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

Abstract

SUMMARYMammals 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.

Advances in understanding chromosome silencing by the long non-coding RNA Xist

“…A-repeat deficient RNA is also able to recruit macroH2A, SAF-A and Ash2L [45]. Either these proteins interact with regions of Xist RNA other than the A-repeats or their recruitment is secondary to the establishment of a transcriptionally silent domain that occurs using A-repeat deficient transgenes [58].…”

“…Thus, the function of the A-repeats would be to allow the silencing factors to spread from common repeat elements into genes. [45] rstb.royalsocietypublishing.org Phil Trans R Soc B 368: 20110325…”

“…This implied that the association of Xist RNA with chromatin of the X chromosome was not mediated by hybridization of DNA and RNA, but probably by an interaction with a protein(s). The finding that scaffold attachment factor-A (SAF-A), also termed hnRNP U, one of the major components of the nuclear scaffold, is enriched on the inactive X chromosome in human and mouse female cells [44,45] suggested a potential role for this protein in targeting of the inactive X to the nuclear periphery via association with Xist RNA. In fact, SAF-A contains two distinct domains, a SAF box and RGG domain, that have been shown to mediate SAF-A binding to DNA and RNA, respectively [44,46].…”

X chromosome inactivation: A silence that needs to be broken

“…While many PcG proteins showed Xi-enrichment, a trxG protein Ash2I (absent, small or homeotic discs 2) showed surprising Xi-enrichment (Pullirsch et al, 2010). However, it is believed that, as a trxG protein, Ash2I only plays a regulatory role unrelated to gene activation.…”

“…However, it did not result in gene silencing and dearth of euchromatin marks on the chromosome (Thorogood and Brown, 2010). When a mutant form of Xist, which lacked gene silencing capacity, was induced in ES cells, it reassembled heterochromatic modifications on the chromosome territory (Pullirsch et al, 2010). Such a chromosome territory, with all the heterochromatic modifications resembling Xi but without gene silencing, was termed as Xiag (Xi with active genes).…”