Zhang Miao-Qing, Zhao Qiong, Zhang Jing-Pu
Key Laboratory of Biotechnology of Antibiotics, the National Health Commission (NHC), Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing, China.
Postdoctoral Scientific Research Workstation, China Resources Sanjiu Medical & Pharmaceutical Co., Ltd ., Shenzhen, China.
Autophagy. 2020 Dec;16(12):2167-2179. doi: 10.1080/15548627.2020.1719681. Epub 2020 Jan 29.
IFNL2 is a potent antiviral interferon, but the regulation of its gene expression is not fully clear. Here, we report the regulation of ATG10S for transcription. Through sequential deletion of the promoter sequence, we found , a fragment of the promoter responding to ATG10S activity. Subcellular localization and DNA immunoprecipitation assays showed ATG10S translocating into the nucleus and binding to . Online prediction for transcription factor binding sites showed an IRF1 targeting locus in . Luciferase assays, RT-PCR, and western blot analysis revealed a core motif (CAAGAC) existing in , which determined ATG10S and IRF1 activity; individual nucleotide substitution showed that the functional nucleotides of ATG10S targeting were C1, A3, and C6, and the ones associated with IRF1 were A3 and G4 within the core motif. Co-immunoprecipitation assays revealed ATG10S combination with KPNA1/importin α, KPNB1/importin β, and IRF1. The knockdown of endogenous IRF1 increased ATG10S activity on transcription. These results indicate that ATG10S as a transcription factor competes with IRF1 for the same binding site to promote gene transcription. ATG10: autophagy related 10; ATG10S: the shorter isoform of autophagy related 10; BD: binding domain; CM: core motif; co-IP: co-immunoprecipitation; GFP: green fluorescent protein; HCV: hepatitis C virus; IF: immunofluorescence; IFN: interferon; IRF: interferon regulatory factor; LP: lambda promoter; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; RLU: relative light unit; SQSTM1: sequestosome 1.
IFNL2是一种强效抗病毒干扰素，但其基因表达的调控尚不完全清楚。在此，我们报道了自噬相关蛋白10的较短亚型（ATG10S）对转录的调控作用。通过对启动子序列进行逐步缺失，我们发现了一段对ATG10S活性有反应的启动子片段。亚细胞定位和DNA免疫沉淀实验表明，ATG10S易位进入细胞核并与……结合。转录因子结合位点的在线预测显示，在……中有一个干扰素调节因子1（IRF1）靶向位点。荧光素酶实验、逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析显示，在……中存在一个核心基序（CAAGAC），该基序决定了ATG10S和IRF1的活性；单个核苷酸替换表明，ATG10S靶向的功能核苷酸为核心基序内的C1、A3和C6，与IRF1相关的为A3和G4。免疫共沉淀实验揭示了ATG10S与核转运蛋白α（KPNA1/importin α）、核转运蛋白β（KPNB1/importin β）和IRF1的结合。内源性IRF1的敲低增加了ATG10S对……转录的活性。这些结果表明，ATG10S作为一种转录因子，与IRF1竞争相同的结合位点以促进……基因转录。 自噬相关蛋白10（ATG10）；自噬相关蛋白10的较短亚型（ATG10S）；结合结构域（BD）；核心基序（CM）；免疫共沉淀（co-IP）；绿色荧光蛋白（GFP）；丙型肝炎病毒（HCV）；免疫荧光（IF）；干扰素（IFN）；干扰素调节因子（IRF）；λ启动子（LP）；微管相关蛋白1轻链3β（MAP1LC3B/LC3B）；相对光单位（RLU）；聚集体自噬受体（SQSTM1）
