Our understanding of the role of epigenetic marks in gene regulation, and the factors that both add and remove these marks, is increasing rapidly. There are, however, many epigenetic modifying proteins that are yet to be identified, with our own analysis suggesting up to 1200 currently uncharacterised proteins could have the capacity to modify the epigenome. As such, we are developing a novel in vitro system with which to screen for unknown epigenetic modifiers.
Using X inactivation in female mouse embryonic stem cells as both a model epigenetic process and a marker of pluripotency, we are performing an RNA interference screen against all 1200 genes that we predict have epigenetic potential. We have created GFP and mCherry knock-in alleles at the X-linked Hprt locus that allow us to independently monitor transcriptional activity from both X chromosomes by FACS, giving us an unprecedented ability to track the silencing status of each X chromosome.
Female embryonic stem cells have two active X chromosomes, however upon differentiation one chromosome is silenced due to X inactivation. Conversely, to reprogram a female somatic cell back to the pluripotent state the silenced X chromosome must be reactivated. Our fluorescent cell system allows us to rapidly monitor these cellular processes, enabling us to screen for epigenetic modifiers of X inactivation, pluripotency and reprogramming. Pilot experiments suggest that our approach is valid and we have now begun to screen our first pool of ~150 genes.