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

研究反式作用长非编码RNA基因组靶向和染色质调控的原理

• 批准号: BB/N005856/1
• 负责人: Keith Vance
• 金额: $ 65.51万
• 依托单位: University of Bath
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FN005856%2F1
• 关键词: Investigating; principles; trans; acting; long

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.

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，但越来越多的长度大于200个核苷酸的长非编码RNA（lncRNA）被认为是基因表达的重要调控因子。这些分子已被证明在广泛的基本生物过程中起作用，并且lncRNA的子集在各种疾病中失调。LncRNA最初被证明控制同一染色体上邻近基因的表达。然而，现在已经确定lncRNA与基因组中多条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型程序。因此，LncRNA在基因表达控制中发挥的作用比以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仍知之甚少。特别是，目前还不清楚lncRNA是如何从其表达位点转移到发现并与远端DNA序列相互作用以控制靶基因表达的。这在很大程度上是由于这样一个事实，即在这个新的和快速增长的研究领域中，到目前为止，只有很少的这些lncRNA的直接靶基因被鉴定出来。我以前的实验表明，Paupar调节培养中神经细胞的生长和分化，不仅在细胞核中局部调节Pax6表达，而且在遥远的基因组位置，通过结合和直接调节多条染色体上的数百个基因。因此，Paupar是一个不断增长的家族的染色质相关的lncRNA与基因表达控制的全基因组功能的成员，并代表了一个很好的测试案例，研究如何lncRNA是针对遥远的结合位点，并调查其模式的染色质regulation.We已经表明，Paupar结合位点的基因组中富集的PAX6和REST关键神经元转录因子的预测DNA结合序列。此外，Paupar直接与N2A神经母细胞瘤细胞中的这两种蛋白质相关，N2A神经母细胞瘤细胞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。由于已经表明lncRNA基因在细胞核中的相对位置在引导表达的lncRNA分子到达其靶位点中起作用，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，我们将测试功能性Paupar结合位点是否位于细胞核三维空间内的Paupar基因附近或远处，我们将分析位于Paupar基因附近的DNA区域的基因组特征和染色质状态。此外，我们还将研究Paupar分子在调控染色质高级结构中的作用，以及Paupar结合的调控区域对染色质的修饰，这一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。这里发现的全基因组lncRNA功能的一般概念将有助于塑造lncRNA生物学和基因组功能的快速发展领域的未来研究方向。

项目成果

Chromatin interaction maps identify Wnt responsive cis -regulatory elements coordinating Paupar-Pax6 expression in neuronal cells

染色质相互作用图谱识别协调神经元细胞中 Paupar-Pax6 表达的 Wnt 响应顺式调节元件

• DOI: 10.1101/2021.05.18.442939
• 发表时间: 2021
• 作者: Pavlaki I

Chromatin interaction maps identify Wnt responsive cis-regulatory elements coordinating Paupar-Pax6 expression in neuronal cells.

染色质相互作用图识别Wnt响应的顺式调节元件，可在神经元细胞中协调PAUPAR-PAX6表达。

• DOI: 10.1371/journal.pgen.1010230
• 发表时间: 2022-06
• 期刊: PLoS genetics
• 影响因子: 4.5

Paupar LncRNA Promotes KAP1 Dependent Chromatin Changes And Regulates Subventricular Zone Neurogenesis

• DOI: 10.1101/187302
• 发表时间: 2017-09
• 期刊: bioRxiv
• 作者: Ioanna Pavlaki;Farah Alammari;Bin Sun;N. Clark;T. Sirey;Sheena Lee;D. Woodcock;C. Ponting;F. Szele;Keith W. Vance