Xist RNA Is a Potent Suppressor
of Hematologic Cancer in Mice
Eda Yildirim,1,2,6James E. Kirby,7Diane E. Brown,3,5Francois E. Mercier,4,8Ruslan I. Sadreyev,1,2,5,6David T. Scadden,4,8
and Jeannie T. Lee1,2,5,6,*
1Howard Hughes Medical Institute
2Department of Molecular Biology
3Center for Comparative Medicine
4Center for Regenerative Medicine
5Department of Pathology, Harvard Medical School
Massachusetts General Hospital, Boston, MA 02114, USA
6Department of Genetics, Harvard Medical School, Boston, MA 02114, USA
7Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
8Department of Stem Cell and Regenerative Biology and Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA
X chromosome aneuploidies have long been associ-
ated with human cancers, but causality has not been
established. In mammals, X chromosome inactiva-
tion (XCI) is triggered by Xist RNA to equalize gene
expression between the sexes. Here we delete Xist
in the blood compartment of mice and demonstrate
that mutant females develop a highly aggressive
myeloproliferative neoplasm and myelodysplastic
syndrome (mixed MPN/MDS) with 100% penetrance.
Significant disease components include primary
myelofibrosis, leukemia, histiocytic sarcoma, and
vasculitis. Xist-deficient hematopoietic stem cells
(HSCs) show aberrant maturation and age-depen-
dent loss. Reconstitution experiments indicate that
MPN/MDS and myelofibrosis are of hematopoietic
rather than stromal origin. We propose that Xist
loss results in X reactivation and consequent
genome-wide changes that lead to cancer, thereby
causally linking the X chromosome to cancer in
mice. Thus, Xist RNA not only is required to maintain
XCI but also suppresses cancer in vivo.
X chromosome inactivation (XCI) transcriptionally silences one X
chromosome in the female mammal to balance gene expression
between the sexes (Lyon, 1961; Payer and Lee, 2008; Lee, 2011;
Wutz, 2011). The random form of XCI occurs only once during
development, on embryonic days 4.5–5.5 (E4.5–5.5), when the
epiblast consists of 10–20 cells. Beyond E5.5, the inactive X
(Xi)enters into a ‘‘maintenance phase’’ in whichthe sameX chro-
mosome is propagated as Xi in subsequent cell divisions for the
remainder of female life. Initiation of XCI depends on Xist, the 17
kb ‘‘X-inactive specific transcript’’ that targets and tethers
Polycomb-repressive complexes to the X chromosome in cis
(Brown et al., 1992; Clemson et al., 1996; Zhao et al., 2008;
Jeon and Lee, 2011). Current viewshold that, while Xist isessen-
tial for initiation of XCI both in an embryonic stem (ES) model
(Penny et al., 1996) and in mice (Marahrens et al., 1997), Xist is
dispensable once the Xi is established. Indeed, deleting Xist
in vitro in post-XCI fibroblasts and somatic cell hybrids does
not cause immediate X reactivation (Brown and Willard, 1994;
Rack et al., 1994; Csankovszki et al., 1999). Furthermore, in ES
models carrying autosomal Xist transgenes, switching off Xist
after autosomal silencing does not lead to reactivation (Wutz
and Jaenisch, 2000).
Nonetheless, Xist is continuously expressed throughout
female life. Notably, recent studies have uncovered stochastic
single-gene reactivation and a loss of Polycomb repression
when Xist is conditionally deleted in mouse fibroblasts (Zhang
et al., 2007). Moreover, inappropriate silencing of human XIST
et al., 2008; Anguera et al., 2012; Mekhoubad et al., 2012).
Whereas Xist has been investigated extensively in cell culture,
in vivo studies have been limited (Marahrens et al., 1997; Savar-
ese et al., 2006; Kalantry et al., 2009; Namekawa et al., 2010).
These findings and the limited exploration of in vivo models led
us to suspect that Xist may have as yet undiscovered functions
after XCI is established in the early embryo. Does X reactivation
occur, and would potential X-overdosage have undesirable
Intriguingly, supernumerary X chromosomes have long been
associated with human cancers (Moore and Barr, 1955; Liao
et al., 2003; Pageau et al., 2007). For example, breast and
ovarian cancer cells frequently lose the Barr body (the Xi)
and duplicate the active X (Xa). The association between X and
cancer also holds in men. For instance, XXY men have a 20- to
50-fold increased risk of breast cancer in a BRCA1 background
(Fentiman et al., 2006), and testicular germ cell tumors often
acquire supernumerary Xs (Kawakami et al., 2003). Neverthe-
less, an association between X and cancer has remained strictly
correlative. Here, we explore the long-standing association in
Cell 152, 727–742, February 14, 2013 ª2013 Elsevier Inc. 727
Sx7E9 Xist RNA
Xist∆/∆ 8 1-1
Xist∆/+ 27 1-1
Stage 1Stage 2
Spleen:Body weight ratio (x10-2)
2 4 5+
Figure 1. Deleting Xist in the Blood Compartment Results in Female Specific Lethality
(A) Map of the Xist2loxand XistDalleles and FISH probes used to distinguish the two alleles. Xist RNA is detected by using (Cy5-Sx9 probe, cyan). Representative
RNA/DNA FISH are shown (n R 50 each). D, deletion. X, XbaI site.
(B) Xist RNA FISH of splenocytes (n R 100). Xist RNA: green, FITC-Sx9 probe.
(C) Female specific lethality: Kaplan-Meier kill curves plotted over 750 days were generated using Prism (GraphPad software). There were no differences
between any control group: Vav-Cre, Xist2lox/+, or Xist2lox/Xist2loxfemales or corresponding male controls. These control genotypes were combined: females,
‘‘Xist2lox/+’’; males, ‘‘Xist2lox/Y’’.
(legend continued on next page)
728 Cell 152, 727–742, February 14, 2013 ª2013 Elsevier Inc.
mice and demonstrate a direct causal link between X and
To address consequences of losing Xist in vivo, we used the
Vav-Cre transgene to excise Xist conditionally in murine hemato-
poietic stem cells (HSC) as they emerge at E10.5, which is after
the establishment of XCI (Mu ¨ller et al., 1994; Csankovszki et al.,
1999; de Boer et al., 2003). All XistD/+, XistD/XistD, and XistD/Y
pups were born at expected frequencies and without obvious
abnormalities. The Xist deletion was confirmed in splenocytes
by RNA-DNA fluorescence in situ hybridization (FISH), in which
the two alleles could be distinguished using probes Sx7 (present
on both alleles) and E9 (absent in deleted allele) (Figure 1A). In
XistD/XistDfemales, Xist was deleted from Xi in 100% of cells
(Figure 1B). In XistD/+ females, Xist was deleted from Xi in
?50% of cells and from Xa in ?50%, consistent with the random
nature of XCI. For two years, we monitored the mutant animals
alongside of control littermates and performed experiments in
accordance with the relevant regulatory standards of animal
policies of MHG Institutional Animal Care and Use Committee
and, at the 2 year mark, only 10% of XistD/+ and XistD/XistDre-
mained alive (Figure 1C). By contrast, XistD/Y males and control
littermates (Xist+/+; Xist2lox/+; Xist2lox/Y; Vav-Cre) remained
was female specific, with XistD/+ and XistD/XistDfemales
behaving indistinguishably. These data demonstrate a striking
in vivo effect of deleting Xist after XCI is established.
Splenomegaly and Multilineage Hyperproliferation
The female mice typically perished after a period of illness char-
acterized by wasting, shaking, rapid breathing, and lethargy.
Nearly all mice showed distended abdomens and/or necks in
the terminal stage (Figure 1D). Upon necropsy, XistD/+ and
(enlargement of spleen). Splenomegaly was progressive and
specific to female mutants (Figures 1D and 1E, p < 0.01).
Histologic analysis revealed that, prior to 2 months, the spleens
were relatively normal (Figure S1, available online). But in stage 1
of the disease (2–5 months), staining for the proliferation
marker, Ki67, showed general hyperplasia of all splenic com-
partments (Figures 1F and S2), including red pulp (where
erythroids [Ter119+], myeloids [MPO+], monocytes/macro-
phages [Mac2+], and megakaryocytes reside [Schmitt et al.,
2001]), and white pulp (where B cells [B220+] and T cells
[CD3+] reside). In later stage 1, the germinal centers (B220+,
a B cell marker) became enlarged (Figure S2). These changes
suggested marked extramedullary hematopoiesis (EMH), the
formation of new blood cells outside of the bone marrow. In
stage 2 (beyond 4–5 months), myeloid cells (MPO+) and mono-
cell types and eradicated the white pulp and germinal centers
(Figure 1F). XistD/+ and XistD/XistDfemales behaved similarly,
whereas male mutants and female controls exhibited no
pathology (Figure S2). Thus, deleting Xist resulted in hyperprolif-
eration of all hematopoietic lineages, but myeloid cells have
a competitive advantage.
Bone Marrow Dysfunction: Myelofibrosis,
Myeloproliferation, and Myelodysplasia
The adult mouse spleen may be an active site of hematopoiesis
and account for ?30% of total hematopoiesis (Suttie, 2006;
vastly exceeded physiological levels and was accompanied by
defects in central hematopoiesis. Reticulin staining of the bone
marrow revealed progressive myelofibrosis (Figure 2A), a patho-
logical condition of unknown etiology in which the marrow is
replaced by fibrotic tissue. In mutant females of % 2 months of
age, marrow cellularity and spleen size were normal. In stage 1,
myeloid hyperplasia became a prominent feature (Figures 2B
and 2C). In stage 2, marrow cellularity decreased dramatically
as myelofibrosis became exuberant (Figure 2A). These changes
paralleled development of splenomegaly (Figures 1E and 1F)
and EMH not only in spleen but also in organs not normally
associated with EMH, such as liver, kidney, lymph nodes, and
cardiac muscle (Figure 2D). With concurrent marrow pathology,
EMH might be partly compensatory.
Importantly, hematopoiesis at neither primary nor extramedul-
lary sites was normal. In XistD/+ and XistD/XistDfemales (but
not in any mutant males or control females), bone marrow aspi-
rates showed multilineage proliferative and dysplastic changes
(Figure 2B and data not shown). For example, findings included
binucleated erythroid precursors, siderocytes containing nonhe-
moglobin iron (revealed by Prussian blue staining), and
increased phagocytosis of red and white blood cells (RBC,
WBC) by macrophages, as well as increased numbers of fibro-
blasts consistent with myelofibrosis. Although hyperproliferative
in early stages (increased Ki67 staining), end-stage animals
demonstrated pancytopenia (loss of all lineages; Figure 2C)
concurrent with exuberant myelofibrosis.
Circulating cells also demonstrated dysplastic changes (Fig-
ure 3A). In the myeloid lineage, we observed hypogranularity of
neutrophils, atypical condensation of chromatin, and abnormal
lobation in neutrophils and myelocytes (e.g., pseudo-Pelger-
Huet anomaly). Marked leukocytosis or leukopenia, erythropha-
gocytosis, and increased numbers of circulating immature mye-
lomonocytic cells (e.g., bands, metamyelocytes) were seen. In
the platelet lineage, we noted megaplatelets with retained nuclei
(D) Female-specific splenomegaly. Top panels: representative mutant females and age-matched controls are shown. Note abdominal swelling in 27-1-1 (XistD/+,
6.3 months old at death) and cervical mass in 8-1-1 (XistD/XistD, 7 months old at death). Animal ID numbers are shown with genotype throughout the figures.
Bottom: representative spleens from each male and female genotype are shown.
(E) Age-dependent increase in the spleen-to-body weight ratio. Ratios taken at 2 (n = 2–6), 4 (n = 6), and 5 to 16 (n = 14–36) months of age. Means ± SEM are
shown. Significance of the differences between mutant and WT spleen were calculated using the Student’s t test. **p % 0.01; ***p % 0.001.
(F) Temporal progression in spleen pathology. Immunostains are as indicated. Scale bars, 100 mm. See also Figures S1 and S2.
Cell 152, 727–742, February 14, 2013 ª2013 Elsevier Inc. 729
a National Institutes of Health grant (HL44851, D.T.S.), and the Howard
Hughes Medical Institute, where J.T.L. is an Investigator.
Received: February 28, 2012
Revised: December 4, 2012
Accepted: January 23, 2013
Published: February 14, 2013
Adolfsson, J., Ma ˚nsson, R., Buza-Vidas, N., Hultquist, A., Liuba, K., Jensen,
C.T., Bryder, D., Yang, L., Borge, O.J., Thoren, L.A., et al. (2005). Identifica-
tion of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic
potential a revised road map for adult blood lineage commitment. Cell 121,
Anguera, M.C., Sadreyev, R., Zhang, Z., Szanto, A., Payer, B., Sheridan, S.D.,
Kwok, S., Haggarty, S.J., Sur, M., Alvarez, J., et al. (2012). Molecular signa-
tures of human induced pluripotent stem cells highlight sex differences and
cancer genes. Cell Stem Cell 11, 75–90.
Bejar, R., Stevenson, K., Abdel-Wahab, O., Galili, N., Nilsson, B., Garcia-Man-
ero, G., Kantarjian, H., Raza, A., Levine, R.L., Neuberg, D., and Ebert, B.L.
(2011). Clinical effect of point mutations in myelodysplastic syndromes. N.
Engl. J. Med. 364, 2496–2506.
Bennett, J.M., and Orazi, A. (2009). Diagnostic criteria to distinguish hypocel-
lular acute myeloid leukemia from hypocellular myelodysplastic syndromes
and aplastic anemia: recommendations for a standardized approach. Haema-
tologica 94, 264–268.
Brecqueville, M., Rey, J., Bertucci, F., Coppin, E., Finetti, P., Carbuccia, N.,
Cervera, N., Gelsi-Boyer, V., Arnoulet, C., Gisserot, O., et al. (2012). Mutation
analysis of ASXL1, CBL, DNMT3A, IDH1, IDH2, JAK2, MPL, NF1, SF3B1,
SUZ12, and TET2 in myeloproliferative neoplasms. Genes Chromosomes
Cancer 51, 743–755.
Brown, C.J., and Willard, H.F. (1994). The human X-inactivation centre is
not required for maintenance of X-chromosome inactivation. Nature 368,
Brown, C.J., Hendrich, B.D., Rupert, J.L., Lafrenie `re, R.G., Xing, Y., Lawrence,
J., and Willard, H.F. (1992). The human XIST gene: analysis of a 17 kb inactive
X-specific RNA that contains conserved repeats and is highly localized within
the nucleus. Cell 71, 527–542.
Clemson, C.M., McNeil, J.A., Willard, H.F., and Lawrence, J.B. (1996). XIST
RNA paints the inactive X chromosome at interphase: evidence for a novel
RNA involved in nuclear/chromosome structure. J. Cell Biol. 132, 259–275.
Csankovszki, G., Panning, B., Bates, B., Pehrson, J.R., and Jaenisch, R.
(1999). Conditional deletion of Xist disrupts histone macroH2A localization
but not maintenance of X inactivation. Nat. Genet. 22, 323–324.
deBoer, J.,Williams,A.,Skavdis,G.,Harker,N., Coles,M.,Tolaini, M.,Norton,
T., Williams, K., Roderick, K., Potocnik, A.J., and Kioussis, D. (2003). Trans-
genic mice with hematopoietic and lymphoid specific expression of Cre.
Eur. J. Immunol. 33, 314–325.
Dewald, G.W., Brecher, M., Travis, L.B., and Stupca, P.J. (1989). Twenty-six
patients with hematologic disorders and X chromosome abnormalities.
Frequent idic(X)(q13) chromosomes and Xq13 anomalies associated with
pathologic ringed sideroblasts. Cancer Genet. Cytogenet. 42, 173–185.
Dierlamm, J., Michaux, L., Criel, A., Wlodarska, I., Zeller, W., Louwagie, A.,
Michaux, J.L., Mecucci, C., and Van den Berghe, H. (1995). Isodicentric
(X)(q13) in haematological malignancies: presentation of five new cases, appli-
cation of fluorescence in situ hybridization (FISH) and review of the literature.
Br. J. Haematol. 91, 885–891.
Doll, D.C., Grogan, T.M., and Greenberg, B.R. (1987). Chronic myelomono-
cytic leukemia terminating as malignant histiocytosis. Hematol. Pathol. 1,
Feldman, A.L., Minniti, C., Santi, M.,Downing, J.R., Raffeld, M.,and Jaffe, E.S.
(2004). Histiocytic sarcoma after acute lymphoblastic leukaemia: a common
clonal origin. Lancet Oncol. 5, 248–250.
Fentiman, I.S., Fourquet, A., and Hortobagyi, G.N. (2006). Male breast cancer.
Lancet 367, 595–604.
Harrison, D.E. (1980). Competitive repopulation: a new assay for long-term
stem cell functional capacity. Blood 55, 77–81.
Heinonen, K., Mahlama ¨ki, E., Riikonen, P., Meltoranta, R.L., Rahiala, J., and
Perkkio ¨, M. (1999). Acquired X-chromosome aneuploidy in children with acute
lymphoblastic leukemia. Med. Pediatr. Oncol. 32, 360–365.
Higgins, M.E., Claremont, M., Major, J.E., Sander, C., and Lash, A.E. (2007).
CancerGenes: a gene selection resource for cancer genome projects. Nucleic
Acids Res. 35(Database issue), D721–D726.
Huang, S., Jean, D., Luca, M., Tainsky, M.A., and Bar-Eli, M. (1998). Loss of
AP-2 results in downregulation of c-KIT and enhancement of melanoma
tumorigenicity and metastasis. EMBO J. 17, 4358–4369.
Huang, W., Sherman, B.T., and Lempicki, R.A. (2009). Systematic and integra-
tive analysis of large gene lists using DAVID bioinformatics resources. Nat.
Protoc. 4, 44–57.
Huarte, M., and Rinn, J.L. (2010). Large non-coding RNAs: missing links in
cancer? Hum. Mol. Genet. 19(R2), R152–R161.
Irizarry, R.A., Bolstad, B.M., Collin, F., Cope, L.M., Hobbs, B., and Speed, T.P.
(2003). Summaries of Affymetrix GeneChip probe level data. Nucleic Acids
Res. 31, e15.
Jeon, Y.,and Lee,J.T.(2011).YY1tethers Xist RNA totheinactive Xnucleation
center. Cell 146, 119–133.
Kalantry, S., Purushothaman, S., Bowen, R.B., Starmer, J., and Magnuson, T.
(2009). Evidence of Xist RNA-independent initiation of mouse imprinted
X-chromosome inactivation. Nature 460, 647–651.
Kawakami, T., Okamoto, K., Sugihara, H., Hattori, T., Reeve, A.E., Ogawa, O.,
and Okada, Y. (2003). The roles of supernumerical X chromosomes and XIST
expression in testicular germ cell tumors. J. Urol. 169, 1546–1552.
Kiel, M.J., Yilmaz, O.H., Iwashita, T., Yilmaz, O.H., Terhorst, C., and Morrison,
S.J. (2005). SLAM family receptors distinguish hematopoietic stem and
progenitor cells and reveal endothelial niches for stem cells. Cell 121, 1109–
Laurencet, F.M., Chapuis, B., Roux-Lombard, P., Dayer, J.M., and Beris, P.
(1994). Malignant histiocytosis in the leukaemic stage: a new entity
(M5c-AML) in the FAB classification? Leukemia 8, 502–506.
Lee, J.T. (2011). Gracefully ageing at 50, X-chromosomeinactivation becomes
a paradigm for RNA and chromatin control. Nat. Rev. Mol. Cell Biol. 12, 815–
Lee, J.T. (2012). Epigenetic regulation by long noncoding RNAs. Science 338,
Li, L., Bailey, E., Greenblatt, S., Huso, D., and Small, D. (2011). Loss of the
wild-type allele contributes to myeloid expansion and disease aggressiveness
in FLT3/ITD knockin mice. Blood 118, 4935–4945.
Liao, D.J., Du, Q.Q., Yu, B.W., Grignon, D., and Sarkar, F.H. (2003). Novel
perspective: focusing on the X chromosome in reproductive cancers. Cancer
Invest. 21, 641–658.
Lyon, M.F. (1961). Gene action in the X-chromosome of the mouse (Mus
musculus L.). Nature 190, 372–373.
Marahrens,Y.,Panning, B.,Dausman, J.,Strauss,W., andJaenisch, R.(1997).
Xist-deficient mice are defective in dosage compensation but not spermato-
genesis. Genes Dev. 11, 156–166.
Mazumdar, M., Sung, M.H., and Misteli, T. (2011). Chromatin maintenance by
a molecular motor protein. Nucleus 2, 591–600.
Mekhoubad, S., Bock, C., de Boer, A.S., Kiskinis, E., Meissner, A., and Eggan,
K. (2012). Erosion of dosage compensation impacts human iPSC disease
modeling. Cell Stem Cell 10, 595–609.
Moore, K.L., and Barr, M.L. (1955). The sex chromatin in benign tumours and
related conditions in man. Br. J. Cancer 9, 246–252.
Mu ¨ller, A.M., Medvinsky, A., Strouboulis, J., Grosveld, F., and Dzierzak, E.
(1994). Development of hematopoietic stem cell activity in the mouse embryo.
Immunity 1, 291–301.
Cell 152, 727–742, February 14, 2013 ª2013 Elsevier Inc. 741
Muramatsu, H., Makishima, H., and Maciejewski, J.P. (2012). Chronic myelo-
monocytic leukemia and atypical chronic myeloid leukemia: novel pathoge-
netic lesions. Semin. Oncol. 39, 67–73.
Namekawa, S.H., and Lee, J.T. (2011). Detection of nascent RNA, single-copy
DNA and protein localization by immunoFISH in mouse germ cells and
preimplantation embryos. Nat. Protoc. 6, 270–284.
Namekawa, S.H., Payer, B., Huynh, K.D., Jaenisch, R., and Lee, J.T. (2010).
Two-step imprinted X inactivation: repeat versus genic silencing in the mouse.
Mol. Cell. Biol. 30, 3187–3205.
Okada, S., Nakauchi, H., Nagayoshi, K., Nishikawa, S., Miura, Y., and Suda, T.
hematopoietic cells. Blood 80, 3044–3050.
Pageau, G.J., Hall, L.L., Ganesan, S., Livingston, D.M., and Lawrence, J.B.
(2007). The disappearing Barr body in breast and ovarian cancers. Nat. Rev.
Cancer 7, 628–633.
Pang, W.W., Price, E.A., Sahoo, D., Beerman, I., Maloney, W.J., Rossi, D.J.,
Schrier, S.L., and Weissman, I.L. (2011). Human bone marrow hematopoietic
stem cells are increased in frequency and myeloid-biased with age. Proc. Natl.
Acad. Sci. USA 108, 20012–20017.
Paulsson, K., Haferlach, C., Fonatsch, C., Hagemeijer, A., Andersen, M.K.,
Slovak, M.L., and Johansson, B.; MDS Foundation. (2010). The idic(X)(q13)
in myeloid malignancies: breakpoint clustering in segmental duplications
and association with TET2 mutations. Hum. Mol. Genet. 19, 1507–1514.
Payer, B., and Lee, J.T. (2008). X chromosome dosage compensation: how
mammals keep the balance. Annu. Rev. Genet. 42, 733–772.
Pellagatti, A.,Cazzola, M., Giagounidis, A.,Perry, J., Malcovati, L., Della Porta,
M.G., Ja ¨dersten, M., Killick, S., Verma, A., Norbury, C.J., et al. (2010). Deregu-
lated gene expression pathways in myelodysplastic syndrome hematopoietic
stem cells. Leukemia 24, 756–764.
Penny, G.D., Kay, G.F., Sheardown, S.A., Rastan, S., and Brockdorff, N.
(1996). Requirement for Xist in X chromosome inactivation. Nature 379,
Percy, D.H., and Barthold, S.W. (2007). Mouse (Ames, IA: Blackwell).
Rack, K.A., Chelly, J., Gibbons, R.J., Rider, S., Benjamin, D., Lafrenie ´re, R.G.,
Oscier, D., Hendriks, R.W., Craig, I.W., Willard, H.F., et al. (1994). Absence of
the XIST gene from late-replicating isodicentric X chromosomes in leukaemia.
Hum. Mol. Genet. 3, 1053–1059.
Sandberg, A.A., ed.(1983).‘‘The Xchromosomeinhuman neoplasia, including
sex chromatin and congenital conditions with X chromosome anomalies.’’
Cytogenetics of the mammalian X-chromosome, Part B: X Chromosome
Anomalies and Their Clinical Manifestations (New York: Alan R Liss, Inc.),
Hematopoietic precursor cells transiently reestablish permissiveness for X
inactivation. Mol. Cell. Biol. 26, 7167–7177.
Schmitt, A., Guichard, J., Masse ´, J.M., Debili, N., and Cramer, E.M. (2001). Of
mice and men: comparison of the ultrastructure of megakaryocytes and plate-
lets. Exp. Hematol. 29, 1295–1302.
Shen, Y., Matsuno, Y., Fouse, S.D., Rao, N., Root, S., Xu, R., Pellegrini, M.,
Riggs, A.D., and Fan, G. (2008). X-inactivation in female human embryonic
stem cells is in a nonrandom pattern and prone to epigenetic alterations.
Proc. Natl. Acad. Sci. USA 105, 4709–4714.
Silva, S.S., Rowntree, R.K., Mekhoubad, S., and Lee, J.T. (2008). X-chromo-
some inactivation and epigenetic fluidity in human embryonic stem cells.
Proc. Natl. Acad. Sci. USA 105, 4820–4825.
Takizawa, T., Gudla, P.R., Guo, L., Lockett, S., and Misteli, T. (2008). Allele-
specific nuclear positioning of the monoallelically expressed astrocyte marker
GFAP. Genes Dev. 22, 489–498.
Tefferi, A. (2011). Primary myelofibrosis: 2012 update on diagnosis, risk
stratification, and management. Am. J. Hematol. 86, 1017–1026.
Vainchenker, W., Delhommeau, F., Constantinescu, S.N., and Bernard, O.A.
(2011). New mutations and pathogenesis of myeloproliferative neoplasms.
Blood 118, 1723–1735.
Wutz, A. (2011). Gene silencing in X-chromosome inactivation: advances in
understanding facultative heterochromatin formation. Nat. Rev. Genet. 12,
Wutz, A., and Jaenisch, R. (2000). A shift from reversible to irreversible X
inactivation is triggered during ES cell differentiation. Mol. Cell 5, 695–705.
Yamamoto, K., Nagata, K., Kida, A., and Hamaguchi, H. (2002). Acquired gain
of an X chromosome as the sole abnormality in the blast crisis of chronic
neutrophilic leukemia. Cancer Genet. Cytogenet. 134, 84–87.
Zhang, L.F., Huynh, K.D., and Lee, J.T. (2007). Perinucleolar targeting of the
inactive X during S phase: evidence for a role in the maintenance of silencing.
Cell 129, 693–706.
Zhao, J., Sun, B.K., Erwin, J.A., Song, J.J., and Lee, J.T. (2008). Polycomb
proteins targetedbyashortrepeatRNA tothe mouse Xchromosome. Science
Zheng, R., and Blobel, G.A. (2010). GATA Transcription Factors and Cancer.
Genes Cancer 1, 1178–1188.
742 Cell 152, 727–742, February 14, 2013 ª2013 Elsevier Inc.