Award details

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

Reference	BB/N005856/1
Principal Investigator / Supervisor	Dr Keith Vance
Co-Investigators / Co-Supervisors
Institution	University of Bath
Department	Biology and Biochemistry
Funding type	Research
Value (£)	514,161
Status	Completed
Type	Research Grant
Start date	03/05/2016
End date	02/05/2019
Duration	36 months

Abstract

Many long non-coding RNAs (lncRNAs) are now recognised as key regulators of gene expression and chromatin organisation with functions in a diverse range of fundamental biological processes. Despite this, the mode of action for the majority of these transcripts is poorly understood. We previously identified the lncRNA Paupar as a novel transcriptional regulator of both local and distal gene expression programmes in neural cells in culture. In this proposal, we will investigate the function of specific lncRNA-transcription factor associations and higher order chromatin structure in guiding Paupar to its target sites genome wide. We will perform chromatin immunoprecipitation (ChIP) and next generation sequencing to map the genome wide binding profiles of the PAX6 and REST key neuronal transcription factors in N2A mouse neuroblastoma cells. These proteins were identified as ideal candidate factors for Paupar genomic targeting. We will then perform Capture Hybridization Analysis of RNA Targets in PAX6 and REST inducible loss of function cell lines to assay for changes in Paupar chromatin association at co-occupied binding sites. This will directly test whether PAX6 and REST can recruit Paupar to chromatin. We will perform genome wide Capture-C to define the relative position of the Paupar locus to its genomic binding sites within the higher order structure of the genome in the nucleus in N2A cells and we will validate a number of identified interactions using DNA-FISH. We will also investigate the mechanisms of lncRNA mediated chromatin regulation at bound locations. We will perform chromosome conformation capture to test whether Paupar is required for initiating and/or maintaining genomic regulatory interactions and ChIP-qPCR to assay whether Paupar can induce histone modification changes at bound regulatory regions using Paupar knockdown N2A cells. This work will generate important molecular insights into lncRNA function in genome wide transcriptional control.

Summary

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.

Impact Summary

The research in this proposal is directly relevant to the BBSRC's Bioscience for Health strategic research priority and has the potential to lead to improved regenerative medicine treatments for neurological diseases and for neuroblastoma, a neural crest cell derived cancer. Long non-coding RNAs (lncRNAs) have been shown to be mis-expressed in patients with Huntington's, Alzheimer's diseases and Parkinson's diseases. The lncRNA studied here, termed Paupar, is conserved between mouse and human and is expressed in adult mouse neural stem cells in vivo where it functions as an important regulator of stem cell maintenance and neurogenesis. We will also map the genome wide binding sites for the PAX6 and REST transcription factors in this proposal and investigate their role in modulating Paupar function. These proteins are key regulators of neural stem cell self-renewal and neurogenesis and may be involved in numerous neurological diseases such as Alzheimer's disease, Huntington's Disease, Parkinson's disease and stroke. Furthermore, mutations in PAX6 are linked to aniridia, a disorder of the eye that is characterised by the absence of the iris. By generating important insights into the molecular mechanisms controlling neural stem cell self-renewal and differentiation this research has the potential to lead to improved regenerative medicine treatments for neurological disorders beyond the end of this grant. LncRNAs are emerging as novel diagnostic/prognostics markers and therapeutic targets for cancer treatment. Our previous research showed that Paupar is required for the growth of neuroblastoma cells in culture. Understanding its mode of action may, in the longer term, lead to improved treatments for neuroblastoma, the second most common solid tumour in children. The diseases that this research may help to better understand have a huge burden on individuals, families and society while the economic burden, including direct healthcare costs and indirect costs incurred such as loss of earnings and inability to work, is a huge global problem. Any potential therapeutic improvements would enhance the quality of life of disease sufferers and would reduce the economic and societal burden imposed by these diseases. We will participate in Brain Awareness Week (BAW), held in March each year, to raise awareness amongst the public, clinicians and BAW partner organisations of the emerging role of lncRNAs as potential new therapeutic targets for the treatment of neurological diseases. We will also attend local events organised by the Aniridia Network UK. These provide opportunities for people to learn about aniridia and related conditions and includes members of the public as well as medical professionals and scientists. Furthermore, we will present our research at meetings of the Cancer Research at Bath (CR@B) network. This is a growing collaboration between the academics at the University of Bath and clinicians at the Royal United Hospital which aims to promote and foster inter-disciplinary research and therapeutic development.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Neuroscience and Behaviour
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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