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.