The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome - PubMed (original) (raw)

. 2013 Aug 16;341(6147):1237973.

doi: 10.1126/science.1237973. Epub 2013 Jul 4.

Amy Pandya-Jones, Patrick McDonel, Alexander Shishkin, Klara Sirokman, Christine Surka, Sabah Kadri, Jeffrey Xing, Alon Goren, Eric S Lander, Kathrin Plath, Mitchell Guttman

Affiliations

The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome

Jesse M Engreitz et al. Science. 2013.

Abstract

Many large noncoding RNAs (lncRNAs) regulate chromatin, but the mechanisms by which they localize to genomic targets remain unexplored. We investigated the localization mechanisms of the Xist lncRNA during X-chromosome inactivation (XCI), a paradigm of lncRNA-mediated chromatin regulation. During the maintenance of XCI, Xist binds broadly across the X chromosome. During initiation of XCI, Xist initially transfers to distal regions across the X chromosome that are not defined by specific sequences. Instead, Xist identifies these regions by exploiting the three-dimensional conformation of the X chromosome. Xist requires its silencing domain to spread across actively transcribed regions and thereby access the entire chromosome. These findings suggest a model in which Xist coats the X chromosome by searching in three dimensions, modifying chromosome structure, and spreading to newly accessible locations.

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Figures

Fig. 1

Fig. 1. RNA Antisense Purification accurately purifies lncRNA-chromatin interactions

(A) Schematic diagram of RAP. Biotinylated probes (blue) are hybridized to the target RNA (red) crosslinked to proteins and DNA. Gray inset: 120-mer antisense capture probes are designed to tile across the entire target RNA. (B) RT-qPCR of the RNA captured using antisense probes to Xist (Xist RAP, black) or sense probes to Xist (Control RAP, gray) in MLFs. Enrichments represent mean +/− SEM (N = 3 replicate experiments) and are normalized to the mean of the sense control experiments. We note that Tsix, an antisense transcript overlapping Xist, is not expressed in MLFs. (C) RNA sequencing reads originating from the Xist transcript (dark gray) and all other cellular RNAs (light gray) for polyA-selected RNA (left) and RNA captured using antisense probes for the Xist RNA (right) (35). (D) DNA sequencing reads aligning to each chromosome after purification with antisense probes (Xist RAP, black) and sense probes (Control RAP, light gray), and input genomic DNA (dark gray) in MLFs.

Fig. 2

Fig. 2. High-resolution mapping of Xist localization on the inactive X-chromosome in differentiated female cells

(A) Xist enrichment over input (dark gray), H3K27me3 enrichment over input (red line), and gene density (blue line) (35) across the entire X-chromosome in female MLFs. (B) Zoom in on a highly-enriched Xist region (light-red box, chrX:95,000,000-99,000,000) shows Xist (black) and H3K27me3 (red) enrichments. (C) Cumulative distribution plot comparing Xist enrichment across all 10-Kb windows on the X-chromosome (black) and on autosomes (gray). Dashed lines demarcate the least- and most-highly enriched regions on the X-chromosome. (D) Correlation between Xist enrichment and the ratio of expression from the inactive X (Xi) and active X (Xa) for known escape genes (39). (E) Xist enrichment (chrX:17,031,000-19,901,000) across two of the least-enriched regions (blue boxes). (F) Xist enrichment around the Xist genomic locus including a least-enriched region (blue box). Xist enrichment at the Xist locus (gray arrow) extends above the _y_-axis maximum.

Fig. 3

Fig. 3. High-resolution view of Xist spreading during initiation of XCI

(A) We replaced the 1,027 bases upstream of Xist with a tetracycline-responsive (TetO) promoter in male mouse ES cells (35). (B) Representative FISH images for the Xist RNA (red) and DAPI staining of the nucleus (blue) after Xist induction with doxycycline. Xist FISH signals were classified into categories – including no signal, spot, small cloud, and large cloud – at each time point (35). (C) Xist RAP enrichments over input across the X-chromosome for the same time points as in (B). The _y_-axis scale (0-50) is the same for all time points. Xist enrichment at the Xist locus extends above _y_-axis maximum. (D) Two proposed models for Xist spreading (gray arrows) from its transcription locus (tick mark) across the chromosome. (E) A zoom in on the _y_-axis of Xist enrichment after one hour of Xist induction. (F) Xist enrichments from RAP in wild-type female ES cells after 6 hours of differentiation. Early sites are defined by significant deviations (P < 0.05) above the local (10-Mb) average (35).

Fig. 4

Fig. 4. Early Xist localization correlates with the 3-D proximity contacts of the Xist transcription locus

(A) Two models for how Xist spreads to early initiation sites (marked by gray arrows in all panels). Gray boxes on the left model represent hypothetical high-affinity interaction sites. (B) Correlation between Xist RNA localization (red) after one hour of Xist induction and Hi-C contact frequencies (blue) between distal sites and the Xist genomic locus (dashed line). Black arrows point to selected regions with lower Xist RNA enrichments than would be expected from the Hi-C contact frequencies. (C) Correlation between Xist transgene RNA localization (red) after three hours of induction of the Xist cDNA transgene in the Hprt locus and Hi-C contact frequencies (blue) between distal sites and the Hprt integration locus (dashed line). Correlation calculations exclude the shaded gray regions (10-Mb on each side). Contact frequencies represent normalized Hi-C interaction counts in male ES cells between each window and the window containing the Xist or Hprt locus (35).

Fig. 5

Fig. 5. The A-repeat deletion of Xist cannot spread over the active regions of the X-chromosome

(A) Xist enrichment over representative genes at chrX:45,252,000-45,408,000. Dashed line denotes the average enrichment for the entire region. Solid line represents the smoothed enrichment in sliding windows across the region. (B) Xist enrichment averaged over all active genes (red line, N = 608), inactive genes (blue line, N = 595), and randomly permuted regions across the X-chromosome (black line) (35). Shaded regions represent 95% confidence intervals for the average enrichment. (C) Localization of wild-type Xist (wt Xist, top) and Xist lacking the A-repeat (ΔA Xist, bottom) by RNA FISH (left) and RAP (right) across the X-chromosome. (D) Comparison of wt and ΔA Xist enrichment across a representative region at chrX:44,600,000-47,100,000. Gray boxes mark regions that are depleted for Xist localization in ΔA versus wt Xist. All panels present data from three hours after Xist induction in undifferentiated male ES cells, where Xist is expressed from its endogenous locus (A-B) or from the Hprt locus (C-D). Genes were classified as active or inactive using RNA-Seq data from undifferentiated male ES cells (35). (D) Model: in the presence of the A-repeat (left), Xist localizes across the entire X-chromosome (red cloud) but is initially excluded from active genes (red arrows). In the absence of the A-repeat (right), Xist accumulates on the periphery of active gene-dense regions but cannot spread to actively transcribed genes (red arrows) or inactive genes (blue arrows) that lie within these regions.

Fig. 6

Fig. 6. A model for how Xist exploits and alters three-dimensional genome architecture to spread across the X-chromosome

Upon induction of expression in ES cells (left), Xist (red) spreads to spatially proximal sites on the periphery of active gene-dense regions (black). Gene-poor regions (blue, purple) contact the Xist transcription locus infrequently, leading to slower spreading to these regions. When encountering a new region (left inset), Xist interacts with chromatin through a degenerate localization mechanism, possibly through the matrix protein hnRNP U (30, 34), and uses its A-repeat domain to spread over active genes. Xist may then recruit PRC2 (14) and other proteins (56) to modify and compact chromatin, thereby repositioning nearby chromosomal regions into the Xist RNA compartment (red cloud, right inset). These structural changes may propagate Xist spreading (right) by pulling new regions (green, yellow) of the chromosome into closer proximity to the Xist genomic locus and the growing Xist compartment.

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