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 tilapia Oreochromis mossambicus (OmB cells) to identify osmolality/salinity-responsive enhancers (OSREs) in the genes of O. mossambicus. Genomic DNA comprising the regulatory regions of two strongly salinity-induced genes, inositol monophosphatase 1 (IMPA1.1) and myo-inositol phosphate synthase (MIPS), was isolated and analyzed with dual luciferase enhancer trap reporter assays. We identified five sequences (two in IMPA1.1 and three in MIPS) that share a common consensus element (DDKGGAAWWDWWYDNRB), which we named "OSRE1." Additional OSREs that were less effective in conferring salinity-induced trans-activation and do not match the OSRE1 consensus also were identified in both MIPS and IMPA1.1. Although 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, OSRE1 trans-activates genes independent of orientation. We conclude that OSRE1 is a cis-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). biochemical evolution | compatible osmolytes | osmotic stress signaling | enhancer | CRE

Novel zebrafish behavioral assay to identify modifiers of the rapid, nongenomic stress response

“…Seawater is at about 600 mM and freshwater in rivers is at about 1 mM NaCl . The salinity in a zebrafish body is about 150 mM, which is similar to that in humans . Although the concentration (100 mM) in our stimulation is higher than that of freshwater, it is far less than that of seawater and below the physiological salinity in zebrafish body.…”

“…52,53 The salinity in a zebrafish body is about 150 mM, which is similar to that in humans. 54,55 Although the concentration (100 mM) in our stimulation is higher than that of freshwater, it is far less than that of seawater and below the physiological salinity in zebrafish body. This would not reverse the normal physiology of zebrafish in which the animal maintains osmotic homeostasis in hypotonic environments.…”

An osmolality/ salinity-responsive enhancer 1 (OSRE1) in intron 1 promotes salinity induction of tilapia glutamine synthetase

“…Systematic bioinformatics searches of the entire intron 1 sequence for the occurrence of a previously identified OSRE1 was performed by utilizing the OSRE1-consensus (DDKGGAAWWDWWYDNRB) and several specific OSRE1 sequences (incl. O. mossambicus IMPA1.1-OSRE1: AGTGGAAAAATACTAAG) that yielded high hyperosmotic induction of reporter activity in a previous study 16 . This approach enabled us to identify a single copy of OSRE1-like sequence (AGTGGAAAAATACAAC) in intron 1 of GS.…”

“…Enhancers such as OSRE1 are generally considered to function independent of whether they occur in the 5' or 3' regulatory regions or in introns 11,17 . Although most enhancers, including OSRE1 in O. mossambicus IMPA1.1 and MIPS genes 16 , are found in the 5' regulatory region, intronic enhancers have been previously reported. For example, using human cell lines, Harris et al have identified a tissue-specific enhancer in intron 1 of the cystic fibrosis transmembrane conductance regulator gene (CFTR) 18,19 .…”

“…In contrast to these findings in mammalian models, a comprehensive understanding of enhancer functions in fish exposed to salinity stress is still very limited. We have recently identified several copies of a CRE, the osmolality/salinity-responsive enhancer 1 (OSRE1) in the inositol monophosphatase (IMPA1.1) and myo-inositol phosphate synthase (MIPS) genes of O. mossambicus 16 . Enhancers such as OSRE1 are generally considered to function independent of whether they occur in the 5' or 3' regulatory regions or in introns 11,17 .…”