title: Osmolality/salinity-responsive enhancers (OSREs) control induction of osmoprotective genes in euryhaline fish.
abstract: Fish respond to salinity stress by transcriptional induction of many genes, but the mechanism of their osmotic regulation is unknown. We developed a reporter assay using cells derived from the brain of the tilapiaOreochromis mossambicus(OmB cells) to identify osmolality/salinity-responsive enhancers (OSREs) in the genes ofOmossambicusGenomic DNA comprising the regulatory regions of two strongly salinity-induced genes, inositol monophosphatase 1 (IMPA1.1) andmyo-inositol phosphate synthase (MIPS), was isolated and analyzed with dual luciferase enhancer trap reporter assays. We identified five sequences (two inIMPA1.1and three inMIPS) that share a common consensus element (DDKGGAAWWDWWYDNRB), which we named "OSRE1." Additional OSREs that were less effective in conferring salinity-inducedtrans-activation and do not match the OSRE1 consensus also were identified in bothMIPSandIMPA1.1Although OSRE1 shares homology with the mammalian osmotic-response element/tonicity-responsive enhancer (ORE/TonE) enhancer, the latter is insufficient to confer osmotic induction in fish. Like other enhancers, OSRE1trans-activates genes independent of orientation. We conclude that OSRE1 is acis-regulatory element (CRE) that enhances the hyperosmotic induction of osmoregulated genes in fish. Our study also shows that tailored reporter assays developed for OmB cells facilitate the identification of CREs in fish genomes. Knowledge of the OSRE1 motif allows affinity-purification of the corresponding transcription factor and computational approaches for enhancer screening of fish genomes. Moreover, our study enables targeted inactivation of OSRE1 enhancers, a method superior to gene knockout for functional characterization because it confines impairment of gene function to a specific context (salinity stress) and eliminates pitfalls of constitutive gene knockouts (embryonic lethality, developmental compensation).
authors.affiliation: Biochemical Evolution Laboratory, Department of Animal Science, University of California, Davis, CA, 95616.|Laboratory of Aquaculture Nutrition and Environmental Health, School of Life Sciences, East China Normal University, Shanghai, 200241, China.
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Wang
authors.affiliation: Biochemical Evolution Laboratory, Department of Animal Science, University of California, Davis, CA, 95616; dkueltz@ucdavis.edu.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Kültz
mesh_terms: D000818:Animals; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009994:Osmolar Concentration; D064587:Osmoregulation / Q000235:genetics*; D013312:Stress, Physiological; D017210:Tilapia / Q000235:genetics / Q000378:metabolism*
chemical_list: D029941:Fish Proteins
keywords: CRE; biochemical evolution; compatible osmolytes; enhancer; osmotic stress signaling