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The X Chromosome Is Organized Into a Gene-Rich Outer Rim and an Internal Core Containing Silenced Nongenic Sequences

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The X Chromosome Is Organized Into a Gene-Rich Outer Rim and an Internal Core Containing Silenced Nongenic Sequences The X chromosome is organized into a gene-rich outer rim and an internal core containing silenced nongenic sequences Christine Moulton Clemson, Lisa L. Hall, Meg Byron, John McNeil, and Jeanne Bentley Lawrence* Department of Cell Biology, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA 01655 Edited by Barbara J. Meyer, University of California, Berkeley, CA, and approved March 27, 2006 (received for review February 8, 2006) We investigated whether genes escape X chromosome inactivation article, we describe experiments to discriminate between these two by positioning outside of the territory defined by XIST RNA. Results models. reveal an unanticipated higher order organization of genes and We examined the distribution of X-linked genes that do or do not noncoding sequences. All 15 X-linked genes, regardless of activity, escape X inactivation as well as a psuedoautosomal marker, ␣-sat- position on the border of the XIST RNA territory, which resides ellite, subtelomeric and Cot-1 sequences and quantified their spatial outside of the DAPI-dense Barr body. Although more strictly relationship to XIST RNA, the Barr body, and the chromosome delineated on the inactive X chromosome (Xi), all genes localized territory. Our results yield unexpected insights regarding sequence predominantly to the outer rim of the Xi and active X chromosome. organization on the Xi and the role that XIST plays in defining the This outer rim is decorated only by X chromosome DNA paints and inactive chromosome territory; they also contribute to an emerging is excluded from both the XIST RNA and dense DAPI staining. The understanding of interphase chromosome organization more gen- only DNA found well within the Barr body and XIST RNA territory erally. Finally, our findings provide evidence that the formation of was centromeric and Cot-1 DNA; hence, the core of the X chromo- the Barr body represents the chromosome-wide transcriptional some essentially excludes genes and is composed primarily of silencing of noncoding RNA. noncoding repeat-rich DNA. Moreover, we show that this core of repetitive sequences is expressed throughout the nucleus yet is Results silenced throughout Xi, providing direct evidence for chromosome- Genes Reproducibly Border the XIST RNA Territory Irrespective of wide regulation of ‘‘junk’’ DNA transcription. Collective results Their Activity. We began asking whether genes that escape inacti- suggest that the Barr body, long presumed to be the physical vation might ‘‘escape’’ XIST RNA by localizing at the periphery, or manifestation of silenced genes, is in fact composed of a core of just outside, the territory defined by XIST RNA. We used dual silenced noncoding DNA. Instead of acting at a local gene level, color fluorescence in situ hybridization to detect single-copy genes XIST RNA appears to interact with and silence core architectural along the length of the X chromosome and quantified their spatial elements to effectively condense and shut down the Xi. relationship to XIST RNA. Initially, we scored the results in three categories (for a detailed Barr body ͉ chromosome territory ͉ nuclear organization ͉ XIST ͉ explanation, see Supporting Materials and Methods, which is pub- noncoding RNA lished as supporting information on the PNAS web site): (i) IN, if the gene was found inside of the XIST RNA territory; (ii) OUT, if inactivation in mammalian females is a prominent example of the gene was outside the XIST RNA; and (iii) BORDER, if the Xthe formation of facultative heterochromatin during early gene was positioned at the edge of the XIST RNA accumulation development, which prevents the deleterious effects of overexpres- (see Fig. 1 for examples). Eight genes subject to X inactivation, sion of X-linked genes. In interphase, the inactive X chromosome seven genes that escape inactivation, and the X centromere were (Xi) is found as a condensed heterochromatic Barr body, usually mapped in no less than 50 cells, and most were scored in multiple positioned at the nuclear or nucleolar periphery (1–4). Because experiments; results are summarized in Fig. 2. Individual genes many genes have been identified that escape X inactivation, pack- were not located randomly with respect to XIST RNA. Irrespective aging differences of sequences at some level within the Xi is of the distance from the site of XIST transcription or activity, all presumed (5, 6). Although it is generally assumed that the Barr body genes positioned at the very border of the XIST RNA territory, is condensed DNA comprising genes normally expressed on the whereas the X centromere sequences were predominantly posi- active X chromosome (Xa), the specific makeup of the Barr body tioned inside. Interestingly, although found to border the XIST has not been investigated. RNA a majority of the time, ZXD was found IN more often than X inactivation is a multistep process initiated by XIST (7–9). Just other genes (perhaps due to its close linkage to the centromere). after XIST RNA sweeps across the chromosome, a defined pattern Although the bulk of our scoring was done on 2D images, 3D of chromatin changes including histone modifications, recruitment analysis was performed on multiple samples and fully corroborated of macroH2A, and methylation occurs (for review see refs. 10 and our 2D analysis (see Movie 1, which is published as supporting 11). We have shown that XIST RNA remains in the nucleus, information on the PNAS web site). Several lines of evidence functionally associated with the inactive chromatin, forming an suggest that hybridization efficiency to the condensed Xi chromatin interphase territory coincident with Xi (12, 13). We incorporated was not a factor in our analysis. Single-copy probes hybridized to Xa our results into two models for the higher-level organization of the and Xi with similar intensity and frequency. In addition, hybrid- inactive X chromosome (12). In each alternate view, XIST RNA ization to nongenic sequences such as centromeric and Cot-1 DNA would induce differences in the packaging of the chromatin to (see Fig. 4) were consistently detected within the Barr body. To rule facilitate inactivation. In the first model, all genes, regardless of whether they escape from or are subject to X inactivation, would be interspersed throughout the chromosome territory and would not Conflict of interest statement: No conflicts declared. be cytologically distinct. In the second model, genes subject to This paper was submitted directly (Track II) to the PNAS office. inactivation would be internal to the X chromosome territory and Abbreviations: Xi, inactive X chromosome; Xa, active X chromosome; RT, room temperature. separable from active genes that escape X inactivation by position- *To whom correspondence should be addressed. E-mail: [email protected]. ing on the outside of the XIST RNA and chromosome. In this © 2006 by The National Academy of Sciences of the USA 7688–7693 ͉ PNAS ͉ May 16, 2006 ͉ vol. 103 ͉ no. 20 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601069103 Downloaded by guest on October 4, 2021 Fig. 1. Examples of gene positions relative to XIST RNA. Gene signals were scored as occurring either IN, OUT, or on the BORDER of the RNA domain. (A) IN: X centromere is green, XIST RNA is red, and overlap appears yellow. (B) OUT: NFIB (red) was found outside of the XIST RNA (green) most of the time in cells with an X;9 translocation. Most of the genes fell into the BORDER category: (C) PGK-1 (red) is found mostly at the periphery of the XIST RNA domain (green). SLC16A2 (green) (D) and TIMP1 (red) (E) show the typical gene position bordering the XIST RNA territory. (F) The XIST gene identified with an intron probe (red) borders the mature XIST RNA (green) and the Barr body (black) (Inset). We subdivided the IN category further to include: (G) IN HOLE: SLC16A2 (green), like most genes, although rarely found inside of the XIST domain (red), when it was inside, it was often in a hole of the XIST signal (Inset). (H) GATA, a marker for the psuedoautosomal region (red), was found to primarily border the XIST RNA (green). (I) Three X-linked genes (green) simultaneously detected relative to the Barr body (black) show that genes are not usually embedded within the Barr body. out possible fixation artifacts, we prepared two different cell lines RNA territory the vast majority of the time (Fig. 2B). The bulk of with our standard technique and a protocol that involves no cell signals scored in this category show a striking position at the very dehydration. Using 3D analysis, we saw no significant difference in edge of the XIST territory, with no cytological separation or gene location or z-axis dimensions of nuclear, chromosome, and extensive overlap between the XIST RNA and gene signal (e.g., see XIST RNA territories. In the course of exhaustively examining the Fig. 1E). All genes examined, regardless of metaphase chromosome location of genes within the interphase X, it became clear that we position or distance from the XIST locus, occupied this precise could expand the IN category. When a gene was scored as IN the localization at the very boundary of the XIST RNA signal. Using XIST territory, it was oftentimes in a ‘‘hole’’ of XIST signal; a an XIST intron probe to discriminate the gene from mature hallmark of this category is that there is no yellow color to indicate message, we found the XIST gene (and hence the XIC locus) was CELL BIOLOGY a merging of the XIST and gene signal (see Fig. 1G). 3D analysis also typically localized at the periphery of XIST RNA (Fig. 1F). The confirmed our ability to discriminate this and the other scoring subtelomeric sequences (Xq Tel and Xp Tel) were also predomi- categories reliably and further suggested that the hole corresponds nantly at the edge of the XIST RNA territory; however, the to an invagination (see Movie 2, which is published as supporting ␣-satellite sequences were rarely found to border the XIST.
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