From atomic to in vivo: a characterisation of ILF3 nucleic acids interactions in the context of eukaryotic transcription regulation

从原子到体内：真核转录调控背景下 ILF3 核酸相互作用的表征

• 批准号: 2454516
• 金额: --
• 依托单位: University of Sussex
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2454516
• 关键词: atomic; vivo; characterisation; ILF3; nucleic

This project builds on the recent in vivo evidence for dual DNA- and RNA-binding proteins (DRBPs) having key roles in transcription regulation by binding to specific DNA structures at promoter regions. As this type of interaction is currently absent from ENCODE, unravelling its mechanism is fundamental to our understanding of the basic rules of eukaryotic gene expression. Interleukin enhancer-binding factor 3 (ILF3) is a DRBP that is known to extensively regulate RNA splicing, stabilization and export but whose role in regulating transcription as a chromatin interacting protein has not been comprehensively characterized. ILF3 binds extensively to chromatin, with an increased occupancy frequency at active promoters. A model has been proposed by which ILF3 would function analogously to a conveyor belt from chromatin to gene, through RNA transcript to ribosome in dynamic regulation of gene expression. ILF3 contains two dsRNA binding domains and two additional domains that can interact with nucleic acids. Still lacking is the understanding of how ILF3 differentially recognizes RNA and DNA structures and specifically associates to promoter regions in chromatin. An example of this is the non-standard A-form DNA structure in the promoter of the critical, developmentally regulated transcription factor gata2. Ilf3 is bound to cytoplasmic RNA in fertilized Xenopus laevis eggs but subsequently translocates to the nucleus where it becomes bound to the gata2 promoter just prior to activation of this gene's transcription. Base substitutions that increase B-form structure propensity in the gata2 promoter decrease both ILF3 binding and transcription whilst mutations that maintain A-form propensity also maintain, or increase, transcription factor binding and transcription. This project aims to unravel structurally and functionally ILF3's versatile interactions with diverse nucleic acids to modulate transcription. Structures of ILF3 will be analysed when bound to RNA and various forms of DNA to compare binding modes and their relevance to function. A larger transcriptional complex containing ILF3 and partner ILF2, which controls transcription at the B-globin locus control region, will also be structurally characterized. Critical residues involved in key interactions will be mutated interrogated in Xenopus laevis to determine the importance of each interaction in the context of gene regulation in vivo.

该项目建立在最近的体内证据的双重DNA和RNA结合蛋白（DRBPs）具有关键作用的转录调控结合到特定的DNA结构在启动子区域。由于这种类型的相互作用是目前缺乏的ENCODE，解开其机制是我们的真核基因表达的基本规则的理解的基础。白细胞介素增强子结合因子3（ILF 3）是一种DRBP，已知其广泛调节RNA剪接、稳定和输出，但其作为染色质相互作用蛋白在调节转录中的作用尚未被全面表征。ILF 3广泛地结合染色质，在活性启动子处具有增加的占据频率。已经提出了一个模型，通过该模型，ILF 3将类似于从染色质到基因的传送带，通过RNA转录物到核糖体，在基因表达的动态调节中发挥作用。ILF 3含有两个dsRNA结合结构域和两个可以与核酸相互作用的额外结构域。仍然缺乏的是了解ILF 3如何区别识别RNA和DNA结构，并特别是相关的启动子区域染色质。这方面的一个例子是在关键的发育调节转录因子gata 2的启动子中的非标准A型DNA结构。Ilf 3与非洲爪蟾受精卵的细胞质RNA结合，但随后易位到细胞核，在那里它与gata 2启动子结合，正好在该基因转录激活之前。增加gata 2启动子中B型结构倾向的碱基取代降低ILF 3结合和转录，而维持A型倾向的突变也维持或增加转录因子结合和转录。该项目旨在揭示ILF 3在结构和功能上与不同核酸的多功能相互作用以调节转录。当与RNA和各种形式的DNA结合时，将分析ILF 3的结构，以比较结合模式及其与功能的相关性。一个更大的转录复合物含有ILF 3和合作伙伴ILF 2，它控制转录的B-珠蛋白基因座控制区，也将在结构上进行了表征。参与关键相互作用的关键残基将在非洲爪蟾中进行突变询问，以确定体内基因调控背景下每个相互作用的重要性。