Ni Dongjiao, Huang Xiang, Wang Zhibo, Deng Lin, Zeng Li, Zhang Yiwei, Lu Dongdong, Zou Xinhua
Key Laboratory of Biological Feed of Ministry of Agriculture and Rural Affairs, Boen Biotechnology Co. Ltd, Guangzhou 511400, China.
Asian-Australas J Anim Sci. 2020 Mar;33(3):398-407. doi: 10.5713/ajas.19.0076. Epub 2019 Jul 2.
The Yip1 domain family (YIPF) proteins were proposed to function in endoplasmic reticulum (ER) to Golgi transport and maintenance of the morphology of the Golgi, which were homologues of yeast Yip1p and Yif1p. YIPF3, the member 3 of YIPF family was a homolog of Yif1p. The aim of present study was to investigate the expression and regulation mechanism of porcine YIPF3.
Quantitative realtime polymerase chain reaction (qPCR) was used to analyze porcine YIPF3 mRNA expression pattern in different tissues and pig kidney epithelial (PK15) cells stimulated by polyinosine-polycytidylic acid (poly [I:C]). Site-directed mutations combined with dual luciferase reporter assays and electrophoretic mobility shift assay (EMSA) were employed to reveal transcription regulation mechanism of porcine YIPF3.
Results showed that the mRNA of porcine YIPF3 (pYIPF3) was widely expressed with the highest levels in lymph and lung followed by spleen and liver, while weak in heart and skeletal muscle. Subcellular localization results indicated that it expressed in Golgi apparatus and plasma membranes. Upon stimulation with poly (I:C), the level of this gene was dramatically up-regulated in a time- and concentration-dependent manner. pYIPF3 core promoter region harbored three cis-acting elements which were bound by ETS proto-oncogene 2 (ETS2), zinc finger and BTB domain containing 4 (ZBTB4), and zinc finger and BTB domain containing 14 (ZBTB14), respectively. In which, ETS2 and ZBTB4 both promoted pYIPF3 transcription activity while ZBTB14 inhibited it, and these three transcription factors all played important regulation roles in tumorigenesis and apoptosis.
The pYIPF3 mRNA expression was regulated by ETS2, ZBTB4, and ZBTB14, and its higher expression in immune organs might contribute to enhancing ER to Golgi transport of proteins, thus adapting to the immune response.
Yip1结构域家族（YIPF）蛋白被认为在内质网（ER）到高尔基体的运输以及高尔基体形态维持中发挥作用，它们是酵母Yip1p和Yif1p的同源物。YIPF家族成员3即YIPF3是Yif1p的同源物。本研究旨在探究猪YIPF3的表达及调控机制。
采用定量实时聚合酶链反应（qPCR）分析猪YIPF3 mRNA在不同组织以及经聚肌苷酸-聚胞苷酸（poly [I:C]）刺激的猪肾上皮（PK15）细胞中的表达模式。运用定点突变结合双荧光素酶报告基因检测和电泳迁移率变动分析（EMSA）来揭示猪YIPF3的转录调控机制。
结果显示，猪YIPF3（pYIPF3）的mRNA广泛表达，在淋巴和肺中表达水平最高，其次是脾脏和肝脏，而在心脏和骨骼肌中表达较弱。亚细胞定位结果表明它在高尔基体和质膜中表达。经poly（I:C）刺激后，该基因的水平以时间和浓度依赖的方式显著上调。pYIPF3核心启动子区域含有三个顺式作用元件，分别与ETS原癌基因2（ETS2）、含锌指和BTB结构域4（ZBTB4）以及含锌指和BTB结构域14（ZBTB14）结合。其中，ETS2和ZBTB4均促进pYIPF3的转录活性，而ZBTB14抑制其转录活性，并且这三种转录因子在肿瘤发生和细胞凋亡中均发挥重要的调控作用。
pYIPF3 mRNA的表达受ETS2、ZBTB4和ZBTB14调控，其在免疫器官中的较高表达可能有助于增强蛋白质从内质网到高尔基体的运输，从而适应免疫反应。
