X Chromosomes Alternate between Two States prior to Random X-Inactivation
Figure 1
X-Chromosomal Loci Display SD FISH Signals Independent of Asynchronous DNA Replication
(A) FISH for Xic genomic sequences (red) demonstrates the three classes of signals in PFA-fixed female ES cells. DNA was stained with DAPI (blue).
(B) ES cells display an elevated proportion of SD signals at X-chromosomal loci. Average data from two to four experiments ( n > 150), scored by two independent scorers, are presented. Error bars indicate one standard deviation. See Figure S1A for complete scoring of SS, SD, and DD signals in these cells.
(C) The Xic and Pgk1 show a single peak of replication in female ES cells. DNA was isolated from cells that were arrested in G1, released into S phase, and pulsed with BrdU at hourly intervals. Progression through S phase was monitored by FACS ( Figure S1B). An equal amount of BrdU-labeled human DNA was added to each time point. BrdU-labeled DNA was immunopurified, and sequences present in each fraction were assessed by PCR. Standard shows amplification of a human sequence, as a control for variability in immunoprecipitations. IgG represents PCR analysis from labeled DNA purified with mouse IgG instead of anti-BrdU antiserum. –BrdU indicates analysis of an anti-BrdU immunoprecipitation from unlabeled DNA. Pre-IP depicts PCR analysis of the input DNA.
(D) Live female ES cells, pulse-labeled with BrdU, were sorted into six fractions by Hoechst staining for DNA content (upper panel). FISH for the Xic (middle panel), and Pgk1 (lower panel) in these fractions shows constant, high proportions of SD signals (red triangles) throughout S-phase. Proportions of SS (black circles) and DD (gray squares) are also shown. The high proportion of BrdU-positive cells in all fractions (bold black line) shows that a substantial proportion of cells in all six fractions are in S phase. Over 80% of cycling ES cells are in S phase and fewer than 10% are in G1 [40], inevitably leading to the inclusion of early S phase cells in fraction 1. Data are representative of two or three independent experiments.
(E) In ES cells, singlet and doublet FISH signals for X-chromosomal loci exhibit equivalent fluorescence intensity. Plots show the ratios of S/D (solid symbols) or D/D (open symbols) FISH signal intensities in individual MEF or ES cell nuclei displaying an SD or DD pattern for Pgk1 or the Xic as indicated. The intensity of both pinpoints in each doublet was summed to calculate the total intensity of doublet signals. When calculating the D/D intensity ratios, the two doublets in a cell were randomly assigned to the numerator or denominator. Mean ratio values and 95% confidence intervals for the means are indicated.
(F) Comparison of the proportions of cells displaying SS (white), SD (black), and DD (gray) signals for an autosomal locus (Fn1) and three X-chromosomal loci (Mecp2, Xic, and Pgk1) in S-phase ES cells upon PFA or MeOH fixation.