territory reorganization in a human disease with altered DNA methylation

Maria R. Matarazzo*, Shelagh Boyle†, Maurizio D’Esposito*‡, and Wendy A. Bickmore†‡

*Institute of Genetics and Biophysics ‘‘Adriano Buzzati Traverso,’’ Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Naples, Italy; and †Medical Research Council Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh EH4 2XU, United Kingdom

Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved September 4, 2007 (received for review April 2, 2007) Chromosome territory (CT) organization and chromatin condensa- nodeficiency centromeric instability facial anomalies (ICF) syn- tion have been linked to expression. Although individual drome patients (14) lead to loss of DNA methylation at specific can be transcribed from inside CTs, some regions that have genomic sites (15, 16). Prominent sites of hypomethylation in constitutively high expression or are coordinately activated loop ICF are satellite DNAs in the juxtacentromeric heterochromatin out from CTs and decondense. The relationship between epige- at chromosome regions 1qh, 16qh, and 9qh, and aberrant netic marks, such as DNA methylation, and higher-order chromatin association of the satellite II-rich 1qh and 16qh regions has been structures is largely unexplored. DNMT3B mutations in immuno- seen in nuclei of ICF cells (17). Sites on the female inactive X deficiency centromeric instability facial anomalies (ICF) syndrome chromosome (Xi), especially CpG islands, are also hypomethy- result in loss of DNA methylation at particular sites, including CpG lated in ICF syndrome. This does not reflect a chromosome-wide islands on the inactive (Xi). This allows the specific defect in gene inactivation on Xi. In female ICF cells, the Xi still effects of DNA methylation on CTs to be examined. Using fluores- forms a Barr body, is associated with the XIST noncoding RNA, cence in situ hybridization, we reveal a differential organization of and mostly replicates late (15). However, two genes, SYBL1 and the human pseudoautosomal region (PAR)2 between the CTs of the G6PD, do show abnormal escape from X chromosome inacti- X and Y in normal males and the active X (Xa) and the Xi in females. vation (XCI) in female ICF cells (16). SYBL1 is located in the There is also a more condensed chromatin structure on Xi com- pseudoautosomal region (PAR)2 of the long arms of the human pared with Xa in this region. PAR2 genes are relocalized toward the X and Y . In this region, genes that are subject to, outside of the Y and Xi CTs in ICF, and on the Xi, we show that this and that escape, XCI are closely linked. Of the two PAR2 genes can extend to genes distant from the site of DNA hypomethylation (SPRY3 and SYBL1) that are subject to XCI, only SYBL1 is itself. This reorganization is not simply a reflection of the tran- modified by DNA methylation (at its CpG island) on both Xi and scriptional activation of the relocalized genes. This report of the Y of male cells. In ICF, hypomethylation of the SYBL1 CpG altered CT organization in a human genetic disease illustrates that island is associated with abnormal expression of both the Xi- and DNA hypomethylation at restricted sites in the genome can lead to Y-linked SYBL1 alleles (14). The inactivation of SPRY3 is more extensive changes in nuclear organization away from the independent of DNA methylation and is unaltered in ICF cell original site of epigenetic change. lines (Fig. 1 and Table 1) (14, 18). Because the hypomethylation defect is restricted to a limited X inactivation ͉ Y chromosome number of loci, ICF cells provide a unique opportunity to examine the specific effect of loss of DNA methylation on the organization of CTs. We used interphase FISH to examine o fully understand the orchestration of gene expression, it is the nuclear organization of PAR2 genes on the long arms of the Tnecessary to understand how chromatin is spatially orga- human X and Y chromosomes (Fig. 1). We show that there is a nized within the cell nucleus. In particular, the position of a gene differential organization of the human PAR2 among the CTs of with respect to its chromosome territory (CT) and the chromatin the X and Y in normal males and the active X (Xa) and the Xi condensation of its genomic region have been linked to gene in females. We also provide direct evidence for a more con- activation and repression (1). Although individual genes can be densed chromatin structure in the PAR2 region on Xi compared transcribed from inside of CTs (2), some regions that have with Xa. In ICF, we show that PAR2 genes are relocalized constitutively high gene expression (3, 4) or are subject to toward the outside of the Y and Xi CTs and that, on Xi, this coordinate gene activation during development (5, 6) or differ- relocalization is not limited to genes that are themselves directly entiation (7) or in response to physiological stimuli (8) locate at subject to hypomethylation and is not just a passive consequence the edge or outside of their CT. This level of nuclear reorgani- of their transcriptional activation. This report of altered CT zation is often accompanied by a visible level of chromatin organization in a human genetic disease illustrates that the decondensation (5, 6, 9). The precise function, if any, of repo- influence of DNA hypomethylation at restricted sites in the sitioning toward the outside of CTs remains unclear (1), but it may allow for genes to access a nuclear environment enriched in the components of the transcription (10) and/or mRNA- Author contributions: M.D. and W.A.B. designed research; M.R.M. and S.B. performed processing machinery (4, 11, 12) and so enhance the efficiency research; M.R.M., M.D., and W.A.B. analyzed data; and M.R.M., M.D., and W.A.B. wrote the of transcription. paper. The interplay between this level of higher-order chromatin The authors declare no conflict of interest. organization and epigenetic mechanisms that act at primary This article is a PNAS Direct Submission. levels of chromatin structure, such as DNA methylation, is a Abbreviations: CI, confidence interval; CT, chromosome territory; ICF, immunodefi- little-explored area. In plants, changes in the architecture of CTs ciency centromeric instability facial anomalies; LCL, lymphoblast cell line; PAR, pseudo- autosomal region; Xa, active X chromosome; XCI, X chromosome inactivation; Xi, can be induced by treatment with inhibitors of DNA methylation inactive X chromosome. (13). However, the genome-wide demethylation that follows ‡To whom correspondence may be addressed. E-mail: [email protected] or 5-azacytidine treatment or genetic deficiency in DNA methyl- [email protected]. transferases makes it difficult to investigate the direct effects of This article contains supporting information online at www.pnas.org/cgi/content/full/ DNA methylation on chromosome organization at specific loci. 0702924104/DC1. Mutations in the DNA methyltransferase DNMT3B in immu- © 2007 by The National Academy of Sciences of the USA

16546–16551 ͉ PNAS ͉ October 16, 2007 ͉ vol. 104 ͉ no. 42 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0702924104 Downloaded by guest on October 1, 2021 Differential Nuclear Organization of the PAR2 Region Between the Xa and Xi. This correlation, between the differential CT position of X- and Y-linked SPRY3 and SYBL1 alleles and their expression, could reflect inherent differences between the organization of the X and Y chromosomes or could be part of a more general allele-specific silencing pathway for these genes. In the latter case, there may also be differential positioning of SPRY3 and Fig. 1. Genomic map of PAR2 of the human sex chromosomes. Map of the SYBL1 relative to the CTs of the Xa and Xi in female cells. PAR2 on Xq and Yq showing, from the centromere to the telomere (tel), the To address this question, we first exploited a female fibroblast positions of the genes (open boxes), and the cosmid probes (gray lines) used cell line (GM07693) with a balanced translocation, 46,X,t(X;10) in FISH experiments. The hatched box indicates the heterochromatin of Yq12. (p11.2;q24.3) (Fig. 2c). X inactivation in this cell line is skewed (Ͼ95:5) such that the normal X chromosome is the Xi, and expression of SPRY3 and SYBL1 originates from the translo- genome is far more extensive than previously imagined, which cated X (14, 24). We distinguished the Xa and Xi in 2D FISH, might, in part, explain why genes whose expression is dysregu- using paints for both 10q and the X (Fig. 2c). Thirty to 40% of lated in ICF are often found not to be the direct targets of DNA the SPRY3 and SYBL1 hybridization signals are located at the hypomethylation themselves (19). edge or outside of the Xa territory, whereas this is seen for only 4–8% of signals on the Xi (SI Fig. 5b). Therefore, both SPRY3 Results and SYBL1 have significantly different positions relative to the Differential Nuclear Organization of the PAR2 Region on the X and Y. Xa and Xi CTs (Fig. 2d). Differential nuclear organization is SYBL1 and SPRY3 are transcribed only on the X in male cells and restricted to the two PAR2 genes with allele-specific expression the Xa in female cells (20, 21). The silent Xi and Y alleles of both profiles. Signals for IL9R, which is not expressed in these genes are associated with repressive histone marks, but only fibroblast cells, occupy similar positions relative to the edge of SYBL1 silencing is associated with DNA methylation (Table 1) the Xa and Xi CTs. (14, 18). We first used 2D FISH to compare how the differen- To exclude any effect of the translocation on the organization tially expressed alleles of SPRY3 and SYBL1 are organized of the Xa in GM07693 and to confirm our findings by 3D FISH relative to territories of the X and Y in cells from a normal male in formaldehyde-fixed cells, we performed combined RNA/ (Fig. 2a). 2D FISH allows for fast and automated image analysis DNA FISH on a female cell line with a normal karyotype (WI38 and can reveal differences in CT organization, even though it fibroblasts), using RNA FISH for XIST to identify the Xi (Fig. exaggerates the absolute distances measured as compared with 2e). In these cells, SPRY3 and SYBL1 are expressed, but IL9R is 3D FISH and so may not fully reflect in vivo structure (6, 22). On not transcribed (data not shown). We first analyzed individual the Y, we found that most (Ϸ70%) SPRY3 and SYBL1 hybrid- CTs in their plane of focus, using the same scripts as for 2D ization signals are inside, or just at the edge (Ϯ0.2 ␮m), of the FISH. This confirmed the more external localization of SPRY3 CT, consistent with the silent state of these genes. There were and SYBL1, but not IL9R, on the territory of the Xa compared Ͻ5% of signals located far (Ͼ0.6 ␮m) outside of the CT of the with the Xi (Fig. 2f and SI Fig. 5c). In addition, we visually Y [see supporting information (SI) Fig. 5a]. In contrast, although examined PAR2 nuclear organization in these cells on z-stacks most of the hybridization signals for the expressed X-linked taken at 0.2-␮m intervals along the z axis and manually scored alleles are still localized inside of the CT, 24–36% are located the alleles as inside or outside of the CT. In 80% of cell signals outside. This may represent the proportion of alleles that are for SPRY3 and SYBL1, Xa alleles were more outside of the CT ϭ actively transcribing, because SYBL1 primary transcript is ob- than their counterparts on Xi (n 25). These data suggest that served in Ϸ35% of cells by RNA FISH (data not shown). This the location of PAR2 genes within the CT is not just a gener- differential organization of the PAR2 region on the X and Y is alized feature of the organization of Xa, Xi, or Y but that it reflected in the average position of the genes relative to each CT. correlates to the specific regulation of gene expression. The X-linked alleles are significantly closer to the edge of the CT than the Y-linked alleles (Fig. 2b). The Xi Is More Condensed Than the Xa at PAR2. Movement away IL9R is weakly expressed in our EBV-transformed lympho- from CTs is often, but not always, accompanied by a visible increase in the interphase distance (d) between probe signals. blast cells (data not shown). However, in cell types where it is 2 expressed, it is transcribed from both sex chromosomes (23). Mean-squared interprobe distances (d ) are related to genomic separation, but differences in d2 for a constant genomic sepa-

Consistent with this, both alleles of IL9R have the same location CELL BIOLOGY ration are thought to represent a decondensation of higher-order relative to the X and Y CTs (Fig. 2b), which suggests that chromatin structure (5, 6, 9, 25). We used 2D FISH to measure transcribed PAR2 alleles are located more externally to the CT d between SPRY3 and SYBL1, SYBL1 and IL9R, and SPRY3 and than inactive ones. IL9R in both male and female (GM07693) cells. The translo- cated X (Xa) in GM07693 female cells was identified with a BAC 2 Table 1. Summary of the transcriptional behavior of the PAR2 probe from 10q25 (Fig. 3a). Differences in d between the X and genes on the Xi and Y chromosomes and the involvement of Y in male cells were not statistically significant (Fig. 3 b and c), DNA methylation in their regulation in both WT and ICF cells even though the PAR2 genes are differentially expressed and spatially organized between these two chromosomes. There is Gene also no significant difference in the SYBL1–IL9R interprobe SPRY3 SYBL1 distances between the two X chromosomes of female cells. Feature Chromosome (WT/ICF) (WT/ICF) However, there is a significant (P Ͻ 0.000) increase in d2 between Ϫ ϪϪϩ the SPRY3 and SYBL1 alleles of the Xa compared with those on Expression Xi / / the Xi, which is also confirmed by the measurement of the Y Ϫ/ϪϪ/ϩ distance between SPRY3 and IL9R genes (Fig. 3 b and c). DNA methylation Xi Ϫ/Ϫϩ/Ϫ Therefore, there is decondensation of the chromatin on the Xa, Y Ϫ/Ϫϩ/Ϫ specifically in the SPRY3–SYBL1 region.

Matarazzo et al. PNAS ͉ October 16, 2007 ͉ vol. 104 ͉ no. 42 ͉ 16547 Downloaded by guest on October 1, 2021 Fig. 2. Nuclear organization of PAR2 in male and female cells. (a) DNA FISH, using Y (top row) or X (bottom row) chromosome paints (green) together with cosmid probes (red) for SPRY3, SYBL1, and IL9R on methanol/acetic acid (MA)-fixed nuclei of normal male LCLs. Nuclei were counterstained with DAPI (blue). (Scale bar, 5 ␮m.) (b) Mean [Ϯ95% confidence interval (CI)] position for each allele of the three PAR2 genes relative to the edge of the X and Y CT in MA-fixed cells. Because the territory of the X chromosome is larger than that of the Y (reflecting their different DNA contents), the position of each cosmid probe signal was normalized relative to the radius of a circle of area equal to that of the corresponding CT. The significance of the differences in position between X- and Y-linked alleles is indicated. *, P Յ 0.01; **, P Յ 0.005. (n Ն 70.) (c) Four-color DNA FISH, using chromosome paints for 10q (red) and the X (green), together with an SYBL1-specific probe (yellow, arrow) on nuclei of the female fibroblast cell line GM07693 that carries a balanced translocation, 46,X,t(X;10). The normal chromosome is Xi. The karyogram of the normal and derivative chromosomes X and 10 is shown at Left. (Scale bar, 5 ␮m.) (d) Mean (Ϯ95% CI) position (in micrometers) for each allele of the three PAR2 genes relative to the edge of the Xa and Xi CT in MA-fixed GM07693 cells. The significance of the differences in position between alleles is indicated. ***, P Յ 0.000. (n Ն 65.) (e)(Left) RNA FISH with XIST probe. (Right) DNA FISH, using SYBL1 and X chromosome paint. Shown are pictures of the same nucleus on two different planes of focus in the z axis to visualize the 3D position of SYBL1 probe (Scale bar, 5 ␮m.). (f) Mean (Ϯ95% CI) position (in micrometers) for each allele of the three PAR2 genes relative to the edge of the Xa and Xi CT in paraformaldehyde-fixed WI38 cells. ***, P Յ 0.000. (n Ն 50.)

Altered CT Organization of the PAR2 Region in ICF Syndrome. Biallelic increase in the interphase distances between SPRY3 and SYBL1 SYBL1 expression in two ICF cell lines, derived from male (PT5) but not between SYBL1 and IL9R (P ϭ 0.22) (Fig. 4d), similar and female (PT3) patients, accompanies reduced CpG methyl- to what we saw when comparing the X and Y or the Xa and Xi ation at its promoter. Additionally, all of the differential histone alleles in normal cells (Fig. 3). The distance measured between modifications tested are erased in the same region (14, 16, 26). the SPRY3 and IL9R in normal and ICF cells is not significantly By contrast, the silencing of SPRY3 Xi and Y alleles is unaltered different. in these ICF cells, consistent with the fact that DNA methylation Because expression of SYBL1 is also altered from the Xi in is not involved in the inactivation of this gene (Table 1) (18). ICF, we analyzed its position within the Xi CT in control female To determine whether the altered gene expression and DNA fibroblasts (WI38) and fibroblasts from the PT3 ICF patient, methylation of SYBL1 affects its nuclear organization, we de- using XIST RNA FISH before 3D DNA FISH to distinguish termined the CT position of PAR2 alleles in ICF (PT5) lym- between the Xa and Xi. Compared with WT, there is significant phoblast cell lines (LCLs) by 2D FISH (Fig. 4a). Whereas SYBL1 relocalization of SYBL1 to the outside of the Xi CT in ICF cells signals are mainly inside or just at the edge of the Y territory in (Fig. 4e and SI Fig. 6b) so that it occupies an average position WT cells (Fig. 2b and SI Fig. 5a), in PT5 cells, Ͼ60% of signals similar to the Xa allele (Fig. 4b and SI Fig. 5e). localize far outside of the territory (Fig. 4a and SI Fig. 6a) and Reorganization of Xi and Y territories in ICF cells could be a indeed seem to be even more external than the X-linked allele direct effect of altered DNA methylation on higher-order chroma- (Fig. 4b and SI Fig. 5d). Compared with WT cells, there is a tin structure, or it could be a downstream consequence of tran- significant relocalization of SYBL1, but not of SPRY3 and IL9R, scriptional activation of SYBL1. However, although there is no to a position external to the Y territory in ICF cells (Fig. 4c). This relocalization of SPRY3 detected within the Y territory of ICF cells, relocalization is also accompanied by a significant (P ϭ 0.001) there is a relocalization of SPRY3 signals toward a more external

16548 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0702924104 Matarazzo et al. Downloaded by guest on October 1, 2021 Fig. 4. PAR2 interphase organization in control and ICF male cells. (a) 2D FISH with probe for SYBL1 (red) and Y chromosome paint (green) in control (Left) and ICF (Right) male LCLs. (b) Mean (Ϯ95% CI) position (in micrometers) for each allele of the three PAR2 genes relative to the edge of the X and Y CT (Left) Fig. 3. Interphase chromatin condensation of PAR2. (a) FISH with probes for and Xa and Xi (Right) in ICF male LCLs and female fibroblasts. The significance SPRY3 and SYBL1 in nuclei from male (Left) or female GM07693 (Right) cells. of differences in position between alleles is indicated. *, P Յ 0.01; **, P Յ 0.005. The X- and Y-linked alleles in male cells were distinguished by cohybridization (n Ն 65.) (c) Mean (Ϯ95% CI) position (in micrometers) for SYBL1-Y, SPRY3-Y, with a Y chromosome paint. The Xa and Xi alleles in GM07693 cells were and IL9R-Y alleles relative to the edge of the Y territory in WT and ICF cells. ***, distinguished by hybridization with a chromosome 10 BAC (RP11 596L14). (b) P Յ 0.000. (n Ն 70.) (d) Distribution of squared interphase distances (in square Distribution of squared interphase distances (in square micrometers) mea- micrometers) measured between SPRY3 and SYBL1, SYBL1 and IL9R, and sured between SPRY3 and SYBL1, SYBL1 and IL9R, and SPRY3 and IL9R probes SPRY3 and IL9R probes on Y chromosomes in WT and ICF cells. (n Ն 50.) (e) on X and Y in male cells (Left) and Xa and Xi in female cells (Right). (n Ն 57.) Mean (Ϯ95% CI) of the position (in micrometers) for SYBL1-Xi, SPRY3-Xi, (c) Mean (Ϯ95% CI) squared interphase distances (in square micrometers) IL9R-Xi, and IDS-Xi alleles relative to the edge of the Xi territory in WT (WI38 measured between SPRY3 and SYBL1 and SYBL1 and IL9R probes on X and Y for SYBL1, SPRY3, and IL9R and GM01871 for IDS) and ICF fibroblasts. ***, P Յ in male cells (Left) and Xa and Xi in female cells. (n Ն 57.) 0.000. (n Ն 50.) CELL BIOLOGY

position within the Xi territory in ICF cells, even though this gene hypomethylation in ICF does not result in a general disorgani- remains transcriptionally silent (Fig. 4e)(P ϭ 0.01). zation of the Xi CT. These data reveal a more extensive effect of DNA hypom- Discussion ethylation on the organization of the Xi than is seen on the Y. In this study, we have shown that nuclear organization depends It has been suggested that nearly all CpG-island associated genes on chromosomal context and function, not on DNA sequence per on Xi may be hypomethylated in ICF (16). To determine whether se. We found that the PAR2, which undergoes a relatively high there is a more generalized effect of DNA hypomethylation on rate of recombination between the X and the Y (27), is differ- the organization of the Xi CT, we examined the gene IDS that ently organized between the CTs of the X and Y in male cells Ϸ is located 6 Mb proximal of PAR2 in Xq28. IDS is hypom- (Fig. 2b) and between the Xa and Xi of female cells (Fig. 2 d and ethylated but not expressed on Xi in PT3 ICF fibroblasts (16). f). In normal male and female cells, CT organization correlates Consistent with its localization in a gene-dense region of the with the expression status of PAR2 alleles, with the expressed (3), in GM01871 control female fibroblasts, IDS alleles located toward the outside of their respective CTs. is localized toward the outside of the Xi CT, even though it Our data, showing that active PAR2 alleles appear positioned remains transcriptionally silent (Fig. 4e and SI Fig. 6c). There is toward the outside of the Xa CT and that their inactive coun- no relocalization of IDS in ICF cells (P ϭ 0.15). Therefore, DNA terparts on Xi are positioned well inside of that CT, are

Matarazzo et al. PNAS ͉ October 16, 2007 ͉ vol. 104 ͉ no. 42 ͉ 16549 Downloaded by guest on October 1, 2021 consistent with some other studies of Xi organization. A study of tional activation of relocated genes if the right transcription X-linked ANT2 (subject to XCI) and the PAR1 gene ANT3, factor environment is available (33). This may explain the failure which escapes inactivation, suggested a differential position to detect DNA hypomethylation at many genes whose expression between inactive and active alleles. The active alleles were is dysregulated in ICF (19). located toward the edge of X chromosome CTs, whereas the inactive ANT2 allele was positioned toward the interior of the Xi Materials and Methods CT (28). CT organization has also been linked to the initiation Cell Culture. Cell lines used in this study were as follows: male of XCI. Very early in mouse ES cell differentiation, genes that EBV-transformed human LCL from a healthy individual; female are yet to be inactivated are found outside of the XIST RNA fibroblast cell line GM07693 (National Institute of General domain that coats the territory of the Xi. They then become Medical Sciences, Bethesda, MD), carrying a balanced localized inside of the XIST domain as they inactivate (29). Even translocation, 46,X,t(X;10),(Xqter3Xp11.2::10q24.3310qter; in differentiated mouse somatic cells, genes that escape XCI 10pter310q24.3::Xp11.13Xpter) (24); normal female diploid remained outside of the XIST domain (29). In contrast, it has fibroblast cell line GM01871 (National Institute of General been suggested in one study that -coding genes of the Medical Sciences); normal female diploid fibroblast (lung) cell human Xi, including the PAR1 gene MIC2, tend to be arranged line WI-38 (American Type Culture Collection, Manassas, VA); at the periphery of the CT, regardless of whether they do or do LCLs from the male ICF patient PT5 (34); and fibroblast cell line not escape XCI, with the internal volume of the Xi composed of from the female ICF patient PT3 (16). noncoding repetitive DNA (30). Although we cannot comment All LCLs were cultured in RPMI medium 1640 supplemented on the distribution of repetitive DNA within the Xi CT, our data with 10% FBS and 2 mM L-glutamine. The growth medium for are consistent with the idea that inactivated alleles are located all fibroblasts was Eagle’s MEM with Earle’s salt, 15% FBS, and well within the Xi CT, whereas active alleles are generally 2mML-glutamine. located at or beyond the edge of the Xa CT (Fig. 2 d and f). We also found evidence for differential chromatin condensation/ Three-Color and Four-Color DNA FISH. Nuclei for 2D FISH were interphase separation between the Xa and Xi (but not between the prepared as described in ref. 3. Cosmid probes (80 ng) encom- X and Y) specifically in the proximal part of PAR2 (SPRY3– passing the PAR2 genes (U160G10, C8.2, and LLJ136; ref. 21) SYBL1) (Fig. 3c). The appearance of the Barr body in the nucleus and paints for the X or Y chromosomes (150 ng) were cohy- of female cells has led many to speculate that there might be bridized together with 5–10 ␮g of human Cot1 and 5 ␮gof increased chromatin condensation on the Xi (31). However, this salmon sperm DNA. Paints and cosmids were labeled by nick idea was questioned by a study that reported equivalent volumes, translation with biotin-16-dUTP and digoxigenin-11-dUTP, re- and hence equivalent levels of overall chromatin condensation, of spectively. Biotinylated probes were detected by using FITC Xa and Xi (32). That study used X chromosome paints prepared followed by biotinylated anti-avidin and a final layer of fluoro- from plasmid libraries and may have lacked the sensitivity to detect chrome-conjugated avidin. Digoxigenin-labeled probe was de- decondensed chromatin located at the periphery of the Xa. We tected with sequential layers of rhodamine-conjugated anti- conclude that there is likely differential chromatin condensation digoxigenin and Texas red-conjugated anti-sheep IgG. between some specific regions of Xa and Xi. In the case of four-color DNA FISH, digoxigenin-labeled In male ICF cells, there is a relocalization to the outside of the probes were detected by using rhodamine anti-digoxigenin and Y CT of the PAR2 gene SYBL1, which is hypomethylated and Texas red anti-sheep IgG (Vector Laboratories, Peterborough, transcriptionally activated. The adjacent Y-linked SPRY3, which U.K.), biotin-labeled probes were detected by using Cy5 strepta- remains silent, is unaltered in its nuclear organization in ICF vidin and biotinylated anti-avidin (Vector Laboratories), and the (Fig. 4c). This differential reorganization is reflected in an X or Y chromosome paints directly labeled with FITC (Cambio, increased interphase separation between Y-linked SYBL1 and Cambridge, U.K.) were detected by using F1 rabbit anti-FITC SPRY3 alleles in ICF cells (Fig. 4d). This would indicate that the and F2 FITC anti-rabbit (Cambio). Hybridization, washing, and effects of DNA hypomethylation on Y chromosome organiza- detection were carried out as described in ref. 3. Slides were tion are restricted to the immediate vicinity of the activated counterstained with 0.5 ␮g/ml DAPI. SYBL1 gene. By contrast, in female ICF cells, both SYBL1 and SPRY3 relocalize to the outside of the territory of the Xi, even Combined RNA and DNA FISH. Cells grown on slides were fixed in though, as in males, only SYBL1 is subject to hypomethylation 4% paraformaldehyde for 10 min, permeabilized in 0.7% Triton and transcriptional activation (Fig. 4e). Hence, in this case, DNA X-100 for 10 min, and stored in 70% ethanol. After progressive hypomethylation causes changes in the interphase organization dehydration in ethanol, the cells were air-dried and hybridized of the Xi that extend far beyond the genes that are immediately overnight at 37°C in 50% formamide/2ϫ SSC with an XIST subject to hypomethylation and transcriptional activation. The probe (provided by C. Brown, University of British Columbia, Xi-allele of SPRY3 appears to be moved toward the periphery of Vancouver, BC, Canada) labeled by nick translation with its CT in the absence of actual activation of gene expression, digoxigenin-11-dUTP. Slides were washed twice in 50% form- reaffirming that a gene’s intra-CT position is not just a passive amide/2ϫ SSC and once in 2ϫ SSC. XIST signal was detected consequence of its transcriptional activation. Analysis of other with rhodamine anti-digoxigenin and Texas red anti-sheep IgG regions of the human genome has suggested that the CT (Vector Laboratories) with washes in 4ϫ SSC/1% Tween 20. organization of a gene is a reflection of its transcriptional After XIST visualization, cells were fixed in 4% paraformal- potential and not activation per se and also depends on the dehyde for 30 min, denatured in 50% formamide/2ϫ SSC at 80°C activity of its surrounding genomic regions (3, 4). In the case of for 15 min, and rinsed in 2ϫ SSC before overnight hybridization the PAR2 on Xi and Y, the different effects of ICF-induced with biotin-16-dUTP-labeled gene-specific probe and chromo- hypomethylation on CT reorganization could be due to the some paint directly labeled with FITC. Probe detection was as nature of the X- and Y-specific sequences just proximal of this that used for DNA FISH. PAR. Although the X-linked PAR2 immediately abuts the gene-rich Xq28 region, the Y-linked PAR2 is adjacent to the Image Capture and Analysis. Slides for 2D FISH were examined by block of heterochromatin at Yq12, and this might restrict CT using an Axioplan fluorescence microscope (Carl Zeiss, Wel- reorganization (Fig. 1). Elsewhere in the genome, altered CT wyn, U.K.) fitted with a Chroma 84000 quadruple-band pass organization encompassing extended regions around hypom- filter set (Chroma Technology, Rockingham, VT). Grayscale ethylated sequences may allow for the inappropriate transcrip- images were captured with an Orca AG CCD camera

16550 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0702924104 Matarazzo et al. Downloaded by guest on October 1, 2021 (Hamamatsu Photonics, Welwyn Garden City, Hertfordshire, 3-axis XYZ stage with integrated piezo Z-drive (Applied Sci- U.K.). Images were collected from 50 to 100 randomly selected entific Instrumentation, Eugene, OR), and a Photometrics Cool- nuclei for each experiment and then analyzed by using a custom snap HQ2 CCD camera (Roper Scientific, Tucson, AZ). The IPLab script that calculates the distance between the probe filterwheels were fitted with a #86000 Sedat quad set with single signals and the nearest CT edge or the distance between two emission filters (Chroma Technology). Image capture was per- signals, as described in refs. 3 and 5. Positive distances indicate formed by using IPLab v4.0 for PC (BD Biosciences, Rockville, that the locus is located inside of the CT, whereas negative values MD). represent signals that are located outside of the detectable limits of the CT. The statistical significance of the data was tested by We thank P. Perry for helpful software assistance; C. Brown for using the nonparametric Mann–Whitney and Kolmogorov– providing XIST probe; and A. Cerase for help in the initial part of the Smirnov tests of the null hypothesis. P Ͻ 0.05 has been taken as work, C. Morey for help with statistical analysis, and both for stimulating statistically significant. discussion. This work was supported by Provincia di Napoli Grant 02 PEG 42 bil.2006 (to M.R.M.), Telethon Grant GGP03208 and a grant For 3D analysis, the imaging system comprised a Zeiss Axio- ϫ from Provincia di Napoli (to M.D.), the U.K. Medical Research Council, vert 200 fluorescence microscope equipped with 100 /1.4 and European Union Sixth Framework Programme Network of Excellence 63ϫ/1.4 plan apochromat objectives, a Lambda LS 300W xenon Epigenome Grant LSHG-CT-2004-503433, and the Short Mobility Pro- source with liquid light guide, 10-position excitation and emis- gram from the National Research Council (M.M.). W.A.B. is a Centen- sion filterwheels (Sutter Instruments, Novato, CA), a PZ2000 nial Fellow of the James S. McDonnell Foundation.

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