In female mammalian cells, dosage compensation for X-linked genes is achieved by the transcriptional silencing, early in development, of many genes on just one of the two X chromosomes. Several properties distinguish the inactive X (Xi) from its active counterpart (Xa). These include expression of Xist, a gene located in the X-inactivation center (Xic), late replication, differential methylation of selected CpG islands and underacetylation of histone H4. The relationship between these properties and transcriptional silencing remains unclear. Female mouse embryonic stem (ES) cells have two active X chromosomes, one of which is inactivated as cells differentiate in culture. We describe here the use of these cells in studying the sequence of events leading to X-inactivation. By immunofluorescent labeling of metaphase chromosome spreads from ES cells with antibodies to acetylated H4, we show that an underacetylated X chromosome appears only after 4 days of differentiation, and only in female cells. The frequency of cells with an underacetylated X reaches a maximum by Day 6. In undifferentiated cells, H4 in centric heterochromatin is acetylated to the same extent as that in euchromatin but has become relatively underacetylated, as in adult cells, by Day 4 of differentiation (i.e. , when deacetylation of Xi is first seen). The overall deacetylation of Xi follows Xist expression and the first appearance of a single, late-replicating X, both of which occur on Day 2. It also follows the silencing of X-linked genes. Levels of mRNA from four such genes, Hprt, G6pd, Rps4, and Pgk-1, had all fallen by approximately 50% (relative to the autosomal gene Aprt) by Days 2-4. The results show that properties that characterize Xi are put in place in a set order over several days. H4 deacetylation occupies a defined place within this sequence, suggesting that it is an intrinsic part of the X-inactivation process. The stage at which a completely deacetylated Xi is first seen suggests that deacetylation may be necessary for the maintenance of silencing but is not required for its initiation. Nor is it required for, or an immediate consequence of, late replication. However, we note that selective deacetylation of H4 on specific genes would not be detected by the microscopical approach we have used and that such selective deacetylation may still be part of the silencing process.

The propagation of X chromosome inactivation is thought to be mediated by the cis- limited spreading of the non-protein coding Xist transcript. In this report we have investigated the localization of Xist RNA on rodent metaphase chromosomes. We show that Xist RNA exhibits a banded pattern on the inactive X and is excluded from regions of constitutive heterochromatin. The banding pattern suggests a preferential association with gene-rich, G-light regions. Analysis of X:autosome rearrangements revealed that restricted propagation of X inactivation into cis -linked autosomal material is reflected by a corresponding limited spread of Xist RNA. We discuss these results in the context of models for the function of Xist RNA in the propagation of X inactivation.

L.P.O'Neill and A.M.Keohane contributed equally to this workWe have investigated the role of histone acetylation in X chromosome inactivation, focusing on its possible involvement in the regulation of Xist, an essential gene expressed only from the inactive X (Xi). We have identified a region of H4 hyperacetylation extending up to 120 kb upstream from the Xist somatic promoter P 1 . This domain includes the promoter P 0 , which gives rise to the unstable Xist transcript in undifferentiated cells. The hyperacetylated domain was not seen in male cells or in female XT67E1 cells, a mutant cell line heterozygous for a partially deleted Xist allele and in which an increased number of cells fail to undergo X inactivation. The hyperacetylation upstream of Xist was lost by day 7 of differentiation, when X inactivation was essentially complete. Wild-type cells differentiated in the presence of the histone deacetylase inhibitor Trichostatin A were prevented from forming a normally inactivated X, as judged by the frequency of underacetylated X chromosomes detected by immunofluorescence microscopy. Mutant XT67E1 cells, lacking hyperacetylation upstream of Xist, were less affected. We propose that (i) hyperacetylation of chromatin upstream of Xist facilitates the promoter switch that leads to stabilization of the Xist transcript and (ii) that the subsequent deacetylation of this region is essential for the further progression of X inactivation.

Underacetylation of histone H4 is thought to be involved in the molecular mechanism of mammalian X chromosome inactivation, which is an important model system for large-scale genetic control in eukaryotes. However, it has not been established whether histone underacetylation plays a critical role in the multistep inactivation pathway. Here we demonstrate differential histone H4 acetylation between the X chromosomes of a female marsupial, Macropus eugenii. Histone underacetylation is the only molecular aspect of X inactivation known to be shared by marsupial and eutherian mammals. Its strong evolutionary conservation implies that, unlike DNA methylation, histone underacetylation was a feature of dosage compensation in a common mammalian ancestor, and is therefore likely to play a central role in X chromosome inactivation in all mammals.