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比最初想象的要广泛得多。现在已经鉴定了数千种长链非编码RNA（lncRNA），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为什么基因在给定的时间表达，并提供深入了解lncRNA在基因调控中的生物学贡献和意义。由于缺乏鉴定真正的lncRNA染色质靶点的可靠方法，该领域的进展受到阻碍。在这里，我们建议使用HOTAIRM 1 lncRNA作为发现工具，以表征这些转录本如何在细胞分化过程中调节基因表达。去年，我们报道了HOTAIRM 1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HOTAIRM 1的表达对于物理分离基因沿着HOXA簇和解偶联它们的表达是必要的。我们初步的荧光原位杂交（FISH）数据显示HOTAIRM 1变体的核定位部分重叠，点状图案表明它们调节整个基因组中许多基因的表达。我们现在建议开发两种新的技术来寻找全基因组的lncRNA基因靶点。我的研究将有助于研究任何类型的非编码RNA，并通过我的计划，我将培训来自各个层次的学生在实验室进行研究，并通过计算分析科学数据。具体目标。目标1.用蒂耶里鉴定新的HOTAIRM 1调节基因。我们建议开发一种新的方法来识别给定lncRNA全基因组的直接基因靶点。我们将这种技术命名为“蒂耶里”，意为“RNA-染色质相互作用的靶向原位提取”，并将其设计为规避当前方法的许多已知缺陷。我们将在NT 2-D1细胞中使用视黄酸诱导的HOTAIRM 1来建立和优化这种技术，并确定由这种lncRNA控制的新的基因组区域。 目标2.调整2C-ChIP以分析HOTAIRM 1沿着靶基因组区域的物理分布。最近，我们设计了碳拷贝-ChIP（2C-ChIP），通过深度测序对来自定义的基因组区域的ChIP信号进行分析。该技术是定量的，并且可以在大的基因组区域上实现非常高的分辨率。我们建议将2C-ChIP与蒂耶里相结合，以定量绘制HOTAIRM 1沿着HOXA簇的实际物理分布以及在Aim 1中发现的新靶区域。

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.