15 research outputs found

    ChIP Mapping of H3K27m3, H4K20m1, H3K9m2, H3K4m3, and H3K4m2 on the <i>Xist</i>-Expressing Chromosome 11 during Differentiation of Clone 36 ES Cells

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    <div><p>A genetic map of Chromosome 11 indicating the loci analysed is given on the left (<i>Xist</i>-TG, approximate integration site of <i>Xist</i> transgene; <i>puro, PGKpuromycin</i> marker).</p> <p>(A to F) Chromatin was prepared from undifferentiated clone 36 ES cells grown for 3 d in the presence (light bars) or absence (dark bars) of doxycycline. H3K27m3 and H4K20m1 were enriched at three intergenic microsatellite sequences at 18.0 (A), 45.5 (C), and 75.2 (D) cM. (B) H3K27m3 was established over the coding sequence of <i>PGKpuromycin</i> in doxycycline-induced cells, which was accompanied by a loss of H3K4m2 and H3K4m3. (E) Tubulin control. (F) Control microsatellite located on Chromosome 15.</p> <p>(G–L) Analysis of H3K27m3, H4K20m1, and H3K9m2 in clone 36 ES cells differentiated for 9 d with (light bars) or without (dark bars) doxycycline. Histone methylation marks were monitored. Experiments were performed in duplicate, and the standard error is indicated in the graphs.</p></div

    Sequences of <i>Xist</i> RNA Required for H3K27m3 Establishment

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    <div><p>(A) Schematic representation of the <i>Xist</i> cDNA (top) indicating repeats A to E, restriction sites, and the locations of deletions (coloured bars) relative to the location of sequences required for localisation (black and hatched boxes; <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020171#pbio-0020171-Wutz2" target="_blank">Wutz et al. 2002</a>).</p> <p>(B) Analysis of H3K27m3 on metaphase chromosome spreads from undifferentiated ES cells after 3 d of <i>Xist</i> induction (see text). The staining patterns (<i>n</i> > 100) were scored as chromosome-wide dense methylation (black), reduced methylation (grey), and a single band (open).</p> <p>(C) Pattern of H3K27m3 triggered by different <i>Xist</i> mutants on metaphase chromosomes after 3 d of induction. Enlarged view of Chromosome 11 (clone 36) or the X chromosome (T20 lines, J1 knock-in line).</p> <p>(D) Focal H3K27m3 staining in interphase nuclei (percentage given; <i>n</i> > 100) of undifferentiated ES cells expressing <i>Xist</i> constructs.</p></div

    Establishment of Chromosomal Memory during ES Cell Differentiation

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    <div><p>(A) Clone 36 ES cells were differentiated for 13 d in the presence of doxycycline (lane 1) or in the absence of inducer (lane 2) and the percentage of cells with H3K27m3 staining was determined (<i>n</i> > 800). At the beginning of differentiation, parallel cultures received either no <i>Xist</i> induction (lane 3) or a pulse of doxycycline for 24 h (lane 4), 36 h (lane 5), 48 h (lane 6), 60 h (lane7), or 72 h (lane 8) followed by withdrawal of inducer and concerted late induction from day 8 to day 13. A dashed red line indicates the 24-h interval of the transition when the chromosomal memory is recruited.</p> <p>(B and C) Establishment of irreversible transcriptional silencing during differentiation.</p> <p>(B) Ectopic inactivation of Chromosome 11 caused by <i>Xist</i> induction in differentiating clone 36 ES cells was assessed by Northern blot analysis of <i>PGKpuromycin (puro)</i> and <i>Gapd</i> as a loading control. Lanes were aligned electronically for better readability. ES cells were differentiated for 13 d in the presence of doxycycline (lane 1) or in the absence of inducer (lane 2). At the start of differentiation, parallel cultures received a <i>Xist</i> pulse for 24, 36, 48, or 60 h followed by withdrawal of inducer for the rest of the time (lanes 3 to 7) or followed by reinduction of <i>Xist</i> at day 8 of differentiation (lanes 8 to 11). All cells were analysed at day 13 of differentiation.</p> <p>(C) A quantitation of the <i>puro</i> expression relative to <i>Gapd</i> was derived from two independent Northern blots using tnimage software. A dashed red line indicates the 24-h interval in which the transition from reversible to irreversible silencing occurs.</p></div

    Early <i>Xist</i> Expression Imparts a Chromosomal Memory Independent of Silencing

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    <p>Transgenic <i>Xist</i> expression was induced from Chromosome 11 in clone 36 ES cells (black bars) or a silencing-deficient <i>Xist</i> RNA from the X in J1:XistΔSX-tetOP ES cells (open bars) at time points during differentiation (see text). The percentage of cells showing H3K27m3 staining is plotted (<i>n</i> > 250). Below, a scheme of <i>Xist</i> induction is given for all cultures, with arrows representing time of analysis.</p

    Epigenetic Imprints at the Initiation of X Inactivation

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    <div><p>(A–H) Indirect immunofluorescence and subsequent DNA FISH analysis on mitotic chromosomes prepared from undifferentiated clone 36 ES cells after 3 d of <i>Xist</i> induction. H3K27m3 (A), H4K20m1 (B), and Ezh2 (D) are enriched on the arms of Chromosome 11 upon ectopic <i>Xist</i> expression. H3K9m2 (C) is not enhanced upon <i>Xist</i> expression. H3K4m2 (E) is reduced on Chromosome 11 upon <i>Xist</i> induction (green box) and absent from pericentric heterochromatin and the Y chromosome (orange arrow). (F) Histone H4 multiple-lysine acetylation is partially reduced (green box, left panel). Hypoacetylation (red) is restricted to chromosomal regions which show high levels of H3-K27 trimethylation (green, right panel). H3K9m3 (G) and H3K27m1 (H) are enriched at constitutive heterochromatin of pericentric regions and the Y (orange arrows).</p> <p>(I–K) Indirect immunofluorescence (upper panels) and subsequent <i>Xist</i> RNA FISH (red, <i>Xist</i> RNA; blue, DAPI) analysis of H3K27m3 (I), H4K20m1 (J), and Ezh2 (K) in interphase nuclei of undifferentiated clone 36 ES cells expressing <i>Xist</i> for 3 d.</p></div

    Restriction of H3K27m3 Establishment and Transcriptional Silencing in Differentiation

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    <div><p>(A) Initiation of H3K27m3 during clone 36 ES cell differentiation. <i>Xist</i> expression was induced at the beginning (+) or at various time points (24 to 120 h) after the start of differentiation, or not induced (−). The percentages of interphase cells showing H3K27m3 (black bars; <i>n</i> > 700) and Ezh2 (grey bars; <i>n</i> > 200) staining were determined at day 12 of differentiation.</p> <p>(B) Initiation of transcriptional silencing during differentiation was assessed by Northern blot analysis of <i>PGKpuromycin (puro)</i> and <i>Gapd</i> as a loading control in parallel cultures as described for (A).</p> <p>(C) Western analysis of Ezh2 and Eed protein levels during differentiation of clone 36 ES cells after induction with retinoic acid. Histones H3 and H4 were used as a loading control.</p> <p>(D) Establishment of H3K27m3 during embryonic development. <i>Xist</i> expression was induced from the single X chromosome of male Xist-tetOP embryos (see text) for 3 d (E9.5–12.5 and E13.5–16.5). The percentage of cells with H3K27m3 staining in interphase (left) and clusters of <i>Xist</i> RNA (right, open bars) are given (<i>n</i> > 300). Grey areas indicate the proportion of H3K27m3-positive cells to <i>Xist</i>-positive cells.</p> <p>(E) <i>Xist</i> RNA FISH (top) and H3K27m3 (bottom) staining of histological sections prepared from neck connective tissue of embryos described in (C).</p></div

    Model for the Transition from Initiation to Maintenance of X Inactivation

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    <div><p>Phases of X inactivation are given relative to days of ES cell differentiation (bottom).</p> <p>(A) In undifferentiated ES cells, efficient chromosome-wide H3K27m3 depends on both <i>Xist</i> RNA localisation to the chromosome in <i>cis</i> and initiation of transcriptional silencing via the A repeat (black triangles).</p> <p>(B) Early in differentiation, silencing becomes dispensable for high-level H3K27m3 (dotted arrow).</p> <p>(C) The beginning of the critical window is specified in that <i>Xist</i> loses its potential to trigger H3K27m3 (dotted arrow) and transcriptional silencing. The critical window is negotiated by sustaining high levels of H3K27m3, which is thought to constitute—together with <i>Xist</i> RNA—the signal for the recruitment of the chromosomal memory (black oval). The memory is established on the Xi exactly when silencing becomes irreversible and <i>Xist</i> independent.</p> <p>(D) During the maintenance phase of X inactivation the chromosomal memory allows <i>Xist</i> RNA to establish H3K27m3 efficiently.</p></div

    Additional file 2: Figure S2. of Glucocorticoid receptor and nuclear factor kappa-b affect three-dimensional chromatin organization

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    Activated p65 induces de novo P300 depositions to latent genomic loci. (A) Pile-up heat map depicting the H3K4me1 and H3K4me3 signal around (±12 kb) all p65-bound promoters and DBSs. (B) Pile-up heat map depicting the p65 and P300 signal at all p65-bound enhancers upon vehicle, DMSO (−), and TNFα (+) treatment. (C) Example screenshot depicting TNFα-induced P300 recruitment at genomic regions (red box) and recruitment of p65 at genomic loci that are pre-marked by P300. (D) Motif occurrence at all p65-bound DBS presented as a function of TNFα-dependent P300 recruitment (x-axis) (top-panel). Level of shared binding of p65 and other TFs at all p65-bound DBS, presented as a function of TNFα-dependent P300 recruitment (bottom panel). (E) Level of H3K27ac, DNase I hypersensitivity, and H3K4me1 at all p65-bound DBSs (induced and constitutive P300 sites). (PDF 909 kb

    Additional file 8: Figure S6. of Glucocorticoid receptor and nuclear factor kappa-b affect three-dimensional chromatin organization

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    (A) Histogram depicting the genomic proximity (localization) of P300 binding sites identified by ChIP-seq in relation to those identified by using ChIA-PET self-ligation PETs. Identical comparison is performed for both DMSO-treated and TA + TNFα-treated data sets. (B) P300 ChIP-seq signal at P300 binding sites commonly identified by ChIP-seq and ChIA-PET and those binding sites that were uniquely detected in the ChIP-seq data set. (C) P300 ChIP-seq signal at P300 binding sites that were either involved (anchor) or not involved (non-anchor) in long-range interaction as identified by ChIA-PET analysis. (D) An example screenshot depicting the P300 interaction subdomains, P300 ChIP-seq binding sites in relation to topological domains as defined by replication timing data ( www.replicationdomain.org ). (E) Localization of all the interaction subdomains identified by ChIA-PET analysis (P300 and POLII) in relation to topological domains. (PDF 1041 kb
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