Strains of M. tuberculosis during infection

This project is now closed

Establishment of chromosome conformation capture techniques (3C)

Project supervisor – Greg Elgar

Division of Systems Biology

Chromosome Conformation Capture (3C and its derivatives) is a well established technique that permits the capture of interactions between regions of DNA via the cross linking of interacting proteins. This is an ideal technique for the capture of enhancer:promoter interactions and has been used extensively in this context to capture interactions for example at the alpha-globin locus (Vernimmen, 2009).

In my lab we work with a set of highly conserved distal regulatory elements that all associate with key developmental regulators and many of these have been shown act as enhancers in reporter assays. Whilst there is an assumption that these elements interact as enhancers with the promoters of their target gene, even though this can be over a megabase away, there is very little 3C data to support this. Furthermore, emerging but scant data suggests that these conserved non-coding elements (CNEs) may interact not only with the promoter but with each other (Robyr, 2011).

We would like to establish the 3C technique in my lab and initially identify which elements are interacting with each other, and the promoter, at a small set of candidate gene loci where we have extensive data on their enhancer activity. Amongst these would be the sox21 gene, where we have assayed all 19 CNE regions, and also the Meis2 locus, a particularly complicated and large locus that we also have a current interest in.

This work would be carried out on zebrafish embryo extracts, collected at a time point where we know that these elements are active. This approach has proved successful and has recently been published (Irimia, 2012). If this proves successful, we would extend the approach to a genome wide screen, using one of the derived 3C techniques that allows high throughput analysis (5C and Hi-C (de Wit, 2012)).

Ultimately the aim is to generate a catalogue of likely interactions between CNEs and promoters that will allow the prediction of likely structures at the ‘enhanceosome’ and provide a first pass model of how different regulatory elements within complex regulatory architectures interact during early development.

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