Investigating the principles of trans-acting long non-coding RNA genomic targeting and chromatin regulation

除了传统的蛋白质编码基因外，哺乳动物基因组还表达数千种非编码RNA分子。尽管这些分子中的大多数功能未知，但越来越多的长度超过200个核苷酸的长链非编码rna （lncrna）被认为是基因表达的重要调节因子。这些分子已被证明在广泛的基本生物学过程中发挥作用，并且在各种疾病中lncrna的子集失调。LncRNAs最初被证明可以控制同一染色体上邻近基因的表达。然而，现在已经确定lncrna与基因组中多个染色体上多达数千个不同位置相互作用，以调节基因表达的大程序。因此，LncRNAs在基因表达控制中发挥的作用比之前预期的要广泛得多。尽管如此，这些分子在调节全基因组基因表达程序中的功能尚不清楚。特别是，lncrna如何从其表达位点转移到发现远端DNA序列并与之相互作用以控制靶基因表达尚不清楚。这在很大程度上是因为到目前为止，在这个快速发展的新研究领域中，只有极少数lncrna的直接靶基因被确定。papar lncRNA基因位于基因组Pax6基因旁边，在序列和表达上在脊椎动物中是保守的。我之前的实验表明，在培养中，Paupar调节神经细胞的生长和分化，不仅在细胞核中局部调节Pax6的表达，而且在遥远的基因组位置，通过结合和直接调节多个染色体上的数百个基因。因此，Paupar是一个不断增长的染色质相关lncRNAs家族的成员，该家族在基因表达控制中具有全基因组功能，并且代表了研究lncRNAs如何靶向远端结合位点并研究其染色质调节模式的绝佳测试案例。我们已经证明，基因组中的PAX6和REST关键神经元转录因子的DNA结合序列富集于PAX6和REST的预测DNA结合位点。此外，在N2A神经母细胞瘤细胞中，Paupar与这两种蛋白直接相关，这是一种广泛使用的神经细胞分化模型。因此，我们将绘制N2A细胞中PAX6和REST蛋白的全基因组结合谱，并直接测试这些特异性lncrna -蛋白相互作用是否在将Paupar带到其全基因组靶点中发挥作用。由于已经提出lncRNA基因在细胞核中的相对位置在引导表达的lncRNA分子到达其靶位点方面起作用，我们将定义Paupar基因相对于其先前描述的基因组结合位点和转录靶基因的位置。然后，我们将在细胞核的三维空间内测试功能性Paupar结合位点是否位于靠近或远离Paupar基因的位置，我们将描述靠近Paupar基因的DNA区域的基因组特征和染色质状态。此外，我们将研究Paupar分子在调节高阶染色质结构中的作用，以及在Paupar结合的调控区域对染色质的修饰。这项工作将对控制神经干细胞生长和分化的分子机制产生重要的见解。在此发现的全基因组lncRNA功能的一般概念将有助于塑造未来快速发展的lncRNA生物学和基因组功能领域的研究方向。

The mammalian genome expresses thousands of non-coding RNA molecules in addition to traditional protein coding genes. Although the majority of these molecules are of unknown function, a growing number of long non-coding RNAs (lncRNAs), greater than 200 nucleotides in length, are being recognised as important regulators of gene expression. These molecules have been shown to function in a wide range of fundamental biological processes and a sub-set of lncRNAs are dysregulated in various diseases. LncRNAs were originally shown to control the expression of nearby genes on the same chromosome. However, lncRNAs have now been identified that interact with up to several thousand different locations across multiple chromosomes in the genome to regulate large programmes of gene expression. LncRNAs therefore play a much more widespread role in gene expression control than previously anticipated. Despite this, the function of these molecules in regulating genome wide gene expression programmes is poorly understood. In particular, it is unclear how lncRNAs are transferred from their site of expression to find and interact with distant DNA sequences to control target gene expression. This is in large part due to the fact that the direct target genes for only a very few of these lncRNAs have so far been identified in this new and rapidly growing area of research.The gene for the Paupar lncRNA is located beside the Pax6 gene in the genome and is conserved in sequence and expression amongst vertebrates. My previous experiments showed that Paupar regulates the growth and differentiation of neural cells in culture and functions not only locally in the nucleus to regulate Pax6 expression but also, at distant genomic locations, by binding and directly regulating hundreds of genes across multiple chromosomes. Paupar is therefore a member of a growing family of chromatin associated lncRNAs with genome wide functions in gene expression control and represents an excellent test case to study how lncRNAs are targeted to distant binding sites and to investigate their mode of chromatin regulation.We have shown that Paupar binding sites in the genome are enriched for predicted DNA binding sequences for the PAX6 and REST key neuronal transcription factors. Furthermore, Paupar directly associates with these two proteins in N2A neuroblastoma cells, a widely used model for neural cell differentiation. We will therefore map the genome wide binding profile of the PAX6 and REST proteins in N2A cells and directly test whether these specific lncRNA-protein interactions play a role in bringing Paupar to its genome wide targets. As it has been suggested that the relative position of a lncRNA gene in the nucleus plays a role in guiding expressed lncRNA molecules to their target sites we will define the position of the Paupar gene relative to its previously described genomic binding sites and transcriptional target genes. We will then test whether functional Paupar binding sites are located in close or distant proximity to the Paupar gene within three dimensional space in the nucleus and we will characterise the genomic features and chromatin status of the DNA regions that are located close to the Paupar gene. Furthermore, we will investigate the role of the Paupar molecule in regulating higher order chromatin structure and the modification of chromatin at Paupar bound regulatory regions.This work will generate important insights into the molecular mechanisms controlling the growth and differentiation of neural stem cells. The general concepts of genome wide lncRNA function discovered here will help shape future research directions in the fast moving field of lncRNA biology and genome function.