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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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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.
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pone-0089110-g003: 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.

Mentions: Among the thousands of genome sites bound by transcription factors, no clear indication has emerged so far that could help recognizing those that have prominent regulatory role. We used our knowledge of Otx2 direct target genes to evaluate whether additional features such as chromatin marks or sequence properties could help predicting the relevance of OBRs. The rationale was the following: if a given criteria is relevant to cis-active function of an OBR, then target-gene OBRs should have a lower rank according to this criteria (Fig. 3A). We used the list of previously identified Otx2 direct target genes to sample criteria. As most of these genes were RPE-specific, we restricted our analysis to the RPE OBR core dataset. Figure 3B shows that only sequence conservation led to a mean rank ratio above 1, indicating a lower mean rank of the selected genes. For all other criteria, the relative mean rank of OBRs close to Otx2 target genes remained equal to or below 1. Thus, sequence conservation is an indicator for OBR relevance in this experiment, and might help distinguishing functional binding sites in ChIP-seq experiments. To assess the predictive value of this criterion, we applied it to the NR core dataset. We tested whether OBRs close to genes with relevant ontology terms were enriched among the most conserved OBRs (Fig. 3C). When considering the 2/3 most conserved OBRs in the NR, we observed that 90% of OBRs close to relevant NR genes were present, a proportion significantly superior to the expected one. When considering the 50% most conserved OBRs, 75% of OBRs close to relevant NR genes were included, an even more remote proportion from the expected one. Khi2 tests supported the statistical significance of these results. Hence, filtering the most conserved OBRs also distinguishes relevant genes in the NR.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3928427&req=5

pone-0089110-g003: 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.
Mentions: Among the thousands of genome sites bound by transcription factors, no clear indication has emerged so far that could help recognizing those that have prominent regulatory role. We used our knowledge of Otx2 direct target genes to evaluate whether additional features such as chromatin marks or sequence properties could help predicting the relevance of OBRs. The rationale was the following: if a given criteria is relevant to cis-active function of an OBR, then target-gene OBRs should have a lower rank according to this criteria (Fig. 3A). We used the list of previously identified Otx2 direct target genes to sample criteria. As most of these genes were RPE-specific, we restricted our analysis to the RPE OBR core dataset. Figure 3B shows that only sequence conservation led to a mean rank ratio above 1, indicating a lower mean rank of the selected genes. For all other criteria, the relative mean rank of OBRs close to Otx2 target genes remained equal to or below 1. Thus, sequence conservation is an indicator for OBR relevance in this experiment, and might help distinguishing functional binding sites in ChIP-seq experiments. To assess the predictive value of this criterion, we applied it to the NR core dataset. We tested whether OBRs close to genes with relevant ontology terms were enriched among the most conserved OBRs (Fig. 3C). When considering the 2/3 most conserved OBRs in the NR, we observed that 90% of OBRs close to relevant NR genes were present, a proportion significantly superior to the expected one. When considering the 50% most conserved OBRs, 75% of OBRs close to relevant NR genes were included, an even more remote proportion from the expected one. Khi2 tests supported the statistical significance of these results. Hence, filtering the most conserved OBRs also distinguishes relevant genes in the NR.

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