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

Chanhee Kim,Dietmar Kültz

doi:10.1038/s41598-020-69090-z

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.

Highlights

Euryhaline fish have evolved the capacity to utilize a suite of osmoresponsive genes for rapidly switching between hypo- and hyper-osmoregulation in response to salinity stress to maintain plasma ionic and osmotic homeostasis[1]
Actinomycin D applied to OmB cells during exposure to hyperosmotic stress prevented glutamine synthetase production, which confirms that glutamine synthetase upregulation is mediated by transcriptional induction (Fig. 1 and Supplementary Fig. S1)
We discovered a novel molecular mechanism where intron 1 harboring an osmoresponsive cis-regulatory elements (CREs) (GS-osmolality/ salinity-responsive enhancer 1 (OSRE1)) positively regulates transcription of the tilapia GS under hyperosmotic stress

Summary

Introduction

Euryhaline fish have evolved the capacity to utilize a suite of osmoresponsive genes for rapidly switching between hypo- and hyper-osmoregulation in response to salinity stress to maintain plasma ionic and osmotic homeostasis[1]. Even more moderate but acute salinity stress occurring during transfer of tilapia from freshwater to 25 g/kg results in plasma osmolality increasing up to 460 mOsmol/kg at 15 h9 This species has evolved molecular mechanisms for rapidly turning on and off genes that encode enzymes and transporters involved in hypo- and hyper-osmoregulation[10,11]. 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[18] Enhancers such as OSRE1 are generally considered to function independent of whether they occur in the 5′ or 3′ regulatory regions or in introns[19]. Mechanistic insight into salinity (hyperosmotic) stress adaptation of euryhaline fish helps elucidate mechanisms that can be targeted to improve aquaculture practices in arid and coastal areas impacted by climate c hange[33]

Methods

Results

Conclusion