Adams David E, Li Siru, Zhen Yuxuan, Kaynak Ahmet, Qi Xiaoyang, Yu Jane J, Shao Wen-Hai
Division of Rheumatology, Allergy & Immunology, Department of Internal Medicine, College of Medicine, University of Cincinnati, Cincinnati, OH 45267, USA.
Division of Immunobiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45267, USA.
Cells. 2025 May 29;14(11):798. doi: 10.3390/cells14110798.
WD40 repeat and FYVE containing protein 1 (WDFY1) functions in membrane trafficking and protein complex scaffolding. WDFY1 has been studied in the immune system and in different oncogenic conditions. Therefore, comprehensive understanding of WDFY1 regulation mechanisms is much desired. In this study, we analyzed the promoter and 5'- and 3'-untranslated regions (UTRs) of and identified critical sequence elements, transcription factors (TFs), and miRNAs that collaboratively regulate gene expression. A 3.5 kb segment of the mouse promoter and 5'-UTR was cloned into a luciferase expression vector and transfected into HeLa cells. Luciferase assays of promoter deletion mutants revealed approximately four-fold increased activity attributed by a 500 bp distal fragment upstream of the 5'-UTR. Four TFs (Sp1, Ap-1, Hes1, and TCF7) were found to be critical for expression with binding sites spread throughout the promoter and 5'-UTR regions. Cloning of a 3.2 kb fragment of 3'-UTR into the luciferase expression vector led to an ~3.5-fold decrease in luciferase activity. Complementary siRNA and luciferase assays mutually confirmed our findings. Most importantly, IL-6, a critical cytokine in organ inflammation, was found to promote WDFY1 expression through the upregulation of Sp1 in primary renal mesangial cells. We, therefore, identified a potential inflammation-driven WDFY1 upregulation in mice.
WD40重复序列和含FYVE结构域蛋白1（WDFY1）在膜运输和蛋白质复合体支架构建中发挥作用。WDFY1已在免疫系统和不同致癌条件下得到研究。因此，人们非常渴望全面了解WDFY1的调控机制。在本研究中，我们分析了WDFY1的启动子以及5'和3'非翻译区（UTR），并鉴定了协同调控该基因表达的关键序列元件、转录因子（TF）和微小RNA（miRNA）。将小鼠WDFY1启动子和5'UTR的3.5 kb片段克隆到荧光素酶表达载体中，并转染至HeLa细胞。对启动子缺失突变体进行荧光素酶检测，结果显示5'UTR上游500 bp的远端片段使活性增加了约四倍。发现四个转录因子（Sp1、Ap-1、Hes1和TCF7）对WDFY1表达至关重要，其结合位点分布在整个启动子和5'UTR区域。将WDFY1 3'UTR的3.2 kb片段克隆到荧光素酶表达载体中，导致荧光素酶活性降低约3.5倍。互补siRNA和荧光素酶检测相互证实了我们的发现。最重要的是，发现器官炎症中的关键细胞因子白细胞介素-6（IL-6）通过上调原代肾系膜细胞中的Sp1来促进WDFY1表达。因此，我们在小鼠中鉴定出一种潜在的炎症驱动的WDFY1上调机制。
