Cells presenting two nuclear domains of accumulation of Ezh2-Venus demonstrate occasional instability in Ezh2-Venus recruitment.

<p>In both A and B, the 12 left panels show wide-field fluorescent maximum projection images of Z-stacks in Venus channel at selected time points during differentiation of the Z8.1 ES cell line. Time is indicated relative to the start of the sequence shown (hours: minutes). The white arrows point to the Ezh2-Venus accumulation foci. When necessary, gray triangles allow unambiguous identification of the cell that could be followed in two successive panels. Transmission images at the start and the end of the sequence are shown in the two panels on the right. It was verified that the nuclei had been fully imaged in Z by examining individual plans of the Z-stacks. Full time-lapses corresponding to these stills are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116109#pone.0116109.s008" target="_blank">S4 Video</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116109#pone.0116109.s009" target="_blank">S5 Video</a>. A) The initial time in this sequence corresponds to 44 hours and 50 minutes after shifting from 2i plus LIF to EpiLCs culture conditions. In this cell with two fluorescent territories (arrowed), one of the two Ezh2-Venus territories of accumulation faded away with a 6 hours time course. 3 hours and 30 minutes after mitosis, one of the daughter cells still presented a single fluorescent territory (arrowed). B) The initial time in this sequence corresponds to 42 hours and 35 minutes after shifting from 2i plus LIF to EpiLCs culture conditions. Daughter cells exhibited only a single Ezh2-Venus territory of accumulation over more than 6 hours despite the fact that the mother cell had two domains (arrows).</p

The Ezh2-Venus protein is recruited at the nascent inactive X chromosome in differentiated ES cells.

<p>A) Schematic strategy for the COOH-tagging of the Ezh2 protein expressed from a mouse BAC DNA. B) Expression of the Ezh2-Venus fusion protein in ES cells. Western blotting using an Ezh2 antibody and nuclear extracts from the parental ES cell line HP3-10 (WT) and from four neomycin-resistant clones transfected with Venus-tagged BAC DNA The arrow points to the migration level of the fusion protein. C) Nuclear localization of the Ezh2-Venus fusion protein in fixed ES cells. Wide-field fluorescent microscopy for Hoechst 33342 (blue, left panel) and Venus (green, right panel) of the Z8.1 ES cells cultured in 2i plus LIF and fixed for 3 minutes with 4% PFA. D) Ezh2-Venus nuclear foci are detectable in differentiated live female ES cells. Live imaging was performed on the Z8.1 ES cells after differentiation for 50 hours. Nuclear fluorescent foci of Venus signal are visible (green, left and central panel). DNA was stained with Hoechst 33342 (pseudo-colored in red) and overlaid on the phase contrast image (right panel) and on the Venus channel image (central panel). E) Ezh2-Venus nuclear foci correspond to <i>Xist</i> RNA clouds. Live cells of the Z8.1 line differentiated during 70 hours were imaged for Venus (top-left panel) and then fixed and processed for <i>Xist</i> RNA-FISH (top-right panel; DAPI blue, <i>Xist</i> green). Despite moderate shifts due to live cell movements prior to fixation, both panels show recognizable nuclei presenting similarly localized Ezh2-Venus and <i>Xist</i> nuclear territories. All nuclear Ezh2-Venus foci detected in live cells corresponded to a <i>Xist</i> RNA cloud (bottom panel, n = 60) although the reciprocal was not the same. F) In the course of differentiation, the kinetics of Ezh2-Venus foci is delayed as compared to the kinetics of <i>Xist</i> RNA accumulation. Duplicate samples at different time-points of the same differentiation experiment using the Z8.1 ES cell line, were processed for live imaging of Ezh2-Venus or for <i>Xist</i> RNA-FISH. Cells were counted for nuclear fluorescent territories after image acquisition. Bars on top of the columns represent standard deviation of the counts of three groups of cells (n>150 each) at each timepoint. Detection of Ezh2-Venus territories is delayed as compared with <i>Xist</i> clouds, which had already reached a plateau by 40 hours of differentiation in this experiment. The data presented in panel C to F have been reproduced with no significant difference for at least two other transgenic Ezh2-Venus ES cell lines.</p

Live-cell imaging showing that the recruitment of Ezh2-Venus to the nascent inactive X chromosome is lost during mitosis and is restored progressively during interphase.

<p>In both A and B, the 12 left panels show wide-field fluorescent maximum projection images of Z-stacks in the Venus channel at selected time points during differentiation of the Z8.1 ES cell line. Time is indicated relative to the start of the sequence shown (hours: minutes). The white arrows point to the Ezh2-Venus accumulation foci. When necessary, gray triangles unambiguously identify the cell, which can be followed in two successive panels. Transmission images at the start and the end of the sequence are shown in the two panels on the right. It was verified that the nuclei had been fully imaged in Z by examining individual plans of the Z-stacks. Full time-lapses corresponding to these stills are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116109#pone.0116109.s006" target="_blank">S2 Video</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116109#pone.0116109.s007" target="_blank">S3 Video</a>. A) The initial time in this sequence corresponds to 50 hours and 50 minutes after shifting from 2i plus LIF to EpiLCs culture conditions. Daughter nuclei lacked fluorescent Ezh2-Venus accumulation for 1 hour after the first mitosis, and for only 12 minutes after the second mitosis. The dashed line at time 10:36 indicates that the image of one of the cells was digitally treated to bring it closer to its sister. B) The initial time shown in this sequence corresponds to 53 hours and 25 minutes after shifting from 2i plus LIF to EpiLCs culture conditions. After the separation of the daughter cells, the nuclei lacked a domain of Ezh2-Venus accumulation for 1 hour and 12 minutes. The daughter cell in the lower region of the image then started to display a single fluorescent domain and only then regained a second domain (arrows).</p

Initiation of X chromosome inactivation can occur on both Xs in the course of differentiation of naive ES cells.

<p>A) Kinetic analysis using quantitative RT-PCR of the expression levels of <i>Nanog</i> and <i>Xist</i> normalized to <i>Actin</i> levels in the course of three days of differentiation of ES cells. Wild-type female cell lines: gC1, HP3-10 and PGK1. Control wild-type male cell lines: CK35 and E14Tg2A. RT- controls required at least 10 cycles more than the RT+ samples in order to exceed threshold. Columns represent the ratio of the means of triplicate measurement of 2^-ct for <i>Nanog</i>, <i>Xist</i> and <i>Actin</i> at each timepoint in each differentiation experiment. Error bars represent standard deviation of the ratios of <i>Xist</i> (or <i>Nanog</i>) to <i>Actin</i> calculated as: sd of <i>Xist</i>/<i>Actin</i>  =  (mean of <i>Xist</i>/<i>Actin</i>) x [(sd of <i>Xist</i>/mean of <i>Xist</i>)² + (sd of <i>Actin</i>/mean of <i>Actin</i>)²]^0,5. Two fully independent differentiation experiments performed on separate days with each female ES cell line are shown. B) Immuno-RNA-FISH of <i>Xist</i> and H3-K27me3 using the HP3-10 cell line differentiated for 3 days: <i>Xist</i> RNA clouds (green, top panel) correspond to domains of enrichment for H3-K27me3 (red, bottom panel). In B, C, E, and F, DAPI blue staining was digitally dampened to favor visualization of the other channels. Nuclei are circled with a dashed line. C) Immuno-RNA-FISH of <i>Xist</i> and H3-K27me3 using the gC1 cell line differentiated for 36 hours. Nuclei showing two <i>Xist</i> clouds (green, top panel) additionally show two domains of enrichment for the H3-K27me3 mark (red, bottom panel). D) Kinetic analysis using <i>Xist</i> RNA-FISH. Cells showing one or two <i>Xist</i> clouds were counted over the course of independent 72-hours differentiation experiments with the gC1, HP3-10 and PGK1 ES cell lines (n>250). E) Differentiated female ES cells presenting two <i>Xist</i> clouds have a normal complement of two X chromosomes. Sequential RNA-FISH for <i>Xist</i> (top left panel) and DNA-FISH using a BAC-561P13 probe which maps within the X inactivation center (bottom left panel) were performed with the HP3-10 cell line. This experiment was similarly performed with the gC1 ES cell line. Cells presenting two Xist clouds were evaluated for their complement of X chromosomes (right panels; gC1 48 h, n = 60; gC1 72 h, n = 25; HP3-10 48 h; n = 45; HP3-10 72 h, n = 30). All or most cells presenting two <i>Xist</i> clouds have a normal complement of two X chromosomes, although some X triplody arouse at 72 hours in this cell population. F) Double RNA-FISH for <i>Xist</i> (green) and <i>Rnf12</i> (red) using the HP3-10 ES cell line differentiated during 40 hours. A cell which did not upregulate <i>Xist</i> expressed <i>Rnf12</i> bi-allelically (left panels) and a cell which upregulated both alleles of <i>Xist</i> silenced <i>Rnf12</i> (right panels). The efficiency of detection of the <i>Rnf12</i> primary transcription site was 86% as determined in cells lacking <i>Xist</i> expression. <i>Rnf12</i> expression was examined in cells with no <i>Xist</i> cloud (n = 159), with one <i>Xist</i> cloud (n = 181) or with 2 <i>Xist</i> clouds (n = 75). Nearly all the cells presenting 2 <i>Xist</i> clouds were functionally nullisomic for <i>Rnf12</i> expression. Repeat experiments with the HP3-10 and gC1 ES cell lines gave essentially identical results.</p