Control Induction of Osmoprotective Genes in Euryhaline Fish

Osmolality/salinity-responsive enhancers (OSREs) PNAS PLUS control induction of osmoprotective genes in euryhaline fish Xiaodan Wanga,b and Dietmar Kültza,1 aBiochemical Evolution Laboratory, Department of Animal Science, University of California, Davis, CA, 95616; and bLaboratory of Aquaculture Nutrition and Environmental Health, School of Life Sciences, East China Normal University, Shanghai, 200241, China Edited by George N. Somero, Stanford University, Pacific Grove, CA, and approved February 17, 2017 (received for review September 1, 2016) Fish respond to salinity stress by transcriptional induction of many that controls the activity and distribution of aquatic animals, such genes, but the mechanism of their osmotic regulation is unknown. changes significantly impact fish biodiversity and distribution. We developed a reporter assay using cells derived from the brain of Most fish (>25,000 extant species) are teleosts and osmor- the tilapia Oreochromis mossambicus (OmB cells) to identify egulators, meaning that they maintain their extracellular body osmolality/salinity-responsive enhancers (OSREs) in the genes of fluids at a relatively constant osmolality of ∼300 mOsmol/kg (is- O. mossambicus. Genomic DNA comprising the regulatory regions osmotic to a salinity of 9 g/kg). Only a small minority of fish (such of two strongly salinity-induced genes, inositol monophosphatase 1 as marine hagfish and elasmobranchs) are osmoconformers (3). (IMPA1.1)andmyo-inositol phosphate synthase (MIPS), was isolated Altering habitat salinity causes stress and evokes compensatory and analyzed with dual luciferase enhancer trap reporter assays. We osmoregulatory responses in fish (3). Fish species that tolerate identified five sequences (two in IMPA1.1 and three in MIPS) that only a narrow salinity range are known as “stenohaline species”; share a common consensus element (DDKGGAAWWDWWYDNRB), those that can tolerate a wide salinity range are referred to as which we named “OSRE1.” Additional OSREs that were less effec- “euryhaline species” (4). Euryhaline fish have evolved special tive in conferring salinity-induced trans-activation and do not match biochemical and physiological mechanisms that allow them to the OSRE1 consensus also were identified in both MIPS and perceive and compensate for changes in the salinity of their EVOLUTION IMPA1.1. Although OSRE1 shares homology with the mammalian aquatic habitat. They can sense osmotic stress, leading to the ac- osmotic-response element/tonicity-responsive enhancer (ORE/TonE) tivation of osmosensory signaling mechanisms that, in turn, con- enhancer, the latter is insufficient to confer osmotic induction in trol osmoregulatory effectors to alleviate osmotic stress (5). A trans fish. Like other enhancers, OSRE1 -activates genes indepen- large number of osmoregulatory effector genes and their protein cis dent of orientation. We conclude that OSRE1 is a -regulatory products are regulated when euryhaline fish experience salinity element (CRE) that enhances the hyperosmotic induction of stress (3, 6, 7). The osmosensory signaling networks that control osmoregulated genes in fish. Our study also shows that tailored these effector genes are most potent and apparent in euryhaline reporter assays developed for OmB cells facilitate the identifica- species with highly dynamic osmoregulatory ability (8). One such tion of CREs in fish genomes. Knowledge of the OSRE1 motif allows species is Oreochromis mossambicus, which has a very wide salinity affinity-purification of the corresponding transcription factor and tolerance range of 0–120 g/kg (9) and is a well-established model computational approaches for enhancer screening of fish genomes. for studies of teleost osmoregulation (10–12). 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 Significance specific context (salinity stress) and eliminates pitfalls of consti- tutive gene knockouts (embryonic lethality, developmental Salinity stress is common in many environments and is predicted compensation). to intensify. Such stress increases the expression of numerous genes in fish, but the corresponding regulatory mechanisms are biochemical evolution | compatible osmolytes | osmotic stress signaling | unknown. Our study provides a toolkit for discovering and func- enhancer | CRE tionally validating cis-regulatory elements (CREs) that control inducible gene expression in fish. This toolkit was used for experimental identification of the first osmotic/salinity-responsive major challenge of biology is understanding the evolutionary/ CREs in fish (OSRE1). Our findings greatly empower novel ap- adaptive significance of genetic variation and the biochemical A proaches for deciphering fish osmosensory signaling and gene mechanisms that govern the phenotypic diversity of organisms. Mi- regulatory networks. Because sequence variation in inducible croevolutionary and functional autecology studies aim to explain CREs is critical for the evolution of stress tolerance, knowledge how organisms adapt to environmental change and stress, but cur- of osmolality/salinity-responsive enhancers is critical for revealing rently they rely heavily on correlations of phenotypes with particular the evolution and function of regulatory networks responsible SNPs or other sequence variations (1). Moreover, holistic systems for euryhalinity of fish. biology approaches aimed at explaining physiological plasticity and acclimatory responses to environmental change and stress often rely Author contributions: D.K. designed research; X.W. performed research; X.W. and D.K. on comprehensive correlations between specific environmental analyzed data; and X.W. and D.K. wrote the paper. conditions and changes in the abundance of particular mRNAs or The authors declare no conflict of interest. proteins. There is great need to complement such approaches with This article is a PNAS Direct Submission. studies that establish causal links between sequence variation, Freely available online through the PNAS open access option. changes in gene expression, and environmental signals to understand Data deposition: The sequences reported in this paper have been deposited in the Gen- Bank database (accession nos. KX649230 and KX649231) and are available at Panorama the mechanistic consequences of global climate change on organis- Public, https://panoramaweb.org/labkey/XW2016-1.url. mal form and function. Climate change accelerates the melting of 1To whom correspondence should be addressed. Email: [email protected]. polar icecaps, the salinization of coastal areas, and the decrease in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. average ocean salinity (2). Because salinity is a major abiotic factor 1073/pnas.1614712114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1614712114 PNAS Early Edition | 1of10 Downloaded by guest on September 29, 2021 Evolutionary differences in the environmental regulation of gene the mechanisms by which osmosensory signaling networks control expression are often the result of altered cis-regulatory elements gene expression during salinity stress. In the current study we de- (CREs) (13, 14). In fact, genetic variation in CREs and/or trans- veloped an enhancer trap reporter assay for the OmB cell line and acting factors represents an efficient and common evolutionary used it to identify and functionally validate an osmotic/salinity- strategy for changing environmentally modulated gene-expression responsive CRE, OSRE1, that is present in multiple copies in patterns (15–18). Changed gene-expression patterns, in turn, lead the IMPA1.1 and MIPS genes of euryhaline fish. to altered phenotypes to achieve biochemical adaptation and the evolution of populations and species (15–18). Such variation Results modifies the highly dynamic nature of gene-regulatory networks, Development of the Luciferase Reporter Assay for Identifying CREs in which embody the interaction of a large number of CREs and trans Tilapia OmB Cells. We developed an enhancer trap reporter assay factors (19–22). Therefore, the identification and experimental for identifying OSREs by using firefly luciferase reporter plasmid, validation of environmentally regulated CREs in organisms that Renilla luciferase normalizer plasmid, and the OmB cell line. are uniquely adapted to particular habitats is a critical prerequisite Transfection of OmB cells was most efficient at 80% cell con- for understanding biochemical evolution (23). fluency using ViaFect reagent (Fig. S1 A and B). The optimal ratio Transcriptional enhancers are CREs that were discovered several of Firefly to Renilla luciferase constructs transfected into OmB cells decades ago, but their identification and the characterization of was 8:1. The highest dynamic range of reporter activity was ac- their environmental, developmental, or tissue-specific activation is complished when using a ratio of 1 μgDNAto3μLoftransfection still challenging (23). A popular and powerful approach for genome- reagent. The optimal time of dosing cells in hyperosmotic medium wide mapping of enhancers is ChiP (24). However, this approach was determined by transfecting OmB cells kept in isosmotic medium requires knowledge of specific epigenetic chromatin states (e.g., a at different confluency (40–90%) with firefly luciferase reporter particular histone posttranslational modification) or enhancer-specific plasmid.

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