Sex chromosome silencing in the marsupial male
Satoshi H. Namekawa*, John L. VandeBerg†, John R. McCarrey‡, and Jeannie T. Lee*§
*Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, and Department of Genetics, Harvard Medical
School, Boston, MA 02114;†Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, TX 78245; and‡Department of Biology,
University of Texas, San Antonio, TX 78249
Edited by Arthur D. Riggs, Beckman Research Institute of the City of Hope, Duarte, CA, and approved April 20, 2007 (received for review January 12, 2007)
In marsupials, dosage compensation involves silencing of the
father’s X-chromosome. Because no XIST orthologue has been
found, how imprinted X-inactivation occurs is unknown. In euth-
erians, the X is subject to meiotic sex chromosome inactivation
(MSCI) in the paternal germ line and persists thereafter as post-
meiotic sex chromatin (PMSC). One hypothesis proposes that the
paternal X is inherited by the eutherian zygote as a preinactive X
and raises the possibility of a similar process in the marsupial germ
line. Here we demonstrate that MSCI and PMSC occur in the
opossum. Surprisingly, silencing occurs before X–Y association.
After MSCI, the X and Y fuse through a dense plate without
obvious synapsis. Significantly, sex chromosome silencing contin-
ues after meiosis, with the opossum PMSC sharing features of
eutherian PMSC. These results reveal a common gametogenic
program in two diverse clades of mammals and support the idea
that male germ-line silencing may have provided an ancestral form
of mammalian dosage compensation.
meiosis ? X-inactivation
X chromosomes (XX) and the male inheriting an X and a Y
(XY). With some variation, the XY scheme of sex determination
can be seen in all three extant clades of mammals, including the
prototherians (monotremes) that evolved some 300 million years
ago, the metatherians (marsupials) that evolved 150–200 million
years ago, and the eutherians (placental mammals) that evolved
100–150 million years ago. In addition to the sexually dimorphic
development of males and females, this system of sex determi-
nation has important consequences for other aspects of mam-
malian development: one relating to the inequality of sex
chromosome gene dosage and the other to the behavior of sex
chromosomes in the germ line. Both stem from the fact that
genetic content on the Y has gradually eroded over 300 million
years of evolution (1–3).
Because the Y carries only a fraction of the genetic material
found on the X, females have nearly twice the sex chromosome
gene dosage as males, often necessitating coevolution of dosage
compensation. In mammals, dosage compensation is achieved by
the transcriptional inactivation of one X in the female. Three
forms of X-chromosome inactivation (XCI) have been reported.
In marsupials XCI is imprinted to occur exclusively on the
paternal X, although the degree of silencing varies among
somatic tissues (1, 4, 5). In contrast, eutherian XCI can be either
imprinted or random (6, 7). In somatic tissues, XCI is random
and can occur on either the maternal or paternal X. However, in
the placental tissues of some eutherian mammals (e.g., mouse
and cow), the paternal X resembles that in marsupials and is
preferentially inactivated (7, 8). A third form of XCI is known
to occur in the male germ line of eutherian mammals. During the
first meiotic prophase, the X and Y become transcriptionally
silenced in a process known as meiotic sex chromosome inacti-
vation (MSCI) (9, 10).
n mammals, sex is determined by the differential inheritance
of the X and Y chromosomes, with the female inheriting two
The process of MSCI is the second significant consequence of
adopting the XY method of sex determination in mammals.
MSCI has so far been documented only in eutherian mammals
in which, during prophase I of meiosis, homologous chromo-
somes pair and exchange genetic material. In the male germ line,
however, the X and Y can pair only through their remaining
mouse it was recently shown that asynapsed regions of the X and
Y become transcriptionally inactivated simply by virtue of their
being unpaired during pachytene of prophase I (12, 13), in a
process that is termed meiotic silencing by unpaired chromatin
(14). Several recent studies have also shown that the effects of
male MSCI unexpectedly extend beyond meiosis I and continue
through the end of spermatogenesis (15–17).
MSCI has long led to questions regarding its raison d’etre. The
enrichment of spermatogenesis genes on the X and Y (18–20)
despite meiotic and postmeiotic silencing raises one of the major
paradoxes in the field. One idea is that MSCI and meiotic
silencing by unpaired chromatin exist only as an evolutionary
relic of meiotic silencing of unpaired DNA, a host defense
mechanism first described in Neurospora crassa (21) with anal-
ogies in metazoans such as Caenorhabditis elegans (22). Other
ideas suggest that silencing is obligatory for the suppression of
recombination between nonhomologous regions of the X and Y
(23), or for preventing asynapsed XY regions from triggering the
meiotic checkpoint (24).
Meiotic and postmeiotic silencing may subserve yet another
purpose: the problem of dosage compensation in the earliest
mammals as the Y-chromosome lost genetic material. Imprinted
paternal X silencing in the early eutherian embryo may at least
(25–27), a hypothesis proposed earlier for the marsupial embryo
(5, 28–30). In support of this, one study finds that the paternal
X is already silent at conception and may be preinactivated (26).
Because meiotic silencing of unpaired DNA/meiotic silencing by
unpaired chromatin would silence any portion of the X that no
longer has homology with the Y (12, 13), MSCI and its after-
effects would provide an immediate stop-gap measure of dosage
compensation at a time of rapid change on the sex chromosomes
(27). However, this hypothesis is opposed by the view that XCI
takes place de novo at the four- to eight-cell stage, which would
Author contributions: J.R.M. and J.T.L. contributed equally to this work; S.H.N., J.R.M., and
J.T.L. designed research; S.H.N. performed research; J.L.V. and J.R.M. contributed new
reagents/analytic tools; S.H.N. and J.T.L. analyzed data; and S.H.N. and J.T.L. wrote the
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations: XCI, X-chromosome inactivation; MSCI, meiotic sex chromosome inactiva-
tion; PMSC, postmeiotic sex chromatin; DP, dense plate; XIC, X-inactivation center; Pol-II,
§To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
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therefore argue against dosage compensation as a beneficiary of
meiotic and postmeiotic silencing (see ref. 31 for full discussion).
Thus, the basis of imprinted XCI in eutherians is currently
Much remains unknown regarding XCI in marsupials and to
what extent mechanisms might be shared with those in euther-
ians. Eutherian XCI is regulated by the X-inactivation center
(XIC/Xic), which contains the noncoding genes Xist (32–34), Tsix
(35–37), and Xite (38). Repeated attempts to find the XIC
orthologue in marsupials have failed and instead find that the
syntenic region is rearranged (39–41). Interestingly, a recent
report suggests that the ancestral XIST was vertebrate LNX3, a
protein-coding gene with functions still extant but unrelated to
dosage compensation in the marsupial (42). Without an XIC,
how would XCI be achieved in the marsupial? One view holds
that dosage compensation may have evolved independently in
the marsupial and eutherian (42). Yet the classic view proposes
that marsupial XCI results from preprogramming events in the
paternal germ line (5, 28, 29). Therefore, a strictly germ-line-
driven process, such as one proposed for imprinted XCI in the
early mouse embryo (25, 26), might function in the marsupial (5,
27, 29, 30). Significantly, MSCI in the mouse does not require
XIST (43, 44). However, there has been no formal evidence of
MSCI so far in the marsupial, although a condensed X and Y has
been reported in prophase I (45). Here we sought to determine
whether a germ-line-driven mechanism might be feasible for
marsupial XCI by investigating spermatogenic events in the
South American opossum, Monodelphis domestica. We report
that meiotic and postmeiotic events are surprisingly well con-
served between metatherian and eutherian mammals.
Results and Discussion
Sex Chromosome Behavior During Opossum Meiosis. During meiosis
a primary spermatocyte proceeds through two division rounds
(meiosis I and II) to generate four haploid spermatids that bear
either an X- or Y-chromosome in addition to a haploid com-
plement of autosomes. During meiosis I, homologous chromo-
somes pair and undergo homologous exchange during prophase
(leptotene, zygotene, pachytene, and diplotene) and are segre-
gated to distinct nuclei through metaphase, anaphase, and
telophase. Primary spermatocytes at these various stages can be
distinguished from each other by SCP3 staining of the axial
elements and by their sex chromosome configuration. They
differ from other cell types in a seminiferous tubular spread by
their diploid chromosome constitution. It is during prophase I
that MSCI takes place.
To determine the behavior of M. domestica chromosomes
during meiosis I, we coimmunostained centromeric proteins and
SCP3 and observed a 2n ? 18 karyotype (Fig. 1), confirming a
previous report of eight autosome pairs and a pair of sex
chromosomes (XX female, XY male) (46). At leptotene, ho-
mologous chromosomes had yet to pair (pale SCP3 staining), but
a bouquet-like arrangement of chromosomes was already evi-
dent with all homologous telomeres clustered at one pole of the
nuclear membrane (data not shown). At zygotene, synapsis (as
revealed by stronger SCP3 staining) first became evident at the
telomeric poles of each autosome pair and moved inward along
the axial elements (Fig. 1A). At this stage, the sex chromosomes
became distinguishable for the first time (without chromosome
painting by FISH), discernible as condensed acrocentric chro-
mosomes (Fig. 1A?). Thus, through leptotene and zygotene, the
behavior of the M. domestica X and Y paralleled that of the
mouse X and Y.
By early pachytene, homologous autosomes appeared fully
synapsed, while the X and Y remained separate and became
progressively more condensed, as evident by their decreased
axial length and increased staining of axial elements (Fig. 1B).
Interestingly, unlike the mouse X and Y, the opossum sex
chromosomes folded into an arc, i.e., looped so that the two ends
came in close contact (Fig. 1B?). Unlike observations in euth-
erians, X–Y association occurred late during pachytene and only
after the autosomes had synapsed (Fig. 1 C and C?), confirming
delayed sex chromosome association described previously in M.
domestica (47) and other marsupials (48). This contrasts with
eutherian pachytene, during which partial homology between
the X and Y enables synaptonemal complex formation and true
homologous pairing, akin to what is observed among autosomal
Some measure of X–Y association could be observed at
resembling that in eutherian spermatocytes (Fig. 1 C and C?).
However, the absence of any obvious homology between the sex
chromosomes appeared to preclude the formation of synaptone-
mal complex (48). Instead, the X and Y associate through a
dense plate (DP) (49) between the X and Y arcs (Fig. 1 C and
C?). SCP3 staining of the XY body became very intense, whereas
staining of autosomes became weaker (Fig. 1C). By late
pachytene, the axial elements of the sex chromosomes (SCP3
staining) became thin and entangled, whereas the DP became
increasingly prominent (Fig. 1 D and D?). The DP appeared to
be attached to the nuclear envelope, consistent with previous
description (48). At diplotene, SCP3 staining of the DP re-
mained intense but became extremely weak on autosomes (Fig.
immunostaining with anti-SCP3 (green) and anti-centromere (CEN; red) pro-
teins.*, polarized direction of bouquet structure. (A–E and A?–D?) Multiple
focal planes are projected. (A?–D?) Higher magnification of sex chromosomes.
(E?) Higher magnification of DP shown in E? (single z-sections). (F and G) DNA
Arrowheads, DPs; arrows, XY bodies.
Meiotic behavior of sex chromosomes in M. domestica. (A–E) Double
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1 E and E?). By DAPI staining alone, the XY body could easily
be identified in the diplotene nucleus as a bright, condensed
structure [Fig. 1E and supporting information (SI) Fig. 6]. By
contrast, the DP was not DAPI-intense, suggesting that the DP
is a proteinaceous structure with little if any chromosomal DNA
(Fig. 1E?) (49). As confirmed by X- and Y-painting, the sex
chromosomes were clearly distinguishable as DAPI-intense
structures from early pachytene to diplotene (Fig. 1 F and G).
Thus, although the opossum also develops an XY body at
mid-pachytene, it differs from the eutherian counterpart by an
absence of any true synapsis and by its association through a DP.
Furthermore, we noted that, although it resides at a peripheral
nuclear location, the opossum XY body does not protrude out of
the nucleus at mid-late pachytene as is characteristic of the
mouse XY body. Overall, the behavior of the M. domestica sex
chromosomes during male meiosis is similar to what has been
described for other marsupials (48).
MSCI in the Opossum. Although MSCI is well established in
eutherians, this phenomenon has not previously been docu-
mented in marsupials. Because some aspects of the opossum XY
body are reminiscent of that in eutherians, we next asked
whether the opossum X and Y are also subject to MSCI. We
performed Cot-1 RNA FISH, a technique whereby new RNA
synthesis can be detected through hybridization to highly repet-
itive elements (Cot-1 fraction) found in the 3? untranslated
regions and introns before splicing (15). Indeed, Cot-1 RNA
FISH showed that the X and Y excluded Cot-1 hybridization by
early pachytene (Fig. 2 A and A?). Immunostaining for the RNA
polymerase II (Pol-II) showed a dearth of Pol-II (Fig. 2B). At
pachytene, the sex chromosomes became decorated with the
heterochromatin-associated proteins, HP1? and HP1? (Fig. 2C
and data not shown), and also by ?H2AX, a protein associated
with the repair of double-strand breaks that is known to be an
early mark of MSCI (50) (Fig. 2D). During this time, the X and
Y appeared to be the prominent DAPI-intense structure in the
early pachytene spermatocyte (Figs. 1F and 2D), even when the
cells were prepared under relatively harsh conditions (hypoton-
ically swollen nuclei) (SI Fig. 6). These characteristics persisted
through diplotene, as Cot-1 and Pol-II signals continued to be
excluded from the XY body (Fig. 2 E and F) and as HP1?, HP1?,
and ?H2AX continued to be enriched on the XY body (Fig. 2 G
consistently labeled by SCP3, other proteins such as HP1?,
HP1?, and ?H2AX could not be detected on this structure at any
time (Fig. 2 H and H?, SI Fig. 7, and data not shown).] Thus, we
to MSCI during pachytene in a manner similar to that observed
for eutherian sex chromosomes.
However, we also observed several interesting differences
between eutherian and marsupial MSCI. First, although it is
thought that eutherian MSCI occurs after the X and Y have
partially synapsed through the pseudoautosomal region (10, 51),
association (Fig. 2 A–D). This implied that MSCI does not
require partial synapsis of the X and Y, consistent with the idea
that MSCI is induced by unpaired DNA (meiotic silencing of
unpaired DNA/meiotic silencing by unpaired chromatin) rather
than by paired elements (12). A second significant difference is
that HP1? and HP1? association in the opossum takes place
earlier than in the mouse. Whereas these proteins are found on
the mouse XY body only late in pachytene (15), they could be
observed on the opossum sex chromosomes by early pachytene
as the chromosomes become looped (Fig. 2C) and decorated by
?H2AX (Fig. 2D).
PMSC in the Opossum. MSCI was previously believed to be specific
to meiosis I. Because it is now known that most genes on the
eutherian sex chromosomes do not reactivate at the end of
meiosis I (15–17), we asked whether silencing also persists into
spermiogenesis in opossum. We first examined secondary sper-
matocytes undergoing meiosis II. Secondary spermatocytes
could usually be observed as two attached or closely juxtaposed
staining with anti-?H2AX (green). (B and F) Double immunostaining with
anti-SCP3 (green) and HP1? (red). (D) Double immunostaining with anti-
?H2AX (green) and HP1? (red). (A? and H?) Higher magnification of sex
chromosome and DP shown in A and H, respectively. (A, A?, B, E, F, and H?)
Single z-sections. (C, D, G, and H) Multiple focal planes are projected. Meiotic
stages are noted above panels. Arrows, sex chromosomes; arrowheads, DPs.
MSCI in M. domestica. (A and E) Cot-1 RNA FISH (red) and immuno-
www.pnas.org?cgi?doi?10.1073?pnas.0700323104Namekawa et al.
daughter cells, one carrying only the X and the other only the Y.
They can be distinguished from round spermatids (which also
have segregated Xs and Ys) by their larger size and presence of
sister chromatids (by centromeric staining; data not shown).
In secondary spermatocytes, we were surprised to find that
?H2AX remained (Fig. 3 A and B; 94% of nuclei with ?H2AX
on sex chromosome, n ? 70), in contrast to its disappearance
from the eutherian XY after diplotene I (15). Because ?H2AX
is thought to be involved specifically with events during prophase
I in eutherian spermatocytes, its continued presence on the
segregated X and Y seemed rather puzzling. Analysis by Cot-1
RNA FISH, Pol-II immunostaining, and DAPI staining showed
that the sex chromosomes remained undertranscribed in the
secondary spermatocytes (Fig. 3 A and B). These results dem-
onstrated that the sex chromosomes remained relatively sup-
pressed in the secondary spermatocyte.
of meiosis, we investigated round spermatids. Round spermatids
could be distinguished from secondary spermatocytes by their
smaller size, tendency to cluster into groups of X-bearing and
Y-bearing daughter cells, and a clear nine-chromosome consti-
tution (e.g., nine centromeric signals) (Fig. 4 K and L). Indeed,
D), retained their DAPI-intense staining, and continued to be
enriched for HP1? and HP1? (Fig. 3 C–F and SI Fig. 8). These
data showed that, just as in eutherians, the postmeiotic X of the
opossum is transcriptionally suppressed. Their epigenetic pro-
files were also similar. Trimethylation of H3-K9 (H3–3meK9)
was initially observed on the sex chromosomes (as marked by
HP1?) in primary spermatocytes. This occurred by the time of
XY body formation in pachytene (meiosis I) (Fig. 4A and SI Fig.
9) and continued in round spermatids (Fig. 4 B and C and SI Fig.
9). In mice, H3–3meK9, HP1?, and HP1? persist longer than any
other chromatin marks and are present until genome-wide
chromosome condensation at the end of spermiogenesis (SI Fig.
10) (15, 17). H3-K27 trimethylation (H3–3meK27), a marker of
the inactive X in the mouse soma (52), was present at relatively
low levels on the XY body as compared with the rest of the
genome in opossum primary spermatocyte (Fig. 4G) and also on
the X in round spermatids (Fig. 4H). These data demonstrated
that the postmeiotic X continues to be transcriptionally sup-
pressed in marsupials, reminiscent of the PMSC described in
eutherians (15–17). Thus, the silencing initiated by marsupial
Some interesting differences between the mouse and opossum
PMSC could also be observed. First, murine PMSC can be
recognized as a DAPI-bright structure attached to the sperma-
tid’s single chromocenter, the DAPI-intense focal cluster of
centric heterochromatin (Fig. 4J) (15–17). By contrast, we did
not observe a chromocenter in the opossum, because immuno-
staining for HP1? and centromere proteins showed that the
centromeres were distributed all over the nuclei and were not
DAPI-intense (Fig. 4 K and L). The opossum PMSC was
therefore uniquely DAPI-intense (Fig. 4 K and L). Other
differences occurred in the profiles of various chromatin-
associated proteins. For example, dimethylation of H3-K9 (H3–
2meK9) was present in primary spermatocytes by pachytene/
diplotene of both species (Fig. 4D) (15–17, 53) but was curiously
absent from (or undetectable on) the X and Y of the opossum
spermatid (Fig. 4E). Furthermore, ?H2AX persisted longer in
the opossum, decorating the X and Y in 94% of secondary
spermatocytes (n ? 70) (Fig. 3 A and B). A further surprise was
that ?H2AX was even enriched on the X and Y in 4% of early
round spermatids (n ? 451) (Fig. 4F and SI Fig. 9) before
disappearing completely in later stages (Fig. 4I and SI Fig. 9).
Thus, although PMSC is conserved in the marsupial, its epige-
netic profile, subject to the vagaries of immunostaining, appears
to differ slightly from that of eutherians.
In conclusion, our study and related work (J. Hornecker, P.
Samollow, E. Robinson, J.L.V., and J.R.M., unpublished data)
show that MSCI and PMSC occur in the marsupial and that the
silencing initiated during the first meiotic prophase continues
through meiosis II and into the postmeiotic period (Fig. 5). Thus,
spermatogenic events regulating transcriptional activity of the
sex chromosomes are very well conserved in the marsupial and
eutherian. This is in striking contrast to the absence of conser-
vation in XIC elements that regulate XCI in the eutherian soma
(39–42). These data are consistent with a mechanism of im-
ing with anti-Pol-II (red) and ?H2AX (green), followed by DNA FISH (X-paint,
green; Y-paint, red). (C and D) Cot-1 RNA FISH (red) and immunostaining with
anti-HP1? (green). (E) DNA FISH (X-paint, green). (F) DNA FISH (Y-paint,
A and B and round spermatids in C–F.
Namekawa et al. PNAS ?
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printed XCI that would occur independent of XIST in the
marsupial and make possible a mechanism that relies instead on
inheritance of a silent X derived from the male germ line (5, 25,
The state of the paternal X upon arrival in the opossum zygote
requires further study. In the absence of any significant cytoplasm
and the replacement of histones for protamines, how might epige-
netic information be transmitted from the sperm to the zygote?
Previous studies of X-linked genes silenced by MSCI in eutherians
have shown that hypermethylation of DNA is not involved (54, 55).
Studies of the active and inactive X-chromosomes in female kan-
garoos have indicated that DNA methylation is also not involved in
somatic XCI (56). Our studies have highlighted three persistent
marks of meiotic silencing (H3–3meK9, HP1?, and HP1?) that are
(15, 17). Interestingly, H3–3meK9, HP1?, and HP1? are also the
last chromatin-associated marks to be detected before protamine-
mediated compaction during mouse spermiogenesis (SI Fig. 10).
These marks are therefore candidates for transgenerational inher-
itance of epigenetic programming associated with the paternal X.
Until recently, spermatozoa were believed to deliver little more
than DNA into the oocyte. However, several studies now lend
credence to paternal inheritance of both RNA and nonprotamine
proteins that may be critical to early embryonic development
(57–60). Given the apparent absence of the XIC, could epigenetic
programming initiated by MSCI and maintained by PMSC survive
protamine packaging to establish imprinted XCI in the marsupial
Materials and Methods
Slide Preparation. M. domestica seminiferous tubules were pre-
pared with slight modifications from previous methods (15).
Testes were dissected in PBS on ice. Several pieces of seminif-
erous tubule were placed in 4% paraformaldehyde in 1? PBS
plus 0.5% Triton X-100 for 10 min at room temperature, rinsed
in 1? PBS, shredded between two forceps, cytospun onto a glass
slide at 2,000 rpm (Cytospin 4; Thermo Fisher Scientific,
Waltham, MA) for 10 min, and air-dried. Hypotonic treatment
was performed as described (61). Mouse testis slides were
prepared as described (15).
Fractionation of M. domestica Cot-1 DNA. Fractionation of highly
repetitive Cot-1 DNA from M. domestica was performed ac-
cording to ref. 62. M. domestica genomic DNA (100 ?g/ml) was
denatured in 250 mM phosphate buffer (pH 6.8), then annealed
for 24 h at 55°C. The double-stranded/repetitive fraction was
purified by hydroxyapatite chromatography.
in mid-pachytene to diplotene (A, D, and G), round spermatids (B, C, E, F, H, and I), mouse round spermatid (J), and Monodelphis round spermatids (K and L).
Arrows, XY bodies. (F and I) Double immunostaining of H3–3meK9 (green) and ?H2AX (red) in the round spermatids. All images in A–I are single z-sections. (J–L)
are superimposed on single z-sections of DAPI and HP1?.*, chromocenter; arrowheads, mouse PMSCs. All images except J are of M. domestica.
Characterization of M. domestica PMSC. (A–E, G, and H) Double immunostaining of various chromatin marks (green) as indicated along with HP1? (red)
Segregation of X, Y
Meiosis II Spermiogenesis
Association of X, Y
X, Y silencing
H3-2meK9 on X, Y
HP1β, HP1γ, H3-3meK9 on X, Y
γH2AX on X, Y
takes place by MSCI at early pachytene and is maintained in spermatids as
PMSC with modifications similar to those seen in eutherian. Barred chromo-
somes represent transcriptionally suppressed chromatin.
www.pnas.org?cgi?doi?10.1073?pnas.0700323104Namekawa et al.
FISH and Immunofluorescence.Cot-1RNAFISHwasperformedas
described (15). For immunofluorescence, slides were incubated
in PBT (0.15% BSA/0.1% Tween 20) plus 5% goat serum for 60
min before overnight incubation at 37°C with the following
antibodies: SCP3 (Novus Biologicals, Littleton, CO), 1:100;
centromere (Antibodies Incorporated, Davis, CA), 1:100; RNA
Pol-II CTD 8WG16 (Upstate, Charlottesville, VA), 1:200; HP1?
(Abcam, Cambridge, MA), 1:100; HP1? (Chemicon, Temecula,
CA), 1:1,000; H3–2meK9 (Upstate), 1:100; H3–3meK9 (Up-
state; used unless otherwise designated), 1:200; H3–3meK9
(Abcam; used only in SI Fig. 10), 1:200; H3–3meK27 (Upstate),
1:200; ?H2AX (kindly provided by R. Scully, Beth Israel Dea-
coness Medical Center, Boston, MA), 1:1,000; and ?H2AX
(Upstate), 1:5,000 (used in 1:1,000 at 4°C after Cot-1 RNA
FISH). Thereafter, slides were washed three times for 5 min in
PBS plus 0.1% Tween 20, incubated with secondary antibodies
(Alexa dyes; Invitrogen, Carlsbad, CA) at 1:500 for 60 min in
PBT, washed in PBS plus 0.1% Tween 20, and mounted in
Vectashield with DAPI. For combined RNA FISH/immuno-
staining, we carried out RNA FISH first, followed by immuno-
fluorescence. DNA FISH was performed by using chromosome
painting for the M. domestica (kindly provided by W. Rens,
Cambridge Resource Centre for Comparative Genomics, Cam-
bridge, U.K.). All images were acquired with the Axioplan
microscope (Zeiss, Thornwood, NY). Z-section images were
acquired by using Openlab (Improvision, Lexington, MA).
We thank W. Rens and the Wellcome Trust for X and Y chromosome
paints for M. domestica, J. Dennis for advice on Cot-1 DNA fraction-
ation, and J. Hornecker and H. Yoshioka for help in preparation of M.
domestica testes. This work was supported by a grant from the Japan
Society for the Promotion of Science (to S.H.N.), a grant from the
Robert J. Kleberg, Jr., and Helen C. Kleberg Foundation (to J.L.V.), and
National Institutes of Health Grants HD-46637 (to J.R.M.) and GM-
58839 (to J.T.L.). J.T.L. is also an Investigator of the Howard Hughes
1. Graves JA (1996) Annu Rev Genet 30:233–260.
2. Charlesworth B (1991) Science 251:1030–1033.
3. Vicoso B, Charlesworth B (2006) Nat Rev Genet 7:645–653.
4. Sharman GB (1971) Nature 230:231–232.
5. McCarrey JR (2001) in Gene Families: Studies of DNA, RNA, Enzymes, and
Proteins, eds Xue G, Xue Z, Xu R, Holmes R, Hammond GL, Lim HA (World
Scientific, Teaneck, NJ), pp 59–72.
6. Lyon MF (1961) Nature 190:372–373.
7. Takagi N, Sasaki M (1975) Nature 256:640–642.
8. Xue F, Tian XC, Du F, Kubota C, Taneja M, Dinnyes A, Dai Y, Levine H,
Pereira LV, Yang X (2002) Nat Genet 31:216–220.
9. Lifschytz E, Lindsley DL (1972) Proc Natl Acad Sci USA 69:182–186.
10. Turner JM (2007) Development (Cambridge, UK) 134:1823–1831.
11. Burgoyne PS (1982) Hum Genet 61:85–90.
12. Turner JM, Mahadevaiah SK, Fernandez-Capetillo O, Nussenzweig A, Xu X,
Deng CX, Burgoyne PS (2005) Nat Genet 37:41–47.
13. Baarends WM, Wassenaar E, van der Laan R, Hoogerbrugge J, Sleddens-
Linkels E, Hoeijmakers JH, de Boer P, Grootegoed JA (2005) Mol Cell Biol
14. Schimenti J (2005) Nat Genet 37:11–13.
15. Namekawa SH, Park PJ, Zhang LF, Shima JE, McCarrey JR, Griswold MD,
Lee JT (2006) Curr Biol 16:660–667.
16. Turner JM, Mahadevaiah SK, Ellis PJ, Mitchell MJ, Burgoyne PS (2006) Dev
17. Greaves IK, Rangasamy D, Devoy M, Marshall Graves JA, Tremethick DJ
(2006) Mol Cell Biol 26:5394–5405.
18. Wang PJ, McCarrey JR, Yang F, Page DC (2001) Nat Genet 27:422–426.
19. Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, Cordum HS, Hillier L, Brown LG,
Repping S, Pyntikova T, Ali J, Bieri T, et al. (2003) Nature 423:825–837.
20. Khil PP, Smirnova NA, Romanienko PJ, Camerini-Otero RD (2004)Nat Genet
21. Shiu PK, Raju NB, Zickler D, Metzenberg RL (2001) Cell 107:905–916.
22. Bean CJ, Schaner CE, Kelly WG (2004) Nat Genet 36:100–105.
23. Jablonka E, Lamb MJ (1988) J Theor Biol 133:23–36.
24. Miklos GL (1974) Cytogenet Cell Genet 13:558–577.
25. Huynh KD, Lee JT (2001) Curr Opin Cell Biol 13:690–697.
26. Huynh KD, Lee JT (2003) Nature 426:857–862.
27. Huynh KD, Lee JT (2005) Nat Rev Genet 6:410–418.
28. Cooper DW (1971) Nature 230:292–294.
29. Vandeberg JL (1983) J Exp Zool 228:271–286.
30. Lyon MF (1999) in Results and Problems in Cell Differentiation, ed Ohlsson R
(Springer, Heidelberg), pp 73–90.
31. Heard E, Disteche CM (2006) Genes Dev 20:1848–1867.
32. Borsani G, Tonlorenzi R, Simmler MC, Dandolo L, Arnaud D, Capra V,
Grompe M, Pizzuti A, Muzny D, Lawrence C, et al. (1991) Nature 351:325–329.
33. Brockdorff N, Ashworth A, Kay GF, Cooper P, Smith S, McCabe VM, Norris
DP, Penny GD, Patel D, Rastan S (1991) Nature 351:329–331.
34. Brown CJ, Ballabio A, Rupert JL, Lafreniere RG, Grompe M, Tonlorenzi R,
Willard HF (1991) Nature 349:38–44.
35. Lee JT (2000) Cell 103:17–27.
36. Sado T, Wang Z, Sasaki H, Li E (2001) Development (Cambridge, UK)
37. Lee JT, Lu N (1999) Cell 99:47–57.
38. Ogawa Y, Lee JT (2003) Mol Cell 11:731–743.
39. Davidow LS, Breen M, Duke SE, Samollow PB, McCarrey JR, Lee JT (2007)
Chromosome Res 15:137–146.
41. Shevchenko AI, Zakharova IS, Elisaphenko EA, Kolesnikov NN, Whitehead
S, Bird C, Ross M, Weidman JR, Jirtle RL, Karamysheva TV, et al (2007)
Chromosome Res 15:127–136.
42. Duret L, Chureau C, Samain S, Weissenbach J, Avner P (2006) Science
43. Turner JM, Mahadevaiah SK, Elliott DJ, Garchon HJ, Pehrson JR, Jaenisch
R, Burgoyne PS (2002) J Cell Sci 115:4097–4105.
44. McCarrey JR, Watson C, Atencio J, Ostermeier GC, Marahrens Y, Jaenisch
R, Krawetz SA (2002) Genesis 34:257–266.
45. Solari AJ (1974) Int Rev Cytol 38:273–317.
46. Reig OA, Bianchi NO (1969) Experientia 25:1210–1211.
47. Solari AJ, Bianchi NO (1975) Chromosoma 52:11–25.
48. Page J, Berrios S, Rufas JS, Parra MT, Suja JA, Heyting C, Fernandez-Donoso
R (2003) J Cell Sci 116:551–560.
49. Page J, Viera A, Parra MT, de la Fuente R, Suja JA, Prieto I, Barbero JL, Rufas
JS, Berrios S, Fernandez-Donoso R (2006) PLoS Genet 2:e136.
50. Mahadevaiah SK, Turner JM, Baudat F, Rogakou EP, de Boer P, Blanco-
Rodriguez J, Jasin M, Keeney S, Bonner WM, Burgoyne PS (2001) Nat Genet
51. Turner JM, Aprelikova O, Xu X, Wang R, Kim S, Chandramouli GV, Barrett
JC, Burgoyne PS, Deng CX (2004) Curr Biol 14:2135–2142.
52. Plath K, Fang J, Mlynarczyk-Evans SK, Cao R, Worringer KA, Wang H, de la
Cruz CC, Otte AP, Panning B, Zhang Y (2003) Science 300:131–135.
53. Khalil AM, Boyar FZ, Driscoll DJ (2004) Proc Natl Acad Sci USA 101:16583–
54. McCarrey JR, Berg WM, Paragioudakis SJ, Zhang PL, Dilworth DD, Arnold
BL, Rossi JJ (1992) Dev Biol 154:160–168.
55. Grant M, Zuccotti M, Monk M (1992) Nat Genet 2:161–166.
56. Loebel DA, Johnston PG (1997) Mamm Genome 8:146–147.
57. Krawetz SA (2005) Nat Rev Genet 6:633–642.
58. Chu DS, Liu H, Nix P, Wu TF, Ralston EJ, Yates JR III, Meyer BJ (2006)
59. Gatewood JM, Cook GR, Balhorn R, Bradbury EM, Schmid CW (1987)
60. Bench GS, Friz AM, Corzett MH, Morse DH, Balhorn R (1996) Cytometry
62. Britten RJ, Kohne DE (1968) Science 161:529–540.
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