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. 2013 Dec 19;504(7480):465-469.
doi: 10.1038/nature12719. Epub 2013 Oct 27.

High-resolution Xist binding maps reveal two-step spreading during X-chromosome inactivation

Matthew D Simon #  1   2 , Stefan F Pinter #  1   3 , Rui Fang #  2 , Kavitha Sarma  1   3 , Michael Rutenberg-Schoenberg  2 , Sarah K Bowman  1 , Barry A Kesner  1   3 , Verena K Maier  1   3 , Robert E Kingston  1 , Jeannie T Lee  1   3
Affiliations

High-resolution Xist binding maps reveal two-step spreading during X-chromosome inactivation

Matthew D Simon et al. Nature. .

Abstract

The Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation (XCI), the process by which mammals compensate for unequal numbers of sex chromosomes. During XCI, Xist coats the future inactive X chromosome (Xi) and recruits Polycomb repressive complex 2 (PRC2) to the X-inactivation centre (Xic). How Xist spreads silencing on a 150-megabases scale is unclear. Here we generate high-resolution maps of Xist binding on the X chromosome across a developmental time course using CHART-seq. In female cells undergoing XCI de novo, Xist follows a two-step mechanism, initially targeting gene-rich islands before spreading to intervening gene-poor domains. Xist is depleted from genes that escape XCI but may concentrate near escapee boundaries. Xist binding is linearly proportional to PRC2 density and H3 lysine 27 trimethylation (H3K27me3), indicating co-migration of Xist and PRC2. Interestingly, when Xist is acutely stripped off from the Xi in post-XCI cells, Xist recovers quickly within both gene-rich and gene-poor domains on a timescale of hours instead of days, indicating a previously primed Xi chromatin state. We conclude that Xist spreading takes distinct stage-specific forms. During initial establishment, Xist follows a two-step mechanism, but during maintenance, Xist spreads rapidly to both gene-rich and gene-poor regions.

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Figures

Figure 1
Figure 1. CHART-seq reveals a two-step mechanism of Xist spreading during de novo XCI
a, Xist RNA is enriched on Xi. Normalized read densities displayed in mus, cas, and composite (comp) tracks. b, Coverage of enriched segments on chrX and autosomes. c, Xist coverage at indicated timepoints relative to gene silencing. Enriched segments shown beneath in gray. Brackets, y-axis scale. Xist peaks at d0 have less amplitude and density, but reflect d3 and d7 patterns, and are Xi-enriched (Extended Data Fig. 2f), consistent with initial Xist spreading to local regions, suggesting initial differentiation in a subfraction of cells. RNAseq of d7 and MEF shown below. Skewed allelic expression consistent with Xi-silencing (value −0.5 = 3-fold expression difference between Xi and Xa). d-e, Xist CHART signals (40 kb bins) from d7 correlate with d3 (d) and MEF (e)(see Extended Data Fig. 3). Regions showing >10-fold differences after normalization are colored purple and displayed on the X (lower panels). f, Depletion of Xist at a representative escapee. g, Xist preferentially targets genes in active chromatin (H3K4me3-marked on d7). Xist densities shown for gene bodies of active (n=532), inactive (n=475), and escapee genes (n=10). Medians are indicated. Individual data points overlaid on boxplot; error bars, 1.5-fold interquartile range. *p<0.05, **p<1×10−8, ***p<2.2×10−16 , Mann-Whitney U tests. h, Xist RNA distribution from d7 cells relative to chromatin features.
Figure 2
Figure 2. Co-spreading of Xist RNA and PRC2
a, Normalized read densities of Xist, EZH2, and H3K27me3 on chrX in d7 cells. b, Xist densities (200 kb bins) correlated with EZH2, H3K27me3 and H3K4me3 signals at different stages of XCI. Pearson's r displayed. EZH2/H3K27me3 R2 values: 0.3/0.37 for d0, 0.77/0.88 for d7, and 0.23/0.66 for MEFs, respectively. H3K4me3 R2 values: < 0.15 across all samples.
Figure 3
Figure 3. Xist knockoff uncovers a distinct spreading method during the maintenance phase
a, RNA FISH shows depletion and recovery of Xist RNA (green) in MEF cells after Xist knockoff. %nuclei with Xist clouds and sample size (n) shown. Scr, scrambled LNA. b, Chromosome-wide recovery of Xist after LNA-4978 knockoff on chrX and chr13. Regions of recovery comparing 8h over 3h LNA-4978 were determined using a maximum likelihood enrichment estimate. c,d, Xist knockoff and recovery across chrX. Colored regions show >10-fold median-normalized differences between samples. (c) Entire chrX (d) zoom of one region with more late domain recovery (right) than the other (left). e,f, Xist CHART signals (40 kb bins) from LNA-4978 8h correlated with MEF (e); LNA-4978 3h correlated against LNA-4978 8h (f). Regions showing >10-fold differences after normalization are colored as shown in panel d. g, Xist recovery in indicated samples, with 40-kb binned Xist densities normalized to median levels of early domains of each sample, to determine how early and late domains recover from knockoff compared to during de novo XCI. Normalized median values for each sample indicated above box. *, p<0.05; ***, p<10−8, Wilcoxon test, as in Extended Data Figs. 7c, 3c. h, Model: distinct methods of Xist spreading during establishment and maintenance.
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