CHX10 Targets a Subset of Photoreceptor Genes

Abstract

The homeobox gene CHX10 is required for retinal progenitor cell proliferation early in retinogenesis and subsequently for bipolar neuron differentiation. To clarify the molecular mechanisms employed by CHX10 we sought to identify its target genes. In a yeast one-hybrid assay Chx10 interacted with the Ret1 site of the photoreceptor-specific gene Rhodopsin. Gel shift assays using in vitro translated protein confirmed that CHX10 binds to Ret1, but not to the similar Rhodopsin sites Ret4 and BAT-1. Using retinal nuclear lysates, we observed interactions between Chx10 and additional photoreceptor-specific elements including the PCE-1 (Rod arrestin/S-antigen) and the Cone opsin locus control region (Red/green cone opsin). However, chromatin immunoprecipitation assays revealed that in vivo, Chx10 bound sites upstream of the Rod arrestin and Interphotoreceptor retinoid-binding protein genes but not Rhodopsin or Cone opsin. Thus, in a chromatin context, Chx10 associates with a specific subset of elements that it binds with comparable apparent affinity in vitro. Our data suggest that CHX10 may target these motifs to inhibit rod photoreceptor gene expression in bipolar cells.The mammalian retina consists of three nuclear layers. The outer nuclear layer houses the cell bodies of photoreceptors (rods and cones), whereas those of horizontal, bipolar, and amacrine interneurons and Müller glia reside in the inner nuclear layer. The innermost layer is the ganglion cell layer, which consists of a mixture of ganglion and amacrine neurons. These three cellular areas are separated by outer and inner plexiform layers that house synaptic connections. This intricate laminated structure develops through the amplification of multipotent progenitor cells, generation of more restricted post-mitotic transition cells, and maturation of these cells into terminally differentiated neurons and glia (1). In rodents, ganglion, horizontal, cone, and amacrine transition cells are born in the prenatal period, bipolar and Müller cells are born post-natally, and rods are born throughout retinal development (2). Transcription factors play critical roles in each of the stages of retinal development, but many gaps remain in our understanding of the specific target genes involved. Here, we focus on one of these factors, the homeobox gene CHX10, and its role in sculpting the characteristics of bipolar interneurons.Homeodomain (HD) 4 proteins regulate retinal development from the earliest stages of optic vesicle formation to the final stages of maturation in the adult (reviewed in Refs. 3 and 4)). Previous molecular analysis linked a naturally occurring mutation in the homeobox of Chx10 to the ocular retardation phenotype in mice (or J ) (5). These mice display a dramatic decrease in retinal progenitor cell (RPC) proliferation and lack bipolar cells, phenotypes that reflect the expression of Chx10 in both of these cell types 5 (5, 6). Molecular and genetic studies have begun to reveal some aspects of the mechanism by which Chx10 regulates retinal development...

Negative regulation of Vsx1 by its paralog Chx10/Vsx2 is conserved in the vertebrate retina

“…We found that Chx10 R200Q does not significantly repress reporter activity, suggesting that the ability of Chx10 to repress transcription is largely dependent on high affinity binding to sequences conforming to the consensus. These findings are in agreement with a previous report showing that mutation of another residue in the DNA binding helix of Chx10 (N51A) also reduces the efficiency of transcriptional repression by Chx10 (Dorval et al, 2005;Dorval et al, 2006). Interestingly, mutation of both consensus sites in the Vsx1 genomic sequence (mut3) causes a significant drop in basal reporter activity.…”

“…These findings combined with the shared DNA binding characteristics and repressive activities of Chx10 and Vsx1 led to the proposal that these genes could function in bipolar cells to prevent inappropriate expression of photoreceptor genes (Dorval et al, 2006). While possible, this scenario is not likely to fully explain how Chx10 and Vsx1 function in bipolar cells.…”

“…Based on a collection of known Chx10 binding sequences, Dorval and colleagues defined the following sequence as a consensus for Chx10 binding: PyTAATTPuPu (Py, pyrimidine; Pu, purine; Dorval et al, 2006). A scan of genomic DNA associated with the Vsx1 locus revealed two potential sites: one at −576 nt (TTAATTAG) and another at −1275 nt (CTAATTGG) relative to the Vsx1 transcriptional start site as predicted by ensembl (www.ensembl.org).…”

“…Chx10 overexpression inhibits photoreceptor differentiation and several phototransduction genes are candidate direct transcriptional targets of negative regulation by Chx10 (Dorval et al, 2006;Livne-Bar et al, 2006;Toy et al, 2002). These findings combined with the shared DNA binding characteristics and repressive activities of Chx10 and Vsx1 led to the proposal that these genes could function in bipolar cells to prevent inappropriate expression of photoreceptor genes (Dorval et al, 2006).…”

Chx10 is required to block photoreceptor differentiation but is dispensable for progenitor proliferation in the postnatal retina

“…Other distinct possibilities are that Chx10 acts downstream of Otx2 to regulate expression of another transcription factor essential for rod genesis, such as Nrl as mentioned above, or that, because paired-like homeodomain proteins can heterodimerize (32), Chx10 modulates Otx2 and͞or Crx target specificity. Although the fate determinants that Chx10 regulates are unknown, we showed that it binds and represses certain late-stage genes involved in photoreceptor differentiation (33). Thus, Chx10 appears to inhibit both photoreceptor specification and subsequent maturation.…”