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.

X inactivation in female mammals is one of the best studied examples of heritable gene silencing and provides an important model for studying maintenance of patterns of gene expression during differentiation and development. The process is initiated by a cis-acting RNA, the X inactive specific transcript (Xist). Xist RNA is thought to recruit silencing complexes to the inactive X, which then serve to establish and maintain the inactive state in all subsequent cell divisions. Most lineages undergo random X inactivation, there being an equal probability of either the maternally (Xm) or paternally (Xp) inherited X chromosome being inactivated in a given cell. In the extraembryonic trophectoderm and primitive endoderm lineages of mouse embryos, however, there is imprinted X inactivation of Xp. This process is also Xist dependent. A recent study has shown that imprinted X inactivation in trophectoderm is not maintained in embryonic ectoderm development (eed) mutant mice. Here we show that Eed and a second Polycomb group protein, Enx1, are directly localized to the inactive X chromosome in XX trophoblast stem (TS) cells. The association of Eed/Enx1 complexes is mitotically stable, suggesting a mechanism for the maintenance of imprinted X inactivation in these cells.

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.

The b-amyloid peptide (Ab) that accumulates in senile plaques in Alzheimer's disease is formed by cleavage of the amyloid precursor protein (APP). The APP gene has several intronic Alu elements inserted in either the sense or antisense orientation. In this study, we demonstrate that binding of SC35 and hnRNPA1 to Alu elements on either side of exon 7 in the transcribed pre-mRNA is involved in alternative splicing of APP exons 7 and 8. Neuronal cells transfected with the full-length form of APP secrete higher levels of Ab than cells transfected with the APP695 isoform lacking exons 7 and 8. Finally, we show that treatment of neuronal cells with estradiol results in increased expression of APP695, SC35 and hnRNPA1, and lowers the level of secreted Ab. An understanding of the regulation of splicing of APP may lead to the identification of new targets for treating Alzheimer's disease.