Chromatin structure analysis of the mouse Xist locus.

The Xist gene is expressed exclusively from the inactive X chromosome and plays a central role in regulating X chromosome inactivation. Here we describe experiments aimed at defining the extent of the active chromatin domain of the expressed Xist allele. By using an allele-specific general DNaseI sensitivity assay we show that there is preferential digestion of the expressed allele at sites within the transcribed locus but not in flanking sites located up to 70 kb 5'. A putative proximal boundary for the Xist domain is located within 10 kb upstream of promoter P1. Chromatin in the expressed domain was found to be acetylated at H4 in XX somatic cells but also in XY cells, where Xist is never expressed. A single clear exception to this was the Xist promoter, which is acetylated only in XX cells. These observations concur with the view that H4 acetylation may not be a general marker of active chromatin domains and further support data implicating local promoter acetylation as being of primary functional significance in vivo.

Xist RNA exhibits a banded localization on the inactive X chromosome and is excluded from autosomal material in cis.

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.

Developmentally regulated Xist promoter switch mediates initiation of X inactivation.

Developmental regulation of the mouse Xist gene at the onset of X chromosome inactivation is mediated by RNA stabilization. Here, we show that alternate promoter usage gives rise to distinct stable and unstable RNA isoforms. Unstable Xist transcript initiates at a novel upstream promoter, whereas stable Xist RNA is transcribed from the previously identified promoter and from a novel downstream promoter. Analysis of cells undergoing X inactivation indicates that a developmentally regulated promoter switch mediates stabilization and accumulation of Xist RNA on the inactive X chromosome.

Stabilization of Xist RNA mediates initiation of X chromosome inactivation.

The onset of X inactivation is preceded by a marked increase in the level of Xist RNA. Here we demonstrate that increased stability of Xist RNA is the primary determinant of developmental up-regulation. Unstable transcript is produced by both alleles in XX ES cells and in XX embryos prior to the onset of random X inactivation. Following differentiation, transcription of unstable RNA from the active X chromosome allele continues for a period following stabilization and accumulation of transcript on the inactive X allele. We discuss the implications of these findings in terms of models for the initiation of random and imprinted X inactivation.