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.
Highlights
Photoreceptor-specific expression of rhodopsin is mediated by multiple cis-acting elements in the proximal promoter region
These clones, with or without the additional sequence from the 5Ј-untranslated region, were retransformed into L40 yeast. The presence of both the bait vector pLex-NRL leucine zipper (NRL-ZIP) and bovine CRX (bCRX)-prey clones was found to be essential for growth on minus His medium (Fig. 3)
We have provided evidence for direct physical interaction of NRL and CRX, two transcription factors implicated in rhodopsin regulation
Summary
CRX, a photoreceptor-specific paired-like homeodomain protein, was identified as the Ret-4 and BAT-1 binding protein by yeast one-hybrid screening and shown to activate the promoters of rhodopsin and other retinal genes [31]. Mutations in the human CRX and NRL genes have been identified in retinopathies, and these mutations result in altered transcriptional synergy in rhodopsin promoter activation assays [37,38,39,40,41] Based on these findings, we hypothesized that the transcriptional synergy between NRL and CRX in rhodopsin regulation results from cooperativity in binding to adjacent NRE and Ret-4 (or BAT-1) sites and/or from direct physical interaction, leading to the formation of a stable enhanceosome and/or initiation complex. We demonstrate direct interaction between the leucine zipper of NRL and the homeodomain of CRX using the yeast two-hybrid interaction trap and in vitro glutathione S-transferase (GST) pull-down assays
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