The role of Xist RNA in the maintenance of X chromosome inactivation

Abstract

The role of Xist RNA in silencing the inactive X of female somatic cells was investigated by generating a conditional allele of the Xist gene. A system was set up in which reactivation of two X-linked genes, the endogenous Hprt gene and an X-linked GFP transgene, can be quantitatively assessed. Mouse embryonic fibroblasts derived from mice carrying the conditional Xist mutation were cultured and infected with an adenovirus vector carrying the gene for Cre recombinase. After Cre mediated deletion of Xist, the inactive X remained transcriptionally silent, late replicating, and hypoacetylated on histone H4, confirming that X-inactivation can be maintained in the absence of Xist RNA. However, the Xist mutant inactive X was no longer enriched in histone macroH2A 1. Furthermore, the reactivation rate of GFP and Hprt increased, indicating Xist RNA does contribute to gene repression on the inactive X. DNA methylation, histone hypoacetylation and Xist RNA were found to act synergistically in X chromosome silencing. To investigate whether Xist RNA can also silence the active X chromosome of male somatic cells, Xist expression on the active X was induced by demethylation. Demethylation was achieved by Cre mediated deletion of a conditional mutant allele of DNA methyltransferase-l (Dnmtl) in male fibroblasts. In these cells, Xist RNA coated the active X chromosome, in a pattern indistinguishable from coating of the inactive X of female cells. Although many Xist expressing chromosomes also transcribed X-linked genes Pgk-i and Hprt, in a small percent of cells Xist expression led to X chromosome inactivation. The proportion of chromosome expressing X-linked genes declined, and occasionally the X became late replicating, indicating that X-inactivation can be initiated in male somatic cells.