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

By J. Engreitz, Amy Pandya-Jones, P. McDonel, Alexander A. Shishkin, Klára Sirokmán, Christine Surka, Sabah Kadri, Jeff Xing, Amir Goren, Eric S. Lander and 2 more. Science, 2013

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Introduction Mammalian genomes encode thousands of large noncoding RNAs (lncRNAs), many of which regulate gene expression, interact with chromatin regulatory complexes, and are thought to play a role in localizing these complexes to target loci across the genome. A paradigm for this class of lncRNAs is Xist, which orchestrates mammalian X-chromosome inactivation (XCI) by coating and silencing one X chromosome in females. Despite the central role of RNA-chromatin interactions in this process, the mechanisms by which Xist localizes to DNA and spreads across the X chromosome remain unknown. Upon activation, Xist spreads from its genomic locus to sites in close three-dimensional proximity. Xist modifies chromatin architecture at these sites, thereby repositioning these regions into the Xist compartment (red cloud) and pulling new regions (green, yellow) of the chromosome into closer proximity. These structural changes allow Xist to access new sites and spread across the entire chromosome. Methods We developed a biochemical method called RNA antisense purification (RAP) to map the localization of a lncRNA across the genome. RAP uses long biotinylated antisense RNA probes to hybridize to and capture a target lncRNA and associated genomic DNA, enabling high-resolution mapping of lncRNA binding sites through high-throughput DNA sequencing. We applied RAP to study the localization of Xist during the initiation and maintenance of XCI. Results We show that during the maintenance of XCI, Xist binds broadly across the X chromosome, lacking defined localization sites. Xist preferentially localizes to broad gene-dense regions and excludes genes that escape XCI. At the initiation of XCI in mouse embryonic stem cells, Xist initially transfers to distal regions across the X chromosome that are not defined by specific sequences. Instead, Xist RNA identifies these regions using a proximity-guided search mechanism, exploiting the three-dimensional conformation of the X chromosome to spread to distal regions in close spatial proximity to the Xist genomic locus. Initially, Xist is excluded from actively transcribed genes and accumulates on the periphery of regions containing many active genes. Xist requires its silencing domain to spread across these regions and access the entire chromosome. Discussion Our data suggest a model for how Xist can integrate its two functions—localization to DNA and silencing of gene expression—to coat the entire X chromosome. In this model, Xist exploits three-dimensional conformation to identify and localize to initial target sites and leads to repositioning of these regions into the growing Xist compartment. These structural changes effectively pull new regions of the chromosome closer to the Xist genomic locus, allowing Xist RNA to spread to these newly accessible sites by proximity transfer. This localization strategy capitalizes on the abilities of a lncRNA to act while tethered to its transcription locus and to interact with chromatin regulatory proteins to modify chromatin structure. Beyond Xist, other lncRNAs may use a similar strategy to locate regulatory targets in three-dimensional proximity and to alter chromatin structure to establish local nuclear compartments containing co-regulated targets. Understanding Xist-ance Large noncoding RNAs (lncRNAs) are increasingly appreciated to play important roles in the cell. A number of lncRNAs act to target chromatin regulatory complexes to their sites of action. Engreitz et al. (p. 10.1126/science.1237973, published online 4 July; see the Perspective by Dimond and Fraser) found that the mouse Xist lncRNA, which initiates X-chromosome inactivation, was transferred from its site of transcription to distant sites on the X chromosome purely through their close three-dimensional proximity to the Xist gene. Xist initially localized to the periphery of active genes on the X chromosome but gradually spread across them using its A-repeat domain, until the Xist RNA bound broadly across the inactive X chromosome in differentiated female cells. A large noncoding RNA uses folds within the chromosome to drive the spread of a chromatin repressive complex. [Also see Perspective by Dimond and Fraser] 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.

Subfields: Cancer Research, Endocrinology, Plant Science

View Paper: https://www.semanticscholar.org/paper/9af550f820fdefa866a68adce939c9721c3bba02

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Citations in the corpus

Systematic allelic analysis defines the interplay of key pathways in X chromosome inactivation (2018, background): “The basis for this effect is not clear but may relate to previous ﬁ ndings, indicating that the spread of Xist RNA from early nucleation sites is abrogated when the A-repeat is deleted 33 .”
Higher-order inter-chromosomal hubs shape 3D genome organization in the nucleus (2018, neutral): “…Models: Cell Lines F1 2-1 hybrid wild-type mouse ES cell line (129 x cast) Kathrin Plath (Engreitz et al., 2013) pSM33 mES cell line Kathrin Plath (Engreitz et al., 2013) GM12878 Human Lymphoblasts Coriell Cell Repositories GM12878, RRID:CVCL_7526 Human HEK293T ATCC Cat# CRL-3216,…”
Xist nucleates local protein gradients to propagate silencing across the X chromosome (2021, methodology): “In agreement with these observations, RNA antisense purification (RAP) of Xist followed by DNA sequencing of associated chromatin (Engreitz et al., 2013) showed that Xist localizes to gene-rich, open chromatin regions of the A-compartment (Figure 2I).”
Genetic Screens in Human Cells Using the CRISPR-Cas9 System (2013, methodology): “The remainder of the library preparation protocol was performed as previously described except that a sgRNA-specific reverse primer was used for library amplification (34).”