Crx, a Novel Otx-like Paired-Homeodomain Protein, Binds to and Transactivates Photoreceptor Cell-Specific Genes

Abstract

The otd/Otx gene family encodes paired-like homeodomain proteins that are involved in the regulation of anterior head structure and sensory organ development. Using the yeast one-hybrid screen with a bait containing the Ret 4 site from the bovine rhodopsin promoter, we have cloned a new member of the family, Crx (Cone rod homeobox). Crx encodes a 299 amino acid residue protein with a paired-like homeodomain near its N terminus. In the adult, it is expressed predominantly in photoreceptors and pinealocytes. In the developing mouse retina, it is expressed by embryonic day 12.5 (E12.5). Recombinant Crx binds in vitro not only to the Ret 4 site but also to the Ret 1 and BAT-1 sites. In transient transfection studies, Crx transactivates rhodopsin promoter-reporter constructs. Its activity is synergistic with that of Nrl. Crx also binds to and transactivates the genes for several other photoreceptor cell-specific proteins (interphotoreceptor retinoid-binding protein, beta-phosphodiesterase, and arrestin). Human Crx maps to chromosome 19q13.3, the site of a cone rod dystrophy (CORDII). These studies implicate Crx as a potentially important regulator of photoreceptor cell development and gene expression and also identify it as a candidate gene for CORDII and other retinal diseases.

Regulation of photoreceptor gene expression by Crx-associated transcription factor network

“…The function of these Otd orthologs has diverged over time with Crx dedicated specifically to the development and maintenance of retinal photoreceptors and pinealocytes in the pineal gland involved in circadian regulation (see below). In terms of the protein sequence, the three mammalian Otd homologs share 87 −88% homology in the homeodomain near the N-terminus and high homology in several discrete regions in the C-terminal portion, including a glutamine-rich region and the Otx-tail [ (Chen et al, 1997;Furukawa et al, 1997); Figure 1]. Their homeodomain belongs to the K50 (lysine at position 50) paired-like class, similar to that of Drosophila bicoid protein, which, based on structure and functional studies, prefers to bind to DNA motifs with TAATCC or TAAGCT sequences (Treisman et al, 1989;Furukawa et al, 1997;Baird-Titus et al, 2006).…”

“…Using RT-PCR with degenerate primers corresponding to the paired-like homeodomain, Furukawa et al cloned a murine Crx gene, which shows a photoreceptor-specific expression pattern . Chen et al reported cloning bovine Crx using a yeast one-hybrid assay with a rhodopsin promoter element, Ret4, as bait (Chen et al, 1997) and demonstrated that Crx can bind to three target sites in the rhodopsin promoter as well as targets in several other rod gene promoters. Furthermore, Crx acts as a transcription activator and synergizes with the bZIP transcription factor Nrl in activating rhodopsin-reporter gene expression, suggesting for the first time that a high level of rhodopsin expression requires the function of at least two photoreceptor transcription factors.…”

“…Using chromatin immunoprecipitation assays, we have shown that Crx activates transcription by directly binding to the promoter and/or enhancer regions of the target genes in photoreceptor cells . However, in transient cell transfection assays with target promoter-luciferase reporters, Crx alone has only a moderate transactivating activity (2−5-fold enhancement), even with the rhodopsin promoter, a wellknown Crx target (Chen et al, 1997). Thus, one mechanism for Crx to activate transcription is to interact with other transcription regulators.…”

“…Nrl was the first protein identified that acts synergistically with Crx to activate the rhodopsin promoter (Chen et al, 1997;Mitton et al, 2000). This synergy, however, is not observed for cone opsin promoters .…”

Characterization of a novel quiescence responsive element downregulated by v-Src in the promoter of the neuroretina specific QR1 gene

“…Interestingly, synergistic stimulation implicating a Maf protein was reported in the case of a neuroretina speci®c gene. Thus, Nrl was shown to cooperate with the Crx, a homeodomain containing transcription factor, to transactivate reporter constructs of the rhodopsin gene promoter carrying adjacent Nrl and Crx binding sites (Chen et al, 1997). Cooperation between A and C box could be mediated by a synergistic binding to the DNA, as illustrated with various Maf proteins.…”