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

Abstract

Euryhaline tilapia (Oreochromis mossambicus) are fish that tolerate a wide salinity range from fresh water to > 3× seawater. Even though the physiological effector mechanisms of osmoregulation that maintain plasma homeostasis in fresh water and seawater fish are well known, the corresponding molecular mechanisms that control switching between hyper-(fresh water) and hypo-osmoregulation (seawater) remain mostly elusive. In this study we show that hyperosmotic induction of glutamine synthetase represents a prominent part of this switch. Proteomics analysis of the O. mossambicus OmB cell line revealed that glutamine synthetase is transcriptionally regulated by hyperosmolality. Therefore, the 5′ regulatory sequence of O. mossambicus glutamine synthetase was investigated. Using an enhancer trapping assay, we discovered a novel osmosensitive mechanism by which intron 1 positively mediates glutamine synthetase transcription. Intron 1 includes a single, functional copy of an osmoresponsive element, osmolality/salinity-responsive enhancer 1 (OSRE1). Unlike for conventional enhancers, the hyperosmotic induction of glutamine synthetase by intron 1 is position dependent. But irrespective of intron 1 position, OSRE1 deletion from intron 1 abolishes hyperosmotic enhancer activity. These findings indicate that proper intron 1 positioning and the presence of an OSRE1 in intron 1 are required for precise enhancement of hyperosmotic glutamine synthetase expression.

Prediction and Experimental Validation of a New Salinity-Responsive Cis-Regulatory Element (CRE) in a Tilapia Cell Line

“…The last criterion we adopted in this study was to rationalize according to our previous publications elucidating where CREs (in particular, osmotically responsive CREs) are located. We previously identified seven osmotically responsive CREs (OSRE1) that were localized between −232 and +56 relative to the TSS and intron 1 in several osmotically regulated genes [ 17 , 18 ]. Thus, emphasis was placed on including this region for each of the 19 genes.…”

“…Then, we examined the regions including 1 kb upstream relative to TSS and 5’ UTR regions (including exon 1 and intron 1) to determine any enrichment pattern of the STREME1 motif in this region. The rationale for first focusing on this region was that proximal promoters, noncoding exon 1, and intron 1 were previously shown to harbor osmoresponsive CREs, which facilitate transcriptional induction during hyperosmolality [ 17 , 18 ]. Chloride intracellular channel 2 ( clic2 ) and uridine phosphorylase 1 ( upp1 ) each had three significant occurrences of the STREME1 motif in this region and were selected for experimental validation of the functionality of this motif during hyperosmotic stress.…”

“…The O. mossambicus brain (OmB) cell line was subjected to all hyperosmotic stress challenges. L-15 medium containing 5% (vol/vol) fetal bovine serum (FBS) and 1% (vol/vol) penicillin-streptomycin at 26 °C was used to grow OmB cells at 2% CO2 as previously described [ 17 , 18 ]. Using a large supply of OmB cell superstock (passage 15; P15), all experiments were conducted on OmB cells between P20 and P27.…”

“…Ligation reactions were prepared as follows: 50 ng of vector, 10–20 ng of insert (1:5 molar ratio), 2 μL of ligase buffer, 1 μL of T4 ligase (1 U/μL) and nuclease-free H 2 O were added to 20 μL of digested materials. Ligation reactions were incubated in a thermocycler (Mastercycler, Eppendorf) at 25 °C for 5 h. The ligation products were transformed into 10-beta-competent E. coli (New England Biolabs) as previously described [ 18 ]. After transformation, an appropriate amount of the bacterial solution was spread onto a prewarmed (37 °C) LB-ampicillin plate.…”

“…Few studies have identified the mechanism of regulation of transcripts and proteins, i.e., whether regulation takes place at the level of transcription (gene expression), posttranscriptional mRNA abundance regulation, translational regulation, or protein turnover. We have previously demonstrated that gene expression control by a specific novel CRE, the osmotic/salinity response element 1 (OSRE1), is largely responsible for the hyperosmotic upregulation of several osmoregulated proteins [ 17 , 18 ].…”