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First published online 11 March 2008
doi: 10.1242/jcs.026104

Journal of Cell Science 121, 1119-1127 (2008)
Published by The Company of Biologists 2008
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The facultative heterochromatin of the inactive X chromosome has a distinctive condensed ultrastructure

Alena Rego, Paul B. Sinclair, Wei Tao*, Igor Kireev and Andrew S. Belmont{ddagger}

Department of Cell and Developmental Biology, University of Illinois, 601 South Goodwin Avenue, Urbana, IL 61801, USA


Figure 1
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  		Fig. 1. Homogeneous Xi size and appearance in confluent female human fibroblasts (WI-38 cells). (A-C) DAPI staining, blue; histone H3-3mK27 immunostaining (mouse antibody) marking Xi, red. (A) 85% of cells show a recognizable, condensed Barr body in non-deconvolved single optical sections (95% if 3D data analyzed). (B,C) The remaining cells either show multiple DAPI-intense bodies of similar size to the Barr body (B) or no distinct DAPI bright body (C). (D) Histogram showing Xi area (µm2), based on H3-3mK27 immunostaining, for confluent versus log-phase cells. (E,F) H3-3mK27 immunostaining of Xi in log-phase cells frequently shows a fibrillar substructure protruding from the Xi core. Original image, top framed area; corresponding deconvolved optical section, lower inset. Arrows indicate the position of the Xi. Scale bar: 2 µm.

 

Figure 2
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  		Fig. 2. TEM appearance of Xi in WI-38 cells using conditions that preserve large-scale chromatin structure. (A) Deconvolved optical section from a live cell expressing histone H2B-GFP (top panel) shows a condensed Barr body (arrow) with a 200- to 400-nm-diameter substructure. Bottom panel: higher-magnification view of the Barr-body region, rotated 90° counterclockwise, from top panel view. (B-D) TEM images of 200-nm Epon sections through nuclei in samples prepared with three different fixation protocols all show a heterochromatic body with a similar 200- to 400-nm-wide chromatin substructure. (B) Direct fixation with 2% GA in phosphate buffer. (C) Cells in media pre-fixed by 8 minutes of UV irradiation in the presence of EtBr with a subsequent permeabilization step before fixation by 2% GA in buffer A*. (D) Fixation by 2% GA 1 minute after permeabilization in buffer A*. Higher-magnification views are shown at the bottom of each panel. Arrows indicate Xi heterochromatin body; Nu, nucleolus.

 

Figure 3
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  		Fig. 3. Unique heterochromatin body visualized by TEM corresponds to the Barr body. (A,B) Serial sections (200 nm) show one and only one heterochromatin body in a WI-38 nucleus with the size, shape and characteristic chromatin ultrastructure of the Barr body (comparable to LM images). (A) Two equatorial sections, 8 and 11 (S8, S11), of the whole nucleus. See supplementary material Fig. S1 for the complete set of serial sections. (B) Details of the heterochromatin body of the above nucleus, sections 5-12 (S5-S12). (C) H3-3mK27 (rabbit antibody) immunogold labeling decorating the Xi heterochromatin periphery. Arrowheads and arrows point to the Barr body; Nu, nucleolus.

 

Figure 4
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  		Fig. 4. Xi and chromocenters have a different ultrastructure. (A) Fluorescent images of a mouse embryonic fibroblast nucleus counterstained by DAPI (blue) show the Xi (arrows) identified by mouse anti H3-3mK27 antibody (pink) with weaker DAPI intensity than observed in chromocenters (arrowheads) but still noticeably above the background DAPI intensity level. (B,C) Electron micrographs of 200-nm sections of two nuclei demonstrate looser chromatin packing of the Xi (arrows) compared with that of chromocenters (some marked by arrowheads). (C) Nanogold staining of histone H3-3mK27 (rabbit antibody) shows the comparison of the labeled Xi facultative heterochromatin (arrow) and heterochromatin of the adjacent chromocenter (arrowhead). Nu, nucleolus.

 

Figure 5
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  		Fig. 5. 3D reconstruction of Xi heterochromatin. (A) Non-processed, 60-nm serial sections. Arrows indicate the Xi; arrowheads in zoomed insets point to specific large-scale Xi chromatin motifs of different dimensions: 30 nm (section 7), 215 nm (section 8) and 550 nm (section 10). Nuclear pores (NP) are present in the nuclear-envelope region adjacent to Xi heterochromatin and connect with interchromatin tunnels of nucleoplasm within the Barr-body volume. (B-D) 21 intensity-inverted and normalized (to be proportional to electron scattering `mass') serial sections were used for 3D reconstruction of the Barr body. See supplementary material Movie 1 for the complete serial section stack. (B) Maximum intensity projection of aligned sections (~1300-nm depth) gives the impression of a more-solid Barr body, similar to that visualized by LM. (C) Orthogonal views show large nucleoplasm channels penetrating throughout the Barr-body interior and interconnecting with NPs. Images for this reconstruction were median filtered and interpolated in z to provide uniform x-y-z voxel dimensions. Green line, y-axis; blue line, x-axis; red line, z-axis. (D) An example of the input image after additional median filtering (originally section 7) used for solid model rendering shown in D' and D'', and for stereo-pair projections shown in D''' (also see supplementary material Fig. S6). All provide further illustration of the porous internal structure of Xi heterochromatin. See rotating solid model of the Barr body in supplementary material Movie 2. Scale bar: 1 µm (three left-most panels in A; B-D).

 

Figure 6
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  		Fig. 6. Serial sections (200 nm) reveal the Xi–nuclear-envelope association even when the Xi appears interior in equatorial sections (confluent WI-38 cells). (A-C) Three sections of the same immunostained (H3-3mK27) nucleus with nanogold-labeled heterochromatin of the Xi (arrows). (A) The equatorial section 4 shows the Xi and adjacent nucleolus (Nu) in the nuclear interior. (B,C) Top-two grazing sections of the above nucleus (section 2, section 1) show the attachment of the Xi and adjacent nucleolus to the overlying nuclear periphery. (D) An example of the apparent intranuclear Xi (arrow) attached to the deep nuclear envelope invagination (Inv).

 

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