Chromatin immunoprecipitation assay on the rainbow trout opsin proximal promoters illustrates binding of NF-κB and c-jun to the SWS1 promoter in the retina ☆

Abstract

Misexpression of opsins has been linked to apoptosis of photoreceptor cells in the vertebrate retina. Salmonid fish lose their ultraviolet-sensitive (UVS) cones through post-natal developmental apoptosis mediated by thyroid hormone (TH). In order to identify genetic mechanisms that may play a role in the loss of UVS cones, the transcriptional regulation of the SWS1 opsin in the rainbow trout ( Oncorhynchus mykiss) was investigated. The Transfac database was interrogated with promoter sequence acquired by genome-walking PCR using MatInspector V2.2 to identify putative transcription factor (TF) binding sites. Putative binding sites for AP-1 (c-jun) and NF-κB were found in the SWS1 opsin promoter and were chosen for further investigation due to their high MatInspector scores, their established role in photoreceptor apoptosis, and their relative exclusion from other opsin promoters. NF-κB and c-jun proteins were visualized in rainbow trout retinal tissue with immunohistochemistry and c-jun was identified in rainbow trout retinal protein homogenate by immunoblot. A chromatin immunoprecipitation–polymerase chain reaction technique was employed to examine the in vivo interaction of c-jun and NF-κB proteins with their proposed binding sites in the opsin promoters. This analysis demonstrated that NF-κB and c-jun bind to the SWS1 opsin promoter, but not to the other rod and cone opsin promoters tested. Given the role of NF-κB and c-jun during photoreceptor apoptosis, the influence of their activity through TH and their selective binding to the SWS1 opsin promoter in rainbow trout, these TFs represent good candidates of mechanisms underlying UVS cone degeneration in salmonids.