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

Abstract

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.

Fig. 1.

Ash2l is a component of the Xi. (A) 3T3 cells were derived from female mouse embryos homozygous for a conditional endogenous Xist allele and heterozygous for the rtTA transactivator and a doxycycline-inducible tetOP-Cre. Focal enrichment of Saf-A on the Xi was observed before Cre induction (—Cre) and was lost after doxycycline-induced deletion of Xist (+Cre). DNA is stained by DAPI (blue). (B) Immunofluorescence staining (IF) of ΔSX ES cells after 6 days of differentiation with Xist induction using serum #516 to detect macroH2A (green) and Ash2l (red) on the X chromosome. Specificity of the staining was confirmed by incubation with an Ash2l blocking peptide (lower panel). A merged picture with DNA stained by DAPI (blue) is shown. Scale bars: 5 μm. (C) Ash2l recruitment was analyzed in clone 36 ES cells harboring an inducible Ash2l RNAi construct or control clones on day 6 of differentiation in the presence of doxycycline. The number of cells showing Ash2l foci and Xist clusters is given (left). Ash2l RNAi clones 1 and 2 contain independent siRNA sequences. Western analysis (right) of clones 1 and 2 containing an inducible Ash2l RNAi cassette targeted downstream of the Col1a1 locus shows that induction of RNAi with doxycycline (dox) reduces Ash2l protein. Lamin B was used as a loading control.

Fig. 2.

A human autoimmune serum recognizes SAF-A on the Xi. (A) IF of HEK293 cells using serum #184 (green) and an antibody specific for H3K27me3 (red). Enrichment of the target antigen is observed in interphase (upper panels) and in mitosis (lower panels). DNA is stained by DAPI. Scale bars: 5 μm. (B) Western analysis of total protein extracted from 293 cells using serum #184 identifies a band of 120 kDa (left). Bands corresponding to transgenic SAF-A proteins are identified in 293 cells transiently transfected with the SAF-A constructs indicated (right). Lamin B was used as a loading control. (C) 3T3 cells homozygous for a conditional Xist allele and carrying an inducible Cre transgene were stably transfected with Saf-A:hrGFP. Focal enrichment of Saf-A on the Xi was observed before Cre induction (—Cre) but was lost after doxycycline-induced deletion of Xist (+Cre). DNA is stained by DAPI (blue).

Fig. 3.

Saf-A recruitment in ES cells requires Xist but not gene silencing. (A) Localization of the Saf-A:hrGFP fusion protein (green) in ΔSX^Saf-A:hrGFP ES cells after 6 days of differentiation in the presence of doxycycline. The X chromosome was identified by co-staining with an H3K27me3-specific antibody (red). (B) Western analysis showing expression of the Saf-A:hrGFP fusion protein in parental and transgenic clone 36^Saf-A:hrGFP and ΔSX^Saf-A:hrGFP ES cells using an α-hrGFP antibody. (C) Saf-A enrichment is rapidly lost after Xist expression is turned off. ΔSX^Saf-A:hrGFP ES cells were differentiated in the presence of doxycycline. Then doxycycline was removed from the medium and the cells were differentiated for 1, 2, 3 or 4 more days without Xist induction and analyzed on day 12. The percentage of cells in parallel cultures showing focal Saf-A:hrGFP signals, H3K27me3 staining and Xist RNA clusters is given. Loss of Saf-A foci follows Xist delocalization, whereas H3K27me3 persists longer. (D) Focal Saf-A enrichment depends on Xist expression in 36^Saf-A:hrGFP cells after stable silencing is established. The percentage of 36^Saf-A:hrGFP cells showing focal Saf-A:hrGFP signals, H3K27me3 staining and Xist RNA clusters is given after differentiation in the presence of doxycycline for 8 days (+), or after differentiation for 4 days in the presence followed by 4 days in the absence of doxycycline (stepped symbol). Stable silencing was confirmed by northern analysis (right) of the repression of a puromycin marker gene co-integrated with the Xist transgene in 36^Saf-A:hrGFP ES cells differentiated for 8 days in the presence (+, lane 1) or absence (−, lane 2) of doxycycline, or for 4 days in the presence followed by 4 days in the absence of doxycycline (stepped symbol, lane 3).

Fig. 4.

Timing of Saf-A and Ash2l recruitment in ΔSX^Saf-A:hrGFP ES cell differentiation. An analysis of H3K27me3 (A), Saf-A (B), Ash2l (C) and macroH2A (D) recruitment by Xist in either undifferentiated ΔSX^Saf-A:hrGFP ES cells or after 1, 2, 3, 4 and 8 days of differentiation in the presence of doxycycline is shown. The percentage of cells showing focal enrichment is given. The experiments were performed twice for macroH2A and Ash2l, and three times for Saf-A and H3K27me3. A representative experiment is shown (n=100).

Fig. 5.

Saf-A, Ash2l and macroH2A recruitment requires Xist expression in early differentiation. (A) Efficient enrichment of Saf-A, Ash2l and mH2A requires Xist induction early in differentiation. ΔSX^Saf-A:hrGFP ES cells were induced to differentiate with retinoic acid and doxycycline was added either at the onset of differentiation (+) or after 1, 3 or 6 days of differentiation. The percentage of cells showing focal H3K27me3, Saf-A:hrGFP, Ash2l and mH2A signals on day 12 of differentiation is given. (B) Xist expression during early differentiation enables efficient factor recruitment upon reinduction of Xist in differentiated cells. Xist was expressed in undifferentiated ES cells for 3 days and turned off during differentiation on day 1, 2, 3, 4 or 5. The percentage of cells showing focal H3K27me3, Saf-A, Ash2l and mH2A signals after reinduction of Xist on day 8 for 4 more days is shown. Reversibility of recruitment was shown in parallel cultures differentiated for 4 days in the presence followed by 4 days in the absence of doxycyline (stepped symbol).

Fig. 6.

Chromosome-wide histone H4 hypoacetylation is triggered by Xist independent of gene silencing. (A) An antibody directed against acetylated histone H4 (green) reveals a hypoacetylated X chromosome identified by a DNA FISH X paint probe (red) in ΔSX ES cells after 3 days of differentiation with Xist induction. A representative image is shown. (B,C) Histone H4 hypoacetylation is observed after 9 days of differentiation with Xist induction (B) and is maintained in ΔSX ES cells that were differentiated for 4 days with and then for 5 days without Xist expression (C). A magnified image showing remaining acetylated bands on the X chromosome is shown to the right in B. The unlabeled arrows indicate Xiag chromosomes.