Linking RNA to chromatin landscape and organization

将 RNA 与染色质景观和组织联系起来

• 批准号: RGPIN-2019-05281
• 负责人: Dostie, Josée
• 金额: $ 3.06万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=715444
• 关键词: Linking; RNA; chromatin; landscape; organization

BACKGROUND. Important technological advances in the genomics field have led to the realization that mammalian genomes are much more extensively transcribed than originally thought. Thousands of long non-coding RNAs (lncRNAs) have now been identified, and although the biological relevance of many of them has been demonstrated, the mechanisms by which they regulate the expression of genes remain mostly unknown. Identifying these will help understand why genes are expressed at a given time, and provide insight into the biological contribution and significance of lncRNAs in gene regulation at large. Progress in this field has been hampered by the lack of robust methods to identify bona fide lncRNA chromatin targets. Here, we suggest using the HOTAIRM1 lncRNA as a discovery tool to characterize how these transcripts may regulate gene expression genome-wide during cellular differentiation. Last year, we reported that HOTAIRM1 controls gene expression through changes in chromatin structure and spatial organization. We found that HOTAIRM1 expression is necessary to physically dissociate genes along the HOXA cluster and uncouple their expression. Our preliminary fluorescence in situ hybridization (FISH) data revealed partially overlapping nuclear localizations of HOTAIRM1 variants, with punctate patterns suggesting that they regulate the expression of many genes across the genome. We now suggest developing two novel technologies to find lncRNA gene targets genome-wide. My research will be useful to study any type of non-coding RNAs, and through my program, I will train students from all levels to conduct research at the bench and analyze scientific data computationally. SPECIFIC AIMS. Aim 1. Identify new HOTAIRM1-regulated genes with TIERI. We propose developing a new methodology to identify direct gene targets of given lncRNAs genome-wide. We named this technique TIERI' for “Targeted In situ Extraction of RNA-chromatin Interactions”, and designed it to circumvent many of the known deficiencies of current methods. We will use retinoic acid-induced HOTAIRM1 in NT2-D1 cells to establish and optimize this technique, and identify new genomic regions controlled by this lncRNA. Aim 2. Adapt 2C-ChIP to profile the physical distribution of HOTAIRM1 along target genomic regions. Recently, we devised carbon copy-ChIP (2C-ChIP) to profile ChIP signal from defined genomic regions with deep sequencing. This technique is quantitative and can achieve very high resolutions over large genomic regions. We propose combining 2C-ChIP with TIERI to quantitatively map the actual physical distribution of HOTAIRM1 along the HOXA cluster and new target regions found in Aim 1 with TIERI.

背景资料。 基因组学领域的重要技术进步导致人们认识到，哺乳动物基因组的转录范围比最初认为的要广泛得多。目前已鉴定出数千个长的非编码RNA(LncRNAs)，虽然已证实其中许多具有生物学意义，但它们调控基因表达的机制大多仍不清楚。识别这些基因将有助于理解为什么基因在给定的时间表达，并提供对lncRNAs在基因调控中的生物学贡献和意义的洞察。由于缺乏可靠的方法来识别真正的lncRNA染色质靶标，这一领域的进展一直受到阻碍。在这里，我们建议使用HOTAIRM1 lncRNA作为发现工具来表征这些转录本如何在细胞分化过程中调节全基因组的基因表达。去年，我们报道了HOTAIRM1通过染色质结构和空间组织的变化来控制基因的表达。我们发现HOTAIRM1的表达是沿着HOXA簇物理分离基因并解偶联它们的表达所必需的。我们的初步荧光原位杂交(FISH)数据显示，HOTAIRM1变体的核定位部分重叠，点状图案表明它们调控基因组中许多基因的表达。我们现在建议开发两种新技术来寻找全基因组范围的lncRNA基因靶点。我的研究将有助于研究任何类型的非编码RNA，通过我的计划，我将培训来自各个层次的学生在实验台上进行研究，并通过计算分析科学数据。 明确的目标。 目的1.利用Tieri技术鉴定新的HOTAIRM1调控基因。我们建议开发一种新的方法来在全基因组范围内识别给定的lncRNAs的直接基因靶标。我们将这项技术命名为Tieri‘，意为“靶向原位提取RNA-染色质相互作用”，并设计它来绕过当前方法的许多已知缺陷。我们将使用维甲酸诱导NT2-D1细胞中的HOTAIRM1来建立和优化这一技术，并识别由该lncRNA控制的新的基因组区域。 目的2.采用2C-CHIP技术研究HOTAIRM1基因在靶基因组区域的物理分布。最近，我们设计了碳拷贝芯片(2C-CHIP)，用于从定义的基因组区域中提取芯片信号并进行深度测序。这项技术是定量的，可以在大的基因组区域上获得非常高的分辨率。我们建议将2C芯片与Tieri相结合，以定量地绘制HOTAIRM1沿着HOXA星系团的实际物理分布以及与Tieri在Aim 1中发现的新靶区。