1 Department of Molecular Biology, University of Wyoming, PO Box 3944, Laramie,
WY 82071-3944, USA
2 Howard Hughes Medical Institute and Department of Molecular, Cellular, and
Developmental Biology, University of Colorado, Boulder, CO 80309-0347,
USA
* Author for correspondence (e-mail: davidfay{at}uwyo.edu)
Accepted 24 April 2003
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SUMMARY |
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Key words: lin-35, Retinoblastoma, ubc-18, Ubiquitin, C. elegans, Pharynx
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INTRODUCTION |
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In addition to cell-cycle regulation, in vitro and tissue culture studies
have shown that pRb associates with a diverse set of proteins, many of which
regulate the expression of genes required for tissue-specific differentiation
(reviewed by Morris and Dyson,
2001). For example, pRb enhances the DNA binding and
transactivation activities of NF-IL6 (Chen
et al., 1996b
) and the C/EBP
(Chen et al., 1996a
) family of
transcription factors to promote adipocyte and leukocyte differentiation,
respectively. pRb also promotes muscle differentiation by augmenting the
activity of MyoD (Gu et al.,
1993
) and through inhibition of the transcriptional repressor HBP1
(Tevosian, 1997; Shih et al.,
1998
). Finally, pRb may bind and regulate the activities of a
number of additional factors, including the paired homeodomain-containing
proteins Pax3, Pax5, Chx10 and Mhox
(Wiggan et al., 1998
;
Eberhard and Busslinger,
1999
); several hormone-responsive transcription factors, including
the glucocorticoid receptor (Singh et al.,
1995
); and the osteoblast transcription and differentiation
factor, CBFA1 (Thomas et al.,
2001
). Whether or not the majority of these reported activities
represent authentic in vivo functions for Rb remains to be
determined.
Acting in concert with transcriptional regulatory factors, the
ubiquitin-mediated degradation pathway has emerged as the other principal
mechanism by which cells control the abundance of individual proteins. The
process is carried out by three classes of enzymes (termed E1, E2 and E3) that
act sequentially to catalyze the attachment of ubiquitin, a highly conserved
76 amino acid protein, to the protein substrate targeted for degradation
(reviewed by Weissman, 2001
).
The process is initiated by E1 enzymes (also known as ubiquitin-activating
enzymes), which form a thiol-ester bond with the C-terminal glycine of
ubiquitin in an ATP-dependent manner. The E2 or UBC (for ubiquitin-conjugating
or ubiquitin-carrier) enzyme then accepts ubiquitin from the E1 via a
transthiolation reaction involving the C terminus of ubiquitin. Finally, the
transfer of ubiquitin from E2 to a lysine on the target protein is catalyzed
by the E3 ubiquitin ligase. E3 enzymes can further be subdivided into two
separate families containing either a HECT or a RING finger domain. E3s with a
HECT domain form thiol-ester intermediates with ubiquitin prior to attachment
to the target protein (Huibregtse et al.,
1995
), whereas E3s with a RING finger mediate the direct transfer
of ubiquitin from E2 to the target protein
(Joazeiro and Weissman, 2000
).
In either case, the majority of ubquitylated proteins are subsequently
degraded by the 26S proteasome.
A recent analysis of the C. elegans genome identified 20 genes
encoding predicted E2/UBC enzymes along with three UBC variants
(Jones et al., 2002). This
compares with 12 UBCs in S. cerevisiae, 25 in Drosophila and
26 that have thus far been identified in the human proteome. Thus, complexity
in the ubiquitylation process begins at the level of UBCs and is further
amplified by the large number of potential E3 genes found in the genomes of
most higher eukaryotes; the C. elegans genome encodes for >150
RING-finger or HECT domain proteins. Interestingly, RNA-mediated interference
(RNAi) experiments of the 23 C. elegans UBC genes revealed functions
for only four of them (Jones et al.,
2002
). RNAi of these genes [let-70 (ubc-2), ubc-9,
ubc-12, and ubc-14], all of which are conserved in yeast,
results in developmental arrest at various stages. Thus, a large proportion of
UBCs in C. elegans may be functionally redundant, either with each
other or with other cellular factors that act to regulate protein levels.
Using a genetic screen to identify mutations causing synthetic phenotypes
with lin-35/Rb in C. elegans, we have previously reported
the identification of mutations in fzr-1, a regulatory subunit of the
APC proteasome (Fay et al.,
2002). lin-35; fzr-1 double mutants display a
hyperproliferation phenotype that affects virtually all cell types examined.
We now describe our analysis of another mutation identified in this screen.
Cloning of the gene reveals that it corresponds to ubc-18, the C.
elegans homolog of human UBCH7 (ARIH1 Human Gene Nomenclature
Database). The coordinate inactivation of lin-35 and ubc-18
leads to several developmental defects, including a failure to execute
pharyngeal morphogenesis properly. The expanding role of lin-35
beyond cell-cycle control and vulval induction in C. elegans should
shed light on the conserved role of Rb in higher eukaryotes.
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MATERIALS AND METHODS |
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Strains used to map ubc-18(ku354) included: lin-35(n745);
dpy-17(e164), unc-32(e189)III [6/19 Unc-non-Dpy (UND) and 9/12
Dpy-non-Unc (DNU) recombinants picked up the ubc-18 mutation]; and
lin-35(n745); sma-3(e491), unc-32(e189) [16/16 UNS and 0/7 SNU picked
up the ubc-18 mutation]. Strains WY2 [dpy-17(e164),
ubc-18(ku354); unc-32(e189)], WY78 and CB4856 were used in SNP mapping
procedures to place ubc-18(ku354) between SNP markers eam34f12.s1@151
on cosmid R01H2 and eam49b07.s1@287 on cosmid T21D11. For this effort, a total
of 98 DNU and UND recombinants were isolated, homozygosed and (in the case of
recombinants derived from WY2) injected with lin-35 dsRNA to
ascertain the status of the ubc-18 mutation. For SNP mapping with
WY78, we used a lin-35 strain that had been backcrossed five times to
CB4856 (WY72) and followed the presence of ubc-18 based on the
requirement for kuEx119. For additional details on genetic mapping,
see Fay et al. (Fay et al.,
2002).
Duplication and deficiency analysis
lin-35; ubc-18/ubc-18; qDp3 animals were generated by crossing
JK867 [dpy-17(e164) ncl-1(e1865) unc-36(e251)III; qDp3(III;f)]
hermaphrodites to lin-35 males, then crossing F1 male progeny to
lin-35; ubc-18, unc-32; kuEx119 hermaphrodites. Putative lin-35;
d17, ncl-1, unc-36/ubc-18, unc-32; qDp3 animals were identified, as well
as lin-35; ubc-18; unc-32; qDp3 animals in the next generation. Such
animals were wild type and generated UNC-32 but not DPY-17, UNC-36 progeny.
The presence of the lin-35 homozygous mutation was confirmed by the
phenotypes of the Unc animals (lin-35; ubc-18; unc-32) as well as
through the use of lin-15a(RNAi) which led to a high penetrance of
the Muv phenotype when lin-35 was homozygous. To generate lin-35; ubc-18,
unc-32/+; qDp3 animals, lin-35; ubc-18; unc-32; qDp3 animals
were crossed to lin-35; dpy-17, unc-32/+ males and wild-type cross
progeny hermaphrodites that gave rise to DpyUnc animals were identified,
indicating the genotype lin-35; ubc-18; unc-32/dpy-17, unc-32; qDp3.
For deficiency mapping, lin-35; dpy-17, ubc-18, unc-32/+ males were
crossed to MT1978 [nDf16/unc-36(e251) dpy-19(e1259)] and lin-35;
nDf16/dpy-17, ubc-18, unc-32; kuEx119 animals were identified. However,
such animals, when fertile, were rapidly out-competed by homozygous
lin-35; ubc-18; kuEx119 sibling progeny. Similar results were
obtained when we attempted to produce animals with the genotype lin-35;
nDf16/+; kuEx119, suggesting that the lin-35 mutation may be
deleterious in conjunction with the deficiency. Direct testing of
nDf16/+ animals for sensitivity to lin-35 reduction using
RNAi led to first generation nDf16/+ progeny that were uniformly
sterile (data not shown). Thus, our inability to isolate stable lin-35;
ubc-18/nDf16:kuEx119 strains stems from an apparent toxic genetic
interaction between lin-35 and nDf16.
Other methods
Cosmid rescue
Rescue was obtained by co-injection of C. elegans genomic cosmids
or PCR products with pRF4 into WY83 using standard procedures
(Mello and Fire, 1995).
Rescued animals were fertile and showed a roller (Rol) phenotype. For
single-gene rescue of WY83 animals, a 2570 bp PCR fragment corresponding to
nucleotides 31,220-33,790 of R01H2 was used. The ubc-18-coding region
spans nucleotides 32,225-32,928 and is encoded by the reverse strand.
RNAi
RNAi was carried out according to standard methods
(Fire et al., 1998;
Fraser et al., 2000
). Feeding
vectors were constructed as previously described
(Fay et al., 2002
). For
lin-37, a 774 bp fragment of C. elegans genomic DNA
corresponding to nucleotides 1763-2555 of cosmid ZK418 (261-816 of
lin-37) was subcloned into the polylinker of PD129.36. For
ubc-18, a 691 bp PCR fragment of C. elegans genomic DNA
corresponding to nucleotides 32,232-32,923 of R01H2 (the entire
ubc-18 coding region) was subcloned into vector pPD129.36.
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RESULTS |
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lin-35; ubc-18; kuEx119 animals are healthy, fertile and generally
appear indistinguishable from wild-type animals
(Fig. 1A,B). By contrast, most
lin-35; ubc-18 animals that fail to inherit the array arrest or die
as larvae, and those animals reaching adulthood are typically small and
sterile (Fig. 1A,B;
Table 1). As predicted for a
synthetically lethal mutation, inactivation of lin-35 (expressed from
the extrachromosomal array) using RNAi caused lin-35; ubc-18; KuEx119
animals to adopt the double-mutant phenotype
(Fig. 1A,B). As previously
described, lin-35 single mutants are viable but show a substantial
reduction in brood sizes compared with wild type
(Table 1)
(Lu and Horvitz, 1998;
Fay et al., 2002
). Similarly,
ubc-18 single mutants exhibit a marked reduction in brood size and
are slightly growth retarded (Table
1; data not shown).
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We also observed a high incidence of larval arrest in animals that were
homozygous for lin-35, but heterozygous for ubc-18, though
the rate of arrest was lower than that observed for homozygous double mutants
(Table 1). In addition,
lin-35; ubc-18/+ animals that reached adulthood showed an 50%
reduction in brood size compared with lin-35 mutants alone
(Table 1). These results
suggest that the ubc-18 mutation could be semi-dominant or
haplo-insufficient, or, based on the method used to generate the heterozygous
animals, ubc-18 could be maternally required. Further experiments
indicated that the effect in heterozygous animals results from a combination
of the latter two possibilities: (1) lin-35 mutant animals that are
heterozygous, but maternally wild type, for ubc-18 show lower rates
of larval arrest then those that are heterozygous but maternally deficient
(Table 1); thus, the presence
of maternal ubc-18 can partially reduce the severity of lin-35;
ubc-18 genetic interactions; (2) overexpression of the ku354
variant of UBC-18 (via an extrachromosomal array), together with
lin-35 inactivation by RNAi, did not lead to a phenocopy of the
double-mutant phenotype (data not shown); (3) in an otherwise wild-type
background, ubc-18/+ heterozygous animals had wild-type brood sizes,
indicating that the effects in heterozygous animals were specific to
ubc-18 genetic interactions with lin-35
(Table 1); and (4) RNAi of
ubc-18 performed on lin-35; ubc-18; kuEx119 animals strongly
enhanced the penetrance of the Pun phenotype in progeny not harboring the
array compared with the array-deficient progeny of untreated lin-35;
ubc-18; kuEx119 animals [Table
1 and see discussion of ubc-18(RNAi) below]. This latter
result also indicates that ku354 represents a partial
loss-of-function allele of ubc-18.
We also carried out further genetic analysis using a regional free
duplication (qDp3) that contains sequences from an 0.5 Mb
region of chromosome III, including ubc-18. Briefly, we found that
lin-35; ubc-18/ubc-18; qDp3 animals displayed a similar phenotype to
lin-35; ubc-18/+ animals (Table
1 and data not shown), further supporting our conclusion that
ku354 is a LOF allele and does not act in a dominant or
dose-dependent manner. We note that we were unable to generate stable strains
that were heterozygous for ubc-18 and nDf16 (a large
chromosomal deficiency that deletes ubc-18) in the lin-35;
KuEx119 background, as the lin-35 mutation surprisingly showed a
synthetic genetic interaction with the deficiency itself (also see Materials
and Methods).
ubc-18 encodes a homolog of the ubiquitin activating enzyme
UbcH7
ku354 was mapped to a small segment of chromosome III between
sma-3 and unc-32 using genetic markers and single-nucleotide
polymorphisms (SNPs). To identify the affected gene, lin-35; ubc-18;
KuEx119 animals were co-injected with regional cosmids and a dominant
marker conferring a Rol phenotype. Rescued lines were identified by the
presence of healthy Rol animals that no longer required the original array
containing lin-35 (kuEx119) for viability. Two overlapping
cosmids in the region, R01H2 and C49B6, were found to rescue the lin-35;
ubc-18 synthetic lethality. A minimal rescuing sequence was identified
that contained only a single predicted open reading frame: that encoding the
C. elegans gene ubc-18/R01H2.6. Six out of eight
independently derived transgenic lines containing the wild-type
ubc-18 gene efficiently rescued the double-mutant phenotype (also see
Materials and Methods).
ubc-18 encodes a predicted protein of 153 amino acids that is
highly similar to known E2-type ubiquitin-conjugating enzymes
(Jones et al., 2002). UBC-18
shows greatest similarity to UbcH7 in humans (29% identical and 86% similar)
and is approximately equally similar to S. cerevisiae proteins Ubc-5p
and Ubc-4p (Fig. 2; data not
shown). The database prediction for ubc-18 is supported by the
existence of multiple sequenced cDNA clones (kindly provided by Y. Kohara)
that confirm both the 3' terminus of the gene as well as the precise
locations of its four exons. In addition, the 5' untranslated region
that was identified by the cDNA clones contains two in-frame stop codons
located just upstream (15 and 24 nucleotides) of the start ATG.
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RNAi of ubc-18 has previously been reported to have no phenotype
in wild-type animals (Jones et al.,
2002). Consistent with this, we observed, at most, a subtle effect
of ubc-18(RNAi) on viability and brood size in N2 animals, although
treated animals generally took an extra day to reach maturity when compared
with control animals (Table 1;
data not shown). Mutations in rrf-3, a predicted RNA-directed RNA
polymerase, have recently been reported to enhance the overall efficacy of
gene inactivation by RNAi feeding methods
(Simmer et al., 2002
).
Consistent with this, RNAi inactivation of ubc-18 in the
rrf-3 mutant background led to an
50% reduction in brood size
when compared with rrf-3 mutants alone
(Table 1).
To see if we could reiterate the double-mutant phenotype, we performed ubc-18(RNAi) on lin-35 and lin-35; rrf-3 animals. RNAi treatment of lin-35; rrf-3 double mutants resulted in first-generation progeny that either arrested as larvae or developed into completely sterile adults (Table 1). These adults often failed to generate sperm and contained abnormal-appearing oocytes, similar to some lin-35; ubc-18 double mutants (data not shown). We note that even without RNAi treatment, lin-35; rrf-3 double mutants were markedly less healthy than either lin-35 or rrf-3 single mutants (Table 1). Treatment of lin-35 mutants with ubc-18(RNAi) led to more subtle defects in the first generation (Table 1), though prolonged exposure resulted in higher rates of larval arrest and adult sterility in subsequent generations (data not shown).
We failed to observe any Pun animals arising from ubc-18(RNAi) treatment of lin-35 or lin-35; rrf-3 animals (Table 1) or in experiments where both genes were simultaneously inactivated by RNAi (data not shown). Such results may reflect a failure to sufficiently inactivate ubc-18 by RNAi methods, or they may occur if strong inactivation of ubc-18 led to sterility of the treated parent prior to the production of Pun progeny.
lin-35; ubc-18 embryos are defective at pharyngeal
morphogenesis
An initial examination of both lin-35; ubc-18 and ubc-18;
lin-35(RNAi) embryos (hereafter referred to as double-mutant embryos)
indicated that the first observed defects in pharyngeal development coincided
with events taking place at or just prior to the 1.5-fold stage of
embryogenesis. At this time in wild-type animals, a subset of anterior
pharyngeal cells reorient their longitudinal axes, form associations with
neighboring cells and then shift their positions towards the anterior region
of the embryo through a localized contraction mechanism, leading to pharyngeal
elongation (Portereiko and Mango,
2001) (see below). By contrast, pharyngeal cells of double-mutant
embryos remain stationary and fail to move towards the anterior
(Fig. 3A-D). In addition,
whereas the anterior border of the basement membrane that encapsulates the
pharyngeal primordium becomes less distinct during elongation in wild-type
embryos, this feature remains prominent in the double mutants
(Fig. 3A,C; see
Fig. 5). The failure of mutant
embryos to undergo pharyngeal elongation could result from several distinct
underlying defects: (1) mutant embryos may generate incorrect numbers of
pharyngeal precursor cells during early embryonic development, leading to
defects at later stages; (2) these precursor cells, once generated, may fail
to differentiate properly; and (3) properly specified pharyngeal cells may
fail to execute one or more steps during morphogenesis of the organ.
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An examination of wild-type embryos at the 1.5-fold stage identified an
average of 100±3 (n=11; range, 95-104) GFP+ cells in the head
region. This figure is in close agreement with previously reported numbers
using antibodies against PHA-4 in similar-stage embryos (97±3)
(Horner et al., 1998)
(Fig. 3A,B). An analysis of Pun
double-mutant embryos revealed an average of 93±13 GFP+ cells
(n=13) with numbers ranging from 66-105. Importantly, 8/13 mutant
embryos contained 95 or more GFP+ cells, whereas only 3/13 contained fewer
than 91 cells (Fig. 3C,D).
Thus, for most embryos examined, the number of cells adopting a pharyngeal
identity (based on this GFP marker) was indistinguishable from that of wild
type. Similar results were also obtained by examining
pha-4::GFP-expressing cells in L1 larvae of double-mutant and
wild-type animals (Fig. 3E-H; data not shown). These findings indicate that gross differences in the number
of pharyngeal cells alone cannot account for the observed defects in a large
subset (perhaps the majority) of the double-mutant embryos. Thus, the failure
to elongate is probably unrelated to early steps of pharyngeal cell
specification or survival.
We next examined mutant animals for their ability to generate terminally
differentiated pharyngeal cell types. The adult pharynx comprises muscle,
epithelial and neuronal cells, as well as marginal and gland cell types
(Albertson and Thomson, 1976;
Sulston and Horvitz, 1977
). We
used several different GFP reporter strains to assay for the presence of
muscle (Okkema et al., 1993
),
epithelial (Mohler et al.,
1998
) and neuronal cells
(Maduro and Pilgrim, 1995
) in
double-mutant embryos and larvae. We found that all three cell types were
produced, indicating that mutants are capable of generating most or all of the
correct pharyngeal cell types (Fig.
4). Consistent with this, we have observed sustained rhythmic
pumping in many severely Pun pharynges of L1 mutant animals, suggesting that
most or all terminal cell types have been specified and are physiologically
functional, although the overall pharyngeal structure is grossly perturbed. We
note that our level of analysis does not allow us to make conclusions
regarding the differentiation status of individual cells within the pharynx.
Therefore, it is possible that double mutants may contain a subset of
improperly or incompletely differentiated pharyngeal cells that could
contribute to the observed defects.
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To aid in our analysis, we used a reporter strain that expresses a
membrane-localized GFP-fusion protein under the control of the pha-4
promoter (kindly provided by S. Mango), which allowed us to follow changes in
pharyngeal cell shape. In wild-type embryos, elongation was completed by the
1.5-fold stage of embryonic development (400 minutes into embryonic
development; Fig. 5A-D). By
contrast, 17 of the 24 double-mutant embryos displayed clear defects in the
ability of anterior epithelial cells to initiate or complete the reorientation
step (Stage I; Fig. 5E-L).
Interestingly, a substantial fraction of the orientation-defective embryos
(9/17) were only partially deficient at this step, such that one or more
epithelial cells appeared to correctly shift their axes, whereas others
remained in their original orientation
(Fig. 5I-L). Other mutant
embryos (7/24) exhibited more complex and variable defects such as a general
disorganization of the leading-edge cells (data not shown). Taken together,
these results indicate that lin-35 and ubc-18 functionally
cooperate to control a crucial and early step of pharyngeal morphogenesis
during C. elegans development.
ubc-18 interactions with SynMuv genes
Previous studies on fzr-1/slr-1 demonstrated a limited set of
genetic interactions with class B synthetic multivulval (SynMuv) gene family
members (Fay et al., 2002). To
determine the spectrum of ubc-18 genetic interactions, we inactivated
class B SynMuv genes in ubc-18 mutants by RNAi feeding methods and
assayed progeny for the appearance of the Pun phenotype
(Table 2). All but one of the
class B genes tested produced substantial numbers of Pun animals following
inactivation by RNAi. These included lin-36
(Thomas and Horvitz, 1999
) and
lin-37 (Boxem and van den Heuvel,
2002
), two genes of unknown function; the RbAp46/48 homolog,
lin-53 (Lu and Horvitz,
1998
); a C. elegans Mi2 homolog and NURD complex
component, let-418 (Solari and
Ahringer, 2000
; von Zelewsky
et al., 2000
); and histone deactylase, hda-1
(Lu and Horvitz, 1998
). The
sole exception in the class B category was efl-1, one of two E2F
homologs in C. elegans (Ceol and
Horvitz, 2001
), which failed to produce significant numbers of Pun
animals either alone (data not shown) or in the ubc-18 mutant
background (Table 2). In
addition, we saw no effect following inactivation of the class A SynMuv gene
lin-15a (Clark et al.,
1994
; Huang et al.,
1994
). In several cases, these experiments were complicated by
non-synthetic RNAi effects as the genes that were being inactivated were
themselves essential for viability (Table
2). Nevertheless, in such cases we were still able to observe a
marked enrichment in the number of animals displaying the Pun phenotype,
demonstrating that inactivation of these genes is interchangeable with
lin-35 for the production of this phenotype.
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DISCUSSION |
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A recent global analysis of gene expression using microarrays in C.
elegans indicates that the ubc-18 transcript is present at
significant levels in both oocytes and embryos, as well as in late stages of
larval development and into adulthood
(Hill et al., 2000). In
addition, using antibodies, LIN-35 has been shown to be expressed throughout
development in most or all cell types (Lu
and Horvitz, 1998
). We were unsuccessful in our attempts to
generate transgenic animals that express GFP under the control of the
ubc-18 promoter using several different reporter constructs (data not
shown). Given that transgenic arrays are typically silenced in the C.
elegans germline, our results may indicate that ubc-18
expression is germline specific. Consistent with this, data available on the
C. elegans expression database
(http:://nematode.lab.nig.ac.jp/db/index.html;
kindly provided by Y. Kohara) suggest that ubc-18 expression may be
largely or completely restricted to germline cells. Germline expression of
UBC-18 is also consistent with our observation that maternal ubc-18
function markedly reduced larval lethality in lin-35; ubc-18/+
animals (Table 1).
A genetic interaction between lin-35/Rb and a ubiquitin
pathway component
ubc-18 is the second gene that we have identified as a
lin-35-synthetic mutation. Simultaneous inactivation of
lin-35 and ubc-18 led to growth arrest, infertility and
defects in pharyngeal morphogenesis. Based on known roles of these proteins,
we believe that LIN-35 and UBC-18 share a set of common targets for
transcriptional repression and ubiquitin-mediated degradation, respectively.
We hypothesize that the abnormal expression of one or more mutual targets is
probably responsible for the pleiotropic defects observed in the double
mutants. Thus, in the absence of LIN-35 function, UBC-18 can promote the
degradation of some subset of derepressed LIN-35 transcriptional targets.
Conversely, UBC-18 function is not essential if LIN-35 is present and actively
repressing the expression of its target genes. It is only when both
lin-35 and ubc-18 are absent, that one or more proteins can
accumulate to levels sufficient to produce the observed developmental
defects.
It is also conceivable that the phenotype observed in the double mutants
may not be due solely to defects resulting from an increase in the abundance
of target proteins. It has become clear in recent years that ubiquitylation
can affect protein function by mechanisms unrelated to protein stability.
These functions include roles in protein trafficking (reviewed by
Pickart, 2001), transcription
(reviewed by Conaway et al.,
2002
) and signal transduction
(Wang et al., 2001
;
Ulrich, 2003
). Nevertheless,
we currently favor a model whereby lin-35 and ubc-18 act
coordinately to downregulate the activity of proteins whose presence would be
deleterious to specific developmental processes or basic life functions.
Our analysis of the double-mutant animals identified a predominant defect
affecting stage I of pharyngeal morphogenesis. This phenotype differs
substantially from defects reported for two previously described pharyngeal
mutants in C. elegans: pha-1 and pha-4. pha-4
mutants fail to specify any pharyngeal cells, which instead adopt alternative
cell fates (Mango et al.,
1994). In the case of pha-1 mutants, the correct number
of pharyngeal cells are initially specified; however, these cells then fail to
undergo terminal differentiation (Schnabel
and Schnabel, 1990
; Granato et
al., 1994
). pha-4 and pha-1 encode transcription
factors of the FoxA/HNF3 and bZIP families, respectively. pha-4
directly activates the transcription of most or all genes required for
pharyngeal identity (Gaudet and Mango,
2002
). It is possible that lin-35 and ubc-18 act
to downregulate one of the early pha-4 target genes in order for
morphogenesis to proceed. Alternatively, lin-35 and ubc-18
may negatively regulate genes that are not normally expressed in this tissue
but whose expression is deleterious to the development of the organ.
A novel role for lin-35/Rb in morphogenesis and
development
At present, relatively little is known about the targets of pRb
(particularly those unrelated to cell cycle progression) and even less is
known about the substrates of most E2 and E3 enzymes. Given the genetic
interactions that we observed between ubc-18 and a number of the
SynMuv genes tested (Table 2),
it is unlikely that the targets of pRB that are involved in pharyngeal
development also function in cell cycle control. Interestingly, we have
isolated a second mutation, slr-9(fd1), that produces a synthetic Pun
phenotype in conjunction with lin-35. slr-9 has been mapped to a
small interval on LGIII that does not contain any predicted proteins with a
known involvement in the protein degradation pathway (E.L. and D.S.F.,
unpublished). Thus, we may be poised to identify either novel components
involved in ubiquitin pathway targeting or, possibly, specific effectors of
pharyngeal development.
Extensive studies have defined a central and clear role for pRb in
regulating entry and progression through the S-phase of the cell cycle.
Genetic studies carried out in Drosophila have further suggested that
pRb may act exclusively through E2F, as LOF mutations in e2f1, one of
two Drosophila E2F homologs, can largely suppress the lethal
phenotype of mutations in rbf, one of two known Drosophila
Rb-family genes (Du, 2000). A
separate role for pRb in differentiation, which is independent of its
interactions with E2F, is, however, suggested by the observation that certain
pRb mutants that are defective at E2F binding can nevertheless induce
tissue-specific gene expression and promote differentiation
(Sellers et al., 1998
). In
addition, using a cell line that overexpresses a non-regulatable form of pRb,
Markey and colleagues (Markey et al.,
2002
) recently identified a large set of putative pRb
transcriptional targets that are not known or predicted to be regulated by E2F
family members. Finally, E2F family members themselves have been shown through
microarray experiments to control the expression of a diverse set of targets
including a number of genes associated with differentiation and development
(Ishida et al., 2001
;
Muller et al., 2001
).
Thus, our synthetic lethal screen has uncovered a function for lin-35/Rb that is distinct from those previously described for pRB in higher eukaryotes. Additional developmental functions of lin-35 should be revealed by the further analysis of lin-35 synthetic mutants and by looking directly for interactions between lin-35 and other putative E2 and E3 enzymes using an RNAi approach. Furthermore, by identifying specific phenotypes for putative E2 and E3 enzymes, it may also be possible to make predictions regarding the functional and biochemical associations between the many E2 and E3 enzymes present in the C. elegans genome.
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ACKNOWLEDGMENTS |
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