Li Zhuanzhuan, Xu Chengxun, Yu Hong, Kong Lingfeng, Liu Shikai, Li Qi
Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, China.
Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China.
Gene. 2023 Apr 20;861:147258. doi: 10.1016/j.gene.2023.147258. Epub 2023 Feb 6.
Previous study has found that b (0, +) -type amino acid transporter 1 (CgB-aat1) plays an essential role on mantle pigmentation in the Pacific oyster Crassostrea gigas. However, the molecular regulation of CgB-aat1 gene expression remains unclear. Herein, three POU domain family members, CgPOU2F1, CgPOU3F4-like and CgPOU4F3-X1 were characterized and they all had POUs and HOX domains, respectively, which were important in transcriptional regulation. CgPOU3F4-like gene expression was the highest in mantle edge. Subsequently, the dual-luciferase reporter result showed that the core regulatory region of CgB-aat1 gene was from -632 to -350 bp of promoter. In transient co-transfection assays, the strongest activity was activated only by CgPOU3F4-like, suggesting CgPOU3F4-like was a valid transcriptional activator of CgB-aat1 gene promoter. And the structural integrity of CgPOU3F4-like was essential for its activation function. In addition, site directed mutagenesis assay was applied to detect three key binding sites between CgPOU3F4-like and core region of CgB-aat1 gene promoter, and this interaction was verified by ChIP test. Furthermore, CgPOU3F4-like knockdown by RNA interference led to obvious decreases in CgB-aat1 and cystathionine beta-synthase (CgCbs) expressions at both mRNA and protein levels. Collectively, these results indicate that CgPOU3F4-like positively regulate CgB-aat1 gene expression and it may be a critical upstream transcriptional regulation factor in pheomelanin synthesis in C. gigas.
先前的研究发现，b(0, +)-型氨基酸转运体1（CgB-aat1）在太平洋牡蛎（Crassostrea gigas）的外套膜色素沉着中起关键作用。然而，CgB-aat1基因表达的分子调控机制仍不清楚。在此，对三个POU结构域家族成员CgPOU2F1、CgPOU3F4-like和CgPOU4F3-X1进行了鉴定，它们分别具有POUs和HOX结构域，这些结构域在转录调控中很重要。CgPOU3F4-like基因在外套膜边缘的表达最高。随后，双荧光素酶报告基因结果表明，CgB-aat1基因的核心调控区域位于启动子的-632至-350 bp处。在瞬时共转染实验中，只有CgPOU3F4-like激活了最强的活性，表明CgPOU3F4-like是CgB-aat1基因启动子的有效转录激活因子。并且CgPOU3F4-like的结构完整性对其激活功能至关重要。此外，应用定点突变实验检测了CgPOU3F4-like与CgB-aat1基因启动子核心区域之间的三个关键结合位点，并且通过染色质免疫沉淀实验验证了这种相互作用。此外，RNA干扰敲低CgPOU3F4-like导致CgB-aat1和胱硫醚β-合酶（CgCbs）在mRNA和蛋白质水平的表达明显降低。总的来说,这些结果表明CgPOU3F4-like正向调控CgB-aat1基因表达，并且它可能是太平洋牡蛎中褐黑素合成的关键上游转录调控因子。
