New Tools to Decipher the Role of lncRNAs and Their Protein Interactomes in Hematopoiesis

项目摘要/摘要 研究长非编码RNA(LncRNAs)及其蛋白质相互作用的生物分析工具迫切需要 需要的。LncRNAs是转录调控基因表达的关键元件。他们一直都是 显示出通过几种不同的机制发挥作用。它们可能直接作为基因调控因子发挥作用， 在它们活动的基因组位置通过转录产生。或者，lncRNAs与蛋白质相互作用以 控制基因表达。例如，lncRNAs可以充当与之相互作用的蛋白质的“分子接收器”。 Dna或rna；在这种情况下，蛋白质与lncRNA的结合与其与其主要成分的相互作用竞争。 目标。LncRNAs还可以引导蛋白质与其DNA靶标结合，从而抑制或激活转录。他们 不仅可以作用于顺式基因或其转录位点附近，还可以作用于反式基因的多个基因组位点。最后， LncRNA可以作为一个平台，分子可以在上面聚集起来，作为一个团队在特定的 位置和时间(例如，组蛋白修饰复合体)。这些机制中的每一个都需要 具有不同蛋白质队列的lncRNA。因此，了解与特定lncRNAs结合的蛋白质是必不可少的。 了解lncRNAs控制基因表达的机制所需的信息。解开这一切 相互作用的巨大多样性需要能够快速识别和量化lncRNA的工具- 相关蛋白质。尽管lncRNAs及其相互作用的蛋白质非常重要，但在 是可用来研究它们的工具的重大限制。我们建议开发和验证一套 强大的新工具，用于发现、识别和量化LncRNA，并用于全面的 它们的蛋白质相互作用的蛋白质组学分析。 已知，lncRNAs直接基因表达来调节造血系统中的细胞命运，从而使 发展过程中基因编程的多样化。LncRNA功能中断也可能导致 特殊的髓系和淋巴系癌症的恶变。GATA因子调控的lncRNA， 在强大的遗传系统中发现(野生型和GATA2增强子突变小鼠，GATA-1基因 补充系统和GATA因子在原代人红系前体细胞中的敲除)，对照 人红系前体细胞功能与红细胞发育。在最初的侧写研究中，我们有 鉴定出74个受GATA因子调控的lncRNA。这里开发的新工具的性能将是 在这74个LncRNA的子集上进行彻底测试，以发现GATA因子依赖的调节电路和 控制造血的网络。 拟议的研究将推动最先进的lncRNA鉴定和蛋白质组学分析。 相互作用组学，应用于造血过程中最重要的调控网络之一。我们将带来一个新的 对与GATA因子相关的lncRNA相互作用体的定义的可见度达到前所未有的水平，同时 开发强大的开源软件工具和详细的实验协议。

Project Summary/Abstract Bioanalytical tools to study long noncoding RNAs (lncRNAs) and their protein interactors are desperately needed. lncRNAs are critical elements in the transcriptional regulation of gene expression. They have been shown to function through several different mechanisms. They may serve directly as gene-regulatory factors, produced by transcription at the genomic site where they act. Alternatively, lncRNAs interact with proteins to control gene expression. For example, lncRNAs can act as a “molecular sink” for proteins that interact with DNA or RNA; in this case, binding of the protein to the lncRNA competes with its interaction with its primary target. lncRNAs can also guide proteins to their DNA targets to either repress or activate transcription. They can act not only in cis, or near their site of transcription, but also in trans, at multiple genomic sites. Finally, lncRNAs can act as platforms upon which molecules can convene to perform a function as a team at a specific location and time (e.g. histone modification complexes). Each of these mechanisms requires the interaction of lncRNAs with a diverse protein cohort. Knowledge of the proteins bound to specific lncRNAs is thus essential information needed to understand mechanisms by which lncRNAs control gene expression. Unraveling this tremendous diversity of interactions demands tools capable of rapid identification and quantification of lncRNA- associated proteins. Despite the great importance of lncRNAs and the proteins that interact with them, there are significant limitations in the tools available to study them. We propose to develop and validate a suite of powerful new tools for the discovery, identification, and quantification of lncRNAs and for the comprehensive proteomic analysis of their protein interactomes. It is known that lncRNAs direct gene expression to modulate cell fate in the hematopoietic system, enabling diversification of gene programming during development. Disrupted lncRNA function can also contribute to malignant transformation in specific myeloid and lymphoid cancers. GATA factor-regulated lncRNAs, discovered in powerful genetic systems (wild type and Gata2 enhancer-mutant mice, GATA-1 genetic complementation system and GATA factor knockdowns in primary human erythroid precursor cells), control human erythroid precursor cell function and erythrocyte development. In initial profiling studies, we have identified 74 GATA factor-regulated lncRNAs. The performance of the new tools developed here will be thoroughly tested on a subset of these 74 lncRNAs to discover GATA factor-dependent regulatory circuits and networks that control hematopoiesis. The proposed research will drive state-of-the-art lncRNA identification and proteomic analysis of the lncRNA interactome, applied to one of the most important regulatory networks in hematopoiesis. We will bring a new and unprecedented level of visibility to the definition of the GATA factor-relevant lncRNA interactome, while developing powerful open-source software tools and detailed experimental protocols.