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DNA和rna结合蛋白（drbp）通过结合启动子区域的特定DNA结构在转录调节中起关键作用。由于这种类型的相互作用目前在ENCODE中不存在，因此揭示其机制对于我们理解真核生物基因表达的基本规则至关重要。白细胞介素增强子结合因子3 （Interleukin enhancer-binding factor 3, ILF3）是一种DRBP，已知广泛调控RNA剪接、稳定和输出，但其作为染色质相互作用蛋白调节转录的作用尚未得到全面表征。ILF3与染色质广泛结合，在活性启动子上占用频率增加。ILF3通过RNA转录到核糖体，类似于染色质到基因的传送带，参与基因表达的动态调控。ILF3包含两个dsRNA结合域和两个可以与核酸相互作用的附加域。仍然缺乏对ILF3如何识别RNA和DNA结构的差异以及特异性地与染色质启动子区域相关联的理解。这方面的一个例子是在关键的，发育调节转录因子gata2的启动子中的非标准a型DNA结构。在非洲爪蟾受精卵中，Ilf3与细胞质RNA结合，但随后易位到细胞核中，在激活该基因的转录之前，它与gata2启动子结合。在gata2启动子中增加b型结构倾向的碱基替换会减少ILF3结合和转录，而维持a型结构倾向的突变也会维持或增加转录因子结合和转录。本项目旨在从结构和功能上揭示ILF3与多种核酸的多种相互作用，以调节转录。当与RNA和各种形式的DNA结合时，将分析ILF3的结构，以比较其结合模式及其与功能的相关性。包含ILF3和伙伴ILF2的更大的转录复合体，控制b -珠蛋白位点控制区的转录，也将进行结构表征。参与关键相互作用的关键残基将在非洲爪蟾中进行突变，以确定每种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.