title: 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: BACKGROUND
  A 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).
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  3D-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.
authors: Biocenter, Department of Biology II, Ludwig Maximilians University (LMU), Martinsried, Germany ; Department of Biochemistry, University of Oxford, Oxford, UK.
authors: Daniel
authors: D
authors: Smeets
authors: Biocenter, Department of Biology II, Ludwig Maximilians University (LMU), Martinsried, Germany.
authors: Yolanda
authors: Y
authors: Markaki
authors: Institute of Statistics, Ludwig Maximilians University (LMU), Munich, Germany.
authors: Volker J
authors: VJ
authors: Schmid
authors: Biocenter, Department of Biology II, Ludwig Maximilians University (LMU), Martinsried, Germany ; Department of Biochemistry, University of Oxford, Oxford, UK.
authors: Felix
authors: F
authors: Kraus
authors: Department of Biochemistry, University of Oxford, Oxford, UK.
authors: Anna
authors: A
authors: Tattermusch
authors: Department of Biochemistry, University of Oxford, Oxford, UK.
authors: Andrea
authors: A
authors: Cerase
authors: Biocenter, Department of Biology II, Ludwig Maximilians University (LMU), Martinsried, Germany.
authors: Michael
authors: M
authors: Sterr
authors: Biocenter, Department of Biology II, Ludwig Maximilians University (LMU), Martinsried, Germany.
authors: Susanne
authors: S
authors: Fiedler
authors: Department of Biochemistry, University of Oxford, Oxford, UK.
authors: Justin
authors: J
authors: Demmerle
authors: Biocenter, Department of Biology II, Ludwig Maximilians University (LMU), Martinsried, Germany.
authors: Jens
authors: J
authors: Popken
authors: Biocenter, Department of Biology II, Ludwig Maximilians University (LMU), Martinsried, Germany.
authors: Heinrich
authors: H
authors: Leonhardt
authors: Department of Biochemistry, University of Oxford, Oxford, UK.
authors: Neil
authors: N
authors: Brockdorff
authors: Biocenter, Department of Biology II, Ludwig Maximilians University (LMU), Martinsried, Germany.
authors: Thomas
authors: T
authors: Cremer
authors: Biocenter, Department of Biology II, Ludwig Maximilians University (LMU), Martinsried, Germany ; Department of Biochemistry, University of Oxford, Oxford, UK.
authors: Lothar
authors: L
authors: Schermelleh
authors: Biocenter, Department of Biology II, Ludwig Maximilians University (LMU), Martinsried, Germany.
authors: Marion
authors: M
authors: Cremer
keywords: Barr body; CT; Chromatin domain; Chromosome territory; Inactive X chromosome; Interchromatin compartment; SAF-A; Super-resolution microscopy; X chromosome inactivation; Xist RNA