Figure 1.
X-linked gene expression in somatic cell hybrids before and after expression of XIST/Xist.
(A–C) Shows the XIST/Xist expression in cell nuclei using RNA FISH probes for the human XIST RNA (green) and the mouse Xist RNA (red). (A) The XIST+/Xist+ (AHA-4C1) somatic cell hybrid; (B) The female mouse fibroblast control cell line, BMSL2; (C) The human female fibroblast line GM04626 (karyotype: 47,XXX). (D) Schematic diagram of both the human and mouse X chromosomes showing the location of the genes analyzed to the left of gel images of RT-PCR products of cDNA from the control female (GM7350 (human) and BMSL2 (mouse)); the hybrid not expressing AHA-11aB1 (XIST−/Xist−); and the derivative hybrid that expresses AHA-4C1 (XIST+/Xist+). (E) Quantitative RT-PCR of 4 X-linked genes. An intermediate hybrid AHA-A5-2b (XIST+/Xist−) was analyzed for expression along with the hybrid AHA-4C1 (XIST+/Xist+). The gene expression was normalized to Actin and expressed as a fold-change relative to the parent hybrid AHA-11aB1 (XIST−/Xist−). The error bars represent the standard deviation of three separate RNA isolations.
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.
Figure 3.
DNA methylation of X-linked gene promoters before and after expression of XIST/Xist.
Genomic DNA was cut with a methylation-sensitive restriction enzyme prior to amplification by PCR with primers flanking the enzyme cut sites (lanes labeled Digest). Mock digested samples were used as a positive control (Mock). If a band occurs in the digest lane, it means that the site/sites within the amplicon are methylated (e.g. XIST/Xist). If no band was observed after PCR then we conclude that there was no intact template to amplify as the internal restriction enzyme sites were not methylated.
Figure 4.
Active histone modifications at the promoters of X-linked genes after expression of XIST/Xist.
H3K4m2 (upper) and H3Ac (lower) marks were assessed by ChIP followed by quantitative PCR for the two human and four mouse genes listed. The %IP is relative to input and error bars indicate error between 2–3 replicate immunoprecipitations.