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First published online 10 January 2007
doi: 10.1242/dev.02771
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1 Department of Biology, Graduate School of Science and Technology, Kobe
University, 1-1 Rokkodaicho, Nadaku, Kobe 657-8501, Japan.
2 Department of Biology, Indiana University, Bloomington, IN 47405, USA.
3 RIKEN Center for Developmental Biology, Chuo-ku, Kobe 650-0047, Japan.
Author for correspondence (e-mail:
sstrome{at}indiana.edu)
Accepted 27 November 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: C. elegans, MRG-1, Germ line, X chromosome silencing
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Studies in the nematode Caenorhabditis elegans have identified
numerous types of factors required during embryogenesis and early larval
stages for the primordial germ cells (PGCs) to develop properly (reviewed by
Strome, 2005
). The maternally
provided factor PIE-1 plays a key role, by blocking RNA polymerase IImediated
transcription in the germline blastomeres and protecting those cells from
following somatic fates (Mello et al.,
1992; Seydoux et al.,
1996; Batchelder et al.,
1999). The C. elegans Nanos homologs NOS-1 and NOS-2 and
several Pumilio-related proteins, probably operating as translational
regulators, ensure that the PGCs become incorporated into the somatic gonad
primordium, remain mitotically quiescent at early stages, and survive at later
stages (Subramaniam and Seydoux,
1999). The `maternal-effect sterile' proteins MES-2, MES-3, MES-4
and MES-6 operate at the level of histone tail modifications to regulate
chromatin organization and gene expression in the germ line; MES-4 cooperates
with MES-2, MES-3 and MES-6 to repress the X chromosomes in the germ line
(Capowski et al., 1991;
Fong et al., 2002;
Bender et al., 2004;
Bender et al., 2006). Their
function is required for PGC proliferation and survival.
The C. elegans mrg-1 gene was previously identified by RNAi as
being required for PGC proliferation
(Fujita et al., 2002). The
predicted C. elegans MRG-1 protein is related to three human
proteins: mortality factor MORF4 and two mortality factor-related proteins
MRG15 and MRGX. MORF4 induces senescence in human tumor cell lines and
therefore appears to oppose immortality
(Bertram et al., 1999). Based
on analysis of MRG knockout mice, MRG15 promotes cell proliferation and is
essential for embryo survival, whereas MRGX is not required for viability or
fertility (Tominaga et al.,
2005a; Tominaga et al.,
2005b). Caenorhabditis elegans MRG-1 is considered to be
an ortholog of MRG15, although MRG-1 shows lower sequence similarity (26%
identity, 50% similarity) to human MRG15 than do the homologs in the other 17
species examined (Bertram and
Pereira-Smith, 2001). Notably, MRG-1, like MRG15, possesses a
chromodomain.
The presence of a chromodomain in MRG-1 suggests that it associates with
chromatin, specifically with methylated histone tails, as has been
demonstrated for several chromodomaincontaining proteins. For example,
heterochromatin protein 1 (HP1) binds H3 tails methylated on Lys9 (H3K9),
Polycomb (Pc) binds methylated H3K27, and Eaf3 binds methylated H3K36
(Bannister et al., 2001;
Lachner et al., 2001;
Cao et al., 2002;
Czermin et al., 2002;
Carrozza et al., 2005;
Keogh et al., 2005). Among the
candidate C. elegans proteins for creating the methyl marks that
recruit MRG-1 are the MES proteins. MES-2 operates in a complex with MES-3 and
MES-6 to methylate H3K27 (Bender et al.,
2004; Ketel et al.,
2005), and MES-4 methylates H3K36
(Bender et al., 2006).
To further understand the role of MRG-1 in cell proliferation and development, we isolated and analyzed three mrg-1 deletion mutants. Loss of maternal MRG-1, like loss of mouse MRG15, leads to significant levels of embryonic lethality. Surviving embryos develop into apparently healthy adults that lack a germ line; the latter is a result of failure of PGCs to proliferate and also PGC degeneration. As predicted, MRG-1 is associated with chromatin. Intriguingly, it is only detected on the autosomes and not on the X chromosomes. This pattern resembles that of MES-4, and yet neither MRG-1 nor MES-4 depends on the other for its chromosomal association. Studies of gene expression patterns suggest that MRG-1 is not essential for activation of germline-expressed genes in mrg-1 mutant larvae but is needed for gene silencing in the germ lines of their mothers. Specifically, transgenes and genes on the X are derepressed in mutant mothers. This finding, and the differential sensitivity of XX and XO worms to loss of MRG-1 function, points to the X chromosome as a likely target of MRG-1 regulation during germline development. MRG-1 also can serve an important role in somatic cells, as loss of MRG-1 function suppresses the ectopic expression of several germline genes and the larval lethality caused by loss of the chromatin regulator MEP-1. Our results provide insights into chromatin-level regulation of germline potential and immortality.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Isolation of mrg-1 deletion alleles
The deletion allele mrg-1(qa6200) was isolated by screening a
library of UV/TMP-mutagenized worms by two sequential rounds of PCR. First
round primers were 5'-AAGGGCATTCTTCACTGTTGGA-3' and
5'-GATAAATGGCCGCTGAAACTTG-3'. Second round primers were
5'-TTGTTCACAACTTTCACCGGCT-3' and
5'-CTCGGCCATGGGCTAGAAAC-3'. The tm1227 and
ok1262 alleles were isolated using similar PCR screens by Dr Shohei
Mitani at the National BioResource Project and by the C. elegans Gene
Knockout Consortium, respectively. Each mrg-1 allele was backcrossed
to wild type ten times and then balanced with either eT1 or
qC1[qIs26].
Sequencing mrg-1 mutations
DNA was prepared from sterile homozygous mrg-1(ok1262) worms. PCR
primers 5'-GAAGATCGTCTGGGATGGAA-3' and
5'-AGCGATGGCAAGGAACTCTA-3' amplified a 
1.5 kb product.
Sequencing primers 5'-GAGCAAATGAGAACGGTCGATGATCTGC-3' and
5'-GTTCGATGGAGCGCGCTTGCATTATTTTC-3' were used in standard ABI
BigDye sequencing reactions. For mrg-1(qa6200) and
mrg-1(tm1227), DNA was prepared from heterozygous worms. From both,
PCR primers 5'-AAGGGCATTCTTCACTGTTGGA-3' and
5'-GATAAATGGCCGCTGAAACTTG-3' amplified a 1.3 kb product and a
0.9 kb product. Both DNA bands were gel purified and sequenced using the
primers used for PCR.
Immunofluorescence analysis
Worms and embryos were fixed using a paraformaldehyde and methanol
procedure (Subramaniam and Seydoux,
1999) or a methanol and acetone procedure
(Strome and Wood, 1983).
Primary antibodies used were: affinity-purified rabbit anti-MRG-1 fusion
protein at 1:400-1:1000 (Fujita et al.,
2002), affinity-purified rat anti-MRG-1 peptide (raised against
the C-terminal 24 amino acids conjugated to keyhole limpet hemocyanin) at 1:5,
affinity-purified rabbit anti-MES-4 at 1:100
(Bender et al., 2006), mouse
monoclonal antibody H5 to RNA Pol II pSer2 at 1:25 (Covance), mouse monoclonal
antibody PA3 at 1:500 [from M. Monestier
(Monestier et al., 1994)],
rabbit anti-H3K27me3 at 1:1000 [from Y. Zhang
(Plath et al., 2003)], rabbit
anti-H3K36me2 at 1:200 [from Y. Zhang
(Tsukada et al., 2006)],
rabbit anti-PGL-1 at 1:100,000 (Kawasaki
et al., 1998), mouse monoclonal anti-PGL-1 antibody K76
(Strome and Wood, 1983) and
affinity-purified rabbit anti-GLH-1 at 1:300
(Gruidl et al., 1996).
Secondary antibodies used were Alexa Fluor 488, 546, 594, 647 and TRITC goat
antimouse, anti-rabbit and anti-rat IgGs (from Molecular Probes). Samples were
mounted in Gelutol or SlowFade (Molecular Probes) and examined by fluorescence
microscopy and Nomarski optics on an Olympus BX51 microscope, a Nikon Eclipse
TE200 microscope with an UltraVIEW LCI spinning-disk confocal laser and
UltraVIEW software (Perkin-Elmer), or a Nikon Eclipse E800 microscope with
Metamorph software (Universal Imaging Corp.). Images were assembled using
Adobe Photoshop 7.0 and Illustrator 10.0.3.
Generation of transgenic animals containing a pie-1 promoter::gfp::mrg-1 transgene
mrg-1 coding sequence, PCR amplified from a mrg-1 cDNA
clone, was transferred using Gateway technology into the vector pID3.01, which
contains the unc-119(+) gene [from G. Seydoux
(Poteryaev et al., 2005)]. The
final clone was bombarded into unc-119 worms
(Praitis et al., 2001).
Transgenic lines were identified by rescue of the Unc phenotype and by
observing GFP expression in the germ line.
Testing for expression of a repetitive extrachromosomal array in the germ line
The extrachromosomal array pBK48.1, which contains many copies of GFPtagged
let-858 driven by its own promoter
(Kelly et al., 1997;
Kelly and Fire, 1998), was
introduced via genetic crosses into dpy-18 mrg-1(ok1262)/eT1.
Expression of the let-858::gfp array in dpy-18 mrg-1
M+Z-adult hermaphrodites was scored at 20°C by fluorescence
microscopy.
Quantification of mRNA levels in larvae and dissected adult gonads
For the analysis shown in Fig.
7, total RNA was isolated from 300 L1 and L2 larvae and
reverse transcribed using oligo dT primer and High-Capacity cDNA Archive Kit
(Applied Biosystems). RT-PCR was performed in triplicate using SYBR Green PCR
Mastermix (Applied Biosystems) and an Applied Biosystems 7300 Real-Time PCR
system. Primer Express 3.0 software (Applied Biosystems) was used to design
primers for pgl-1, glh-1, nos-1, myo-2 and rpa-1. All data
were normalized to rpa-1, which encodes ribosomal subunit protein P1.
The 2-
Ct method was used to calculate relative fold
changes, as described in an Applied Biosystems user bulletin.
For the analysis shown in Fig.
6, wild-type and mrg-1(qa6200) M+Z-hermaphrodites were
incubated in a drop of M9 buffer overnight. Approximately 3000 of their
wild-type and mrg-1(qa6200) M-Z-L1 larvae were collected and fed for
6 hours. cDNA preparation, RT-PCR performed in triplicate and Pfaffl analysis
were done as described in Bender et al.
(Bender et al., 2006), but
using a Stratagene MX3000p QPCR system.
For the analysis shown in Fig.
8, 50 gonad arms were dissected from wild-type and
mrg-1(qa6200) M+Z-young adult hermaphrodites and total RNA isolated
as previously described (Chi and Reinke,
2006; Bender et al.,
2006). cDNA preparation, primer design, RT-PCR performed in
triplicate and Pfaffl analysis were done as described in Bender et al.
(Bender et al., 2006), but
using a Stratagene MX3000p QPCR system.
Western blot analysis
Approximately 100 N2, dpy-18 mrg-1(ok1262) M+Z-, and
mrg-1(tm1227) M+Z-homozygous worms boiled in SDS sample buffer were
separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose
membrane. Antibodies used were affinity-purified rabbit anti-MRG-1 (1:500),
mouse monoclonal anti-
-tubulin (1:2000, Sigma) and horseradish
peroxidaseconjugated goat secondaries (1:10,000, Jackson Laboratories).
Horseradish peroxidase was detected using SuperSignal West Pico
Chemiluminescent Substrate (Pierce).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). To gain
insights into the zygotic and maternal requirements for MRG-1 function and to
enable in-depth analysis, we obtained three deletion alleles of mrg-1
(Fig. 1A). The tm1227
allele lacks 401 bp surrounding the initiation codon. The qa6200
allele lacks 450 bp of the first two exons and first intron of the gene and is
predicted to shift the translational reading frame. The ok1262 allele
lacks 1443 bp extending from the middle of intron 2 to the middle of intron 3.
After immunostaining using anti-MRG-1 antibody, nuclear signal was detected in
wild-type embryos, but not in mrg-1 mutant embryos
(Fig. 1B and data not shown).
By western blot analysis using anti-MRG-1 antibody, a protein of the predicted
size (37 kD) was observed in wild-type worm extracts; in the two mutant
alleles tested, signal at 37 kD was reduced to <5% of wild-type level,
which may be residual maternal product in the worms analyzed
(Fig. 1C). These results
suggest that all three mrg-1 alleles are strong loss-offunction and
perhaps null alleles.
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30-50%); all
surviving embryos developed into sterile M-Z-adults. Thus, a maternal supply
or maternal function of mrg-1 gene product is important for proper
embryo development and especially crucial for the germ line.
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In mrg-1 mutants the PGCs fail to proliferate and degenerate
In wild-type worms, the two PGCs (Z2 and Z3) present in newly hatched L1
larvae divide to generate over 1000 germ nuclei. In mrg-1 M-Z-L1s,
two PGCs are present, and P granules appear to have been inherited normally
(data not shown). However, the PGCs do not proliferate normally
(Fig. 2A and see Table S1 in
the supplementary material). The average number of germ nuclei in
mrg-1(qa6200) larvae increased slightly to 6 (range 2-8) in L2 larvae
and then decreased to 1 (range 0-3) by the L4 stage. The number of nuclei in
mrg-1(tm1227) was 2-6 in L2/L3 and 0-7 by L4. These results indicate
that mrg-1 sterility results from failure of the PGCs to proliferate,
and from death of germ cells in at least some worms.
Two types of cell death have been observed in the C. elegans germ
line: programmed cell death (apoptosis), as observed in nos-1; nos-2
mutants (Subramaniam and Seydoux,
1999), and degenerative cell death (necrosis), as observed in
mes-2, mes-3, mes-4 and mes-6 mutants
(Paulsen et al., 1995;
Garvin, 1998). Loss of
ced-3 or ced-4, two genes required for programmed cell death
(Ellis and Horvitz, 1986), did
not suppress the germline proliferation defect and sterility of
mrg-1(RNAi) worms (data not shown), suggesting that programmed cell
death is not responsible for the tiny germ lines in mrg-1 mutants.
Instead, it is likely that necrosis-like death contributes. Consistent with
this, mrg-1 mutant germ lines contain enlarged nuclei
(Fig. 2B,C), similar to those
observed in degenerating somatic cells
(Chalfie and Wolinsky, 1990;
Hall et al., 1997) and in
mes mutant germ lines (Paulsen et
al., 1995).
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), and rat
antibody raised against the C-terminal 24 amino acids of MRG-1. Both
antibodies are specific for MRG-1 in embryonic stages (e.g. see
Fig. 1B). In adult stages, the
rabbit antibody shows some nonspecific staining of nucleoli in mrg-1
mutant adults (data not shown). MRG-1 is highly enriched in nuclei and concentrated on chromatin. In early embryos, MRG-1 is present in the nuclei of all blastomeres (Fig. 3A-F). In late embryos and young larvae, MRG-1 staining is higher in the nuclei of the two PGCs, Z2 and Z3, than in somatic blastomeres (Fig. 3G-L). In larvae and adults, MRG-1 staining is seen primarily in the nuclei of germ cells, although it is also faintly visible in the nuclei of several somatic cell types, including intestinal cells. In the adult germ line, all germ nuclei in the mitotic and meiotic regions are stained (Fig. 3M-O). These results demonstrate that MRG-1 is present in the germ line at all stages of development and is maternally loaded into embryos. In addition, zygotically expressed MRG-1 is produced in all cells by at least the 100-cell stage; it accumulates to higher levels in the PGCs than in somatic tissues (see Fig. S1 in the supplementary material).
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; Bender et al.,
2006), revealed that GFP::MRG-1 associates with the same five
chromosomes as MES-4 (Fig.
4A-D). Thus, the chromosome lacking MRG-1 is the X.
The observation that both MES-4 and MRG-1 are concentrated on autosomes and
the finding in yeast that the MRG-1-related chromodomain protein Eaf3 binds
preferentially to histone H3 tails methylated on Lys36
(Carrozza et al., 2005;
Keogh et al., 2005) suggested
the attractive possibility that MRG-1 association with chromatin requires
MES-4 and its H3K36 methylation activity. To test this, we imaged MRG-1 in
early mes-4 M-Z-null mutant embryos, which lack detectable MES-4 and
H3K36me2 (Bender et al., 2006).
MRG-1 showed robust chromosomal association in mes-4 null early
embryos (Fig. 4K-M). MRG-1 also
appeared to be associated with chromosomes in the PGCs of mes-4
M-Z-L1s, but the small size of those nuclei, the high level of nucleoplasmic
MRG-1 present even in wild type (see above), and the poor staining by
anti-MRG-1 of samples fixed for optimal preservation of chromosomes made it
difficult to assess with confidence the dependence of MRG-1 on MES-4 in those
cells. In other epistasis experiments, we observed a normal-appearing pattern
of MRG-1 in mes-2 M-Z-embryos
(Fig. 4H-J), of MES-4 and
H3K36me2 in mrg-1 M-Z-embryos
(Fig. 4Q-S, and see Fig. S2A-F
in the supplementary material), and of MES-2-catalyzed H3K27me3 in
mrg-1 M-Z-embryos (see Fig. S2G-J in the supplementary material).
Thus, the results of molecular epistasis tests do not support the notion that
the recruitment of MRG-1 to chromatin depends on the MES system, or vice
versa.
MRG-1 is not required for PGCs to initiate expression of several germline genes
Because MRG-1 binds to chromosomes, and the Mrg-1 sterile phenotype
reflects defective development of the PGCs, we sought to determine whether the
gene expression capabilities of the PGCs are impaired. Transcription is
repressed in the germline blastomeres, and is thought to commence shortly
after P4 divides into Z2 and Z3, at about the 100-cell stage
(Seydoux et al., 1996;
Seydoux and Dunn, 1997). One
type of evidence for transcriptional turn-on in Z2 and Z3 is the appearance of
the elongating form of RNA polymerase II (Pol II), assessed by staining with
the H5 antibody to Pol II phosphorylated on Ser2 in the C-terminal domain
(Seydoux and Dunn, 1997). In
early mrg-1 embryos, as in wild-type embryos, H5 staining is detected
in the nuclei of somatic blastomeres but not of the germline blastomere
(Fig. 5A-D). In 100-cell and
older mrg-1 embryos, as in wild type, H5 staining is detected in the
nuclei of Z2 and Z3 (Fig.
5E-H). Thus, MRG-1 is not required for the appearance of
elongating Pol II in PGCs, suggesting that transcriptional turn-on occurs, at
least for some genes.
To assess whether MRG-1 is needed for activation of germline-expressed
genes, we examined the accumulation in L1s of several transcripts whose
zygotic synthesis is known to commence during embryogenesis in Z2 and Z3. The
pgl-1 and glh-1 genes encode protein components of
germline-specific P granules (Kawasaki et
al., 1998; Gruidl et al.,
1996). nos-1 encodes a Nanos homolog that participates in
regulation of PGC development (Subramaniam
and Seydoux, 1999). For all three genes, transcript levels drop to
undetectable in early-mid stages of embryogenesis and then increase
specifically in Z2 and Z3 in late-stage embryos
(Kawasaki et al., 2004;
Subramaniam and Seydoux, 1999)
(Y. Kohara, personal communication). Transcript levels were measured in
wild-type L1s and mrg-1 M-Z-L1s by RT-PCR. As shown in
Fig. 6A, although the
expression levels varied somewhat, all three transcripts accumulated to
wild-type levels in mrg-1 mutant L1s.
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). We
mated pgl-1 mutant hermaphrodites (lacking maternal pgl-1
mRNA and protein) with pgl-1(+) males bearing a GFP transgene, and
determined whether GFP+ outcross L1s (pgl-1/+) could express the
paternal pgl-1(+) allele. When pgl-1 mothers were treated
with mrg-1 dsRNA, PGL-1 was observed in nine of 22 L1s analyzed
(Fig. 6C); all L1s that were
allowed to grow up developed into sterile adults, confirming the effectiveness
of mrg-1 RNAi. Thus, at least some mrg-1(RNAi) larvae
activated zygotic expression of pgl-1. The absence of detectable
PGL-1 in the remaining mrg-1(RNAi) L1s may reflect impairment in some
larvae of transcription activation or of posttranscriptional accumulation of
PGL-1 protein. Appearance of newly synthesized GLH-1 and GLH-4 could be
assessed more directly. In wild type, the maternal load of both GLHs and (of
glh-1 mRNA) drops to undetectable during embryogenesis (Z.L., T.T.
and S.S., unpublished; Y. Kohara, personal communication). Synthesis from
newly transcribed mRNA is presumed to be responsible for the restored levels
of both GLHs in newly hatched L1s. mrg-1 M-Z-embryos and larvae
showed the same pattern of GLH-1 and GLH-4 staining as wild type,
disappearance in mid-late embryos and reappearance in L1s
(Fig. 6E,F and data not shown),
revealing that MRG-1 is not required for turn-on of these two glh
genes. These protein-staining results are consistent with the mRNA
quantification results in mrg-1 mutants.
The above results demonstrate that mrg-1 mutant PGCs resemble
wild-type PGCs in their acquisition of transcriptional competence in
100-cell embryos and their ability to activate expression of at least
some germline genes in late embryos and early larvae. Thus, MRG-1 does not
appear to be required for correct specification of PGC identity or for
initiation of the germline program of gene expression.
MRG-1 is required for somatic cells to misexpress several germline genes in mep-1 mutant larvae
As MRG-1 does not appear to be required to activate expression of germline
genes in the PGCs, we investigated its involvement in the aberrant activation
of germline genes in the somatic cells of synMuv B mutants. A subset of synMuv
B mutants (e.g. mep-1, lin-35/Rb) display ectopic expression of
pgl-1 in the somatic cells of young larvae
(Unhavaithaya et al., 2002;
Wang et al., 2005;
Andersen et al., 2006).
Strikingly, simultaneous loss of MES-4 function suppresses this `ectopic PGL-1
in the soma' phenotype, and also suppresses the larval lethality of
mep-1 (Unhavaithaya et al.,
2002; Wang et al.,
2005). We tested whether loss of MRG-1 also suppresses these
dramatic Mep-1 mutant phenotypes. RNAi of mrg-1 suppressed the
ectopic expression of PGL-1 protein and the overexpression of pgl-1
RNA in mep-1(RNAi) larvae (Fig.
7A-C,G), as recently observed by Cui et al.
(Cui et al., 2006). Analysis
of two other germline genes yielded similar results - depletion of MRG-1
reduced the ectopic expression of GLH-1
(Fig. 7D-F) and the
overexpression of glh-1 and nos-1 transcripts
(Fig. 7G) caused by MEP-1 loss.
mRNA levels for myo-2, a gene normally expressed in somatic cells,
were not increased by mep-1 RNAi or decreased by simultaneous RNAi of
mep-1 and mrg-1 (Fig.
7G). Thus, MRG-1(+) function is required for aberrant expression
of germline genes in mep-1 larvae.
MRG-1(+) function is also required for the larval arrest phenotype of
mep-1. Only 1.6% of the progeny from mep-1(RNAi) mothers
grew to adult worms, whereas 100% of the progeny from mep-1(RNAi);
mrg-1(RNAi) mothers grew to adult, albeit sterile, worms. Our findings
and similar findings of Cui et al. (Cui et
al., 2006) show that loss of MRG-1 suppresses multiple
mep-1 mutant defects, including aberrant patterns of gene expression
and larval arrest.
MRG-1 is required for silencing of transgenes and X-linked genes in the maternal germ line
The mes mutants display gene expression defects in the germ line
of fertile M+Z-mothers (Kelly and Fire,
1998; Bender et al.,
2006). The similar phenotypes displayed by mrg-1 and
mes mutants prompted us to test whether gene expression defects in
the mother's (M+Z-) germ line might contribute to the Mrg-1 maternal-effect
sterile phenotype. As one test, we examined gene expression from an
extrachromosomal array containing many copies of a GFP-tagged ubiquitously
expressed gene, let-858. In wild-type germ lines, such arrays are
silenced (Kelly et al., 1997)
(Fig. 8A); in fertile
mes-4 and mes-3 M+Z-germ lines, they are desilenced
(Kelly and Fire, 1998). We
observed desilencing also in the germ lines of fertile mrg-1
M+Z-mutants (Fig. 8C). Cui et
al. (Cui et al., 2006)
observed similar transgene desilencing after RNAi depletion of mrg-1.
These results demonstrate that MRG-1 is required for transgene silencing in
the germ line.
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). RT-PCR analysis confirmed the microarray results for six
upregulated X-linked genes, three upregulated autosomal genes and five
unaffected genes (Bender et al.,
2006). We dissected 50 gonads from mrg-1(qa6200) M+Z- and
from wild-type adult hermaphrodites and used RT-PCR to examine the levels of
nine of the mRNAs examined by RT-PCR in mes-4 mutant germ lines. In
two independent experiments (Fig.
8E), five X-linked genes upregulated in mes-4 were also
upregulated at least twofold in mrg-1 compared with wild type; four
genes unaffected in mes-4 were also unaffected (less than twofold
difference) in mrg-1 compared to wild type. These and the transgene
results suggest that MRG-1 cooperates with the MES system to achieve
transcriptional repression, and that the X chromosome is a target for
repression.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The causes of sterility in mrg-1 M-Z-hermaphrodites appear to be
failure of PGCs to proliferate combined with degeneration of the few germ
cells present. Among other characterized mutants that display maternal-effect
`tiny germline' phenotypes, mrg-1 most closely resembles the
mes mutants, mes-2, mes-3, mes-4 and mes-6
(Capowski et al., 1991;
Paulsen et al., 1995). This
resemblance extends to the differential sensitivity of XX and XO animals to
mrg-1 and mes mutations
(Garvin et al., 1998).
MRG-1 associates with autosomes and does so independently of autosomal MES-4
MRG-1 joins MES-4 in showing the unique property of associating selectively
with autosomes (Bender et al.,
2006). This along with the similar maternal-effect sterile
phenotypes of mrg-1 and mes-4 mutants suggested that they
might function together. Based on the finding that Saccharomyces
cerevisiae Set2-catalyzed methylation of H3K36 is required for
association of the MRG-1 homolog Eaf3 with nucleosomes
(Keogh et al., 2005), we
predicted that MES-4-catalyzed methylation of H3K36 would be required for
MRG-1 association with chromosomes. This requirement was not observed, arguing
against a model in which MRG-1 associates with MES-4-deposited methyl marks
and serves as a downstream effector of MES-4. Other tests of MES-MRG
relationships gave similar negative results: MES-2 function is not required
for the chromosomal association of MRG-1, and MRG-1 is not required for the
chromosomal association of MES-4 or for MES-mediated methylation of H3K36 or
H3K27. It remains possible that MES-4 functions redundantly with other
chromatin regulators to recruit MRG-1 to autosomes and/or that MES-mediated
methylation regulates the activity of MRG-1.
MRG-1 and activation of expression of germline genes
PGCs in mrg-1 mutants appeared normal in their acquisition of the
elongating form of RNA Pol II at the 100-cell stage of embryogenesis and
in their ability to turn on expression of the germline genes pgl-1, glh-1,
glh-4 and nos-1 in late embryos and L1s. We conclude that
mrg-1 PGCs have germline identity and are competent to express at
least some genes characteristic of the early germline program.
Important insights into possible roles for MRG-1 have emerged from genetic
interactions between mrg-1 and mep-1. MEP-1, known to exist
in a complex with the NuRD subunits LET-418 and HDA-1, is required to prevent
expression of germ cell traits in somatic cells; in larvae depleted of MEP-1
or LET-418, somatic cells express germline genes such as pgl-1
(Unhavaithaya et al., 2002).
We and Cui et al. (Cui et al.,
2006) have found that loss of MRG-1 suppresses the ectopic
expression of germline genes in mep-1 mutant larvae and suppresses
mep-1 larval arrest. Loss of MES function also suppresses these
mep-1 phenotypes (Unhavaithaya et
al., 2002). These findings lead to a model in which the MES
proteins and MRG-1 confer germline competence on all cells; in somatic cells
the NuRD complex antagonizes the functions of the MES proteins and MRG-1 to
protect somatic cells from expressing germline traits
(Unhavaithaya et al., 2002;
Shin and Mello, 2003;
Strome, 2005) (this study). It
is interesting that MRG-1 is needed for mep-1 somatic cells, but not
for wild-type PGCs, to express germline genes. One possibility is that a
single MRG-1-requiring mechanism causes mep-1 somatic cells to
inappropriately express germline genes, but that redundant mechanisms ensure
the germline fate of PGCs and launch their gene expression program. Another
possibility is that MRG-1 has different functions in somatic cells versus
PGCs. It has been reported that the MRG-1 homologs yeast Eaf3 and human MRG15
exist in both histone acetyltransferase (HAT)-containing and histone
deacetylase (HDAC)-containing complexes
(Gavin et al., 2002;
Doyon et al., 2004). By
analogy, MRG-1 may exist in different complexes and regulate different target
genes in somatic versus germline cells.
MRG-1 and silencing transgenes and the X chromosomes
Tests for whether gene-silencing mechanisms are operating properly in early
PGCs have not been developed. Consequently, to investigate if mrg-1
mutants are defective in silencing genes in the germ line, we analyzed gene
expression in fertile mrg-1 M+Z-mothers, as done previously for
fertile mes M+Z-mothers (Kelly
and Fire, 1998; Bender et al.,
2006). Known targets of silencing in the germ line are transgenes
present in repetitive extrachromosomal arrays
(Kelly et al., 1997), and the
X chromosomes (Kelly et al.,
2002; Fong et al.,
2002; Bender et al.,
2006). Evidence for X silencing comes from microarray analysis
comparing mRNA accumulation in germline-containing versus germline-lacking
hermaphrodites (Reinke et al.,
2000; Reinke et al.,
2004) and from staining germline chromosomes with antibodies that
recognize marks of actively expressed chromatin
(Kelly et al., 2002;
Fong et al., 2002). The X
chromosomes lack marks of active chromatin, and X-linked genes are
significantly under-represented in the germline mRNA pool.
We observed that mrg-1 M+Z-germ lines are defective in silencing
both types of targets. Similar to mes-3 and mes-4 M+Z-germ
lines (Kelly and Fire, 1998),
mrg-1 M+Z-germ lines display expression of a repetitive GFP
transgene. Similar to mes-4 M+Z-germ lines
(Bender et al., 2006),
mrg-1 M+Z-germ lines upregulate at least some genes on the X
chromosome. We specifically sampled X-linked genes known to be upregulated in
mes-4 mutant germ lines; all five of those genes were also
upregulated in mrg-1 mutant germ lines. Importantly, four autosomal
genes that were not upregulated in mes-4 were also not upregulated in
mrg-1. Future microarray analysis will reveal whether the profile of
up- and downregulated genes in mrg-1 overlaps extensively or not with
the profile in mes-4.
How can autosomally concentrated MRG-1 participate in repressing genes on
the X chromosomes? Two models were proposed for MES-4
(Bender et al., 2006). One
model invokes that MES-4 activates expression of an autosomal gene that
encodes a repressor of the Xs and repetitive arrays. The other model proposes
that autosomally concentrated MES-4 or its H3K36me mark repels a repressor,
and focuses its action on the Xs and repetitive arrays. Similar models can be
proposed for MRG-1. In fact, MRG-1 and MES-4 may operate together, even though
they do not display dependence on one another for recruitment to chromosomes.
We speculate that desilencing of genes on the X may underlie the PGC defects
and death observed in mrg-1 M-Z-worms. Desilencing of genes on the X
may also contribute to suppression of mep-1 mutant phenotypes in
somatic cells (P. Raghavan and T. H. Shin, personal communication).
Elucidating the gene targets of MRG-1 in PGCs and in somatic cells are
important future directions.
The biochemical activities of MRG-1 are currently unknown. The dosage
compensation complex (DCC) in Drosophila, the NuA4 complex in S.
cerevisiae and the NuA4-related Tip60 complex in humans all contain a
MRG-1 homolog (fly MSL3, yeast Eaf3 and human MRG15) and also a HAT subunit
(Smith et al., 2000;
Eisen et al., 2001;
Akhtar, 2003;
Cai et al., 2003). We speculate
that MRG-1 may exist in a complex similar to the DCC and NuA4 HAT complexes
and may regulate chromatin organization and gene expression through regulation
of histone acetylation. Consistent with this, MRG-1 interacts with ZK1127.3
(Li et al., 2004), the
predicted C. elegans homolog of the yeast NuA4 subunit Eaf7, and
recent studies have revealed that RHA-1, a C. elegans homolog of the
Drosophila DCC component MLE, is required for germline gene silencing
(Walstrom et al., 2005), as is
MRG-1. Either of the two models proposed above for how MRG-1 participates in
repressing genes on the X could involve histone acetylation by autosomally
concentrated MRG-1-containing HAT complexes.
Temporal requirement for MRG-1 in PGC development
We hypothesize that a chromatin state induced by MRG-1 in the maternal germ
line is crucial for the normal early program of PGC development and to ensure
germline immortality. Relevant to this, we noticed that in mrg-1 M-Z+
worms, some germ lines showed normal proliferation, whereas others showed
severe underproliferation. This `all or nothing' effect suggests that MRG-1
participates in some stochastic event or decision, and that the outcome is
normal proliferation and fertility or underproliferation and sterility. An
attractive model is that MRG-1 acts epigenetically to induce a `germline'
chromatin state that is passed from generation to generation of germ cells.
Normally, that state must be inherited from the maternal germ line, but at low
frequency it can be induced de novo (i.e. in mrg-1 M-Z+ worms).
Resolving the temporal requirement for MRG-1 function will shed light on the
important issue of how germline properties are maintained in perpetuity.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/4/757/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Department of Pediatrics, Kobe University Graduate School
of Medicine, 7-5-1 Kusunokicho, Chuo-ku, Kobe, 650-0017 Japan
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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