2006_11_Diaz-Perez

Copyright Ó 2006 by the Genetics Society of America

DOI: 10.1534/genetics.105.051375







A Deletion at the Mouse Xist Gene Exposes Trans-effects That Alter the

Heterochromatin 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, 2005

Accepted for publication August 26, 2006





ABSTRACT

The inactive X chromosome of female mammals displays several properties of heterochromatin

including late replication, histone H4 hypoacetylation, histone H3 hypomethylation at lysine-4, and

methylated CpG islands. We show that cre-Lox-mediated excision of 21 kb from both Xist alleles in female

mouse 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 phase

than 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 in

S phase. Extreme late replication is a property of fragile sites and the 21-kb deletions destabilized the DNA

of 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 of

g-H2AX, a histone involved in DNA repair, on the X chromosome. The Xist locus therefore maintains the

DNA 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

The Xist RNA is quite stable and colocalizes exclusively

with the Xi (Brown et al. 1992; Clemson et al. 1996). In

addition to the role of Xist, the spread of X-inactivation

and Riggs 1983). X-inactivation requires a region called correlates with high concentrations of LINE-1 elements

the X-inactivation center (Xic) (Lyon 1996). Physical on the X chromosome (Lyon 1998). Accordingly, X-

homologous association of the two copies of the Xic has linked genes that escape X-inactivation are found in re-

been proposed to trigger X-inactivation (Marahrens gions with reduced concentrations of LINE-1 sequence

1999) and such an association has recently been shown (Bailey et al. 2000). In cells deficient for the DNA

to mark the onset of X-inactivation (Bacher et al. methyltransferase Dnmt3b, the DNA of LINE-1 ele-

2006; Xu et al. 2006). The X-linked Xist gene, which ments on the Xi, but not on the Xa, is hypomethylated

resides in the Xic (Brown et al. 1991), plays a central (Hansen 2003) and X-inactivation is either incomplete

role in the subsequent heterochromatin formation or not fully maintained (Hansen et al. 2000).

(Penny et al. 1996) and Xist knockout mice die early in Another feature that distinguishes the Xi from the Xa

embryogenesis due to a failure to undergo X-inactiva- and from autosomes is that it is replicated later in S

tion (Marahrens et al. 1997). Xist encodes an un- phase (Taylor 1968; Taylor and Miner 1968). The

translated RNA that is expressed from the inactive X replication timing of the Xi reflects a general trend

chromosome (Xi) but not from the active X chromo- where later replication times are associated with gene

some (Xa) (Brockdorff et al. 1991; Brown et al. 1991). repression and early replication with transcriptional

competence (Gilbert 2002). The available evidence

1

indicates that the same replication origins are utilized

Corresponding author: Department of Human Genetics, UCLA, Gonda

Center, 695 Charles E. Young Dr., South Los Angeles, CA 90095-7088. on the active and inactive X chromosomes (Cohen et al.

E-mail: ymarahrens@mednet.ucla.edu 2003; Gomez and Brockdorff 2004), thus suggesting



Genetics 174: 1115–1133 (November 2006)

1116 S. V. Diaz-Perez et al.



that the replication timing differences between the two H2AX to produce g-H2AX (Burma et al. 2001). g-H2AX

X chromosomes stem from the times in S phase that associates with the Xi in the absence of experimentally

their origins are activated. While in female human cells incurred DNA damage, but this is restricted to late S

the Xi is replicated much later in S phase than the Xa phase (Chadwick and Lane 2005). The phosphoryla-

(Priest et al. 1967), the Xi is not replicated nearly as late tion of p53 stabilizes and activates the protein, which

in S phase in female mouse cells as in human cells signals for either cell cycle arrest or apoptosis (Attardi

(Evans et al. 1965; Galton and Holt 1965; Tiepolo 2005). g-H2AX has been proposed to recruit additional

et al. 1967). This has led to the Xi in mouse cells being proteins to sites of DNA damage (Bassing and Alt

distinguished by its absence of label incorporation in 2004). Deficiency in either ATR or ATM disturbs the

early S phase rather than by its being disproportionately maintenance of X-inactivation (Ouyang et al. 2005).

replicated late in S phase (Nesbitt and Gartler 1970). Excision of the transcribed Xist allele from the Xi

Nevertheless, there is always a consistent trend of the leads to the loss of the Xist RNA and absence of

mouse Xi displaying more label incorporation late in S macroH2A from the Xi (Csankovszki et al. 1999) and

phase than the Xa in both primary and transformed to a destabilization of X chromosomal gene silencing

female fibroblasts (Diaz-Perez et al. 2005). (Csankovszki et al. 2001) but does not abolish late

The protein composition of the Xi also distinguishes replication (Csankovszki et al. 1999) or result in an

it from other chromosomes. The histone H2A homolog, acetylated Xi (Csankovszki et al. 1999). The tran-

macrohistone H2A, is present along the length of the scribed Xist allele, therefore, functions in cis to main-

Xi but not the Xa (Costanzi and Pehrson 1998). In tain a subset of the features of the Xi heterochromatin.

addition, nearly all of the nucleosomes of the Xi are Excision of 21 kb from the nontranscribed Xist locus of

hypoacetylated at the N-terminal tail of histone H4 the Xa results in the Xa being replicated later in S phase

(Jeppesen and Turner 1993). Histone tail acetylation is (Diaz-Perez et al. 2005). Both Xist alleles therefore dis-

a widespread characteristic of euchromatin and histone play biological activity. Here we show that element(s)

deacetylation is a general characteristic of heterochro- at both copies of the Xist gene control the chromatin

matin ( Jenuwein and Allis 2001). Furthermore the structure of the Xi and influence the replication time of

nucleosomes of the Xi are methylated at histone H3 both X chromosomes. Xist deficiency furthermore de-

lysine-9 (Peters et al. 2001; Chadwick and Willard stabilizes both X chromosomes, leading to deletions

2004) or lysine-27 (Plath et al. 2003; Chadwick and and translocations, the phosphorylation of p53 at serine-

Willard 2004), and both are histone modifications 15, and the increased association of the DNA repair/

associated with heterochromatin. Methylation at H3 genome maintenance protein g-H2AX with the Xi. Xist

lysine-4, a euchromatic histone modification that ap- deletions therefore reveal trans-interactions that occur

pears to be mutually exclusive to lysine-9 methylation, is subsequent to the initiation of X-inactivation.

conspicuously absent from the Xi (Boggs et al. 2001).

Yet another feature that distinguishes the Xi from other

chromosomes is that it is associated with high concen- MATERIALS AND METHODS

trations of the BRCA1 protein that associates with XIST Fibroblasts and growth conditions: All of the mice used in

RNA (Ganesan et al. 2002). In BRCA1-deficient cells, this study had a 129 genetic background. Mouse primary

XIST RNA, macroH2A, and H3 lysine-9 methylation all fibroblasts were obtained from wild-type 129 mice and also

failed to concentrate on the Xi (Ganesan et al. 2002, from crosses involving previously described mouse strains

(Csankovszki et al. 1999, 2001) by trypsinization of 13-day

2004). embryos, culture, and immortalization with SV40 T-antigen

In addition to its role in X-inactivation, BRCA1 func- ( Jat et al. 1986). Three immortalized fibroblast cell lines were

tions as a tumor suppressor that plays a role in cell obtained from three E13.5 mouse embryos (one embryo per

cycle checkpoints, in multiple types of DNA repair, cell line), in which 21 kb of sequence at the Xist locus were

and in the maintenance of genome stability (Scully flanked by Lox sites (floxed) on both the Xa and Xi (XaXist-flox

XiXist-flox). Three additional immortalized fibroblast cell lines

and Livingston 2000; Welcsh et al. 2000; Narod and were obtained from three wild-type 129 embryos (XaXist-WTXiXist-WT).

Foulkes 2004). Stalled DNA replication forks as well The three XaXist-floxXiXist-flox cell lines (XaXist-floxXiXist-flox-1, -2, and -3)

as various types of DNA damage, including UV dam- and three XaXist-WTXiXist-WT cell lines (XaXist-WTXiXist-WT-1, -2, and -3)

age, cause the ataxia-telangiectasia-mutated and Rad3- were infected with adenovirus expressing cre recombinase

related (ATR) kinase to phosphorylate various targets and GFP (Tan et al. 1999) and plated out in 24-well plates at

less than one cell per well (limiting dilution) to recover clonal

including BRCA1 (Tibbettset al. 2000), p53 at serine-15 cell lines from each progenitor line. GFP expression was used

(Tibbetts et al. 1999), and H2AX (to produce g-H2AX) to identify infected cells. Starting from the fibroblasts that

(Ward and Chen 2001; Ward et al. 2004). Double- arise during the limiting dilution procedure, each clonal cell

strand breaks cause the related ataxia-telangiectasia line was passaged five times. During this passaging, lines that

mutated (ATM) kinase to phosphorylate many of the were 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 the

same targets including BRCA1 (Cortez et al. 1999; floxed Xist allele were identified using PCR. RNA FISH for Xist

Gatei et al. 2000), p53 at serine-15 (Banin et al. 1998; transcript was used to determine that, in the heterozygous line,

Canman et al. 1998; Khanna et al. 1998), and histone the deletion was on the Xi (XaXist-floxXiXist-D21-kb-1.1) (not shown).

Xist Affects DNA Stability 1117



After the aforementioned five passages, the three clonal XaXist-D21-kb 6-cm-diameter dishes with 106 cells with virus in 200 ml of

XiXist-D21-kb cell lines and the three clonal XaXist-WTXiXist-WT cell Dulbecco modified Eagle’s minimum essential medium

lines (from six different embryos) were each subjected to a (DMEM) with 5% fetal bovine serum (FBS) at 37° for 1 hr

BrdU pulse (see below) and metaphase spread chromosomes followed by the addition of 3.0 ml of DMEM with10% FBS.

were prepared. These spreads were analyzed for evidence of Primary and transformed mouse fibroblast cell lines were

chromosomal deletions and translocations using chromo- grown in DMEM supplemented with 10% fetal bovine serum

some paint, BrdU immunostaining, and spectral karyotyping (GIBCO, Grand Island, NY), penicillin (100 mg/ml), and

(see below). Note that the three XaXist-D21-kbXiXist-D21-kb (-1.1, -2.1, streptomycin (100 mg/ml). The PCR primers 59 LoxF (59-TTT

and -3.1) and three XaXist-WTXiXist-WT (-1.1, -2.1, and -3.1) cell lines CTG GTC TTT GAG GGC AC-39), 59 LoxR (59-ACC CTT GCC

used in the analysis for deletions and translocations were gen- TTT TCC ATT TT-39), and Xint3R (59-CAC TGG CAA GGT

erated using identical procedures. Primary XaXist-D21-kbXiXist-D21-kb GAA TAG CA-39) were used to identify the Xist-flox (612-bp

did not grow well enough to perform immunostaining or PCR product), Xist-D21kb (513 bp), and Xist-WT (427 bp)

replication timing experiments; we are exploring approaches alleles. XaXist-D21kbXiWT fibroblasts were distinguished from

to remedy this. XaWTXiXist-D21kb fibroblasts using RNA FISH against the Xist

In addition, cell lines XaXist-floxXiXist-flox-1, -2, and -3 were RNA (see below).

infected with adenovirus expressing only GFP and limiting RNA FISH: Fibroblasts were grown on coverslips for 24 hr

dilution was used to recover clonal cell lines XaXist-floxXiXist-flox- and then fixed in 4% formaldehyde for 15 min at room

1.1, -2.1, and -3.1 using the same procedure and the same temperature (RT). The cells were permeabilized in PBS

number of passages as was used to obtain lines XaXist-D21-kb containing 0.5% Triton-X for 5 min on ice and washed in

XiXist-D21-kb-1.1, -2.1, and -3.1. Two additional cell lines used in PBS and 23 SSC. RNA FISH hybridization was carried out as

this study were obtained by infecting fibroblasts that were previously described (Spector and Goldman 1998.). The Xist

heterozygous for the Xist-flox allele with adenovirus express- probe was labeled by nick translation with biotin-21-dUTP.

ing cre recombinase and GFP and using limiting dilution, PCR, After overnight hybridization at 37° posthybridization washes

and RNA FISH to recover and identify clonal cell lines that were done as previously described (Spector and Goldman

carried the 21-kb deletion on the Xi (XaXist-WTXiXist-D21-kb-1.1 and 1998). The probe was detected with a 500-fold dilution of

XaXist-WTXiXist-D21-kb-2.1). Finally, three XaXist-flox,Hprt-D XiXist-flox,Hprt-WT avidin-FITC ( Jackson ImmunoResearch, Westgrove, PA) at RT

cell lines and derivative XaXist-D21-kb,Hprt-D XiXist-D21-kb,Hprt-WT cells were for 1 hr. The nuclei were counterstained with 49,6-diamidino-2-

obtained using the same procedure, except that a series of phenylindole (DAPI).

additional mouse matings were first performed to enable the Two-color DNA FISH: A combination probe, composed of a

production of fibroblasts that also included a published Hprt mixture of seven different small probes, was used to identify

deletion (Hooper et al. 1987) on the Xa. The generation of the the inactive X chromosome. This combination probe, hence-

Hprt-heterozygous cell lines is described in detail elsewhere forth referred to as 6.8-kb probe, contains PCR products of

( J. L. Salstrom, C. Wang, C. Wang, D. Dutta, S. Zeitlin, G. sizes (in base pairs) 412, 605, 609, 850, 1011, 1560, and 1755.

Csankovszki, C. D. Eller, S. Diaz-Perez, J. Wang, A. Chess, Each of these PCR products was amplified separately using Taq

S. Huang, B. Kaltenboeck and Y. Marahrens, unpublished Polymerase (Promega, Madison, WI) from a region extend-

data). ing up to 14.7 kb upstream of the 59 end of exon 2 of the Hprt

A large proportion of the immortalized cells in each culture gene and that is deleted in the Hprt-D allele used in this study

contained either three or four X chromosomes. Limiting (Thompson et al. 1989). The mouse BAC RP23-412J16 [pur-

dilution was also used to obtain clonal cell lines containing chased from Invitrogen (Carlsbad, CA)] served as template for

predominantly two X chromosomes and an approximately the PCR reactions. The sequences of primers are: GCA AGC

diploid number of chromosomes. To this end, the same three ATA AGG ACC AGA GC (412R), TTC CAC AAG AAA TAT TAC

immortalized XaXist-floxXiXist-flox (-1, -2, and -3) cell lines de- ACA AAA CA (412L), CCT AAC CAT TGA GCC GTC TT

scribed in the previous paragraph were infected with adeno- (605R), GGT CTC TGA ACT ACC AAT TGC AC (605L), GCA

virus expressing cre recombinase and/or GFP (Tanet al. 1999), ATG ACA AAT GTT TTG TGG (609R), TGC TTA TTA GCA

and limiting dilution was used to recover numerous clonal cell CAA GAC CTC AAG (609L), ATC ACC CTA TTC CCA GTG

lines from each progenitor line. PCR was used to identify XaXist- GA (850R), GCA GAT GAT AAG CTA TCC TTG AGA (850L),

D21-kb

XiXist-D21-kb cell lines and metaphase spread chromosomes CAT CAC TGA GTC TTG CTG GTT T (1011R), CAA TTT AGG

and flow cytometry was used to identify XaXist-D21-kbXiXist-D21-kb and GGA AGG AAG CA (1011L), TGG TAG CTG GGC ATA AAA

XaXist-floxXiXist-flox lines that were derived from diploid cells. A GC (1560R), AAT GGG AGA AAA GGC AGG AT (1560L), CAG

number of these diploid cell lines were subjected to X chromo- GAA AGG GTG TGT GTG TG (1755R), and TAC GCT CTG

some paint to confirm the presence of two X chromosomes. GCA GTT TTC AA (1755L). The PCR products were gel

We chose six approximately diploid cell lines containing two X extracted using the Wizard PCR Preps DNA purification

chromosomes each that were derived from three embryos, system (Promega). To mark both the active and the inactive

with lines XaXist-D21-kbXiXist-D21-kb-1.2 and XaXist-floxXiXist-flox-1.2 from X chromosomes, mouse BAC RP23-298N24 (obtained from

the same embryo, XaXist-D21-kbXiXist-D21-kb-2.2 and XaXist-floxXiXist-flox- Invitrogen) was used to obtain a second probe. For fluorescent

2.2 from the same embryo, and XaXist-D21-kbXiXist-D21-kb-3.2 and probe preparations, 1 mg of DNA was direct labeled with

XaXist-flox XiXist-flox-3.2 from the same embryo. Using the identical either FluorX-dCTP (for the whole BAC probe) or Cy3-dCTP

procedure, approximately diploid cell lines containing two X (for the Xi-specific 6.8-kb combination probe), using the Nick

chromosomes each were also derived from the XaXist-WTXiXist-D21-kb Translation kit (Amersham Biosciences, Arlington Heights,

and XaXist-floxXiXist-D21-kb cells (XaXist-WTXiXist-D21-kb-1.2 and XaXist-flox IL). To prepare the 6.8-kb probe, equimolar concentrations of

XiXist-D21-kb-1.2). The diploid lines thus established were all used the seven different probes were mixed together, to a total DNA

at equally low passage numbers because they all became in- content of 1 mg, for labeling. Labeled probes were purified

creasingly tetraploid with extended passaging. Diploid XaXist- using NucAway Spin columns (Ambion, Austin, TX) and

flox,Hprt-D

XiXist-flox,Hprt-WT and XaXist-D21-kb,Hprt-D XiXist-D21-kb,Hprt-WT cells precipitated with 40 mg mouse Cot-1 DNA, 100 mg salmon

were also obtained. sperm, and 100 mg tRNA; washed in 75% ethanol followed by

Infection of fibroblasts with adenovirus expressing cre 100% ethanol; and resuspended in 100 ml hybridization buffer

recombinase and/or adenovirus expressing GFP (Tan et al. (50% formamide, 10% dextran sulfate, 13 SSC). Cells were

1999) was performed at 50 multiplicities of infection (MOI) in treated for FISH as described previously (Singh et al. 2003).

1118 S. V. Diaz-Perez et al.



Briefly, cells were fixed with 3:1 methanol–acetic acid, dropped representation of each color in the final image was adjusted

on poly-l-lysine-coated slides (Sigma, St. Louis) in a humid using the software setting of the gain for that color. The BrdU

chamber, and denatured for 2 min at 69°–72° in 70% incorporation studies were not done simultaneously with Xist

formamide/23 SSC. An 18-ml aliquot of the two probes (12 RNA FISH because the Xist RNA is lost from the Xi during

ml of 6.8-kb probe, 6 ml of BAC probe) was prehybridized (90° mitosis. The Xist RNA signal that can be seen on the Xi in early

for 5 min, followed by 10 min at 37°) and then hybridized mitotic cells is very fragile and the treatments that occur

overnight with cells at 37°. Next, cells were washed three times during the BrdU incorporation assay caused the Xi to lose Xist

with 50% formamide/23 SSC at 42° followed by three washes RNA signal.

with 13 SSC also at 42°. The following washes were done at Spectral karyotyping: The spectral karyotyping (SKY) probe

room temperature: 13 SSC (10 min), 43 SSC (5 min), 43 mixture (Applied Spectral Imaging) was applied according to

SSC/0.1% Tween-20 (5 min), and 43 SSC (5 min). The cells the manufacturer’s recommendations for metaphase chromo-

were mounted in Vectashield mounting medium containing some spreads (MCSs) prepared as described for the BrdU

DAPI (Vector Laboratories, Burlingame, CA) to counterstain incorporation assay. Chromosomal aberrations were quanti-

the nuclei. Cells were viewed with a Nikon E600 fluorescent fied using an Olympus BX-61 microscope equipped with an

microscope. Images were captured with a CCD camera using Applied Spectral Imaging interferometer and 403 and 633

SPOT Advanced software. objectives, driven by a desktop computer with SKY acquisition

Quantitative determination of Xist mRNA: Quantitative and analysis software.

determination of Xist mRNA was performed by one-step Quantitation of incorporated BrdU: X chromosome paints

reverse transcription (RT) fluorescence resonance energy (Cambio, Cambridge, UK) were used to identify the X chro-

transfer (FRET) real-time PCR of 1:100 diluted poly(A) RNA mosomes and the X chromosome displaying the higher level

samples in a Lightcycler modeled after the duplex PCR of BrdU incorporation within a spread was always assumed to

approach described earlier (Wang et al. 2004). Xist primers be the inactive one. Images obtained using Quips mFISH were

(muXISTmRNAUP, 59-CCC TAC ATC AAA GTA GGA GAA transferred to NIH image (http:/ /rsb.info.nih.gov/nih-image)

AAG CTG CTG-39; muXISTmRNADN, 59-GAA GGG TAA TAT and the numbers of pixels occupied by the X chromosomes

TTG GTA GAT GGC ATT GTG T-39) transversed the bound- (DAPI) and by fluorescently labeled BrdU (Texas Red) were

aries of exons 4 and 5 and of exons 5 and 6, respectively, and then calculated for each MCS. Fisher’s exact test was used to

the FRET probes (muXISTFLU, 59-CCT AGC TTC TGG AGA compare the percentages of X chromosomes displaying BrdU

GAG AAC CAA ATA GAG-6-FAM-39; muXISTBOD, 59-Bodipy incorporation between different cell lines. Box plots were used

630/650-AGA ATG GCT TCC TCG AAG GTC AGT GC- to visualize the distributions of BrdU measurements across

Phosphate-39) detected exon 5. Thermal cycling conditions categorical groupings. Box plots labeled ‘‘% BrdU signal’’

of this PCR were 30 min reverse transcription at 55°, followed represent measurements of the number of pixels of BrdU

by 2 min denaturation at 95°, followed by thermal cycling: 6 signal on a chromosome divided by the number of pixels of

times at 95°, 0 sec/68°, 12 sec/72°, 8 sec; 9 times at 95°, 0 sec/ DAPI signal occupied by the same chromosome multiplied by

66°, 12 sec/72°, 8 sec; 3 times at 95°, 0 sec/64°, 12 sec/72°, 8 sec; 100. NIH Image was also used to record the intensity of each

25 times at 95°, 0 sec/56°, 12 sec followed by fluorescence pixel. ‘‘BrdU area 3 intensity’’ represents the % BrdU signal

acqusition/72°, 10 sec. Quantitative standards were produced multiplied by the average intensity of the pixels representing

by PCR amplification with dTTP, gel purification, and quan- BrdU. The statistical analyses were performed using the

tification of the fragment by Pico-Green assay (Invitrogen). software package R (Ihaka and Gentleman 1996), which

Reaction chemistry was as published (Wang et al. 2004). The can be downloaded from http:/ /cran.r-project.org/. Differ-

internal autosomal reference gene transcript (porphobilino- ences in measurements were tested across categorical group-

gen deaminase, PBGD) was amplified from undiluted poly(A) ings using the Kruskal–Wallis test (Kruskal 1964) and the

RNA in a separate reaction following the duplex PCR proto- P-values obtained from this test are displayed above the

col as described (Wang et al. 2004). All analyte transcript corresponding box plots.

concentrations are expressed as copies per PBGD reference X chromosome paint: A total of 10 ml of mouse X

transcripts. chromosome-specific biotinylated probe (Cambio) were used

Fluorescent immunostaining for BrdU in metaphase to detect the X chromosomes by fluorescent in situ hybridiza-

chromosome spreads: BrdU (30 mm) was added to asynchro- tion (DNA FISH) to ethanol-dehydrated cells according to

nous actively growing fibroblasts at 80% confluence. Several manufacturer’s instructions. The probe was detected using

BrdU pulse lengths were performed on multiple cell lines and streptavidin–FITC ( Jackson ImmunoResearch) and anti-

these data were used to determine that 4.5 hr is the appro- streptavidin FITC (Vector Laboratories) at 1:50 dilution each

priate duration of BrdU incorporation for each replication- in blocking solution (13 PBS, 10% human serum, 0.05%

timing experiment (data not shown). Exponentially growing Tween 20). The chromosomes were counterstained with DAPI

asynchronous fibroblasts were cultured for 4.5 hr in the and viewed with a Leica DMR fluorescent microscope. Images

presence of BrdU and 0.050 mg/ml Colcemid (Life Technol- were captured with Quips mFISH software (Vysis).

ogies, Grand Island, NY) was added 1 hr before harvesting. Determining sequence composition across the X chromo-

Cell suspensions were incubated 13–15 min in 0.4% KCl at 37° some: We identified the types and positions of repetitive

followed by fixation with 3:1 methanol:acetic acid. Metaphase sequences from the RepeatMasker output provided by the

spreads were prepared by dropping the BrdU-treated cells UCSC genome browser (http:/ /genome.ucsc.edu). For suc-

onto coverslips followed by DNA denaturation in 70% form- cessive 1-Mb intervals, we then obtained a value for each re-

amine/23 SSC at 73° for 2 min. Following preincubation with peat type representing the percentage of the 1-Mb sequence

blocking buffer (13 PBS, 10% FBS, 0.2% Tween 20), in- occupied by that repeat type.

corporated BrdU was detected using 1:20 dilution in blocking Histone immunolabeling and whole-chromosome paint:

solution of monoclonal anti-BrdU antibody (Sigma) followed Indirect immunofluorescence with anti-acetyl-histone H4

by 1:150 dilution in blocking solution of Texas-Red anti-mouse antibody (Serotec, Oxford) and histone H3 (trimethyl-K4)

antibody ( Jackson ImmunoResearch) in blocking buffer. antibody (Abcam) was carried out on asynchronous cultures.

Images were captured using Quips mFISH software (Vysis, Fibroblasts were grown at 80% confluence, Colcimid (0.05

Adelphia, NJ). The individual colors of a recorded image were mg/ml) was added for 1 hr, and fibroblasts were harvested and

stored separately by the Vysis Quips mFISH software and the then swollen in 0.4% KCl at 37° for 13 min. Fibroblasts were

Xist Affects DNA Stability 1119



dropped onto coverslips. Cell membranes were solubilized by KCM for 15 min and washed three times with KCM. The nuclei

immersion in KCM buffer [120 mm KCl, 20 mm NaCl, 10 mm were counterstained with DAPI and viewed with a Leica DMR

Tris–HCl, pH 8, 0.5 mm EDTA, 0.1% (v/v) Triton X-100] in a fluorescent microscope. Images were captured with Quips

petri dish (35 3 10 mm). The coverslips were transferred to mFISH software (Vysis).

blocking solution (10% FBS in KCM) at 37° for 1 hr and then Simultaneous immunolabeling of histone H3 trimethyl-

incubated with 20 ml of 1:10 of anti-acetylated H4 (Serotec) or lysine 4 and DNA FISH for sequence flanking the Xist gene:

1:20 anti-methH3-K4 (Abcam) in a humidified chamber for We detected histone H3 (trimethyl-K4) using a rabbit anti-

2 hr at RT or overnight at 4°. The coverslips were then washed body to H3 (Abcam), followed by a goat antibody to rabbit

three times for 5 min in KCM and transferred to blocking conjugated with Texas Red ( Jackson ImmunoResearch). The

solution and incubated at 37° for 1 hr. Primary antibody was immunodetection of histone was combined with DNA FISH as

detected with anti-rabbit Texas Red ( Jackson ImmunoRe- described (Brown et al. 2001) with the following modifica-

search) at 1:100 dilution in blocking solution and incubated tions: we labeled the P1 clone ppJL1 (Lee et al. 1999) with

for 1 hr at RT. The coverslips were washed three times with digoxigenin by nick translation (Dig–Nick translation mix;

KCM buffer and fixed at RT with 4% paraformaldehyde Roche, Indianapolis) and detected the label with sheep anti-

(Electron Microscopy Sciences, Fort Washington, PA) in Digoxigenin-Fluorecein (Roche) and rabbit anti-sheep Fluo-

KCM for 20 min and washed three times with KCM at RT. To recein (Vector Laboratories).

detect the X chromosomes, the coverslips were incubated two Simultaneous immunolabeling of g-H2AX and DNA FISH

times in methanol:acetic acid (1:3 v/v) at À20° for 20 min for sequence flanking the Xist gene: Biotinylated dUTP-

each. DNA was denatured with 70% formamide/23 SSC at 73° labeled DNA FISH probes were prepared by nick translation

for 8 min, dehydrated, and then hybridized to 10 ml of the from BAC DNA and P1 DNA, both of which include the Xist

biotinylated probe (Cambio) according to the manufacturer’s gene and flanking DNA sequence. Cell lines XaXist-D21-kbXiXist-

D21-kb

instructions. The probe was detected using streptavidin–FITC -1.1 and XaXist-D21-kbXiXist-D21-kb-2.1 were maintained in DMEM

( Jackson ImmunoResearch) and anti-streptavidin FITC (Vec- containing 10% FBS and seeded on coverslips 1 day before the

tor Laboratories) at 1:50 dilution each in blocking solution experiment. Cells on coverslips were fixed with 4% para-

(13 PBS, 10% human serum, 0.05% Tween 20). The chromo- formaldehyde in PBS for 10 min followed by 5 min permeabi-

somes were counterstained with DAPI and viewed with a Leica lization with 0.5% Triton X-100 at room temperature. Primary

DMR fluorescent microscope. Images were captured with antibody recognizing g-H2AX (Upstate Biotechnology) was

Quips mFISH software (Vysis). applied for 1 hr. Cells were washed with PBS three times before

Western analysis of p53: Proteins from whole-cell extracts incubation with secondary antibody that was conjugated with

were separated by sodium dodecyl sulfate (SDS) polyacryli- FITC ( Jackson ImmunoResearch Laboratories). Cells were

mide gel electrophoresis (PAGE), transferred to a charged then fixed again with methanol:acetic acid (3:1) at À20° for

PVDF membrane, and blocked with 5% nonfat milk in TBST 40 min followed by dehydration in 70, 90, and 100% ethanol

(20 mm Tris pH 7.4, 150 mm NaCl, 0.05% Tween-20). Primary for 3 min each. Cells were denatured in 70% formamide/23

and secondary antibody incubations were performed in 5% SSC at 85° for 30 min, cooled down with cold 70% ethanol, and

nonfat milk in TBST. Proteins were detected using the ECL dehydrated with 90 and 100% ethanol. The probe derived

Plus Western blotting detection reagent (GE Healthcare, from BAC DNA or from P1 DNA was simultaneously dena-

Piscataway, NJ). Primary reagents included: rabbit anti-p53- tured at 75° for 10 min in a hybridization mixture (DNA probe,

Ser15 (Cell Signaling Technology, Beverly, MA), rabbit anti- Cot1DNA, hybridization buffer, and 50% formamide) and

p53(CM5) (Vector Laboratories), mouse anti-b actin (Sigma), prehybridized at 37° for 20 min. The prehybridized probe was

and mouse anti-ATM-Ser1981 (Rockland, Gilbersville, PA). applied to the pretreated coverslip and incubated at 37° over-

Donkey anti-rabbit IgG-horseradish peroxidase (HRP) (GE night. After a series of stringent washes, avidin–Texas Red was

Healthcare) and sheep anti-goat IgG-HRP (GE Healthcare) added to the coverslip and incubated for 1 hr. Signal was

were used as the secondary antibodies. visualized using a Nikon Eclipse E800 microscope equipped

BRCA1, g-H2AX, and MeCP2 immunolabeling: Fibroblasts with a SenSys cooled CCD camera (Photometrics, Tucson,

were grown at 80% confluence and dropped onto coverslips. AZ). Images were captured using Metamorph image acquisi-

The cells were fixed with 2% of paraformaldehyde in PBS for tion software (Universal Imaging, Downingtown, PA).

5 min and washed three times with 13 PBS. The cells were

incubated in permeabilized solution (13 PBS, 0.5% Triton X-

100) for 10 min at 4° and washed three times with KCM buffer

RESULTS

[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 to Histone H4 acetylation and histone H3 lysine-4 meth-

blocking solution (10% FBS in KCM) at 37° for 1 hr and then

ylation accumulate on the inactive X chromosome when

incubated with anti-Brca1 1:2.5 dilution at 37° for 2 hr. The

coverslips were washed three times with KCM buffer and 21 kb are excised from both copies of the Xist gene:

blocking again at 37° for 30 min. The Brca1 antibody was To examine the role of the Xist locus (Figure 1A, top)

detected with anti-mouse Texas Red at 1:150 dilution and in the maintenance of chromatin structure, three fe-

incubated at room temperature for 1 hr and then washed male immortalized murine embryonic fibroblast (MEF)

three times and the process repeated for histone gH2AX cell lines were obtained, from three different E13.5

(Upstate Biotechnology, Charlottesville, VA) that was detected

using anti-rabbit FITC. The immunodetections were also embryos, in which 21 kb of sequence at the Xist gene

performed in the reverse order (Brca1 last) with similar re- were flanked by Lox sites (Figure 1A, middle) on both

sults. In other experiments, the g-H2AX antibody (Upstate the Xa and the Xi (lines XaXist-floxXiXist-flox-1, XaXist-flox

Biotechnology) was detected using either anti-rabbit FITC or XiXist-flox-2, and XaXist-floxXiXist-flox-3). The three lines were

anti-rabbit Texas Red. In yet other experiments, MeCP2 treated with adenovirus expressing cre recombinase

antibody (Upstate Biotechnology) was detected using anti-

rabbit FITC and in a subset of experiments the process was and GFP and were also infected with adenovirus ex-

repeated using Brca1 antibody detected with anti-mouse Texas pressing only GFP. Limiting dilution was used to obtain

Red. Finally, the cells were fixed with 4% paraformaldehyde in clonal cells that were homozygous for the 21-kb deletion

1120 S. V. Diaz-Perez et al.









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

D21-kb

XiXist-D21-kb cells. (A) Map of the Xist-WT, Xist-flox, and Xist-D21-kb alleles (left) and identification of the three alleles by PCR

genotyping (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 on

histone H3 and were also stained with DAPI and subjected to X chromosome paint or DNA FISH using an X chromosome-specific

probe as indicated. Arrowheads mark X chromosomes. X chromosome paint hybridized throughout the X chromosome and

also to the centromeres of all chromosomes in MCS. (B) XaXist-floxXiXist-flox MCSs fluorescently immunostained for acetylated lysines

on 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 was

also subjected to X chromosome paint (green) (a, right ). (C) XaXist-D21-kbXiXist-D21-kb MCSs immunostained for acetylated lysines on

histone 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. MCS

from 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)

Xist Affects DNA Stability 1121



(XaXist-D21-kbXiXist-D21-kb) (Figure 1A, bottom) or were XaXist-flox for a region of the X chromosome in combination with

XiXist-flox (from the adeno-GFP infections). The cell lines immunofluorescence indicated that one of the two X

were chosen such that each pair of cell lines with the chromosomes was hypomethylated at lysine-4 on his-

same number was derived from the same embryo (e.g., tone H3 in XaXist-flox, Hprt-DXiXist-flox, Hprt-WT cells (Figure 1J),

lines XaXist-floxXiXist-flox-1.1 and XaXist-D21-kbXiXist-D21-kb-1.1 are indicating that the Hprt deletion did not disrupt H3

derived from GFP-adenovirus and cre-adenovirus infec- lysine-4 hypomethylation on the Xi. In contrast, an X

tions of line XaXist-floxXiXist-flox-1, respectively). Using the chromosome that was hypomethylated at H3 lysine-4

same procedure, MEF lines XaXist-D21-kbXiXist-WT-1.1, XaXist-WT was absent from in the corresponding XaXist-D21-kb,Hprt-D

XiXist-D21-kb-1.1, and XaXist-D21-kbXiXist-WT-1.1 were obtained. PCR XiXist-D21-kb,Hprt-WT cells (Figure 1K), as expected. Two-color

(Figure 1A, right) and FISH (see below) were used to DNA FISH was performed to verify the presence of the

determine the genotypes of the cell lines (see materials inactive X chromosome in the XaXist-D21-kb,Hprt-DXiXist-D21-kb,

Hprt-WT

and methods). cells. A Cy3-labeled (red) combination probe,

We examined the state of histone acetylation in these comprising a mixture of seven separate probes to the

cell lines by fluorescence immunostaining of MCSs. deleted region of Hprt, was used to identify the inactive

Similar to previously published results for wild-type X chromosome (see materials and methods for

female cells ( Jeppesen and Turner 1993), the Xi was details). A FluorX (green)-labeled mouse BAC (RP23-

readily distinguishable from the other chromosomes 298N24) was used as a probe to mark both the inactive

due to its severe hypoacetylation of histone H4 in MCSs and the active X chromosomes. Cells, hybridized with

from cell lines XaXist-floxXiXist-flox-1, -2, and -3 (Figure 1B) both these probes together, when examined confirmed

and in MCSs from lines XaXist-floxXiXist-flox-1.1, -2.1, and -3.1 the presence of the inactive X chromosome. For one

(not shown). The Xi was also hypoacetylated on histone predominantly diploid XaXist-D21-kb,Hprt-DXiXist-D21-kb,Hprt-WT cell

H4 in MCSs from XaXist-D21-kbXiXist-WT cells (Figure 1D), line, 94 of 100 cells examined showed red hybridization

XaXist-WTXiXist-D21-kb cells (Figure 1E), and XaXist-floxXiXist-D21-kb signal in their nuclei (Figure 1L, a). Similarly, 96 of 100

cells (not shown) in agreement with previously pub- cells showed a red hybridization dot in another XaXist-D21-kb,

Hprt-D

lished results (Csankovszki et al. 1999). In contrast, in XiXist-D21-kb,Hprt-WT cell line derived from a different em-

MCSs from the three XaXist-D21-kbXiXist-D21-kb cell lines (1.1, bryo (Figure 1L, b). For both cell lines, most cells had

2.1, and 3.1), an inactive X chromosome could no one red dot near one of the two green dots. Also, we

longer be distinguished from the other chromosomes occasionally observed cells ($5%) that were tetraploid

on the basis of hypoacetylation (Figure 1C). for the X chromosome—having four green dots and two

The Xi was also severely hypomethylated at lysine 4 red dots near two of the green dots (not shown). We

of histone H3 in MCSs from the cell lines XaXist-floxXiXist-flox conclude that the Xi is still present in cells that have

-1, -2, and -3 (Figure 1F) and in the cell lines XaXist-flox excised 21 kb of Xist sequence from both the Xa and

XiXist-flox-1.1, 2.1, and 3.1 (not shown), as was previously the Xi and the homozygous deletion of Xist sequence

reported for wild-type female cells (Boggs et al. 2001). results in the Xi acquiring H3 lysine-4 methylation and

In contrast, in MCSs from all three XaXist-D21-kbXiXist-D21-kb H4 acetylation along its length. Additional lines of evi-

cell lines (1.1, 2.1, and 3.1), a Xi could not be distin- dence that indicate that the Xi is retained in XaXist-D21-kb

guished from the other chromosomes on the basis of XiXist-D21-kb cells are presented below.

hypomethylation (Figure 1G). RNA FISH revealed that, The dramatic chromatin changes observed on the

in contrast to the Xist RNA seen in XaXist-WTXiXist-WT cells inactive X chromosome in XaXist-D21-kbXiXist-D21-kb cells

(Figure 1H), no XIST RNA signal whatsoever was seen raised the question of whether Xist RNA was expressed

in XaXist-D21-kbXiXist-D21-kb cells (Figure 1I). To determine from the 39 undeleted portion of the Xist gene of either

whether the excision of 21 kb from both Xist alleles X chromosome in XaXist-D21-kbXiXist-D21-kb cells. To deter-

causes the Xi to be lost from cells, we examined XaXist-D21-kb mine whether a truncated Xist RNA was expressed,

XiXist-D21-kb cell lines from three embryos that carried a quantitative determination of Xist mRNA was per-

deletion at the Hprt locus (Hooper et al. 1987) on the formed by RT–FRET real-time PCR using PCR primers

Xa, while the Hprt locus on the Xi was intact. DNA FISH that 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 using

a P1-derived probe that recognizes the X chromosome and simultaneously stained with DAPI (top, blue) and immunostained

for trimethylated lysine-4 on histone H3 (bottom, red). (L) XaXist-D21-kb,Hprt-DXiXist-D21-kb,Hprt-WT MEFs were subjected to DNA FISH

using a probe that recognizes DNA sequence that is exclusively on the inactive X chromosome (red) and a probe that recognizes

both X chromosomes (green). The XaXist-D21-kb,Hprt-DXiXist-D21-kb,Hprt-WT MEFs ‘‘a’’ and ‘‘b’’ represented in K and L are derived from

the XaXist-flox,Hprt-DXiXist-flox,Hprt-WT MEFs ‘‘a’’ and ‘‘b’’ respectively represented in J via exposure to cre recombinase.

1122 S. V. Diaz-Perez et al.



respectively, and a FRET probe that recognized exon 5. than most or all autosomes. Although the mouse Xi

The internal autosomal reference gene transcript used does not replicate nearly as late in S phase in mouse cells

was PBGD (Wang et al. 2004). In two XaXist-WTXiXist-WT cell as in human cells, it nevertheless replicates later in S

lines, the Xist mRNA/PBGD mRNA ratios were 720.3 phase than the active X chromosome (Evans et al. 1965;

and 699.1. In contrast, the Xist mRNA/PBGD mRNA Galton and Holt 1965; Tiepolo et al. 1967). We

ratios in two XaXist-D21-kbXiXist-D21-kb cell lines were 0.0 and consequently inferred that the later replicating X chro-

0.0 because only PBGD mRNA, but no Xist RNA, was mosome in our female cultures was the Xi and that

detected in these samples. A comprehensive and de- the earlier replicating X chromosome was the Xa. The

tailed analysis of Xist RNA levels in cells bearing vari- level of BrdU signal that was recorded in a photograph

ous Xist genotypes will be published elsewhere ( J. L. of an X chromosome in a MCS depended, in part, on

Salstrom, C. Wang, C. Wang, A. Datta, S. Zeitlin, G. the gain setting of the red channel of the mQuips Vysis

Csankovszki, C. D. Eller, S. Diaz-Perez, J. Wang, A. software that was used to display the BrdU signal. At low

Chess, S. Huang, B. Kaltenboeck and Y. Marahrens, gain, only the fluorescent signal representing incorpo-

unpublished data). rated BrdU that exceeds a high intensity threshold is

Altered replication time on the inactive X chromo- recorded in the image of an X chromosome (Figure 2E,

some in response to 21-kb deletions: Since the Xi had top). At intermediate gain, the fluorescent BrdU signal

acquired two euchromatic properties (H4 acetylation exceeding an intermediate intensity threshold is re-

and H3 lysine-4 methylation) in XaXist-D21-kbXiXist-D21-kb cells corded in the image (Figure 2E, middle). At high gain,

we predicted that the deletions would also cause the Xi even small amounts of incorporated BrdU will be dis-

to replicate earlier in S phase since euchromatin gen- played among the pixels representing the BrdU signal

erally replicates earlier in S phase than heterochromatin (Figure 2E, bottom). To normalize the BrdU signal,

(Gilbert 2002). To determine if this was the case, we all photographs of metaphase chromosome spreads

purified approximately diploid lines XaXist-D21-kbXiXist-D21-kb- were taken at a standardized ‘‘low-gain’’ setting where

1.2, -2.2, and -3.2 from cre-adenovirus-infected XaXist-flox only five autosomes display BrdU signal and one of these

XiXist-flox progenitor lines (1, 2, and 3) using limiting five autosomes displays only one pixel of BrdU. This

dilution. We also purified predominantly diploid lines method of normalizing the gain using BrdU signal on

XaXist-floxXiXist-flox-1.2, -2.2, and -3.2 from GFP-adenovirus- autosomes was previously used to show that portions of

infected XaXist-floxXiXist-flox progenitor lines (1, 2, and 3). the Xa replicate later in S phase if 21 kb are deleted from

Each pair of diploid cell lines with the same first num- the Xist locus of the Xa and the method is described in

ber was derived from the same embryo (e.g., lines more detail in this earlier study (Diaz-Perez et al. 2005).

XaXist-floxXiXist-flox-1.2 and XaXist-D21-kbXiXist-D21-kb-1.2 are de- In addition, only MCSs displaying two X chromosomes

rived from line XaXist-floxXiXist-flox-1). Using the same pro- were considered. At the measurements taken at low

cedure, the predominantly diploid lines XaXist-D21-kbXiXist-WT- gain, all three clonal XaXist-flox XiXist-flox fibroblast lines

1.2, XaXist-WTXiXist-D21-kb-1.2, and XaXist-D21-kbXiXist-WT-1.2 MEF (1.2, 2.2, and 3.2) displayed modest BrdU signal on one

lines were also obtained. All diploid lines in this study X chromosome and either very little (Figure 2, C and D)

were used at a low passage number (with respect to or no (Figure 2, F and G) BrdU signal on the other X

limiting dilution) in our analyses because they increas- chromosome and therefore closely resembled the signal

ingly accumulated cells that had lost their diploid char- levels seen in XaXist-WTXiXist-WT MCSs (Diaz-Perez et al.

acter with repeated passages. 2005). Contrary to expectation, more BrdU signal (late

The predominantly diploid cell lines were subjected replication) was observed on the inactive X chromo-

to a replication-timing assay that uses fluorescence some in XaXist-WTXiXist-D21-kb-1.2 cells (Figure 2, H–K) and

immunostaining to detect chromosomal regions that XaXist-floxXiXist-D21-kb-1.2 cells (not shown) than in the

incorporated BrdU late in S phase. Cells were pulse XaXist-floxXiXist-flox cells. Quantitation of the BrdU signal

labeled with BrdU for 4.5 hr and metaphase chromo- on the Xi in 40 XaXist-WTXiXist-D21-kb spreads and 40 XaXist-flox

some spreads were prepared. Cells that were in mid-S XiXist-flox spreads using NIH IMAGE software revealed

phase at the onset of BrdU addition (Figure 2B) significantly more BrdU signal on the XiXist-D21-kb than on

incorporated BrdU in mid- and late S phase but did the XiXist-flox by percentage of area displaying BrdU signal

not reach mitosis in the 4.5-hr interval (Figure 2A) and (P ¼ 5.45 3 10À7) (Figure 2L) or when multiplying this

therefore were not represented among the MCSs. Cells area by the average intensity of the signal (P ¼ 5.30 3

that were in late S phase at the onset of BrdU addition 10À8) (Figure 2M). In contrast, no significant difference

(Figure 2B) incorporated BrdU in late S phase, reached was detected on the Xa in the same spreads regardless

mitosis in the 4.5-hr interval, and the incorporated of whether percentage of area occupied by BrdU signal

BrdU was detected in the MCSs (Figure 2C). Identifica- (P ¼ 0.655) or area times intensity (P ¼ 0.377) was

tion of X chromosomes using X chromosome paint considered (box plots not shown). However, this quan-

(Figure 2D) revealed that one of the two X chromo- titation was from a single XaXist-WTXiXist-D21-kb cell line and

somes in female cells consistently displayed more BrdU should therefore be considered preliminary. No signif-

signal than the other X chromosome and more signal icant difference in BrdU incorporation levels on the

Xist Affects DNA Stability 1123









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. Actively

growing 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 is

always assumed to be the X chromosome displaying the lower proportion of BrdU incorporation and the two X chromosomes

are marked by arrowheads. In each photograph of a MCS displaying BrdU, the gain has been adjusted such that five autosomes

display BrdU incorporation. (A–D) XaXist-floxXiXist-flox-1.2: (A) interphase cell; (B) possible times in the cell cycle that the indicated

fibroblasts were inferred to be exposed to BrdU; (C and D) XaXist-floxXiXist-flox-1.2 MCS. (E) XiXist-flox from an XaXist-floxXiXist-flox MCS

displayed at low gain (top), intermediate gain (middle), and high gain (bottom). Only the most intense BrdU signal is visible

at 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 40

XaXist-floxXiXist-flox MCSs (.10 MCSs from each of the three lines) and 17 XaXist-WTXiXist-WT MCSs (7 and 10 MCSs from each of

two lines) summarized in box plots that display percentage of BrdU signal on the Xi. (O and P) XaXist-D21XiXist-D21-1.2 MCS. (Q and

R) 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 40

XaXist-WTXiXist-D21 spreads and 40 XaXist-D21XiXist-D21 spreads. P-values were obtained using the Kruskal–Wallis test (Kruskal 1964).





inactive X chromosome was seen when comparing may replicate overall later in S phase when it is har-

XaXist-floxXiXist-flox cells to XaXist-WTXiXist-WT cells (P ¼ 0.407) boring the 21-kb deletion. Furthermore, heterozygosity

(Figure 2N), indicating that the presence of the Lox for the Xist deletion causes the X chromosome harbor-

sites in the absence of the 21-kb deletion did not have a ing the deletion to be replicated later than normal in

measurable effect on replication time. The Xi therefore S phase but does not affect the replication time of the

1124 S. V. Diaz-Perez et al.



Figure 3.—Relationship between the pattern

of 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 40

XaXist-D21-kbXiXist-D21-kb spreads used in Figure 2 were

obtained at a gain that was higher than that used

in Figure 2. Red, BrdU; blue, DAPI stain of chro-

mosomal DNA; green, X chromosome paint. (A

and B) Active and inactive X chromosomes of

a 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-1

elements along the X chromosome superim-

posed on an image of a Xa displaying BrdU sig-

nal. The graph was generated using a Loess

curve applied to RepeatMasker output provided

by the UCSC genome browser. The DNA se-

quence of the centromere and pericentromeric

region were not available and the coordinates

along the X chromosome (x-axis) were measured

starting at the centromere-proximal starting

point of the available DNA sequence.





wild-type X chromosome, regardless of whether the mu- (not shown). Four of the five regions displaying late

tation is on the Xa (Diaz-Perez et al. 2005) or on the Xi. replication on the Xa were found to correspond to the

The effect of deleting the 21 kb from both Xist alleles four regions along the Xa that are most heavily enriched

was next investigated using the same assay. The earlier for LINE-1 elements (Figure 3D). The fifth region on

replicating Xa displayed more BrdU signal in spreads the Xa that displayed late replication was the pericen-

from the three XaXist-D21-kbXiXist-D21-kb lines (Figure 2, O–R) tromeric region for which the genome sequence was

than in the XaXist-floxXiXist-flox cell lines (Figure 2, C, D, F, unavailable. In contrast to this reproducible pattern of

and G), in agreement with earlier results obtained using BrdU signal seen on the Xa in XaXist-D21-kbXiXist-D21-kb cells, a

XaXist-D21-kbXiXist-WT cells (Diaz-Perez et al. 2005). In XaXist- consistent pattern was not readily apparent on the Xa in

D21-kb

XiXist-D21-kb MCSs the Xi also displayed higher levels wild-type cells or when 21 kb was deleted only from one

of BrdU incorporation (Figure 2, O–R) than in XaXist-flox Xist allele (not shown) (Diaz-Perez et al. 2005). There-

XiXist-flox cell lines (Figure 2, C, D, F, and G). This fore, although deletion of 21 kb exclusively from the Xist

difference was readily apparent when 40 inactive X allele on the Xa causes the Xa to be replicated later in S

chromosomes were quantitated from each cell line phase (Diaz-Perez et al. 2005), the excision of 21 kb

(Figure 2, S and T). We conclude that excision of 21 from both Xist copies further altered the replication

kb from the Xist gene of the Xi causes the Xi to be timing in a manner that resulted in the regions with the

replicated later in S phase. Finally, preliminary data highest concentrations of LINE-1 elements on the Xa

indicated that the Xi displayed a significantly higher being replicated later in S phase.

proportion of incorporated BrdU when 21 kb was Evidence that the 21-kb Xist deletions destabilize the

deleted from both Xist alleles (Figure 2, O–R and U) DNA of both X chromosomes: During our prelimi-

than when the deletion was exclusively on the Xi in nary analyses we had encountered numerous XaXist-D21-kb

XaXist-WTXiXist-D21-kb-1.2 (Figure 2, H–K and U) and XaXist-flox XiXist-D21-kb MCSs displaying evidence of deletions or trans-

XiXist-D21-kb-1.2 cells (not shown). This suggested that ele- locations involving the X chromosome. Such spreads

ments at both Xist alleles may influence the extent of were excluded from subsequent analyses of histone

BrdU signal on the Xi in the assay for late replication. modifications and BrdU incorporation. To investigate

Relationship between the pattern of late S-phase the influence of the 21-kb deletion in the Xist gene on

BrdU incorporation and the concentration of LINE-1 the incidence of deletions and translocations, it was

sequence on the XaXist-D21-kb of XaXist-D21-kbXiXist-D21-kb cells: necessary to prepare three XaXist-WTXiXist-WT MEF cell lines

When the XaXist-D21-kbXiXist-D21-kb MCSs were examined at a using the identical procedure as was used to produce

higher gain than in Figure 2, the Xi displayed BrdU XaXist-D21-kbXiXist-D21-kb cell lines. To this end, three XaXist-WT

signal throughout its length (Figure 3, A and B) while XiXist-WT MEF cell lines were obtained from three E13.5

the Xa displayed four to six regions of concentrated 129 embryos using the same procedure as was used

BrdU signal in 33 of the 40 XaXist-D21-kbXiXist-D21-kb-2.2 MCSs to produce the three XaXist-floxXiXist-flox lines. All six lines

examined (Figure 3, A–C). This pattern was also prev- were infected with adenovirus expressing cre recombi-

alent on the Xa in XaXist-D21-kbXiXist-D21-kb -1.2 and -3.2 cells nase and GFP and subjected to limiting dilution, and

Xist Affects DNA Stability 1125









Figure 4.—Evidence that deletion of 21 kb from the Xist gene destabilizes the X chromosome. MCSs from fibroblasts were

either subjected to an assay that identifies X chromosomes using X chromosome paint (B and D–K, green) and late replicating

regions 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 for

small X chromosomal fragments (A–D and L), X chromosomal DNA within autosomes [E, F, O, and P (arrow)], truncated X

chromosomes (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 times

with SKY hybridization (left), DAPI (middle), and computer-classified color (right). Note that SKY cannot identify the origin of

centromeric/pericentromeric regions (oriented at the top of each chromosome), leading to frequent discolorations that do not

represent translocations. The software also displays discolored regions at the edges (left or right sides) of chromosomes, which is a

staining artifact rather than translocated material.





derivative cell lines were expanded from infected (GFP- the X chromosome paint was seen to display a charac-

expressing) cells using equal numbers of passages (see teristic pattern along the X chromosome that was highly

materials and methods). The cell lines were sub- reproducible among wild-type spreads (Figure 4G).

jected to the replication-timing assay used for Figure 2 Using this pattern as a guide, we observed that a fre-

to distinguish the active and inactive X chromosomes. quent abnormality was the truncation of a Xi (not

Inspection of MCSs from three XaXist-WTXiXist-WT cell lines shown) or a Xa (Figure 4H) due to the loss of the

derived from cre-adenovirus-infected cells revealed evi- centromere-distal tip of the X chromosome. We also

dence for X chromosome deletions or translocations observed a chromosome that appeared to be a duplica-

in 0 of 60 MCSs. Similarly, 0 of 68 MCSs from XaXist- tion of the X chromosome (Figure 4I), an X chromo-

flox

XiXist-flox cells showed signs of aberrations in the X some that seemed to be linked to an autosome (Figure

chromosome. In contrast, we saw evidence for deletions 4J), and what appeared to be dicentric chromosomes

or translocations in 14 of 84 spreads (16.6%) from where a portion (Figure 4K) or all (not shown) of the

cultures of the three XaXist-D21-kbXiXist-D21-kb cell lines (1.1, chromosome was derived from the X chromosome.

2.1, and 3.1). One of the most common abnormalities To confirm these findings and to determine whether

was a small fragment of the X chromosome (Figure 4, autosomes in the XaXist-D21-kbXiXist-D21-kb cell lines also

A and B). We also saw evidence of a ring X chromo- displayed abnormalities, SKY (Liyanage et al. 1996)

some (Figure 4, C and D) and small portions of the X was performed on spreads from cell lines XaXist-D21-kb

chromosome integrated into autosomes (Figure 4, E XiXist-D21-kb-1.1 and XaXist-D21-kbXiXist-D21-kb-1.3. In agreement

and F) but were not confident of the latter findings with the X chromosome paint, the size of the X chro-

because the signal could be due to X chromosome paint mosome varied within spreads, reflecting deletions or

hybridizing to autosomal material. By reducing the gain, rearrangements of the X chromosome (Figure 4, L and

1126 S. V. Diaz-Perez et al.



M). In some metaphases structural rearrangements signal is still present in XaXist-D21-kbXiXist-D21-kb cells. Brca1

involving the X were identified. These included a simple has been reported to concentrate on the Xi primarily in

nonreciprocal translocation involving chromosomes X S-phase cells (Ganesan et al. 2002; Chadwick and Lane

and 13 (Figure 4N) and a dicentric structure resulting 2005). Immunostaining of XaXist-floxXiXist-flox cells for

from fusion between two X chromosomes (Figure 4P, Brca1 revealed a large domain of concentrated Brca1

arrowhead). Involvement of the X in more complex protein (Figure 5B) in 29/90 XaXist-floxXiXist-flox cells

rearrangements was also suggested by SKY. These in- (32.2%) and in 24/90 XaXist-WTXiXist-WT cells (26.7%).

cluded a dicentric chromosome involving segments of XaXist-D21-kbXiXist-D21-kb cells displayed the concentrated

chromosomes 3 and 9 flanking two or more small Brca1 signal (Figure 5C) in 28/90 cells (31.1%), suggest-

segments of X (Figure 4O) and a translocation linking ing that loss of the Xist sequence (including Xist RNA)

portions of chromosomes 1 and 6 with a small segment did not affect the concentration of Brca1 to the Xi.

of X chromosome material at the junction (Figure 4P, The delayed replication times and DNA instability on

arrow). Because the MCSs displaying specific chromo- both X chromosomes in XaXist-D21-kbXiXist-D21-kb cells may be

somal anomalies were not examined as clonal cell lines, due to replication stress. A number of expressed fragile

no material exists to allow confirmation of the complex sites are known to be replicated late in S phase (Hansen

rearrangements, so it is a formal possibility that the X et al. 1993; Wang et al. 1999; Hellman et al. 2000; Arlt

signal arose from intermixing between two SKY colors at et al. 2003) and the ATR kinase maintains fragile site

the junction. A tally of the definitive simple rearrange- stability (Casper et al. 2002). Replication stress triggers

ments and deletions provided clear evidence of X the ATR kinase to phosphorylate the histone H2A

chromosome instability in XaXist-D21-kbXiXist-D21-kb MCSs: in variant H2AX to produce g-H2AX and also causes

total, 13/80 karyotypes (16%) analyzed by SKY dis- DNA instability (Ward et al. 2004). Immunofluores-

played structural abnormalities involving the X chro- cence failed to detect large regions of concentrated

mosome. In the same spreads, 0/80, 1/80, and 1/80 g-H2AX resembling the Brca1 signal in the three XaXist-flox

karyotypes displayed abnormalities involving chromo- XiXist-flox cell lines (Figure 5D) or in XaXist-WTXiXist-WT cells

somes 3, 4, and 5, respectively, and did not involve the X (not shown) except in a low proportion of cells (4/90

chromosome. The abnormal chromosome 4 was a small XaXist-floxXiXist-flox cells and 4/90 XaXist-WTXiXist-WT cells), con-

fragment and the abnormal chromosome 5 was slightly sistent with a report that g-H2AX associates with the Xi

shorter than normal and not a definitive abnormality. in wild-type cells exclusively in late S phase (Chadwick

Phosphorylation of p53 and H2AX and localization and Lane 2005). In contrast, a region of g-H2AX con-

of g-H2AX to the Xi in cells carrying the 21-kb centration was observed in 58/90 (64%) and 59/90

deletion: The frequent rearrangements suggested the (65%) in two XaXist-D21-kbXiXist-D21-kb cell lines examined

presence of DNA damage on the X chromosome that (Figure 5E, a and b, respectively) and in 50/90 (56%)

might have arisen from replication stress. DNA damage XaXist-WTXiXist-D21-kb cells (Figure 5F). Twenty-six of 125

would cause the ATR (Tibbettset al. 1999) and/or ATM (21%) XaXist-D21-kbXiXist-D21-kb cells displaying the g-H2AX

(Banin et al. 1998; Canman et al. 1998; Khanna et al. signal also displayed a Brca1 signal, a marker of the Xi

1998) protein kinases to phosphorylate the p53 protein (Ganesan et al. 2002) (Figure 6A). In an attempt to

at serine-15. To determine whether the 21-kb deletion is identify a heterochromatic feature other than Brca1 or

associated with the phosphorylation of the p53, extracts late replication that was retained by the Xi in XaXist-D21-kb

were prepared from independent cell lines of each of XiXist-D21-kb cells we also considered MeCP2, which binds

the following genotypes: XaXist-WTXiXist-WT, XaXist-D21-kb methylated DNA (Meehan et al. 1992) but for which

XiXist-WT, XaXist-WTXiXist-D21-kb, and XaXist-D21-kbXiXist-D21-kb. Western localization to the hypermethylated Xi has not been

blots using these extracts revealed elevated levels of addressed. We found a distinct domain of concentrated

serine-15 phosphorylated p53 protein in all cell lines MeCP2 in 83/90 (92.5%) of diploid (not shown) and

that carried the XiXist-D21-kb compared to XaXist-WTXiXist-WT tetraploid (not shown) XaXist-WTXiXist-WT cells. Twenty-four

cells (Figure 5A) while overall levels of p53 remained of 120 (20%) of XaXist-WTXiXist-WT cells displaying a MeCP

approximately the same (Figure 5A). Although the signal also displayed a Brca1 signal (Figure 6B). XaXist-WT

simplest explanation is that p53 is phosphorylated by XiXist-D21-kb (not shown) and XaXist-D21-kbXiXist-D21-kb (Figure

ATR, we acknowledge that ATM (Paull et al. 2000; 6C) cells retained the MeCP2 signal in approximately

Burma et al. 2001; Stiff et al. 2004) or another kinase the same proportion of cells (84/90, 93.5% and 80/90,

might play a role. 88.9% respectively), indicating that the 21-kb deletions

Brca1 localizes to the Xi, associates with Xist RNA, did not notably affect the intense localization of MeCP2.

fosters the Xist RNA signal at the Xi, and is involved in Since MeCP2 is not an established marker of the Xi

X-inactivation (Ganesan et al. 2002, 2004). Brca1 is also these data did not conclusively show that the g-H2AX

involved in the maintainance of genome stability signal localized to the X chromosome.

(Moynahan et al. 2001; Weaver et al. 2002; Bruun et al. To definitively determine whether the g-H2AX sig-

2003). Our finding that Xi DNA is unstable in XaXist-D21-kb nal in XaXist-D21-kbXiXist-D21-kb cells colocalized with the X

XiXist-D21-kb cells raised the issue of whether the Brca1 chromosome, DNA FISH specific to the Xic (a region on

Xist Affects DNA Stability 1127



Figure 5.—Phosphoryla-

tion of p53 and H2AX

and increased localization

of g-H2AX to the Xi in

cells carrying the 21-kb

deletion in the Xist gene.

(A) p53 is phosphorylated

on serine-15 in cells that

carry the Xist-D21-kb allele

on the Xi. Western blot

probed for p53 phosphory-

lated at serine-15 (top),

for total p53 (middle),

and for b-actin (loading

control, 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) Brca1

or g-H2AX immunolocali-

zation in female cells using

FITC (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 displaying

DAPI (left) and Brca1

(right); (C) XaXist-D21-kbXiXist-

D21-kb

-1.2 cell displaying

DAPI (left), and Brca1

(right). (D–F) g-H2AX dis-

plays a concentrated signal

in 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-1.2

cell (a) and a XaXist-floxXiXist-

flox

-2.2 cell (b) displaying

DAPI (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 the

g-H2AX images to show that the signal representing the g-H2AX domain was not present at reduced intensity in D.





the X chromosome that encompasses the Xist gene) was the Xic DNA FISH signal (not shown), bringing the over-

performed in conjunction with immunostaining for all g-H2AX–Xic association to 46% among cells display-

g-H2AX. A total of 300 XaXist-D21-kbXiXist-D21-kb cells displaying ing a large g-H2AX signal. The high proportion of cells

the large g-H2AX signal (as reported earlier in Figure with immediately adjacent g-H2AX and DNA FISH

5E) were scored for the additional presence of a super- signals may be due to g-H2AX being associated with a

imposed Xic DNA FISH signal. Ninety of the 300 XaXist- subset of the X chromosome that does not include the

D21-kb

XiXist-D21-kb cells displaying the large g-H2AX signal Xic. Finally, 44 of the 300 cells (15%) showed a g-H2AX

(30.0%) displayed a superimposed Xic DNA FISH signal domain that was clearly separate from the Xic DNA FISH

(Figure 6D). Interestingly, 5 of these 90 XaXist-D21-kb signals (not shown). The remaining cells could not be

XiXist-D21-kb cells displayed one greatly enlarged Xic signal scored with confidence due to uneven signals, uncertain

that always colocalized with the g-H2AX domain, suggest- signal number, or absence of DNA FISH signal. A total

ing that multiple duplications of Xic sequence had oc- of 19.5% of all XaXist-D21-kbXiXist-D21-kb cells displayed a

curred (Figure 6E). In addition to the 90 cells displaying clear g-H2AX signal that encompassed (90/703) or

superimposed signals, 47 of the 300 cells (16%) displayed touched (47/703) a clear Xic DNA FISH signal. We

a g-H2AX signal that touched but did not encompass conclude that the X chromosome instability, delayed

1128 S. V. Diaz-Perez et al.









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 is

retained 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 (center

left ), 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-2.2

cell is shown. The fractions represent the proportion of the cells displaying a g-H2AX or a MeCP2 signal that simultaneously

display a colocalized BRCA1 or X-chromosome-specific DNA FISH signal.



replication timing, and p53 phosphorylation that is We consider the aforementioned colocalization num-

brought on by excision of 21 kb from both Xist alleles bers to be very conservative because, when g-H2AX

are accompanied by an increase in g-H2AX on the X immunostaining was combined with X chromosome-

chromosome. specific DNA FISH, the g-H2AX signal appeared to be

Xist Affects DNA Stability 1129



TABLE 1

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



Property XaXist-WT XiXist-WT XaXist-flox XiXist-flox XaXist-WT XiXist-D21-kb XaXist-flox XiXist-D21-kb XaXist-D21-kb XiXist-WT XaXist-D21-kb XiXist-D21-kb

Xist transcription Yes Yesa Nob Noa Yesb No

Xi H4 hypoacetylation Yes Yes Yesc Yesc Yesc No

Xi H3-lys4 Yes Yes ND ND ND No

hypomethylation

Xi replication time Late Late Very lated Very lated Latee Very very late

X deletions or None None (few)f (few)f ND Many

translocations

p53-ser15 Low Low Elevatedd Elevatedd Low Elevated

phosphorylation

g-H2AX signal Infrequent Infrequent Frequentd Frequentd ND Frequent

MeCP2 signal Yes Yes Yesd Yesd ND Yes

Brca1 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).





degraded in a subset of cells and cells with degraded proportion of inactivated genes, like the X chromo-

g-H2AX signal were not scored as ‘‘superimposed’’ or some. Since in Xist-WT cells g-H2AX associates with the

‘‘immediately adjacent’’. [In contrast .90% (654/703) Xi exclusively in late S phase (Chadwick and Lane

of all cells displayed DNA FISH signals, so FISH was 2005), we cannot exclude the possibility that g-H2AX

relatively unaffected.] Consistent with the idea that can also transiently form a strong signal on an autosome

degradation reduced our percentages, g-H2AX/DNA that has a high abundance of inactivated genes.

FISH colocalization was markedly higher when only the

36 cells (of the 300 cells) with the most pronounced

g-H2AX domains were considered. Among these 36 cells,

DISCUSSION

19 (53%) had the Xic DNA FISH signal encompassed

by the g-H2AX signal, 9 (25%) displayed directly adja- We show that in female mouse cells, the excision of

cent g-H2AX and Xic DNA FISH signals, and 8 (22%) 21 kb from the Xist gene of both the Xa and the Xi (XaXist-

D21-kb

showed separated g-H2AX and DNA FISH signals. How- XiXist-D21-kb) resulted in the appearance of two histone

ever, the increased colocalization of X-chromosome- modifications throughout the Xi that are generally

specific DNA FISH signal with the 36 most pronounced associated with euchromatin: histone H4 acetylation

g-H2AX domains should be interpreted with caution and methylation on lysine-4 of histone H3. Despite the

as the level of colocalization involving these g-H2AX appearance of these euchromatic histone modifica-

domains may not be representative of all g-H2AX tions, the inactive X chromosome of XaXist-D21-kbXiXist-D21-kb

domains but may reflect a qualitatively distinct sub- cells displayed abundant DNA replication that was

population that exhibits higher colocalization for an- very late in S phase and stood in contrast with the

other reason(s). For example, they may represent cells moderately late replication that normally predominates

in a particular portion of the cell cycle that are char- on the Xi in mouse cells (Nesbitt and Gartler 1970).

acterized by resistance to signal degradation and g-H2AX The active X chromosome of XaXist-D21-kbXiXist-D21-kb cells

domain colocalization with the Xic. Cells with spacially also displayed a shift to later replication that was pre-

distinct g-H2AX and Xic DNA FISH signals may contain dominantly in chromosomal regions associated with

X chromosomes that have lost the Xic region (a plau- high concentrations of LINE-1 elements. The X chro-

sible explanation since the Xic appears to be unstable; mosomes of XaXist-D21-kbXiXist-D21-kb cells were unstable and

Figure 6E). In addition, some g-H2AX domains may be prone to deletions and translocations. The X chro-

associated with autosomes. For example, there is evi- mosome instability was accompanied by the phosphor-

dence that 8–23% of autosomal genes are subject to a ylation of p53 at serine-15 and an increase in the

random but coordinated inactivation process that re- proportion of cells that bear a high concentration of

sembles X-inactivation (Gimelbrant and Chess 2006). g-H2AX on the X chromosome. These findings are

It is not known whether any autosome contains a higher summarized in Table 1.

1130 S. V. Diaz-Perez et al.



We previously showed that the nontranscribed Xist to DNA repair or renders repetitive sequences unstable

allele on the Xa has biological activity as the 21-kb Xist and prone to DNA damage. Increased DNA damage

deletion altered Xa replication timing in cis; however, would not only slow replication but also lead to the

this deletion did not significantly affect the overall observed X chromosome instability.

replication timing of the Xi in trans (Diaz-Perez et al. The late-shifted replication of the two X chromo-

2005). Here we show that the nontranscribed Xist allele somes in XaXist-D21-kbXiXist-D21-kb cells was associated with an

on the Xa has more far-reaching biological activities that increased incidence of deletions and translocations

were not evident in heterozygous cells due to redun- involving the X chromosome, p53 phosphorylation at

dancy with the transcribed Xist allele on the Xi. We show serine-15, and the appearance of high levels of phos-

that element(s) at the nontranscribed Xist allele on the phorylated histone H2AX (g-H2AX) on the Xi. Un-

Xa (possibly promotor elements) function in trans to usually late replication and a predisposition to form

help maintain hypoacetylation on histone H4 and also deletions and translocations are also properties of rare

to control the replication timing of the Xi. Under the (Hansen et al. 1993; Wang et al. 1999; Hellman et al.

assumption that the later replicating X chromosome 2000) and common (Arlt et al. 2003) fragile sites. Rare

was the Xi, the Xi was much later replicating in XaXist-D21- fragile sites are caused by triplet repeat expansions

kb

XiXist-D21-kb cells than in cells without the deletion on the (Sutherland 2003) while common fragile sites are

Xa. Deletions at both Xist alleles also resulted in a highly broader regions (with poorly defined sequence deter-

distinctive replication-timing pattern on the other X minants) whose fragility can be induced (or ‘‘activated’’)

chromosome (in all likelihood the Xa): the LINE-1-rich by the partial inhibition of DNA replication using DNA

regions were replicated later in S phase than the rest polymerase inhibitors such as aphidicolin (Arlt et al.

of the X chromosome. This pattern, which was not 2003). Activated fragile sites are therefore thought to be

apparent in heterozygous or wild-type cells, provides a regions that cause DNA polymerase to stall and a subset

link between the Xist gene and LINE-1 elements that of these stalling events escapes cell cycle checkpoints

had previously been proposed to play a role in X- leading to chromosome breaks (Arlt et al. 2003).

inactivation (Lyon 1998) and had also been proposed Aphidicolin treatment has been associated with the

to interact with the Xist locus by heterochromatin appearance of numerous g-H2AX foci (Musio et al.

association (Marahrens 1999). LINE-1 elements dis- 2005), which may be associating not only with double-

play the heterochromatic property of DNA methylation strand breaks but also with stalled replication forks

on both the Xa and the Xi in wild-type cells (Hansen (Ward et al. 2004). An attractive possibility therefore is

2003). Our findings therefore indicate that both Xist that the deletion of 21 kb from both Xist gene copies

alleles function in cis and in trans to influence the state creates an abnormal chromatin structure on the X

of heterochromatic regions on both X chromosomes. chromosome that causes DNA polymerases to stall,

Our data reveal nonredundant cis- and trans-effects by resulting in the widespread accumulation of g-H2AX,

the two alleles on replication time as the double dele- delayed DNA replication, and the appearance of fragile

tion caused both X chromosomes to display altered rep- sites. Interestingly, both Brca1 (Ganesan et al. 2002)

lication times and/or patterns that were distinct from and Atr (Ouyang et al. 2005) have been implicated in

the times and/or patterns in heterozygous or wild-type the control of the heterochromatin of the Xi and the

cells. In contrast, the two Xist alleles were redundant disruption of either Brca1 (Arlt et al. 2004) or Atr

in the control of histone H4 deacetylation since the (Casper et al. 2002) activity has been reported to lead to

presence of either Xist deletion resulted in little or no the appearance of common fragile sites throughout the

H4 acetylation while the double deletion resulted in H4 human genome. As chromatin is increasingly impli-

acetylation appearing throughout the Xi. cated in the maintenance of genome stability and in

What are possible explanations/mechanisms for the DNA repair and BRCA1 associates with XIST RNA, it is

delayed replication timing of the Xi in the doubly not inconceivable that a common pathway to maintain

deleted cells? A protein has been reported that increases X chromosome stability may involve Xist, Brca1, and Atr.

replication fork progression through chromatin and Cells homozygous for the Xist deletion retained the

also helps resolve paused forks (Szyjka et al. 2005). Brca1 signal while loss of Brca1 has previously been

Other studies have identified chromatin-associated reported to lead to loss of Xist RNA signal (Ganesan

proteins that also maintain chromosome stability by et al. 2002), suggesting that Brca1 functions upstream of

preventing the stalling and collapse of replication forks Xist. Consistent with this are the similar effects of Brca1

(Krings and Bastia 2004; Kai et al. 2005; Sommariva and Xist deficiencies on the Xi: loss of macroH2A

et al. 2005). The abnormal chromatin in XaXist-D21-kb (Csankovszki et al. 1999; Ganesan et al. 2002), altered

XiXist-D21-kb cells may impede the association or function of replication timing (Ganesan et al. 2002), altered H3

such proteins. Another possibility is that the XaXist-D21-kb methylation (Ganesan et al. 2002), and DNA instability

XiXist-D21-kb condition increases the amount of DNA dam- (Narod and Foulkes 2004). The proportion of cells

age that arises on the X chromosome, perhaps by that displayed a BRCA1 signal on the X chromosome

fostering a chromatin structure that either is refractory was previously reported to vary considerably among

Xist Affects DNA Stability 1131



human cell lines (Ganesan et al. 2002) and may be However, we find that the 39 undeleted portion of the

particularly high in MEFs. Differences in species and Xist-D21 allele is not transcribed from either the Xa or

cell type likely play a role in these differences. It should the Xi. Homologous pairing could not be detected in

be noted that in human cells the BRCA1 signal on the Xi wild-type MEFs in asynchronous cells (Xu et al. 2006).

is restricted to late S phase when the Xi is replicated Although this may be due to pairing being restricted to a

(Chadwick and Lane 2005). In mouse cells, the in- small portion of the cell cycle in wild-type cells such as

active X chromosome begins replicating much earlier late S phase (LaSalle and Lalande 1996), we wonder

in S phase (Evans et al. 1965; Galton and Holt 1965; whether the loss of Xist may also increase the homolo-

Tiepolo et al. 1967). Brca1 may associate with the Xi gous pairing of the Xa and Xi. Interestingly, there is

while it replicates and the extension of the Brca1 signal evidence that H3 lysine-4 methylation is required for

to a broader portion of the cell cycle in MEFs may, at homologous pairing during male meiosis (Hayashi

least in part, reflect the replication of the Xi over a et al. 2005). It will be interesting to see whether

larger time interval. We should also point out that, in homologous pairing of the X chromosomes is increased

mouse, the Xist RNA signal associated with the Xi in XaXist-D21-kbXiXist-D21-kb cells and whether such an increase

persists into metaphase (Lee and Jaenisch 1997; occurs at many positions along the X chromosomes and

Duthie et al. 1999) while in humans the RNA signal requires the H3 lysine-4 methylation that arises on the

disappears at the onset of mitosis (Clemson et al. 1996). Xi in these cells.

In MEFs, Brca1 association with the Xi might also be We thank Shridar Ganesan and David M. Livingston for their

extended to additional sections of the cell cycle. generous gift of anti-Brca1 antibody. Adenoviruses expressing cre

Our finding that an intense MeCP2 signal that recombinase (Tan et al. 1999) and GFP were kindly provided by Carol

colocalizes with the Brca1 signal is retained in XaXist-D21-kb Eng and Arnold J. Berk. We thank Elaine Wong for generating some

of the primary fibroblasts, Moira Regelson for performing statistical

XiXist-D21-kb cells suggests that DNA methylation is retained

analysis, Jeannie T. Lee (Department of Molecular Biology, Massachu-

on the Xi. Indeed, we have found that DNA methyla- setts General Hospital) for providing the Xist P1 mouse clone ppJL1,

tion plays an important role in gene silencing on the and Barbara Panning (Department of Biochemistry and Biophysics,

inactive X chromosome in XaXist-D21-kbXiXist-D21-kb cells ( J. L. University of California, San Francisco) for providing a Xist-containing

Salstrom, C. Wang, C. Wang, A. Datta, S. Zeitlin, G. BAC. This work was supported by a March of Dimes Basil O’Connor

Starter Scholar research award (5-FY99-819) (Y.M.), a seed grant from

Csankovszki, C. D. Eller, S. Diaz-Perez, J. Wang, A.

the University of California, Los Angeles, Jonsson Cancer Center

Chess, S. Huang, B. Kaltenboeck and Y. Marahrens, Foundation (Y.M.), and the National Institutes of Health [R01

unpublished results). Interestingly, observations in HD41451:01 (Y.M.) and R01CA107300 (M.A.T.)].

fungi (Tamaru and Selker 2001), plants (Jackson

et al. 2002), and mammals (Lehnertz et al. 2003)

indicate that H3 lysine-9 methylation drives DNA

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