Figure 4. RLIM-independent XCI in ESCs in vitro.

A) Xist levels (ssRT-qPCR) in ESCs, EB-differentiated and cultured for 6d in 7.5% O₂. Results represent three independent experiments. Xist levels of differentiated WTp cells are set to 1. B, C) Summary of comparison of Xist cloud formation in ESCs (EB-differentiated in 7.5% O₂) cultured for 6d as determined by RNA FISH using Xist as probe. Genotypes are indicated. Xist clouds were counted in two independent experiments (10 EBs with >100 cells counted per EB). D) Representative images of RNA FISH on 6d EB-differentiated ESCs (7.5% O₂). Lower left panel shows Rlim16p co-hybridized with Xist (red) and Rlim (green) probes in higher magnification. E) Rlim-independent chromosome wide X-silencing in vitro. Log2 transformed RNA-seq data obtained from two biological replicates of undifferentiated and differentiated (6d EB-differentiation; 7.5% O₂) each of WT, Rlim16p and E14 ESCs were compared for F/M expression level ratios from 550 X-linked genes (left panel) and 13526 autosomal genes (right panel). F) Summary of XCI during female mouse embryogenesis and the dependence on Rlim. Embryonic stages, iXCI (light grey) and rXCI (red) and XCR (dark grey) as well as embryonic cell lineages are indicated (grey, totipotent cell lineage; black, extraembryonic cell lineages; blue, epiblast cell lineage). Error bars in A–C indicate standard error of the mean (SEM). See also Figure S3.