The repeat E of Xist RNA is required for the loading of ASH2L to the Xi but not for the deposition of the repressive epigenetic modifications.

<p>(A) Immuno-FISH for Xist RNA (green) and H3K27me3 (red) at day 0 and 8 upon differentiation. Nuclei were counterstained by DAPI. Arrowhead indicates typical dispersed Xist RNA cloud. Scale bar is 10 μm. (B) Frequency of Xist RNA cloud and H3K27me3 positive cells during differentiation from three independent experiments. More than 300 nuclei in each ES cell line at each time point were counted. (C) Immunostaining for ASH2L (green) and H3K27me3 (red) at day 0 and 8 upon differentiation. Nuclei were counterstained by DAPI. (D) Co-localization frequency of H3K27me3 and ASH2L from two independent experiments. More than 200 nuclei in each ES cell lines at each differentiation time were counted.</p

Xist induction occurs normally in the Xist repeat E mutant female ES cells upon differentiation.

<p>(A) A map of <i>Xist</i>/<i>Tsix</i> locus to show the targeted deletion of the <i>Xist</i> repeat E (XistΔrepE) on 129 allele. The targeting vector is shown above the map. SA, Splice acceptor; IRES, internal ribosomal entry site; Hyg, hygromycin resistance gene; tpA, tandem polyadenylation signals. The positions of primer pairs used for allele-specific <i>Xist</i> expression analysis (B and C) are shown as asterisks. (B) Allele-specific RT-PCR and genomic PCR analysis for <i>Xist</i> using the primer pair (X7E) which amplifies the 3′-end of the Xist repeat E in the XistΔrepE mutant cell lines. (C) Allele-specific RT-qPCR of the <i>Xist</i> expression across exon 1–3 (X1-3) in the XistΔrepE mutant. Gapdh was used as an internal control for normalization. Each value was also normalized to that of the undifferentiated wildtype 16.7 cells which is set to 1. The mean ± SD from three independent experiments is shown. <i>P</i>-values were calculated by an unpaired t-test (*p<0.05, **p<0.01). (D) A map of <i>Xist</i>/<i>Tsix</i> locus to show the targeted truncation of the <i>Tsix</i> in XistΔrepE cells on 129 allele. The positions of primer pairs used for allele-specific <i>Tsix</i> and <i>Xist</i> expression analysis (E and F) are shown as asterisks. (E) Allele-specific RT-qPCR of the <i>Tsix</i> expression at exon 4 (T) in the XistΔrepE /TST mutants. Gapdh was used as an internal control for normalization. The expression values were normalized to Gapdh and those of the parental cells. (F) Allele-specific RT-qPCR of the <i>Xist</i> expression across exon 1–3 (X1-3) in the XistΔrepE mutant. The expression values were normalized to Gapdh and those of the undifferentiated wildtype 16.7 cells (D). The mean ± SD from three independent experiments is shown. (G) Half-life assay for Xist RNA in the TST and XistΔrepE/TST mutant cells. The mean ± SD from two independent experiments is shown.</p

Xist RNA repeat E is essential for ASH2L recruitment to the inactive X and regulates histone modifications and escape gene expression

<div><p>Long non-coding RNA Xist plays a crucial role in establishing and maintaining X-chromosome inactivation (XCI) which is a paradigm of long non-coding RNA-mediated gene regulation. <i>Xist</i> has Xist-specific repeat elements A-F which are conserved among eutherian mammals, underscoring their functional importance. Here we report that Xist RNA repeat E, a conserved <i>Xist</i> repeat element in the <i>Xist</i> exon 7, interacts with ASH2L and contributes to maintenance of escape gene expression level on the inactive X-chromosome (Xi) during XCI. The Xist repeat E-deletion mutant female ES cells show the depletion of ASH2L from the Xi upon differentiation. Furthermore, a subset of escape genes exhibits unexpectedly higher expression in the repeat E mutant cells than the cells expressing wildtype <i>Xist</i> during X-inactivation, whereas the silencing of X-linked non-escape genes is not affected. We discuss the implications of these results to understand the role of ASH2L and Xist repeat E for histone modifications and escape gene regulation during random X-chromosome inactivation.</p></div

H3K27me3 level at X-linked gene promoter is reduced on the Xi in XistΔE mutant.

<p>(A) Allele-specific ChIP experiments of X-linked genes with antibodies against H3K4me3, H3K27me3, and control mouse IgG. The mean ± SD from two independent experiments is shown. Statistical significance was calucuated by an unpaired t-test (*p<0.05, **p<0.01). (B) ChIP experiments of autosomal genes with antibodies against H3K4me3, H3K27me3, and control mouse IgG. The mean ± SD from two independent experiments is shown. <i>P</i>-values were derived from an unpaired t-test (*p<0.05, **p<0.01).</p

ASH2L recruitment to the Xi depends on the repeat E of Xist RNA.

<p>(A) Map of primer pairs across <i>Xist</i> for the RIP analysis. White boxes indicate <i>Xist</i> exons. The <i>Xist</i> repeats A-F are shown by black boxes. The positions of the primer pairs across <i>Xist</i> are shown as arrowheads. (B) Formaldehyde-crosslinked Xist RIP using anti-ASH2L antibody. A relative amount of Xist RNA immunoprecipitated by anti-ASH2L antibody to input was quantified by RT-qPCR. The means ± standard deviation (SD) from three independent experiments are shown. (C) UV-crosslinked Xist RIP using anti-ASH2L antibody. The mean ± SD from three independent experiments is shown. (D) Schematics of tet-inducible full-length and mutant Xist cDNAs used in this study. (E) ImmunoFISH and immunostaining of the tet-inducible <i>Xist</i> T20 ES cells at day 2 in the presence of Dox. Nuclei were counterstained by DAPI. Scale bar is 5 μm. (F) Summary of the tet-inducible mutant <i>Xist</i> cDNA experiment. Colocalization percentage was calculated by immunoFISH and immunostaining experiments in Fig 1E. More than 100 nuclei for each transgenic ES cell line in more than two independent experiments were counted.</p

Binding of NF-κB to remodeled and slid nucleosomes.

<p>(<b>A</b>) Nucleosomes, RSC-remodeled nucleosomes (remosomes), slid nucleosomes and naked 601_D₀ DNA were incubated with the indicated amount of NF-κB and separated on a 5% native PAGE. The positions of the different particles are shown on the left part of the gel. (<b>B</b>) UV laser footprinting of the indicated distinct NF-κB bound particles. The experiment was carried out as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003830#pgen-1003830-g002" target="_blank">Figure 2</a>. The NF-κB binding site is shown as vertical black line and the black arrow indicates the nucleosomal dyad; M, 10 bp DNA molecular marker (<b>C</b>) top, “zoom” of the NF-κB binding region from the footprints shown in (B); bottom, scan of the footprints; red, in presence NF-κB; green in absence of NF-κB.</p

Dilution driven H2A–H2B dimer eviction allows binding of NF-κB to Nucleosome Core Particle.

<p>(<b>A</b>) 152 bp DNA fragment derived from <i>X. borealis</i> somatic 5 S RNA gene containing single NF-κB site near the dyad NF1 (53–56) was amplified by PCR and uniquely 3′ end labeled with α-³²P by Klenow. Nucleosomes were reconstituted on this labeled fragment as described previously. The DNA and nucleosomes at a concentration 40 nM were incubated with increasing amounts of NF-κB as indicated to allow the formation of stable complexes which were subsequently irradiated by a single high intensity UV laser pulse (<i>E</i>_pulse∼0.1 J/cm²). The formation of complexes was checked by EMSA (upper panel, DNA lane 1–3, nucleosomes lane 4–9), the positions of free DNA and nucleosomes are indicated, “cplx.” represents NF-κB – DNA/nuc complexes. The samples were split into two parts, DNA was purified and treated with Fpg glycosylase to cleave 8-oxoG (represented by ▸,lower panel, DNA lane 1–3 and nucleosome lane 4–9) and with T4 endonuclease V to cleave CPDs (represented by ◊, DNA lane 1′–3′ and nucleosome lane 4′–9′). The cleaved DNA fragments were visualized by 8% sequencing gel. (<b>B</b>) The same 152 bp 5S fragment was 5′ end labeled with γ-³²P by T4 polynucleotide kinase and used for nucleosome reconstitution. DNA and nucleosomes, at 10 nM final concentration, were incubated with increasing amounts of NF-κB as indicated to allow the formation of complexes. The assembly of the complexes was checked by EMSA (upper panel, DNA lane 1–5, nucleosomes lane 1′–5′). The samples were irradiated with a single high intensity UV laser pulse (<i>E</i>_pulse∼0.1 J/cm²), treated with a mix of Fpg glycosylase and T4 endonuclease V to cleave both the 8-oxoG (▸) and CPDs (◊). Finally, the cleaved products were visualized by 8% sequencing gel (DNA, lane 1–5; nucleosomes lane 1′–5′). The NF-κB binding sites (vertical bold lines) and the NF-κB recognition sequences are shown. The arrows designated the nucleosomal dyad.</p

NF-κB displaces H1 from the chromatosome and binds to its recognition sequence.

<p>(<b>A, upper panel</b>), schematics of the substrates used in each experiment. (<b>A, lower panel</b>), EMSA of the binding of NF-κB to depicted substrates. The bottom strand of the 255 bp 601_D₈ DNA (Supplementary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003830#pgen.1003830.s001" target="_blank">figure S1</a>) was uniquely 5′-end labeled by ^32-P and used to reconstitute centrally positioned nucleosomes. Chromatosomes were assembled by using the NAP-1/H1 complex to properly deposit H1 on the nucleosome in H1/nuc ratio of ∼1.5. The first two panels show the NF-κB-DNA (lanes 1–4) and NF-κB-nucleosome (lanes 5–8) complexes formed upon incubation with increasing amount of NF-κB. The last panel illustrates both the interaction of chromatosomes with the indicated increasing amount of NF-κB (lanes 1′–5′) and the deposition of H1 on the already assembled (at increasing NF-κB concentration) NF-κB nucleosome complexes (lanes 6′–9′). (<b>B</b>) UV laser (upper panel) and •OH (lower panel) footprinting of the NF-κB binding region of NF-κB-DNA and distinct NF-κB-nucleosome complexes.</p

Characterization of the reconstituted nucleosomes.

<p>(<b>A</b>) Schematics of the reconstituted nucleosomes. The canonical NF-κB site was inserted in the 255 bp 601 DNA fragment either at the dyad of the nucleosome (601_D₀ DNA) or at the nucleosomal end (601_D₇ DNA) or in the linker DNA (601_D₈ DNA); bold lines, free DNA arms; dashed line, core particle region. The vertical black line represents the dyad. The NF-κB binding sites (BS) are depicted by the red line. The length of each region is shown on top of the constructs. The very bottom schematics shows the location of the NF-κB binding site inserted in the 5S rDNA fragment of <i>Xenopus borealis</i> used for nucleosome reconstitution. (<b>B</b>) Electrophoretic analysis of the indicated purified recombinant histones and histone octamer. (<b>C</b>) Electrophoretic analysis of purified recombinant NF-κB (p50); M, molecular marker; p50, the p50 subunit of NF-κB. (<b>D</b>) Nucleosome reconstitution check by 5% native PAGE. (<b>E</b>) •OH radical and (<b>F</b>) DNase I footprinting of free 601 DNA and the indicated reconstituted nucleosomes.</p

FACT facilitates both RSC-induced remodeling and mobilization of nucleosomes.

<p>(<b>A)</b> DNase I footprinting. End-positioned nucleosomes, reconstituted on ³²P 5’-labeled 241 bp 601 DNA fragment, were incubated with 0.2 units of RSC in the absence (lane 3) or in the presence of 1.6 pmol of FACT (lane 4) for 50 min at 30°C; lane 5, same as lane 3, but with 1 unit of RSC; After arresting the remodeling reaction, the samples were digested with 0.1 units of DNase I for 2 min, the cleaved DNA was isolated and run on 8% PAGE under denaturing conditions; lanes 1 and 2, controls showing the DNase I cleavage pattern of nucleosomes (lane 1) alone or incubated with 1.6 pmol FACT under the conditions described above. (<b>B</b>) The presence of FACT increases the efficiency of RSC-induced nucleosome mobilization. Centrally positioned nucleosomes were incubated with 0.2 units of RSC in the presence of increasing amount of FACT, the reaction was arrested and the samples were run on native PAGE. The position of the non-mobilized and the slid end-positioned nucleosomes were indicated; lane 1 control nucleosomes; lane 2, nucleosomes incubated with RSC alone (in the absence of FACT). (<b>C</b>) Quantification of the data presented in (B).</p