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

破译lncRNA及其蛋白质相互作用组在造血作用中作用的新工具

• 批准号: 10368117
• 负责人: Emery H Bresnick
• 金额: $ 44.43万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10368117
• 关键词: Ablation; Adult; Anemia; Binding Proteins; Biological; Biological Process; Blood Cells; CRISPR/Cas technology; Cell Line; Cell Maintenance; Cell physiology; Cells; Childhood; Chromatin; Complex; Computer software; DNA; Data; Development; Distant; Dysmyelopoietic Syndromes; Elements; Enhancers; Erythroblasts; Erythrocytes; Erythroid; Erythroid Cells; Erythroid Progenitor Cells; Evaluation; GATA1 gene; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Transcription; Genomics; Glean; Hematopoiesis; Hematopoietic; Hematopoietic System; Histones; Human; Immunologic Deficiency Syndromes; Knowledge; Location; Malignant - descriptor; Malignant lymphoid neoplasm; Mass Spectrum Analysis; Measurement; Mediating; Molecular; Mus; Mutant Strains Mice; Myelogenous; Oligonucleotides; Pathology; Performance; Protein Analysis; Protein Databases; Proteins; Proteomics; Protocols documentation; RNA; RNA analysis; Regulator Genes; Research; Role; Sampling; Site; Software Tools; System; Testing; Time; Transcriptional Regulation; Untranslated RNA; Validation; cell growth; cell type; cohort; complement system; design; differential expression; experimental study; histone modification; knock-down; leukemia/lymphoma; mutant; novel; open source; protein complex; protein expression; proteogenomics; small hairpin RNA; software development; technology development; tool; transcriptome

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.

项目摘要/摘要 研究长不编码RNA（LNCRNA）及其蛋白质相互作用的生物分析工具拼命 需要。 LNCRNA是基因表达转录调控中的关键元素。他们曾经 显示通过几种不同的机制发挥作用。它们可以直接用作基因调节因素， 通过转录在其作用的基因组部位产生。或者，LNCRNA与蛋白质相互作用 控制基因表达。例如，lncRNA可以充当与与 DNA或RNA；在这种情况下，蛋白质与lncRNA的结合与其主要的相互作用竞争 目标。 LNCRNA还可以引导蛋白质到其DNA靶标，以抑制或激活转录。他们 不仅可以在顺式或在其转录部位附近起作用，而且可以在多个基因组部位作用。最后， lncrnas可以充当平台，分子可以方便地在特定方面执行团队的功能 位置和时间（例如Hisstone修饰复合体）。这些机制中的每一个都需要相互作用 LNCRNA具有潜水蛋白队列的LNCRNA。因此，对与特定lncRNA结合的蛋白质的了解至关重要 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，但 是可用于研究它们的工具的重大局限性。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和综合 其蛋白质相互作用的蛋白质组学分析。 众所周知，lncRNA直接基因表达调节造血系统中的细胞命运，使得能够 开发过程中基因编程的多样化。破坏的lncRNA功能也会有助于 特定髓样和淋巴癌的恶性转化。 GATA因子调节的LNCRNA， 在强大的遗传系统（野生型和GATA2增强子突变小鼠，GATA-1遗传中发现）发现 原代人红细胞前体细胞中的互补系统和GATA因子敲低）对照 人红细胞前体细胞功能和红细胞发育。在最初的分析研究中，我们有 鉴定出74个GATA因子调节的LNCRNA。这里开发的新工具的性能将是 在这74个LNCRNA的子集上进行了彻底的测试，以发现GATA因子依赖性调节电路和 控制造血的网络。 拟议的研究将推动LNCRNA的最新lncRNA鉴定和蛋白质组学分析 Interactome，应用于造血中最重要的调节网络之一。我们将带一个新的 以及对与GATA因子相关的lncRNA相互作用的定义的前所未有的可见性水平，而 开发强大的开源软件工具和详细的实验协议。