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

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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.