转录因子STAT6识别靶基因启动子N4位点DNA的分子机制研究

Host cells have evolved a wide array of fundamental mechanisms to recognize and eliminate invading microbial pathogens, including developing the ability to recognize pathogen-associated molecular patterns (PAMPs) by receptors named pattern recognition receptors (PRRs) and subsequently invoke powerful cellular signaling events that stimulate the production of type I interferons and pro-inflammatory cytokines. Our understanding of innate immune recognition of DNA have made great progress, such as the STING activation by cyclic dinucleotides (c-di-GMP and cGAMP). .Signal transducers and activators of transcription 6 (STAT6) plays a prominent role in adaptive immunity by transducing signals from extracellular cytokines through the canonical Janus kinase JAK-STAT pathway. Specifically, IL-4 induces the phosphorylation of IL-4 receptor, which in turn recruits cytosolic STAT6 by its SH2 domain; the recruited STAT6 is phosphorylated on tyrosine 641 (Y641) by JAK1, which results in the dimerization and nuclear translocation of STAT6 to activate target genes. However, recently, it is reported that STAT6 is required for innate immune signaling in response to virus infection. Viruses or cytoplasmic nucleic acids trigger STING to recruit STAT6 to the endoplasmic reticulum (ER), leading to STAT6 phosphorylation on Tyr641 by TBK1, independent of JAKs. The phosphorylated STAT6 (p-STAT6) then dimerizes and translocates to the nucleus to bind with specific target gene promoters and induce specific gene expression which responsible for immune cell homing..It is an important open question in STAT proteins that how STAT recognize N4 or N3 site DNA on its target gene promoters. Specifically, it is unknown that how STAT6 recognize N4 site DNA on its specific target gene promoters. We have solved the crystal structures of phosphorylated STAT6 core fragment and its complex with DNA containing N4 site. Structural analysis reveals a clear rotational conformation change of STAT6 upon DNA binding. Combining both structural biology technologies, cell biology and other biophysical methods, molecular dynamics simulation, single-molecule FRET experiment and small angle X-ray scattering (SAXS), we propose to get insights into the molecular mechanisms of STAT6 recognize N4 site DNA on its specific target gene promoters.

病毒与宿主在长期进化过程中相互作用导致机体出现复杂而精细的免疫机制，天然免疫反应就是宿主抵抗病毒的第一道屏障。转录因子STAT6以往的研究主要集中于IL4/IL13通路在过敏性和自身免疫性疾病中的作用。最新研究表明病毒感染后STAT6通过与STING相互作用而被TBK1磷酸化激活，激活的STAT6迁移至细胞核中特异识别靶基因启动子N4位点DNA导致一些趋化因子表达，从而参与抗病毒天然免疫反应。目前靶基因启动子N4和N3位点DNA的识别机制是STAT蛋白研究领域亟待解决的科学问题。申请人长期从事病毒感染与天然免疫信号通路的结构与功能研究，经过多年的准备和摸索，在已解析磷酸化STAT6 core fragment二聚体结构(2.7Å)和获得其与N4位点DNA复合物结构的基础上，拟综合运用结构生物学和细胞生物学、分子动力学模拟等手段，致力于阐明STAT6识别靶基因启动子N4位点DNA的分子机制。

病毒与宿主在长期进化过程中相互作用导致机体出现复杂而精细的免疫机制，天然免疫反应就是宿主抵抗病毒的第一道屏障。有关 STAT6 作为转录因子在免疫系统中的功能研究已有多年，但有关STAT 蛋白如何区分启动子区域N4 和N3 位点DNA 的分子机制一直不清楚。因此，目前靶基因启动子N4和N3位点DNA的识别机制是STAT蛋白研究领域亟待解决的科学问题。. 申请人采用结构生物学方法已解析了STAT6核心区的磷酸化二聚体晶体结构，同时完成其与N4位点DNA和N3位点DNA两个复合物三维精细结构的解析工作，这是其它STAT家族蛋白中从来没有过的。在此基础上采用细胞生物学和分子动力学模拟等方法，揭示STAT6 识别靶基因启动子区域N4 位点DNA的分子机制，进而可为全面认识STAT6 在免疫系统中的作用奠定基础，为病毒感染诱导机体免疫反应的分子机制研究提供必要的补充。.

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