The Leucine Zipper of NRL Interacts with the CRX Homeodomain

Abstract

Photoreceptor-specific expression of rhodopsin is mediated by multiple cis-acting elements in the proximal promoter region. NRL (neural retina leucine zipper) and CRX (cone rod homeobox) proteins bind to the adjacent NRE and Ret-4 sites, respectively, within this region. Although NRL and CRX are each individually able to induce rhodopsin promoter activity, when expressed together they exhibit transcriptional synergy in rhodopsin promoter activation. Using the yeast two-hybrid method and glutathione S-transferase pull-down assays, we demonstrate that the leucine zipper of NRL can physically interact with CRX. Deletion analysis revealed that the CRX homeodomain (CRX-HD) plays an important role in the interaction with the NRL leucine zipper. Although binding with the CRX-HD alone was weak, a strong interaction was detected when flanking regions including the glutamine-rich and the basic regions that follow the HD were included. A reciprocal deletion analysis showed that the leucine zipper of NRL is required for interaction with CRX-HD. Two disease-causing mutations in CRX-HD (R41W and R90W) that exhibit reduced DNA binding and transcriptional synergy also decrease its interaction with NRL. These studies suggest novel possibilities for protein-protein interaction between two conserved DNA-binding motifs and imply that cross-talk among distinct regulatory pathways contributes to the establishment and maintenance of photoreceptor function.Gene activation is a stringently controlled process, involving combinatorial and cooperative action of multiple regulatory proteins with promoter and enhancer DNA elements (1-6). Recent studies, including reconstitution experiments, have suggested that target specificity and transcriptional synergy are achieved by specific and precise interactions among various activator proteins during the assembly of higher order nucleoprotein complexes, including the "enhanceosome" (7-11). The elucidation of these protein-protein and protein-DNA interactions is critical to our understanding of the mechanisms of cell type-and tissue-specific gene expression.Generation of multiple neuronal cell types during retinal development is an evolutionarily conserved biological process, which offers a convenient model system to investigate tissuespecific gene regulation. More than 30 transcription factors representing several classes of DNA-binding proteins are expressed in developing and mature mammalian retina; nevertheless, the precise function of a majority of these proteins remains to be elucidated (12-14). Rhodopsin, the G-proteincoupled light receptor, is expressed specifically in the rod photoreceptors of retina and is a pivotal protein for visual function. Its expression is correlated to rod differentiation and maintained at high levels afterward, throughout life (15). Altered expression of rhodopsin and mutations that affect its function in mature rods result in retinal degeneration (15,16). Regulation of rhodopsin expression is primarily at the level of transcription and is mediated by two distinct...

Conserved Transcriptional Activators of the Xenopus Rhodopsin Gene

“…Nrl is expressed specifically in developing and mature rods (32), and has been shown to activate expression of rhodopsin both in non-retinal and retinal cell cultures (24 -26). Furthermore, Nrl has been shown to bind to Crx (33), and with Crx, can synergistically activate expression of the rhodopsin promoter (24). We show here that Xenopus XOtx5 (34,35) and XL-maf (36) are the functional Xenopus counterparts of mammalian Crx and Nrl.…”

Proximal and Distal Sequences Control UV Cone Pigment Gene Expression in Transgenic Zebrafish

“…Multiple transcription factors (and their binding sites) have been defined, including the transcription factors Crx and NRL, both of which have been shown to be essential for normal photoreceptor development in mammals (23)(24)(25)(26)(27)(28)(29)(30). In Drosophila, multiple transcription factors and cis-acting controlling sequences have been defined for several visual pigment genes, and these have been shown in vivo to control the differential activation of visual pigment genes in the appropriate photoreceptor types (31,32).…”

The Minimal Transactivation Domain of the Basic Motif-Leucine Zipper Transcription Factor NRL Interacts with TATA-binding Protein

“…Yeast Transformations-Yeast strains used in the study were L40 (22) for the pHyblexZeo vector, and Y187 (MATa, ura3-52, his3-200, ade2-101, trp1-901, leu2-3,112, gal4D, metϪ, gal80D, URA3::GAL1 UAS-GAL1 TATA-lacZ) (BD Biosciences) for the pGBKT7 vector. Yeast transformations were performed as described (31) and plated onto YPDA (yeast extract, peptone, glucose, adenine, and agar) ϩ20 g/ml Zeocin and -Trp plates to select for the pHybLexZeo and pGBKT7 vectors, respectively.…”

“…Protein extracts (as indicated for a specific experiment) were co-incubated at 37°C for 1 h, and immunoprecipitation was performed in the presence of phosphate-buffered saline and protease inhibitors using protein A-or G-Sepharose beads. Beads were washed, resuspended in Laemmli sample buffer, boiled for 10 min, and subjected to SDS-PAGE, followed by autoradiography (22,24).…”