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Defining the mechanism and biological role of R-loop promoter activity in mammalian gene expression


Sir William Dunn School of Pathology

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Prof N J Proudfoot No more applications being accepted Competition Funded PhD Project (Students Worldwide)

About the Project

R-loops are triplex nucleic acids that form when RNA invades double stranded DNA, displacing single strand DNA by forming an RNA:DNA hybrid. These structures commonly form near transcription start and end site regions of many protein coding genes and have been implicated in a range of gene regulatory processes (1). We have recently shown that R-loops have the capacity to act as transcriptional promoters (2). In effect the single stranded DNA of the R-loop recruits a set of RNA polymerase II (Pol II) general transcription factors. These in turn recruit Pol II which then initiates antisense transcription. In particular R-loop promoter activity may play a critical role in Pol II enhancer function. These enhancers activate protein coding gene genes, even though they are often located far from their associated genes. Furthermore they generate transcripts (called eRNA) which we show are highly prone to form R-loops. The potential role of eRNA in enhancer function remains unresolved. It is the aim of this research project to investigate these R-loop promoters.
This project will focus on the characterisation of R-loop promoter sequence and factor determinants. This will involve setting up in vitro transcription systems using purified transcription components (transcription factors, Pol II, different sized R-loops structures engineered synthetically). Role of R-loop promoter activity in enhancer function will also be investigated. Specific eRNA associated with particular enhancers that activate well defined protein coding genes will be selectively removed by CRISPR targeting experiments. This will involve the use of hybrid Cas9-RNaseH targeted to specific R-loops generated by eRNA. These experiments will allow the selective removal of specific eRNA associated R-loops and will lead on to an analysis of enhancer mediated gene activation. Such an approach is aimed at providing a better understanding of how transcriptional enhancers work in mammalian cells.



Funding Notes

4 Year DPhil Prize Studentships cover full University fees, a tax free enhanced stipend of ~£17,285 pa, and up to £5,300 pa for research costs and travel. The competition is open to applicants from all countries. See https://www.path.ox.ac.uk/content/prospective-graduate-students for full details and to apply.

References

(1) http://www.genesdev.org/cgi/doi/10.1101/gad.242990.114
(2) https://doi.org/10.1016/j.molcel.2019.10.002
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