Linking RNA to chromatin landscape and organization

背景。基因组学领域的重要技术进步使人们认识到，哺乳动物基因组的转录范围比原先认为的要广泛得多。成千上万的长链非编码rna （lncrna）已经被鉴定出来，尽管其中许多的生物学相关性已经被证明，但它们调节基因表达的机制仍然是未知的。识别这些将有助于理解基因在特定时间表达的原因，并深入了解lncrna在基因调控中的生物学贡献和意义。由于缺乏可靠的方法来鉴定真正的lncRNA染色质靶标，这一领域的进展受到阻碍。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在细胞分化过程中如何调节全基因组的基因表达。去年，我们报道了HOTAIRM1通过改变染色质结构和空间组织来控制基因表达。我们发现HOTAIRM1的表达对于物理解离HOXA簇上的基因和解耦它们的表达是必要的。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，点状模式表明它们调节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的lncRNA基因靶点。我的研究对研究任何类型的非编码rna都是有用的，通过我的项目，我将培养各个层次的学生在台前进行研究，并对科学数据进行计算分析。具体的目标。目的1。利用TIERI鉴定新的hotairm1调控基因。我们建议开发一种新的方法来确定给定lncrna的全基因组直接基因靶点。我们将这项技术命名为“TIERI”，即“靶向rna -染色质相互作用的原位提取”，并设计它来规避当前方法的许多已知缺陷。我们将在NT2-D1细胞中使用视黄酸诱导的HOTAIRM1来建立和优化该技术，并鉴定由该lncRNA控制的新的基因组区域。目标2。利用2C-ChIP分析HOTAIRM1沿目标基因组区域的物理分布。最近，我们设计了碳复制芯片（2C-ChIP），通过深度测序来分析特定基因组区域的ChIP信号。这项技术是定量的，可以在大的基因组区域实现非常高的分辨率。我们建议将2C-ChIP与TIERI相结合，定量绘制HOTAIRM1沿着HOXA簇和Aim 1中发现的新目标区域的实际物理分布。

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.