Primary non-random X inactivation associated with disruption of Xist promoter regulation.

In this report we demonstrate primary non-random X chromosome inactivation following targeted mutagenesis of a region immediately upstream of XIST promoter P(1). In heterozygous animals there is a preferential inactivation of the targeted X chromosome in 80--90% of cells. The phenotype correlates with inappropriate activation of XIST in a proportion of the mutant XY embryonic stem cells.

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'.

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.

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.

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.