Figure 5. Xist Silencing Is Necessary, but Not Sufficient, for XCR.
(A) (i) Representative image of an immunoFISH analysis for NANOG (green) and Xist RNA (red) at day 14 of reprogramming with MEFs carrying a deletion of Tsix on the Xi and of Xist on the Xa, illustrating that NANOG+ cells lose Xist RNA accumulation on the Xi even in the absence of Tsix on the Xi. (ii) iPSCs derived from the experiment in (i) were stained for NANOG (green) and Tsix RNA (red), confirming monoallelic expression of Tsix due to deletion on one X chromosome (arrowheads).
(B) Kinetics of Xist RNA loss in the absence of Tsix on the Xi. Proportion of NANOG+ cells with XiXist+ in reprogramming time courses performed with MEFs with (gray bars) and without (blue bars) Tsix on the Xi. Just like Tsix deletion, the additional deletion of Xist on the Xa (dark versus lighter bars) does not affect the kinetics of Xist RNA loss in NANOG+ cells.
(C) Diagram of Xist overexpression reprogramming experiments using MEFs in which the promoter of Xist on the Xi is replaced with a tet-inducible promoter.
(D) Proportion of NANOG+ cells with XiXist+ in reprogramming cultures described in (C) with and without ectopic Xist induction conditions (+/−dox), based on immunoFISH analysis.
(E) As (D), but for NANOG+ cells with biallelic Atrx expression.
(F) As (D), but for the number of ESRRB+ colonies.
(G) Diagram of the Xist deletion reprogramming experiments with female conditional Xist MEFs.
(H) Xist RNA FISH for MEFs described in (G) under control (−dox) and +dox conditions, the latter leading to Xist RNA loss in the majority of cells.
(I) Number of NANOG+ colonies at various time points of reprogramming for the experiment described in (G) under control (no dox/−Cre) and the Xist deletion (+dox/+Cre) conditions.
(J) As in (I), but quantitation of NANOG+ cells with biallelic Atrx expression based on immunoFISH analysis.