Understanding the role of epigenetic marks in gene regulation, and the factors that add and remove these marks, is critical to understanding both normal development and disease. There are however, many epigenetic modifiers yet to be identified, with our own analysis suggesting up to 1200 genes remain to be discovered. Using X chromosome inactivation (XCI) in female embryonic cells as a model epigenetic process, we are performing RNAi screens against all 1200 candidate genes.

Our initial screen in mouse embryonic fibroblasts (MEFs) revealed the H3K9 methyltransferase Setdb1 is required for maintenance of XCI. MEFs exhibit only the maintenance phase of XCI, so to screen our list of 1200 candidate genes more thoroughly have developed a screening method in embryonic stem cells, which undergo all phases of XCI upon differentiation. To make this process amenable to high throughput screening we have created GFP and mCherry knock-in alleles at the X-linked Hprt locus, allowing us to independently monitor transcriptional activity from each X chromosome, thereby giving us an unprecedented ability to track XCI.

 Our initial screen yielded many putative factors required for XCI and our current attempts to validate candidates are proving promising. Moreover, we have developed protocols for improved culturing of primary female ES cells.