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Systems approach to identify the origins of transcriptional bursting

Principal Supervisor: Daniel Hebenstreit - SLS

Co-supervisor: Barbel Finkenstadt, Department of statistics

PhD project title: Systems approach to identify the origins of transcriptional bursting

University of Registration: Warwick

Project outline:

Expression levels of the same mRNA or protein vary strongly among the cells of an otherwise identical population as recent, novel experimental techniques have revealed. Such biological noise has great functional implications [1] and is largely due to transcriptional bursting [2]. This process refers to the episodic production of mRNAs in short, intense bursts, interspersed by periods of transcriptional inactivity. Bursting has been demonstrated in a wide range of pro- and eukaryotic species, attesting to its universal importance. However, the mechanistic origins of bursting remain elusive [3].

It will be the aim of this project to investigate the sub-processes of transcription with regards to their roles in transcriptional bursting. We will use a mammalian cell line and will introduce various gene constructs into its genome. By inserting mutations into these, we will be able to perturb and analyse individual mechanisms involved in transcription. We will then use the RNA-FISH assay [4] to measure in large numbers of single cells the absolute numbers of mRNA that are expressed from the gene constructs. The characteristics of the singe-cell mRNA distributions can be used to infer the absence or presence of transcriptional bursting and its kinetic parameters [2]. Integrating this information with the types of mutations we used will greatly advance our understanding of the mechanistic origins of bursting.

References:

  1. Balazsi, G., A. van Oudenaarden, and J.J. Collins, Cellular decision making and biological noise: from microbes to mammals. Cell, 2011. 144(6): p. 910.
  2. Munsky, B., G. Neuert, and A. van Oudenaarden, Using gene expression noise to understand gene regulation. Science, 2012. 336(6078): p. 183.
  3. Hebenstreit D. Are gene loops the cause of transcriptional noise? Trends in Genetics. 2013. 29(6): p. 333. 4. Raj, A., et al., Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods, 2008. 5(10): p. 877.

BBSRC Strategic Research Priority: Molecules, cells and systems

  • Techniques that will be undertaken during the project:
  • Mammalian cell culture
  • Genome editing
  • qPCR, RNA-FISH
  • Advanced data analysis in collaboration with specialists
  • Flow cytometry
  • Potentially programming

Contact: Dr Daniel Hebenstreit, University of Warwick