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First published online 3 August 2006
doi: 10.1242/dev.02500
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1 Department of Molecular Genetics and Microbiology, PO Box 100266, University
of Florida, Gainesville, FL 32610-0266, USA.
2 Howard Hughes Medical Institute and Department of Cell and Developmental
Biology, University of Pennsylvania School of Medicine, Philadelphia, PA
19104, USA.
3 Epigenetics Program, Models of Disease Center, Novartis Institute for
Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, USA.
* Author for correspondence (e-mail: resnick{at}mgm.ufl.edu)
Accepted 19 June 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Mouse, Primordial germ cells
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Lawson et al., 1999;
Ying et al., 2000;
Ying and Zhao, 2001). PGCs can
first be detected in the extra-embryonic mesoderm at 7.25 days post-coitus
(dpc) (Ginsburg et al., 1990).
By 8.5 dpc, PGCs enter the embryo proper and actively migrate through the
hindgut endoderm, colonizing the developing gonads between 10.5 and 11.5 dpc.
During this time, PGCs proliferate from an initial population of 45 cells at
7.5 dpc to 25,000 cells at 13.5 dpc when proliferation ceases
(Tam and Snow, 1981). Sexual
differentiation of the germline begins at 13.5 dpc with female germ cells
entering prophase I of meiosis. Within the male gonad, a signal thought to
originate from the testis cords prevents entry into meiosis and male PGCs
enter a mitotic arrest by 14.5 dpc
(McLaren, 1983). Prior to
these changes, male and female PGCs are sexually indifferent, capable of
following either the male or female pathway
(McLaren and Southee,
1997).
Shortly after PGCs enter the urogenital ridges, both male and female germ
cells undergo a common set of changes independent of sexual differentiation.
Changes in cell morphology and cell-adhesion properties occur as the germ
cells transition to a nonmigratory state
(De Felici et al., 1992;
Donovan et al., 1986;
Garcia-Castro et al., 1997).
Male and female PGCs also cease proliferating, have decreased potential to
form pluripotent stem cell lines (Matsui
et al., 1992; McLaren,
1984; Resnick et al.,
1992), and undergo a wave of apoptosis
(Coucouvanis et al., 1993;
Wang et al., 1998). These
differentiation events are accompanied by changes in gene expression as some
germ cell marker genes, such as Tnap (Akp2 - Mouse Genome
Informatics) and Zfp148, are downregulated
(Donovan et al., 1986;
Hahnel et al., 1990;
Takeuchi et al., 2003). Other
genes, including Mvh (Ddx4 - Mouse Genome Informatics),
Scp3 (Sycp3 - Mouse Genome Informatics), Dazl,
Mageb4 and Gcna1 are upregulated during this time
(Cooke et al., 1996;
Di Carlo et al., 2000;
Fujiwara et al., 1994;
Osterlund et al., 2000).
In addition to the differentiation events mentioned above, PGCs mediate two
essential epigenetic processes. First, female PGCs reactivate their silenced X
chromosome, thereby ensuring that each oocyte carries an active X chromosome
(Monk and McLaren, 1981;
Tam et al., 1994).
Interestingly, the ability to reactivate the inactive X chromosome is not
confined to female germ cells, as XXY male germ cells also possess this
reactivation capability (Mroz et al.,
1999). Second, migratory germ cells carry
parent-of-origin-specific imprinting marks and high levels of allele-specific
methylation that contribute to monoallelic expression in migratory PGCs. These
differentially methylated regions become hypomethylated as PGCs colonize the
gonads, leading to a loss of imprinting and biallelic gene expression
(Hajkova et al., 2002;
Lee et al., 2002;
Szabo et al., 2002). However,
this wave of demethylation is not restricted to imprinted loci and genes of
the X chromosome, as several non-imprinted genes and repetitive sequences also
show decreased methylation at this time
(Hajkova et al., 2002;
Lane et al., 2003;
Lees-Murdock et al.,
2003).
We have been investigating regulatory mechanisms underlying postmigratory
germ cell differentiation. Several studies suggest that continuing PGC
development is regulated by a cell intrinsic program rather than by inductive
signals from the gonads. PGCs located in ectopic locations enter meiosis and
initiate expression of the postmigratory marker GCNA1 on schedule without
exposure to the urogenital ridges (Wang et
al., 1997). Embryonic stem cells have been shown to differentiate
to form PGC-like cells that can go on to form cells resembling both oocytes
and spermatocytes, further demonstrating that PGC differentiation can occur
independently of the gonadal environment
(Geijsen et al., 2004;
Hubner et al., 2003;
Toyooka et al., 2003). Last,
cessation of germ cell proliferation has also been suggested to be cell
intrinsic (Ohkubo et al.,
1996).
We previously tested the potential of premigratory germ cells to
differentiate in culture and reported that 8.5 dpc premigratory PGCs in feeder
culture can differentiate to express GCNA1 on the correct temporal schedule
(Richards et al., 1999).
Surprisingly, the rate of differentiation in culture increased when PGCs were
exposed to the DNA demethylating agent 5-azacytidine or the histone
deacetylase inhibitor trichostatin A
(Maatouk and Resnick, 2003).
This suggests that epigenetic mechanisms may contribute to the regulation of
germ cell differentiation.
Here, we further investigate the role of DNA methylation in the process of PGC differentiation. We present evidence that several postmigratory germ cell-specific genes are demethylated in germ cells as they colonize the genital ridges and that DNA demethylation controls the temporal expression of these genes in vivo. In addition, we show that these postmigratory germ cell-specific genes are ectopically expressed in DNA methyltransferase mutant embryos, suggesting that DNA methylation is a mechanism of silencing germ cell-specific genes in somatic tissues. These results provide the first in vivo evidence of tissue-specific embryonic gene regulation mediated by dynamic changes in DNA methylation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) or
Dnmt1c allele (Lei et
al., 1996) were maintained on the B6(CAST7) mixed background
(Mann et al., 2003).
Dnmt1c embryos used for immunostaining were also
maintained on a mixed background (129/SvJae x C57BL/6).
Primordial germ cell isolation and purification
Gonads were collected from 10.5 dpc and 13.5 dpc embryos. At 13.5 dpc,
embryos were sex segregated based on the presence of testis cords in the male
gonad. PGCs were immunomagnetically purified using the TG-1 antibody as
described (Pesce and De Felici,
1995). Purified fractions were greater than 85% (10.5 dpc) and 90%
(13.5 dpc) germ cells, as judged by alkaline phosphatase staining.
Immunodepleted fractions contained less than 1% PGCs and primarily contained
somatic cells from the gonad and mesonephros.
Bisulfite conversion and DNA sequencing
Genomic DNA isolated from both purified and immunodepleted fractions was
subjected to bisulfite conversion as described
(Clark et al., 1994). Bisulfite
primers were designed against the converted DNA sequences and are listed in
Table 1. PCR amplification was
performed on 10% of one purification (approximately one embryo equivalent)
with HotStar Taq (Qiagen) using the following cycling conditions: 95°C for
15 minutes followed by 35 cycles of 95°C for 45 seconds, 53°C for 30
seconds and 72°C for 1.5 minutes. Bisulfite PCR amplifications were
performed on two independent germ cell purifications to avoid inconsistencies
that might arise from conducting PCR on small amounts of DNA. PCR products
were gel purified using Wizard DNA Clean-up System (Promega) and cloned using
the pGEM-T Easy Vector System (Promega). Plasmid sequencing was carried out
using ABI Prism BigDye terminator (PerkinElmer) by the Center for Mammalian
Genetics DNA Sequence Core.
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-[32PO4]-dCTP labeled probes. Probe fragments
were generated by gel purification of the RT-PCR products obtained from testis
cDNA.
Immunochemical methods
Embryos were collected at 8.5 and 9.5 dpc and fixed overnight in
methanol:dimethyl sulfoxide (4:1) at 4°C. Endogenous peroxidase activity
was inactivated by a 2-hour incubation in methanol:dimethyl sulfoxide: 30%
hydrogen peroxide (4:1:1) at room temperature. Embryos were stored in 100%
methanol at -20°C.
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). Whole-mount embryos were immunostained using the same
procedure with several adjustments. Blocking was performed in PBSMT (PBS, 2%
w:v nonfat dry milk, 0.1% Triton X-100) and a horseradish peroxidase
(HRP)-conjugated mouse antirat IgM (Zymed) secondary antibody was used. Color
development was performed using the Liquid DAB (3,3'-diaminobenzidine)
Substrate Kit following the manufacturer's instructions (Zymed). Paraffin
sections were stained for SSEA1 immediately after GCNA1 staining as previously
described (Richards et al.,
1999). |
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Furthermore, we found that the DNA demethylating agent
5-azacytidine accelerates the rate and extent of germ cell differentiation in
culture (Maatouk and Resnick,
2003). Because the expression of GCNA1 in cultured PGCs is
sensitive to changes in DNA methylation, we explored whether induction of
other postmigratory germ cell genes could be regulated by a demethylation
event. As the gene encoding GCNA1 remains unknown, we investigated the
methylation status of several postmigratory germ cell-specific genes that
share a similar expression pattern with GCNA1. Mvh, Scp3 and
Dazl are exclusively expressed by both male and female germ cells as
they enter the urogenital ridges between 10.5-11.5 dpc
(Cooke et al., 1996;
Di Carlo et al., 2000;
Fujiwara et al., 1994).
Fig. 1 shows that the first
exon of each of these genes is contained within a CpG island. To examine the methylation status of these postmigratory germ cell genes, genomic DNA obtained from immunomagnetically purified 10.5 and 13.5 dpc PGCs was subjected to bisulfite sequence conversion. Approximately 20 CpG residues were analyzed for the presence of methylation. For each gene, 10.5 dpc PGCs showed high levels of methylation, but by 13.5 dpc all three genes showed a significant loss of methylation with most clones being completely unmethylated (Fig. 2). No significant differences in methylation were observed between male and female PGCs, consistent with the idea that PGCs are indifferent prior to 12.5 dpc. These results suggest that DNA demethylation of the germline between 10.5 and 12.5 dpc functions not only to reprogram imprinted loci and reactivate the X chromosome, but may contribute to additional postmigratory differentiation events.
Although most clones isolated from the PGC fraction were significantly
hypomethylated at 13.5 dpc, some clones retained high levels of methylation.
Some of these clones may be explained by the presence of contaminating somatic
cells in the purified germ cell preparations, as cells of the immunodepleted
fraction remained highly methylated at 13.5 dpc
(Fig. 2). However, because 13.5
dpc PGC preparations are routinely more than 90% pure, we favor the
explanation that some PGC genomes have not undergone demethylation by 13.5
dpc. Consistent with this interpretation, some germ cells initiate GCNA1
expression by 11.5 dpc, but many germ cells do not express the marker until
14.5 dpc (Enders and May,
1994).
Fig. 2 demonstrates that the
germ cell-specific genes Mvh, Dazl and Scp3 exhibit loss of
methylation as they first become expressed. In contrast to this pattern of
expression, several genes are negatively regulated in PGCs as they
differentiate into gonocytes. Tnap is expressed as germ cells are
initially allocated in the extra-embryonic mesoderm at 7.25 dpc, but
expression is lost between 13.5 and 14.5 dpc
(Donovan et al., 1986;
Ginsburg et al., 1990). We
examined the Tnap locus to explore any potential role of dynamic DNA
methylation changes on a gene that is negatively regulated in gonocytes.
Bisulfite analysis of eight CpG dinucleotides upstream of exon 1 shows that
this region of Tnap is unmethylated at 10.5, 13.5 and 14.5 dpc in
both germ cells and somatic cells. This result is consistent with the notion
that DNA demethylation occurs in genes that are positively regulated as germ
cells transition into gonocytes, rather than a characteristic of all genes
expressed in germ cells.
Expression of GCNA1 is sensitive to DNA methylation in primordial germ cells
Primordial germ cells in culture express the postmigratory germ cell marker
GCNA1 on an accelerated schedule when exposed to the DNA demethylating agent
5-azacytidine (Maatouk and Resnick,
2003). Although many genes respond to this agent in culture, Walsh
and Bestor (Walsh and Bestor,
1999) found that few genes are subject to dynamic DNA methylation
changes in vivo. To determine whether GCNA1 expression is regulated by DNA
methylation in vivo, we next tested whether GCNA1 is prematurely expressed by
PGCs in DNA methyltransferase 1 (Dnmt1) mutant embryos. Dnmt1
maintains methylation patterns during DNA replication and the null
Dnmt1c mutation results in a 98% loss of genomic
methylation (Lei et al.,
1996). Dnmt1c/c mutant embryos were
cross-sectioned and immunostained for the PGC marker SSEA1 and for the
postmigratory germ cell marker GCNA1. GCNA1 is normally not detectable prior
to 10.5 dpc (Enders and May,
1994). In 8.5 dpc Dnmt1c/c embryos, PGCs
located in the yolk sac endoderm simultaneously expressed GCNA1 and SSEA1,
with GCNA1 being detected 2-3 days earlier than expected
(Fig. 4B). PGCs in the
posterior region of 9.5 dpc embryos also prematurely expressed GCNA1
(Fig. 4C). These results
indicate that in vivo expression of GCNA1 is temporally controlled by DNA
methylation.
|
). Surprisingly, not only was GCNA1 prematurely
expressed in PGCs, but ectopic expression was observed in somatic cells
scattered throughout the entire embryo
(Fig. 5C,D). Because these
cells do not express the PGC markers SSEA1
(Fig. 4) or OCT4
(Hattori et al., 2004), we
suggest that they are not PGCs that have migrated to aberrant locations. As only a small number of cells ectopically expressed GCNA1, it seemed likely that the low levels of functional Dnmt1 enzyme present in the hypomorphic Dnmt1n/n mutants might attenuate promiscuous gene activation. To test this idea GCNA1 immunostaining was also performed on 9.5 dpc Dnmt1c/c embryos. The more severe mutation consistently caused much higher levels of ectopic expression than observed in the Dnmt1n/n embryos (Fig. 5F,G). As expected, wild-type and heterozygous embryos at these stages exhibited no GCNA1 expression (Fig. 5A,E). Development of Dnmt1-deficient mutants is frequently retarded such that 9.5 dpc Dnmt1c/c embryos more closely resemble wild-type 8.5 dpc embryos. Fig. 5H demonstrates that GCNA1 expression is not readily detected in a more closely stage-matched 8.5 dpc Dnmt1+/c embryo. Together, these results indicate that the postmigratory germ cell marker GCNA1 is ectopically expressed both temporally and spatially in embryos lacking a functional Dnmt1 enzyme.
Premature expression of postmigratory primordial germ cell genes in Dnmt1 mutant embryos
Several postmigratory germ cell-specific genes are demethylated as the germ
cells colonize the developing gonads (Fig.
2). Additionally, GCNA1 is ectopically expressed under conditions
of reduced methylation. To determine if additional postmigratory germ
cell-specific genes are prematurely expressed in Dnmt1 mutant
embryos, RT-PCR analysis was performed to examine the expression profiles of
Mvh, Scp3, Dazl and another PGC-specific gene that shares a similar
expression pattern, Mageb4
(Osterlund et al., 2000)
(Fig. 6). As these genes are
expressed only after PGCs enter the developing gonads, little or no expression
was detected in wild-type and heterozygous 9.5 dpc embryos, as expected.
However, embryos homozygous for either the Dnmt1n or
Dnmt1c mutation precociously expressed each of the germ
cell genes analyzed. In addition, expression seemed to be greater in the more
severe Dnmt1c mutant. These results support the notion
that postmigratory PGC gene expression is dependent upon the genome-wide
demethylation event that occurs during colonization of the gonads.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Lane et al.,
2003; Lee et al.,
2002; Lees-Murdock et al.,
2003). Although most CpG islands are unmethylated regardless of
tissue or expression status (Ioshikhes and
Zhang, 2000; Rollins et al.,
2006), we found that several germ cell-specific genes are highly
methylated at 10.5 dpc and are included in this wave of germ cell
demethylation as they are first expressed
(Fig. 2). These genes remain
methylated and silent in somatic cells. Loss of methylation, as observed in
Dnmt1 mutants, correlates with their premature expression in germ
cells and ectopic expression in somatic cells. Together, these results
strongly suggest that the naturally occurring demethylation of these genes in
germ cells is rate limiting for their expression, and that DNA methylation is
necessary to maintain silencing of these genes in somatic cells.
The rapid rate of demethylation and the presence of nuclear Dnmt1 protein
led Hajkova et al. to propose that germ cell demethylation results from an
active mechanism, rather than the passive process of replication without
further methylation (Hajkova et al.,
2002). Our results are consistent with this proposal as we
observed individual PGC genomes having intermediate levels of methylation,
probably representing PGCs in the process of being actively demethylated
(Fig. 2). Precocious expression
of germ cell-specific genes, presumably owing to passive demethylation in
Dnmt1 mutants, suggests that the hypomethylated state is sufficient
for transcription and does not require the process of active
demethylation.
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;
Li et al., 1993;
Sado et al., 2000;
Walsh et al., 1998).
Bdnf expression is increased in neurons of mice bearing a
conditionally deleted Dnmt1 gene
(Martinowich et al., 2003).
Although numerous genes are ectopically expressed in Dnmt1-deficient
cells in culture, we are aware of only one previous report of ectopic gene
expression of a single copy gene in Dnmt1 mutant embryos. OCT4, a
transcription factor present in premeiotic germ cells, was found to be
ectopically expressed in placentas of Dnmt1 mutant embryos
(Hattori et al., 2004). Here,
we report extensive ectopic expression of germ line-specific genes resulting
from loss of DNA methylation.
Expression of postmigratory germ cell genes is attenuated in DNA-deficient mutants
If DNA methylation is necessary to silence germ cell genes in somatic
cells, why are only some cells positive for GCNA1 in the
Dnmt1-deficient embryos? Although Dnmt1c/c
embryos lack detectable Dnmt1 activity, Dnmt1n/n embryos
produce low levels of functional Dnmt1 enzyme and retain about 30% of genomic
methylation (Lei et al.,
1996). The experiments reported here were not performed under
directly comparable conditions; however, Mvh, Dazl, Scp3 and
Mageb4 all show greater expression in the
Dnmt1c/c compared with the Dnmt1n/n
mutants relative to the Hprt control
(Fig. 6). This was also
observed for the expression of GCNA1 in Dnmt1n/n compared
with Dnmt1c/c embryos
(Fig. 5). Repressive chromatin
structure or compensation by de novo DNA methyltransferases may maintain
silencing in non-expressing cells. Alternatively, DNA demethylation in mutant
embryos, which occurs by a passive replication-dependent mechanism, may occur
more slowly in some cells as loss of Dnmt1 may decrease the rate of
cell proliferation (Jackson-Grusby et al.,
2001; Milutinovic et al.,
2003). Slower cell cycles could lengthen the time it takes to
passively demethylate, causing delayed gene activation. This may account for
the large number of cells observed in the Dnmt1c/c mutant
that do not initiate GCNA1 expression.
How could DNA methylation silence germ cell-specific genes in both germ and
somatic lineages? Several mechanisms, including restricted expression of
positive acting transcription factors, steric interference with transcription
factor binding sites, attraction of methyl DNA binding proteins and DNA
methylation induced changes in histone modifications have been proposed
(Jaenisch and Bird, 2003).
Interestingly, recent reports suggest that the repressive transcription factor
E2F6 is necessary to silence several spermatogenic genes in somatic cells, and
that promoters of these genes are hypomethylated in E2F6-deficient cells
(Pohlers et al., 2005;
Storre et al., 2005). We are
currently investigating whether E2F6 and DNA methylation share a common
pathway to repress germ cell-specific genes.
DNA methylation mediated regulation of germ cell development
DNA methylation has previously been proposed to regulate the expression of
tissue-specific genes; however, the lack of substantial in vivo evidence has
narrowed the proposed role of methylation to silencing of endogenous
retrotransposons and maintaining monoallelic gene expression within imprinted
loci and on the inactive X chromosome in females
(Jaenisch, 1997;
Walsh and Bestor, 1999). Our
data provide strong evidence that methylation may indeed control
tissue-specific gene expression for a set of germ cell-specific genes that are
coordinately activated upon germ cell entry into the gonads.
Seki et al. (Seki et al.,
2005) recently investigated genome-wide changes in chromatin
modifications during primordial germ cell development. Using antibodies to
5-methylcytosine, they observed that PGCs at the base of the allantois at 8.0
dpc have similar methylation levels as somatic cells; however, migrating PGCs
in the hindgut displayed lower methylation levels. This first wave of germ
cell demethylation may signify the transition from a somatic cell fate to a
more pluripotent state, as germ cells at this stage resemble cells of the
inner cell mass in their expression profiles and their ability to give rise to
pluripotent stem cell lines (Donovan and
de Miguel, 2003; Matsui et
al., 1992; Resnick et al.,
1992).
Our data suggest that the second wave of demethylation, which temporally coincides with entry into the gonads, controls the expression of several genes required for gametogenesis, as well as contributing to imprint erasure, reactivation of the inactive X chromosome and expression of IAP retrotransposons. Other aspects of PGC differentiation may also be linked to DNA dimethylation; however, the lethality of Dnmt1 mutant embryos prior to 10.5 dpc prevents the examination postmigratory germ cell differentiation events. Conditional deletion of Dnmt1 in PGCs might allow for further analysis of other changes that temporally overlap this wave of demethylation.
Cancer testis antigens
Efforts to identify cancer-derived gene products as targets for
immunotherapy have revealed an association between genes normally expressed
only in germ cells, but ectopically activated in tumors. Currently, 89
transcripts grouped into 44 families are recognized as cancer testis (CT)
antigens (Scanlan et al.,
2004). Boon and colleagues (De
Smet et al., 1996; De Smet et
al., 2004) have demonstrated lower levels of promoter methylation
in tumors expressing the MAGEA1 cancer testis antigen compared with
non-expressing cells. Furthermore, MAGEA1 expression could be induced
in response to demethylating agents. This led to the suggestion that the loss
of DNA methylation that accompanies tumor progression may be responsible for
MAGE gene expression.
Koslowski et al. (Koslowski et al.,
2004) reported that more than half of CT genes are expressed in
premeiotic germ cells and that several could be induced in peripheral blood
leukocytes by 5-azacytidine treatment. Similarly we found that several
premeiotic germ cell-specific genes are expressed following loss of DNA
methylation, including Mageb4, the murine homolog of a human CT
antigen. Our data provide direct in vivo evidence that premeiotic gene
expression is linked to hypomethylation, and provides a likely explanation for
the frequent appearance of germ cell-specific genes in certain tumors.
Evolution of the germ cell lineage
Boule and Vasa, the Drosophila homologs of
Dazl and Mvh,were originally identified as components of
Drosophila germ plasm, and are highly conserved in germ cell
development. While organisms with a mosaically determined germ line inherit
these gene products as maternal factors, Dazl and Mvh are
expressed in postmigratory germ cells in the mouse, 3-4 days after the germ
line is specified. Interestingly, divergent mechanisms of germ cell
specification operate within the amphibian class. The Xenopus
germline is mosaically determined, while salamanders (Axolotl)
specify germ cells by an inductive mechanism similar to mammals
(Johnson et al., 2003). As
salamanders delay expression of axdazl and axvh until germ
cells arrive at the gonad (Bachvarova et
al., 2004), it would be interesting to investigate the potential
role of methylation in the expression of axdazl and axvh.
The observation that methylation were to regulate expression of these genes in
salamanders would suggest that control of germ cell differentiation by DNA
methylation may be a widely conserved mechanism among species that use
inductive signals to specify the germ cell lineage. Additionally, this would
suggest that DNA methylation in the germ line initially arose to regulate the
timing of germ cell differentiation rather than epigenetic processes such as
genomic imprinting.
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ACKNOWLEDGMENTS |
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