College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
Fujian Engineering Research Center of Aquatic Breeding and Healthy Aquaculture, Xiamen, 361021, China.
BMC Mol Cell Biol. 2020 Nov 24;21(1):83. doi: 10.1186/s12860-020-00323-9.
The effects of diverse stresses ultimately alter the structures and functions of proteins. As molecular chaperones, heat shock proteins (HSPs) are a group of highly conserved proteins that help in the refolding of misfolded proteins and the elimination of irreversibly damaged proteins. They are mediated by a family of transcription factors called heat shock factors (HSFs). The small abalone Haliotis diversicolor is a species naturally distributed along the southern coast of China. In this study, the expression of HdHSF1 was inhibited by RNAi in hemocytes in order to further elucidate the regulatory roles of HdHSF1 on heat shock responsive genes in abalone. Meanwhile, to understand the transcriptional regulation of the HdHSF1 gene, the 5'-upstream regulatory region of HdHSF1 was characterized, and the relative promoter activity was examined by dual-luciferase reporter gene assay system in HEK293T cell lines.
After the inhibition of the H. diversicolor HSF1 gene (HdHSF1) by dsRNA (double-stranded RNA), the expression of most heat shock related-genes was down-regulated (p < 0.05). It indicated the importance of HdHSF1 in the heat shock response of H. diversicolor. Meanwhile, 5'-flanking region sequence (2633 bp) of the HdHSF1 gene was cloned; it contained a putative core promoter region, TATA box, CAAT box, CpG island, and many transcription elements. In HEK293T cells, the 5'-flanking region sequence can drive expression of the enhanced green fluorescent protein (EGFP), proving its promoter function. Exposure of cells to the high-temperature (39 °C and 42 °C) resulted in the activation of HdHSF1 promoter activity, which may explain why the expression of the HdHSF1 gene participates in heat shock response. Luciferase activity of different recombinant plasmids, which contained different truncated promoter fragments of the HdHSF1 gene in HEK293T cells, revealed the possible active regions of the promoter. To further identify the binding site of the critical transcription factor in the region, an expression vector with the site-directed mutation was constructed. After being mutated on the GATA-1 binding site, we found that the luciferase activity was significantly increased, which suggested that the GATA-1 binding site has a certain weakening effect on the activity of the HdHSF1 promoter.
These findings suggest that GATA-1 may be one of the transcription factors of HdHSF1, and a possible signaling pathway mediated by HdHSF1 may exist in H. diversicolor to counteract the adverse effects of heat shock stress.
各种应激的影响最终会改变蛋白质的结构和功能。热休克蛋白（HSPs）作为分子伴侣，是一组高度保守的蛋白质，有助于蛋白质的重折叠和不可逆损伤蛋白质的清除。它们由一类称为热休克因子（HSFs）的转录因子介导。皱纹盘鲍（Haliotis diversicolor）是一种自然分布在中国南部沿海的物种。在这项研究中，通过 RNAi 抑制血细胞中的 HdHSF1 表达，以进一步阐明 HdHSF1 对鲍鱼热休克反应基因的调控作用。同时，为了了解 HdHSF1 基因的转录调控，对 HdHSF1 的 5'-上游调控区进行了特征描述，并通过双荧光素酶报告基因检测系统在 HEK293T 细胞系中检测相对启动子活性。
dsRNA（双链 RNA）抑制皱纹盘鲍 HSF1 基因（HdHSF1）后，大多数热休克相关基因的表达下调（p<0.05）。这表明 HdHSF1 在皱纹盘鲍的热休克反应中非常重要。同时，克隆了 HdHSF1 基因的 5'-侧翼区序列（2633bp）；它包含一个推定的核心启动子区、TATA 盒、CAAT 盒、CpG 岛和许多转录因子。在 HEK293T 细胞中，5'-侧翼区序列可驱动增强型绿色荧光蛋白（EGFP）的表达，证明其具有启动子功能。细胞暴露于高温（39°C 和 42°C）会激活 HdHSF1 启动子活性，这可能解释了为什么 HdHSF1 基因的表达参与了热休克反应。在 HEK293T 细胞中，不同重组质粒的荧光素酶活性表明，该基因的 5'-侧翼区序列可能包含启动子的活性区域。为了进一步鉴定该区域关键转录因子的结合位点，构建了带有定点突变的表达载体。在 GATA-1 结合位点发生突变后，我们发现荧光素酶活性显著增加，这表明 GATA-1 结合位点对 HdHSF1 启动子活性具有一定的减弱作用。
这些发现表明，GATA-1 可能是 HdHSF1 的转录因子之一，皱纹盘鲍中可能存在由 HdHSF1 介导的信号通路，以抵消热休克应激的不利影响。
