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First published online 4 October 2006
doi: 10.1242/dev.02614
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Department of Molecular Genetics, Ohio State University, Columbus, OH 43210 USA
* Author for correspondence (e-mail: chamberlin.27{at}osu.edu)
Accepted 6 September 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: Apoptosis, ced-9, Caenorhabditis elegans, Transcriptional regulation, egl-38, pax-2
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). It provides a regulated process to refine organ structure
or remove cells that are damaged or no longer required. Apoptosis is mediated
by a core biochemical pathway that regulates the activation of caspases
(reviewed by Danial and Korsmeyer,
2004; Yan and Shi,
2005). These enzymes act to digest cellular proteins
systematically, and thus their activity can result in irreversible damage to
cells. Caspase activity is regulated by the proteolytic processing of a
procaspase to its active form, as well as by inhibitor of apoptosis proteins
(IAPs). The importance of tight regulation of caspase activity and the
cell-death pathway is evident from the fact that an inappropriate increase or
decrease in apoptosis levels is a hallmark of many human diseases, including
cancer, neurodegenerative disorders and autoimmune disease. Biochemical and
genetic studies have been central to identifying the components and
mechanistic details of the core apoptotic pathway. However, less is understood
about the potential regulatory inputs that promote survival or trigger
apoptosis, especially during normal development.
The nematode C. elegans has served as a pioneering model for gene
discovery and functional analysis of apoptosis in vivo (reviewed by
Lettre and Hengartner, 2006).
Genetic studies in C. elegans have identified a core apoptotic
pathway that shares molecular components with other organisms. Both somatic
cells that die during the normal process of embryonic and larval development,
as well as germ cells that die in the adult as part of gamete formation and
germline homeostasis, use this core pathway. However, the regulatory inputs
that influence somatic and germline cell death differ. Cell lineage and
developmental cell fate specification mechanisms play an important role in
regulating which somatic cells will die, and the pattern of cell death is
highly reproducible from one animal to the next
(Sulston and Horvitz, 1977;
Sulston et al., 1983). By
contrast, there is a variable pattern of cell death in the germline, and the
regulatory processes include DNA damage-induced checkpoint controls, the
activity of signal transduction pathways and bacterial infection
(Aballay and Ausubel, 2001;
Gartner et al., 2000;
Gumienny et al., 1999).
Although many genes that are important for regulating either somatic or
germline cell death have been identified, it is clear that new genes remain to
be discovered (e.g. Lettre et al.,
2004). Importantly, aside from core apoptotic genes (the caspase
gene ced-3, the Apaf-1 gene ced-4, the Bcl-2 gene
ced-9 and the BH3-only gene egl-1; see
Fig. 4A), regulatory genes that
impact apoptosis in both somatic and germline cells have not been
identified.
This work focuses on the role of Pax2/5/8 proteins in regulating apoptosis.
Pax proteins are transcription factors identified by the presence of the
DNA-binding paired domain (reviewed by Chi
and Epstein, 2002). Pax proteins fall into four subgroups based on
their DNA-binding specificity, sequence similarity within the paired domain
and the presence or absence of other sequence motifs. The Pax2/5/8 subgroup is
defined by the mammalian Pax2, Pax5 and Pax8 gene products.
Genetic studies of Pax2/5/8 genes in a range of animals indicate that
they are important for the normal development of subsets of cells and tissues,
and that they can be key regulators in the development of organs. At the
cellular level, Pax2/5/8 proteins can promote proliferation and cell survival,
and influence differentiation. This effect is observed in both gain- and
loss-of-function conditions. For example, increased expression of
Pax2 in mouse kidney results in renal hyperplasia
(Dressler et al., 1993), and
inappropriate expression of Pax genes is associated with primary cancer cells
and cancer cell lines (Muratovska et al.,
2003; Wu et al.,
2005). By contrast, Pax2 mutant mice and human
individuals with PAX2 mutations exhibit renal hypoplasia
(Sanyanusin et al., 1995;
Torres et al., 1995).
Importantly, interfering with Pax gene expression in cancer cells promotes
apoptosis, showing that Pax gene expression protects cells from cell death
(Bernasconi et al., 1996;
Buttiglieri et al., 2004;
Muratovska et al., 2003).
Similarly, the hypoplastic defect in Pax2/+ mutant mice can be
suppressed by caspase inhibitors, demonstrating that the reduced kidney
development results, at least in part, from increased apoptosis
(Clark et al., 2004;
Dziarmaga et al., 2003).
Although the evidence that at least some Pax genes act to protect cells from
apoptosis is strong, the molecular mechanism for this function is not well
understood.
In order to better understand how Pax2/5/8 genes influence cell
death, we have studied their function in C. elegans. This organism
has two genes of the Pax2/5/8 class: egl-38 and
pax-2. Although these genes result from a recent gene duplication,
both have some non-redundant features
(Wang et al., 2004). Here, we
show that the C. elegans Pax2/5/8 genes act similarly to the
mammalian genes to promote cell survival. The two genes influence both somatic
and germline cell death, demonstrating that they have a global role in
modulating apoptosis. We have used molecular and genetic experiments to
demonstrate that these Pax proteins affect the core apoptotic pathway by
directly modulating the transcription of ced-9, the only C.
elegans pro-survival bcl-2 gene. This work defines a mechanism
for Pax2/5/8 factors in influencing cell death, and identifies transcriptional
regulation of bcl-2/ced-9 as a potential regulatory point at which to
modulate cell death during normal development.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Mutations are described in wormbase
(http://www.wormbase.org/).
Mutations used are as follows. Linkage group I (LG I): cep-1(gk138).
LG III: mpk-1(oz140), ced-4(n1162), ced-6(n1813), ced-9(n1950 n2161), unc-119(e2498).
LG IV: pax-2(ok935), egl-38(n578), egl-38(sy294), egl-38(sy287), egl-38(gu22), let-60(n1046), dpy-20(e1282), ced-3(n717).
LG X: lin-2(e1309).
Integrated transgenes were saIs21 (hsp::egl-38)
(Zhang et al., 2005),
wdIs5 (unc-4::gfp)
(Lickteig et al., 2001;
Pflugrad et al., 1997). The
alleles chosen for epistasis analysis with pax-2 and egl-38
were selected because they have been used for cell death assays in similar
experiments (Gumienny et al.,
1999; Lettre et al.,
2004), and, except for let-60(n1046), are considered to
be genetically null or strong reduction-of-function alleles. The
pax-2(ok935) allele is a deletion that includes exons coding for the
DNA-binding domain, and is considered a genetic null. The
egl-38(n578) allele is a mis-sense mutation that alters the sequence
of the EGL-38 DNA-binding domain
(Chamberlin et al., 1997). This
allele preferentially disrupts a subset of egl-38 functions, and is
considered to be a non-null hypomorph. This allele was selected for our cell
death assays because it exhibits the strongest defect in germline apoptosis,
yet is homozygous viable.
RNA-mediated gene interference
cDNA fragments corresponding to ced-10, egl-1, ced-3 and
ced-4 were amplified using RT-PCR and cloned into pBluescript (all
primer sequences not listed are available on request). The cDNA inserts were
transferred to pPD129.36 (pPD vectors were a gift from A. Fire), and the
resultant clones were transformed into HT115. Bacteria were cultured on NGM
plates containing 25 µg/ml carbenicillin, 10 µg/ml tetracyclin and 1 mM
IPTG, according to the method of Kamath et al. (Kameth et al., 2001). Three to
four L4 hermaphrodites were incubated on plates to produce offspring, and then
removed after 24 hours. Their offspring were maintained on the RNAi feeding
plates until being analyzed for cell death, described below. For germline cell
death, L4 animals from treated mothers were transferred to fresh RNAi-feeding
plates, and germline cell death was examined after 36 hours.
Apoptosis assays
Germline apoptosis was assessed in staged adult animals (generally 24 hours
post L4/adult molt) and stained with SYTO12 according to the protocols of
Gumienny et al. (Gumienny et al.,
1999). Each genetic or experimental condition included at least 20
animals. For induced expression of PAX-2, EGL-38, CED-9 or MEV-1, animals
bearing hsp::pax-2, hsp::egl-38, hsp::ced-9 or hsp::mev-1
transgenes were selected as L4 larvae, as above, and allowed to mature for 24
hours. Protein expression was induced for 30 minutes at 35°C, and the
animals were recovered to room temperature for 30 minutes and stained with
SYTO12 for 4 hours. For experiments in engulfment-defective strains, animals
were selected as above. However, the number of germ cell corpses was scored
using the characteristic cell morphology (described below) under Nomarski
optics. Data are reported as mean number of corpses or of SYTO12-positive
cells ±the standard error of the mean (s.e.m.).
Somatic cell apoptosis was assessed in comma-stage embryos using Nomarski
optics. In general, dying cells appear as refractile discs that are clearly
distinct from other cell types (Sulston
and Horvitz, 1977). Each genetic condition included at least 45
animals. The number of VC motoneurons was scored as number of ventral cord
unc-4::gfp-positive cells in L2 larvae. Each genetic condition
included at least 35 animals.
Genetic mosaic analysis
egl-38(n578) animals were injected with a mixture of 10 ng/µl
of C04G2 cosmid (egl-38 rescuing cosmid)
(Chamberlin et al., 1997), 15
ng/µl of a cytoplasmically localized transformation marker
(myo-2::gfp) (Okkema et al.,
1993) and 100 ng/µl of a ubiquitously expressed nuclear marker
(sur-5::gfp) (Gu et al.,
1998; Yochem et al.,
1998). Mosaic analysis for egl-38 was conducted by
selecting mid-L4 stage animals and scoring a panel of cells derived from
different lineage precursors for the presence or absence of
sur-5::gfp (see Fig.
5). These animals were recovered, allowed to mature for 24 hours,
and were then stained with SYTO12 and analyzed for apoptosis, as described
above. The offspring of each mosaic animal were subsequently assayed for GFP
to determine whether the transgene was present in the germline.
Construction of hsp::pax-2, hsp::ced-9, hsp::mev-1 and ced-9::gfp reporter transgenes
The heat-inducible clones (hsp::pax-2, hsp::ced-9 and
hsp::mev-1) were constructed in the same manner as
hsp::egl-38 (Zhang et al.,
2005). cDNA coding for each gene was amplified by RT-PCR of
wild-type RNA and cloned downstream of the hsp16-41 promoter in
pPD49.83. The pax-2 cDNA was incorporated into a modified pPD49.83
vector that includes 3' sequences coding for the FLAG epitope. All of
the clones were sequenced to confirm that no mutations were introduced by PCR.
Transgenes were produced by the microinjection of 50-100 ng/µl hsp
plasmid DNA together with either 15 ng/µl pDP#MM016 [unc-119(+)
plasmid] (Maduro and Pilgrim,
1995) into the mitotic germline of unc-119(e2498)
animals, or 9 ng/µl myo-2::gfp
(Okkema et al., 1993) into the
mitotic germline of wild type (N2) or pax-2(ok935) egl-38(n578)
mutants, according to the method of Mello et al.
(Mello et al., 1991). The
function associated with the hsp::pax-2 transgene used in epistasis
with ced-9 (Fig. 4B,C)
was confirmed by assaying its effects in non-Ced-9 siblings. To
detect induced protein expression from clones with the FLAG tag
(Fig. 2B), transgenic animals
were heat-shocked, as above, and total protein was isolated as described
(Zhang et al., 2005). Total
protein (40 µg) was used for western blot analysis. The blot was probed
with AP-conjugated monoclonal anti-FLAG antibody (diluted 1:700; Sigma M2) and
detected with BCIP/NBT.
Genomic clones (Fig. 7) of the ced-9 gene were constructed using PCR, with the T07C4 cosmid DNA as template (construction details are available upon request). The full-length clone was confirmed to rescue the defects associated with ced-9(n1950 n2161). Sequences corresponding to the gfp gene were amplified using PCR from the vector pPD95.69, and cloned into a unique PstI site of exon 3 of the ced-9 gene, producing a translational fusion clone, ced-9::gfp. The insert was sequenced to verify it, and ced-9::gfp was introduced into animals as transgenes, as described above, using 40 ng/µl ced-9::gfp and 9 ng/µl myo-2::gfp as a transgene marker. The deletion clones were derivatives of the full-length clone. The mutant clone was generated by following the QuickChange site-directed mutagenesis protocol (Stratagene) with primers LP (5'-ACAAACCCCGACTCTAGATCTCTGCTCCGAAACAGATTTTC-3') and RP (5'-GAAAATCTGTTTCGGAGCAGAGATCTAGAGTCGGGGTTTGT-3'). The introduced BglII site was confirmed by restriction digestion, and the clone was confirmed by sequencing. At least 150 of various stages of embryos from each line were assessed for the pattern and strength of GFP expression.
Quantitative RT-PCR
Worms were harvested from NGM plates, with or without heat shock treatment
(described above), and lysed by sonication. Total RNA was extracted with
Trizol (Invitrogen) and cleaned according to the RNeasy protocol (Qiagen).
Total RNA (2 µg) from each sample was used for reverse transcription using
random hexamer priming and SuperScript II reverse transcriptase (Invitrogen).
The resulting first-strand cDNA was used for PCR analysis (specific primers
are available upon request). Quantitative PCR was performed using iCycler iQ
Real-Time PCR (BioRad) with BioRad iQ SYBR Green supermix reagents. The PCR
was performed for 35 cycles of 95°C for 30 seconds, 56°C for 30
seconds and 72°C for 30 seconds. The abundance of ced-9, mev-1, ced-3,
ced-4 and egl-1 was normalized to the levels of act-2,
and the fold induction was calculated by dividing the relative abundance of
each genotype by that of wild-type and non-heat shocked animals. Each
experimental condition was replicated three times, from two independent
batches of RNA (six replicates total).
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), with the following modifications. Transgenic lines
harboring the hsp::egl-38::FLAG or hsp::pax-2::FLAG
transgene were heat shocked at 35°C for 45 minutes and harvested 1 hour
after heat shock treatment. Worms were subjected to a 1% formaldehyde solution
for 30 minutes at room temperature to promote the cross-linking of DNA and
proteins. After chelating the formaldehyde by adding 500 µl of 2.5 M
glycine, worms were harvested in 1 ml of ChIP buffer [50 mM HEPES (pH 7.6), 1
mM EDTA, 140 mM NaCl, 0.5% NP-40, 10% glycerol, 5 mM DTT and protease
inhibitor cocktail (Calbiochem)] and sonicated five times for 30 seconds. The
cross-linked chromosomal DNA was sheared by sonication (30 seconds for 10
cycles) and the resulting lysates were used for immunoprecipitation. One
quarter of the solution was reserved to assay input DNA. Immunoprecipitation
was performed by incubating the lysate with either anti-FLAG (Sigma, 1:100) or
anti-Histone (Upstate, 1:100) antibody for 1 hour at 4°C followed by
binding of antibody with protein A/G-agarose beads (Calbiochem). Beads without
antibody were used as a negative control. The immune complexes were washed
once with low-salt ChIP buffer (above), once with high-salt ChIP buffer (as
above, but 1 M NaCl), once with LiCl buffer [10 mM Tris-Cl (pH 8.0), 0.25 M
LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA] and twice with TE buffer. The
immunoprecipitates were eluted twice with 250 µl of elution buffer [100 mM
NaHCO3, 2%(w/v) SDS]. At this point, 10 µl of 10 mg/ml of RNAse
A was added in order to degrade RNA. The solution was incubated at 65°C
overnight to reverse cross-links, and treated with 5 µl of Proteinase K (10
mg/ml) to degrade any remaining proteins. After precipitating DNA with ethanol
for 30 minutes at -80°C, the precipitate was dissolved in 100 µl of TE
buffer and purified using a PCR-DNA purification column (Qiagen). To detect
precipitated DNA, PCR was performed using 36 cycles of 95°C for 45
seconds, 50°C for 30 seconds and 72°C for 45 seconds with primer sets
for ced-9 upstream region (LP,
5'-ATAGTTGATATACCAAATTGTGGGC-3'; RP,
5'-GGACAAAACTTCATTCCGACGT-3') and ced-9 coding region
(LP, 5'-CGGACAACTCGCTGACGAAT-3'; RP,
5'-GGCTCTTCCCAATCATTGAT-3'). |
RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Animals
homozygous for a null mutation in one of the genes (pax-2) are viable
and fertile, whereas animals homozygous for a null mutation in the other
(egl-38) die as young larvae. However, several reduction-of-function
but non-null alleles of egl-38 have been identified, allowing its
function to be assessed in a range of larval and adult tissues
(Chamberlin et al., 1999;
Chamberlin et al., 1997;
Trent et al., 1983;
Zhang et al., 2005). To
investigate whether C. elegans Pax2/5/8 genes affect apoptosis, we
assessed germline apoptosis in pax-2 and egl-38 mutants, and
found that animals mutant for either gene exhibit an increase in cell death
(Fig. 1). In addition, double
mutants between pax-2(ok935) and egl-38(n578), the
egl-38 allele with the strongest apoptosis defect, display an
increase in germline apoptosis that is approximately additive
(Fig. 1G-I). Thus, increased
germline apoptosis is a defect associated with mutants of both of the C.
elegans Pax2/5/8 genes.
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C. elegans Pax2/5/8 mutants exhibit inappropriate apoptosis of somatic cells
In order to determine whether pax-2 and egl-38 affect
somatic, as well as germline, cell survival, we observed double-mutant
embryos. We found that they exhibit a striking increase in the number of cell
corpses that are characteristic of apoptotic cell death
(Fig. 3A-E). Animals homozygous
for mutations in only one of the two genes also exhibit a small but
significant increase in cell corpses. As an additional assay for loss of
somatic cells, we used an unc-4::gfp reporter transgene that is
expressed in the VA motoneurons (Lickteig
et al., 2001; Miller and
Niemeyer, 1995). The VA motoneurons derive from the postembryonic
division of ventral cord P cells, a lineage that exhibits increased apoptosis
in other mutants, such as ced-9
(Hengartner et al., 1992). We
find that pax-2 egl-38 double mutants have fewer
unc-4::gfp-positive cells than do the wild type
(Fig. 3F). Consistent with an
increase in apoptosis, the pax-2(ok935) egl-38(n578) double mutants
are uncoordinated, and exhibit a level of embryonic and larval lethality (data
not shown). We conclude that the C. elegans Pax2/5/8 genes protect
somatic, as well as germline, cells from inappropriate apoptosis. Although
single mutants exhibit some somatic cell defects, the effect is significantly
enhanced when both pax-2 and egl-38 are mutant, indicating
that there is partial redundancy between the two genes for this function.
egl-38 and pax-2 act upstream of ced-9 in the core apoptotic pathway
To determine genetically where egl-38 and pax-2 act to
influence cell death, we constructed double mutants and assayed germline
apoptosis (Fig. 4B). We find
that apoptosis in egl-38 and pax-2 mutants is suppressed by
mutations in either the caspase gene ced-3 or the Apaf-1-related gene
ced-4, indicating that the cell deaths in Pax2/5/8 mutants
use the caspase-mediated apoptotic pathway. ced-3 and ced-4
are also epistatic to egl-38 and pax-2 in somatic cells
(Fig. 3G). As Pax2/5/8
and ced-9 loss-of-function mutants each exhibit the same defect of
increased apoptosis, we tested epistasis between these genes by comparing the
effect of induced hsp::pax-2 expression in wild type with that in
ced-9 mutants. We find that the germline cell survival effects
resulting from induced expression of PAX-2 require functional ced-9,
showing that the protection from apoptosis is mediated through the activity of
ced-9. Consistent with this gene order, induced expression of
ced-9 is also sufficient to bypass the cell-death defect in pax-2
egl-38 double mutants (see below).
Two main regulatory pathways influence germline apoptosis
(Fig. 4A). Normal physiological
germ-cell death requires the activity of a ras/MAPK pathway
(Gumienny et al., 1999).
Genotoxic treatments, such as ionizing radiation, also promote germline cell
death, and response to such treatments is mediated through checkpoint controls
and p53 (Derry et al., 2001;
Gartner et al., 2000;
Schumacher et al., 2001). We
constructed double mutants between key downstream genes in each of these
pathways, and in the Pax2/5/8 genes
(Fig. 4B,C). In general, we
found that the double mutants between pax-2 or egl-38 and
the MAP kinase gene mpk-1 or the p53 gene cep-1 exhibit
germline cell death patterns intermediate to either single mutant. Although
this result does not provide a strict epistatic relationship, we can conclude
that blocking either of these two regulatory pathways does not block the
production of dying cells in Pax2/5/8 mutants, arguing that the
Pax2/5/8 genes do not act upstream in either of these defined
regulatory pathways. Double mutant analysis between ced-9 and
regulatory genes such as mpk-1 and cep-1 has yielded similar
intermediate phenotypes, and it is possible that the intermediate pattern of
apoptosis reflects other germline functions for the genes under study
(Gartner et al., 2000;
Gumienny et al., 1999;
Lettre et al., 2004).
Collectively, we interpret that the Pax2/5/8 genes act upstream of
ced-9 in the core apoptotic pathway, but downstream or in parallel to
other regulatory pathways known to influence germline apoptosis.
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), into
egl-38(n578) animals. This transgene can be lost at cell division,
resulting in genetically mosaic animals. We selected mid-L4 animals from this
strain, and observed a panel of somatic cells derived from different embryonic
precursors to infer where the transgene was lost during development
(Fig. 5). These animals were
recovered and examined 24 hours later for germline apoptosis. As
sur-5::gfp can be detected in most somatic cells, but not germline
cells, we determined whether the transgene was present or absent in the
germline precursor (P4) by observing whether GFP was expressed in the
offspring. Animals with the transgene present in all somatic, as well as in
germline, cells exhibited germline apoptosis levels similar to wild type,
whereas animals with the transgene absent from all cells exhibited germline
apoptosis levels similar to egl-38(n578) mutants (an average of
3.04±0.18 and 6.93±0.21 corpses per gonad arm, respectively).
Mosaic animals (lacking the transgene in one or more embryonic precursor) that
retained the transgene in the germline were similar to wild type. In
particular, we focused on cells of the MS lineage (precursor to the somatic
gonad) and the E lineage (precursor to the intestine), as these cells surround
the germline cells and represent potential sources of survival signal(s).
However, we found that all 16 animals that retained the transgene in the
germline but lost it in either the MS lineage, or the E lineage, or both,
exhibited normal patterns of apoptosis (2.75±0.43 corpses per gonad
arm). Thus, egl-38 is not required in these somatic tissues to
promote germline cell survival. Animals that had lost the transgene in the
germline but retained it in somatic cells exhibited increased apoptosis
(4.03±0.28 corpses per gonad arm), although apoptosis levels were less
than those observed in animals lacking the transgene in all cells. This result
suggests that egl-38 may be able to influence germline survival by
functioning in somatic cell(s), in addition to its role in the germline.
Altogether, however, the mosaics support the idea that this Pax2/5/8
gene has a role within germline tissue to promote cell survival.
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)
(mev-1 is also known as cyt-1;
Fig. 7A). In operons of this
type, the genes are initially transcribed as a single RNA, which is then
processed through trans-splicing into separate mRNAs (reviewed by
Blumenthal and Gleason, 2003).
Thus, genes within an operon are subject to the same transcriptional
regulatory inputs. We characterized the mev-1 transcript in the
different genetic backgrounds, and found that the abundance of this gene is
altered in response to Pax2/5/8 activity in a manner similar to
ced-9. These results are consistent with a model in which the
Pax2/5/8 proteins alter the transcription of the mev-1 ced-9
operon.
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As mev-1 and ced-9 transcripts are altered in response to
Pax2/5/8 gene activity, we tested whether the operon could be a
direct target for the Pax2/5/8 proteins. Starting with a clone of genomic DNA
that includes both mev-1 and ced-9, we created a reporter
gene by introducing the coding sequences for GFP into the third exon of
ced-9 (Fig. 7). We
found that when this clone is introduced into animals as part of a transgene,
the GFP-tagged CED-9 is expressed broadly in pre-elongation embryos in a
cytoplasmic lattice-like pattern, as has been reported for the CED-9 protein
(Chen et al., 2000)
(Fig. 7B-I). In addition, we
found that CED-9::GFP is enriched in the gonadal region where germ cell
apoptosis is observed (Fig.
6E,I). A shorter reporter clone that lacks 
500 bp upstream of
mev-1 fails to show robust CED-9::GFP expression
(Fig. 6L,Q). This result
identifies that there are regulatory sequences upstream of mev-1 that
are important for the normal expression of ced-9, and is consistent
with mutant rescue experiments that showed sequences upstream of
mev-1 were required for optimal rescue of ced-9 mutant
defects (Hengartner and Horvitz,
1994). Within this functionally defined upstream regulatory
region, we identified one sequence with similarity to sequences bound by
Pax2/5/8 proteins. Mutant clones that delete or disrupt this site fail to
express abundant CED-9::GFP (Fig. 6L-N,
Q-S), showing that the site is necessary for the activity
associated with the full-length clone. We conclude that this site is important
for the normal expression of ced-9. The simplest interpretation based
on gene organization and the location of the functional sequence, is that the
site affects the transcription of the mev-1 ced-9 operon.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Transcriptional regulation of the mev-1 ced-9 operon by Pax2/5/8 factors
ced-9 is the downstream gene in a two-gene operon that includes
the succinate dehydrogenase cytochrome b subunit gene mev-1
(Hengartner and Horvitz,
1994). Our work suggests that altered ced-9 transcription
is sufficient to account for the cell death phenotype associated with the
Pax2/5/8 mutants, although the Pax2/5/8 proteins may also influence
the expression of other genes that affect cell survival. Mutations in
mev-1 confer reduced stress tolerance, and can promote inappropriate
somatic apoptosis, which is dependent on the caspase CED-3
(Senoo-Matsuda et al., 2003;
Senoo-Matsuda et al., 2001).
Although the specific role for mev-1 in apoptosis has not been
determined, mitochondria play a role in regulating cell death in C.
elegans, as they do in other organisms
(Jagasia et al., 2005), and,
thus, mev-1 may influence aspects of mitochondrial function important
for normal cell survival. Co-transcriptional regulation of the operon by the
Pax2/5/8 proteins provides a mechanism to coordinate the expression of genes
that have a protective role for the cell, or that contribute to mitochondrial
biogenesis.
Dynamic spatial and temporal regulation of ced-9 expression
We and others have found that ced-9 is not expressed uniformly in
all cells throughout the development of C. elegans
(Chen et al., 2000;
Hill et al., 2000). Indeed, in
contrast to what might be predicted with respect to its anti-apoptotic
function, ced-9 is enriched at times when the majority of normal
apoptosis takes place. Endogenous CED-9 protein, as well as our CED-9::GFP, is
most abundant in embryos as cells begin to leave the cell cycle and
differentiate or initiate apoptosis. We also observe enriched CED-9::GFP in
the region of the gonad where germline apoptosis takes place. Germline
expression has not been reported for the CED-9 protein
(Chen et al., 2000), but is
observed using ced-9 RNA in situ analysis (the Nematode Expression
Pattern DataBase,
http://nematode.lab.nig.ac.jp/),
suggesting that the difference between CED-9::GFP and CED-9 detected by
antibody may reflect a difference of stability or post-transcriptional
regulation between the endogenous and the GFP-tagged proteins. To explain why
ced-9 transcription is upregulated at times and locations where cell
death is most prevalent, we envisage that there are developmental conditions
or signals that promote apoptosis, and that CED-9 expression is raised at
these times to limit which cells ultimately die.
Transcription as a mechanism to influence cell death
The decision of a cell to undergo apoptosis is mediated by a regulatory
cascade that modulates the activity of proteins. However, the relative
abundance of these proteins is crucial to the activity of the cascade,
providing important parallel mechanisms to regulate the output of the pathway.
Increased transcriptional regulation of bcl-2 and related
pro-survival genes protect cells from apoptosis in mammals and C.
elegans (McDonnell et al.,
1989; Vaux et al.,
1988; Vaux et al.,
1992), and the bcl-2 gene was originally identified as a
constitutively overexpressed oncogene that contributes to follicular B-cell
lymphoma (Tsujimoto et al.,
1984). Our results demonstrate that transcriptional regulation of
ced-9/bcl-2 by Pax2/5/8 proteins contributes to normal cell survival,
and that cells are sensitive to the level of pax-2 and
egl-38 activity. This sensitivity identifies Pax2/5/8 protein
activity as a potential regulatory point to influence cell death, although
future experiments are required to test whether these factors normally respond
to regulatory input in vivo, or rather contribute more towards maintaining
basal levels of ced-9 transcription. We hypothesize that PAX-2 and
EGL-38 transcription of ced-9 provides a mechanism to influence cell
death decisions, as this mechanism is also observed for other cell death
pathway components. For example, in C. elegans, transcriptional
regulation of the pro-apoptotic BH3 domain-only gene egl-1 in both
germline and somatic cells influences whether a cell will die
(Hofmann et al., 2002;
Thellmann et al., 2003). As
EGL-1 inhibits CED-9 activity, the balance of expression between these two
genes can determine the cell death decision. We anticipate that, during normal
development, altering the transcription of core apoptosis pathway genes will
be a widely employed mechanism to reproducibly link cell fate and
apoptosis.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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-FLAG antibody. PCR of DNA recovered from
immunoprecipitation using