NR2E3, a photoreceptor-specific orphan nuclear receptor, is believed to play a pivotal role in the differentiation of photoreceptors. Mutations in the human NR2E3 gene and its mouse ortholog are associated with enhanced S-cones and retinal degeneration. In order to gain insights into the NR2E3 function, we performed temporal and spatial expression analysis, yeast two-hybrid screening, promoter activity assays and co-immunoprecipitation studies. The Nr2e3 expression was localized preferentially to the rod, and not to the cone, photoreceptor nuclei in rodent retina. The yeast two-hybrid screening of a retinal cDNA library, using NR2E3 as the bait, identified another orphan nuclear receptor NR1D1 (Rev-erbalpha). The interaction of NR2E3 with NR1D1 was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation experiments. In transient transfection studies using HEK 293 cells, both NR2E3 and NR1D1 activated the promoters of rod phototransduction genes synergistically with neural retina leucine zipper (NRL) and cone-rod homeobox (CRX). All four proteins, NR2E3, NR1D1, NRL and CRX, could be co-immunoprecipitated from the bovine retinal nuclear extract, suggesting their existence in a multi-protein transcriptional regulatory complex in vivo. Our results demonstrate that NR2E3 is involved in regulating the expression of rod photoreceptor-specific genes and support its proposed role in transcriptional regulatory network(s) during rod differentiation.

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...

Inhibition of VEGF in developing eyes has the short-term effect of delayed vascular growth and the long-term effects of decreased function with persistent changes in the neuroretinal structures.

Eukaryotic DNA is organized as a nucleoprotein polymer termed chromatin with nucleosomes serving as its repetitive architectural units. Cellular differentiation is a dynamic process driven by activation and repression of specific sets of genes, partitioning the genome into transcriptionally active and inactive chromatin domains. Chromatin architecture at individual genes/loci may remain stable through cell divisions, from a single mother cell to its progeny during mitosis, and represents an example of epigenetic phenomena. Epigenetics refers to heritable changes caused by mechanisms distinct from the primary DNA sequence. Recent studies have shown a number of links between chromatin structure, gene expression, extracellular signaling, and cellular differentiation during eye development. This review summarizes recent advances in this field, and the relationship between sequence-specific DNA-binding transcription factors and their roles in recruitment of chromatin remodeling enzymes. In addition, lens and retinal differentiation is accompanied by specific changes in the nucleolar organization, expression of non-coding RNAs, and DNA methylation. Epigenetic regulatory mechanisms in ocular tissues represent exciting areas of research that have opened new avenues for understanding normal eye development, inherited eye diseases and eye diseases related to aging and the environment.

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Background: Mef2c gene expression is significantly diminished in the retinas of NRL (neural retina leucine zipper) knockout mice. Results: NRL binding, RNA polymerase II association, and acetylation of histone H3, revealed a novel alternate Mef2c promoter. Conclusions: Activation of the retinal Mef2c promoter is NRL-dependent and specific to rod photoreceptor cells. Significance: Mef2c represents a novel regulatory node downstream of NRL in adult rod photoreceptor cells.

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