Development of a reporter assay for identifying osmotically responsive enhancers in euryhaline fish

Abstract

Euryhaline fish respond to salinity stress by transcriptional regulation of a large number of genes and their corresponding protein products. While many of such genes have been identified, the mechanism of their osmotic regulation is unknown. To obtain mechanistic insight into osmosensory signaling pathways we developed a reporter assay for identifying osmoresponsive enhancers in osmotically regulated genes. An immortal Mozambique tilapia cell line (OmB cells) was used for expressing a luciferase gene under the control of genomic DNA isolated from Mozambique tilapia. The genomic DNA was isolated from various regions near the inositol monophosphatase 1 (IMPA1) gene, which encodes a robustly hyperosmotically induced key enzyme of the myo-inositol biosynthesis (MIB) pathway. IMPA1 converts glucose-6-phosphate to the compatible osmolyte myo-inositol, which protects cells from hyperosmotic stress. IMPA1 activity, mRNA, and protein are all highly upregulated during salinity stress. We have optimized the use of a dual luciferase enhancer trap assay with genomic DNA from Mozambique tilapia. Bacterial propagation of the corresponding plasmids containing genomic tilapia DNA was shown to be efficient with Top10 but not JM109 cells. Transfection of tilapia OmB cells was shown to be most efficient with 80% confluent of OmB and Viafect Transfection Reagent. The range of luminesce generated by luciferase expression was optimized to be well above background but allow for a high dynamic range to be quantified in the absence and presence of an osmoresponsive enhancer. Data obtained with this assay using controls (315 mOsm/kg) and hyperosmotically treated OmB cells (620 mOsm/kg) far indicate that large portions of the IMPA1 5′UTR (6.4kb upstream, 3.4kb downstream, and 600bp inside the gene) do not contain an osmoresponsive enhancer. However, some gaps in sequence coverage still have to be accounted for and it is also possible that the enhancer is located outside this region. Therefore, future experiments will expand the sequence range screened for IMPA1 and also apply this assay to additional osmoregulated genes of euryhaline tilapia. Support or Funding Information This work was supported by National Science Foundation, IOB 1355098. Figure 1Open in figure viewerPowerPoint Dual luciferase reporter assays of various genomic IMPA1 sequences. The columns in yellow color are the results of 5′UTR sequences; the purple one is the fragment inside the IMPA1 gene; the green column represents data for 3′UTR; blue columns indicate the corresponding control group. None of these genomic DNA regions significantly increased the ratio of Firefly/Renilla luciferase activity.