The Trithorax group protein Ash2l and Saf-A are recruited to the inactive X chromosome at the onset of stable X inactivation

Mammals compensate X chromosome gene dosage between the sexes by silencing of one of the two female X chromosomes. X inactivation is initiated in the early embryo and requires the non-coding Xist RNA, which encompasses the inactive X chromosome (Xi) and triggers its silencing. In differentiated cells, several factors including the histone variant macroH2A and the scaffold attachment factor SAF-A are recruited to the Xi and maintain its repression. Consequently, in female somatic cells the Xi remains stably silenced independently of Xist . Here, we identify the Trithorax group protein Ash2l as a novel component of the Xi. Ash2l is recruited by Xist concomitantly with Saf-A and macroH2A at the transition to Xi maintenance. Recruitment of these factors characterizes a developmental transition point for the chromatin composition of the Xi. Surprisingly, expression of a mutant Xist RNA that does not cause gene repression can trigger recruitment of Ash2l, Saf-A and macroH2A to the X chromosome, and can cause chromosome-wide histone H4 hypoacetylation. This suggests that a chromatin configuration is established on non-genic chromatin on the Xi by Xist to provide a repressive compartment that could be used for maintaining gene silencing. Gene silencing is mechanistically separable from the formation of this repressive compartment and, thus, requires additional pathways. This observation highlights a crucial role for spatial organization of chromatin changes in the maintenance of X inactivation.