An in vitro compartmentalisation screen for mammalian DNA demethylases

Our genome carries epigenetic information on top of the genetic one which is important for normal development and function of a healthy organism. Some epigenetic marks in DNA are based on addition of a small chemical group to the DNA. These marks occur in different organs and make them work properly. The marks however have to be removed again in germ cells, so that babies start their development with a clean slate . Also, these marks have to be reprogrammed when cells are taken from patients and transformed back into stem cells for treatment, which is a new and potentially revolutionary concept in regenerative medicine. This grant application proposes to identify factors with which epigenetic marks can be removed from DNA, which might conceivably lead to big improvements in stem cell generation from patients, and hence regenerative medicine.

Genome wide epigenetic reprogramming occurs in germ cells and early embryos in mammalian development. Such reprogramming is important for immortality of the germ cell lineage, genomic imprinting, the erasure of epimutations, the transmission of pluripotency, and early lineage commitment events. Experimental reprogramming through iPS cells also requires epigenetic reprogramming, and this is often inefficient or incomplete, thus limiting the potential clinical applications of this promising techniques. A cardinal mechanism of reprogramming is demethylation of 5-methylcytosine in DNA by active and passive mechanisms. Despite efforts to identify active demethylases in mammals there is no confirmed enzyme that can either directly or indirectly (involving DNA repair) demethylate DNA. The identification of demethylases in mammals would be a critical advance in our progress in understanding reprogramming and in harnessing this mechanism for regenerative medicine, especially for the efficient generation of iPS cells. We have devised, constructed and validated a universal screen for demethylases which is based on an in vitro compartmentalisation assay. This assay links, in emulsion droplets, clones from cDNA expression libraries to a methylated DNA sequence; upon expression of a demethylase in a droplet this sequence becomes demethylated and renders this particular cDNA clone amplifiable by PCR. We have validated the screen and found it to be exquisitely sensitive and selective for demethylated molecules. Biochemical reaction conditions can be adjusted at will by using different extracts for the emulsions, and can be adapted to direct or repair mediated demethylation. Any number of cDNA libraries can be screened quickly and effectively. Candidate clones isolated from screens will be tested in our established mammalian cell and animal systems, including the iPS system, in order to reveal demethylase and reprogramming functions in vivo.