PMC

The H3-K27me3 mark of the Xi is lost in XIST⁻ cells. (A) ImmunoFISH combining immunostaining for H3-K27me3 (red) and X chromosome painting (green). (Scale bar, 5 μm.) (B) Immunostaining of H3-K27me3 (red) combined with XIST RNA FISH (green).

RT-PCR and RNA FISH analysis of XIST expression during hESC differentiation. (A) Quantitative real-time RT-PCR for XIST expression on d0 (undifferentiated), d3, d5, d8, and d12 of differentiation compared with primary female fibroblasts (WI-38). XIST is normalized to GAPDH. (B) XIST RNA FISH in undifferentiated (d0) and differentiated (d12) hESC. Nuclei are stained with DAPI (blue). Arrows point to XIST clouds (green). (Scale bar, 10 μm.)

Cot-1 RNA FISH reveals that XIST⁻ nuclei of class II and III lines have Xi. (A–D) Combined RNA/DNA FISH reveals that class III lines have undergone XCI but lost XIST expression. Arrows point to Xi associated with a Cot-1 hole. (E) XIST⁻ cells of p.23 HUES5 class II line also exhibit one Xi. Asterisk denotes XIST-coated Xi. (F) XIST⁻ d0 cells of the class I line HUES9 largely do not exclude Cot-1 hybridization. Double arrowheads indicate Cot-1⁺ X chromosomes. (Scale bar, 5 μm.)

Karyotype analysis and Cot-1 RNA FISH. (A) Karyotype analysis by G-banding was performed at Cell Line Genetics on 20 metaphase spreads from d0 HUES9, HUES5, and HUES6. All are 46XX, although HUES9 and HUES5 appeared to carry a small but cytogenetically visible pericentric inversion on one chromosome 9. (B) Two XIC DNA FISH signals (green) are evident in a vast majority of nuclei in all class III lines (d0). Note: that after d14–d15 of differentiation, the frequency of nuclei with 2 XIC signals did not change significantly, e.g., 89% for HUES6 (n = 108), 89% for HUES15 (n = 104), 92% for H9 (n = 114), and 92% for HES-2 (n = 126). Percentage with XIC PAC signals and sample sizes (n) are shown. (Scale bar, 5 μm.) (C) RNA FISH shows that XIST RNA domains (green) exclude Cot-1 hybridization (red), indicating transcriptional silencing of the X chromosome in WI-38 fibroblasts and class I and II lines indicated. Arrows point to examples of Xs in Cot-1 holes. (Scale bar, 10 μm.)