Transcription is central to life, but many things remain unclear. In eukaryotes, transcription of mRNA is carried out by RNA polymerase II (Pol2) and is connected to several little-understood phenomena, such as the ‘pausing’ of Pol2 after initiating transcription, the prevalence of ‘aborted transcription’ events, or the role of macromolecular assemblies that include Pol2 itself.
In general, the spatial dynamics of nuclear factors including Pol2 appear key to understanding transcription, yet remain enigmatic. This is largely due to a disconnect between methodological approaches to study these subjects; next generation sequencing (NGS) and/or genomics-based approaches offer genome-wide readouts but have limited resolution in terms of spatial dynamics. Imaging-based approaches, on the other hand, are reversed in these two regards.
In this project, we want to pursue a novel strategy that will bridge gaps in the approaches outlined above. Based on a system we have developed recently, we will track in vivo the position and function of (ideally) a single, tagged Pol2 from its release at a specified timepoint and genomic location.
The tagging of Pol2 will allow its detection/isolation, enabling us to conduct next generation sequencing assays such as ChIP-seq to map genomic positions and transcriptional activity of Pol2, as well as microscope imaging to track its diffusion in the nucleus.
The resulting data will help us analyse and understand better the occurrence of abortive transcription, the frequency of reinitiated transcription at a local gene, the timing of individual steps of transcription, and the role of the chromatin state for transcription.
The PhD project is mainly aimed at processing and analysing the resulting data, but contributions to the experimental side are highly appreciated if the applicant has the necessary skills and/or background.
Key experimental skills involved:
Bioinformatics, genome wide data analysis, potentially quantitative modelling; depending on skills, additional techniques to be used include experimental molecular biology, next generation sequencing, imaging
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