Fig. 1.
H3K27me3 decreases and XIST RNA remains with Xi at all passages in HGPS cells. (A) Cells were processed for immunofluorescence with antibodies against LA/C (a and d) and H3K27me3 (b and e; overlay, c and f). Control cell nuclei at p9 displayed a distinct LA/C nuclear rim (a) associated with a compact Xi (b). The HGPS cells showed a change in nuclear morphology by p21 by using anti-LA/C (d), and there was no obvious Xi revealed by anti-H3K27me3 (e). (Scale bars, 5 μm.) (B) There was a decrease in H3K27me3 by Western blot analysis of HGPS compared with control total cell extracts at p20. (C) In early-passage HGPS cells stained with anti-H3K27me3, the Xi appeared either as a uniformly stained compact domain (a and d) or a loose array of closely spaced granules (b and e), or it could not be detected (a typical lamina region in a nucleus with no Xi staining is depicted in the white box; c and f). d–f are enlargements (×4) of the regions in the white boxes in a–c. (Scale bars, 5 μm.) (D) Nuclear contour ratios (CR; see also ref. 1) were determined for control and HGPS cells at early and late passages (nuclei with a CR ≥ 0.7 were considered nonlobulated, and those with a CR < 0.7 were considered lobulated). In early-passage HGPS cells, ≈43% of the nuclei lost their H3K27me3 Xi mark, and ≈80% of these were normally shaped. In late-passage cells, ≈63% lost this mark, whereas ≈20% of these nuclei were normally shaped. In early-passage controls, ≈6% of the nuclei did not contain an obvious Xi, as distinguished by anti-H3K27me3. At late passages, this number increased to ≈20%. (E) Control (a–d) and HGPS cells (e–l) were prepared for XIST RNA FISH and stained with anti-H3K27me3. (a–d) In controls at early and late passages (p13–20), XIST RNA was associated with the Xi, and ≈93% (n = 102) also contained the H3K27me3 mark. (e–l) In HGPS cells, XIST FISH revealed an Xi at all passages, whereas by p17–18, the H3K27me3 staining on Xi was lost (≈53%; n = 82). (i–l) In lobulated HGPS nuclei lacking the H3K27me3 mark on Xi, the XIST RNA staining was more dispersed. d, h, and l are enlargements (×5.6) of the overlay regions in the white boxes (c, g, and k). (Scale bars, 10 μm.) (F) Western blotting shows a decrease in EZH2 in HGPS cells compared with controls at p14. Actin was used as a loading control.