Investigating the role of enhancers in the recruitment of RNA polymerase II to the  α-globin promoter in live cells in real time 

Supervisors Prof Doug Higgs and Dr Danuta Mariola Jeziorska

Live cell imaging has shown that transcription is a dynamic process consisting of a series of bursts of activity, interrupted by off stages when no transcript is produced. This so-called “transcriptional bursting” is associated with RNA synthesis by multiple engaged RNA polymerases II (RNAPII) that accumulate in a dynamic fashion at the site of actively transcribed genes. Transcription is known to be regulated by enhancers; however, their role in modulating the burst dynamics and RNAPII recruitment to the promoter of the transcribed gene is not clear and may uncover new regulatory properties of enhancers. To investigate the role of enhancers in this process, we will employ a live cell imaging system that allows direct correlation of the dynamics of transcriptional bursting with RNAPII recruitment at the well-characterised alpha globin locus. We have established an experimental system to visualise transcription of the α-globin gene during haematopoiesis using the Pseudomonas aeruginosa PP7 bacteriophage system. Using this system, we can detect alpha globin specific signal with a very good signal to noise ratio. To correlate the transcription dynamics with RNAPII recruitment, we will tag the endogenous RNAPII catalytic subunit (RPB1) with a fluorescent reporter in this system. This will allow us to monitor the kinetics of RPB1-FP and alpha globin transcription in clones with and without the key regulatory elements of the gene in order to uncover their role in this process. 

Training opportunities:
The project offers training in a wide range of molecular biology and genetic engineering techniques including: cell culture, cloning, qPCR, and CRISPR/Cas9 genome editing. Significant experience will also be gained in the use of live cell super-resolution imaging techniques and image data analysis. 

References:

  1. Hay D. et al Nature Genetics 2016
  2. Larson D.R. et al Science 2011
  3. Cho W.K. et al eLife 2016

For further information, please contact: Prof Doug Higgs