Active Chromatin Marks Are Retained on X Chromosomes Lacking Gene or Repeat Silencing Despite XIST/Xist Expression in Somatic Cell Hybrids
Figure 2
Analysis of Cot-1 hybridization in somatic cell hybrids expressing Xist/XIST by dual RNA FISH hybridization.
Cot-1 RNA (mouse (m) or human (h)) signal is red, Xist/XIST RNA signal is green and DAPI is blue. (A–D) Lines were drawn in ImageJ (NIH) and pixel intensities of each signal was plotted and shown to the right of the images. Yellow arrow heads indicate where the Xist signal corresponds to reductions in the Cot-1 signal. (A) Human female cell line, GM04626, with two inactive X chromosomes and two hCot-1 holes corresponding to the two XIST RNA signals. (B) Mouse female cell line, BMSL2, showing one mCot-1 hole corresponding to the mouse Xist signal, this pattern was observed in 59% of cells (n = 100). (C) XIST+/Xist+ somatic cell hybrid, AHA-4C1, showing a mCot-1 hole corresponding to the mouse Xist signal, this pattern was observed in 7% of cells (n = 100). (D) AHA-4C1 without a mCot-1 hole. In panels C and D bright foci of mCot-1 staining are observed (white arrows). (E) hCot-1 RNA expression (red) in the XIST−/Xist− hybrid cell, AHA-11aB1, containing only one human chromosome (the X chromosome). (F) hCot-1 RNA expression (red) in the XIST+/Xist+ hybrid cell, AHA-4C1, containing only one human chromosome (the X chromosome), and XIST RNA (green) drifting from the hCot-1 domain. (G) An Xi-hybrid cell line, t86-B1maz1b-3a, containing two human chromosome (X+15). Both human chromosomes form an hCot-1 transcriptional domain, one with the XIST signal drifting away.