Otx2 ChIP-seq Reveals Unique and Redundant Functions in the Mature Mouse Retina

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.

Stimulation of functional neuronal regeneration from Müller glia in adult mice

“…Black arrows mark open chromatin in promoter regions that are bound by Otx2 in ANT-treated but not control Ascl1 cells, and are appropriate regions of binding based on a whole-retina Otx2 ChIP–seq dataset. d , e , Otx2 ChIP–seq peaks from ANT-treated cells show central enrichment at Otx2 ChIP–seq sites from whole adult retina 15 , but this is not present in the Otx2 ChIP–seq from MG that expressed Ascl1 but received none of the other treatments (TSA, NMDA). f , Top-scoring transcription factor binding motif enrichment (HOMER software suite) from ANT-treated Otx2 ChIP–seq peak calls along with the closest matching transcription factor motif (TF match) and P value for de novo motif enrichment.…”

“…ANT-treated retinas were collected 46 days post-TSA. c , IGV browser tracks at bipolar genes Crx , Cplx4 and Cabp5 showing: Otx2 ChIP–seq from whole adult mouse retina 15 (grey); Otx2 ChIP–seq from FACS-purified ANT-treated and Ascl1-only cells (blue); and DNA accessibility from ATAC–seq of FACS-purified ANT-treated cells (red). Scale for all tracks shown to the right as reads per million.…”

“…To determine whether these were associated with cis -regulatory elements of retinal neurons, we FACS-purified bipolar cells from adult Grm6-tdTomato mice and carried out ATAC–seq. We compared Otx2 ChIP–seq data from whole retina 15 with the ATAC–seq from bipolar cells to generate a set of bipolar Otx2 peaks (Fig. 4h, i).…”

Photoreceptor cell fate specification in vertebrates

“…The deletion of Otx2 from the retina prevents the development of photoreceptors and bipolar cells, whereas its overexpression can promote the formation of both cell types (Nishida et al, 2003;Koike et al, 2007;Sato et al, 2007;Wang et al, 2014). Accordingly, chromatin immunoprecipitation (ChIP) and enhancer characterization experiments show that Otx2 associates with the promoters and enhancers of genes expressed in both photoreceptors and bipolar cells (Kim et al, 2008;Brzezinski et al, 2013;Samuel et al, 2014). The initiation of Otx2 expression in progenitors leads to the activation of additional transcription factors required for correct fate specification.…”

“…Deletion of Crx in mice does not prevent photoreceptor specification like Otx2 deletion, but instead leads to a dramatic reduction in the expression of photoreceptor genes. A comparison of Crx and Otx2 ChIP-seq datasets shows that their transcriptional targets largely overlap in the retina, although there are also some unique targets (Samuel et al, 2014). Crx is expressed in both rods and cones and, as a result, mutations in this gene can lead to diseases that affect one or both photoreceptor types, such as Leber's congenital amaurosis and cone-rod dystrophy (Box 2) (Freund et al, 1997;Swaroop et al, 1999;Tran and Chen, 2014 The distinction between rod and cone gene expression profiles occurs downstream (or independently) of Otx2 and Crx, and two key factors that control this specification event are Rorβ and Nrl (Swaroop et al, 2010).…”