xist gene exposes trans-effects that alter the ...xist rna, macroh2a, and h3 lysine-9 methylation...

Copyright � 2006 by the Genetics Society of AmericaDOI: 10.1534/genetics.105.051375

A Deletion at the Mouse Xist Gene Exposes Trans-effects That Alter theHeterochromatin of the Inactive X Chromosome and the Replication

Time and DNA Stability of Both X Chromosomes

Silvia V. Diaz-Perez,* David O. Ferguson,† Chen Wang,‡ Gyorgyi Csankovszki,§ Chengming Wang,**Shih-Chang Tsai,* Devkanya Dutta,†† Vanessa Perez,* SunMin Kim,* C. Daniel Eller,*

Jennifer Salstrom,* Yan Ouyang,* Michael A. Teitell,‡‡ Bernhard Kaltenboeck,**Andrew Chess,†† Sui Huang‡ and York Marahrens*,1

*Department of Human Genetics and ‡‡Department of Pathology and Laboratory Medicine, University of California, Los Angeles,California 90095, †Department of Pathology and §Department of Molecular, Cellular, and Developmental Biology, University

of Michigan, Ann Arbor, Michigan 48109, ‡Department of Cell and Molecular Biology, Northwestern University,Chicago, Illinois 60611, **Department of Pathobiology, Auburn University, Auburn, Alabama 36849 and

††Center for Human Genetic Research, Harvard Medical School, Boston, Massachusetts 02114

Manuscript received September 22, 2005Accepted for publication August 26, 2006

ABSTRACT

The inactive X chromosome of female mammals displays several properties of heterochromatinincluding late replication, histone H4 hypoacetylation, histone H3 hypomethylation at lysine-4, andmethylated CpG islands. We show that cre-Lox-mediated excision of 21 kb from both Xist alleles in femalemouse fibroblasts led to the appearance of two histone modifications throughout the inactive X chro-mosome usually associated with euchromatin: histone H4 acetylation and histone H3 lysine-4 methylation.Despite these euchromatic properties, the inactive X chromosome was replicated even later in S phasethan in wild-type female cells. Homozygosity for the deletion also caused regions of the active X chro-mosome that are associated with very high concentrations of LINE-1 elements to be replicated very late inS phase. Extreme late replication is a property of fragile sites and the 21-kb deletions destabilized the DNAof both X chromosomes, leading to deletions and translocations. This was accompanied by the phos-phorylation of p53 at serine-15, an event that occurs in response to DNA damage, and the accumulation ofg-H2AX, a histone involved in DNA repair, on the X chromosome. The Xist locus therefore maintains theDNA stability of both X chromosomes.

X-INACTIVATION in female mammals is the for-mation of heterochromatin throughout one of

two X chromosomes early in development (Gartler

and Riggs 1983). X-inactivation requires a region calledthe X-inactivation center (Xic) (Lyon 1996). Physicalhomologous association of the two copies of the Xic hasbeen proposed to trigger X-inactivation (Marahrens

1999) and such an association has recently been shownto mark the onset of X-inactivation (Bacher et al.2006; Xu et al. 2006). The X-linked Xist gene, whichresides in the Xic (Brown et al. 1991), plays a centralrole in the subsequent heterochromatin formation(Penny et al. 1996) and Xist knockout mice die early inembryogenesis due to a failure to undergo X-inactiva-tion (Marahrens et al. 1997). Xist encodes an un-translated RNA that is expressed from the inactive Xchromosome (Xi) but not from the active X chromo-some (Xa) (Brockdorff et al. 1991; Brown et al. 1991).

The Xist RNA is quite stable and colocalizes exclusivelywith the Xi (Brown et al. 1992; Clemson et al. 1996). Inaddition to the role of Xist, the spread of X-inactivationcorrelates with high concentrations of LINE-1 elementson the X chromosome (Lyon 1998). Accordingly, X-linked genes that escape X-inactivation are found in re-gions with reduced concentrations of LINE-1 sequence(Bailey et al. 2000). In cells deficient for the DNAmethyltransferase Dnmt3b, the DNA of LINE-1 ele-ments on the Xi, but not on the Xa, is hypomethylated(Hansen 2003) and X-inactivation is either incompleteor not fully maintained (Hansen et al. 2000).

Another feature that distinguishes the Xi from the Xaand from autosomes is that it is replicated later in Sphase (Taylor 1968; Taylor and Miner 1968). Thereplication timing of the Xi reflects a general trendwhere later replication times are associated with generepression and early replication with transcriptionalcompetence (Gilbert 2002). The available evidenceindicates that the same replication origins are utilizedon the active and inactive X chromosomes (Cohen et al.2003; Gomez and Brockdorff 2004), thus suggesting

1Corresponding author: Department of Human Genetics, UCLA, GondaCenter, 695 Charles E. Young Dr., South Los Angeles, CA 90095-7088.E-mail: [email protected]

Genetics 174: 1115–1133 (November 2006)

that the replication timing differences between the twoX chromosomes stem from the times in S phase thattheir origins are activated. While in female human cellsthe Xi is replicated much later in S phase than the Xa(Priest et al. 1967), the Xi is not replicated nearly as latein S phase in female mouse cells as in human cells(Evans et al. 1965; Galton and Holt 1965; Tiepolo

et al. 1967). This has led to the Xi in mouse cells beingdistinguished by its absence of label incorporation inearly S phase rather than by its being disproportionatelyreplicated late in S phase (Nesbitt and Gartler 1970).Nevertheless, there is always a consistent trend of themouse Xi displaying more label incorporation late in Sphase than the Xa in both primary and transformedfemale fibroblasts (Diaz-Perez et al. 2005).

The protein composition of the Xi also distinguishesit from other chromosomes. The histone H2A homolog,macrohistone H2A, is present along the length of theXi but not the Xa (Costanzi and Pehrson 1998). Inaddition, nearly all of the nucleosomes of the Xi arehypoacetylated at the N-terminal tail of histone H4(Jeppesen and Turner 1993). Histone tail acetylation isa widespread characteristic of euchromatin and histonedeacetylation is a general characteristic of heterochro-matin (Jenuwein and Allis 2001). Furthermore thenucleosomes of the Xi are methylated at histone H3lysine-9 (Peters et al. 2001; Chadwick and Willard

2004) or lysine-27 (Plath et al. 2003; Chadwick andWillard 2004), and both are histone modificationsassociated with heterochromatin. Methylation at H3lysine-4, a euchromatic histone modification that ap-pears to be mutually exclusive to lysine-9 methylation, isconspicuously absent from the Xi (Boggs et al. 2001).Yet another feature that distinguishes the Xi from otherchromosomes is that it is associated with high concen-trations of the BRCA1 protein that associates with XISTRNA (Ganesan et al. 2002). In BRCA1-deficient cells,XIST RNA, macroH2A, and H3 lysine-9 methylation allfailed to concentrate on the Xi (Ganesan et al. 2002,2004).

In addition to its role in X-inactivation, BRCA1 func-tions as a tumor suppressor that plays a role in cellcycle checkpoints, in multiple types of DNA repair,and in the maintenance of genome stability (Scully

and Livingston 2000; Welcsh et al. 2000; Narod andFoulkes 2004). Stalled DNA replication forks as wellas various types of DNA damage, including UV dam-age, cause the ataxia-telangiectasia-mutated and Rad3-related (ATR) kinase to phosphorylate various targetsincluding BRCA1 (Tibbettset al. 2000), p53 at serine-15(Tibbetts et al. 1999), and H2AX (to produce g-H2AX)(Ward and Chen 2001; Ward et al. 2004). Double-strand breaks cause the related ataxia-telangiectasiamutated (ATM) kinase to phosphorylate many of thesame targets including BRCA1 (Cortez et al. 1999;Gatei et al. 2000), p53 at serine-15 (Banin et al. 1998;Canman et al. 1998; Khanna et al. 1998), and histone

H2AX to produce g-H2AX (Burma et al. 2001). g-H2AXassociates with the Xi in the absence of experimentallyincurred DNA damage, but this is restricted to late Sphase (Chadwick and Lane 2005). The phosphoryla-tion of p53 stabilizes and activates the protein, whichsignals for either cell cycle arrest or apoptosis (Attardi

2005). g-H2AX has been proposed to recruit additionalproteins to sites of DNA damage (Bassing and Alt

2004). Deficiency in either ATR or ATM disturbs themaintenance of X-inactivation (Ouyang et al. 2005).

Excision of the transcribed Xist allele from the Xileads to the loss of the Xist RNA and absence ofmacroH2A from the Xi (Csankovszki et al. 1999) andto a destabilization of X chromosomal gene silencing(Csankovszki et al. 2001) but does not abolish latereplication (Csankovszki et al. 1999) or result in anacetylated Xi (Csankovszki et al. 1999). The tran-scribed Xist allele, therefore, functions in cis to main-tain a subset of the features of the Xi heterochromatin.Excision of 21 kb from the nontranscribed Xist locus ofthe Xa results in the Xa being replicated later in S phase(Diaz-Perez et al. 2005). Both Xist alleles therefore dis-play biological activity. Here we show that element(s)at both copies of the Xist gene control the chromatinstructure of the Xi and influence the replication time ofboth X chromosomes. Xist deficiency furthermore de-stabilizes both X chromosomes, leading to deletionsand translocations, the phosphorylation of p53 at serine-15, and the increased association of the DNA repair/genome maintenance protein g-H2AX with the Xi. Xistdeletions therefore reveal trans-interactions that occursubsequent to the initiation of X-inactivation.

MATERIALS AND METHODS

Fibroblasts and growth conditions: All of the mice used inthis study had a 129 genetic background. Mouse primaryfibroblasts were obtained from wild-type 129 mice and alsofrom crosses involving previously described mouse strains(Csankovszki et al. 1999, 2001) by trypsinization of 13-dayembryos, culture, and immortalization with SV40 T-antigen(Jat et al. 1986). Three immortalized fibroblast cell lines wereobtained from three E13.5 mouse embryos (one embryo percell line), in which 21 kb of sequence at the Xist locus wereflanked by Lox sites (floxed) on both the Xa and Xi (XaXist-flox

XiXist-flox). Three additional immortalized fibroblast cell lineswere obtained from three wild-type 129 embryos (XaXist-WTXiXist-WT).The three XaXist-floxXiXist-flox cell lines (XaXist-floxXiXist-flox-1, -2, and -3)and three XaXist-WTXiXist-WTcell lines (XaXist-WTXiXist-WT-1, -2, and -3)were infected with adenovirus expressing cre recombinaseand GFP (Tan et al. 1999) and plated out in 24-well plates atless than one cell per well (limiting dilution) to recover clonalcell lines from each progenitor line. GFP expression was usedto identify infected cells. Starting from the fibroblasts thatarise during the limiting dilution procedure, each clonal cellline was passaged five times. During this passaging, lines thatwere homozygous for the 21-kb deletion (XaXist-D21-kbXiXist-D21-kb-1.1, -2.1, and -3.1) and one line that was heterozygous for thefloxed Xist allele were identified using PCR. RNA FISH for Xisttranscript was used to determine that, in the heterozygous line,the deletion was on the Xi (XaXist-floxXiXist-D21-kb-1.1) (not shown).

1116 S. V. Diaz-Perez et al.

After the aforementioned five passages, the three clonal XaXist-D21-kb

XiXist-D21-kb cell lines and the three clonal XaXist-WTXiXist-WT celllines (from six different embryos) were each subjected to aBrdU pulse (see below) and metaphase spread chromosomeswere prepared. These spreads were analyzed for evidence ofchromosomal deletions and translocations using chromo-some paint, BrdU immunostaining, and spectral karyotyping(see below). Note that the three XaXist-D21-kbXiXist-D21-kb (-1.1, -2.1,and -3.1) and three XaXist-WTXiXist-WT (-1.1, -2.1, and -3.1) cell linesused in the analysis for deletions and translocations were gen-erated using identical procedures. Primary XaXist-D21-kbXiXist-D21-kb

did not grow well enough to perform immunostaining orreplication timing experiments; we are exploring approachesto remedy this.

In addition, cell lines XaXist-floxXiXist-flox-1, -2, and -3 wereinfected with adenovirus expressing only GFP and limitingdilution was used to recover clonal cell lines XaXist-floxXiXist-flox-1.1, -2.1, and -3.1 using the same procedure and the samenumber of passages as was used to obtain lines XaXist-D21-kb

XiXist-D21-kb-1.1, -2.1, and -3.1. Two additional cell lines used inthis study were obtained by infecting fibroblasts that wereheterozygous for the Xist-flox allele with adenovirus express-ing cre recombinase and GFP and using limiting dilution, PCR,and RNA FISH to recover and identify clonal cell lines thatcarried the 21-kb deletion on the Xi (XaXist-WTXiXist-D21-kb-1.1 andXaXist-WTXiXist-D21-kb-2.1). Finally, three XaXist-flox,Hprt-D XiXist-flox,Hprt-WT

cell lines and derivative XaXist-D21-kb,Hprt-D XiXist-D21-kb,Hprt-WTcells wereobtained using the same procedure, except that a series ofadditional mouse matings were first performed to enable theproduction of fibroblasts that also included a published Hprtdeletion (Hooper et al. 1987) on the Xa. The generation of theHprt-heterozygous cell lines is described in detail elsewhere(J. L. Salstrom, C. Wang, C. Wang, D. Dutta, S. Zeitlin, G.Csankovszki, C. D. Eller, S. Diaz-Perez, J. Wang, A. Chess,S. Huang, B. Kaltenboeck and Y. Marahrens, unpublisheddata).

A large proportion of the immortalized cells in each culturecontained either three or four X chromosomes. Limitingdilution was also used to obtain clonal cell lines containingpredominantly two X chromosomes and an approximatelydiploid number of chromosomes. To this end, the same threeimmortalized XaXist-floxXiXist-flox (-1, -2, and -3) cell lines de-scribed in the previous paragraph were infected with adeno-virus expressing cre recombinase and/or GFP (Tanet al. 1999),and limiting dilution was used to recover numerous clonal celllines from each progenitor line. PCR was used to identify XaXist-

D21-kbXiXist-D21-kb cell lines and metaphase spread chromosomesand flow cytometry was used to identify XaXist-D21-kbXiXist-D21-kb andXaXist-floxXiXist-flox lines that were derived from diploid cells. Anumber of these diploid cell lines were subjected to X chromo-some paint to confirm the presence of two X chromosomes.We chose six approximately diploid cell lines containing two Xchromosomes each that were derived from three embryos,with lines XaXist-D21-kbXiXist-D21-kb-1.2 and XaXist-floxXiXist-flox-1.2 fromthe same embryo, XaXist-D21-kbXiXist-D21-kb-2.2 and XaXist-floxXiXist-flox-2.2 from the same embryo, and XaXist-D21-kbXiXist-D21-kb-3.2 andXaXist-flox XiXist-flox-3.2 from the same embryo. Using the identicalprocedure, approximately diploid cell lines containing two Xchromosomes each were also derived from the XaXist-WTXiXist-D21-kb

and XaXist-floxXiXist-D21-kb cells (XaXist-WTXiXist-D21-kb-1.2 and XaXist-flox

XiXist-D21-kb-1.2). The diploid lines thus established were all usedat equally low passage numbers because they all became in-creasingly tetraploid with extended passaging. Diploid XaXist-

flox,Hprt-D XiXist-flox,Hprt-WT and XaXist-D21-kb,Hprt-D XiXist-D21-kb,Hprt-WT cellswere also obtained.

Infection of fibroblasts with adenovirus expressing crerecombinase and/or adenovirus expressing GFP (Tan et al.1999) was performed at 50 multiplicities of infection (MOI) in

6-cm-diameter dishes with 106 cells with virus in 200 ml ofDulbecco modified Eagle’s minimum essential medium(DMEM) with 5% fetal bovine serum (FBS) at 37� for 1 hrfollowed by the addition of 3.0 ml of DMEM with10% FBS.Primary and transformed mouse fibroblast cell lines weregrown in DMEM supplemented with 10% fetal bovine serum(GIBCO, Grand Island, NY), penicillin (100 mg/ml), andstreptomycin (100 mg/ml). The PCR primers 59 LoxF (59-TTTCTG GTC TTT GAG GGC AC-39), 59 LoxR (59-ACC CTT GCCTTT TCC ATT TT-39), and Xint3R (59-CAC TGG CAA GGTGAA TAG CA-39) were used to identify the Xist-flox (612-bpPCR product), Xist-D21kb (513 bp), and Xist-WT (427 bp)alleles. XaXist-D21kbXiWT fibroblasts were distinguished fromXaWTXiXist-D21kb fibroblasts using RNA FISH against the XistRNA (see below).

RNA FISH: Fibroblasts were grown on coverslips for 24 hrand then fixed in 4% formaldehyde for 15 min at roomtemperature (RT). The cells were permeabilized in PBScontaining 0.5% Triton-X for 5 min on ice and washed inPBS and 23 SSC. RNA FISH hybridization was carried out aspreviously described (Spector and Goldman 1998.). The Xistprobe was labeled by nick translation with biotin-21-dUTP.After overnight hybridization at 37� posthybridization washeswere done as previously described (Spector and Goldman

1998). The probe was detected with a 500-fold dilution ofavidin-FITC (Jackson ImmunoResearch, Westgrove, PA) at RTfor 1 hr. The nuclei were counterstained with 49,6-diamidino-2-phenylindole (DAPI).

Two-color DNA FISH: A combination probe, composed of amixture of seven different small probes, was used to identifythe inactive X chromosome. This combination probe, hence-forth referred to as 6.8-kb probe, contains PCR products ofsizes (in base pairs) 412, 605, 609, 850, 1011, 1560, and 1755.Each of these PCR products was amplified separately using TaqPolymerase (Promega, Madison, WI) from a region extend-ing up to 14.7 kb upstream of the 59 end of exon 2 of the Hprtgene and that is deleted in the Hprt-D allele used in this study(Thompson et al. 1989). The mouse BAC RP23-412J16 [pur-chased from Invitrogen (Carlsbad, CA)] served as template forthe PCR reactions. The sequences of primers are: GCA AGCATA AGG ACC AGA GC (412R), TTC CAC AAG AAA TAT TACACA AAA CA (412L), CCT AAC CAT TGA GCC GTC TT(605R), GGT CTC TGA ACT ACC AAT TGC AC (605L), GCAATG ACA AAT GTT TTG TGG (609R), TGC TTA TTA GCACAA GAC CTC AAG (609L), ATC ACC CTA TTC CCA GTGGA (850R), GCA GAT GAT AAG CTA TCC TTG AGA (850L),CAT CAC TGA GTC TTG CTG GTT T (1011R), CAATTTAGGGGA AGG AAG CA (1011L), TGG TAG CTG GGC ATA AAAGC (1560R), AAT GGG AGA AAA GGC AGG AT (1560L), CAGGAA AGG GTG TGT GTG TG (1755R), and TAC GCT CTGGCA GTT TTC AA (1755L). The PCR products were gelextracted using the Wizard PCR Preps DNA purificationsystem (Promega). To mark both the active and the inactiveX chromosomes, mouse BAC RP23-298N24 (obtained fromInvitrogen) was used to obtain a second probe. For fluorescentprobe preparations, 1 mg of DNA was direct labeled witheither FluorX-dCTP (for the whole BAC probe) or Cy3-dCTP(for the Xi-specific 6.8-kb combination probe), using the NickTranslation kit (Amersham Biosciences, Arlington Heights,IL). To prepare the 6.8-kb probe, equimolar concentrations ofthe seven different probes were mixed together, to a total DNAcontent of 1 mg, for labeling. Labeled probes were purifiedusing NucAway Spin columns (Ambion, Austin, TX) andprecipitated with 40 mg mouse Cot-1 DNA, 100 mg salmonsperm, and 100 mg tRNA; washed in 75% ethanol followed by100% ethanol; and resuspended in 100 ml hybridization buffer(50% formamide, 10% dextran sulfate, 13 SSC). Cells weretreated for FISH as described previously (Singh et al. 2003).

Xist Affects DNA Stability 1117

Briefly, cells were fixed with 3:1 methanol–acetic acid, droppedon poly-l-lysine-coated slides (Sigma, St. Louis) in a humidchamber, and denatured for 2 min at 69�–72� in 70%formamide/23 SSC. An 18-ml aliquot of the two probes (12ml of 6.8-kb probe, 6 ml of BAC probe) was prehybridized (90�for 5 min, followed by 10 min at 37�) and then hybridizedovernight with cells at 37�. Next, cells were washed three timeswith 50% formamide/23 SSC at 42� followed by three washeswith 13 SSC also at 42�. The following washes were done atroom temperature: 13 SSC (10 min), 43 SSC (5 min), 43SSC/0.1% Tween-20 (5 min), and 43 SSC (5 min). The cellswere mounted in Vectashield mounting medium containingDAPI (Vector Laboratories, Burlingame, CA) to counterstainthe nuclei. Cells were viewed with a Nikon E600 fluorescentmicroscope. Images were captured with a CCD camera usingSPOT Advanced software.

Quantitative determination of Xist mRNA: Quantitativedetermination of Xist mRNA was performed by one-stepreverse transcription (RT) fluorescence resonance energytransfer (FRET) real-time PCR of 1:100 diluted poly(A) RNAsamples in a Lightcycler modeled after the duplex PCRapproach described earlier (Wang et al. 2004). Xist primers(muXISTmRNAUP, 59-CCC TAC ATC AAA GTA GGA GAAAAG CTG CTG-39; muXISTmRNADN, 59-GAA GGG TAA TATTTG GTA GAT GGC ATT GTG T-39) transversed the bound-aries of exons 4 and 5 and of exons 5 and 6, respectively, andthe FRET probes (muXISTFLU, 59-CCT AGC TTC TGG AGAGAG AAC CAA ATA GAG-6-FAM-39; muXISTBOD, 59-Bodipy630/650-AGA ATG GCT TCC TCG AAG GTC AGT GC-Phosphate-39) detected exon 5. Thermal cycling conditionsof this PCR were 30 min reverse transcription at 55�, followedby 2 min denaturation at 95�, followed by thermal cycling: 6times at 95�, 0 sec/68�, 12 sec/72�, 8 sec; 9 times at 95�, 0 sec/66�, 12 sec/72�, 8 sec; 3 times at 95�, 0 sec/64�, 12 sec/72�, 8 sec;25 times at 95�, 0 sec/56�, 12 sec followed by fluorescenceacqusition/72�, 10 sec. Quantitative standards were producedby PCR amplification with dTTP, gel purification, and quan-tification of the fragment by Pico-Green assay (Invitrogen).Reaction chemistry was as published (Wang et al. 2004). Theinternal autosomal reference gene transcript (porphobilino-gen deaminase, PBGD) was amplified from undiluted poly(A)RNA in a separate reaction following the duplex PCR proto-col as described (Wang et al. 2004). All analyte transcriptconcentrations are expressed as copies per PBGD referencetranscripts.

Fluorescent immunostaining for BrdU in metaphasechromosome spreads: BrdU (30 mm) was added to asynchro-nous actively growing fibroblasts at 80% confluence. SeveralBrdU pulse lengths were performed on multiple cell lines andthese data were used to determine that 4.5 hr is the appro-priate duration of BrdU incorporation for each replication-timing experiment (data not shown). Exponentially growingasynchronous fibroblasts were cultured for 4.5 hr in thepresence of BrdU and 0.050 mg/ml Colcemid (Life Technol-ogies, Grand Island, NY) was added 1 hr before harvesting.Cell suspensions were incubated 13–15 min in 0.4% KCl at 37�followed by fixation with 3:1 methanol:acetic acid. Metaphasespreads were prepared by dropping the BrdU-treated cellsonto coverslips followed by DNA denaturation in 70% form-amine/23 SSC at 73� for 2 min. Following preincubation withblocking buffer (13 PBS, 10% FBS, 0.2% Tween 20), in-corporated BrdU was detected using 1:20 dilution in blockingsolution of monoclonal anti-BrdU antibody (Sigma) followedby 1:150 dilution in blocking solution of Texas-Red anti-mouseantibody (Jackson ImmunoResearch) in blocking buffer.Images were captured using Quips mFISH software (Vysis,Adelphia, NJ). The individual colors of a recorded image werestored separately by the Vysis Quips mFISH software and the

representation of each color in the final image was adjustedusing the software setting of the gain for that color. The BrdUincorporation studies were not done simultaneously with XistRNA FISH because the Xist RNA is lost from the Xi duringmitosis. The Xist RNA signal that can be seen on the Xi in earlymitotic cells is very fragile and the treatments that occurduring the BrdU incorporation assay caused the Xi to lose XistRNA signal.

Spectral karyotyping: The spectral karyotyping (SKY) probemixture (Applied Spectral Imaging) was applied according tothe manufacturer’s recommendations for metaphase chromo-some spreads (MCSs) prepared as described for the BrdUincorporation assay. Chromosomal aberrations were quanti-fied using an Olympus BX-61 microscope equipped with anApplied Spectral Imaging interferometer and 403 and 633objectives, driven by a desktop computer with SKY acquisitionand analysis software.

Quantitation of incorporated BrdU: X chromosome paints(Cambio, Cambridge, UK) were used to identify the X chro-mosomes and the X chromosome displaying the higher levelof BrdU incorporation within a spread was always assumed tobe the inactive one. Images obtained using Quips mFISH weretransferred to NIH image (http://rsb.info.nih.gov/nih-image)and the numbers of pixels occupied by the X chromosomes(DAPI) and by fluorescently labeled BrdU (Texas Red) werethen calculated for each MCS. Fisher’s exact test was used tocompare the percentages of X chromosomes displaying BrdUincorporation between different cell lines. Box plots were usedto visualize the distributions of BrdU measurements acrosscategorical groupings. Box plots labeled ‘‘% BrdU signal’’represent measurements of the number of pixels of BrdUsignal on a chromosome divided by the number of pixels ofDAPI signal occupied by the same chromosome multiplied by100. NIH Image was also used to record the intensity of eachpixel. ‘‘BrdU area 3 intensity’’ represents the % BrdU signalmultiplied by the average intensity of the pixels representingBrdU. The statistical analyses were performed using thesoftware package R (Ihaka and Gentleman 1996), whichcan be downloaded from http://cran.r-project.org/. Differ-ences in measurements were tested across categorical group-ings using the Kruskal–Wallis test (Kruskal 1964) and theP-values obtained from this test are displayed above thecorresponding box plots.

X chromosome paint: A total of 10 ml of mouse Xchromosome-specific biotinylated probe (Cambio) were usedto detect the X chromosomes by fluorescent in situ hybridiza-tion (DNA FISH) to ethanol-dehydrated cells according tomanufacturer’s instructions. The probe was detected usingstreptavidin–FITC (Jackson ImmunoResearch) and anti-streptavidin FITC (Vector Laboratories) at 1:50 dilution eachin blocking solution (13 PBS, 10% human serum, 0.05%Tween 20). The chromosomes were counterstained with DAPIand viewed with a Leica DMR fluorescent microscope. Imageswere captured with Quips mFISH software (Vysis).

Determining sequence composition across the X chromo-some: We identified the types and positions of repetitivesequences from the RepeatMasker output provided by theUCSC genome browser (http://genome.ucsc.edu). For suc-cessive 1-Mb intervals, we then obtained a value for each re-peat type representing the percentage of the 1-Mb sequenceoccupied by that repeat type.

Histone immunolabeling and whole-chromosome paint:Indirect immunofluorescence with anti-acetyl-histone H4antibody (Serotec, Oxford) and histone H3 (trimethyl-K4)antibody (Abcam) was carried out on asynchronous cultures.Fibroblasts were grown at 80% confluence, Colcimid (0.05mg/ml) was added for 1 hr, and fibroblasts were harvested andthen swollen in 0.4% KCl at 37� for 13 min. Fibroblasts were


dropped onto coverslips. Cell membranes were solubilized byimmersion in KCM buffer [120 mm KCl, 20 mm NaCl, 10 mm

Tris–HCl, pH 8, 0.5 mm EDTA, 0.1% (v/v) Triton X-100] in apetri dish (35 3 10 mm). The coverslips were transferred toblocking solution (10% FBS in KCM) at 37� for 1 hr and thenincubated with 20 ml of 1:10 of anti-acetylated H4 (Serotec) or1:20 anti-methH3-K4 (Abcam) in a humidified chamber for2 hr at RT or overnight at 4�. The coverslips were then washedthree times for 5 min in KCM and transferred to blockingsolution and incubated at 37� for 1 hr. Primary antibody wasdetected with anti-rabbit Texas Red (Jackson ImmunoRe-search) at 1:100 dilution in blocking solution and incubatedfor 1 hr at RT. The coverslips were washed three times withKCM buffer and fixed at RT with 4% paraformaldehyde(Electron Microscopy Sciences, Fort Washington, PA) inKCM for 20 min and washed three times with KCM at RT. Todetect the X chromosomes, the coverslips were incubated twotimes in methanol:acetic acid (1:3 v/v) at �20� for 20 mineach. DNA was denatured with 70% formamide/23 SSC at 73�for 8 min, dehydrated, and then hybridized to 10 ml of thebiotinylated probe (Cambio) according to the manufacturer’sinstructions. The probe was detected using streptavidin–FITC(Jackson ImmunoResearch) and anti-streptavidin FITC (Vec-tor Laboratories) at 1:50 dilution each in blocking solution(13 PBS, 10% human serum, 0.05% Tween 20). The chromo-somes were counterstained with DAPI and viewed with a LeicaDMR fluorescent microscope. Images were captured withQuips mFISH software (Vysis).

Western analysis of p53: Proteins from whole-cell extractswere separated by sodium dodecyl sulfate (SDS) polyacryli-mide gel electrophoresis (PAGE), transferred to a chargedPVDF membrane, and blocked with 5% nonfat milk in TBST(20 mm Tris pH 7.4, 150 mm NaCl, 0.05% Tween-20). Primaryand secondary antibody incubations were performed in 5%nonfat milk in TBST. Proteins were detected using the ECLPlus Western blotting detection reagent (GE Healthcare,Piscataway, NJ). Primary reagents included: rabbit anti-p53-Ser15 (Cell Signaling Technology, Beverly, MA), rabbit anti-p53(CM5) (Vector Laboratories), mouse anti-b actin (Sigma),and mouse anti-ATM-Ser1981 (Rockland, Gilbersville, PA).Donkey anti-rabbit IgG-horseradish peroxidase (HRP) (GEHealthcare) and sheep anti-goat IgG-HRP (GE Healthcare)were used as the secondary antibodies.

BRCA1, g-H2AX, and MeCP2 immunolabeling: Fibroblastswere grown at 80% confluence and dropped onto coverslips.The cells were fixed with 2% of paraformaldehyde in PBS for5 min and washed three times with 13 PBS. The cells wereincubated in permeabilized solution (13 PBS, 0.5% Triton X-100) for 10 min at 4� and washed three times with KCM buffer[120 mm KCl, 20 mm NaCl, 10 mm Tris–HCl, pH 8, 0.5 mm

EDTA, 0.1% (v/v) Triton X-100]. The cells were transferred toblocking solution (10% FBS in KCM) at 37� for 1 hr and thenincubated with anti-Brca1 1:2.5 dilution at 37� for 2 hr. Thecoverslips were washed three times with KCM buffer andblocking again at 37� for 30 min. The Brca1 antibody wasdetected with anti-mouse Texas Red at 1:150 dilution andincubated at room temperature for 1 hr and then washedthree times and the process repeated for histone gH2AX(Upstate Biotechnology, Charlottesville, VA) that was detectedusing anti-rabbit FITC. The immunodetections were alsoperformed in the reverse order (Brca1 last) with similar re-sults. In other experiments, the g-H2AX antibody (UpstateBiotechnology) was detected using either anti-rabbit FITC oranti-rabbit Texas Red. In yet other experiments, MeCP2antibody (Upstate Biotechnology) was detected using anti-rabbit FITC and in a subset of experiments the process wasrepeated using Brca1 antibody detected with anti-mouse TexasRed. Finally, the cells were fixed with 4% paraformaldehyde in

KCM for 15 min and washed three times with KCM. The nucleiwere counterstained with DAPI and viewed with a Leica DMRfluorescent microscope. Images were captured with QuipsmFISH software (Vysis).

Simultaneous immunolabeling of histone H3 trimethyl-lysine 4 and DNA FISH for sequence flanking the Xist gene:We detected histone H3 (trimethyl-K4) using a rabbit anti-body to H3 (Abcam), followed by a goat antibody to rabbitconjugated with Texas Red (Jackson ImmunoResearch). Theimmunodetection of histone was combined with DNA FISH asdescribed (Brown et al. 2001) with the following modifica-tions: we labeled the P1 clone ppJL1 (Lee et al. 1999) withdigoxigenin by nick translation (Dig–Nick translation mix;Roche, Indianapolis) and detected the label with sheep anti-Digoxigenin-Fluorecein (Roche) and rabbit anti-sheep Fluo-recein (Vector Laboratories).

Simultaneous immunolabeling of g-H2AX and DNA FISHfor sequence flanking the Xist gene: Biotinylated dUTP-labeled DNA FISH probes were prepared by nick translationfrom BAC DNA and P1 DNA, both of which include the Xistgene and flanking DNA sequence. Cell lines XaXist-D21-kbXiXist-

D21-kb-1.1 and XaXist-D21-kbXiXist-D21-kb-2.1 were maintained in DMEMcontaining 10% FBS and seeded on coverslips 1 day before theexperiment. Cells on coverslips were fixed with 4% para-formaldehyde in PBS for 10 min followed by 5 min permeabi-lization with 0.5% Triton X-100 at room temperature. Primaryantibody recognizing g-H2AX (Upstate Biotechnology) wasapplied for 1 hr. Cells were washed with PBS three times beforeincubation with secondary antibody that was conjugated withFITC (Jackson ImmunoResearch Laboratories). Cells werethen fixed again with methanol:acetic acid (3:1) at �20� for40 min followed by dehydration in 70, 90, and 100% ethanolfor 3 min each. Cells were denatured in 70% formamide/23SSC at 85� for 30 min, cooled down with cold 70% ethanol, anddehydrated with 90 and 100% ethanol. The probe derivedfrom BAC DNA or from P1 DNA was simultaneously dena-tured at 75� for 10 min in a hybridization mixture (DNA probe,Cot1DNA, hybridization buffer, and 50% formamide) andprehybridized at 37� for 20 min. The prehybridized probe wasapplied to the pretreated coverslip and incubated at 37� over-night. After a series of stringent washes, avidin–Texas Red wasadded to the coverslip and incubated for 1 hr. Signal wasvisualized using a Nikon Eclipse E800 microscope equippedwith a SenSys cooled CCD camera (Photometrics, Tucson,AZ). Images were captured using Metamorph image acquisi-tion software (Universal Imaging, Downingtown, PA).

RESULTS

Histone H4 acetylation and histone H3 lysine-4 meth-ylation accumulate on the inactive X chromosome when21 kb are excised from both copies of the Xist gene:To examine the role of the Xist locus (Figure 1A, top)in the maintenance of chromatin structure, three fe-male immortalized murine embryonic fibroblast (MEF)cell lines were obtained, from three different E13.5embryos, in which 21 kb of sequence at the Xist genewere flanked by Lox sites (Figure 1A, middle) on boththe Xa and the Xi (lines XaXist-floxXiXist-flox-1, XaXist-flox

XiXist-flox-2, and XaXist-floxXiXist-flox-3). The three lines weretreated with adenovirus expressing cre recombinaseand GFP and were also infected with adenovirus ex-pressing only GFP. Limiting dilution was used to obtainclonal cells that were homozygous for the 21-kb deletion


Figure 1.—Accumulation of histone H4 acetylation and histone H3 lysine-4 methylation on the inactive X chromosome in XaXist-

D21-kbXiXist-D21-kb cells. (A) Map of the Xist-WT, Xist-flox, and Xist-D21-kb alleles (left) and identification of the three alleles by PCRgenotyping (right) (see materials and methods for details regarding genotyping). (B–G, J, and K) Chromosomes were immu-nostained using an antibody that recognizes acetylated lysines on histone H4 or an antibody against trimethylated lysine-4 onhistone H3 and were also stained with DAPI and subjected to X chromosome paint or DNA FISH using an X chromosome-specificprobe as indicated. Arrowheads mark X chromosomes. X chromosome paint hybridized throughout the X chromosome andalso to the centromeres of all chromosomes in MCS. (B) XaXist-floxXiXist-flox MCSs fluorescently immunostained for acetylated lysineson histone H4 (Texas Red, red): (a) XaXist-floxXiXist-flox-1; (b) XaXist-floxXiXist-flox-2; (c) XaXist-floxXiXist-flox-2. The XaXist-floxXiXist-flox-1 MCS wasalso subjected to X chromosome paint (green) (a, right ). (C) XaXist-D21-kbXiXist-D21-kb MCSs immunostained for acetylated lysines onhistone H4 (Texas Red, red): (a) XaXist-D21-kbXiXist-D21-kb-1.1, (b) XaXist-D21-kbXiXist-D21-kb-2.1, and (c) XaXist-D21-kbXiXist-D21-kb-3.1. MCSfrom a XaXist-D21-kbXiXist-WT-1 cell (D) and a XaXist-WTXiXist-D21-kb-1 cell (E) immunostained for acetylated lysines on histone H4 (FITC,green). (F) MCSs from XaXist-floxXiXist-flox cells immunostained for methylated lysine-4 on histone H3 (red): (a) XaXist-floxXiXist-flox-1;

(continued)


(XaXist-D21-kbXiXist-D21-kb) (Figure 1A, bottom) or were XaXist-flox

XiXist-flox (from the adeno-GFP infections). The cell lineswere chosen such that each pair of cell lines with thesame number was derived from the same embryo (e.g.,lines XaXist-floxXiXist-flox-1.1 and XaXist-D21-kbXiXist-D21-kb-1.1 arederived from GFP-adenovirus and cre-adenovirus infec-tions of line XaXist-floxXiXist-flox-1, respectively). Using thesame procedure, MEF lines XaXist-D21-kbXiXist-WT-1.1, XaXist-WT

XiXist-D21-kb-1.1, and XaXist-D21-kbXiXist-WT-1.1 were obtained. PCR(Figure 1A, right) and FISH (see below) were used todetermine the genotypes of the cell lines (see materials

and methods).We examined the state of histone acetylation in these

cell lines by fluorescence immunostaining of MCSs.Similar to previously published results for wild-typefemale cells (Jeppesen and Turner 1993), the Xi wasreadily distinguishable from the other chromosomesdue to its severe hypoacetylation of histone H4 in MCSsfrom cell lines XaXist-floxXiXist-flox-1, -2, and -3 (Figure 1B)and in MCSs from lines XaXist-floxXiXist-flox-1.1, -2.1, and -3.1(not shown). The Xi was also hypoacetylated on histoneH4 in MCSs from XaXist-D21-kbXiXist-WT cells (Figure 1D),XaXist-WTXiXist-D21-kb cells (Figure 1E), and XaXist-floxXiXist-D21-kb

cells (not shown) in agreement with previously pub-lished results (Csankovszki et al. 1999). In contrast, inMCSs from the three XaXist-D21-kbXiXist-D21-kb cell lines (1.1,2.1, and 3.1), an inactive X chromosome could nolonger be distinguished from the other chromosomeson the basis of hypoacetylation (Figure 1C).

The Xi was also severely hypomethylated at lysine 4of histone H3 in MCSs from the cell lines XaXist-floxXiXist-flox

-1, -2, and -3 (Figure 1F) and in the cell lines XaXist-flox

XiXist-flox-1.1, 2.1, and 3.1 (not shown), as was previouslyreported for wild-type female cells (Boggs et al. 2001).In contrast, in MCSs from all three XaXist-D21-kbXiXist-D21-kb

cell lines (1.1, 2.1, and 3.1), a Xi could not be distin-guished from the other chromosomes on the basis ofhypomethylation (Figure 1G). RNA FISH revealed that,in contrast to the Xist RNA seen in XaXist-WTXiXist-WT cells(Figure 1H), no XIST RNA signal whatsoever was seenin XaXist-D21-kbXiXist-D21-kb cells (Figure 1I). To determinewhether the excision of 21 kb from both Xist allelescauses the Xi to be lost from cells, we examined XaXist-D21-kb

XiXist-D21-kb cell lines from three embryos that carried adeletion at the Hprt locus (Hooper et al. 1987) on theXa, while the Hprt locus on the Xi was intact. DNA FISH

for a region of the X chromosome in combination withimmunofluorescence indicated that one of the two Xchromosomes was hypomethylated at lysine-4 on his-tone H3 in XaXist-flox, Hprt-DXiXist-flox, Hprt-WT cells (Figure 1J),indicating that the Hprt deletion did not disrupt H3lysine-4 hypomethylation on the Xi. In contrast, an Xchromosome that was hypomethylated at H3 lysine-4was absent from in the corresponding XaXist-D21-kb,Hprt-D

XiXist-D21-kb,Hprt-WT cells (Figure 1K), as expected. Two-colorDNA FISH was performed to verify the presence of theinactive X chromosome in the XaXist-D21-kb,Hprt-DXiXist-D21-kb,

Hprt-WT cells. A Cy3-labeled (red) combination probe,comprising a mixture of seven separate probes to thedeleted region of Hprt, was used to identify the inactiveX chromosome (see materials and methods fordetails). A FluorX (green)-labeled mouse BAC (RP23-298N24) was used as a probe to mark both the inactiveand the active X chromosomes. Cells, hybridized withboth these probes together, when examined confirmedthe presence of the inactive X chromosome. For onepredominantly diploid XaXist-D21-kb,Hprt-DXiXist-D21-kb,Hprt-WT cellline, 94 of 100 cells examined showed red hybridizationsignal in their nuclei (Figure 1L, a). Similarly, 96 of 100cells showed a red hybridization dot in another XaXist-D21-kb,

Hprt-DXiXist-D21-kb,Hprt-WT cell line derived from a different em-bryo (Figure 1L, b). For both cell lines, most cells hadone red dot near one of the two green dots. Also, weoccasionally observed cells (�5%) that were tetraploidfor the X chromosome—having four green dots and twored dots near two of the green dots (not shown). Weconclude that the Xi is still present in cells that haveexcised 21 kb of Xist sequence from both the Xa andthe Xi and the homozygous deletion of Xist sequenceresults in the Xi acquiring H3 lysine-4 methylation andH4 acetylation along its length. Additional lines of evi-dence that indicate that the Xi is retained in XaXist-D21-kb

XiXist-D21-kb cells are presented below.The dramatic chromatin changes observed on the

inactive X chromosome in XaXist-D21-kbXiXist-D21-kb cellsraised the question of whether Xist RNA was expressedfrom the 39 undeleted portion of the Xist gene of eitherX chromosome in XaXist-D21-kbXiXist-D21-kb cells. To deter-mine whether a truncated Xist RNA was expressed,quantitative determination of Xist mRNA was per-formed by RT–FRET real-time PCR using PCR primersthat transversed the boundaries of exons 4, 5, and 6,

(b) XaXist-floxXiXist-flox-2; (c) XaXist-floxXiXist-flox-3. The XaXist-floxXiXist-flox-1 MCS was also subjected to X chromosome paint (green) (a, right).(G) MCSs from XaXist-D21-kbXiXist-D21-kb cells immunostained for methylated lysine-4 on histone H3 (red): (a) XaXist-D21-kbXiXist-D21-kb-1.1; (b)XaXist-D21-kbXiXist-D21-kb-2.1; (c) XaXist-D21-kbXiXist-D21-kb-3.1. The XaXist-D21-kbXiXist-D21-kb-1.1 MCS was also subjected to X chromosome paint(green) (a, right). Xist RNA FISH against a XaXist-WTXiXist-WT cell (H) and a XaXist-D21-kbXiXist-D21-kb cell (I). XaXist-flox, Hprt-DXiXist-flox, Hprt-WT

( J) and XaXist-D21-kb,Hprt-DXiXist-D21-kb,Hprt-WT (K) MEFs from two different mouse matings (a and b) were subjected to DNA FISH usinga P1-derived probe that recognizes the X chromosome and simultaneously stained with DAPI (top, blue) and immunostainedfor trimethylated lysine-4 on histone H3 (bottom, red). (L) XaXist-D21-kb,Hprt-DXiXist-D21-kb,Hprt-WT MEFs were subjected to DNA FISHusing a probe that recognizes DNA sequence that is exclusively on the inactive X chromosome (red) and a probe that recognizesboth X chromosomes (green). The XaXist-D21-kb,Hprt-DXiXist-D21-kb,Hprt-WT MEFs ‘‘a’’ and ‘‘b’’ represented in K and L are derived fromthe XaXist-flox,Hprt-DXiXist-flox,Hprt-WT MEFs ‘‘a’’ and ‘‘b’’ respectively represented in J via exposure to cre recombinase.


respectively, and a FRET probe that recognized exon 5.The internal autosomal reference gene transcript usedwas PBGD (Wang et al. 2004). In two XaXist-WTXiXist-WT celllines, the Xist mRNA/PBGD mRNA ratios were 720.3and 699.1. In contrast, the Xist mRNA/PBGD mRNAratios in two XaXist-D21-kbXiXist-D21-kb cell lines were 0.0 and0.0 because only PBGD mRNA, but no Xist RNA, wasdetected in these samples. A comprehensive and de-tailed analysis of Xist RNA levels in cells bearing vari-ous Xist genotypes will be published elsewhere (J. L.Salstrom, C. Wang, C. Wang, A. Datta, S. Zeitlin, G.Csankovszki, C. D. Eller, S. Diaz-Perez, J. Wang, A.Chess, S. Huang, B. Kaltenboeck and Y. Marahrens,unpublished data).

Altered replication time on the inactive X chromo-some in response to 21-kb deletions: Since the Xi hadacquired two euchromatic properties (H4 acetylationand H3 lysine-4 methylation) in XaXist-D21-kbXiXist-D21-kb cellswe predicted that the deletions would also cause the Xito replicate earlier in S phase since euchromatin gen-erally replicates earlier in S phase than heterochromatin(Gilbert 2002). To determine if this was the case, wepurified approximately diploid lines XaXist-D21-kbXiXist-D21-kb-1.2, -2.2, and -3.2 from cre-adenovirus-infected XaXist-flox

XiXist-flox progenitor lines (1, 2, and 3) using limitingdilution. We also purified predominantly diploid linesXaXist-floxXiXist-flox-1.2, -2.2, and -3.2 from GFP-adenovirus-infected XaXist-floxXiXist-flox progenitor lines (1, 2, and 3).Each pair of diploid cell lines with the same first num-ber was derived from the same embryo (e.g., linesXaXist-floxXiXist-flox-1.2 and XaXist-D21-kbXiXist-D21-kb-1.2 are de-rived from line XaXist-floxXiXist-flox-1). Using the same pro-cedure, the predominantly diploid lines XaXist-D21-kbXiXist-WT-1.2, XaXist-WTXiXist-D21-kb-1.2, and XaXist-D21-kbXiXist-WT-1.2 MEFlines were also obtained. All diploid lines in this studywere used at a low passage number (with respect tolimiting dilution) in our analyses because they increas-ingly accumulated cells that had lost their diploid char-acter with repeated passages.

The predominantly diploid cell lines were subjectedto a replication-timing assay that uses fluorescenceimmunostaining to detect chromosomal regions thatincorporated BrdU late in S phase. Cells were pulselabeled with BrdU for 4.5 hr and metaphase chromo-some spreads were prepared. Cells that were in mid-Sphase at the onset of BrdU addition (Figure 2B)incorporated BrdU in mid- and late S phase but didnot reach mitosis in the 4.5-hr interval (Figure 2A) andtherefore were not represented among the MCSs. Cellsthat were in late S phase at the onset of BrdU addition(Figure 2B) incorporated BrdU in late S phase, reachedmitosis in the 4.5-hr interval, and the incorporatedBrdU was detected in the MCSs (Figure 2C). Identifica-tion of X chromosomes using X chromosome paint(Figure 2D) revealed that one of the two X chromo-somes in female cells consistently displayed more BrdUsignal than the other X chromosome and more signal

than most or all autosomes. Although the mouse Xidoes not replicate nearly as late in S phase in mouse cellsas in human cells, it nevertheless replicates later in Sphase than the active X chromosome (Evans et al. 1965;Galton and Holt 1965; Tiepolo et al. 1967). Weconsequently inferred that the later replicating X chro-mosome in our female cultures was the Xi and thatthe earlier replicating X chromosome was the Xa. Thelevel of BrdU signal that was recorded in a photographof an X chromosome in a MCS depended, in part, onthe gain setting of the red channel of the mQuips Vysissoftware that was used to display the BrdU signal. At lowgain, only the fluorescent signal representing incorpo-rated BrdU that exceeds a high intensity threshold isrecorded in the image of an X chromosome (Figure 2E,top). At intermediate gain, the fluorescent BrdU signalexceeding an intermediate intensity threshold is re-corded in the image (Figure 2E, middle). At high gain,even small amounts of incorporated BrdU will be dis-played among the pixels representing the BrdU signal(Figure 2E, bottom). To normalize the BrdU signal,all photographs of metaphase chromosome spreadswere taken at a standardized ‘‘low-gain’’ setting whereonly five autosomes display BrdU signal and one of thesefive autosomes displays only one pixel of BrdU. Thismethod of normalizing the gain using BrdU signal onautosomes was previously used to show that portions ofthe Xa replicate later in S phase if 21 kb are deleted fromthe Xist locus of the Xa and the method is described inmore detail in this earlier study (Diaz-Perez et al. 2005).In addition, only MCSs displaying two X chromosomeswere considered. At the measurements taken at lowgain, all three clonal XaXist-flox XiXist-flox fibroblast lines(1.2, 2.2, and 3.2) displayed modest BrdU signal on oneX chromosome and either very little (Figure 2, C and D)or no (Figure 2, F and G) BrdU signal on the other Xchromosome and therefore closely resembled the signallevels seen in XaXist-WTXiXist-WT MCSs (Diaz-Perez et al.2005). Contrary to expectation, more BrdU signal (latereplication) was observed on the inactive X chromo-some in XaXist-WTXiXist-D21-kb-1.2 cells (Figure 2, H–K) andXaXist-floxXiXist-D21-kb-1.2 cells (not shown) than in theXaXist-floxXiXist-flox cells. Quantitation of the BrdU signalon the Xi in 40 XaXist-WTXiXist-D21-kb spreads and 40 XaXist-flox

XiXist-flox spreads using NIH IMAGE software revealedsignificantly more BrdU signal on the XiXist-D21-kb than onthe XiXist-flox by percentage of area displaying BrdU signal(P ¼ 5.45 3 10�7) (Figure 2L) or when multiplying thisarea by the average intensity of the signal (P ¼ 5.30 3

10�8) (Figure 2M). In contrast, no significant differencewas detected on the Xa in the same spreads regardlessof whether percentage of area occupied by BrdU signal(P ¼ 0.655) or area times intensity (P ¼ 0.377) wasconsidered (box plots not shown). However, this quan-titation was from a single XaXist-WTXiXist-D21-kb cell line andshould therefore be considered preliminary. No signif-icant difference in BrdU incorporation levels on the


inactive X chromosome was seen when comparingXaXist-floxXiXist-flox cells to XaXist-WTXiXist-WT cells (P ¼ 0.407)(Figure 2N), indicating that the presence of the Loxsites in the absence of the 21-kb deletion did not have ameasurable effect on replication time. The Xi therefore

may replicate overall later in S phase when it is har-boring the 21-kb deletion. Furthermore, heterozygosityfor the Xist deletion causes the X chromosome harbor-ing the deletion to be replicated later than normal inS phase but does not affect the replication time of the

Figure 2.—Deletion of 21 kb from the Xist gene on the Xa and the Xi cause the Xi to be replicated later in S phase. Activelygrowing MEF cultures were exposed to BrdU for 4.5 hr, and MCSs were prepared and immunostained using anti-BrdU (red),hybridized to X-chromosome paint (green), and counterstained using DAPI (blue). In all MCSs, the active X chromosome isalways assumed to be the X chromosome displaying the lower proportion of BrdU incorporation and the two X chromosomesare marked by arrowheads. In each photograph of a MCS displaying BrdU, the gain has been adjusted such that five autosomesdisplay BrdU incorporation. (A–D) XaXist-floxXiXist-flox-1.2: (A) interphase cell; (B) possible times in the cell cycle that the indicatedfibroblasts were inferred to be exposed to BrdU; (C and D) XaXist-floxXiXist-flox-1.2 MCS. (E) XiXist-flox from an XaXist-floxXiXist-flox MCSdisplayed at low gain (top), intermediate gain (middle), and high gain (bottom). Only the most intense BrdU signal is visibleat a standardized low-gain setting that was used throughout (see text). (F and G) XaXist-floxXiXist-flox-3.2. (H and I) XaXist-WTXiXist-

D21-1.2. ( J and K) XaXist-WTXiXist-D21-1.2. (L and M) Quantitation of BrdU signal on the Xi in 40 XaXist-floxXiXist-flox and 40 XaXist-WT

XiXist-D21 MCSs (.10 MCSs from each of the lines) summarized in box plots that display percentage of BrdU signal on the Xi,which represents the number of pixels of BrdU signal divided by the number of pixels of DAPI signal multiplied by 100 (L)or percentage of BrdU signal multiplied by average intensity of the BrdU signal (M). (N) Quantitation of BrdU signal in 40XaXist-floxXiXist-flox MCSs (.10 MCSs from each of the three lines) and 17 XaXist-WTXiXist-WT MCSs (7 and 10 MCSs from each oftwo lines) summarized in box plots that display percentage of BrdU signal on the Xi. (O and P) XaXist-D21XiXist-D21-1.2 MCS. (Q andR) XaXist-D21XiXist-D21-3.2 MCS. (S and T) Quantitation of BrdU signal in the Xi from 40 XaXist-floxXiXist-flox and 40 XaXist-D21XiXist-D21 spreads(.10 MCSs from each of the six lines) using box plots that display percentage of BrdU signal (S) and percentage of BrdU sig-nal multiplied by average intensity of the BrdU signal for the Xi (T). (U) Box plots that compare the Xi BrdU signal between 40XaXist-WTXiXist-D21 spreads and 40 XaXist-D21XiXist-D21 spreads. P-values were obtained using the Kruskal–Wallis test (Kruskal 1964).


wild-type X chromosome, regardless of whether the mu-tation is on the Xa (Diaz-Perez et al. 2005) or on the Xi.

The effect of deleting the 21 kb from both Xist alleleswas next investigated using the same assay. The earlierreplicating Xa displayed more BrdU signal in spreadsfrom the three XaXist-D21-kbXiXist-D21-kb lines (Figure 2, O–R)than in the XaXist-floxXiXist-flox cell lines (Figure 2, C, D, F,and G), in agreement with earlier results obtained usingXaXist-D21-kbXiXist-WT cells (Diaz-Perez et al. 2005). In XaXist-

D21-kbXiXist-D21-kb MCSs the Xi also displayed higher levelsof BrdU incorporation (Figure 2, O–R) than in XaXist-flox

XiXist-flox cell lines (Figure 2, C, D, F, and G). Thisdifference was readily apparent when 40 inactive Xchromosomes were quantitated from each cell line(Figure 2, S and T). We conclude that excision of 21kb from the Xist gene of the Xi causes the Xi to bereplicated later in S phase. Finally, preliminary dataindicated that the Xi displayed a significantly higherproportion of incorporated BrdU when 21 kb wasdeleted from both Xist alleles (Figure 2, O–R and U)than when the deletion was exclusively on the Xi inXaXist-WTXiXist-D21-kb-1.2 (Figure 2, H–K and U) and XaXist-flox

XiXist-D21-kb-1.2 cells (not shown). This suggested that ele-ments at both Xist alleles may influence the extent ofBrdU signal on the Xi in the assay for late replication.

Relationship between the pattern of late S-phaseBrdU incorporation and the concentration of LINE-1sequence on the XaXist-D21-kb of XaXist-D21-kbXiXist-D21-kb cells:When the XaXist-D21-kbXiXist-D21-kb MCSs were examined at ahigher gain than in Figure 2, the Xi displayed BrdUsignal throughout its length (Figure 3, A and B) whilethe Xa displayed four to six regions of concentratedBrdU signal in 33 of the 40 XaXist-D21-kbXiXist-D21-kb-2.2 MCSsexamined (Figure 3, A–C). This pattern was also prev-alent on the Xa in XaXist-D21-kbXiXist-D21-kb -1.2 and -3.2 cells

(not shown). Four of the five regions displaying latereplication on the Xa were found to correspond to thefour regions along the Xa that are most heavily enrichedfor LINE-1 elements (Figure 3D). The fifth region onthe Xa that displayed late replication was the pericen-tromeric region for which the genome sequence wasunavailable. In contrast to this reproducible pattern ofBrdU signal seen on the Xa in XaXist-D21-kbXiXist-D21-kb cells, aconsistent pattern was not readily apparent on the Xa inwild-type cells or when 21 kb was deleted only from oneXist allele (not shown) (Diaz-Perez et al. 2005). There-fore, although deletion of 21 kb exclusively from the Xistallele on the Xa causes the Xa to be replicated later in Sphase (Diaz-Perez et al. 2005), the excision of 21 kbfrom both Xist copies further altered the replicationtiming in a manner that resulted in the regions with thehighest concentrations of LINE-1 elements on the Xabeing replicated later in S phase.

Evidence that the 21-kb Xist deletions destabilize theDNA of both X chromosomes: During our prelimi-nary analyses we had encountered numerous XaXist-D21-kb

XiXist-D21-kb MCSs displaying evidence of deletions or trans-locations involving the X chromosome. Such spreadswere excluded from subsequent analyses of histonemodifications and BrdU incorporation. To investigatethe influence of the 21-kb deletion in the Xist gene onthe incidence of deletions and translocations, it wasnecessary to prepare three XaXist-WTXiXist-WT MEF cell linesusing the identical procedure as was used to produceXaXist-D21-kbXiXist-D21-kb cell lines. To this end, three XaXist-WT

XiXist-WT MEF cell lines were obtained from three E13.5129 embryos using the same procedure as was usedto produce the three XaXist-floxXiXist-flox lines. All six lineswere infected with adenovirus expressing cre recombi-nase and GFP and subjected to limiting dilution, and

Figure 3.—Relationship between the patternof late S-phase BrdU incorporation and the con-centration of LINE-1 sequence on the XaXist-D21-kb

of XaXist-D21-kbXiXist-D21-kb MCSs. Images of the 40XaXist-D21-kbXiXist-D21-kb spreads used in Figure 2 wereobtained at a gain that was higher than that usedin Figure 2. Red, BrdU; blue, DAPI stain of chro-mosomal DNA; green, X chromosome paint. (Aand B) Active and inactive X chromosomes ofa XaXist-D21-kbXiXist-D21-kb-2.2 spread displaying X chro-mosome paint (A) and BrdU signal (B). (C) Four-teen active X chromosomes from XaXist-D21-kb

XiXist-D21-kb-2.2 spreads displaying BrdU signal. (D)Graph displaying the concentration of LINE-1elements along the X chromosome superim-posed on an image of a Xa displaying BrdU sig-nal. The graph was generated using a Loesscurve applied to RepeatMasker output providedby the UCSC genome browser. The DNA se-quence of the centromere and pericentromericregion were not available and the coordinatesalong the X chromosome (x-axis) were measuredstarting at the centromere-proximal startingpoint of the available DNA sequence.


derivative cell lines were expanded from infected (GFP-expressing) cells using equal numbers of passages (seematerials and methods). The cell lines were sub-jected to the replication-timing assay used for Figure 2to distinguish the active and inactive X chromosomes.Inspection of MCSs from three XaXist-WTXiXist-WT cell linesderived from cre-adenovirus-infected cells revealed evi-dence for X chromosome deletions or translocationsin 0 of 60 MCSs. Similarly, 0 of 68 MCSs from XaXist-

floxXiXist-flox cells showed signs of aberrations in the Xchromosome. In contrast, we saw evidence for deletionsor translocations in 14 of 84 spreads (16.6%) fromcultures of the three XaXist-D21-kbXiXist-D21-kb cell lines (1.1,2.1, and 3.1). One of the most common abnormalitieswas a small fragment of the X chromosome (Figure 4,A and B). We also saw evidence of a ring X chromo-some (Figure 4, C and D) and small portions of the Xchromosome integrated into autosomes (Figure 4, Eand F) but were not confident of the latter findingsbecause the signal could be due to X chromosome painthybridizing to autosomal material. By reducing the gain,

the X chromosome paint was seen to display a charac-teristic pattern along the X chromosome that was highlyreproducible among wild-type spreads (Figure 4G).Using this pattern as a guide, we observed that a fre-quent abnormality was the truncation of a Xi (notshown) or a Xa (Figure 4H) due to the loss of thecentromere-distal tip of the X chromosome. We alsoobserved a chromosome that appeared to be a duplica-tion of the X chromosome (Figure 4I), an X chromo-some that seemed to be linked to an autosome (Figure4J), and what appeared to be dicentric chromosomeswhere a portion (Figure 4K) or all (not shown) of thechromosome was derived from the X chromosome.

To confirm these findings and to determine whetherautosomes in the XaXist-D21-kbXiXist-D21-kb cell lines alsodisplayed abnormalities, SKY (Liyanage et al. 1996)was performed on spreads from cell lines XaXist-D21-kb

XiXist-D21-kb-1.1 and XaXist-D21-kbXiXist-D21-kb-1.3. In agreementwith the X chromosome paint, the size of the X chro-mosome varied within spreads, reflecting deletions orrearrangements of the X chromosome (Figure 4, L and

Figure 4.—Evidence that deletion of 21 kb from the Xist gene destabilizes the X chromosome. MCSs from fibroblasts wereeither subjected to an assay that identifies X chromosomes using X chromosome paint (B and D–K, green) and late replicatingregions by fluorescent immunostaining for BrdU (H, J, and K, red) or subjected to spectral karyotyping (SKY) (L–P). Chromo-somes from XaXist-D21-kbXiXist-D21-kb MCSs are shown, except in G, which shows an X chromosome from a wild-type MCS. Evidence forsmall X chromosomal fragments (A–D and L), X chromosomal DNA within autosomes [E, F, O, and P (arrow)], truncated Xchromosomes (H and M), X chromosomal duplication (I), X–autosome translocations ( J, K, and N), and a dicentric X chromo-some (P, arrowhead) is shown. (P) Full karyotype of a XaXist-D21-kbXiXist-D21-kb MCS where each chromosome is represented three timeswith SKY hybridization (left), DAPI (middle), and computer-classified color (right). Note that SKY cannot identify the origin ofcentromeric/pericentromeric regions (oriented at the top of each chromosome), leading to frequent discolorations that do notrepresent translocations. The software also displays discolored regions at the edges (left or right sides) of chromosomes, which is astaining artifact rather than translocated material.


M). In some metaphases structural rearrangementsinvolving the X were identified. These included a simplenonreciprocal translocation involving chromosomes Xand 13 (Figure 4N) and a dicentric structure resultingfrom fusion between two X chromosomes (Figure 4P,arrowhead). Involvement of the X in more complexrearrangements was also suggested by SKY. These in-cluded a dicentric chromosome involving segments ofchromosomes 3 and 9 flanking two or more smallsegments of X (Figure 4O) and a translocation linkingportions of chromosomes 1 and 6 with a small segmentof X chromosome material at the junction (Figure 4P,arrow). Because the MCSs displaying specific chromo-somal anomalies were not examined as clonal cell lines,no material exists to allow confirmation of the complexrearrangements, so it is a formal possibility that the Xsignal arose from intermixing between two SKY colors atthe junction. A tally of the definitive simple rearrange-ments and deletions provided clear evidence of Xchromosome instability in XaXist-D21-kbXiXist-D21-kb MCSs: intotal, 13/80 karyotypes (16%) analyzed by SKY dis-played structural abnormalities involving the X chro-mosome. In the same spreads, 0/80, 1/80, and 1/80karyotypes displayed abnormalities involving chromo-somes 3, 4, and 5, respectively, and did not involve the Xchromosome. The abnormal chromosome 4 was a smallfragment and the abnormal chromosome 5 was slightlyshorter than normal and not a definitive abnormality.

Phosphorylation of p53 and H2AX and localizationof g-H2AX to the Xi in cells carrying the 21-kbdeletion: The frequent rearrangements suggested thepresence of DNA damage on the X chromosome thatmight have arisen from replication stress. DNA damagewould cause the ATR (Tibbettset al. 1999) and/or ATM(Banin et al. 1998; Canman et al. 1998; Khanna et al.1998) protein kinases to phosphorylate the p53 proteinat serine-15. To determine whether the 21-kb deletion isassociated with the phosphorylation of the p53, extractswere prepared from independent cell lines of each ofthe following genotypes: XaXist-WTXiXist-WT, XaXist-D21-kb

XiXist-WT, XaXist-WTXiXist-D21-kb, and XaXist-D21-kbXiXist-D21-kb. Westernblots using these extracts revealed elevated levels ofserine-15 phosphorylated p53 protein in all cell linesthat carried the XiXist-D21-kb compared to XaXist-WTXiXist-WT

cells (Figure 5A) while overall levels of p53 remainedapproximately the same (Figure 5A). Although thesimplest explanation is that p53 is phosphorylated byATR, we acknowledge that ATM (Paull et al. 2000;Burma et al. 2001; Stiff et al. 2004) or another kinasemight play a role.

Brca1 localizes to the Xi, associates with Xist RNA,fosters the Xist RNA signal at the Xi, and is involved inX-inactivation (Ganesan et al. 2002, 2004). Brca1 is alsoinvolved in the maintainance of genome stability(Moynahan et al. 2001; Weaver et al. 2002; Bruun et al.2003). Our finding that Xi DNA is unstable in XaXist-D21-kb

XiXist-D21-kb cells raised the issue of whether the Brca1

signal is still present in XaXist-D21-kbXiXist-D21-kb cells. Brca1has been reported to concentrate on the Xi primarily inS-phase cells (Ganesan et al. 2002; Chadwick and Lane

2005). Immunostaining of XaXist-floxXiXist-flox cells forBrca1 revealed a large domain of concentrated Brca1protein (Figure 5B) in 29/90 XaXist-floxXiXist-flox cells(32.2%) and in 24/90 XaXist-WTXiXist-WT cells (26.7%).XaXist-D21-kbXiXist-D21-kb cells displayed the concentratedBrca1 signal (Figure 5C) in 28/90 cells (31.1%), suggest-ing that loss of the Xist sequence (including Xist RNA)did not affect the concentration of Brca1 to the Xi.

The delayed replication times and DNA instability onboth X chromosomes in XaXist-D21-kbXiXist-D21-kb cells may bedue to replication stress. A number of expressed fragilesites are known to be replicated late in S phase (Hansen

et al. 1993; Wang et al. 1999; Hellman et al. 2000; Arlt

et al. 2003) and the ATR kinase maintains fragile sitestability (Casper et al. 2002). Replication stress triggersthe ATR kinase to phosphorylate the histone H2Avariant H2AX to produce g-H2AX and also causesDNA instability (Ward et al. 2004). Immunofluores-cence failed to detect large regions of concentratedg-H2AX resembling the Brca1 signal in the three XaXist-flox

XiXist-flox cell lines (Figure 5D) or in XaXist-WTXiXist-WT cells(not shown) except in a low proportion of cells (4/90XaXist-floxXiXist-flox cells and 4/90 XaXist-WTXiXist-WT cells), con-sistent with a report that g-H2AX associates with the Xiin wild-type cells exclusively in late S phase (Chadwick

and Lane 2005). In contrast, a region of g-H2AX con-centration was observed in 58/90 (64%) and 59/90(65%) in two XaXist-D21-kbXiXist-D21-kb cell lines examined(Figure 5E, a and b, respectively) and in 50/90 (56%)XaXist-WTXiXist-D21-kb cells (Figure 5F). Twenty-six of 125(21%) XaXist-D21-kbXiXist-D21-kb cells displaying the g-H2AXsignal also displayed a Brca1 signal, a marker of the Xi(Ganesan et al. 2002) (Figure 6A). In an attempt toidentify a heterochromatic feature other than Brca1 orlate replication that was retained by the Xi in XaXist-D21-kb

XiXist-D21-kb cells we also considered MeCP2, which bindsmethylated DNA (Meehan et al. 1992) but for whichlocalization to the hypermethylated Xi has not beenaddressed. We found a distinct domain of concentratedMeCP2 in 83/90 (92.5%) of diploid (not shown) andtetraploid (not shown) XaXist-WTXiXist-WT cells. Twenty-fourof 120 (20%) of XaXist-WTXiXist-WT cells displaying a MeCPsignal also displayed a Brca1 signal (Figure 6B). XaXist-WT

XiXist-D21-kb (not shown) and XaXist-D21-kbXiXist-D21-kb (Figure6C) cells retained the MeCP2 signal in approximatelythe same proportion of cells (84/90, 93.5% and 80/90,88.9% respectively), indicating that the 21-kb deletionsdid not notably affect the intense localization of MeCP2.Since MeCP2 is not an established marker of the Xithese data did not conclusively show that the g-H2AXsignal localized to the X chromosome.

To definitively determine whether the g-H2AX sig-nal in XaXist-D21-kbXiXist-D21-kb cells colocalized with the Xchromosome, DNA FISH specific to the Xic (a region on


the X chromosome that encompasses the Xist gene) wasperformed in conjunction with immunostaining forg-H2AX. A total of 300 XaXist-D21-kbXiXist-D21-kb cells displayingthe large g-H2AX signal (as reported earlier in Figure5E) were scored for the additional presence of a super-imposed Xic DNA FISH signal. Ninety of the 300 XaXist-

D21-kbXiXist-D21-kb cells displaying the large g-H2AX signal(30.0%) displayed a superimposed Xic DNA FISH signal(Figure 6D). Interestingly, 5 of these 90 XaXist-D21-kb

XiXist-D21-kb cells displayed one greatly enlarged Xic signalthat always colocalized with the g-H2AX domain, suggest-ing that multiple duplications of Xic sequence had oc-curred (Figure 6E). In addition to the 90 cells displayingsuperimposed signals, 47 of the 300 cells (16%) displayeda g-H2AX signal that touched but did not encompass

the Xic DNA FISH signal (not shown), bringing the over-all g-H2AX–Xic association to 46% among cells display-ing a large g-H2AX signal. The high proportion of cellswith immediately adjacent g-H2AX and DNA FISHsignals may be due to g-H2AX being associated with asubset of the X chromosome that does not include theXic. Finally, 44 of the 300 cells (15%) showed a g-H2AXdomain that was clearly separate from the Xic DNA FISHsignals (not shown). The remaining cells could not bescored with confidence due to uneven signals, uncertainsignal number, or absence of DNA FISH signal. A totalof 19.5% of all XaXist-D21-kbXiXist-D21-kb cells displayed aclear g-H2AX signal that encompassed (90/703) ortouched (47/703) a clear Xic DNA FISH signal. Weconclude that the X chromosome instability, delayed

Figure 5.—Phosphoryla-tion of p53 and H2AXand increased localizationof g-H2AX to the Xi incells carrying the 21-kbdeletion in the Xist gene.(A) p53 is phosphorylatedon serine-15 in cells thatcarry the Xist-D21-kb alleleon the Xi. Western blotprobed for p53 phosphory-lated at serine-15 (top),for total p53 (middle),and for b-actin (loadingcontrol, bottom) in ex-tracts from XaXist-WTXiXist-WT-1.2 (i), XaXist-D21-kbXiXist-WT-1.2(ii), XaXist-WTXiXist-D21-kb-1.1(iii), XaXist-WTXiXist-D21-kb-2.1(iv), XaXist-D21-kbXiXist-D21-kb-2.2(v), and XaXist-D21-kbXiXist-D21-

kb-3.2 (vi) cells. (B–F) Brca1or g-H2AX immunolocali-zation in female cells usingFITC (green) or Texas Red(red) conjugated antibod-ies. Blue, DAPI. (B and C)Brca1 signal in XaXist-D21-kb

XiXist-D21-kb cells: (B) XaXist-flox

XiXist-flox-1.2 cell displayingDAPI (left) and Brca1(right); (C) XaXist-D21-kbXiXist-

D21-kb-1.2 cell displayingDAPI (left), and Brca1(right). (D–F) g-H2AX dis-plays a concentrated signalin the majority of XaXist-D21-kb

XiXist-D21-kb and XaXist-WTXiXist-

D21-kb cells but only in 4.4%of XaXist-floxXiXist-flox cells:(D) a XaXist-floxXiXist-flox-1.2cell (a) and a XaXist-floxXiXist-

flox-2.2 cell (b) displayingDAPI (left side for each)and g-H2AX (right side

for each); (E) a XaXist-D21-kbXiXist-D21-kb-1.2 cell (a) and a XaXist-D21-kbXiXist-D21-kb-2.2 cell (b) displaying DAPI (left) and g-H2AX (right);(F) a XaXist-WTXiXist-D21-kb-1.2 cell displaying DAPI (left) and g-H2AX (right). Note that a higher gain was used in D than in E for theg-H2AX images to show that the signal representing the g-H2AX domain was not present at reduced intensity in D.


replication timing, and p53 phosphorylation that isbrought on by excision of 21 kb from both Xist allelesare accompanied by an increase in g-H2AX on the Xchromosome.

We consider the aforementioned colocalization num-bers to be very conservative because, when g-H2AXimmunostaining was combined with X chromosome-specific DNA FISH, the g-H2AX signal appeared to be

Figure 6.—Concentrated g-H2AX signal observed in XaXist-D21-kbXiXist-D21-kb cells localizes to the inactive X chromosome. (A)g-H2AX signal colocalizes with the Brca1 signal in XaXist-D21-kbXiXist-D21-kb cells: (a) a XaXist-D21-kbXiXist-D21-kb-1.2 cell displaying DAPI (left),Brca1 (center), and g-H2AX (right); (b) a XaXist-D21-kbXiXist-D21-kb-2.2 cell displaying DAPI (left), Brca1 (center), and g-H2AX (right).(B) A concentrated MeCP2 signal in XaXist-floxXiXist-flox cells colocalizes with the Brca1 signal. (C) A concentrated MeCP2 signal isretained in XaXist-D21-kbXiXist-D21-kb cells. (D) The concentrated g-H2AX signal in XaXist-D21-kbXiXist-D21-kb cells colocalizes with X chromo-somal DNA: a XaXist-D21-kbXiXist-D21-kb-1.2 cell (a) and a XaXist-D21-kbXiXist-D21-kb-2.2 cell (b) display, in each case, DAPI (left), g-H2AX (centerleft ), DNA FISH signal recognizing the X chromosome (center right), and the g-H2AX and DNA FISH signals merged (right). (E)Example of a rare XaXist-D21-kbXiXist-D21-kb cell where the DNA FISH signal recognizing the Xic region on the X chromosome and co-localizing with the g-H2AX domain is greatly enlarged, suggesting multiple duplications involving the Xic; a XaXist-D21-kbXiXist-D21-kb-2.2cell is shown. The fractions represent the proportion of the cells displaying a g-H2AX or a MeCP2 signal that simultaneouslydisplay a colocalized BRCA1 or X-chromosome-specific DNA FISH signal.


degraded in a subset of cells and cells with degradedg-H2AX signal were not scored as ‘‘superimposed’’ or‘‘immediately adjacent’’. [In contrast .90% (654/703)of all cells displayed DNA FISH signals, so FISH wasrelatively unaffected.] Consistent with the idea thatdegradation reduced our percentages, g-H2AX/DNAFISH colocalization was markedly higher when only the36 cells (of the 300 cells) with the most pronouncedg-H2AX domains were considered. Among these 36 cells,19 (53%) had the Xic DNA FISH signal encompassedby the g-H2AX signal, 9 (25%) displayed directly adja-cent g-H2AX and Xic DNA FISH signals, and 8 (22%)showed separated g-H2AX and DNA FISH signals. How-ever, the increased colocalization of X-chromosome-specific DNA FISH signal with the 36 most pronouncedg-H2AX domains should be interpreted with cautionas the level of colocalization involving these g-H2AXdomains may not be representative of all g-H2AXdomains but may reflect a qualitatively distinct sub-population that exhibits higher colocalization for an-other reason(s). For example, they may represent cellsin a particular portion of the cell cycle that are char-acterized by resistance to signal degradation and g-H2AXdomain colocalization with the Xic. Cells with spaciallydistinct g-H2AX and Xic DNA FISH signals may containX chromosomes that have lost the Xic region (a plau-sible explanation since the Xic appears to be unstable;Figure 6E). In addition, some g-H2AX domains may beassociated with autosomes. For example, there is evi-dence that 8–23% of autosomal genes are subject to arandom but coordinated inactivation process that re-sembles X-inactivation (Gimelbrant and Chess 2006).It is not known whether any autosome contains a higher

proportion of inactivated genes, like the X chromo-some. Since in Xist-WT cells g-H2AX associates with theXi exclusively in late S phase (Chadwick and Lane

2005), we cannot exclude the possibility that g-H2AXcan also transiently form a strong signal on an autosomethat has a high abundance of inactivated genes.

DISCUSSION

We show that in female mouse cells, the excision of21 kb from the Xist gene of both the Xa and the Xi (XaXist-

D21-kbXiXist-D21-kb) resulted in the appearance of two histonemodifications throughout the Xi that are generallyassociated with euchromatin: histone H4 acetylationand methylation on lysine-4 of histone H3. Despite theappearance of these euchromatic histone modifica-tions, the inactive X chromosome of XaXist-D21-kbXiXist-D21-kb

cells displayed abundant DNA replication that wasvery late in S phase and stood in contrast with themoderately late replication that normally predominateson the Xi in mouse cells (Nesbitt and Gartler 1970).The active X chromosome of XaXist-D21-kbXiXist-D21-kb cellsalso displayed a shift to later replication that was pre-dominantly in chromosomal regions associated withhigh concentrations of LINE-1 elements. The X chro-mosomes of XaXist-D21-kbXiXist-D21-kb cells were unstable andprone to deletions and translocations. The X chro-mosome instability was accompanied by the phosphor-ylation of p53 at serine-15 and an increase in theproportion of cells that bear a high concentration ofg-H2AX on the X chromosome. These findings aresummarized in Table 1.

TABLE 1

Summary of the properties of MEFs bearing the 21-kb deletion in the Xist gene

Property XaXist-WTXiXist-WT XaXist-floxXiXist-flox XaXist-WTXiXist-D21-kb XaXist-floxXiXist-D21-kb XaXist-D21-kbXiXist-WT XaXist-D21-kbXiXist-D21-kb

Xist transcription Yes Yesa Nob Noa Yesb NoXi H4 hypoacetylation Yes Yes Yesc Yesc Yesc NoXi H3-lys4

hypomethylationYes Yes ND ND ND No

Xi replication time Late Late Very lated Very lated Latee Very very lateX deletions or

translocationsNone None (few)f (few)f ND Many

p53-ser15phosphorylation

Low Low Elevatedd Elevatedd Low Elevated

g-H2AX signal Infrequent Infrequent Frequentd Frequentd ND FrequentMeCP2 signal Yes Yes Yesd Yesd ND YesBrca1 signal Yes Yes Yesd Yesd ND Yes

a S. Diaz-Perez and Y. Marahrens (unpublished data).b Csankovszki et al. (1999).c Preliminary evidence for a slight increase in H4 acetylation near the telomeres has been obtained (S. Diaz-Perez and

Y. Marahrens, unpublished data).d Data were obtained from only one XaXist-WTXiXist-D21-kb and one XaXist-floxXiXist-D21-kb cell line; however, the XaXist-WTXiXist-D21-kb and XaXist-flox

XiXist-D21-kb cell lines produced very similar results.e Preliminary evidence indicates an altered pattern of DNA replication (S. Diaz-Perez and Y. Marahrens, unpublished results).f Preliminary data (S. Diaz-Perez and Y. Marahrens, unpublished results).


We previously showed that the nontranscribed Xistallele on the Xa has biological activity as the 21-kb Xistdeletion altered Xa replication timing in cis; however,this deletion did not significantly affect the overallreplication timing of the Xi in trans (Diaz-Perez et al.2005). Here we show that the nontranscribed Xist alleleon the Xa has more far-reaching biological activities thatwere not evident in heterozygous cells due to redun-dancy with the transcribed Xist allele on the Xi. We showthat element(s) at the nontranscribed Xist allele on theXa (possibly promotor elements) function in trans tohelp maintain hypoacetylation on histone H4 and alsoto control the replication timing of the Xi. Under theassumption that the later replicating X chromosomewas the Xi, the Xi was much later replicating in XaXist-D21-

kbXiXist-D21-kb cells than in cells without the deletion on theXa. Deletions at both Xist alleles also resulted in a highlydistinctive replication-timing pattern on the other Xchromosome (in all likelihood the Xa): the LINE-1-richregions were replicated later in S phase than the restof the X chromosome. This pattern, which was notapparent in heterozygous or wild-type cells, provides alink between the Xist gene and LINE-1 elements thathad previously been proposed to play a role in X-inactivation (Lyon 1998) and had also been proposedto interact with the Xist locus by heterochromatinassociation (Marahrens 1999). LINE-1 elements dis-play the heterochromatic property of DNA methylationon both the Xa and the Xi in wild-type cells (Hansen

2003). Our findings therefore indicate that both Xistalleles function in cis and in trans to influence the stateof heterochromatic regions on both X chromosomes.Our data reveal nonredundant cis- and trans-effects bythe two alleles on replication time as the double dele-tion caused both X chromosomes to display altered rep-lication times and/or patterns that were distinct fromthe times and/or patterns in heterozygous or wild-typecells. In contrast, the two Xist alleles were redundantin the control of histone H4 deacetylation since thepresence of either Xist deletion resulted in little or noH4 acetylation while the double deletion resulted in H4acetylation appearing throughout the Xi.

What are possible explanations/mechanisms for thedelayed replication timing of the Xi in the doublydeleted cells? A protein has been reported that increasesreplication fork progression through chromatin andalso helps resolve paused forks (Szyjka et al. 2005).Other studies have identified chromatin-associatedproteins that also maintain chromosome stability bypreventing the stalling and collapse of replication forks(Krings and Bastia 2004; Kai et al. 2005; Sommariva

et al. 2005). The abnormal chromatin in XaXist-D21-kb

XiXist-D21-kb cells may impede the association or function ofsuch proteins. Another possibility is that the XaXist-D21-kb

XiXist-D21-kb condition increases the amount of DNA dam-age that arises on the X chromosome, perhaps byfostering a chromatin structure that either is refractory

to DNA repair or renders repetitive sequences unstableand prone to DNA damage. Increased DNA damagewould not only slow replication but also lead to theobserved X chromosome instability.

The late-shifted replication of the two X chromo-somes in XaXist-D21-kbXiXist-D21-kb cells was associated with anincreased incidence of deletions and translocationsinvolving the X chromosome, p53 phosphorylation atserine-15, and the appearance of high levels of phos-phorylated histone H2AX (g-H2AX) on the Xi. Un-usually late replication and a predisposition to formdeletions and translocations are also properties of rare(Hansen et al. 1993; Wang et al. 1999; Hellman et al.2000) and common (Arlt et al. 2003) fragile sites. Rarefragile sites are caused by triplet repeat expansions(Sutherland 2003) while common fragile sites arebroader regions (with poorly defined sequence deter-minants) whose fragility can be induced (or ‘‘activated’’)by the partial inhibition of DNA replication using DNApolymerase inhibitors such as aphidicolin (Arlt et al.2003). Activated fragile sites are therefore thought to beregions that cause DNA polymerase to stall and a subsetof these stalling events escapes cell cycle checkpointsleading to chromosome breaks (Arlt et al. 2003).Aphidicolin treatment has been associated with theappearance of numerous g-H2AX foci (Musio et al.2005), which may be associating not only with double-strand breaks but also with stalled replication forks(Ward et al. 2004). An attractive possibility therefore isthat the deletion of 21 kb from both Xist gene copiescreates an abnormal chromatin structure on the Xchromosome that causes DNA polymerases to stall,resulting in the widespread accumulation of g-H2AX,delayed DNA replication, and the appearance of fragilesites. Interestingly, both Brca1 (Ganesan et al. 2002)and Atr (Ouyang et al. 2005) have been implicated inthe control of the heterochromatin of the Xi and thedisruption of either Brca1 (Arlt et al. 2004) or Atr(Casper et al. 2002) activity has been reported to lead tothe appearance of common fragile sites throughout thehuman genome. As chromatin is increasingly impli-cated in the maintenance of genome stability and inDNA repair and BRCA1 associates with XIST RNA, it isnot inconceivable that a common pathway to maintainX chromosome stability may involve Xist, Brca1, and Atr.

Cells homozygous for the Xist deletion retained theBrca1 signal while loss of Brca1 has previously beenreported to lead to loss of Xist RNA signal (Ganesan

et al. 2002), suggesting that Brca1 functions upstream ofXist. Consistent with this are the similar effects of Brca1and Xist deficiencies on the Xi: loss of macroH2A(Csankovszki et al. 1999; Ganesan et al. 2002), alteredreplication timing (Ganesan et al. 2002), altered H3methylation (Ganesan et al. 2002), and DNA instability(Narod and Foulkes 2004). The proportion of cellsthat displayed a BRCA1 signal on the X chromosomewas previously reported to vary considerably among


human cell lines (Ganesan et al. 2002) and may beparticularly high in MEFs. Differences in species andcell type likely play a role in these differences. It shouldbe noted that in human cells the BRCA1 signal on the Xiis restricted to late S phase when the Xi is replicated(Chadwick and Lane 2005). In mouse cells, the in-active X chromosome begins replicating much earlierin S phase (Evans et al. 1965; Galton and Holt 1965;Tiepolo et al. 1967). Brca1 may associate with the Xiwhile it replicates and the extension of the Brca1 signalto a broader portion of the cell cycle in MEFs may, atleast in part, reflect the replication of the Xi over alarger time interval. We should also point out that, inmouse, the Xist RNA signal associated with the Xipersists into metaphase (Lee and Jaenisch 1997;Duthie et al. 1999) while in humans the RNA signaldisappears at the onset of mitosis (Clemson et al. 1996).In MEFs, Brca1 association with the Xi might also beextended to additional sections of the cell cycle.

Our finding that an intense MeCP2 signal thatcolocalizes with the Brca1 signal is retained in XaXist-D21-kb

XiXist-D21-kb cells suggests that DNA methylation is retainedon the Xi. Indeed, we have found that DNA methyla-tion plays an important role in gene silencing on theinactive X chromosome in XaXist-D21-kbXiXist-D21-kb cells (J. L.Salstrom, C. Wang, C. Wang, A. Datta, S. Zeitlin, G.Csankovszki, C. D. Eller, S. Diaz-Perez, J. Wang, A.Chess, S. Huang, B. Kaltenboeck and Y. Marahrens,unpublished results). Interestingly, observations infungi (Tamaru and Selker 2001), plants (Jackson

et al. 2002), and mammals (Lehnertz et al. 2003)indicate that H3 lysine-9 methylation drives DNAmethylation. H3 lysine-4 methylation arises throughoutthe inactive X chromosome in XaXist-D21-kbXiXist-D21-kb cellsand this modification is thought to be mutually exclu-sive to H3 lysine-9 methylation (Boggs et al. 2001). Ourdata therefore suggest that, in XaXist-D21-kbXiXist-D21-kb cells,DNA methylation is uncoupled from histone H3 lysine-9methylation. Tiling arrays previously revealed an incom-plete correspondence between H3 lysine-9 methylationand DNA methylation in plants (Lippman et al. 2004).

Our findings raise the issue of how the trans-effectsthat we detect between the Xa and Xi are achieved. Wehad previously invoked Occam’s razor to propose thatthe initiation of X-inactivation is triggered by the tran-sient homologous pairing of the two copies of the Xic(Marahrens 1999). In support of this model, transienthomologous pairing of the Xic has recently beendemonstrated to mark the initiation of X-inactivation(Bacher et al. 2006; Xu et al. 2006). Our findings thatdeletions at the Xist locus result in trans-effects are mostparsimonious with a model in which the loss of Xist fromboth alleles alters the frequency or the consequences ofhomologous pairing. An alternative explanation is thatthe Xist deletions cause the undeleted 39 portion of theXist gene to be transcribed in XaXist-D21-kbXiXist-D21-kb cellsthat, in contrast to wild-type Xist RNA, function in trans.

However, we find that the 39 undeleted portion of theXist-D21 allele is not transcribed from either the Xa orthe Xi. Homologous pairing could not be detected inwild-type MEFs in asynchronous cells (Xu et al. 2006).Although this may be due to pairing being restricted to asmall portion of the cell cycle in wild-type cells such aslate S phase (LaSalle and Lalande 1996), we wonderwhether the loss of Xist may also increase the homolo-gous pairing of the Xa and Xi. Interestingly, there isevidence that H3 lysine-4 methylation is required forhomologous pairing during male meiosis (Hayashi

et al. 2005). It will be interesting to see whetherhomologous pairing of the X chromosomes is increasedin XaXist-D21-kbXiXist-D21-kb cells and whether such an increaseoccurs at many positions along the X chromosomes andrequires the H3 lysine-4 methylation that arises on theXi in these cells.

We thank Shridar Ganesan and David M. Livingston for theirgenerous gift of anti-Brca1 antibody. Adenoviruses expressing crerecombinase (Tan et al. 1999) and GFP were kindly provided by CarolEng and Arnold J. Berk. We thank Elaine Wong for generating someof the primary fibroblasts, Moira Regelson for performing statisticalanalysis, Jeannie T. Lee (Department of Molecular Biology, Massachu-setts General Hospital) for providing the Xist P1 mouse clone ppJL1,and Barbara Panning (Department of Biochemistry and Biophysics,University of California, San Francisco) for providing a Xist-containingBAC. This work was supported by a March of Dimes Basil O’ConnorStarter Scholar research award (5-FY99-819) (Y.M.), a seed grant fromthe University of California, Los Angeles, Jonsson Cancer CenterFoundation (Y.M.), and the National Institutes of Health [R01HD41451:01 (Y.M.) and R01CA107300 (M.A.T.)].

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Communicating editor: M. Justice


xist gene exposes trans-effects that alter the ...xist rna, macroh2a, and h3 lysine-9 methylation...

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