Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci

Abstract

BackgroundA Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super-resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs).ResultsWe demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an ‘autosomal Barr body’ with less compacted chromatin and incomplete RNAP II exclusion.Conclusions3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform compaction of the Barr body at the nucleosome level. The localization of distinct Xist RNA foci at boundaries of the rudimentary ANC may be considered as snap-shots of a dynamic interaction with silenced genes. Enrichment of SAF-A within Xi territories and its close spatial association with Xist RNA suggests their cooperative function for structural organization of Xi.

The Xist RNA-PRC2 complex at 20-nm resolution reveals a low Xist stoichiometry and suggests a hit-and-run mechanism in mouse cells

“…1A) (6) and as broad discontinuous bands on mitotic chromosomes (16, 17), with broad overlapping staining of PRC2 and other chromatin epitopes (9,13,18,19). To push the resolving power of light microscopy, two recent studies used structured illumination microscopy (SIM) with a resolving power of ∼100 nm to examine the relationship of Xist RNA to nuclear markers (20,21). One report surprisingly concluded that Xist RNA does not colocalize with PRC2 and H3K27me3 and called into question the idea that Xist RNA and PRC2 are functionally tethered (20).…”

Mapping Molecular Function to Biological Nanostructure: Combining Structured Illumination Microscopy with Fluorescence Lifetime Imaging (SIM + FLIM)

“…SIM has previously been applied to study changes in nuclear structure, e.g., to study chromatin territories [13], but many phenomena occur on spatial scales <<100 nm and remain elusive. One example is the compaction of chromatin in the cell nucleus.…”

“…However, this lateral resolution is accompanied by an improved axial resolution (<300 nm) compared to wide-field microscopy [11] and SIM offers the significant advantages of working with many existing fluorophores and sample preparations (including live cells) and providing relatively high acquisition speeds at low intensities as is typical for wide-field microscopy. The experimental convenience and relatively low phototoxicity of SIM have led it to be widely applied for biological studies where superior resolution compared to confocal microscopy is required, e.g., [12,13].…”

Long noncoding RNAs as Organizers of Nuclear Architecture

“…It should be noted that these studies were based on large cell populations and the data are average patterns for millions of cells. A recent paper explored the ultrastructure of the Barr body at the single-cell level, using 3D super-resolution microscopy (Smeets et al, 2014). They found that there are less than 100 distinct Xist RNA foci in a Barr body, which are enriched on the boundaries of collapsed active nuclear compartments, suggesting a non-uniform "coating" of Xist on the inactive X chromosome (Smeets et al, 2014).…”