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. information on the PNAS web site). RNA fluorescence in situ hybridization was used to confirm the With the alternate cell preparation and expanded scoring, we still expression profile of the two cell lines in this study. We detected a found that the genes were scored as on the BORDER of the XIST single RNA foci only from genes subject to inactivation, whereas

Fig. 2. Genes border XIST RNA at interphase. Genomic probes for genes, ␣-satellite, and subtelomeric sequences were cohybridized with XIST RNA in normal diploid ﬁbroblasts, WI38 (CCL 75) and TIG-1 (AG06173). The signals were scored relative to XIST RNA (see Fig. 1 A–C for examples of each category). Genes that escape X inactivation are denoted by an asterisk; TIMP1 escapes X inactivation weakly and is denoted by an *.(A) Clearly, all Xi-linked genes examined, regardless of activity and position on the metaphase chromosome, maintain a bordering relationship with XIST RNA the majority of the time, whereas the ␣-satellite sequences were found mostly inside of the XIST RNA domain. (B) By using a different protocol to preserve the 3D architecture of the nucleus (see Ma- terials and Methods), the IN category was subdivided to include IN (Hole) (see Fig. 1G for an example). The genes were scored as touching the border of XIST RNA the majority of the time, whereas the ␣-satellite sequences were found mostly inside of the XIST RNA domain.

Clemson et al. PNAS ͉ May 16, 2006 ͉ vol. 103 ͉ no. 20 ͉ 7689 Downloaded by guest on October 4, 2021 Fig. 3. Genes are on the periphery of the X chromosome at interphase. (A) Although the genes generally bordered the X chromosome territory, they were more often found IN or IN HOLE than when scored against XIST RNA. Additionally, the genes were more often found inside of Xa than Xi. (B and C) Representative example of gene vs. X paint. (B) SLC16A2 (red) is inside the outer edge of the Xi territory [(Lower Inset) shows the Barr body by DAPI staining] but is found IN HOLE in the Xa Paint (Upper Inset) (see arrow). (C) The Xa is often more dispersed with irregular boundaries compared with Xi. The ZFX gene (red) is found IN HOLE (Inset) (see arrow) of DNA density at Xa, and just inside the Xi. (D–G) Pooled X probes show the characteristic positioning at the edge of the X territory. ZFX, MIC2, SL16A2, and HPRT (all in green) were pooled together and hybridized with X paint (red), examples of Xa are in D and E, and examples of Xi are in F and G.

transcripts from both Xa and Xi were detected for genes that escape in the DNA paint signal and were scored as IN (Hole) (see Fig. 3 X inactivation (see Fig. 6, which is published as supporting infor- B and C), whereas this was not true for the centromere signals, the mation on the PNAS web site). Occasionally, MIC2, which resides majority of which were found inside the territory for both Xa and in the pseudoautosomal region, seemed to be expressing from a Xi. Although we did notice a difference in the frequency of ‘‘gap’’ in the XIST RNA signal, raising the question of whether centromeres inside the territory between the Xi and Xa (81% vs. XIST RNA may associate less tightly with that region. We mapped 59%), it was not as pronounced as reported in ref. 18. Centromere the interphase location of a probe to GATA repeats, which we have positioning may well reflect cell-type-specific differences because recently shown to be strikingly enriched throughout the psuedoau- other studies report centromeres locating peripherally to (19) or tosomal region (14). The pseudoautosomal region was found inside of (20) chromosome territories. primarily to border the XIST RNA (Fig. 1H); furthermore, in cells In these experiments, we used DAPI staining to highlight the where the XIST RNA territory appeared incomplete (see Discus- Barr body (Fig. 3B), allowing us to directly compare the interphase sion), the pseudoautosomal region was not consistently located in organization of Xa and Xi. In general, Xa has a similar but less a gap. pronounced positioning of genes in the outer rim of the territory We also examined the distribution of genes in unbalanced (genes were on average found a distance of 0.2 ␮m more internal translocations in which the Xi and autosomal domains show dif- on Xa than Xi). We found the appearance of the active and inactive ferences in association with XIST RNA (15) and found that territories at interphase to be markedly different: Xa is more spread autosomal genes distant from the breakpoint and not silenced are out, with a less intense signal and diffuse boundaries, whereas Xi is outside of the XIST RNA territory (see Fig. 7, which is published more compact, with a brighter signal with more distinct boundaries as supporting information on the PNAS web site). (Fig. 3 B and C and see Movie 3, which is published as supporting information on the PNAS web site). The apparent projections and Genes Are Found on the Periphery of the Chromosomal DNA Territory increased surface area of the Xa allows for invaginations of the X as Defined by Chromosome Paints. The results presented above show paint signal, as suggested in refs. 21 and 22. We found that Xa genes that escaping vs. nonescaping genes do not occupy different posi- were in a hole depleted of DNA density more often than on Xi. In tions relative to the XIST RNA territory but rather position at the many cases, these holes appeared to be Xa surface invaginations outer edge. To determine the location of genes in the interphase such that that the genes appeared inside but did not overlap the X chromosome territory, we mapped X sequences relative to X paints. paint DNA signal (Fig. 3C). We also frequently found that genes Results are summarized in Fig. 3A. Genes were found at the border appeared to be on extensions of chromatin from both the Xi and Xa a significant fraction of the time and rarely scored as OUT of the territory (Fig. 3 D–G). X territory. The gene sequences were found IN the chromosome Although 3D analysis confirmed our cell preparation techniques territory more often than when compared with XIST RNA; how- and scoring categories, determining the gene position relative to ever, genes scored IN often appeared to be within the outermost XIST and X territories was surprisingly straightforward by using 2D rim (0.15 radius) of the X paint signal (e.g., see Xi in Fig. 3B). image analysis. In these relatively flat cells and with the optics used Additionally, as has been reported in other studies (12, 16, 17), we (see Supporting Materials and Methods), the gene signals fell into just found that a significant fraction of gene signals overlapped a hole one or two planes of focus. Only 2% of gene signals were detected

7690 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601069103 Clemson et al. Downloaded by guest on October 4, 2021 Fig. 4. The Barr body is composed of silent repetitive elements. (A) Cot-1 DNA (red) and Cot-1 RNA (green) were simultaneously detected in normal diploid fibro- blasts (TIG-1). Cot-1 DNA (C) is found throughout the nucleoplasm but is more intense over the DAPI-stained Barr body (B). (D) Cot-1 RNA is also found throughout the nucleoplasm but is not transcribed from the Barr body. (E) A line scan through the Xi shows that the pixel intensity and location of the DAPI and Cot-1 DNA signal overlap, whereas the Cot-1 RNA signal is depleted throughout the Barr body. (F) Cot-1 DNA (red) and Cot-1 RNA (green) is detected inside the Barr body. (H) Cot-1 DNA is more intense over the DAPI stained Barr body [It is not clear exactly why the interphase Xi is stained differen- tially with DAPI. Some evidence suggests the intense staining of the Xi DNA represents differences in folding rather than just condensation (24) (G), whereas the Cot-1 RNA is depleted in this region (I). (J) A line scan through the Barr body shows that the Cot-1 DNA signal is more intense over the Barr body. (K–M) X paint (green) (L) vs. Cot-1 RNA (M). Although the Barr body identified by DAPI (K) (arrowhead) and chromosome painting (L) (arrowhead) is devoid of Cot-1 transcription (M) (arrowhead), Xa (L) (arrow) is actively transcribing Cot-1 RNA (M) (arrow). (N) Ongoing RNA Pol II-mediated transcription is shown using the H5 antibody against the elongating form of the RNA Pol II enzyme (red). The DAPI DNA fluorescence (P) reveals that the heterochromatic Barr body is deficient in RNA Pol II activity (O).

above or below the focus of the territory (i.e., on the top or the Ϸ30% of human genomic DNA) (23, 25) is readily detected bottom of the X territory), and we rarely had to resort to 3D within the Barr body and in the majority of cells shows an acquisition to determine a questionable sample. It was apparent increased signal consistent with the increased DNA density of from experiments in which multiple genes were detected relative to the Barr body (Fig. 4 C and H).

XIST RNA (see Movie 4, which is published as supporting infor- We next asked: if the heterochromatic Barr body is essentially CELL BIOLOGY mation on the PNAS web site) and the X DNA (see Fig. 3 D–G) nongenic sequences, is this DNA silenced? Hybridization to the that genes were not found in every position along the periphery but Cot-1 RNA fraction has been developed as an assay for chromo- were most narrowly restricted to the X–Y plane (lateral edge) of the some inactivation (26) and used by others for this purpose (e.g., ref. territory periphery. The volume occupied by this ‘‘outer rim’’ is 27). Cot-1 transcription is detected throughout the nucleus and estimated to occupy Յ28% of the chromosome territory (see Fig. exhibits a very intense nucleoplasmic staining that avoids the 8, which is published as supporting information on the PNAS web cytoplasm (27); as shown in Fig. 4 D and I, however, Cot-1 RNA is site). We next asked whether different positions along the lateral notably silent over the Xi (defined by DAPI staining) and is readily edge of the chromosome territory are equivalent. We found that detected throughout the Xa (Fig. 4 K–M). To confirm that there is most genes had preferred locations within the Xi periphery, yet no transcription from the Barr body, we used an antibody against there was no strict pattern of intranuclear position that correlated the elongating form of the RNA Pol II enzyme (28) to detect sites with transcriptional activity (see Fig. 9, which is published as of nascent RNA (Fig. 4 N–P). The demonstration here that supporting information on the PNAS web site). protein-coding genes are largely not within the Barr body provides strong evidence that what is being silenced on Xi is heterogeneous Regulation of Junk Transcription: Cot-1 DNA Is Present Throughout the nuclear RNA (26), composed largely of nongenic repetitive DNA, Barr Body and Is Silenced. In cells in which the Barr body could be not repeats within introns. This finding has profound implications well delineated by DAPI staining, we found genes typically just for genome organization and function that will be considered outside and rarely inside of the Barr body (see Fig. 1I). An further in Discussion. important question follows: what is inside the Barr body? Given that protein-coding genes comprise only a small frac- Discussion tion of the genome, it is theoretically possible that the chromo- In this article, we examined the interphase positions of many some interior could be composed of noncoding DNA. We used X-linked sequences by using multiple preparation methods and the Cot-1 DNA fraction as a probe to detect largely nongenic provide strong evidence for a surprising organization of the Xi. We sequences. Although all constituents of this DNA fraction have find that all or most genes are arranged on the periphery of the X not been defined, it is composed of the more highly repetitive chromosome, with largely or entirely noncoding sequences residing elements, consisting largely of LINES, SINES, microsatellite, within the interior of the territory. Given that the painting of Xi by and other repetitive elements (23). When a labeled Cot-1 probe XIST RNA is the signal that induces Xi silencing, we began by is used under DNA-denaturing conditions, highly repetitive asking whether genes that escape inactivation might be distinctly DNA is detected at the Xa (data not shown), the Barr body, and organized to lie outside of the XIST RNA territory. Interestingly, throughout the nucleoplasm (Fig. 4). A line scan through the neither model we previously proposed (Fig. 5 A and B) was correct Barr body of representative cells show that the increase in pixel because we presumed, as the field generally does, that inactivated intensity of the DAPI and Cot-1 DNA signal overlap (4 E and protein-coding genes reside within the Barr body. Remarkably, our J). Thus, the more repetitive fraction of the genome (comprising results support an alternate model (Fig. 5C) in which genes,

Clemson et al. PNAS ͉ May 16, 2006 ͉ vol. 103 ͉ no. 20 ͉ 7691 Downloaded by guest on October 4, 2021 components of the Cot-1 DNA, of particular interest would be the involvement of LINE transcription because this sequence is en- hanced on Xi (14, 39). The question of how XIST RNA directly facilitates X inactivation is an important one. Because XIST RNA encompasses chromatin that extends beyond the Barr body, we do not rule out the possibility that it may interact with individual genes (41); however, our findings argue against it acting primarily at a local scale. Our results indicate that some structural elements of the chromosome provide a frame- work that positions the ‘‘important’’ DNA (genes) in a reproducible position at or very near the surface of the chromosome. The dominion of XIST RNA is apparently rigorously constrained relative to the gene-rich outer chromatin providing evidence that it has an architectural relationship to the chromosome. We speculate Fig. 5. Models of higher level organization of the inactive X chromosome. that rather than transcriptionally silencing individual genes, XIST Previously, we proposed two alternate views: (A) genes that escape inactiva- RNA acts more globally to interact with structural elements to alter tion (green) are interspersed rather than cytologically separate from the the overall chromosome structure, resulting in the silencing of the genes that are subject to inactivation (red); or (B) genes that escape X chromosome. This idea is supported by evidence that XIST RNA inactivation would be organized at the chromosome periphery, outside of the is retained with the insoluble nuclear scaffold even upon removal Barr body. Our work here supports a different model in which genes, regardless of activity, are found at the periphery at the chromosome territory (C). The of DNA (12) and that the longer lived protein scaffold on Xi is genes are found outside of the XIST RNA (yellow), which extends beyond the enriched in matrix-attachment proteins (42, 43). Barr body, the visible manifestation of the heterochromatin. A recent report suggests that some region of the Xi may not consistently associate with XIST RNA (44). It will be interesting to determine how this relates to the organization of genes at the irrespective of activity, are separated from noncoding sequences periphery of the Xi chromatin that we report here. Although we and and positioned on the surface of the XIST RNA or rim (outer 0.15 others (12, 45, 46) typically see that XIST RNA paints most of the radius) of the DNA territory, suggesting that XIST RNA is not Xi DNA and heterochromatin markers (such as UbH2A, methyl- acting at a cytological level to sequester inactive genes from ‘‘active’’ K20, and methyl-K27) at interphase, we do occasionally see gaps in regions of chromatin. Our findings suggest an emerging view of the the interphase XIST RNA territory (see Fig. 1D for an example and interphase inactive X (see Fig. 10, which is published as supporting ref. 12). Although it has been reported that during mitosis XIST information on the PNAS web site) in which the Barr body RNA avoids the centromere on telocentric rodent X chromosomes comprises inactive noncoding sequences, and genes, silenced or (47), we generally find the human X centromere inside of the active, are relegated to a narrow region at the surface of the XIST periphery of the XIST RNA territory at interphase with no RNA territory. These findings not only point to a potential role for consistent hole of RNA signal. We have reported that XIST RNA XIST RNA in an unanticipated architecture of the Xi but also may selectively falls off the human chromosome late in interphase such help clarify gene organization on all chromosomes more generally. that wide gaps and ultimately discrete bands can sometimes be seen The metaphase chromosome has long been known to be pack- as the cell progresses through mitosis (12, 13, 46–48), suggesting aged into gene-rich and gene-poor bands (29). The bulk of the that specific chromatin may have high-affinity binding sites for chromosome (Ͼ90%) is composed of nongenic sequences that may XIST RNA. Our transcriptional inhibition studies reveal a selective help organize the interphase chromosome by separating genes from retention of XIST RNA as a ring around the Barr body (see Fig. 11, noncoding sequences. Because the XIST RNA territory overlaps which is published as supporting information on the PNAS web site, and distributes throughout the Barr body (12) results here demon- and ref. 12), which may have some relationship to the mitotic strate that the bulk of what the XIST RNA associates with in nuclei banding pattern of XIST in gene-rich R bands. is not genes per se, but the core of repeat-rich DNA. Our present Other studies of gene organization on active autosomes have (Fig. 4 K–M) and previous results (26, 30) demonstrate that Cot-1 yielded varying results. Although a body of evidence supports that RNA is prevalent throughout active chromosome territories. These genes are found on the border of chromosome territories (19, 20, findings help to reconcile the apparent discrepancy between de- 49–51); specific genes have been shown to express from either tecting transcription throughout the nucleoplasm (31) and the inside, on the periphery or well outside of the territory (52–54). concept that genes are organized at the periphery of the territory. Other reports show that sequences (particularly large clusters of Results in this study strongly support that this Cot-1 RNA does not genes) move from a more internal to an external location upon represent just repeats within introns of precursor mRNAs, but the transcriptional activation (53, 55–57). A previous study of two silencing of copious nongenic repetitive transcripts throughout the X-linked genes suggested that a gene subject to inactivation was chromosome. Hybridization kinetic analysis suggests that large- more internal than a gene that escaped X inactivation (57). Al- scale ongoing transcription of nongenic sequences (including both though we find that genes are positioned strikingly at the periphery noncoding and repetitive elements) represent anywhere from one- of the XIST RNA and the Xi chromatin; these positions are not as half to two-thirds of all transcription occurring in the nucleus (23, sharply defined relative to the Xa chromatin. However, we do 32–37). The absence of this transcription in the Barr body makes the observe a strong tendency for genes to be positioned near the edge important point that it is regulated and not merely promiscuous of Xa, and this tendency is even more pronounced when one transcription. Ongoing transcription of this ‘‘junk’’ DNA may serve considers that the bulk of the signals conservatively scored as IN to facilitate gene expression on an euchromatic chromosome; were within a hole, a likely invagination, and as such, are technically conversely, silencing of these repetitive elements may serve to on the border of the territory. Because genes may move a fraction create an inactive nuclear microenvironment. We speculate that it of a micron to interact with ‘‘transcription factories’’ (52), we do not is the ability of XIST RNA to bind and condense the repetitive rule out some differences at that level. heterochromatin found in the inner core of Xi that results in Our conclusions go beyond the relevance for Xi chromosome chromosome-wide silencing, an idea consistent with both evidence structure to the organization of genes within interphase chromo- that gene-poor DNA is found more interior to chromosome terri- some structure more generally. These results are consistent with tories (38) and that noncoding repetitive motifs may be involved in previous studies of gene organization that suggest that chromo- X inactivation (14, 39, 40). Although Alu and LINEs are prominent somal boundaries are specific compartments in which genes (19, 31,

7692 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601069103 Clemson et al. Downloaded by guest on October 4, 2021 49, 50, 58, 59) and splicing and transcription factors localize (22, 49, Detection of the whole X library, ␣-satellite, and subtelomeric 51, 60, 61). The act of silencing and condensing the inner core of sequences was done according to manufacturers protocols with Xi may make the existing concentration of protein-coding genes in minor modifications. Cells were extracted and fixed according to the peripheral region more pronounced and observable. Therefore, the no air drying technique described above. A detailed description we suggest that localization of genes to the outer border of can be found in Supporting Materials and Methods. chromatin is not unique, but Xi makes this organization, which may For detection of bulk repetitive primarily nongenic sequences, be representative of how all interphase chromosomes are orga- human Cot-1 DNA was nick-translated and used to detect DNA nized, more striking. sequences with or without denaturation as described in ref. 26. For oligonucleotide hybridization, a biotin incorporated GATA and Materials and Methods FITC-direct-labeled XIST oligo probe were combined and diluted Cells and Cell Culture. Normal female diploid fibroblast (lung) cell in hybridization buffer with 10% formamide, heat-denatured at lines used were CCL 75 (WI-38; American Type Culture Collec- 80°C for 10 min, and hybridized to denatured cells prepared and tion) and AG06173A (TIG1; Coriell Cell Repositories, Camden, fixed according to our standard protocols. Hybridization was per- NJ). Cells were routinely cultured in DMEM with 15% FBS. formedfor3hat37°C. The initial wash was performed at reduced stringency: 10% formamide͞2ϫ SSC for 10 min at 37°C. A detailed Cell Preparation for in Situ Hybridization. For experiments described description of DNA probes used can be found in Supporting in Figs. 4, 6, 7, 11, cells were prepared for in situ hybridization Materials and Methods. according to our standard protocol (62–64). For all other experiments, an alternate technique with no air or ethanol dehydration Immunofluorescence. For detection of nascent RNA transcription, was used (21, 50). Cells were fixed before extraction in 4% cells were incubated with the H5 (Covance, Princeton, NJ) antibody paraformaldehyde͞PBS for 10 min at room temperature (RT), (1:100dilution)in1%BSAfor1hat37°C. Slides were rinsed in PBS rinsed in PBS, incubated in 0.1 M HCl for 10 min at RT, extracted 5min,PBS͞0.01% Triton X-100 for 5 min, and finally in PBS 5 min ͞ in 0.5% triton-X-100 0.5% saponin in PBS for 10 min at RT and before detection. then equilibrated for 20 min in 20% glycerol͞PBS at RT. Cells were further permeabilized through a freeze–thaw cycle consisting of Image Analysis and Morphometrics. Optics and image analysis was dipping the coverslip quickly into liquid nitrogen (21). The cells performed as described in refs. 12, 13, and 46. A detailed descrip- were allowed only to thaw and, if not used immediately, were stored tion of the scoring categories can be found in Supporting Materials in PBS at 4°C. and Methods.

In Situ Hybridization and DNA Probes Used. DNA hybridization was We thank Kelly Smith for helpful discussions and reading the manuscript performed as described in refs. 12 and 64–66. A detailed protocol and Chris Lynch and John Butler for experimental help. This work was for dual RNA͞DNA detection can be found in Supporting Materials supported in part by National Institutes of Health Grants GM53234 and and Methods. GM68138.

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