Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

Abstract

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

Recent Advances in Understanding the Reversal of Gene Silencing During X Chromosome Reactivation

“…In the maintenance phase of XCI, additional repressive modifications, such as DNA methylation, accumulation of macroH2A and hypoacetylation of histone H4, ‘lock in’ gene silencing on the Xi (Csankovszki et al, 2001). More recently, several screens have been reported, which aimed to further identify factors required to induce or maintain XCI (Minkovsky et al, 2015; Moindrot et al, 2015; Monfort et al, 2015; Sripathy et al, 2017; Carrette et al, 2018; Leko et al, 2018). The maintenance phase also includes weakening of self-interacting chromatin regions called topologically associating domains (TADs), and folding into two mega domains (reviewed in Jégu et al, 2017; Froberg et al, 2018; Galupa and Heard, 2018).…”