Disruption of a conserved region of Xist exon 1 impairs Xist RNA localisation and X-linked gene silencing during random and imprinted X chromosome inactivation

In XX female mammals a single X chromosome is inactivated early in embryonic development, a process that is required to equalise X-linked gene dosage relative to XY males. X inactivation is regulated by a cis-acting master switch, the Xist locus, the product of which is a large non-coding RNA that coats the chromosome from which it is transcribed, triggering recruitment of chromatin modifying factors that establish and maintain gene silencing chromosome wide. Chromosome coating and Xist RNAmediated silencing remain poorly understood, both at the level of RNA sequence determinants and interacting factors. Here, we describe analysis of a novel targeted mutation, Xist^INV, designed to test the function of a conserved region located in exon 1 of Xist RNA during X inactivation in mouse. We show that Xist^INV is a strong hypomorphic allele that is appropriately regulated but compromised in its ability to silence X-linked loci in cis. Inheritance of Xist^INV on the paternal X chromosome results in embryonic lethality due to failure of imprinted X inactivation in extra-embryonic lineages. Female embryos inheriting Xist^INV on the maternal X chromosome undergo extreme secondary non-random X inactivation, eliminating the majority of cells that express the Xist^INV allele. Analysis of cells that express Xist^INV RNA demonstrates reduced association of the mutant RNA to the X chromosome, suggesting that conserved sequences in the inverted region are important for Xist RNA localisation.