Screening for epigenetic modifiers of X chromosome inactivation

Abstract

Epigenetic regulation describes heritable changes in gene expression, but not the genome sequence. X chromosome inactivation is a model epigenetic process that involves the silencing of one of the two X chromosomes in female mammals. Much remains unknown about the mechanisms involved in X inactivation, as about epigenetic control in general, and there is scope for the identification of novel genes or the ascription of new functions to known genes.

This project involved the development of different RNA interference screening approaches to identify epigenetic regulators required at different stages of X inactivation. A custom short hairpin RNA library targeting epigenetic regulators was used to perform screens in mouse embryonic fibroblasts for genes involved in the maintenance of X inactivation, by examining reactivation of a green fluorescent protein (GFP) transgene reporter present on the inactive X chromosome.

From these screens, knockdown of the H3K9 methyltransferase Setdb1 resulted in reactivation of the silent GFP, implicating it in the maintenance of X inactivation. H3K9 methylation is a modification made to the chromatin of the inactive X, but the enzyme responsible for its presence is currently unknown. Targeting of other H3K9 methyltransferase genes with short hairpin RNAs did not lead to GFP reactivation. Setdb1-null female embryos were found to fail X inactivation as they showed aberrant expression of the transgenic GFP reporter. Some endogenous X-linked genes also showed low levels of reactivation in Setdb1-depleted mouse embryonic fibroblasts by RNA sequencing.

An alternative system for simultaneously monitoring the activity of both X chromosomes by flow cytometry was also established, using knock-in alleles of GFP and a red fluorescent protein (mCherry) at the endogenous X-linked housekeeping gene Hprt. Female embryonic stem cells were derived with the knock-in alleles; these cells have two active X chromosomes and undergo X inactivation when differentiated. A screen was commenced to examine the initiation and establishment of X inactivation by monitoring the expression of the two fluorochromes during differentiation. Setdb1 depletion in these cells did not alter the early stages of X inactivation, suggesting that Setdb1 is more important for the maintenance of X chromosome silencing.