Multiple spatially distinct types of facultative heterochromatin on the human inactive X chromosome - PubMed (original) (raw)

Multiple spatially distinct types of facultative heterochromatin on the human inactive X chromosome

Brian P Chadwick et al. Proc Natl Acad Sci U S A. 2004.

Abstract

Heterochromatin is defined classically by condensation throughout the cell cycle, replication in late S phase and gene inactivity. Facultative heterochromatin is of particular interest, because its formation is developmentally regulated as a result of cellular differentiation. The most extensive example of facultative heterochromatin is the mammalian inactive X chromosome (Xi). A variety of histone variants and covalent histone modifications have been implicated in defining the organization of the Xi heterochromatic state, and the features of Xi heterochromatin have been widely interpreted as reflecting a redundant system of gene silencing. However, here we demonstrate that the human Xi is packaged into at least two nonoverlapping heterochromatin types, each characterized by specific Xi features: one defined by the presence of Xi-specific transcript RNA, the histone variant macroH2A, and histone H3 trimethylated at lysine 27 and the other defined by H3 trimethylated at lysine 9, heterochromatin protein 1, and histone H4 trimethylated at lysine 20. Furthermore, regions of the Xi packaged in different heterochromatin types are characterized by different patterns of replication in late S phase. The arrangement of facultative heterochromatin into spatially and temporally distinct domains has implications for both the establishment and maintenance of the Xi and adds a previously unsuspected degree of epigenetic complexity.

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Figures

Fig. 1.

Fig. 1.

Spatial relationship of two major Xi heterochromatin types at metaphase. Images represent typical distributions obtained from three independent female cell lines. (a) Partial metaphase spread of RPE1 cells showing the spatial distribution of H3TrimK9 (green, FITC) and H3TrimK27 (red, rhodamine) and four additional higher-magnification images of the Xi showing the merged H3TrimK9 and H3TrimK27 distributions. The white arrow indicates the major H3TrimK27 band centered at Xq23. (b) Distributions of H3TrimK9 and H3TrimK27 in HME1 cells. The location of the Xi in the partial metaphase spreads is indicated by white arrowheads. The white arrow indicates the major H3TrimK27 band centered at Xp11. All images were obtained by indirect immunofluorescence.

Fig. 2.

Fig. 2.

Correlation of H3TrimK27 (a Upper and b Upper) and H3TrimK9 (a Lower and b Lower) heterochromatin with the Xi replication pattern. (a) Metaphase chromosomes prepared from RPE1 cells that were incubated with BrdUrd for 4 h before metaphase arrest (corresponding approximately to the last 2 h of S phase). (b) Metaphase chromosomes prepared from HME1 cells that were incubated with BrdUrd for 3 h before metaphase arrest (corresponding approximately to the last 2 h of S phase). In each section, Top shows a partial metaphase spread, whereas Middle and Bottom show the Xi from independent spreads at higher magnification.

Fig. 3.

Fig. 3.

Characterization of Xi chromatin territories at interphase. All images are of the RPE1 cells and represent typical observations made in at least four independent female cell lines. The white box in each interphase nucleus (top row) represents the Barr body region examined at higher magnification in the other images below. The feature examined by indirect immunofluorescence is labeled above each image. Overlapping red and green signals appear yellow.

Fig. 4.

Fig. 4.

Schematic model showing how heterochromatin of the Xi could transition between metaphase and interphase to be organized into the two nonoverlapping heterochromatin territories and to explain how XIST RNA could rapidly spread in cis outward from the X inactivation center (XIC) along only part of the Xi. See main text for details.

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