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 for identifying osmolality/ salinity responsive enhancers (OSREs) in Oreochromis mossambicus genes in OmB cells. Genomic DNA comprising regulatory regions of two strongly salinity-induced genes, inositol monophosphatase 1 (IMPA1) and myo-inositol phosphate synthase (MIPS), was isolated and analyzed using dual luciferase enhancer trap reporter assays. Using targeted proteomics, salinity-induction of these genes was shown to penetrate to the protein level. Salinity-induction of both genes and upregulation of the corresponding proteins was completely inhibited in the presence of actinomycin D, which indicates that the mechanisms of induction is transcriptional. Five sequences were identified that are responsible for salinity-induced transactivation and share a common consensus (DDKGGAAWWDWWYDNRB). This element was named OSRE1. Additional OSREs that were less effective in conferring salinity-induced transactivation and do not match the OSRE1 consensus were identified in both MIPS and IMPA1 genes. Although OSRE1 shares a core sequence with mammalian ORE/TonE the latter is insufficient to confer osmotic induction in fish.

Tryptic peptides from each sample (200 ng on column) were injected with a nanoAcquity sample manager (Waters), trapped for 1 min at 15 µL/min on a Symmetry trap column (Waters 186003514), and separated on a 1.7µm particle size BEH C18 column (250mm x 75µm, Waters) by reversed phase LC using a nanoAcquity binary solvent manager (Waters). A 125 min linear gradient ranging from 3% to 35% acetonitrile was used. Peptides were ionized by nano-ESI using a pico-emitter tip (New Objective) and analyzed by an ImpactHD UHR-QTOF mass spectrometer (Bruker). Batch-processing of samples was controlled with Hystar 3.2 (Bruker). The mass range was set to 490 – 910 m/z at 15 Hz scan rate with an isolation width of 11 m/z (1 m/z overlap). Spectral libraries were created from prior precursor-dependent MS1 data (< 20 ppm mass error). DIA data for targeted proteins were statistically analyzed using Skyline 3.5 (MacCossLab, UW).

For experiment XW0001, tilapia OmB cells were dosed for 72 h in either hyperosmotic (650 mosm/kg) or isosmotic (315 mosm/kg control) media. Replicates are different dishes from which proteins were extracted and processed independently. For experiment XW0003, tilapia OmB cells were dosed for 24 h in either hyperosmotic (650 mosm/kg) or isosmotic (315 mosm/kg control) media. Two additional treatment groups were exposed to 10 µM actinomycin D during dosing for 24 h in either hyperosmotic (650 mosm/kg) or isosmotic (315 mosm/kg control) media. Replicates are different dishes from which proteins were extracted and processed independently.