Research Article 1119 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‡ Department of Cell and Developmental Biology, University of Illinois, 601 South Goodwin Avenue, Urbana, IL 61801, USA *Present Address: Department of Cell Biology, College of Life Sciences, Peking University, Beijing 100871, Peopleʼs Republic of China ‡Author for correspondence (e-mail: [email protected])

Accepted 8 January 2008 J. Cell Sci. 121, 1119-1127 Published by The Company of Biologists 2008 doi:10.1242/jcs.026104

Summary The mammalian inactive X chromosome (Xi) is a model for condensed regions of the Xi. Serial-section analysis also reveals facultative heterochromatin. Increased DNA compaction for the extensive contacts of the Xi with the nuclear envelope and/or Xi, and for facultative heterochromatin in general, has long nucleolus, with nuclear envelope association being observed been assumed based on recognition of a distinct Barr body in all cells. Implications of our results for models of Xi using nucleic-acid staining. This conclusion has been challenged gene silencing and chromosome territory organization are by a report revealing equal volumes occupied by the inactive discussed. and active X chromosomes. Here, we use light and electron microscopy to demonstrate in mouse and human fibroblasts a unique Xi ultrastructure, distinct from euchromatin and Supplementary material available online at constitutive heterochromatin, containing tightly packed, http://jcs.biologists.org/cgi/content/full/121/7/1119/DC1 heterochromatic fibers/domains with diameters in some cases approaching that of prophase chromatids. Significant space Key words: Facultative heterochromatin, Large-scale chromatin, between these packed structures is observed even within Electron microscopy, Chromocenters, The X chromosome

Introduction usually positioned at the nuclear or nucleolar periphery (Barr and

Journal of Cell Science Interphase chromatin is typically thought to be decondensed at Carr, 1962; Belmont et al., 1986; Puck and Johnson, 1996; Zhang locations in which genes are transcribed and condensed where genes et al., 2007). Inactivation of one of the X chromosomes in female are silent (Wegel and Shaw, 2005). However, this generalization is cells is the mechanism of mammalian dosage compensation of X- contradicted by observations that inactive genes may reside in linked genes (Lyon, 1961). domains of open chromatin, whereas active genes in regions of low A long-standing assumption has been that sequential epigenetic gene density can be embedded within compact chromatin fibers modifications occurring during X inactivation directly lead to Xi (Gilbert and Bickmore, 2006; Gilbert et al., 2004). A diversity of DNA compaction and increased condensation per se might theoretical models for the nuclear organization of active and silent contribute to gene silencing (Arney and Fisher, 2004; Chow and chromatin (reviewed in Cremer et al., 2006; Spector, 2003) stem Brown, 2003). This assumption has been challenged by comparison from our limited understanding of higher-order chromatin structure of the Xi and the active X chromosome (Xa) conformation using beyond the 30-nm fiber, referred to as large-scale chromatin 3D fluorescence in situ hybridization with X-specific whole- structure. This limited understanding results from technical chromosome probes. In human amniotic cells, Xi and Xa territories limitations in imaging of chromatin by light microscopy (LM) and were observed to occupy similar volumes but differed in shape and electron microscopy (EM) (Horowitz-Scherer and Woodcock, surface area (Eils et al., 1996), suggesting the same average 2006), including sensitivity of higher levels of chromatin folding chromatin compaction for both the Xi and Xa (Pollard and to buffers and EM preparation methods (Belmont et al., 1989) and Earnshaw, 2004). lack of suitable high-contrast DNA-specific staining methods for It has been proposed that the Xi in the interphase nucleus is EM. organized into a core of repetitive sequences surrounded by genic Traditional cytology classifies chromatin into less-condensed regions (Chaumeil et al., 2006; Clemson et al., 2006), but a detailed euchromatin and more-condensed heterochromatin. ultrastructural analysis of the Xi has so far been lacking (Heard and Heterochromatin has been further subdivided into permanently Disteche, 2006; Straub and Becker, 2007). Here, we have combined condensed constitutive heterochromatin and facultative LM and EM of the Xi in human and mouse female fibroblasts to heterochromatin, which becomes condensed/decondensed at some demonstrate that the Xi has a unique ultrastructure, containing point during development (Wegel and Shaw, 2005). X inactivation condensed, large-scale chromatin fibers/domains clearly distinct in female mammals is a classic example of the formation of from those observed in euchromatic or constitutive heterochromatin facultative heterochromatin. The inactive X chromosome (Xi) regions. In addition, we demonstrate a distinct position of this appears within interphase nuclei as a heteropycnotic Barr body facultative heterochromatin in the nucleus. 1120 Journal of Cell Science 121 (7)

Results in Xi conformation or orientation resulted in comparable DAPI The Xi has a homogeneous size and appearance in confluent contrast as the surrounding chromatin (Fig. 1C). Inspection of single, WI-38 cells unprocessed optical sections by two independent observers revealed For ultrastructural studies, we wanted a homogeneous cell that, in ~85% of confluent cells (n=100), the Barr-body/Xi population in which the Xi was fully silenced and showed minimal condensed core could be identified solely by the presence of a DAPI- variation in appearance. The original concept of increased chromatin dense body. This number rose to 95% for confluent cells when compaction of the Xi comes from its appearance at the LM level optical section stacks were inspected (n=131). as a distinct nuclear Barr body, which stains more intensely with Xi/Barr-body area, as measured by the H3-3mK27 signal, was nucleic-acid stains than the surrounding chromatin, implying a smaller and showed a more uniform size distribution in confluent higher DNA compaction. versus log-phase cells (2.7 versus 3.4 μm2) (Fig. 1D). We therefore We used human female primary fibroblasts, WI-38 cells, which used confluent cells for most of our ultrastructural analysis. have a high percentage of cells showing a distinct Barr body. This percentage peaked in confluent cells, 10 days after passage. The Identifying sample-preparation methods that preserved Xi Barr body was equated to a DAPI bright body with a unique intensity structure and size distinct from all other DAPI bright regions. Immunostaining A major problem in viewing chromatin ultrastructure by against trimethylated histone H3 on lysine 27 (H3-3mK27) provided transmission electron microscopy (TEM) is the lack of an adequate a robust, independent identification of the Xi/Barr body, with a DNA-specific stain. To obtain satisfactory contrast of chromatin discrete, unique, high-contrast stained body recognizable in nearly and to improve accessibility during pre-embedding immunogold 100% of cells. In cells with a clearly defined Barr body, as defined staining, detergent extraction of the nucleoplasm prior to fixation by DAPI staining, the DAPI dense region colocalized nearly is frequently used. However, the extreme sensitivity of large-scale completely with the region of elevated H3-3mK27 staining (Fig. chromatin structure to minor changes in buffer conditions leads to 1A). significant changes in ultrastructure dependent on the choice of We used H3-3mK27 staining to confirm the identification of a permeabilization buffer (Belmont et al., 1989). DAPI-dense body as the Xi, to visualize the Xi in cells without a Here, we used the Barr-body appearance in live cells to optimize recognizable DAPI-dense body, and to assay the variability in Xi sample preparation and verify EM ultrastructural preservation. In size and conformation. Failure to recognize a Barr body exclusively cells expressing GFP-histone H2B, the GFP fluorescence by DAPI staining was either due to the presence of multiple DAPI- distribution was similar to the DAPI staining distribution, which dense bodies elsewhere in the nucleus (Fig. 1B), or because a change we assume was due to its proportional enrichment based on DNA content. In deconvolved optical sections, the Barr body visualized in live cells by GFP-H2B fluorescence displayed obvious fiber substructures measuring 200-400 nm in width (Fig. 2A). After live- cell imaging, the same samples were then fixed or permeabilized in various buffers prior to fixation in 2% glutaraldehyde (GA) and a repeat optical sectioning. Comparison of images before and after

Journal of Cell Science fixation (data not shown) identified buffer A, used in our previous work to preserve large-scale chromatin structures, as most suitable for Barr-body structural preservation after permeabilization (Belmont et al., 1989). As a higher-resolution test, we used the TEM appearance of Xi large-scale chromatin fibers in unextracted cells (Fig. 2B) to guide selection of buffer and fixation conditions preserving these fibers during nucleoplasm extraction and immunostaining. To minimize buffer-induced alterations in chromatin ultrastructure, we also explored the use of a UV pre-fixation procedure prior to detergent permeabilization and aldehyde fixation. Cross-linking by UV alone or after blue-light irradiation of ethidium bromide (EtBr)-stained samples is reported to stabilize higher levels of chromatin structure (Maeshima et al., 2005; Sheval et al., 2004). By combining these two approaches, we rapidly fixed higher- order chromatin structure by UV in the presence of EtBr. This procedure provided higher-intensity pre-embedding immunogold Fig. 1. Homogeneous Xi size and appearance in confluent female human labeling while better preserving large-scale chromatin fibers fibroblasts (WI-38 cells). (A-C) DAPI staining, blue; histone H3-3mK27 as compared to a light aldehyde-fixation step prior to immunostaining (mouse antibody) marking Xi, red. (A) 85% of cells show a immunostaining. recognizable, condensed Barr body in non-deconvolved single optical sections (95% if 3D data analyzed). (B,C) The remaining cells either show multiple A distinct heavy-metal-stained heterochromatic body, of similar DAPI-intense bodies of similar size to the Barr body (B) or no distinct DAPI size and substructure to that observed for the Barr body by LM in bright body (C). (D) Histogram showing Xi area (μm2), based on H3-3mK27 live cells, was observed by EM in cells fixed directly in 2% GA immunostaining, for confluent versus log-phase cells. (E,F) H3-3mK27 (Fig. 2B). We observed similar ultrastructure and improved contrast immunostaining of Xi in log-phase cells frequently shows a fibrillar of chromatin with (Fig. 2C) or without (Fig. 2D) UV/EtBr cross- substructure protruding from the Xi core. Original image, top framed area; corresponding deconvolved optical section, lower inset. Arrows indicate the linking, followed by permeabilization in buffer A* and fixation with position of the Xi. Scale bar: 2 μm. 2% GA. Subsequent histone H3-3mK27 immunogold staining and Ultrastructure of the Xi 1121

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.

serial sectioning confirmed that these heterochromatin structures spaces of 50-400 nm. This was also true for the Xi as visualized from corresponded to the Barr bodies visualized by LM (see below). single sections from an additional 189 cells prepared by various protocols (Fig. 2). When using buffer conditions preserving large- The Barr body as visualized by EM shows a unique scale fibers, the Xi could be visualized as a tightly folded accumulation ultrastructure distinct from constitutive heterochromatin or of large-scale chromatin fibers/domains with noticeably larger surrounding euchromatin diameters than observed elsewhere throughout most of the nucleus. The larger Xi examples visualized by H3-3mK27 We wished to compare the appearance of facultative and immunofluorescence from log-phase cells often showed distinct constitutive heterochromatin. In human cells, regions of constitutive large-scale chromatin fibers/domains near to or protruding from a heterochromatin are small and not easily identified. We therefore denser core (Fig. 1E,F). These structures were similar in diameter examined female mouse embryonic fibroblasts (MEFs) containing to chromatin substructures observed within the more compact Barr large readily identifiable chromocenters formed by coalescence of bodies of live confluent cells (Fig. 2A). Similarly, the Xi pericentric heterochromatin from several chromosomes. The Barr

Journal of Cell Science heterochromatin substructures visualized at higher resolution by bodies in MEFs were identified by immunostaining against H3- TEM (Fig. 2B-D) were comparable to those visualized by LM. The 3mK27. Although they showed above-background DAPI staining, overall shape and size of the entire heterochromatin domain the DAPI-staining intensity was considerably lower than over visualized by TEM correlated with Barr-body size and shape as chromocenters, including those of similar or even smaller size (Fig. defined by H3-3mK27 or DAPI staining. Individual heterochromatin 4A). fibers/domains of the Xi appeared larger in diameter compared with Complete serial-section sets through 11 nuclei showed very substructures observed in the surrounding bulk chromatin by both similar MEF Barr-body ultrastructure to that observed in WI-38 LM and EM. cells. MEF Xi ultrastructure, with relatively loose packing of To confirm that the heterochromatin structures visualized within heterochromatin fibers/domains, contrasted sharply with the denser, individual sections by TEM corresponded to Barr bodies, we tested more uniform texture of chromocenters (Fig. 4). H3-3mK27 whether they were present exactly once per nucleus and had a immunogold staining clearly distinguished the facultative higher-than-background level of H3-3mK27 immunogold staining. heterochromatin of the Xi from the constitutive heterochromatin of Serial 200-nm sections through the entire volume of 13 randomly the chromocenters (Fig. 4C). Because of the high chromatin packing selected WI-38 nuclei (seven confluent, six log phase) revealed that density within the chromocenters, we cannot resolve whether large- 12 nuclei contained one and only one heterochromatic body (Fig. scale chromatin fibers of similar diameter as in the Xi are also 3A,B, supplementary material Fig. S1), similar to the examples present in the chromocenters, but not recognizable owing to their shown in Fig. 2. The remaining nucleus was unusually large, very closer packing, from the alternative possibility that the large-scale likely tetraploid and contained two joined heterochromatin bodies, chromatin organization in chromocenters is fundamentally different each of similar shape to the bodies seen in the other 12 nuclei. No from that in the Xi. other condensed-chromatin region within these nuclei was comparable in size and overall chromatin compaction. Some of these The Barr body is not a solid mass of chromatin but instead 13 nuclei were from H3-3mK27-immunostained samples and each contains folded heterochromatin fibers/domains with large of these showed higher-than-background immunogold staining regions of interchromatin spaces over the heterochromatic body (Fig. 3C). Reconstructions of four WI-38 nuclei were assembled using In all 13 serial-section data sets, the Xi heterochromatin appeared nominally 40-nm-thick serial thin sections; based on the appearance as a non-solid volume containing spatially separated chromatin of large-scale chromatin fibers in x-z and y-z orthogonal views (see substructures that were 30-400 nm in diameter, with intervening below), 60 nm was a better estimate of section thickness. Within 1122 Journal of Cell Science 121 (7)

individual sections, the overall nuclear chromatin distribution neighboring domains could not be determined unambiguously. The appeared relatively sparse or sponge-like, with the area occupied most common structural motif was ~200 nm in diameter, larger by chromatin representing a small fraction of the total nuclear area than the characteristic diameter of large-scale chromatin fibers in (Fig. 5A, supplementary material Fig. S2A). Similarly, the the surrounding euchromatin. Interchromatin regions up to 400 nm condensed region of the Xi also appeared significantly less solid in dimension separated Xi heterochromatin substructures. These than impressions of the Barr body from LM (Fig. 1A) or thicker chromatin-free subvolumes were contiguous with nuclear pores and TEM sections (Figs 2-5). Maximal intensity projections (Fig. 5B) with the surrounding nucleoplasm (Fig. 5A, supplementary material or additive intensity projections (supplementary material Fig. Movie 1). Deep extensions of nucleoplasm into the Barr-body core S2B,C) of aligned serial thin sections, however, showed a more could be clearly demonstrated by orthogonal views of interpolated solid appearance of the Xi and a crowded nuclear interior, consistent stacks (Fig. 5C). Moreover, orthogonal views revealed a connection with single optical sections of DAPI/H3-3mK27 staining (Fig. 1A) of nuclear pores with intrachromosomal spaces found within the or H2B-GFP fluorescence of live cells (supplementary material Fig. Barr body (Fig. 5C; xz and yz slices, NP). A solid model of the Xi S2D). With ~20 sections per image stack, projections corresponded formed from density-threshold segmentation of the serial section to an ~1.0- to 1.5-μm depth of volume, close to the expected LM reconstruction was calculated (Fig. 5D). 3D visualizations of the depth of field for a high-NA objective lens (±0.75 μm). porous sponge-like substructure of the Xi is provided by different EM images of single thin sections showed that Xi views of the reconstructed solid model (Fig. 5DЈ,DЉ, supplementary .(heterochromatin is not a solid structure but rather an open structure material Movie 2) as well as by stereo pairs (Fig. 5Dٞ of chromatin fibers/domains of variable diameters, from 30 to 600 nm (Fig. 5A). In some regions, actual fiber segments could be The Xi is invariably attached to the nuclear envelope in human visualized as spatially distinct structures, with no overlap with other and mouse fibroblasts with frequent, close connection to the chromatin domains, in adjacent serial sections. In other regions, nucleolus fiber-like regions/domains had multiple contacts with neighboring Consistent with previous findings in the literature (Belmont et al., chromatin condensed regions so the connectivity of DNA between 1986; Bourgeois et al., 1985; Zhang et al., 2007), we observed ~60% of Barr bodies in WI-38 cells associated with the nuclear envelope, based on analysis of single optical sections. However, our initial TEM experiments showed connections of the Xi with the nuclear envelope in all examples examined. Therefore, we investigated the relationship of the Xi to the nuclear envelope using a combination of 3D deconvolution LM and 3D EM analysis. Using H3-3mK27 staining to mark the Xi and nuclear-pore staining to mark the nuclear envelope, we found that 21/23 of Xi examples that were scored

Journal of Cell Science as ‘interior’ based on single optical sections showed some association with the nuclear envelope after analysis of 3D LM data. In the remaining two cells, the Xi position relative to the nuclear envelope was unclear because of the extreme flatness of the nuclear envelope adjacent to the coverslip and poor resolution of LM in the z direction. In total, 14/23 cells showed the Xi adjacent to the top surface of the nuclear envelope, facing away from the coverslip (supplementary material Fig. S3A). Deep invaginations of nuclear envelope reaching the Xi territory were found in 6/23 cells (supplementary material Fig. S3B). Two cells showed a connection to the nuclear envelope through the attachment of extended, H3-3mK27-stained large-scale chromatin

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. Ultrastructure of the Xi 1123

fibers protruding from the Xi (supplementary material Fig. S3B). One cell showed both an attachment to a nuclear envelope invagination and an extended fiber reaching the main nuclear periphery. 3D analysis by TEM using 200-nm serial sections revealed an attachment of the Xi to the nuclear envelope in essentially 100% of cells – 24/24 complete serial-section data sets and 7/7 incomplete serial-section data sets. These 31 randomly selected cells included examples from confluent and log-phase WI-38 cells and MEFs. In total, 20/31 cells showed close association of the Xi with the nuclear envelope in sections parallel to the nuclear equatorial plane (Figs 2-4). 8/31 cells showed the Xi attached to the nuclear envelope in sections far from the equatorial plane, mostly on the top of the nucleus (Fig. 6A-C). The remaining 3/31 cells showed an internally located Xi attached to the nuclear envelope via an invagination of the nuclear envelope (Fig. 6D). The observed association of the Xi with the nuclear envelope does not appear to be specific to the Xi facultative heterochromatin, because a similar association was observed for constitutive heterochromatin of mouse chromocenters. 3D TEM analysis of serial sections through MEF nuclei (n=11) revealed that all observed chromocenters were also attached to the nuclear envelope (supplementary material Fig. S4 and Movie 3). Consistent with a reported ~50% of Xi association with the Fig. 4. Xi and chromocenters have a different ultrastructure. (A) Fluorescent nucleolus in G0 and log-phase MEFs (Zhang et al., 2007), we found images of a mouse embryonic fibroblast nucleus counterstained by DAPI a 40% attachment (n=108) of the Xi to nucleoli by LM and 38% (blue) show the Xi (arrows) identified by mouse anti H3-3mK27 antibody by TEM using serial sections through entire nuclei (9/24) in (pink) with weaker DAPI intensity than observed in chromocenters confluent, quiescent WI-38 cells (supplementary material Fig. S5). (arrowheads) but still noticeably above the background DAPI intensity level. (B,C) Electron micrographs of 200-nm sections of two nuclei demonstrate All Xi in contact with the nucleolus maintained some contact with looser chromatin packing of the Xi (arrows) compared with that of the nuclear envelope. chromocenters (some marked by arrowheads). (C) Nanogold staining of histone H3-3mK27 (rabbit antibody) shows the comparison of the labeled Xi Discussion facultative heterochromatin (arrow) and heterochromatin of the adjacent The mammalian Xi has been a classic model for facultative chromocenter (arrowhead). Nu, nucleolus. heterochromatin for nearly 50 years. Throughout most of this time, Journal of Cell Science

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 DЈ and DЉ, and for stereo-pair projections shown in Dٞ (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). 1124 Journal of Cell Science 121 (7)

regions. In contrast to its appearance as a solid mass by wide-field LM without deconvolution, the Barr body at the ultrastructural level clearly shows a relatively porous structure, formed by the tight folding of large-scale chromatin fibers/domains within a compact volume. The mean density of chromatin compaction is significantly higher than euchromatic chromosome regions but lower than that observed for mouse chromocenters. 3D ultrastructural analysis also revealed that essentially all Barr bodies maintain some contact with the nuclear periphery, which in mammalian cells is correlated with repression of gene expression (Taddei et al., 2004), with ~40% of Barr bodies also in contact with the nucleolus in confluent WI-38 cells. Our results showing a distinctive, condensed ultrastructure for the mouse and human Barr bodies appear to contradict a FISH analysis that found similar volumes for both the Xa and Xi in human amniotic cells (Eils et al., 1996), implying similar mean compaction for both chromosomes (Pollard and Earnshaw, 2004). Several possibilities may account for this apparent contradiction. The limited resolution of LM relative to the size of the Barr body, combined with chromosomal volume changes induced by the FISH procedure, might have led to errors in Xi and Xa volume estimation. Alternatively, our study examined the ultrastructure of the condensed region of the Xi, corresponding to the Barr body visualized by DAPI staining, whereas the FISH study measured the total Xi volume, which includes both the Barr body plus the chromosome regions and active genes that escape silencing. In fact, the authors of the FISH study noted that the Xi chromosome territory appeared frequently to have a denser core FISH signal, whereas the Xa territory showed a more uniform FISH density throughout the territory. The focus of the FISH study on volume comparison might Fig. 6. Serial sections (200 nm) reveal the Xi–nuclear-envelope association therefore have missed the significant differences in chromatin even when the Xi appears interior in equatorial sections (confluent WI-38 structure assumed by the condensed region of the Xi. cells). (A-C) Three sections of the same immunostained (H3-3mK27) nucleus How higher levels of chromatin folding and chromosome territory with nanogold-labeled heterochromatin of the Xi (arrows). (A) The equatorial shape influence gene expression remains unclear. Based on low- section 4 shows the Xi and adjacent nucleolus (Nu) in the nuclear interior.

Journal of Cell Science (B,C) Top-two grazing sections of the above nucleus (section 2, section 1) resolution FISH chromosome paints, an interchromosome domain show the attachment of the Xi and adjacent nucleolus to the overlying nuclear (ICD) model was proposed in which decondensed active genes are periphery. (D) An example of the apparent intranuclear Xi (arrow) attached to positioned on the surface of compact chromosome domains, the deep nuclear envelope invagination (Inv). bordering an interchromosome space containing macromolecular complexes and nuclear bodies important for transcriptional activity (Zirbel et al., 1993). Additional LM analysis of chromosome an increased chromatin compaction, as inferred by the appearance territories have instead shown less-solid chromosome territory of the pycnotic Barr body, has been assumed. Moreover, during the structures with more substructure, while also localizing transcription past 10 years, a set of repressive epigenetic marks has been and active genes within chromosome territories (Mahy et al., 2002b; associated with X inactivation, correlating changes in chromatin Osborne et al., 2004). This has prompted several refinements of the histone composition with X inactivation (Chow and Brown, 2003; original ICD model, so that more or less solid chromosome Heard and Disteche, 2006). However, a FISH analysis revealed territories contain extensive invaginations, leading to more-extensive similar volumes for the Xa versus Xi, emphasizing instead contact with an interchromatin space (Cremer et al., 2006; Williams, differences in shape rather than overall compaction as the major 2003). In all of these models, however, the positioning of active or distinguishing feature of the Xa versus Xi (Eils et al., 1996). potentially active genes to the exterior of a dense chromosome Surprisingly, no careful ultrastructural analysis of the Barr body territory such that they are accessible to the interchromosome space was available to address this apparent contradiction. Here, we remains a key concept. Based on these models, the rounder, identified buffer and sample-preparation conditions preserving Xi smoother shape of the Xi versus Xa might have functional large-scale chromatin structure close to that visualized in live cells. significance in gene repression by reducing the territory surface A novel procedure was developed to provide improved preservation area in contact with the interchromosome space. of Xi large-scale chromatin structure during immunogold-staining By contrast, our results show that chromatin occupies a relatively procedures. low fraction of the nuclear volume in mouse and human fibroblasts, Using these methods, here we show for the first time that the with the possibility for extensive space, not occupied by chromatin, condensed region of the Xi assumes a unique ultrastructure, distinct for movement of large macromolecular complexes and even nuclear from surrounding euchromatin or constitutive heterochromatin, bodies. Even the relatively condensed Barr body still shows containing large-scale chromatin fibers/domains with noticeably considerable intrachromosomal space separating distinct large-scale larger diameter than observed in the surrounding euchromatin chromatin fibers/domains and contiguous with the nuclear pores Ultrastructure of the Xi 1125

and nuclear interior. Recent experiments have revealed looping of part of the Xi, but it is possible that these chromatin regions Mbp-sized gene-rich regions outside of chromosome territories correspond to X-linked genes, or more likely clusters of X-linked accompanying gene activation (Mahy et al., 2002a; Ragoczy et al., genes, that escape silencing. 2003; Volpi et al., 2000). Therefore, the rounder shape reported for Clearly, a large problem in extrapolating from the above-cited the Xi versus Xa (Eils et al., 1996) might be at least partly a FISH studies to our own work is the large mismatch in structural consequence rather than a cause of Xi gene silencing, with the preservation between the associated protocols. We have used elimination of the looping of active regions outside of the core Xi sample-preservation conditions that preserve the Barr-body size and chromosome territory. internal structure so that they are very close to that observed by In considering how Barr-body ultrastructure might influence gene live-cell imaging, as well as by EM of unextracted cells under silencing, we suggest the proper focus instead should be on optimal chemical-fixation methods used in conventional EM. distinctly higher-level compaction of large-scale chromatin Unfortunately, these fixation methods are too stringent to allow fibers/domains observed within the Barr body as compared to localization of specific genes or chromosome regions by FISH surrounding euchromatin regions. This region of the Xi has been methods. By contrast, the best 3D FISH procedures produce near- linked with transcriptional silencing (Chaumeil et al., 2006; Clemson complete loss of large-scale chromatin ultrastructure as visualized et al., 2006), with a movement of genes towards the interior of the by EM (Solovei et al., 2002) and, at least in some cases, these Xist-coated Xi core region accompanying gene silencing during structural perturbations are quite apparent even by LM (Robinett embryonic stem (ES) cell differentiation (Chaumeil et al., 2006). et al., 1996). Therefore, it is difficult at this time to reconcile Our results show that this core also corresponds to the region of differences in possible models of Xi organization created by FISH H3K27 trimethyl modification and assumption of a distinctive, methods versus ultrastructural analysis. large-scale chromatin-folding motif. These correlations suggest a In conclusion, we have demonstrated a distinct condensation of probable functional link between the altered large-scale chromatin Xi chromatin and association of the Xi in all cells with the nuclear packing and gene silencing. envelope. These results are consistent with the idea that spatial Based on the biased localization of X-linked inactive genes segregation of Xi chromatin helps maintain Xi silencing by limiting towards the periphery of the condensed Xist-coated Barr body by access to transcription factors (Heard and Disteche, 2006). Future FISH, together with the localization of Cot-1 DNA largely within experiments directly comparing the ultrastructure of X-linked the Barr body, Clemson and colleagues have suggested extensive active versus inactive genes, and correlating these differences with looping of both active and inactive genes outside or on the edge of regulation of gene silencing, will require the development of the Barr body, which they propose is formed largely by methods for visualizing the location of specific genes and transcriptionally repressed repetitive DNA (Clemson et al., 2006). components of the transcriptional machinery without perturbing However, Chaumeil and colleagues observed X-linked genes chromatin ultrastructure. protruding from the Xist-coated Xi core very early during Xi silencing accompanying ES cell differentiation, with genes Materials and Methods undergoing silencing shifting later during differentiation to a more Cell culture interior position largely at the edge of the Xist-coated Xi core Human female fibroblasts (WI-38) obtained from the American Type Culture Collection were maintained in minimum essential medium (Invitrogen) supplemented

Journal of Cell Science (Chaumeil et al., 2006). with 10% FBS (HyClone Laboratories) at 37°C in 5% CO2. WI-38 cells at cumulative In the Clemson et al. model, all X-linked genes, not just those population doublings between 20 and 40 were used 3 days (log-phase cells) or 10 that escape silencing, loop out from the dense Barr-body core days (confluent cells) after plating. Female mouse embryonic fibroblasts (MEFs) were provided by Edith Heard (Curie Institute, Paris, France). MEFs were maintained in (Clemson et al., 2006). Moreover, in this model, only centromeric Dulbecco’s modified Eagle’s medium (Invitrogen), 10% FBS (HyClone Laboratories) and Cot-1 repetitive DNA localizes within the dense Barr-body core, and 0.001% 2-mercaptoethanol (Sigma) at 37°C in 8% CO2. MEFs were cultured implying extensive looping of repetitive DNA sequences located for 3 days before they were placed in low serum (0.1% FBS) for an additional 72 in intergenic regions back into the Barr-body core. Therefore, this hours to obtain quiescent cells. model would predict extensive looping on the same DNA distance Live-cell observation scale as gene spacing – typically tens to hundreds of kb. Previously, WI-38 cells were transfected by FuGene6 (Roche) with plasmid pH2BGFP-N1 (Kanda we estimated a compaction of approximately 3 Mbp per μm for et al., 1998). Cells were plated in delta-T dishes (Biotechs) or coverslips 2 days after ~100 nm large-scale chromatin fibers (Tumbar et al., 1999), with transfection and cultured for another 10 days before live imaging. the expected compaction of the thicker, ~200 nm diameter, large- Immunofluorescence scale chromatin fibers/domains in the Xi condensed regions Staining was performed essentially as described previously (Tumbar and Belmont, described in this paper expected to be substantially higher. 2001) with the following modifications. Cells were permeabilized in 0.1% Triton, fixed in 1.6% formaldehyde (Polysciences) at room temperature for 10 minutes, and Therefore, with compactions corresponding to greater than 300 kb blocked for 10 minutes with 5% NGS and 5% donkey serum. For Xi labeling, we per 100-nm lengths of large-scale chromatin fibers, we should expect applied mouse anti-tri-methyl histone H3Lys27 (H3-3mK27) antibody (Danny to see many looped-out genes protruding from the surfaces of the Reinberg, NYU College of Medicine, NY) diluted 1:2000. For double-staining of condensed, large-scale chromatin fibers that make up the Barr body. the Xi and nuclear membrane, rabbit anti H3-3mK27 antibody (Upstate) diluted 1:1000 was used together with mouse monoclonal antibody RL1 against nuclear pore O- However, in our serial thin sections, we did not see the extensive linked glycoprotein (ABR-Affinity BioReagents) diluted 1:750. For double-staining looping of decondensed chromatin protruding outwards from the of the Xi and nucleoli, mouse anti-H3-3mK27 antibody diluted 1:2000 was used Barr body as would be expected by the Clemson et al. model together with rabbit polyclonal anti-fibrillarin (Santa Cruz Biotechnology) antibody diluted 1:100. Secondary antibodies (Texas-red-labeled donkey anti mouse, FITC- (Clemson et al., 2006). However, we did see chromatin domains labeled goat anti rabbit) antibodies were diluted 1:500 and obtained from Jackson packaged similarly to surrounding euchromatin – and therefore Laboratory. Cells were counterstained by DAPI. estimated as being 100’s to 1000’s of kb in size – in close contact with but extending outwards from the condensed, H3K27-trimethyl- Light microscopy Immunostained samples were observed with a 60ϫ objective (NA=1.4) using an labeled Barr body. Without any specific label, we could not Olympus IMT-2 microscope. Live cells were imaged with a 100ϫ objective unambiguously determine whether these chromatin domains were (NA=1.40) using a Zeiss Axiovert 100M within 10 minutes after removal from the 1126 Journal of Cell Science 121 (7)

incubator or maintained at 37°C in a closed chamber system (FCS2; Bioptechs). Data Netherlands). AVI movies were created using a Camera Sequence MATLAB script acquisition and 3D deconvolution methods were processed as previously described (Olivier Salvado, CSIRO). ImageJ was used to convert movies to a compressed (Chuang et al., 2006; Tumbar et al., 1999). QuickTime format using QT Movie Writer plugin.

EM preparation of non-immunostained samples We thank Edith Heard (Curie Institute, France) for her comments Cells were grown on glass coverslips. Several procedures designed to preserve large- on the manuscript as well as for providing MEFs. We thank Teru Kanda scale chromatin fibers were used: (a) live cells were fixed directly in 0.1 phosphate and Geoffrey Wahl (The Salk Institute for Biological Studies, USA) buffer, pH 7.4, in 2% GA (Polysciences) overnight at 4°C; (b) cells were permeabilized with 0.1% Triton X-100 (Pierce) in buffer A* (80 mM KCl, 20 mM NaCl, 2 mM for providing the H2B-GFP expression vector (pH2BGFP-N1), as well EDTA, 0.5 mM EGTA, 15 mM Pipes, 0.5 mM spermidine, 0.2 mM spermine, 10 as Danny Reinberg (NYU, College of Medicine, USA) for the anti- μg/ml turkey egg white inhibitor, pH 7.0) for 30-180 seconds followed by fixation H3-3mK27 mouse antibody. Electron microscopy was performed in in 2% GA (25% solution added dropwise) overnight at 4°C; (c) cells on ice in media The Visualization, Media and Imaging Laboratory at the Beckman 2 were UV irradiated (9000 J/m ) for 8 minutes (Stratalinker 1800 UV crosslinker, Institute, UIUC. This work was supported by HFSP RGP0019/2003 Stratagene). EtBr (20 μg/ml) was added to media before UV treatment. After UV fixation, coverslips were permeabilized with 0.1% Triton X-100 in buffer A* for 5 and grant number R01 GM42516 from the National Institute of General minutes and then post-fixed overnight at 4°C in 2% GA in buffer A*. Medical Sciences (A.S.B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the Immunostaining procedure for EM National Institute of General Medical Sciences or the National Institutes Two UV/EtBr cross-linking fixation methods were used prior to immunostaining: of Health. cells were permeabilized for 30 seconds with 0.1% Triton X-100 in buffer A* in the presence of 20 μg/ml EtBr followed by 4 minutes of UV irradiation on ice (4500 J/m2). Alternatively, live cells in media were placed on ice and 20 μg/ml EtBr was References added prior to 8 minutes of UV irradiation (9000 J/m2). Arney, K. L. and Fisher, A. G. (2004). Epigenetic aspects of differentiation. J. Cell Sci. Samples fixed by either method were washed in 0.1% Triton X-100 buffer A*. 117, 4355-4363. All following steps were performed in the same buffer unless specified otherwise. Barr, M. L. and Carr, D. H. (1962). Correlations between sex chromatin and sex Cells were blocked in 1% BSA for 1 hour at room temperature and then rabbit antibody chromosomes. Acta Cytol. 6, 34-45. (Upstate) against histone H3-3mK27 diluted 1:1000 in blocking buffer was applied Belmont, A. S. and Bruce, K. (1994). Visualization of G1 chromosomes: a folded, twisted, supercoiled chromonema model of interphase chromatid structure. J. Cell Biol. 127, at 4°C overnight. Specimens were washed three times followed by a second blocking 287-302. step for 30 minutes at room temperature with 0.1% fish gelatin (Sigma). Incubation Belmont, A. S., Bignone, F. and Ts’o, P. O. (1986). The relative intranuclear positions with secondary nanogold anti-rabbit antibody (Nanoprobes) was performed overnight of Barr bodies in XXX non-transformed human fibroblasts. Exp. Cell Res. 165, 165- at 4°C (antibody diluted in 0.1% fish gelatin, 1% BSA, 1:500). Specimens were washed 179. three times and post-fixed in 2% GA for 1 hour at room temperature. Samples were Belmont, A. S., Braunfeld, M. B., Sedat, J. W. and Agard, D. A. (1989). Large-scale quenched with 150 mM glycine (3ϫ 5 minutes) and then blocked with 0.1% fish chromatin structural domains within mitotic and interphase chromosomes in vivo and gelatin in blocking buffer for 10 minutes at room temperature. Specimens were washed in vitro. Chromosoma 98, 129-143. (5ϫ 2 minutes) in double-distilled H2O before silver or gold enhancement of nanogold Bourgeois, C. A., Laquerriere, F., Hemon, D., Hubert, J. and Bouteille, M. (1985). by HQ Silver (Nanoprobes) for 6-10 minutes or by Gold Enhancement (Nanoprobes) New data on the in-situ position of the inactive X chromosome in the interphase nucleus for 2 minutes. of human fibroblasts. Hum. Genet. 69, 122-129. All EM samples were en bloc stained with uranyl acetate (1% in H2O) for 1 hour Chaumeil, J., Le Baccon, P., Wutz, A. and Heard, E. (2006). A novel role for Xist RNA at room temperature, followed by dehydration in an ethanol series, and infiltration in the formation of a repressive nuclear compartment into which genes are recruited and embedding in Epon 812 (Polysciences). Embedded cells were removed from when silenced. Genes Dev. 20, 2223-2237. Chow, J. C. and Brown, C. J. (2003). Forming facultative heterochromatin: silencing of glass coverslips after boiling the Epon samples in H2O for several minutes. Sections of 60 or 200 nm on formvar support films were stained with uranyl acetate (2% in an X chromosome in mammalian females. Cell. Mol. Life Sci. 60, 2586-2603. H O) for 15 minutes and lead citrate (0.02% in 0.01 M NaOH) for 10 minutes. Carbon Chuang, C. H., Carpenter, A. E., Fuchsova, B., Johnson, T., de Lanerolle, P. and 2 Belmont, A. S. (2006). Long-range directional movement of an interphase chromosome coating was applied after staining to grids used for serial section reconstructions. site. Curr. Biol. 16, 825-831. Journal of Cell Science Clemson, C. M., Hall, L. L., Byron, M., McNeil, J. and Lawrence, J. B. (2006). The Fixation and preparation conditions for specific figure panels X chromosome is organized into a gene-rich outer rim and an internal core containing For Fig. 2B, Fig. 6D and supplementary material Fig. S5C, samples were fixed in silenced nongenic sequences. Proc. Natl. Acad. Sci. USA 103, 7688-7693. 2% GA in 0.1 M phosphate buffer. For Fig. 2C, the sample was fixed by UV/EtBr Cremer, T., Cremer, M., Dietzel, S., Muller, S., Solovei, I. and Fakan, S. (2006). cross-linking for 8 minutes prior to permeabilization and GA post-fixation. For Fig. Chromosome territories-a functional nuclear landscape. Curr. Opin. Cell Biol. 18, 307- 2D, Fig. 3A,B, Fig. 4B, Fig. 6, and supplementary material Fig. S1, Fig. S2A-C, Fig. 316. S4 and Fig. S5B, samples were fixed by 2% GA in buffer A* after partial Eils, R., Dietzel, S., Bertin, E., Schrock, E., Speicher, M. R., Ried, T., Robert-Nicoud, permeabilization for 30-90 seconds in buffer A*. For Fig. 3C, sample was fixed by M., Cremer, C. and Cremer, T. (1996). Three-dimensional reconstruction of painted 10 minutes of UV irradiation with EtBr in medium. For Fig. 4C, Fig. 6A-C and human interphase chromosomes: active and inactive X chromosome territories have supplementary material Fig. S5D, samples were fixed after quick permeabilization similar volumes but differ in shape and surface structure. J. Cell Biol. 135, 1427- in EtBr/buffer A* by 4 minutes of UV irradiation. 1440. Gilbert, N. and Bickmore, W. A. (2006). The relationship between higher-order chromatin structure and transcription. Biochem. Soc. Symp. 73, 59-66. Electron microscopy Gilbert, N., Boyle, S., Fiegler, H., Woodfine, K., Carter, N. P. and Bickmore, W. A. Sections were examined with a Philips CM 200 electron microscope operating at 120 (2004). Chromatin architecture of the human genome: gene-rich domains are enriched kV. Images were acquired using a Tietz Video and Image Processing Systems GmbH in open chromatin fibers. Cell 118, 555-566. ϫ 2k 2k Peltier-cooled CCD camera and software. Heard, E. and Disteche, C. M. (2006). Dosage compensation in mammals: fine-tuning the expression of the X chromosome. Genes Dev. 20, 1848-1867. Image processing Horowitz-Scherer, R. A. and Woodcock, C. L. (2006). Organization of interphase LM and EM images were adjusted for brightness/contrast, superimposed, chromatin. Chromosoma 115, 1-14. pseudocolored and assembled using Adobe Photoshop CS. Alignment of 200-nm Kanda, T., Sullivan, K. F. and Wahl, G. M. (1998). Histone-GFP fusion protein enables serial sections was done manually in Photoshop and movies were created in WCIF sensitive analysis of chromosome dynamics in living mammalian cells. Curr. 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