Max-Planck-Institute of Neurobiology, Am Klopferspitz 18a, D-82152 Planegg-Martinsried, Germany
Author for correspondence (e-mail:
barbara.conradt{at}dartmouth.edu)
Accepted 13 May 2003
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SUMMARY |
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Key words: Apoptosis, C. elegans, egl-1, Snail-like transcription factor, bHLH proteins
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INTRODUCTION |
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Programmed cell death is also a fundamental feature of neurogenesis in
invertebrates, including the nematode Caenorhabditis elegans. The
nervous system of a wild-type C. elegans hermaphrodite is composed of
302 neurons of at least 118 different types
(Sulston and Horvitz, 1977;
Sulston et al., 1983
). During
the development of a C. elegans hermaphrodite, 131 somatic cells die
by programmed cell death, a process that is determined by the essentially
invariant C. elegans cell lineage. Most of these 131 cells are
sisters of cells that differentiate into neurons and, when prevented from
undergoing programmed cell death, often adopt a neuronal fate themselves
(Ellis and Horvitz, 1986
;
Avery and Horvitz, 1987
).
Hence, more than a quarter of the cells that are initially generated and that
have the potential to form neurons are eliminated by programmed cell death
during the development of the C. elegans nervous system.
Genetic analyses of programmed cell death in C. elegans have been
instrumental for our current understanding of the conserved, molecular
mechanisms of this essential process (reviewed by
Horvitz, 1999). Four genes,
egl-1 (egl, egg-laying defective), ced-9
(ced, cell-death defective), ced-4 and ced-3, have
been identified, which, when mutated, can block programmed cell death during
development. These four genes act in a simple genetic pathway, in which
egl-1 negatively regulates ced-9, ced-9 negatively regulates
ced-4 and ced-4 positively regulates ced-3, the
activity of which is required for programmed cell death. The ced-3
gene encodes a pro-caspase, ced-4 an Apaf1-like adaptor,
ced-9 a Bcl-2-like cell-death inhibitor and egl-1 a
pro-apoptotic member of the Bcl-2 family, a BH3-only protein. Genetic and cell
biological observations indicate that CED-9, CED-4 and proCED-3 proteins are
present in most if not all cells during C. elegans embryogenesis. It
has been proposed that in cells that live, CED-9 blocks the activity of CED-4
and, hence, the CED-4-dependent activation of proCED-3. Conversely, in cells
destined to die, EGL-1 negatively regulates CED-9 thereby allowing the
activation of proCED-3 (Chen et al.,
2000
).
Little is known thus far about how the central cell-death pathway and, in
particular, the activity of its most upstream component, the BH3-only protein
EGL-1, is regulated to specifically cause the death of the 131 cells that are
destined to die during C. elegans development. EGL-1 is likely to be
regulated by cell-specific factors because genes have been identified that act
upstream of egl-1 and that, when mutated, block specific cell deaths.
For example, mutations in the genes ces-2 and ces-1
(ces, cell-death specification) block specifically the death of the
NSM sister cells, and of the NSM sister cells and the I2 sister cells
respectively (Ellis and Horvitz,
1991). Furthermore, a mutation in the gene tra-1
(tra, transformer) prevents the death of the hermaphrodite-specific
neurons (HSNs) in males (Conradt and
Horvitz, 1999
). In addition to being involved in the specification
of the male-specific death of the HSNs, the tra-1 gene has a more
general role during C. elegans development. tra-1 functions
as the terminal regulator of all somatic sexual fates in C. elegans
(reviewed by Goodwin and Ellis,
2002
). The cell-specific pathways that regulate EGL-1 activity
therefore may not be cell-death specific pathways but rather pathways that
play additional, important roles during development. tra-1 encodes a
zinc-finger DNA-binding protein, TRA-1A, which directly represses the
transcription of egl-1 in the HSNs of hermaphrodites but not in the
HSNs of males. At least in the HSNs, in which the life-versus-death decision
is determined by somatic sex, EGL-1 activity is therefore regulated at the
transcriptional level (Conradt and Horvitz,
1999
).
In contrast to the male-specific death of the HSNs, the death of the NSM
sister cells appears to be determined solely by lineage. Whether the signal
that triggers this particular death also does so by regulating egl-1
transcription has not yet been determined. As described above, two genes,
ces-1 and ces-2, have been identified that, when mutated,
can block the death of the NSM sister cells and therefore cause a cell-death
specification or Ces phenotype (Ellis and
Horvitz, 1991). A loss-of-function (lf) mutation in the gene
ces-2 blocks the death of the NSM sister cells, indicating that
ces-2 is required for their programmed death. The death of the NSM
sister cells is also blocked by a gain-of-function (gf) mutation in
ces-1, which suggests that ces-1 has cell-death protective
activity and that it can function to prevent the death of the NSM sister
cells. A ces-1(lf) mutation causes no obvious phenotype; however, it
suppresses the ability of the ces-2(lf) mutation to block the death
of the NSM sister cells, suggesting that ces-2 causes the NSM sister
cells to die by negatively regulating the cell-death protective activity of
ces-1. ces-2 encodes a DNA-binding protein most closely related to
the proline- and acid-rich (PAR) subfamily of basic leucine-zipper (bZIP)
transcription factors of vertebrates
(Metzstein et al., 1996
).
ces-1 encodes a DNA-binding protein most similar to members of the
Snail family of zinc-finger transcription factors
(Metzstein and Horvitz, 1999
).
cis-regulatory regions upstream of the ces-1 transcription
unit include a potential CES-2 binding site, which suggests that CES-2 might
be a direct, negative regulator of ces-1 transcription. The
ces-1(gf) mutation is located adjacent to this potential CES-2
binding site, which suggests that this mutation results in NSM sister cell
survival by causing overexpression of ces-1 in the NSM sister cells.
This hypothesis is supported by the observation that overexpression of
ces-1 from extrachromosomal arrays carrying the wild-type
ces-1 locus causes NSM sister cell survival
(Metzstein and Horvitz,
1999
).
Members of the Snail family of zinc-finger DNA-binding proteins act
predominantly as repressors of transcription (reviewed by
Hemavathy et al., 2000). It
has therefore been proposed that in ces-1(gf) animals, CES-1 blocks
the death of the NSM sister cells by blocking the transcription of a
proapoptotic gene (Metzstein and Horvitz,
1999
). As ces-1 acts upstream of the cell-death activator
gene egl-1, egl-1 is a candidate target of ces-1
(Conradt and Horvitz, 1998
). In
this paper we present data indicating that the basic helix-loop-helix (bHLH)
proteins HLH-2 and HLH-3 are at least partially required for the death of the
NSM sister cells and that a heterodimer composed of HLH-2 and HLH-3,
HLH-2/HLH-3, acts as a direct activator of egl-1 transcription.
Furthermore, we describe studies that suggest that in ces-1(gf)
animals, CES-1 acts as a direct repressor of egl-1 transcription by
antagonizing the function of HLH-2/HLH-3.
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MATERIALS AND METHODS |
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Molecular analysis and PCR mutagenesis
Standard molecular biology protocols were used unless otherwise noted.
Primers used throughout this study were based on sequences determined by the
C. elegans Sequencing Consortium
(The C. elegans Sequencing
Consortium, 1998). Plasmid pBC105 was generated by amplifying
his24-gfp from plasmid pJH2.19 (M. Dunn and G. Seydoux, personal
communication), using the polymerase chain reaction (PCR) and appropriate
primers. The PCR product was digested with SmaI and NcoI and
used to replace the SmaI-NcoI fragment of plasmid pBC99
(Conradt and Horvitz, 1999
).
Plasmid pBC08 (Conradt and Horvitz,
1998
) was used to generate plasmids pBC119, pBC13, pBC11, and
pBC149. Plasmid pBC148, which is based on pBluescript and contains a 2.9 kb
PstI-XhoI fragment of pBC08, including Region B, was used to
mutagenize the four Snail-binding sites/E-boxes in Region B. PCR-mediated
mutagenesis was performed. The sequence of the resulting plasmids carrying
four Snail-/E-box- sites (5'-CATATA-3')
(pBC170) or four Snail-/E-box+ sites
(5'-CATATA-3') (pBC180) was confirmed by sequence analysis using
an automated ABI sequencer (Applied Biosystems). A 1.4 kb
HpaI-PstI fragment, which includes the four Snail-binding
sites/E-boxes, of pBC170 and pBC180 was used to replace the
HpaI-PstI fragment of pBC08 and to generate plasmids pBC181
(Snail-/E-box-) and pBC182
(Snail-/E-box+). The plasmid for the expression in
E. coli of dsRNA of hlh-2 (pBC132A) was constructed by
subcloning the corresponding cDNA from plasmid pKM1199 into the BamHI
site of vector L4440 (Krause et al.,
1997
; Timmons et al.,
2001
). The plasmid for the expression of dsRNA of hlh-3
in vitro was obtained by subcloning the corresponding cDNA from pKM1195 into
the BamHI site of pBluescript to generate pBC226. Plasmids for the
expression of hlh-4, hlh-6, hlh-12 or hlh-14 in E.
coli were obtained by PCR-amplifying coding regions of the genes from
genomic DNA using the following primers: 5'-gaa ggg atc ctg ttc tga aac
aac atc ttcc aac g-3' and 5'-gaa ggg atc cgc agt tga tgg ttg ata
gaa ata tg-3' for hlh-4, 5'-gaa ggg atc caa ttc cac att
cca act tcc-3' and 5'-gaa ggg atc ccc aaa ctg atg agc tga aaa
tt-3' for hlh-6, 5'-gaa ggg atc cag cca cct ctt aca taa
ttc-3' and 5'-gaa ggg atc cat ata aac att ggt ttg ggg-3' for
hlh-12, 5'-gaa ggg atc cct gag ctc aga ttt tca g-3' and
5'-gaa ggg atc ctg cgt tct ctc tca ttt ctg-3' for hlh-14.
PCR fragments were digested with BamHI and ligated into pBluescript
to generate plasmids pBC227 (hlh-4), pBC228 (hlh-6), pBC229
(hlh-12) and pBC230 (hlh-14).
Transgenic animals
Germline transformation was performed as described by Mello and Fire
(Mello and Fire, 1995). For
transformation with the Pegl-1his24-gfp reporter,
ced-3(n717); lin-15(n765) animals were
injected with plasmid pBC105 (Pegl-1his24-gfp)
(2.5 ng/µl) and the co-injection marker pL15-EK (50 ng/µl), which
rescues the lin-15(n765) multivulva or Muv phenotype
(Clark et al., 1994
). Injected
animals were shifted to 25°C, and non-Muv F1 animals were picked to
establish transgenic lines. The line carrying the extrachromosomal array
bcEx78 was used to integrate the array into the genome.
ced-3(n717); lin-15(n765); bcEx78
animals were mutagenized using ethyl methanesulfonate (EMS)
(Brenner, 1974
) and transgenic
F2 animals were selected that gave rise to 100% non-Muv progeny at 25°C.
The strain carrying the integration bcIs37 V was backcrossed five
times to ced-3(n717); lin-15(n765). For
transformation with the Ptph-1gfp reporter, we
injected lin-15(n765) animals with plasmid pBY668
(Ptph-1gfp)
(Rohrig et al., 2000
) (50
ng/µl) and pL15-EK (50 ng/µl). Transgenic lines were selected and
maintained at 25°C. The line carrying the array bcEx113 was used
for integration. The strains carrying the integrations bcIs24, bcIs25
and bcIs30 were backcrossed four times to N2. For NSM sister cell
death rescue, egl-1(n1084 n3082)
unc-76(e911); lin-15(n765) bcIs24
animals were injected with the egl-1 rescuing plasmids pBC08 (2.0
ng/µl), pBC119 (1.5 ng/µl), pBC13 (1.1 ng/µl), pBC11 (1.0 ng/µl),
or pBC149 (1.1 ng/µl) and the co-injection marker p76-16B (75 ng/µl),
which rescues the uncoordinated or Unc phenotype of
unc-76(e911) animals
(Bloom and Horvitz, 1997
).
(egl-1 rescuing fragments are toxic and were therefore injected at
such low concentrations.) Non-Unc F1 animals were selected to establish
transgenic lines. ces-2(n732); bcIs25;
egl-1(n1084 n3082) unc-76(e911) animals were
injected with the egl-1 rescuing plasmids pBC08 (2 ng/µl), pBC181
(2 ng/µl) or pBC182 (2 ng/µl) and p76-16B (75 ng/µl). bcIs1
is an integrated array of plasmid pBC99
(Pegl-1gfp) (2.0 ng/µl)
(Conradt and Horvitz, 1999
) and
the co-injection marker pL15-EK (50 ng/µl).
Electrophoretic mobility shift assays and protein production
For electrophoretic mobility shift assays, probes were generated by PCR
amplification in the presence of 10 µCi [32P]-dATP using the
primers 5'-aac tca tcc acg tca cca aa-3' and 5'-ttg tcc act
cgt tta cca ca-3' and plasmids pBC08 (wild-type), pBC181
(Snail-/E-box-) or pBC182
(Snail-/E-box+) as templates. The labeled PCR products
were purified on a 6% acrylamide/TBE gel. A GST-CES-1 zinc-finger fusion
protein construct, expressing GST fused to amino acids 117-270 of CES-1
(referred to as GST-CES-1), was made as described by Metzstein et al.
(Metzstein et al., 1999). GST-CES-1 was produced and purified from E.
coli as described by Ip et al. (Ip et
al., 1992) (4.25 ng represents 1x10-13 moles).
Expression plasmids for His6-HLH-2 (pKM1199) and
His6-HLH-3 (pKM1195) fusion proteins were provided by M. Krause
(Krause et al., 1997
).
His6-HLH-2 and His6-HLH-3 fusion proteins were produced
in E. coli strain BL21-CodonPlus(DE3)-RIL (Stratagene) and purified
as described (Portman and Emmons,
2000
). 5.0 ng and 3.2 ng of His6-HLH-2 and
His6-HLH-3 fusion protein, respectively, represents
1x10-13 mol. The purity and concentration of the fusion
proteins were assessed by SDS-PAGE and the BioRad protein assay (BioRad).
EMSAs were performed as described for HLH-2 and HLH-3, and CES-1
(Krause et al., 1997
;
Metzstein and Horvitz, 1999
).
Binding was quantified using a phosphoimager (Fujifilm BAS-2500) and
appropriate software (Aida Image Analyzer V. 3.21).
RNAi experiments
For RNAi experiments using feeding as the method of delivering dsRNA,
plasmids pBC132A, pBC124A, pBC188, pBC189, pBC190 and pBC191 were transformed
into E. coli strain HT115
(Timmons et al., 2001). NGM
plates containing 6 mM IPTG, 50 µg/ml ampicillin and 12.5 µg/ml
tetracycline were inoculated with transformed HT115 bacteria. The expression
of dsRNA was induced overnight at room temperature. The plates were
subsequently inoculated with L4 larvae. Animals were cultured at 15°C and
their progeny was analysed. For RNAi experiments using injection as the method
of delivering dsRNA, plasmids pBC226, pBC227, pBC228, pBC229 and pBC230 were
used to PCR-amplify their inserts flanked by the T3 and T7
promoter using appropriate primers. PCR products were used as templates for in
vitro transcription using the T3 and T7 polymerases
(Promega). Reactions contained 100 U T3 or T7 polymerase, 2
mM each rATP, rCTP, rGTP, rUTP, 10 mM DTT in a final volume of 100 µl in
DEPC-H2O buffered with transcription buffer. After incubating for 2
hours at 37°C, five units DNase was added and the reaction incubated for
another 20 minutes at 37°C. The RNA was purified by phenol/chloroform
extraction and resuspended in 20 µl DEPC-H2O. Single stranded
sense RNA was annealed with an equal amount of the corresponding antisense RNA
at 37°C for 20 minutes, centrifuged at 4°C for 10 minutes and injected
into the gonad of young adult worms, which were subsequently incubated at
25°C. The progeny of injected animals was analysed.
Analysis of Pegl-1 his24-gfp expression
and NSM sister cell survival
For the analysis of Pegl-1 his24-gfp
expression in the NSMs and NSM sister cells, the NSMs and NSM sister cells
were identified in L1 larvae using Nomarski microscopy as described
(Ellis and Horvitz, 1991) and
analysed for his24-gfp expression using epifluorescence. Percent NSM
sister cell survival was determined in L3 or L4 larvae carrying the
Ptph-1gfp reporter using a combination of
Nomarski microscopy and epifluorescence. To obtain the number of surviving NSM
sister cells per animal, the total number of GFP-positive cells in the
anterior pharynx of an animal was determined and subtracted by two. Percent
NSM sister cell survival represents the percent of NSM sister cells that
survived and n the maximum number of NSM sister cells that could have survived
in the number of animals analysed.
Immunohistochemistry
Embryos were prepared in 10 µl H2O on poly L-Lysine coated
slides and allowed to develop until the 1.5-fold stage in a moist chamber.
They were fixed with 5% paraformaldehyde and stained as described
(Krause et al., 1990;
Krause et al., 1997
). HLH-2
was detected using a polyclonal anti-HLH-2 antibody raised in rabbits
(provided by Mike Krause) and GFP was detected using a monoclonal anti-GFP
antibody (Clontech). Immunofluorescence was viewed using a Leica TCS NT
confocal microscope.
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RESULTS |
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In the case of another specific cell death, the death of the HSNs in males,
it has been shown that the decision between life and death is specified by
egl-1 expression (Conradt and
Horvitz, 1999). To determine whether egl-1 expression
also specifies the cell-death fate of the NSM sister cells, we monitored the
expression of egl-1 in these cells, using an integrated
Pegl-1his24-gfp transgene. Pegl-1his24-gfp expresses a
nuclearly localized fusion of the His24 protein and the green fluorescent
protein (GFP) under the control of the cis-regulatory regions of the
egl-1 gene. The NSM sister cells normally die during a stage called
the 1.5-fold stage of embryogenesis. At this stage of development it is
difficult to identify cells on the basis of their position within the embryo.
We therefore analysed the expression of Pegl-1his24-gfp in the
background of the ced-3 lf mutation n717.
ced-3(n717) blocks programmed cell death, including the death of
the NSM sister cells (Ellis and Horvitz,
1986
). However, because ced-3 acts downstream of
egl-1 genetically, ced-3(n717) should not interfere
with the expression of the Pegl-1his24-gfp transgene. This
experimental design enabled us to analyse the expression of
Pegl-1his24-gfp in the NSMs and in surviving `undead' NSM sister
cells in animals of the first larval stage of development (L1 larvae), in
which these cells are identifiable by position in the anterior pharynx using
Nomarski differential interference contrast (Nomarski microscopy)
(Ellis and Horvitz, 1991
). In
ced-3(n717); Pegl-1his24-gfp larvae, we observed GFP in 88%
of the NSM sister cells, which normally die, and in 0% of the NSMs, which
normally survive (n=51) (Fig.
1A). This indicates that the activity of EGL-1 is regulated at the
transcriptional level in the NSMs and NSM sister cells and, hence, that the
cell-death fate of these cells is specified by egl-1 expression.
|
Region B of the egl-1 locus is required for the death of the
NSM sister cells in vivo
The egl-1 lf mutation n1084 n3082 blocks programmed cell
death, including the death of the NSM sister cells. A 7660 bp genomic fragment
of cosmid C01G9, pBC08, which includes the egl-1 transcription unit,
1036 bp of its upstream region and 5575 bp of its downstream region, when
introduced as an extrachromosomal array, can rescue the cell-death defect of
egl-1(n1084 n3082) animals, including the death of the NSM
sister cells (Fig. 2)
(Conradt and Horvitz, 1998). We
therefore conclude that pBC08 contains the cis-regulatory region or
regions required for the expression of egl-1 in the NSM sister cells.
To determine which region or regions of pBC08 are specifically required for
the expression of egl-1 in these cells, we analysed subclones of
pBC08 for their ability to rescue the death of the NSM sister cells in
egl-1(n1084 n3082) animals
(Fig. 2A).
|
As mentioned above, in wild-type animals, 100% of the NSM sister cells die;
however, 96% of them survive in egl-1(n1084 n3082) animals.
In transgenic egl-1(n1084 n3082) animals carrying
extrachromosomal arrays of the egl-1 rescuing fragment pBC08, between
35% and 5% of the NSM sister cells survived (6/6 lines), indicating that pBC08
rescues the death of the NSM sister cells
(Fig. 2B). (Rescue was defined
as 40% or less NSM sister cell survival.) Similarly, subclone pBC119, which
includes 3447 bp of the region downstream of the egl-1 transcription
unit, rescued the cell-death defect of egl-1(n1084 n3082)
animals (8/8 lines). By contrast, subclones pBC13 (806 bp of downstream
region) and pBC11 (377 bp of downstream region) failed to rescue (0/4 lines
and 0/5 lines). pBC13 lacks 2641 bp of pBC119 that includes a 352 bp region,
called Region B, which is conserved between the egl-1 locus of C.
elegans and C. briggsae (Fig.
2A,C) (see below). As sequence conservation between these two
Caenorhabditis species suggests functional relevance
(Heschl and Baillie, 1990), we
tested whether addition of Region B alone can restore the rescuing activity of
pBC11 (pBC149). We found that pBC149 rescued NSM sister cell death in four out
of seven lines (4/7). Conversely, pBC08 lacking only Region B (pBC165) failed
to rescue (0/6 lines). These results indicate that Region B of the
egl-1 locus is required to rescue the death of the NSM sister cells
in egl-1(n1084 n3082) animals. As the death of the NSM
sister cells is dependent on the expression of egl-1 in the NSM
sister cells, Region B most probably contains the cis-regulatory regions
necessary for the transcription of egl-1 in these cells.
Region B contains four conserved Snail-binding sites to which a
GST-CES-1 fusion protein can bind in vitro
An alignment of the sequence of Region B of the C. elegans egl-1
locus with the sequence of the corresponding region of the C. briggsae
egl-1 locus, revealed extensive sequence conservation at the nucleotide
level. Over their entire length, the sequences are 76% identical
(Fig. 2C). Sequence inspection
revealed that Region B contains four closely spaced Snail-binding sites,
DNA-binding sites for members of the Snail family of zinc-finger transcription
factors (Hemavathy et al.,
2000). The core motif of three of these binding sites is
completely conserved in C. briggsae (binding sites I, II and IV; 6/6
bases identical), and one of them has one base change in C. briggsae
(binding site III; 5/6 bases identical). The sequence of binding sites I and
II of C. elegans is a perfect match to the sequence of the core motif
of a consensus Snail-binding site (5'-CACCTG-3') whereas the
sequences of binding sites III and IV have one mismatch to the consensus
sequence (5'-CATCTG-3' and
5'-CAGCTG-3', respectively).
CES-1 is a member of the Snail family of DNA-binding proteins and has
previously been shown to bind to Snail-binding sites in vitro
(Metzstein and Horvitz, 1999).
In addition, in ces-1(gf) animals, CES-1 prevents the death of the
NSM sister cells by blocking egl-1 expression. To determine whether
in ces-1(gf) animals, CES-1 might block the transcription of
egl-1 directly by binding to the four Snail-binding sites in Region
B, we tested whether CES-1 can bind to these sites in vitro, using
electrophoretic mobility shift assays (EMSAs). A bacterially produced,
affinity-purified CES-1 fusion protein consisting of the C-terminal half of
CES-1 (which includes all five zinc fingers of CES-1) fused to glutathione
S-transferase (GST) could bind and shift a radioactively labeled 390 bp DNA
fragment consisting of wild-type Region B, including the four Snail-binding
sites (Fig. 3, lanes 1-6). Long
exposures and the use of various fragments of Region B indicate that CES-1 can
bind to at least three of the four Snail-binding sites in vitro (data not
shown). Binding of CES-1 to Region B was severely reduced after the
introduction of point mutations that destroy the core motif of the four
Snail-binding sites (5'-CACCTG-3' to
5'-CATATA-3')
(Fig. 3, lanes 7-12). Using an
amount of CES-1 that binds about 50% of the wild-type probe (set to 100%
binding), CES-1 binding was reduced to on average 6% (n=2)
(Fig. 3, compare lanes 4 and
10). Intact Snail-binding sites are therefore required for the ability of
CES-1 to bind to Region B in vitro. The specificity of the observed binding
was furthermore confirmed by competition experiments, using the wild-type
Region B as a probe and a wild-type or mutant Snail-binding site as competitor
(data not shown).
|
The C. elegans bHLH genes hlh-2 and hlh-3
are required for the programmed death of the NSM sister cells
Region B of the egl-1 locus is required to rescue the death of the
NSM sister cells in egl-1(lf) mutants and is therefore most likely to
be necessary for the expression of egl-1 in these cells. We therefore
sought to identify transcriptional activators that are required for the death
of the NSM sister cells and that act through Region B. The core motif of a
Snail-binding site (5'-CACCTG-3') also represents an E-box motif
(5'-CANNTG-3'), the DNA-binding site for members of the family of
bHLH DNA-binding proteins, many of which function as transcriptional
activators (reviewed by Massari and Murre,
2000). Indeed, it has been suggested that members of the Snail
family of transcription factors can functionally antagonise bHLH proteins by
competing for binding to Snail-binding sites/E-boxes
(Fuse et al., 1994
;
Kataoka et al., 2000
;
Nakayama et al., 1998
). We
therefore set out to test whether C. elegans bHLH proteins are
involved in the specification of the NSM sister cell death in vivo. We were
particularly interested in the C. elegans homologues of neuronal bHLH
proteins, which can be divided into two families: the Achaetescute
complex-related proteins and the Atonal-related proteins. The Atonal-related
proteins are further subdivided into three groups: the Neurogenin group, the
NeuroD group and the ATO group (reviewed by
Hassan and Bellen, 2000
;
Lee, 1997
). As tissue-specific
bHLH proteins form DNA-binding heterodimers with ubiquitously expressed E
proteins or Daughterless-like proteins, we also analysed C. elegans
homologues of this class of bHLH proteins. The C. elegans genome
contains at least 35 bHLH genes, including one
daughterless-like, five achaete-scute-like, one
atonal-like and one NeuroD-like gene (reviewed by
Ledent and Vervoort, 2001
).
Using existing mutants and RNA-mediated interference (RNAi)
(Fire et al., 1998
), we tested
whether these genes are involved in specifying the death of the NSM sister
cells.
The only C. elegans daughterless-like gene, hlh-2, has so
far only been defined by weak lf mutations, such as bx108, which were
identified in a screen for enhancers of the phenotype caused by a weak lf
mutation of lin-32, the only C. elegans atonal-like gene
(Portman and Emmons, 2000).
bx108 is a missense mutation in the helix-loop-helix dimerization
domain of HLH-2 and is predicted to affect the ability of the protein to form
heterodimers with other bHLH proteins such as LIN-32
(Portman and Emmons, 2000
).
However, bx108 has so far not been shown to cause a phenotype in an
otherwise wild-type background. The inactivation of hlh-2 by RNAi
results in embryonic lethality, indicating that hlh-2 is essential
for development (Krause et al.,
1997
). To determine whether reducing hlh-2 function has
an effect on the survival of the NSM sister cells, we analysed
hlh-2(bx108) animals and `escapers' of
hlh-2(RNAi) for the survival of NSM sister cells using the
Ptph-1gfp reporter. We found that hlh-2(bx108)
leads to the survival of up to 5% of the NSM sister cells, an effect that is
temperature sensitive (Table
1A). Furthermore, we found that 15% of the NSM sister cells
survived in hlh-2(RNAi) embryos that escaped early
developmental arrest and developed to a stage, at which the Ptph-1gfp
reporter is expressed (Table
1A).
|
The C. elegans atonal and NeuroD-like genes
lin-32 (lin, lineage abnormal) and cnd-1 (cnd,
C. elegans NeuroD), respectively, have been defined by non-lethal, strong
lf mutations (Zhao and Emmons,
1995; Hallam et al.,
2000
). To determine whether these two genes are involved in
specifying the NSM sister cell death, we analysed NSM sister cell survival in
animals carrying lf mutations in either gene. In cnd-1(ju29)
animals, 0% of the NSM sister cells survived (n=60), indicating that
the C. elegans NeuroD homologue is probably not required for the NSM
sister cell death. Similarly, lf mutations of lin-32 did not affect
NSM sister cell survival in an otherwise wild-type background or in a
hlh-2(bx108) background (data not shown).
The five C. elegans achaete-scute-like genes (hlh-3, hlh-4, hlh-6, hlh-12 and hlh-14) have so far not been defined by mutations. For this reason, we used RNAi to analyse their potential role in the specification of the NSM sister cell death. No effect on NSM sister cell survival was observed in hlh-4(RNAi), hlh-6(RNAi), hlh-12(RNAi) or hlh-14(RNAi) animals (Table 1B and data not shown). hlh-3(RNAi), however, caused 7% of the NSM sister cells to survive (n=178), indicating that hlh-3 is at least partially required for the death of the NSM sister cells (Table 1B). Moreover, hlh-3(RNAi) [but not hlh-4(RNAi), hlh-6(RNAi), hlh-12(RNAi) or hlh-14(RNAi)] increased NSM sister cell survival in hlh-2(bx108) animals from 4% to 30%. hlh-2 and hlh-3 therefore might act together to cause the death of the NSM sister cells.
To determine whether reducing hlh-2 and hlh-3 function
causes a general block in programmed cell death or specifically results in the
survival of the NSM sister cells, we analysed the survival of other cells that
normally die in hlh-2(bx108); hlh-3(RNAi)
animals using Nomarski microscopy. During the development of the anterior
pharynx, 16 cells undergo programmed cell death and mutations that block
programmed cell death in general, such as egl-1(n1084
n3082), block many of these cell deaths. egl-1(n1084
n3082) animals therefore have on average about 12 extra cells in this
part of the pharynx (Conradt and Horvitz,
1998). We found that hlh-2(bx108);
hlh-3(RNAi) animals have on average 1.3 extra cells
(n=16). 57% of these extra cells were undead NSM sister cells, as
confirmed by the position of their nuclei and by Ptph-1gfp
expression, and 23% possibly were undead m2 sister cells, as determined by the
position of their nuclei. We were unable to determine the identity of 20% of
the extra cells. Therefore, the majority of the surviving cells are NSM sister
cells. Reducing the activity of hlh-2 and hlh-3, hence,
results in the survival predominantly of the NSM sister cells.
hlh-2 and hlh-3 act downstream of or in parallel to
ces-1 to kill the NSM sister cells
The ability of the ces-2 lf mutation n732 to cause NSM
sister cell survival depends on a functional ces-1 gene, which
suggests that ces-1 acts downstream of ces-2. To determine
whether the ability of hlh-2(RNAi) and
hlh-3(RNAi) to cause NSM sister cell survival similarly
depends on a functional ces-1 gene, we tested whether the
ces-1 lf mutation n703 n1434 can block the NSM sister cell
survival observed in hlh-2(RNAi) and
hlh-3(RNAi) animals. In hlh-2(RNAi)
animals, 14% of the NSM sister cells survived and in ces-1(n703
n1434lf) animals treated with control RNA, 0% survived (dsRNA delivered
by feeding) (Timmons et al.,
2001) (Fig. 4A). In
ces-1(n703 n1434lf); hlh-2(RNAi) animals,
14% of the NSM sister cells survived. Similarly, in
hlh-3(RNAi) animals, 6% of the NSM sister cells survived and
in ces-1(n703 n1434lf) animals treated with control RNA, 1%
survived (dsRNA delivered by injection)
(Fire et al., 1998
). In
ces-1(n703 n1434lf); hlh-3(RNAi) animals,
5% of the NSM sister cells survived (Fig.
4B). These data indicate that a functional ces-1 gene is
not required for the ability of hlh-2(RNAi) or
hlh-3(RNAi) to cause NSM sister cell survival, which
suggests that ces-1 is not acting downstream of hlh-2 and
hlh-3. hlh-2 and hlh-3 therefore act downstream of or in
parallel to ces-1 to kill the NSM sister cells.
|
|
|
As shown above, CES-1 and HLH-2/HLH-3 bind to wild-type Region B with the
four intact Snail-binding sites/E-boxes (referred to as `wild-type' sites) in
vitro but fail to efficiently bind to mutant Region B with the four
Snail-binding sites/E-boxes mutated to
5'-CATATA-3' (referred to as
`Snail-/E-box-' sites)
(Fig. 3, Fig. 5B). In contrast to the
consensus sequence for Snail binding (5'-CACCTG-3'), bases 3 and 4
of the consensus sequence for bHLH binding can be variable
(5'-CANNTG-3') (Massari and
Murre, 2000). We therefore tested whether mutating the four
Snail-binding sites/E-boxes in Region B to 5'-CATATG-3'
(referred to as `Snail-/E-box+' sites) would disrupt
CES-1 binding but still allow the binding of bHLH proteins such as HLH-2/HLH-3
in vitro. As shown in Fig. 3, CES-1 binding to a probe consisting of Region B, in which the four
Snail-binding sites/E-boxes have been mutated to
Snail-/E-box+, is severely reduced when compared with
CES-1 binding to the wild-type probe (Fig.
3, compare lanes 1-6 with lanes 13-18). Using an amount of CES-1
protein that is sufficient to bind about 50% of the wild-type probe (100%
binding), the introduction of the Snail-/E-box+ mutation
reduced binding to on average 6% (n=2) (compare
Fig. 3, lanes 4 and 16). On the
contrary, using an amount of HLH-2/HLH-3 that was sufficient to bind about 50%
of the wild-type probe (100% binding), the introduction of the
Snail-/E-box+ mutation only reduced binding to on
average 17% (n=3) (Fig.
5B; compare lanes 4 and 16). At least in vitro, the introduction
into Region B of Snail-/E-box+ mutations therefore
affects the binding of CES-1 more dramatically than the binding of
HLH-2/HLH-3.
Functional E-boxes in Region B are required to kill the NSM sister
cells and functional Snail-binding sites for the ability of CES-1 to cause
their survival
To determine the effect of bHLH binding and CES-1 binding to the four
Snail-binding sites/E-boxes in Region B in vivo, we introduced the
Snail-/E-box- and Snail-/E-box+
mutations into Region B of the rescuing fragment pBC08 and analysed the
ability of the resulting fragments to rescue NSM sister cell death in
ces-2(n732ts); egl-1(n1084 n3082) animals.
n732ts is a temperature sensitive lf mutation of ces-2: 13%
of the NSM sister cells survive in ces-2(n732ts) animals
raised at 15°C, the permissive temperature; and 73% survive in animals
raised at 25°C, the non-permissive temperature
(Fig. 7)
(Ellis and Horvitz, 1991).
ces-2(n732ts) has been proposed to cause NSM sister cell
survival as a result of ces-1 overexpression in the NSM sister cells
(Metzstein and Horvitz, 1999
).
Culturing transgenic ces-2(n732ts); egl-1(n1084
n3082) animals at 15°C and 25°C therefore allowed us to analyse
the ability of the fragments to rescue the NSM sister cell death defect caused
by egl-1(n1084 n3082) in the presence of slightly or
strongly elevated levels of CES-1 protein in the NSM sister cells,
respectively.
|
The results obtained with the Snail-/E-box+ fragment furthermore indicate that the ability of high levels of CES-1 to block the activator of egl-1 transcription is dependent on functional Snail-binding sites in Region B of the egl-1 locus. As CES-1 and HLH-2/HLH-3 therefore act through overlapping DNA-binding sites, the NSM sister cells might survive in ces-2(n732ts) animals grown at 25°C because high levels of CES-1 protein successfully compete with HLH-2/HLH-3 for binding to Region B in the egl-1 locus.
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DISCUSSION |
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hlh-2 and hlh-3 have so far not been defined by null
mutations. It is therefore unclear whether the low penetrance of the Ces
phenotype observed is due to additional factors that can kill the NSM sister
cells in the absence of hlh-2 and hlh-3 or residual
hlh-2 and hlh-3 activity in the NSM sister cells. It has
been shown that C. elegans neurons are more resistant to the
inactivation of gene function by RNAi than other cell types
(Timmons et al., 2001). As
this also appears to be the case for the NSMs and NSM sister cells (J.H. and
B.C., unpublished), we favour the possibility that the low penetrance of the
Ces phenotype observed is the result of the incomplete inactivation of
hlh-2 and hlh-3 in the NSM sister cells. The inactivation by
mutation of the Snail-binding sites/E-boxes in the egl-1 locus,
through which we propose HLH-2/HLH-3 activates egl-1 transcription in
the NSM sister cells, results in the complete failure to kill NSM sister
cells. This observation suggests that, if additional factors exist that can
kill the NSM sister cells in the absence of hlh-2 and hlh-3,
they are most likely to be additional E-box binding proteins, i.e. additional
bHLH proteins.
So far one lf mutation of ces-2, three gf mutations of
ces-1 (which were found to carry the identical molecular lesion) and
one lf mutation of ces-3, a gene that acts upstream of or in parallel
to ces-1 and that remains to be characterized at the molecular level,
have been identified in genetic screens for mutants, in which the NSM sister
cells survive (Ellis and Horvitz,
1991). However, these screens failed to recover mutations in
hlh-2 and hlh-3. The failure to identify mutations in these
two genes is possibly the result of one or more of the following observations.
First, the fact that ces-2 and ces-3 have so far only been
defined by one lf allele suggests that the screens performed to date were not
saturating. Second, hlh-2(RNAi) results in embryonic
lethality (Krause et al.,
1997
). However, previous screens were performed in a way that did
not allow the identification of mutations in essential genes. Finally, it is
possible that the complete inactivation of hlh-2 and hlh-3
results in a NSM sister cell survival phenotype with low penetrance,
decreasing the likelihood of identifying mutations in these genes
accordingly.
bHLH proteins play key roles in the specification and execution of cell
fates, including neuronal fates, in various organisms (reviewed by
Lee, 1997;
Massari and Murre, 2000
). For
example, in C. elegans, the daughterless-like gene
hlh-2 and the atonal-like gene lin-32 function at
multiple steps during the establishment of a specific neuronal sublineage,
which gives rise to two neurons and one support cell
(Portman and Emmons, 2000
). To
our knowledge, however, it has not yet been demonstrated that bHLH proteins
play a direct role in the activation of programmed cell death. Our results
indicate that the daughterless-like and achaete-scute-like
genes hlh-2 and hlh-3 of C. elegans are at least
partially required for the execution of the programmed cell death fate of the
NSM sister cells. Furthermore, hlh-2 and hlh-3 may not be
required for the specification and execution of the neuronal fate of the NSMs,
as the Ptph-1gfp reporter continues to be expressed in the NSMs in
animals, in which hlh-2 and/or hlh-3 have been at least
partially inactivated. If hlh-2 and hlh-3 function in the
specification or execution of the NSM fate as well, the requirement for their
function for this process is less stringent than the requirement for their
function in promoting the programmed death of the NSM sister cells.
The NSM sister cells might survive in ces-2(lf) and
ces-1(gf) animals, because high levels of CES-1 prevent HLH-2/HLH-3
from binding to the Snail-binding sites/E-boxes in the egl-1
locus
It has been proposed that the NSM sister cells survive in
ces-2(lf) or ces-1(gf) animals as a result of ces-1
overexpression in the NSM sister cells
(Metzstein and Horvitz, 1999).
We have shown that CES-1 can bind to Snail-binding sites/E-boxes in the
egl-1 locus in vitro, which are required for the ability of the
ces-2 lf mutation n732 to block the death of the NSM sister
cells in vivo. This suggests that in ces-2(lf) and ces-1(gf)
animals, CES-1 blocks the death of the NSM sister cells by directly repressing
egl-1 transcription.
Based on our finding that an activator of egl-1 transcription in the NSM sister cells, most probably HLH-2/HLH-3, acts through the identical Snail-binding sites/E-boxes in the egl-1 locus, we propose a molecular model for how CES-1 can repress egl-1 transcription (Fig. 8). We propose that in wild-type animals, in which CES-1 most likely is absent or present at low levels in the NSM sister cells, HLH-2/HLH-3 binds to the Snail-binding sites/E-boxes in the egl-1 locus thereby activating egl-1 transcription in the NSM sister cells, which results in the death of these cells. In ces-2(lf) or ces-1(gf) animals, in which CES-1 levels in the NSM sister cells most likely are elevated, sufficient CES-1 protein is present to successfully bind to the Snail-binding sites/E-boxes thereby preventing HLH-2/HLH-3 from binding to these sites and from activating egl-1 transcription. This molecular model implies that in wild-type animals, CES-1 might play no role in specifying the cell-death fate of the NSM sister cells. This is supported by the fact that the ces-1 lf mutation n703 n1434 does not cause a phenotype in the NSM sister cells. Our model also suggests that in ces-1(gf) animals, CES-1 acts by blocking the function of HLH-2/HLH-3, which is supported by our finding that genetically ces-1 acts upstream of or in parallel to hlh-2 and hlh-3.
|
It has previously been shown that the function of bHLH proteins can be
antagonized by Snail-like proteins. For example, the Snail-like protein
Escargot of Drosophila can repress the E-box-dependent
transcriptional activation of a reporter gene by the bHLH proteins
Daughterless and Scute in transfection assays
(Fuse et al., 1994). In
addition, it has been shown that the murine Snail-like protein mSna competes
with a homodimer composed of the Daughterless-like protein E47 and with a
heterodimer composed of E47 and the Achaetescute-like protein MASH-2 for
binding to E-boxes in vitro and in cultured cells
(Nakayama et al., 1998
).
Finally, the murine Snail-like protein Smuc can block the binding of a
heterodimer composed of the Daughterless-like protein E12 and the bHLH protein
MyoD to E-boxes in vitro and represses E12/MyoD-dependent activation of a
reporter gene in transfection experiments
(Kataoka et al., 2000
). Our
results now demonstrate that by binding to the Snail-binding sites/E-boxes in
cis-regulatory regions of the egl-1 locus in vivo, the
Snail-like protein CES-1 of C. elegans blocks the ability of
HLH-2/HLH-3 to activate the cell-death activator gene egl-1 and to
execute the cell-death fate of the NSM sister cells.
Transcriptional activation of the BH3-only gene
egl-1 is a common mechanism of cell-death execution
The cell death of at least two types of neurons in C. elegans is
dependent on the transcriptional activation of the BH3-only gene
egl-1: the male-specific death of the HSN neurons and the death of
the NSM sister cells. Transcriptional regulation might therefore be an
important mechanism through which the activity of EGL-1 is regulated. The
activity of at least four of the 10 mammalian BH3-only proteins identified to
date is regulated at the transcriptional level (reviewed by
Puthalakath and Strasser,
2002). For example, after DNA damage, the BH3-only genes
Noxa and Puma/Bbc3 are transcriptionally upregulated in a
p53-dependent manner in thymocytes and fibroblasts
(Han et al., 2001
;
Nakano and Vousden, 2001
;
Oda et al., 2000
;
Yu et al., 2001
). Furthermore,
the BH3-only genes Hrk/DP5 and Bim have been found
to be upregulated in cultured neurons after NGF withdrawal, a process that
appears to be dependent on the activation of the c-jun N-terminal kinase (JNK)
(Harris and Johnson, 2001
;
Imaizumi et al., 1999
;
Putcha et al., 2001
;
Whitfield et al., 2001
). In
sympathetic neurons, it could further be shown that the upregulation of
Bim after NGF-withdrawal is required for their programmed death
(Putcha et al., 2001
;
Whitfield et al., 2001
).
Hence, the observation that NGF-withdrawal induced death of sympathetic
neurons in culture is dependent on the synthesis of macromolecules can be
explained by the requirement for Bim expression. The transcriptional
activation of BH3-only genes therefore is crucial for the programmed
death of neurons not only in C. elegans, in which neuronal death is
specified by cell lineage, but also for the programmed death of neurons in
mammals, in which neuronal death is predominantly triggered by cell
non-autonomous signals.
The cell-death activating function of HLH-2 might be conserved
The cell-death function of ces-2 and ces-1 has been
conserved through evolution. The homologue of ces-2 in humans is the
proto-oncogene HLF (HLF, hepatic leukaemia factor)
(Inaba et al., 1996). The
oncogenic form of HLF, the E2A-HLF fusion protein, found in patients carrying
the t(17; 19) (q22;p13) chromosomal translocation, is composed of the
trans-activation domain of the Daughterless-like bHLH protein E2A and the
DNA-binding domain of the CES-2-like protein HLF. E2A-HLF has been shown to
block the programmed death of pro-B cells thereby allowing their leukaemic
transformation and it has been proposed that it does so by inappropriately
activating the transcription of a gene with anti-apoptotic function. The
ces-1-like gene SLUG of humans was found to be a target of
E2AHLF and the overexpression of SLUG can block the programmed death
of leukaemic pro-B cells (Inukai et al.,
1999
). Furthermore, hematopoietic progenitor cells from mice
lacking a functional SLUG gene are more sensitive to DNA-damage
induced programmed cell death, supporting an anti-apoptotic role for
SLUG in hematopoietic lineages
(Inoue et al., 2002
). A
mammalian counterpart of the genetic pathway involved in the specification of
the NSM sister cell death in C. elegans might therefore play an
important role in the regulation of the programmed death of pro-B cells in
mammals. We have shown that the Snail-like CES-1 protein can prevent the death
of the NSM sister cells in C. elegans by antagonizing the function of
HLH-2 and HLH-3 thereby directly blocking the transcription of the
BH3-only gene egl-1 (Fig.
8). It is therefore feasible that homologues of C.
elegans HLH-2 and HLH-3 in mammals act downstream of or in parallel to
SLUG to activate a mammalian BH3-only gene in pro-B cells. Indeed,
the Daughterless-like bHLH protein E2A, a mammalian homologue of HLH-2, is
expressed in B-cell lineages and has been shown to have tumour suppressor
activity (Massari and Murre,
2000
). Furthermore, a number of BH3-only genes have been
shown to be expressed in hematopoietic cells in mammals, including
Hrk/DP5 and Bim
(Puthalakath and Strasser,
2002
). Whether Hrk/DP5 or Bim are targets of the
conserved, HLF- and SLUG-dependent cell-death pathway in pro-B cells and
whether E2A plays a role in their activation remains to be determined.
The BH3-only gene egl-1 of C. elegans is required for most if not all of the 131 programmed cell deaths that occur during C. elegans development. The studies described resulted in the identification of two new factors, hlh-2 and hlh-3, that are involved in specifying the death of two of these 131 cells, the death of the NSM sister cells. The death of the NSM sister cells is specified by lineage. Like their counterparts in higher organisms, hlh-2 and hlh-3, which encode a Daughterless-like and an Achaete-scute-like bHLH protein, respectively, have been implicated in cell fate determination. The lineage-dependent signal required to kill the NSM sister cells might therefore be transduced by hlh-2 and hlh-3.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Present address: Dartmouth Medical School, Department of Genetics, 7400
Remsen, Hanover, NH 03755-3837, USA
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