and non-relevant Lamin-B1 antibody on wild-type mice failed to show significant sequence enrichment above background across the genome, indicating that Otx2 and GFP antibodies were both specific.

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