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

该项目建立在最近的体内证据的基础上，即双重DNA和RNA结合蛋白(DRBPs)通过与启动子区域的特定DNA结构结合在转录调控中发挥关键作用。由于目前ENCODE中还没有这种相互作用，因此了解其机制对于我们理解真核基因表达的基本规律是至关重要的。白细胞介素型增强因子结合因子3(ILF3)是一种广泛调控RNA剪接、稳定和输出的DRBP，但其作为染色质相互作用蛋白在转录调控中的作用尚未得到全面研究。ILF3广泛地与染色质结合，在活性启动子上的占位频率增加。已经提出了一个模型，在该模型中，ILF3的功能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。ILF3含有两个dsRNA结合域和两个可与核酸相互作用的额外结构域。仍然缺乏对ILF3如何区别识别RNA和DNA结构以及如何与染色质中的启动子区域特异性关联的理解。这方面的一个例子是关键的、发育调节的转录因子GATA2启动子中的非标准A型DNA结构。ILF3与非洲爪哇受精卵中的细胞质RNA结合，但随后移位到细胞核，在激活该基因的转录之前，它与GATA2启动子结合。增加GATA2启动子中B-型结构倾向的碱基替换减少了ILF3的结合和转录，而保持A-型倾向的突变也维持或增加了转录因子的结合和转录。该项目旨在从结构和功能上解开ILF3‘S与不同核酸的多种相互作用，以调节转录。当ILF3与RNA和各种形式的DNA结合时，将分析其结构，以比较结合模式及其与功能的相关性。包含ILF3和伴侣ILF2的更大的转录复合体也将在结构上得到表征，该复合体控制着B-珠蛋白基因位点控制区的转录。关键相互作用涉及的关键残基将在非洲爪哇进行突变询问，以确定每个相互作用在体内基因调控背景下的重要性。

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.