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

项目总结/摘要 研究长链非编码RNA（lncRNA）及其蛋白质相互作用物的生物分析工具非常缺乏。 needed. lncRNA是基因表达的转录调控中的关键元件。他们一直 通过几种不同的机制发挥作用。它们可以直接作为基因调节因子， 通过在它们作用的基因组位点转录产生。或者，lncRNA与蛋白质相互作用， 控制基因表达。例如，lncRNA可以充当与蛋白质相互作用的蛋白质的“分子库”。 在这种情况下，蛋白质与lncRNA的结合与其与其主要的 目标lncRNA还可以将蛋白质引导到它们的DNA靶标以抑制或激活转录。他们 不仅可以顺式或在其转录位点附近起作用，而且可以反式在多个基因组位点起作用。最后， lncRNA可以作为一个平台，在这个平台上，分子可以聚集在一起，在特定的环境中发挥团队的作用。 位置和时间（例如组蛋白修饰复合物）。这些机制中的每一个都需要以下方面的相互作用： lncRNA与不同的蛋白质组。因此，了解与特定lncRNA结合的蛋白质是至关重要的 了解lncRNA控制基因表达的机制所需的信息。解开这个 相互作用的巨大多样性需要能够快速识别和定量lncRNA的工具， 相关蛋白质尽管lncRNA和与它们相互作用的蛋白质非常重要， 在研究它们的工具上有很大的局限性。我们建议开发和验证一套 用于发现、鉴定和定量lncRNA的强大新工具， 蛋白质组学分析它们的蛋白质相互作用体。 已知lncRNA指导基因表达以调节造血系统中的细胞命运，使得能够在造血系统中调节细胞命运。 在发展过程中基因编程的多样化。lncRNA功能的破坏也会导致 在特定的骨髓和淋巴癌中的恶性转化。加塔因子调节的lncRNA， 在强大的遗传系统（野生型和Gata 2增强子突变小鼠，加塔-1遗传 原代人红系前体细胞中的互补系统和加塔因子敲低），对照 人红系前体细胞功能和红细胞发育。在最初的分析研究中，我们有 鉴定了74个加塔因子调节的lncRNA。这里开发的新工具的性能将是 对这74种lncRNA的一个子集进行了彻底的测试，以发现加塔因子依赖的调节回路， 控制造血的网络。 这项研究将推动最先进的lncRNA鉴定和lncRNA的蛋白质组学分析。 相互作用组，应用于造血中最重要的调节网络之一。我们将带来一个新的 以及对加塔因子相关lncRNA相互作用组的定义的前所未有的可见性水平， 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.