Biochemical Evolution Laboratory, Department of Animal Science, University of California, Davis, CA, 95616.
Laboratory of Aquaculture Nutrition and Environmental Health, School of Life Sciences, East China Normal University, Shanghai, 200241, China.
Proc Natl Acad Sci U S A. 2017 Mar 28;114(13):E2729-E2738. doi: 10.1073/pnas.1614712114. Epub 2017 Mar 13.
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 using cells derived from the brain of the tilapia (OmB cells) to identify osmolality/salinity-responsive enhancers (OSREs) in the genes of Genomic DNA comprising the regulatory regions of two strongly salinity-induced genes, inositol monophosphatase 1 () and -inositol phosphate synthase (), was isolated and analyzed with dual luciferase enhancer trap reporter assays. We identified five sequences (two in and three in ) that share a common consensus element (DDKGGAAWWDWWYDNRB), which we named "OSRE1." Additional OSREs that were less effective in conferring salinity-induced -activation and do not match the OSRE1 consensus also were identified in both and Although OSRE1 shares homology with the mammalian osmotic-response element/tonicity-responsive enhancer (ORE/TonE) enhancer, the latter is insufficient to confer osmotic induction in fish. Like other enhancers, OSRE1 -activates genes independent of orientation. We conclude that OSRE1 is a -regulatory element (CRE) that enhances the hyperosmotic induction of osmoregulated genes in fish. Our study also shows that tailored reporter assays developed for OmB cells facilitate the identification of CREs in fish genomes. Knowledge of the OSRE1 motif allows affinity-purification of the corresponding transcription factor and computational approaches for enhancer screening of fish genomes. 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 specific context (salinity stress) and eliminates pitfalls of constitutive gene knockouts (embryonic lethality, developmental compensation).
鱼类通过许多基因的转录诱导来应对盐度应激，但它们的渗透调节机制尚不清楚。我们开发了一种使用来自罗非鱼脑的细胞的报告基因检测方法（OmB 细胞），以鉴定两个强烈盐度诱导基因（肌醇单磷酸酶 1 () 和肌醇磷酸合酶 ()）的基因中的渗透压/盐度反应增强子（OSRE）。我们分离并分析了包含这两个基因调控区的基因组 DNA，使用双荧光素酶增强子陷阱报告基因检测。我们鉴定了五个序列（两个在 中，三个在 中），它们共享一个共同的核心元件（DDKGGAAWWDWWYDNRB），我们将其命名为“OSRE1”。在 和 中，还鉴定了其他不太有效的赋予盐度诱导 -激活作用且不符合 OSRE1 共识的 OSRE。尽管 OSRE1 与哺乳动物渗透响应元件/渗透压响应增强子（ORE/TonE）增强子具有同源性，但后者不足以在鱼类中赋予渗透压诱导作用。与其他增强子一样，OSRE1 独立于方向激活基因。我们得出结论，OSRE1 是鱼类渗透压调节基因的 -调节元件（CRE），可增强其对高渗的诱导。我们的研究还表明，为 OmB 细胞开发的定制报告基因检测方法有助于鉴定鱼类基因组中的 CRE。OSRE1 基序的知识允许相应转录因子的亲和纯化以及鱼类基因组增强子筛选的计算方法。此外，我们的研究使 OSRE1 增强子的靶向失活成为可能，这种方法优于基因敲除用于功能表征，因为它将基因功能的损伤限制在特定的环境（盐度应激）中，并消除了组成型基因敲除的陷阱（胚胎致死，发育代偿）。
