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First published online 19 September 2007
doi: 10.1242/dev.004606
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Dartmouth Medical School, Department of Genetics, Norris Cotton Cancer Center, 7400 Remsen, Hanover, NH 03755, USA.
* Author for correspondence (e-mail: barbara.conradt{at}dartmouth.edu)
Accepted 1 August 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: C. elegans, Apoptosis, Germ line, lin-35 RB
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Liu et al., 2004;
Stevaux and Dyson, 2002). The
tumor-suppressing activity of Rb has been attributed to its ability
to block cell proliferation. Specifically, the RB protein can bind to
heterodimers composed of a member of the DP protein family and an `activating'
member of the E2F family of sequence-specific DNA-binding proteins (E2F1,
E2F2, E2F3) (E2F-DP); this blocks the ability of E2F-DP to activate the
transcription of genes, the products of which are required for G1-S phase
transition (reviewed by Attwooll et al.,
2004; Dimova and Dyson,
2005). Independently of its role in cell proliferation,
Rb also plays a role in other processes such as differentiation and
apoptosis (reviewed by Chau and Wang,
2003; Lipinski and Jacks,
1999; Liu et al.,
2004; Stevaux and Dyson,
2002).
The inactivation of the apoptotic pathway contributes to tumorigenesis in
many types of cancers (reviewed by Cory
and Adams, 2002; Danial and
Korsmeyer, 2004). In mice lacking a functional Rb gene
(Rb-/-), ectopic apoptosis is observed in the peripheral
and central nervous system as well as the ocular lens, indicating that
Rb can block apoptosis in these tissues. The fact that Rb
has anti-apoptotic activity seems counterintuitive for a tumor suppressor, as
it suggests that the loss of Rb function not only results in
increased proliferation, but also increased apoptosis. Indeed, in many
Rb-deficient tumors, the apoptotic pathway is found to be inactivated
by mutation as well, thus suggesting that the loss of Rb function
induces tumorigenesis through a mechanism that is dependent on the
inactivation of the apoptotic pathway
(Chau and Wang, 2003).
However, in certain tissues, the loss of Rb function appears to be
sufficient for tumorigenesis, which indicates that Rb might not have
anti-apoptotic activity in all tissues and that its role in apoptosis might
therefore be tissue-specific (reviewed by
Sherr and McCormick,
2002).
The genetic analysis of the role of mammalian Rb in apoptosis has
been hampered by the fact that apart from Rb, two Rb-like
genes [p107 (Rbl1) and p130 (Rbl2)] exist.
Furthermore, mammals have at least three Dp-like genes and eight
E2f-like genes (Attwooll et al.,
2004; Christensen et al.,
2005; Dimova and Dyson,
2005; Logan et al.,
2005; Maiti et al.,
2005; Milton et al.,
2006). By contrast, the C. elegans genome encodes one
ortholog of RB, LIN-35, one ortholog of DP, DPL-1, and two E2F-like proteins,
EFL-1 and EFL-2 (Ceol and Horvitz,
2001; Lu and Horvitz,
1998). lin-35, dpl-1 and efl-1 are members of
the class B synthetic multivulval (synMuv) genes, which were originally
identified because of their role in vulval development: synMuv B genes act
redundantly with class A and class C synMuv genes to antagonize
let-60 RAS, mpk-1 MAPK signaling in the vulval precursor
cells (VPCs), thereby blocking vulval differentiation
(Ceol and Horvitz, 2004;
Ferguson and Horvitz, 1989).
The fact that the loss of lin-35, dpl-1 or efl-1 function
causes similar rather than opposite phenotypes suggests that, at least during
vulval differentiation, lin-35, dpl-1 and efl-1 function
together to repress gene transcription.
Animals lacking lin-35 function are viable and overall have a
wild-type appearance, although they are less fertile and show enhanced
sensitivity to the inactivation of gene function by RNA interference
(Boxem and van den Heuvel,
2001; Fay et al.,
2002; Lu and Horvitz,
1998; Thomas and Horvitz,
1999; Wang et al.,
2005). Furthermore, lin-35 functions redundantly in a
number of processes other than vulval differentiation, such as cell cycle
progression, larval development, somatic gonad development, pharynx
differentiation and transgene expression
(Bender et al., 2004;
Boxem and van den Heuvel, 2001;
Cardoso et al., 2005;
Chesney et al., 2006;
Cui et al., 2004;
Fay et al., 2002;
Fay et al., 2003;
Fay et al., 2004;
Hsieh et al., 1999;
Wang et al., 2005). Therefore,
like mammalian Rb, C. elegans lin-35 plays a role in cell
proliferation and differentiation. Whether lin-35 also plays a role
in apoptosis has not previously been investigated.
During C. elegans development, 131 of the 1090 somatic cells that
are formed undergo apoptosis, a process referred to as `developmental
apoptosis' (Sulston and Horvitz,
1977; Sulston et al.,
1983). Developmental apoptosis is determined by the essentially
invariant somatic cell lineage of C. elegans and is executed through
a conserved apoptotic pathway (reviewed by
Horvitz, 2003;
Lettre and Hengartner, 2006).
Specifically, developmental apoptosis is dependent on the pro-apoptotic genes
ced-4 and ced-3, which encode an APAF1-like adaptor protein
and a pro-caspase, respectively. In cells destined to live, the ability of the
CED-4 protein to induce pro-CED-3 activation and, hence, the execution of
apoptosis, is blocked by the anti-apoptotic protein CED-9, the C.
elegans ortholog of the human proto-oncoprotein BCL2. In cells destined
to die, the pro-apoptotic gene egl-1, which encodes a BH3-only (BH3,
BCL2 homology domain 3) protein, is upregulated at the transcriptional level.
EGL-1 protein can interact with and block CED-9, thereby allowing CED-4
activation.
Apoptosis also occurs in the germ line of adult C. elegans
hermaphrodites (Sulston,
1988), where more than 50% of all germ cells in the pachytene
stage of prophase of meiosis I undergo apoptosis, a process referred to from
hereon as `constitutive germ cell apoptosis'
(Gumienny et al., 1999). Like
developmental apoptosis, constitutive germ cell apoptosis is dependent on the
genes ced-4 and ced-3 and is blocked by ced-9.
However, unlike developmental apoptosis, constitutive germ cell apoptosis is
not dependent on egl-1 (Gumienny
et al., 1999). Finally, the exposure of adult C. elegans
hermaphrodites to genotoxic agents, such as ionizing radiation (IR), causes
large numbers of germ cells in the pachytene stage to undergo apoptosis, a
process referred to as `DNA damage-induced germ cell apoptosis'
(Gartner et al., 2000). DNA
damage-induced germ cell apoptosis is at least partially dependent on
egl-1, which is transcriptionally upregulated in response to DNA
damage in a manner that is dependent on the C. elegans ortholog of
the mammalian p53 gene (also known as Tp53), cep-1
(Derry et al., 2001;
Hofmann et al., 2002;
Schumacher et al., 2001).
In this article, we report that lin-35 RB as well as dpl-1 DP, efl-1 E2F and efl-2 E2F, are required for germ cell apoptosis in C. elegans. Surprisingly, however, the pro-apoptotic role of lin-35 appears to be distinct from the pro-apoptotic roles of dpl-1, efl-1 and efl-2.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The wild-type
strain used was C. elegans var. Bristol (N2). Alleles used are listed
below and are described (Riddle,
1997), except where noted otherwise. LGI:
lin-35(n745, n2996) (Lu
and Horvitz, 1998), cep-1(lg12501)
(Schumacher et al., 2001),
hus-1(op241) (Hofmann et
al., 2002), ppw-1(pk1425)
(Tijsterman et al., 2002),
rrf-1(pk1417) (Simmer et
al., 2003). LGII: dpl-1(n3643, n3316)
(Ceol and Horvitz, 2001),
unc-4(e120), unc-52(e444),
dpy-10(e128), rrf-3(pk1426)
(Simmer et al., 2002),
efl-2(tm2359) (National BioResource Project, Tokyo, Japan)
(tm2359 is a 332 bp deletion with a 47 bp insertion that removes
sequences 5' to the predicted transcriptional start site of the
efl-2 gene and most of exon 1. Exon 1 encodes the putative
dimerization domain and DNA-binding domain of the EFL-2 protein. For this
reason, tm2359 is predicted to be a strong loss-of-function or null
allele of the efl-2 gene). LGIII: ced-9(n1653ts,
n2812) (Hengartner and Horvitz,
1994), ced-6(n2095),
dpy-19(e1259), glp-1(q339)
(Edgley et al., 1995),
ceh-13(sw1) (Brunschwig
et al., 1999), dpy-17(e164), nDf20,
nDp2[unc-86(e1416)] (III;f)
(Finney et al., 1988). LGIV:
dpy-20(e1282). LGV: bcIs39
(Plim-7ced-1::gfp)
(Schumacher et al., 2005;
Zhou et al., 2001),
dpy-21(e428), unc-76(e911),
efl-1(n3318) (Ceol and
Horvitz, 2001), rde-1(ne300)
(Tabara et al., 1999).
Quantification of germ cell apoptosis
For constitutive germ cell apoptosis, hermaphrodites were synchronized at
the fourth larval (L4) stage and analyzed 12, 24, 36 and 48 hours post the L4
stage. Animals were anesthetized using 80 mM sodium azide and mounted on
slides using 2% agarose pads. Apoptotic germ cells were detected using DIC and
epifluorescence. For ced-9(n1653ts)-induced germ cell
apoptosis, animals were synchronized at the L4 stage, cultivated at 20°C
for 12 hours and shifted to 25°C for 24 hours before being analyzed. For
ced-9(n2812)/+-induced germ cell apoptosis, animals were
synchronized at the L4 stage and analyzed by DIC 40 hours post the L4 stage.
For DNA damage-induced germ cell apoptosis, hermaphrodites were synchronized
at the L4 stage and exposed to 120 Gy using a 137Cs source (J. L.
Shepherd, San Fernando, CA) with a dose rate of 10 Gy/minute. 24 hours
post-irradiation, apoptotic germ cells were scored using DIC.
efl-1(n3318) and efl-2(tm2359) animals
were irradiated 12 hours post the L4 stage and analyzed 24 hours
post-irradiation.
Transgenic animals
The Plim-7lin-35 plasmid was constructed by fusing the
lin-35 cDNA to the lim-7 promoter (2.23 kb upstream of
transcriptional start site) (Hall et al.,
1999). This fusion was blunt-cloned into the EcoRI site
of vector pPD95.67 (gift from Dr A. Fire, Stanford University School of
Medicine, CA). The resulting plasmid was injected at a concentration of 1
ng/µl using the plasmid pRF4 [rol-6(dm)] as co-injection marker at
a concentration of 100 ng/µl (Kramer et
al., 1990). The cosmid C32F10 [lin-35(+)] was
injected at a concentration of 10 ng/µl using the plasmid pPD93.97
(Pmyo-3gfp) (gift from Dr A. Fire) as co-injection marker
at a concentration of 50 ng/µl.
RNAi experiments
RNAi experiments were performed by feeding as described
(Timmons et al., 2001).
Briefly, animals were exposed to RNAi plates containing 6 mM IPTG. Animals of
the F1 generation were synchronized at the L4 larval stage, transferred to
fresh RNAi plates and analyzed after the indicated time. An unrelated gene
(F53B3.2) was used as control RNAi.
Semi-quantitative and quantitative expression analyses
For semi-quantitative RT-PCR, hermaphrodites were synchronized at the L4
stage and irradiated with 100 Gy 24 hours post the L4 stage. Total RNA was
isolated from 200 animals 3 hours post-irradiation using Trizol (Invitrogen)
and purified by chloroform extraction and isopropanol precipitation. 1 µg
of total RNA was treated with DNase and reverse transcribed into cDNA using
the SuperScript III First Strand Kit (Invitrogen) and oligo-dT primers. Equal
amounts of cDNA were used as template for gene-specific PCR with appropriate
primers. A plasmid containing the egl-1 cDNA was used as positive
control. Water was used as negative control. tbg-1 (encoding
-tubulin) RT-PCR was performed as a control. For quantitative real-time
RT-PCR, total RNA was isolated from about 40 gonads dissected from
hermaphrodites 27 hours post the L4 stage as described for semi-quantitative
RT-PCR. 400 ng of total RNA was reverse transcribed into cDNA. Gene-specific
PCR reactions using equal amounts of cDNA, appropriate primers and TaqMan
probes were performed with an Opticon DNA Engine (MJ Research). tbg-1
was used as external control. Test gene CT values were normalized
to tbg-1 CT values and relative expression levels were
derived with the comparative CT method
(Livak and Schmittgen,
2001).
Western analysis
About 100 (wild-type) or 120 [lin-35(n745) and
dpl-1(n3643)] gonads were dissected in PBS from synchronized
hermaphrodites 27 hours post the L4 stage, transferred into 2x sample
buffer and boiled for 5 minutes. Proteins were separated on 10% SDS-PAGE gels.
Western analysis was performed using CED-4- and CED-9-specific antibodies as
described (Chen et al., 2000)
and HRP-coupled secondary antibodies. Loading controls were detected with
antibodies specific for ß-actin (AC15, Sigma) and ß-tubulin (N357,
Amersham). NIH image software was used for quantification.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We
found no defect in developmental apoptosis in lin-35(n745)
animals, indicating that lin-35 RB is not required for developmental
apoptosis at least in a wild-type background (see Table S1 in the
supplementary material; data not shown). However, it has recently been
demonstrated that in a sensitized genetic background, lin-35 promotes
developmental apoptosis (Reddien et al.,
2007).
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). Therefore, constitutive germ cell apoptosis was quantified
using a combination of DIC and CED-1::GFP (Plim-7ced-1::gfp,
bcIs39) fluorescence microscopy. As a result of the efficient engulfment
of apoptotic germ cells by the sheath cells, the number of apoptotic germ
cells per gonad arm that can be detected at any given time is very low [4.0
apoptotic germ cells on average in hermaphrodites 48 hours post the fourth
larval (L4) stage] (Gumienny et al.,
1999). For this reason, we analyzed constitutive germ cell
apoptosis in the background of ced-6(n2095). n2095
is a loss-of-function mutation in ced-6, a gene which is required for
the efficient engulfment of apoptotic germ cells
(Ellis et al., 1991). In the
ced-6 mutant background, apoptotic germ cells accumulate, resulting
in a greatly increased number of apoptotic germ cells that can be detected at
any given time (55.3±5.4 apoptotic germ cells on average in
hermaphrodites 48 hours post the L4 stage)
(Fig. 1A, +/+). We found that
lin-35(n745) reduced the number of apoptotic germ cells in
ced-6(n2095) animals by more than 50%
[Fig. 1A,
lin-35(n745)]. Similarly, the lin-35
loss-of-function mutation n2996, a nonsense mutation predicted to
truncate the LIN-35 protein after amino acid 304, reduced the number of
apoptotic germ cells in ced-6(n2095) animals by more than
50%. For comparison, in ced-6(n2095) animals homozygous for
a loss-of-function mutation in the ced-3 gene, almost no apoptotic
germ cells were detected [Fig.
1A, ced-3(n717)].
When compared with wild-type hermaphrodites, lin-35(n754)
hermaphrodites are less fertile (Boxem and
van den Heuvel, 2001; Fay et
al., 2002; Lu and Horvitz,
1998; Thomas and Horvitz,
1999). Therefore, the decrease in the number of apoptotic germ
cells in lin-35(n745) animals might be the result of a
decrease in constitutive germ cell apoptosis or, alternatively, a decrease in
germ cell proliferation. To distinguish between these two possibilities, we
assessed constitutive germ cell apoptosis in animals homozygous for
e1282, a loss-of-function mutation in the dpy-20 gene, which
encodes a novel, BED zinc-finger protein required for normal body morphology
(Clark et al., 1995). Germ
cell proliferation in dpy-20(e1282) animals is compromised
to a degree similar to that in lin-35(n745) animals (see
Table S2 in the supplementary material); however, the
dpy-20(e1282) mutation had no effect on the number of
apoptotic germ cells [Fig. 1A,
dpy-20(e1282)]. This finding indicates that the decrease in
the number of apoptotic germ cells detected in lin-35(n745);
ced-6(n2095) animals is a result of a decrease in
constitutive germ cell apoptosis rather than a decrease in germ cell
proliferation. This conclusion is supported by the following two observations:
first, a partial loss-of-function mutation in the gene dpl-1, n3643,
does not compromise germ cell proliferation but, like
lin-35(n745), decreases the number of apoptotic germ cells
in ced-6(n2095) animals by more than 50% (see below);
second, the loss of lin-35 function completely blocks DNA
damage-induced germ cell apoptosis (see below). Based on these observations,
we conclude that the lin-35/Rb gene promotes constitutive
germ cell apoptosis.
To determine where lin-35 function is required to promote
constitutive germ cell apoptosis, we generated transgenic
lin-35(n745) animals carrying extrachromosomal arrays
composed of cosmid C32F10, which contains the wild-type
lin-35 locus [lin-35(+)], or a transgene expressing the
wild-type lin-35 cDNA under the control of the lim-7
promoter (Plim-7lin-35). We found that lin-35(+),
which has been shown to rescue lin-35 function in the somatic gonad
as well as the germ line (Fay et al.,
2002), but not Plim-7lin-35, which presumably
rescues lin-35 function specifically in the somatic gonad
(Hall et al., 1999), was able
to rescue the germ cell apoptosis phenotype of lin-35(n745)
animals (Table 1A).
Furthermore, reducing lin-35 function by RNAi in a rrf-1
mutant background, which is defective for RNAi in somatic tissues, or a
ppw-1 mutant background, which is defective for RNAi in the germ
line, reduced constitutive germ cell apoptosis by about 50%
(Table 1B and C). These results
demonstrate that lin-35 expression in either somatic gonad or germ
line is insufficient to promote constitutive germ cell apoptosis. Therefore,
we propose that lin-35 function is required in both the somatic gonad
and the germ line to promote constitutive germ cell apoptosis.
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;
Hengartner et al., 1992). We
found that lin-35(n745) reduced the number of gonad arms
with severely damaged germ lines in ced-9(n1653ts) animals
from 83% [n=30; ced-9(n1653ts)] to 20%
[n=40; lin-35(n745);
ced-9(n1653ts)] (Fig.
2A). In addition, in gonad arms with undamaged germ lines,
lin-35(n745) reduced the number of apoptotic germ cells
detectable from 26.0±7.2 [ced-9(n1653ts);
n=14] to 4.1±2.8 [lin-35(n745);
ced-9(n1653ts); n=47]. Furthermore, we found that
lin-35(n745) completely suppresses germ cell apoptosis
induced by the loss of one copy of the ced-9 gene (see below). The
potential ced-9-null allele, n2812, is a nonsense mutation
predicted to truncate the 280 amino acid CED-9 protein after amino acid 45
(Hengartner and Horvitz,
1994). ced-9(n2812) causes massive germ cell
apoptosis resulting in 100% (n>40) gonad arms with severely
damaged germ lines at all growth temperatures
(Gumienny et al., 1999;
Hengartner et al., 1992).
lin-35(n745) failed to suppress the massive germ cell
apoptosis in ced-9(n2812) hermaphrodites and did not affect
the percentage of gonad arms with severely damaged germ lines (100%;
n>40). The finding that lin-35(n745) suppresses the partial but not complete loss of ced-9 function is consistent with the model that lin-35 acts upstream of ced-9 to block ced-9 function. Alternatively, lin-35 might act downstream of ced-9 and the failure of lin-35(n745) to suppress the complete loss of ced-9 function might be due to irreversible germ line damage. To distinguish between these two possibilities, we analyzed germ cell apoptosis in ced-9(n2812) animals homozygous for n2427, a weak loss-of-function mutation of the ced-3 gene, which acts downstream of ced-9. ced-3(n2427) partially suppressed the germ line phenotype caused by ced-9(n2812): in ced-3(n2427); ced-9(n2812) animals, 0% of the gonad arms were severely damaged (n>40), but when compared with wild-type animals (+/+), an elevated level of germ cell apoptosis could be detected [Table 2, ced-3(n2427); ced-9(n2812)]. We found that lin-35(n745) failed to suppress the elevated level of germ cell apoptosis in ced-3(n2427); ced-9(n2812) animals [Table 2, lin-35(n745); ced-3(n2427); ced-9(n2812)]. Therefore, the ability of lin-35(n745) to reduce constitutive germ cell apoptosis is dependent on the presence of a ced-9 gene that is at least partially functional. Based on these results, we conclude that the lin-35 RB gene acts upstream of ced-9 BCL2 to inhibit its function.
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The dosage of ced-9 determines the level of germ cell apoptosis
Hermaphrodites trans-heterozygous for the potential ced-9-null
mutation n2812 and the balancer chromosome qC1
[ced-9(n2812)/qC1] overall are wild type. However,
we found that, when compared with wild-type animals (+/+) or animals
heterozygous for qC1 (+/qC1),
ced-9(n2812)/qC1 animals had an increased number of
apoptotic germ cells (Table 3).
This observation indicates that the ced-9 gene is
haploinsufficient for the repression of constitutive germ cell apoptosis. To
further characterize the effect of ced-9 dosage on germ cell
apoptosis, we analyzed germ cell apoptosis in engulfment-defective animals
with one (+), two (+/+) or three (+/+/+) copies of the ced-9 gene.
With increasing ced-9 dosage, we detected decreasing numbers of
apoptotic germ cells (Fig. 2B).
Thus, changes in the dosage of ced-9 BCL2 and, hence, most likely in
the expression of ced-9 BCL2, strongly affect the level of germ cell
apoptosis. Furthermore, the increase in apoptotic germ cells observed in
animals of the genotype ced-9(n2812)/qC1 was
completely suppressed by lin-35(n745)
(lin-35(n745); ced-9(n2812)/qC1
(Table 3). Therefore, we
conclude that the decreased level of constitutive germ cell apoptosis detected
in animals lacking lin-35 RB function is a consequence of increased
ced-9 expression in the germ line.
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).
Therefore, we determined the effect of decreasing dpl-1, efl-1 or
efl-2 function on constitutive germ cell apoptosis. [As observed for
the loss of lin-35 function, decreasing dpl-1, efl-1 or
efl-2 function did not affect developmental apoptosis (see Table S1
in the supplementary material).] We found that a partial (n3643) or
strong (n3316) loss-of-function mutation in the dpl-1 gene
reduced constitutive germ cell apoptosis in a ced-6(n2095)
background by more than 50% [Fig.
1A, dpl-1(n3643),
dpl-1(n3316)]. Furthermore, strong loss-of-function
mutations in efl-1, n3318,or efl-2, tm2359, decreased
constitutive germ cell apoptosis in a ced-6(n2095)
background by about 40-50% [Fig.
1A, efl-1(n3318),
efl-2(tm2359)]. To determine whether efl-1 and
efl-2 have partially redundant functions in constitutive germ cell
apoptosis, we attempted to construct a C. elegans strain lacking both
efl-1 and efl-2 function. However, we were unable to obtain
such a strain (our unpublished observations). For this reason, we consider it
likely that efl-1 and efl-2 have redundant functions in an
essential process. dpl-1(n3316) and
efl-1(n3318) animals are sterile; however,
dpl-1(n3643) and efl-2(tm2359) animals are
fertile and their fertilities are comparable to the fertility of wild-type
animals (Ceol and Horvitz,
2001) (see Table S2 in the supplementary material; data not
shown). Therefore, we propose that, like lin-35 RB, the genes
dpl-1 DP, efl-2 E2F and most likely also efl-1 E2F
promote constitutive germ cell apoptosis. Reducing the function of dpl-1 in lin-35(n745); ced-6(n2095) animals (lin-35; dpl-1) or efl-2(tm2359); ced-6(n2095) animals (dpl-1; efl-2) or reducing the function of lin-35 in efl-2(tm2359); ced-6(n2095) animals (lin-35; efl-2) did not result in a further decrease in the number of apoptotic germ cells (Fig. 1B). These observations suggest that the inactivation of C. elegans orthologs of components of the RB complex reduces constitutive germ cell apoptosis by about 50%.
The roles of dpl-1 DP and efl-2 E2F in constitutive germ cell apoptosis are distinct from the role of lin-35 RB
In contrast to lin-35(n745), reducing the function of
dpl-1 or efl-2 decreased the number of apoptotic germ cells
detected in ced-9(n2812); ced-3(n2427)
animals by 56% and 60%, respectively (Table
2). [We have been unable to construct a strain of the genotype
efl-1(n3318); ced-9(n2812);
ced-3(n2427), and consider it likely that animals of this
genotype are not viable (our unpublished observations).] Furthermore, we found
that the levels of ced-9 mRNA and CED-9 protein in the germ line of
dpl-1(n3643) animals were not greater than in the germ line
of wild-type (+/+) animals (Fig.
3). These results demonstrate that dpl-1 and
efl-2 do not promote constitutive germ cell apoptosis by inhibiting
ced-9 expression. Therefore, the pro-apoptotic roles of
dpl-1 DP and efl-2 E2F (and possibly efl-1 E2F) are
distinct from that of lin-35 RB.
By contrast, we found that dpl-1(n3643) reduced the levels of ced-3 and ced-4 mRNAs in the germ line by about 40% and 50% (Fig. 3A), respectively, and the level of CED-4 protein by 40% (Fig. 3B,C). To determine whether a reduction of ced-3 or ced-4 expression by about 50% has an effect on constitutive germ cell apoptosis, we analyzed engulfment-defective animals heterozygous for strong loss-of-function mutations in ced-3 [ced-3(n717)/+], ced-4 [ced-4(n1162)/+] or ced-3 and ced-4 [ced-4(n1162)/+; ced-3(n717)/+]. We found that the lack of one functional copy of either ced-3 or ced-4 was not sufficient to reduce constitutive germ cell apoptosis, but the lack of one functional copy of both ced-3 and ced-4 reduced constitutive germ cell apoptosis by 75% (Table 2). Therefore, the simultaneous reduction by 50% of the dosage of ced-3 and ced-4 and, hence, most likely of the expression of ced-3 and ced-4, strongly reduces constitutive germ cell apoptosis. Based on these results, we conclude that the reduction in constitutive germ cell apoptosis observed in animals lacking dpl-1 function is a result of decreased levels of ced-3 and ced-4 mRNA. Therefore, dpl-1 DP, and most likely also efl-1 E2F and efl-2 E2F, promotes constitutive germ cell apoptosis by inducing the expression of the genes ced-4 APAF1 and ced-3 caspase in the germ line.
lin-35 RB, dpl-1 DP and efl-2 E2F are required for DNA damage-induced germ cell apoptosis
Next, we determined whether lin-35, dpl-1, efl-1 and
efl-2 also promote DNA damage-induced germ cell apoptosis
(Gartner et al., 2000). Since
the exposure to genotoxic agents induces massive germ cell apoptosis, DNA
damage-induced germ cell apoptosis was analyzed in a wild-type background
rather than the ced-6(n2095) background. The gene
cep-1 is required for DNA damage-induced germ cell apoptosis and
encodes the C. elegans ortholog of p53
(Derry et al., 2001;
Schumacher et al., 2001). We
found that, like the loss of cep-1 function, reducing lin-35,
dpl-1 or efl-2 function completely blocked DNA damage-induced
germ cell apoptosis [Fig. 4A,
lin-35(n745), dpl-1(n3643),
efl-2(tm2359)]. By contrast, reducing efl-1
function had no effect on this process [efl-1(n3318)].
Furthermore, the loss of lin-35, dpl-1 or efl-2 function did
not result in a defect in DNA damage-induced cell-cycle arrest, indicating
that lin-35, dpl-1 and efl-2 are not required for the
DNA-damage checkpoint (see Fig. S2 and Table S3 in the supplementary material;
data not shown). Based on these results, we conclude that lin-35 RB,
dpl-1 DP and efl-2 E2F, but not efl-1 E2F, are
specifically required for DNA damage-induced germ cell apoptosis.
|
). To determine whether the block in DNA damage-induced
germ cell apoptosis caused by the loss of lin-35, dpl-1 or
efl-2 function is a result of a defect in the upregulation of
egl-1, we analyzed the level of egl-1 mRNA in the germ line
of lin-35(n745), dpl-1(n3643) and
efl-2(tm2359) animals. Using semi-quantitative and
quantitative real-time RT-PCR, we found that in response to DNA damage the
levels of egl-1 mRNA increased to similar levels in the germ line of
wild-type (+/+) and lin-35(n745) animals
(Fig. 4B,C). Similarly, using
semi-quantitative real-time RT-PCR, we found that the levels of egl-1
mRNA increased to similar levels in the germ line of wild-type (+/+),
dpl-1(n3643) and efl-2(tm2359) animals
(Fig. 4B). Therefore, the loss
of lin-35, dpl-1 or efl-2 function does not affect the
cep-1-dependent upregulation of egl-1 transcription. These
results are consistent with the model that lin-35 RB, dpl-1
DP and efl-2 E2F act downstream of or in parallel to cep-1
p53 and egl-1 BH3-only to promote DNA damage-induced germ cell
apoptosis. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Furthermore, our results indicate that the pro-apoptotic
activity of lin-35 in the germ line is dependent on the expression of
lin-35 not only in the germ line but also the somatic gonad. Hence,
lin-35 acts in both a cell-autonomous and cell non-autonomous manner
to promote constitutive germ cell apoptosis. lin-35 appears to be
expressed in the germ line (Reinke et al.,
2004) and it has previously been shown to play a role in the
somatic gonad (Bender et al.,
2004). Furthermore, lin-35 has been shown to act in a
cell non-autonomous manner to antagonize vulval differentiation in the VPCs
(Cui et al., 2006;
Myers and Greenwald, 2005).
Therefore, the LIN-35 protein might function in the germ line to directly or
indirectly cause the repression of ced-9 transcription in the germ
line and it might also affect an activity of the somatic gonad that
non-autonomously promotes the repression of ced-9 transcription in
the germ line. The loss of lin-35 function also consistently resulted in an increase in the level of ced-3 mRNA and a decrease in the level of CED-4 protein. We speculate that rather than being a specific effect of the loss of lin-35 function, the changes in ced-3 and ced-4 expression observed might be the result of lin-35-dependent changes in the somatic gonad. Furthermore, the increase in ced-3 expression observed in lin-35(n745) animals might explain why the loss of lin-35 function only blocks 50% of constitutive germ cell apoptosis and why the defect in constitutive germ cell apoptosis observed in lin-35(n745); dpl-1(n3643) animals is not stronger than in lin-35(n745) animals.
|
).
However, we believe that this failure can be explained by the relatively small
decrease in the levels of ced-4 and ced-3 mRNAs caused by
the loss of dpl-1 or efl-1 function and the threshold level
used in this study. efl-1 and dpl-1 both are expressed in
the germ line (Ceol and Horvitz,
2001; Page et al.,
2001). Therefore, we propose that the DPL-1, EFL-1 and EFL-2
proteins act in a cell-autonomous manner to either directly or indirectly
enhance the transcription in the germ line of ced-4 and
ced-3.
The overexpression of the mammalian E2f1 gene in tissue culture
cells can induce apoptosis. Furthermore, E2f1 overexpression results
in the transcriptional activation of a number of pro-apoptotic genes, such as
the genes encoding the BH3-only proteins NOXA and PUMA, APAF1 and Caspase 3,
7, 8 and 9 (Attwooll et al.,
2004; Dimova and Dyson,
2005). Furthermore, studies on the role of the Drosophila
E2F-like protein dE2F1 in apoptosis indicate that it has pro-apoptotic
activity, at least in cells of the intervein region of wing discs, where dE2F1
promotes apoptosis in response to DNA damage, which probably results from the
transcriptional upregulation of a pro-apoptotic gene(s)
(Moon et al., 2005).
Therefore, the activation of apoptosis by the E2F-DP-dependent transcriptional
activation of pro-apoptotic genes is a mechanism conserved among C.
elegans, Drosophila and mammals.
The pro-apoptotic function of lin-35 RB in constitutive germ cell apoptosis is independent of dpl-1 DP and efl-2 E2F
The function of lin-35 RB in constitutive germ cell apoptosis is
independent of the functions of dpl-1 DP, efl-2 E2F and
probably also of efl-1 E2F. This notion is supported by
microarray-based expression profiling of germ lines, which revealed that there
is extensive overlap between the target genes of DPL-1 and EFL-1, but not
between the target genes of LIN-35 and DPL-1 or EFL-1
(Chi and Reinke, 2006).
Mammalian RB protein is thought to control gene expression almost exclusively
through binding to E2F-DP complexes. However, mutant RB proteins that are
unable to bind to E2F-DP complexes retain certain aspects of RB function
(Sellers et al., 1998). This
observation suggests that RB could have functions that are independent of
E2F-DP activity. Indeed, RB can interact with a variety of transcription
factors other than E2F-DP, such as MyoD, AP-2, C/EBPs and Pax5
(Eberhard and Busslinger,
1999; Macaluso et al.,
2006; Macleod,
1999; Sato et al.,
2001). However, the significance of these interactions is unclear.
Interestingly, egl-38 and pax-2, two C. elegans
genes related to mammalian Pax2/5/8, promote
ced-9 expression in somatic tissues and in the germ line and possibly
encode direct activators of ced-9 transcription
(Park et al., 2006). Thus, we
speculate that the LIN-35 RB protein might interact with the EGL-38 and PAX-2
proteins and antagonize their ability to promote ced-9 BCL2
transcription.
The levels of CED-9 BCL2, CED-4 APAF1 and CED-3 caspase control constitutive germ cell apoptosis
In contrast to developmental apoptosis, which is determined by the
essentially invariant somatic cell lineage and is dependent on the
pro-apoptotic gene egl-1 BH3-only, constitutive germ cell apoptosis
is a stochastic event that is coupled to meiotic cell-cycle progression and
which occurs in an egl-1 BH3-only-independent manner
(Conradt and Horvitz, 1998;
Gumienny et al., 1999). We
found that the levels of CED-9, CED-4 and CED-3 proteins are critical for
constitutive germ cell apoptosis: lowering the dosage of the ced-9
gene increases constitutive germ cell apoptosis, whereas increasing the dosage
of ced-9 or lowering the dosage of ced-4 and ced-3
decreases constitutive germ cell apoptosis. Therefore, the amount of CED-9
protein is a limiting factor in a germ cell's quest for survival and,
conversely, the amounts of CED-4 and CED-3 proteins are limiting factors in a
germ cell's quest for demise. We propose that the combination of
lin-35-dependent repression of ced-9 transcription and
dpl-1-dependent enhancement of ced-4 and ced-3
transcription ensure that the relative levels of CED-9, CED-4 and CED-3
proteins are such that more than 50% of the germ cells undergo apoptosis. How
the pro-apoptotic activities of lin-35 and dpl-1 are
regulated remains to be determined. Furthermore, it remains to be determined
whether lin-35 and dpl-1 are targets of apoptotic signaling
pathways that determine the extent of germ cell apoptosis or whether they are
components of a general machinery that sets the level of ced-9, ced-3
and ced-4 mRNAs in the germ line. Finally, because the loss of
lin-35 and dpl-1, efl-1 or efl-2 function causes a
50% decrease in constitutive germ cell apoptosis, the activities of
additional, as yet uncharacterized factors must contribute to the
life-versus-death decision within germ cells
(Fig. 5A).
The regulation at the transcriptional level of members of the family of
pro- and anti-apoptotic BCL2-like protein is a well-established mechanism to
control apoptosis (Cory and Adams,
2002). However, at least to our knowledge, the regulation at the
transcriptional level of APAF1-like proteins and caspases has so far not been
demonstrated to be a physiologically important mechanism for apoptosis
regulation (Adams, 2003;
Danial and Korsmeyer, 2004).
It will be of interest to determine whether the transcriptional regulation of
Apaf1-like or caspase genes controls apoptosis in species other than
C. elegans.
lin-35 RB, dpl-1 DP and efl-2 E2F are required for DNA damage-induced germ cell apoptosis
lin-35, dpl-1 and efl-2, but not efl-1, are
required for DNA damage-induced germ cell apoptosis. Interestingly, in
response to DNA damage, the level of ced-9 mRNA increases about
2-fold and the levels of ced-4 and ced-3 mRNAs decrease by
about 50% in the germ line of wild-type hermaphrodites (our unpublished
observations). Therefore, the transcriptional regulation of ced-9,
ced-4 and ced-3 does not appear to be a determinant of DNA
damage-induced germ cell apoptosis. Instead, we propose that in response to
DNA damage, lin-35, dpl-1 and efl-2 control the expression
of different target genes that encode critical determinants of DNA
damage-induced germ cell apoptosis (Fig.
5B). Candidate genes are cep-1 p53 and egl-1
BH3-only, which are required for DNA damage-induced germ cell apoptosis.
However, we found that lin-35, dpl-1 and efl-2 act
downstream of or in parallel to cep-1 and egl-1 to cause DNA
damage-induced germ cell apoptosis (Fig.
5B). Finally, we have evidence that apart from lin-35 and
dpl-1, additional synMuvB genes are required for DNA damage-induced
germ cell apoptosis (our unpublished observations). Orthologs of synMuvB
proteins in other species have been implicated in chromatin remodeling and
transcriptional repression (reviewed by
Korenjak and Brehm, 2005;
Lipsick, 2004). Therefore, we
speculate that in response to DNA damage, LIN-35, DPL-1, EFL-2 and additional
synMuvB proteins might assemble to form a transcriptional repressor complex,
which represses the transcription of a gene or genes, the product(s) of which
can block DNA damage-induced germ cell apoptosis
(Fig. 5B).
Implications for the role in apoptosis of mammalian Rb
Currently, the prevailing model is that mammalian Rb has
anti-apoptotic activity. This model is based on the observation that mice
lacking Rb function exhibit ectopic apoptosis. However, the fact that
ectopic apoptosis in Rb-/- mice is observed only in a
limited number of tissues suggests that the role of Rb in apoptosis
might be tissue-specific. This notion is supported by observations indicating
that mutations in the Rb gene are sufficient to cause pituitary and
thyroid tumors in mice and small-cell lung cancer in humans, and that
tumorigenesis in these types of tumors does not appear to depend on the
concomitant inactivation by mutation of the apoptotic pathway
(Chau and Wang, 2003;
Hitchens and Robbins, 2003;
Hu et al., 1994;
Lipinski and Jacks, 1999;
Sherr and McCormick, 2002;
Williams et al., 1994). Based
on these facts and on our finding that C. elegans lin-35 RB has
pro-apoptotic activity in the germ line, we hypothesize that mammalian
Rb functions to promote apoptosis in certain tissues, such as tissues
giving rise to pituitary and thyroid tumors, and small-cell lung cancer.
Furthermore, we hypothesize that in these tissues, the pro-apoptotic activity
of Rb contributes to the tumor-suppressing activity of Rb,
which so far has mainly been attributed to its anti-proliferative activity. We
also speculate that, in analogy to LIN-35 RB, the putative pro-apoptotic
activity of the RB protein could be mediated by the transcriptional repression
of anti-apoptotic Bcl2-like genes
(Cory and Adams, 2002). A
comprehensive understanding of the role in apoptosis of mammalian Rb
in different tissues and contexts will almost certainly improve our knowledge
of the tumor-suppressing activity of Rb and, hence, tumorigenesis in
Rb-deficient tumors.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/20/3691/DC1
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