Expression And Epigenetic Regulation Of Water Channel Protein 1b(AQP1b) Under Cold Pressure

Aquaporin(aquaporin, AQP) is a membrane embedded protein superfamily, its function is to improve the water molecules and small molecules to the passive, rapid transport across the membrane, the proteins play an important role in maintaining the water balance in the body.There are 13 kinds of aquaporins gene in mammals, but 18 aquaporins gene were found in zebrafish, part of which exists in the form of tandem repeat. In the process of the research in aquaporins gene, AQP1 gene expression is considered related to the cold temperature stress. AQP1 has two tandem repeats in teleosts, AQP1 b gene and AQP1 a gene. AQP1 b gene is thought to have evolved new features to adapt to high salt environment in seawater fish.Our study found that zebrafish embryo fibroblast cells(ZF4) AQP1 b gene expression level is significantly increased under cold stress, suggest that AQP1 b genes may involved in the fish to adapt to cold environment, but the epigenetic mechanism is not clear. For further study of zebrafish Aquaporin1b(AQP1b) gene expression regulation mechanism under cold stress, ZF4 cells were cultured at low temperature 18°C(experimental group) and normal temperature 28°C(control group), and then they were used to research. Chromatin Immunoprecipitation-Real time fluorescent quantitative PCR(ChIP-qPCR) and Methylated DNA Immunoprecipitation-Real time fluorescent quantitative PCR(MeDIP-qPCR)were used to detect AQP1 b gene promoter regions histone modification and DNA methylation level.Results: Sonication result suggest that 140 W, 5% duty cycle, total sonicate 20 min and 25 min can get 200 ~ 1000 bp and mainly focus on 500 bp. Epitope integrity analysis using western blot suggested that the immunogenicity of sonicating 24 min is 3 times less than 20 min. So we chose 20 min as the optimal sonication time. Ch IP-qPCR data indicated that the enrichment of Histone modification H3 lysine 4 trimethylation(H3K4me3) at the promoter of AQP1 b gene under cold stress is 3.1 times higher than control sample(P<0.05), but the enrichment of Histone modification H3 lysine 27 trimethylation(H3K27me3) is 2.1 times less then control(P<0.05). MeDIP-qPCR data indicated that DNA methylation at the promoter of AQP1 b gene under cold stress is 7.3 times lower than control sample(P<0.05). Histone H3K4me3 modification at gene promoter regions is usually thought to alter chromatin structure, thereby promoting gene transcription, and histone H3K27me3 modification are generally considered negatively correlated with the expression of genes. Our study explores the best sonication conditions for ChIP in ZF4 cell, and detects AQP1 b gene promoter histone modification and DNA methylation change to changing expression in response to cold stress. For further study AQP1 b gene relate to cold stress laid a foundation.