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Tsix, a gene antisense to Xist at the X-inactivation centre
Author: Kira Zhaurova
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"letter 400 nature genetics ? volume 21 ? april 1999 Tsix, a gene antisense to Xist at the X-inactivation centre Jeannie T. Lee, Lance S. Davidow & David Warshawsky Department of Molecular Biology, Massachusetts General Hospital; and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. Correspondence should be addressed to J.T.L. (e-mail: lee@molbio.mgh.harvard.edu). In mammals, dosage compensation is achieved by X inactivation 1 and is regulated in cis by the X-inactivation centre 2 (Xic) and Xist (refs 3- 5). The Xic controls X-chromosome counting, choice of X to inactivate and initiation of silencing. Xic action culminates in a change in Xist RNA property from a scarce, unstable RNA (refs 6,7) to highly expressed Xist RNA that coats the future inactive X (ref. 8). Deleting a 65-kb region downstream of Xist results in constitutive Xist expression and X inactivation, implying the presence of a cis-regulatory element 9 . In this region, we now report the discovery of a gene antisense to Xist. Tsix is a 40-kb RNA originating 15 kb downstream of Xist and transcribed across the Xist locus. Tsix sequence is conserved at the human XIC. Tsix RNA has no conserved ORFs, is seen exclusively in the nucleus and is localized at Xic. Before the onset of X inactivation, Tsix is expressed from both X chromosomes. At the onset of X inactiva- tion, Tsix expression becomes monoallelic, is associated with the future active X and persists until Xist is turned off. Tsix is not found on the inactive X once cells enter the X-inactivation path- way. Tsix has features suggesting a role in regulating the early steps of X inactivation, but not the silencing step. We found evidence for an RNA antisense to Xist when examining Xist expression in embryonic stem (ES) cells using strand-specific probes by RNA fluorescence in situ hybridization (FISH). The antisense signal was reminiscent of Xist RNA in that it was pin- point, localized to the Xic and found in male, female and trans- genic ES nuclei (>95%, n>2,000; Fig. 1). Antisense signals were consistently brighter than sense signals, suggesting either greater abundance or accessibility of the antisense RNA. We named this novel transcript Tsix in reference to its antisense orientation. We further characterized Tsix in ES cells, a developmental model for X inactivation 10 . To map the extent of antisense tran- scription, we generated strand-specific probes across a 60-kb region (Fig. 2). In the antisense (?as?) orientation, probes 1as- 11as gave strong signals (>95%, n>2,000 nuclei), whereas probes 12as and 13as gave none. In the sense (?s?) orientation, probes 3s- 8s yielded robust signals (>95%, n>2,000 nuclei), whereas probe 9s gave faint signals in 20- 30% of cells (n>2,000 nuclei). The alternatively spliced exon 7 (ref. 11) of Xist lies within probe 9, so the faint signal may represent its use in some cells. The size and intensity of sense and antisense signals suggested that Xist and Tsix may be expressed at comparably low levels. Analysis of male, female and transgenic ES cells gave identical results. Thus, anti- sense transcription spans 40 kb, including all of Xist, and sense transcription agrees with the published Xist map 4 . Strand-specific RT-PCR confirmed the map of Tsix (Fig. 3a,b). RT-PCR of randomly primed cDNA was used to determine whether a transcript of either sense or antisense orientation existed at 19 positions. No RT-PCR product was detected at posi- tions 1, 18, 19 (Fig. 3) or regions beyond (data not shown). At positions 2- 17, where RT-PCR gave products, strand-specific cDNA was used to determine RNA orientation. In the sense orien- tation (Xist), a PCR product was detected at positions 5- 12. In the antisense orientation (Tsix), a PCR product was seen at positions 2- 17. Analyses of male, female and transgenic ES cells revealed identical results and were consistent with RNA FISH data. To map the 5� end of Tsix, we performed 5�-RACE, sequenced the products and found transcription start sites approximately 14,940 bp and 14,990 bp downstream of Xist in a CpG island 12 Fig. 1 Strand-specific FISH probes reveal an antisense RNA crossing the Xist locus. RNA FISH using fluorescein- labelled (green) strand-specific probes (derived from Xist bp 4,512- 14,458) in male, female and transgenic ES cells. The female line is a mosaic of 40 XX (cells with 2 dots) and 39 XO cells (sin- gle dots). In 116.6, X represents the X- linked locus, whereas T is derived from the 20-copy transgenic locus (deter- mined by RNA/DNA FISH). All nuclei were counterstained with DAPI (blue). � 1999 Nature America Inc. ? http://genetics.nature.com � 1999 Nature America Inc. ? http://genetics.nature.com letter nature genetics ? volume 21 ? april 1999 401 (Fig. 3f,g). These results agreed with primer extension analysis, which yielded a product of approximately 600 bp with a primer positioned at +14,392 bp (Fig. 3e). To determine if Tsix was one continuous RNA, we performed long-range reverse transcription with subsequent PCR at downstream positions (Fig. 3c). An RT- PCR product would be detected only if cDNA extended from the primer to the amplified region. With extension from primer I, specific RT-PCR products were observed up to 9 kb downstream of Xist and were not observed at random locations upstream (position a, Fig. 3c; other positions, data not shown). Using primer II, specific products were detected up to 7 kb into Xist exon 1. Our results linked upstream and downstream antisense transcription with that crossing Xist. Together with the lack of gaps detected by FISH and RT-PCR mapping, this argued that Tsix is a continuous 40-kb antisense RNA that spans all of Xist. RNA slot blot analysis indicated that Xist and Tsix were expressed at comparably low levels in undifferentiated ES cells (Fig. 3d; Tsix at relative levels of 1.0, 1.7 and 7.5 in male, female and transgenic cells, respectively; Xist at 1.0, 1.7 and 5.5). Northern analysis failed to detect Tsix RNA in ES cells. We questioned whether some developmental features previ- ously ascribed to Xist might belong to Tsix, because Xist had his- Fig. 2 Mapping Tsix by RNA FISH. a, Strand-specific probes across a 60-kb Xic region were generated and gel-purified to eliminate double-stranded DNA conta- mination. Probes were labelled with fluo- rescein (green) and hybridized to undenatured ES cells (blue). The chart is a compilation of results from male, female and transgenic experiments, all of which gave identical results. Probes 1- 11 were strand-specific, whereas 12 and 13 were double-stranded (ds). b, Representative RNA FISH results of (a). Transgenic nuclei are shown because the large transgenic RNA clusters are easier to visualize. Cells were prepared without Triton X-100 to visualize cytoplasmic RNAs. c, Slot blot hybridization of single-stranded phage DNA was carried out to verify strand- specificity of templates used to make FISH probes. In the examples shown, sense (1s, 9s) and antisense (1as, 9as) phage DNA templates were hybridized to end- labelled, strand-specific oligonucleotides. Positive control, double-stranded (ds) replicative forms of phage DNA; negative control, pGEM11Zf(+) vector. Fig. 3 Tsix begins 15 kb downstream of Xist and spans 40 kb of sequence. a, RT- PCR was performed at 19 densely spaced intervals to confirm the map of Tsix. The chart is compiled from results of male, female and transgenic ES cells, all of which gave identical results. Ran- domly primed cDNA was first used to determine whether a transcript of either orientation existed at each posi- tion. In such instances, strand-specific cDNA was subsequently tested to determine orientation (positions 2- 17). For sense transcript, an antisense primer (for example, 4as) was used to prime cDNA synthesis; for antisense transcripts, a sense primer (for exam- ple, 4s) was used. First strand cDNA was then amplified with paired sense and antisense primers (for example, 4s+4as). b, Representative strand-spe- cific RT-PCR at select positions shown in (a). s, sense; as, antisense; r, random- primed (double-stranded). Positive control, Rrm2, a housekeeping gene. c, Long-range reverse transcription: first strand Tsix cDNA was synthesized from primers I (4s) and II (11s) and amplified with primer pairs at indi- cated positions. d, RNA slot blot analy- sis of Tsix, Xist and DNA methyltransferase (Dnmt1) was per- formed as described 22 . Total RNA from undifferentiated male, female and transgenic ES cells was slot blotted, hybridized to 10 7 cpm of strand-spe- cific probe and exposed to film overnight; probe 4as (Fig. 2a) was used for Tsix and probe 4s was used for Xist. Since probes were of the same size and from the same region, an approximation of relative Xist and Tsix levels was achieved. Results were quantitated by phosphorimaging with normalization to Dnmt1 values. e, Primer extension was performed to map the 5� end of Tsix. A Southern blot of primer extension product hybridized to a nested primer probe is shown. f, 5�-RACE after first round PCR (lane 1) and nested PCR (lane 2). g, Hybridization of first round RACE products to a nested primer probe to demonstrate specificity. a b a ? ? ? ? ? ? ? ? ? ? ? ? ? ? ??? ? ? ? ?? c d e f g bp ? ? c � 1999 Nature America Inc. ? http://genetics.nature.com � 1999 Nature America Inc. ? http://genetics.nature.com letter 402 nature genetics ? volume 21 ? april 1999 torically been examined by double-stranded probes. Using differ- entially labelled strand-specific probes, we studied the temporal and spatial expression patterns of Xist and Tsix in ES cells in which X inactivation can be induced by differentiation into embryoid bodies 10 (EB). Time-course analysis of EB cells revealed a dynamic relationship correlating with early events of X inactivation (Fig. 4). Before differentiation in females, Tsix and Xist were biallelically expressed at low levels (species 1). During differentiation, transient expression patterns appeared in three forms: species 2, retaining low level Xist and Tsix on one X but lacking Tsix on the other X; species 3, with high level Xist but lacking Tsix on one X and low level Xist and Tsix on the other X; and species 4, with high level Xist and no Tsix on one X and low level Tsix on the other X. From days 2- 9, the transient species increased sharply in number as the undifferentiated species 1 diminished, but began to decline as species 5 became dominant. In species 5, high levels of Xist RNA are expressed on one X and no Tsix is expressed. Species 5 was the somatic pattern of Xist and Tsix expression, as it was the only pattern observed in differenti- ated cells. Although Xist RNA was upregulated on the future inactive X, we did not detect Tsix by RNA FISH (Fig. 4a) or RT- PCR (data not shown) in cells of day 12.5 embryos, fibroblasts and adult tissue including brain, skeletal muscle, heart, kidney, liver and adrenal glands (data not shown). In male ES cells, Xist and Tsix RNA were also expressed in more than 95% of undiffer- entiated cells (Fig. 4b). Upon differentiation, we observed three species: species 1, in which Xist and Tsix persisted (undifferenti- ated form); species 2, expressing only Tsix (transient); and species 3, expressing neither. Species 2 was an apparent interme- diate, appearing on day 2 and disappearing with time. Species 3 was the somatic form, as it became the only pattern observed in fibroblasts (Fig. 4b) and adult tissue (data not shown). Fig. 4 Tsix is dynamically regulated during female and male EB differen- tiation. Xist and Tsix RNAs were simultaneously visualized by differ- ential labelling of sense (probe 7s+8s, green, Xist) and antisense (probes 7as+8as or 11as, red, Tsix) probes. Experiments were performed 2- 4 times by scoring 100- 300 nuclei. Since female X chromosomes are unstable in culture, only those cells with inferred 40XX constitution were scored. 40XX cells were identified by simultaneous RNA/DNA FISH using an X-specific Zfx probe. Approximately 30% were XX and 70% were XO (data not shown). a, Patterns of Tsix and Xist expression in female ES cells, embryoid bodies and fibroblasts. b, Patterns of Tsix and Xist expression in male ES, EB and fibroblasts. c, Rela- tive proportions of each pattern dur- ing differentiation. sp, species. Fig. 5 Tsix RNA co-localizes with Xist RNA and is exclusively nuclear. a, Two-colour RNA FISH of transgenic undifferentiated ES cells using differentially labelled Xist (7s+8s, red) and Tsix (7as+8as, green) probes. b, Metaphase chro- mosomes of a female EB cell showing that only Xist (green, 7s+8s) coats the X. Tsix probes (red, 7as+8as) gave no signal. The DAPI-stained image is in black and white. Arrows, inactive X. c,d, RNA/DNA FISH on denatured transgenic nuclei showed that both Xist and Tsix RNAs (red) are cis-localized to the Xic. In (c), the small RNA clusters co-localized with Zfx (green), a gene tightly linked to the Xic. In (d), larger Xist and Tsix RNA clusters co-localized with vector- specific DNA sequences (green; YAC vector), demonstrating that ectopic Tsix RNA retained its cis property. This co-localization was seen using probes 4as, 7as, 8as and 11as. e, RNA FISH was performed on cells prepared without Triton X-100 to retain cytoplasmic RNAs (ref. 8). Tsix probes 7as, 8as and 11as did not reveal cytoplasmic hybridization, but gave nuclear signals. The serum-induced Fos expression (green, arrows) was seen in the cytoplasm, whereas the uninduced Fos expression was not detected. f, RT-PCR to examine Xist, Tsix and Rrm2 expression was performed on nuclear (N) and cytoplasmic (C) fractions of male (M) and female (F) ES cells and fibroblasts (fib) with either primer pairs 16 or 17 (same results). (- ), negative water control; T, total female ES RNA. a b c a b c d e f % cells � 1999 Nature America Inc. ? http://genetics.nature.com � 1999 Nature America Inc. ? http://genetics.nature.com letter nature genetics ? volume 21 ? april 1999 403 The dynamic relationship between Xist and Tsix in female cells is as follows: (i) before the onset of X inactivation, Xist and Tsix are biallelically co-expressed; (ii) at the onset of X inactivation (differentiation), Tsix becomes monoallelic, preceding upregu- lation of Xist RNA on the future inactive X (species 2); (iii) in the monoallelic form, Tsix RNA associates only with the future active X; and (iv) once X inactivation is established, Tsix is repressed. Tsix expression is specific to undifferentiated cells regardless of sex, persists briefly at the onset of X inactivation, appears to associate only with the future active X and disappears after X inactivation is established. This profile suggests that Tsix may regulate events at the onset of X inactivation. Its antisense nature raises the possibility that Tsix directly blocks Xist RNA action on the X chromosome. Localization of Tsix RNA is consistent with this hypothesis (Fig. 5). In ES cells, Tsix RNA was found only in the nuclear com- partment (Fig. 5e,f) and co-localized with Xist RNA to the Xic (Fig. 5a,c,d). Co-localization was seen only in undifferentiated ES cells (male, female, transgenic) and at the onset of X inactivation when the two RNAs were coexpressed (Fig. 4). Once X inactiva- tion was established, Tsix became undetectable, whereas Xist RNA coated the inactive X. Unlike Xist RNA, Tsix RNA never spread beyond the Xic at any time during differentiation (Fig. 5b). A role for Tsix was further supported by its conservation between mouse Xic and human XIC (Fig. 6). Using BLAST, GCG, GRAIL and Exon Finder, comparative sequence analyses revealed no conserved ORFs in Tsix. Together with its nuclear localization, this suggests that, similar to Xist, Tsix encodes an untranslated nuclear RNA. We have identified a second gene in the Xic. Tsix is not required for the silencing step itself because a 65-kb deletion including Tsix sequences results in constitutive X inactivation 9 . Instead, Tsix features suggest an earlier role, such as in X-chromosome counting, choice or initiation, a role supported by available genetic evidence. First, Tsix lies partially within a 65-kb region containing a putative Xist repressor which is hypothesized to be the counting element 9 ; targeted deletion of this region leads to constitutive X inactivation in cis. Second, Tsix is contained within the 80-kb Xic region (with Xist and 30 kb of downstream sequence), which can recapitulate the regulation of X inactiva- tion in a transgene-based assay 13 . Furthermore, Tsix is expressed by all minimal Xic transgenes capable of recapitulating X inacti- vation (J.T.L., unpublished data). Finally, a cosmid transgene car- rying Xist but lacking the 5� end of Tsix can express Xist RNA and silence a linked reporter gene 14 , consistent with the existence of a repressor, deletion of which results in constitutive Xist expres- sion. At least three modes of Tsix action can be envisioned: (i) Tsix RNA may base-pair with and mask functional domains in Xist RNA; (ii) Tsix transcription may alter chromatin structure and thereby modulate Xist expression; and (iii) Tsix may regulate Xist through expression competition 15 . In mammals, antisense genes have also been found for two imprinted loci, Igf2r (ref. 16) and UBE3A at the Angelman syndrome locus 17 . Thus, antisense regulation may represent a more common mechanism of long- range transcriptional regulation than previously recognized. Methods ES cells and culture. ES cells: male J1, 40XY, 129Sv/J (ref. 18); female EL16, 30%-40XX/70%-39X0, 129x(M.castaneus� 129) (provided by E. Li); and transgenic 116.6, carrying 20 copies of a 450-kb Xic transgene on chromo- some 12 (ref. 2). EL16 is karyotypically unstable and stochastically loses one X during culture. We maintained ES cells as described 18 and induced differentiation by growing in suspension culture without LIF (ref. 19). Female and male fibroblasts came from ed13.5 or adult 129Sv/J mice. Serum-induction of Fos in fibroblasts was performed by growing in DMEM without serum overnight followed by 10- 15-min incubation in DMEM+20% FBS. FISH. FISH probes were labelled as described 2 . Detailed procedures for RNA and DNA FISH have been described 2,20,21 . Small probes were used for detection of RNA (multicopy) and larger probes were used for DNA (single copy) to ensure signal specificity. RNA specificity was confirmed by destruction of signals when samples were pretreated with RNase. RT-PCR. For strand-specific RT-PCR, total cellular RNA (10 m g) isolated by RNAzol B reagent (Teltest) was treated with RNase-free DNase I (2 U) for 1 h at 37 o C. Samples were divided into two for +RT and - RT reactions. Strand-specific Xist primers (3 pmol) were added to total RNA (0.2- 2 m g), heated to 70 o C for 5 min and equilibrated to 50 o C. First strand cDNA was synthesized using either sense or antisense primers with Superscript II reverse transcriptase (200 U; GibcoBRL) for 1 h at 50 o C. Controls without reverse transcriptase were processed in parallel. The enzyme was heat-inac- tivated at 80 o C for 45 min. First strand cDNA was amplified with paired sense and antisense primers using Taq polymerase for 27- 32 cycles with an annealing temperature of 52 o C. For long-range reverse transcription, first strand cDNA was synthesized with 11s (0.5 pmol; extension I) or 4s (3 Fig. 6 The mouse Tsix sequence is conserved at the human XIC. Dotplot analysis of mouse Xic and human XIC sequences using GCG software. Sliding 21-bp win- dows and a stringency of 16 in 21 were used so that a 76% nucleotide identity generated a dot. Significant cross-species homology was detected not only in Xist/XIST as previously reported 3,4 , but also in the 5� third of Tsix, including regions 1, 2 and 3 (circled). Region 1 was 56.7% identical to the human sequence (GCG software penalizing for creating but not for elongating gaps; gap weight of 50, length weight of 0). Regions 2 and 3 were 45.3% identical. No obvious homology lay beyond the CpG island. The identity within the Xist/XIST gene body (63.5% from bp 6,200- 10,200 of Xist) was higher than in regions 1- 3. Overall, the conservation within Xist/XIST and Tsix/TSIX was lower than for average protein-coding exons (b -globin, 82.4% over 444 positions 26 ), but was higher than for random X-linked sequences (38.2% at 50- 53 kb down- stream of Xist) and similar to introns of X-linked protein-coding genes (Tsx/TSX 50.6% over 1,700 positions). � 1999 Nature America Inc. ? http://genetics.nature.com � 1999 Nature America Inc. ? http://genetics.nature.com letter 404 nature genetics ? volume 21 ? april 1999 pmol; extension II) and amplified with Taq polymerase for 33 cycles. Nuclear and cytoplasmic RNAs were prepared as described 22 and first strand cDNA was made by random priming. RT-PCR of fractionated material was carried out to 30 cycles with either primer pairs 16 or 17. Probes. The 250-kb BAC-Zfx genomic clone 2 , vector pYAC4 (ref. 23) and Fos probe p302-356pBS(- ) (ref. 24) have been described. The following probes are described in relation to +1 bp of the Xist cDNA sequence: 1 (?5,216 to - 1,652), 2 (- 1,652 to - 58), 3 (- 1,652 to +1,877), 4 (+4,512 to +6,944), 5 (+6,948 to +8,338), 6 (+7,779 to +10,200), 7 (+10,085 to +12,354) and 8 (+11,601 to +14,458). The following are the indicated dis- tance (bp) from the end of Xist exon 6: 9 (2,301- 5,251), 10 (5,052- 6,150), 11 (10,733- 15,047), 12 (16,282- 21,502) and 13 (21,183- 26,942). To gen- erate strand-specific probes, these sequences were cloned into pGEM-Zf plasmids (Promega) and single-stranded phage DNA was isolated as rec- ommended by Promega. Probe template was gel-purified to eliminate con- tamination by double-stranded plasmid and verified as strand-specific by DNA slot blot hybridization with end-labelled oligonucleotides. The num- bers for sense probes are followed by ?s? and those for antisense by ?as?. Primers. The following primer sequences are described in relation to +1 bp of Xist cDNA: 1s (- 8,534 to - 8,514), 1as (- 8,183 to - 8,202), 2s (- 5,470 to - 5,452), 2as (- 4,872 to - 4,891), 3s (- 402 to - 383), 3as (- 58 to - 77), 4s (- 364 to - 344), 4as (- 154 to - 173), 5s (+272 to +294), 5as (+775 to +755), 6s (+6,760 to +6,779), 6as (+6,944 to +6,926), 7s (+9,407 to +9,426), 7as (164- 145 bp of intron 1), 8s (+9,922 to +9,940), 8as (+10,048 to +10,030), 9s (+11,601 to +11,620), 9as (+12,354 to +12,335), 10s (+13,391 to +13,411) and 10as (+14,500 to +14,484). The following span the indicated distance from the end of Xist exon 6: 11s (49- 66), 11as (301- 284), 12s (2,301- 2,321), 12as (2,478- 2,460), 13s (5,052- 5,072), 13as (5,251- 5,233), 14s (5,982- 6,000), 14as (6,150- 6,132), 15s (9,823- 9,842), 15as (10,181- 10,161), 16s (12,538- 12,556), 16as (12,876- 12,857), 17s (14,416- 14,435), 17as (14,872- 14,854), 18s (15,155- 15,174), 18as (15,476- 15,455), 19s (15,155- 15,174) and 19as (16,015- 15,994). Rrm2A and Rrm2C were as described 25 . 5� end mapping. For primer extension, total RNA (10 m g) from transgenic ES cells were annealed to a primer at position +14,392 to +14,409 bp (downstream of Xist) and extended with Superscript II (GibcoBRL) at 50 o C. The extension product was resolved on a denaturing agarose gel and detected with a nested probe. For 5�-RACE (Marathon kit, Clontech), dou- ble-stranded cDNA was synthesized from 116.6 ES total RNA (8 m g) using a primer at +14,392 to +14,409 bp with AMV reverse transcriptase at 42 o C, and RACE linkers were ligated onto cDNA ends. First round PCR prod- ucts were generated using a primer positioned at +14,416 to +14,435 bp and linker primer ap1 and then probed with a nested primer (+14,677 to +14,659 bp downstream) to confirm specificity. Second round PCR was carried out with nested primers ap2 and nes1 (+14,792 to +14,811 bp downstream). PCR products were cloned and sequenced. GenBank accession numbers. Mouse Xic, L04961, U41394, U41395, U41396, X99946; human XIC, U80460. Acknowledgements We thank E. Li for the generous gift of EL16; J.E. Kirby, B. Kingston, M. Oettinger, N. Stavropoulos and Y. Ogawa for critique of the manuscript. This work was supported by an American Cancer Society Grant (ACS-IRG 173- H) and a Hoechst AG award to J.T.L. Received 19 January; accepted 22 February 1999. 1. Lyon, M.F. Gene action in the X-chromosome of the mouse (Mus musculus L.). Nature 190, 372?373 (1961). 2. Lee, J.T., Strauss, W.M., Dausman, J.A. & Jaenisch, R. A 450 kb transgene displays properties of the mammalian X-inactivation center. Cell 86, 83?94 (1996). 3. Brown, C.J. et al. The human XIST gene: Analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus. Cell 71, 527?542 (1992). 4. Brockdorff, N. et al. The product of the mouse Xist gene is a 15 kb inactive X- specific transcript containing no conserved ORF and located in the nucleus. Cell 71, 515?526 (1992). 5. Penny, G.D., Kay, G.F., Sheardown, S.A., Rastan, S. & Brockdorff, N. Requirement for Xist in X chromosome inactivation. Nature 379, 131?137 (1996). 6. Panning, B., Dausman, J. & Jaenisch, R. X chromosome inactivation is mediated by Xist RNA stabilization. Cell 90, 907?916 (1997). 7. Sheardown, S.A. et al. Stabilization of Xist RNA mediate initiation of X chromosome inactivation. Cell 91, 99?107 (1997). 8. Clemson, C.M., McNeil, J.A., Willard, H. & Lawrence, J.B. XIST RNA paints the inactive X chromosome at interphase: Evidence for a novel RNA involved in nuclear/chromosome structure. J. Cell Biol. 132, 259?275 (1996). 9. Clerc, P. & Avner, P. Role of the region 3� to Xist in the counting process of X- chromosome inactivation. Nature Genet. 19, 249?253 (1998). 10. Martin, G.R. et al. X-chromosome inactivation during differentiation of female tertocarcinoma stem cells in vitro. Nature 271, 329?333 (1978). 11. Simmler, M.-C. et al. A 94 kb genomic sequence 3� to the murine Xist gene reveals an AT rich region containing a new testis specific gene Tsx. Hum. Mol. Genet. 5, 1713?1726 (1996). 12. Courtier, B., Heard, E. & Avner, P. Xce haplotypes show modified methylation in a region of the active X chromosome lying 3� to Xist. Proc. Natl Acad. Sci. 92, 3531?3535 (1995). 13. Lee, J.T., Lu, N.F. & Han, Y. Genetic analysis of the mouse X-inactivation center reveals an 80kb multifunction domain. Proc. Natl Acad. Sci. USA (in press). 14. Herzing, L.B.K., Romer, J.T., Horn, J.M. & Ashworth, A. Xist has properties of the X- chromosome inactivation centre. Nature 386, 272?275 (1997). 15. Bartolomei, M.S., Webber, A.L., Brunkow, M.E. & Tilghman, S. Epigenetic mechanisms underlying the imprinting of the mouse H19 gene. Genes Dev. 7, 1663?1673 (1993). 16. Wutz, A. et al. Imprinted expression of the Igf2r gene depends on an intronic CpG island. Nature 389, 745?749 (1997). 17. Rougeulle, C., Cardoso, C., Fontes, M., Colleaux, L. & Lalande, M. An imprinted antisense RNA overlaps UBE3A and a second maternally expressed transcript. Nature Genet. 19, 15?16 (1998). 18. Li, E., Bestor, T.H. & Jaenisch, R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915?926 (1992). 19. Martin, G.R. & Evans, M.J. Differentiation of clonal lines of teratocarcinoma cells: Formation of embryoid bodies in vitro. Proc. Natl Acad. Sci. USA 72, 1441?1445 (1975). 20. Lawrence, J.B., Singer, R.H. & Marselle, L.M. Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization. Cell 57, 493?502 (1989). 21. Panning, B. & Jaenisch, R. DNA hypomethylation can activate Xist expression and silence X-linked genes. Genes Dev. 10, 1991?2002 (1996). 22. Ausubel, F.M. et al. Current Protocols in Molecular Biology (J. Wiley & Sons, New York, 1994). 23. Burke, D.T., Carle, G.F. & Olson, M.V. Cloning of large DNA segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236, 806?812 (1987). 24. Huang, S. & Spector, D.L. Nascent pre-mRNA transcripts are associated with nuclear regions enriched in splicing factors. Genes Dev. 5, 2288?2302 (1991). 25. Lee, J.T. & Jaenisch, R. Long-range cis effects of ectopic X-inactivation centres on a mouse autosome. Nature 386, 275?279 (1997). 26. Hendrich, B.D., Brown, C.J. & Willard, H.F. Evolutionary conservation of possible functional domains of the human and murine Xist genes. Hum. Mol. Genet. 2, 663?672 (1993). � 1999 Nature America Inc. ? http://genetics.nature.com � 1999 Nature America Inc. ? http://genetics.nature.com "
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