title: Chromatin immunoprecipitation assay on the rainbow trout opsin proximal promoters illustrates binding of NF-kappaB 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-kappaB 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-kappaB 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-kappaB proteins with their proposed binding sites in the opsin promoters. This analysis demonstrated that NF-kappaB 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-kappaB 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.
authors.affiliation: Department of Biology, University of Victoria, P.O. Box 3020 Stn CSC, Victoria, BC, Canada V8W 3N5.
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Dann
authors.forename: W Ted
authors.initials: WT
authors.lastname: Allison
authors.forename: Kathy
authors.initials: K
authors.lastname: Veldhoen
authors.forename: Trudi
authors.initials: T
authors.lastname: Johnson
authors.forename: Craig W
authors.initials: CW
authors.lastname: Hawryshyn
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D002843:Chromatin; D005786:Gene Expression Regulation; D016328:NF-kappa B / Q000378:metabolism; D017686:Oncorhynchus mykiss / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D012160:Retina / Q000378:metabolism*; D017299:Rod Opsins / Q000235:genetics* / Q000378:metabolism
chemical_list: D002843:Chromatin; D016328:NF-kappa B; D016755:Proto-Oncogene Proteins c-jun; D017299:Rod Opsins