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First published online January 25, 2006
doi: 10.1242/10.1242/dev.02235
1 Eidgenoessische Technische Hochschule Zuerich, Institute of Cell Biology,
CH-8093 Zuerich, Switzerland.
2 University of Zuerich, Institute of Molecular Biology, CH-8057 Zuerich,
Switzerland.
3 Eidgenoessische Technische Hochschule Zuerich, Institute of Biochemistry,
CH-8093 Zuerich, Switzerland.
Authors for correspondence (e-mail:
monica.gotta{at}bc.biol.ethz.ch;
wilhelm.krek{at}cell.biol.ethz.ch)
Accepted 5 December 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: DNA damage, Germline, Prefoldin, Proliferation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-class PFD-1), PFD2
and PFD4; the core subunit of RNA polymerase II, RPB5; and the ATPases TIP48
and TIP49, which are components of various chromatin-remodeling complexes.
Genetic and biochemical studies in human and in yeast cells have demonstrated
that URI is phosphorylated in a TOR-dependent manner and is required for
nutrient-sensitive TOR-dependent transcriptional programs
(Gstaiger et al., 2003
). In
contrast to other known PFD family proteins, URI contains additional conserved
protein domains and is more than double the size of other PFDs
(Cowan and Lewis, 1999;
Geissler et al., 1998;
Vainberg et al., 1998),
indicating that URI might have multiple functions. More recently, URI has been
shown to interact with the tumor suppressor protein parafibromin, a component
of the PAF1 complex [which is involved in histone methylation and cell cycle
control (Yart et al.,
2005)].
Cellular proliferation requires the accurate replication of DNA to ensure
the viability of cells and the survival of the species. Different types of DNA
damage, such as collapsed replication forks (for a review, see
Lambert and Carr, 2005) and
chromatin-based defects (for reviews, see
Ehrenhofer-Murray, 2004;
Koundrioukoff et al., 2004),
accumulate constitutively during this process and therefore cells have several
mechanisms to ensure that the damage is rapidly recognized and repaired to
maintain genomic integrity. Repair processes are particularly important in
germline cells where any damage can be transmitted to the progeny.
The C. elegans germline has been used extensively to dissect the
signaling pathways that regulate DNA damage responses. The two germline
progenitor cells that are present in newly hatched larvae, will eventually
give rise to 
1000 germ cells per gonad arm in the adult hermaphrodite
(Riddle, 1997). Germ cell
apoptosis and transient mitotic cell cycle arrest are often triggered upon
genotoxic stresses such as DNA damage
(Ahmed et al., 2001;
Gartner et al., 2000) to
prevent the propagation of gametes with damaged genomes. DNA damage triggers
cell cycle arrest in the mitotic part of the germline, a response that is
abrogated in mutants of the DNA damage sensors HUS-1, MRT-2 and RAD-5
(reviewed by Stergiou and Hengartner,
2004). ATM-1 and ATL-1, the homologues of the ATM and ATR kinases,
which have been shown to trigger cell cycle arrest upon DNA damage in all
eukaryotic cells studied so far, have also been suggested to be key components
of the DNA damage signaling in C. elegans
(Boulton et al., 2002). In the
meiotic part of the germline, DNA damage induces p53/CEP-1-mediated apoptosis,
which involves the core apoptotic machinery [the anti-apoptotic Bcl2-like
protein CED-9, the Apaf1-like adaptor protein CED-4 and the pro-caspase CED-3
(reviewed by Stergiou and Hengartner,
2004)].
In this report, we describe a novel role for URI-1 in the maintenance of DNA stability in the absence of exogenous DNA damage. We show that loss of uri-1 results in aberrant DNA damage in the C. elegans germline. The increased DNA breaks that are induced due to the loss of uri-1 trigger a HUS-1 response, which is associated with cell cycle arrest in the mitotic germline and p53/CEP-1-dependent apoptosis in the meiotic germline.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Bristol strain N2 was used as the standard wild-type
strain. The following strains were also used: uri-1(tm939), uri-1(tm939)
dpy-5(e61)/dpy-5(e61) unc-14(e57), dpy-5(e61) unc-14(e57), ced-3(n1286),
ced-9(n1950), gld-2(q497) gld-1(q485); unc-32(e189)
(Kadyk and Kimble, 1998),
hus-1(op241); unc-119(ed3); opIs34
(Hofmann et al., 2002),
glp-4(bn2), fem-1(hc17) (Nelson et
al., 1978), fem-3(q20)
(Barton et al., 1987),
glp-1(q231), atm-1(gk186), spo-11(ok79)/nT1[unc-?(n754)let-?],
hus-1(op241), and cep-1(gk138). The uri-1(tm939) mutant
allele was isolated and kindly provided by the National Bioresource
Project-Japan. Primers used for PCR screening of the tm939 allele
were 5'-CGCGGATCCATCATGAGCGAACTCTACGTTGC-3' and
5'-CCGCTCGAGCGGTCAATTTCTATGCCTGGAAGC-3'. As the homozygous mutant
exhibits a sterile phenotype and/or embryonic lethality, it was marked with
the recessive dpy-5 mutation and cultivated as heterozygous
uri-1(tm939) dpy-5(e61)/dpy-5(e61) unc-14(e57).
Characterization of brood size
L4 hermaphrodites of the desired genotype were individually cloned onto
agar plates and cultured at 25°C. The hermaphrodites were transferred to
fresh plates every 24 hours. The brood size of each animal is the sum of
non-hatched and hatched progeny.
RNAi analysis
Double-stranded RNA was applied to worms by feeding or injection
(Timmons et al., 2001). A
L4440 plasmid that contains a fragment of the C55B7.5 gene was used
(Fraser et al., 2000). The
bacteria were seeded on NGM agar plates containing 6 mM IPTG and 5 µg/ml
carbencillin. Worms were added as L1 larvae on the following day and raised at
25°C. Temperature-sensitive strains were raised at 15°C in P0
generation and shifted to 25°C as L1 larvae. Control animals were fed with
bacteria carrying an empty L4440 construct. Phenotypes were observed in the P0
and F1 progeny of worms that were fed with the respective dsRNA at the
indicated time points.
Northern blot analysis
To prepare RNA, the fem-1(hc17), fem-3(q20), glp-4(bn2) and
wild-type strains were grown at 15°C, synchronized at the L1 larval stage
and afterwards raised at 25°C, the restrictive temperature for the
above-mentioned temperature-sensitive mutants. Under these conditions
glp-4(bn2) (Beanan and Strome,
1992) contains almost no germ cells (12) and is considered a
germline-free animal, fem-3(q20) animals produce sperm but no oocytes
(Barton et al., 1987) and
fem-1(hc17) animals generate oocytes but no sperm
(Nelson et al., 1978). One day
after reaching the L4 larval stage total mRNA was prepared using the TRIZOL
(GIBCO) method (Hope, 1999).
Total RNA was separated in formaldehyde-agarose gels by electrophoresis,
transferred to a Hybond-N+ membrane and hybridized in ExpressHyb
Hybridization Solution (BD Biosciences) according to manufacturer's protocol
with a full length 32P-labeled uri-1 cDNA probe (using the
Prime-It II Random Primer Labeling Kit; Stratagene). The amount of total mRNA
was normalized using rRNA as standard.
Germline apoptosis and germ cell counts
Apoptotic germ cell corpses were counted by morphology under DIC optics as
previously described (Lettre et al.,
2004).
Synchronized wild-type L1 larvae were fed on uri-1(RNAi) and the vector control L4440(RNAi) plates at 25°C until adulthood. The worms where then transferred every 2 hours to new plates and the F1 generation (also fed on RNAi) was collected and methanol fixed (-20°C for at least 10 minutes) at the indicated time points. Worms were washed twice in PBS, and they were suspended in PBS including DAPI at 0.1 µg/ml for 30 minutes at room temperature. After two washes with PBS 3 µl Vectashield was added to the worms and samples were mounted on slides. Germ cells that were identified by nuclear morphology according to DAPI staining were counted. The different developmental stages of the worms were determined by vulval morphology and differentiation of somatic gonads.
Immunostaining
For the anti-phospho-histone H3 (PH3) staining, one day post-L4 adult
gonads were dissected in PBST (0.2% Triton-X) on POLYSINE slide, fixed for 4
minutes in 1% formaldehyde in PBST and freeze-cracked. The slides were
transferred to -20°C cold methanol for 6 minutes and washed three times in
PBS each time for 5 minutes. They were blocked for 30 minutes in 3% BSA in
PBST at 37°C and incubated overnight at 4°C with the rabbit polyclonal
anti-PH3 antibody (1:500 in 3% BSA in PBST, Upstate). The next day the gonads
were washed 3 times in PBST each for 10 minutes at RT and incubated for 2
hours with the secondary antibody (anti-rabbit cy3, 1:200) in PBST at RT.
Gonads where washed three times in PBST each for 10 minutes (0.5 µg/ml DAPI
was added in the first wash) and mounted with 3 µl Vectashield per sample
for further analysis.
For the anti-RAD-51 staining one day post-L4 adult gonads were dissected in PBS on POLYSINE slide and freeze-cracked. The slides were transferred to -20°C cold methanol, methanol:acetone (1:1) and acetone each for 5 minutes and washed three times in PBS for 5 minutes each. They were blocked for 30 minutes in 3% BSA in PBST (0.05% Triton-X) at 37°C and incubated overnight at 4°C with the rabbit polyclonal anti-RAD-51 antibody (1:1000 in 3% BSA in PBST). The next day, the gonads were washed three times in PBST each for 5 minutes at room temperature and incubated with the secondary antibody as described above. Gonads were washed three times in PBST each for 10 minutes (0.5 µg/ml DAPI was added in the first wash) and mounted with 3 µl Vectashield per sample for further analysis.
DNA breakage detection methods
For the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick
end-labeling) assay, which detects DNA strand breaks (nicks) and DNA
fragmentation (staggered DNA ends), 1 day post-L4 gonads were dissected in
PBS, transferred to a 96-well plate and fixed in 4% formaldehyde in PBS at
room temperature for 20 minutes. Gonads were rinsed in PTX (PBS, 0.4% Triton
X-100) three times and incubated in 100 mM sodium-citrate, 0.1% Triton X-100
at 65°C for 20 minutes, followed by two washes in PTX. Gonads were then
incubated for 30 minutes at room temperature in 0.1 M Tris/HCl (pH 7.5)
containing 3% BSA and 20% normal bovine serum. The gonads were rinsed twice
with PBS at room temperature and excess fluid was drained off. Gonads were
afterwards incubated in TUNEL reaction mixture (Roche) at 37°C for 1.5
hours. The reaction was stopped by washing the gonads three times in PTX. DAPI
(1 µg/ml) was added into each well for 5 minutes within the second washing
step. Vectashield (3 µl) was added to each sample and gonads were mounted
on to a cover slip for further analyses. The HUS-1::GFP foci and their nuclear
re-localization following DNA damage were scored as previously described
(Hofmann et al., 2002).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
(Fig. 1A). We obtained a
uri-1(tm939) deletion mutant that lacks the second -helix of
the PFD domain and the entire RPB5-binding region. Furthermore, this deletion
places the remaining 3' coding region out of frame
(Fig. 1A). Phenotypical analysis revealed that loss of uri-1 function causes multiple and variable somatic defects such as protruding vulva (8%±3, n=200), rupture (23%±21, n=200), embryonic lethality (8%±3, n=200), path-finding defects (12%±8, n=73), molting problems (8%±4, n=200) and L3 larval arrest (46%±6, n=41). However, the most penetrant phenotype that is observed upon loss of uri-1 function is sterility. In this report we characterize the germline defects observed in uri-1(lf) mutant and uri-1(RNAi) worms.
The homozygous uri-1(lf) mutants and the progeny of worms fed with uri-1 dsRNA [referred to as uri-1(RNAi)F1] develop into sterile adults (Table 1), indicating that URI-1 is essential for fertility. Both the uri-1(lf) mutants and the uri-1(RNAi)F1 develop a small germline because of a severe reduction in germ cell number (Fig. 1B and Table 1). This dramatic reduction in germ cell number is not due to increased apoptosis as depletion of uri-1 in an apoptosis-deficient strain also results in a small germline (see Table S2 in the supplementary material). Male uri-1(RNAi)F1 animals also suffer from a reduction of germ cells (data not shown), indicating that the function of URI-1 is sex independent.
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In wild-type hermaphrodites, the first germ cells that enter the meiotic
cycle in each gonad arm (40) differentiate as primary spermatocytes, then
secondary spermatocytes, spermatids and finally form activated spermatozoa
(160). Thereafter, a switch in sexual fate occurs so that all germ cells
develop as oocytes (Riddle et al.,
1997). A complete lack of oocytes is observed in
uri-1(lf) mutants and this phenotype is fully penetrant. About 35%
(n=30) of uri-1-depleted germlines do not show any sign of
gametogenesis and when gametogenesis occurs it leads to the development of
sperm but no oocytes in the corresponding gonads. However, the spermatocytes
do not always complete the meiotic divisions, because both spermatocytes
(Fig. 1B, arrowheads) and
mature sperm (Fig. 1B, arrows)
were observed in the gonads of adult hermaphrodites. Moreover, the amount of
sperm in the adult germlines of homozygous uri-1(lf) mutants is
reduced (13±10, n=10) compared with wild type (160).
The adults arising from feeding L1 larvae with uri-1(RNAi) (referred to as (uri-1(RNAi)P0) display a reduced brood size compared with wild type. Moreover, the germ cell number of these animals is reduced to 73% of the wild-type number (Table 1). Heterozygous uri-1 hermaphrodites were also subfertile with significantly decreased brood size (Table 1). This shows that URI-1 is haplo-insufficient in ensuring normal germ cell number and brood size. Interestingly, wild-type worms arising from an uri-1+/- heterozygous mother give rise to approximately half of the brood size of wild-type worms from a wild-type mother, indicating that zygotically provided URI-1 is insufficient for its role in germline cells and that uri-1 has to be maternally supplied to ensure normal brood size.
The observation that homozygous uri-1(lf) mutants have defects in oogenesis and spermatogenesis, but the heterozygous worms appear normal, indicates that these processes are less sensitive to the loss of uri-1 levels than are brood size and overall germ cell number. uri-1(RNAi)F1 animals contain an average of 89 germ cells (Table 1). However, the most severely affected animals of the uri-1(RNAi)F1 contain only few germ cells (around 18) without any visible gametes, most probably reflecting a stronger loss of URI-1 function.
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URI-1 is required for proliferation of germ cells
As the uri-1 mutant germlines are smaller because of a severe
reduction in germ cell number, we wondered whether URI-1 might have a role in
germ cell proliferation. As a hermaphrodite develops from the L1 larval stage
to adulthood, the number of somatic cell nuclei roughly doubles (556 to 1090)
and the number of germ cells increases from 2 to 2000 germ cells per gonad
(Riddle et al., 1997).
Germline cells located at the distal end of the gonad arm constitute a
proliferating stem cell population. During the larval development of C.
elegans, the number of germ cells gradually increases, up to the L3
larval stage, when a transition occurs to a period of rapid proliferation
(Capowski et al., 1991).
Therefore, germline development requires extensive proliferation of cells and
URI-1 might be required for germline development because of a role in germ
cell proliferation.
A time course analysis of germline development in wild-type worms compared with uri-1(RNAi)F1 worms revealed that the rapid increase in germ cell number at the end of the L3 larval stage does not occur in the uri-1(RNAi)F1 animals (Fig. 2A). This defect led to a significant difference in germ cell number between uri-1-depleted and wild-type worms.
To confirm that uri-1 is required for germ cell proliferation, we
performed uri-1(RNAi)F1 in gld-2(q497) gld-1(q485)double
mutants, which have an over-proliferative germline that contains mainly
mitotic cells, owing to meiotic entry defects
(Hansen et al., 2004;
Kadyk and Kimble, 1998). Loss
of uri-1 in the gld-2(q497) gld-1(q485)double mutant
suppresses the over-proliferation defect, and gives rise to small germlines
with a dramatically reduced cell number
(Fig. 2B). Therefore, our data
indicate that uri-1 is required for proliferation of mitotic germ
cells.
Cell cycle progression is inhibited in the homozygous uri-1(lf) mutant
To further investigate the potential effect of loss of uri-1
function on cell cycle progression, we examined the nuclear morphology of
germline cells in wild-type, uri-1(lf) and uri-1(RNAi)F1
animals stained with DAPI and with the anti-phospho-histone H3 antibody (PH3),
which labels metaphase and telophase cells in the C. elegans germline
(Hsu et al., 2000;
Wei et al., 1999). The DAPI
staining revealed the enrichment of nuclei that were arrested at two naturally
occurring stages of the cell cycle. First, some nuclei have condensed
chromosomes that are not yet aligned to the metaphase plate and are not
labeled by the anti-PH3 antibody (Fig.
3A, see arrowheads). This morphology has been described as nuclei
in the G2/M phase and could represent G2, prophase or prometaphase nuclei. A
similar morphology has been observed in the germline of
hoe-1(RNAi)-treated animals and has been proposed to represent
prometaphases (Smith and Levitan,
2004). Second, uri-1-depleted worms also show an
enrichment of nuclei that display a metaphase-like morphology and are labeled
by the anti-PH3 antibody (Fig.
3B). These nuclei might have escaped the prometaphase-like block
and progressed to the next phase of the cell cycle where they finally arrest.
This type of cell cycle arrest has been observed in the gonads of the progeny
of animals deficient for both the DNA helicase RecQ and topoisomerase
III (Kim et al., 2002),
which exhibit extensive DNA breakage.
As mentioned above, some germlines are less affected than others after
reduction of the URI-1 function. In germlines that contain gametes, we observe
spermatids that are also stained with the PH3 antibody, indicating an
additional cell cycle block at the M-phase of spermatocyte meiosis I
(Fig. 3C)
(Golden et al., 2000). This
phenotype is unlikely to reflect a role for URI-1 in spermatogenesis, because
RNA-mediated inactivation of uri-1 in the glp-1(q231) mutant
[in which germ cells enter meiosis early and consequently proliferate far less
than in wild-type worms (Austin and Kimble,
1987)] suppresses the URI-1 phenotype, and gives rise to the
glp-1 phenotype without any visible defects in spermatogenesis (see
Fig. S1 and Table S1 in the supplementary material). We therefore propose that
the defect in spermatogenesis in the URI-1-deficient germlines is a
consequence of the cell proliferation defect rather than a direct effect of
URI-1 on spermatogenesis.
So far, our data show that loss of URI-1 activity leads to cell cycle blocks that occur in both mitotic and meiotic cells. The suppression of the uri-1(RNAi)F1 sperm phenotype by glp-1(lf) and the proliferation defect of uri-1(RNAi)F1 germlines in the gld-2(lf)gld-1(lf) double mutants indicates an important function of URI-1 in mitotic cells.
Loss of uri-1 function results in increased DNA breaks
Block at the G2/M border is often due to activation of the DNA
checkpoint machinery by damaged or incompletely replicated DNA (for a review,
see Hartwell and Weinert,
1989). Because we observed a mitotic cell cycle block in the
germline of the homozygous uri-1 mutants, we used three independent
assays to investigate whether this block is triggered by damaged DNA.
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Second, we stained uri-1 depleted worms with antibodies to RAD-51
which has been shown to bind double strand breaks (DSBs)
(Alpi et al., 2003;
Reddy and Villeneuve, 2004).
Anti-RAD-51 staining detected an increased number of DNA breaks in
uri-1(RNAi)F1 germlines compared with wild type, indicating that
uri-1 depletion results in increased formation of DSBs. It has been
demonstrated that the C. elegans type II topoisomerase SPO-11 is
required to generate meiotic DSBs
(Dernburg et al., 1998). We
therefore wanted to determine if induction of DSBs requires SPO-11 function.
As shown in Table 2, loss of
uri-1 in this mutant background results in an enhancement of DSBs,
indicating that a subset of uri-1(lf)-induced DSBs are SPO-11
independent.
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). To
test whether the uri-1(lf)-induced DNA damage could be detected by
the checkpoint machinery, we performed uri-1(RNAi) in the HUS-1::GFP
strain. uri-1(RNAi) resulted in the formation of nuclear HUS-1::GFP
foci in the germline (Fig. 4B),
consistent with the results obtained with the TUNEL assay and the anti-RAD-51
staining. Our results clearly show that in the absence of exogenous DNA damage stimuli, loss of URI-1 function results in the formation of DNA breaks. This defect results in the activation of the cellular DNA damage sensing machinery in the germline, as indicated by the formation of HUS-1 foci.
HUS-1 and ATL-1 are required for the cell cycle arrest caused by uri-1 depletion
Studies in yeast and human cells characterized Hus1 as a sensor of DNA
damage that functions in concert with the checkpoint control proteins ATM and
ATR to regulate the G2/M transition (for a review, see
Helt et al., 2005). As DSBs in
the germline are known to activate the cell cycle checkpoint machinery, we
investigated if this DNA damage signaling pathway is activated in response to
uri-1 depletion leading to the mitotic cell cycle arrest described
above. To address this issue, we depleted uri-1 in the
hus-1(lf) mutant strain and analyzed the DAPI stained germlines of
hus-1(op241); uri-1(RNAi)F1 animals. As shown in
Fig. 5A, inactivation of the
hus-1 cell cycle checkpoint gene prevents
uri-1(RNAi)-induced enrichment of germ cell nuclei blocked at the
G2/M transition. Interestingly, although depletion of the
atm-1 gene had no effect (data not shown), depletion of
atl-1, the C. elegans homolog of ATR, in the
apoptosis-deficient ced-3 strain also resulted in rescue of the
uri-1-induced cell cycle arrest, phenocopying the rescue observed in
the hus-1;uri-1(RNAi)F1 germline
(Fig. 5B). The observation that
inactivation of the checkpoint genes atl-1 and hus-1 rescues
the cell cycle arrest observed in uri-1-depleted worms indicates that
this phenotype is mediated by the hus-1/atl-1 checkpoints of the
C. elegans DNA damage signaling pathway. However, the decrease in
germ cell number (see Fig. 5)
and the sperm defect observed in uri-1-depleted germlines (data not
shown) are not suppressed by loss of hus-1 or atl-1,
indicating that loss of URI-1 induces cell proliferation defects that are both
hus-1/atl-1 dependent and independent.
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), but
dispensable for physiological germ cell death or somatic cell death
(Schumacher et al., 2001). The
gla phenotype of uri-1(RNAi)P0 is completely suppressed in
the cep-1(lf) background (Fig.
6B). This indicates that the gla phenotype in
uri-1 animals is due to DNA damage-induced activation of CEP-1.
Therefore, reduction of the uri-1 gene sensitizes meiotic germ cells
to cep-1-mediated DNA damage-induced germ cell apoptosis in the
absence of exogenous DNA damage. |
DISCUSSION |
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-irradiation
(Hofmann et al., 2002). This
highlights the severity of the endogenous damage caused by the loss of URI-1
function. The increased numbers of HUS-1::GFP foci formation, the inhibition
of mitotic cell cycle progression and the p53-dependent apoptosis indicate
that sensing, transduction and execution of DNA damage signals are functional
in URI-1 depleted worms. We propose that URI-1 is involved in preventing
and/or repairing endogenous, genotoxic DNA damage to maintain genome
integrity.
Endogenous DNA breaks can arise from replication errors, repair
intermediates and alterations in chromatin remodeling (for reviews, see
Abraham, 2001;
Koundrioukoff et al., 2004;
Lindahl and Wood, 1999;
Osborn et al., 2002;
Sancar et al., 2004). Several
links between DNA repair, replication and chromatin remodeling have been
established (Morrison and Shen,
2005; Shen et al.,
2000). For example, PCNA has been shown to be involved in
chromatin remodeling, replication and repair (for a review, see
Majka and Burgers, 2004).
Interestingly, human and C. elegans URI are part of a multi-protein
complex that contains, among other proteins, the ATPases TIP48 and TIP49
(Gstaiger et al., 2003), which
are established components of several chromatin remodeling/modifying
complexes, including the human TIP60 HAT complex
(Frank et al., 2003).
Recently, the C. elegans homologs of TIP48 and TIP49, and URI-1 were
shown to be required for the process of RNA silencing
(Kim et al., 2005). Moreover,
our laboratory showed that URI-1 binds a component of the Paf1 complex in
human cells, which is important for histone modification and cell cycle
control (Yart et al., 2005).
Given these links, it is tempting to speculate that at least one function of
URI-1 is, directly or indirectly, dedicated to chromatin remodelling and/or
replication and repair, and that defects in these processes could possibly
explain the endogenous DNA damage in uri-1-depleted animals.
Interestingly, S. cerevisiae rad27
(Tong et al., 2001) is
synthetically lethal with the S. cerevisiae ortholog of
uri-1 (bud27) (Reagan et
al., 1995). The human ortholog of rad27, the potential
cancer susceptibility gene human flap endonuclease 1 (FEN1)
(Harrington and Lieber, 1994;
Lieber, 1997;
Stillman, 1989), is known to
have a role in the maintenance of genetic stability in eukaryotic genomes (for
reviews, see Henneke et al.,
2003; Kucherlapati et al.,
2002). It is also known to function in DNA repair (BER, NIR, HR
and NHEJ) (Ishchenko et al.,
2003; Klungland and Lindahl,
1997; Lieber,
1997; Tishkoff et al.,
1997; Wu et al.,
1999) and DNA replication, two processes essential for proper
proliferation.
In mammalian and yeast cells, URI has been shown to have a central role in
the regulation of nutrient-sensitive TOR-dependent gene expression programs.
Interestingly, a link between genomic stability and nutrient status has been
previously suggested (Fiorentino and
Crabtree, 1997; Kurz and
Lees-Miller, 2004). One of these reports shows that the yeast dna2
mutant, which displays all of the characteristics of cells blocked at the
G2/M border, can be rescued by the overexpression of the nutrient
sensor Tor1p (Fiorentino and Crabtree,
1997). In addition, dna-2 is a player in cellular
processes like germline development and DNA repair in C. elegans
(Lee et al., 2003) and has
been shown to be involved in DNA replication in yeast
(Kao et al., 2004). Moreover,
a dna-2 mutant in combination with mre-11, a gene that
encodes a protein required for meiotic recombination and DNA repair
(Chin and Villeneuve, 2001),
exhibits a small germline phenotype similar to uri-1(lf)
(Lee et al., 2003). Finally,
mutants of genes implicated in nutrient sensing, starvation and mitochondrial
respiratory chain-deficiency in general display a L3 larval arrest
(Long et al., 2002;
Tsang et al., 2001), similar
to the arrest observed in uri-1(lf) mutants, pointing to the
possibility that the function of URI as a mediator of nutrient signals is
conserved in C. elegans. The transition from L3 to L4, which entails
an increase in mtDNA copy number, has been speculated to involve an
energy-sensing decision or checkpoint
(Tsang et al., 2001) and is
around the developmental time point at which the uri-1(lf)
proliferation defects emerge. All these data indicate a possible link between
nutrient status, DNA metabolism and DNA damage. In this respect, it is
interesting to mention that the size of the germ cell nuclei in
uri-1(RNAi)F1 animals is enlarged (see arrowheads in
Fig. 3A), a phenotype that is
also observed during radiation-induced cell proliferation arrest and results
from growth without proliferation (Gartner
et al., 2000). However, unlike the effects of a single
-irradiation dose, which induces homogeneous increase in size of
nuclei, loss of URI-1 function induces heterogeneous enlargement. The
different extent of enlargement could indicate that individual cells receive
proliferation-blocking stimuli at different times or stages.
In summary, our findings identify a novel role for URI-1 in the maintenance of DNA integrity by affecting directly or indirectly DNA metabolism. As DNA breaks are damaging for cells and bear a mutagenic potential, it will be interesting to test if this novel and essential function of the evolutionary conserved protein URI-1 is conserved in higher organisms. As the mammalian URI-1 ortholog co-exists in a biochemical complex with the human PAF-1 complex, it will be interesting to test if URI-1 maintains DNA stability by affecting the state of chromatin. URI-1 may represents a novel link between the epigenetic regulation of chromatin structure and genomic integrity with implications for human cancer.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/4/621/DC1
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