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