Howard Hughes Medical Institute, and Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA
* Author for correspondence (e-mail: pws{at}caltech.edu)
Accepted 4 October 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: EGF, RAS, LET-23, SOP-1, DPY-22, Mediator, Vulva, C. elegans
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Given the likely diffusible nature of LIN-3, mechanisms must exist to
ensure that normally only a subset of the VPCs responds to the growth factor.
A ligand-independent gain-of-function allele of let-23, sa62, confers
ectopic vulval fate transformations in P3.p and P4.p at high frequency
(Katz et al., 1996),
indicating that receptor activation is sufficient to drive cell
differentiation. Under physiological conditions, positional selectivity for
cell fate determination might be achieved by dependency on a threshold level
of LET-23 pathway activation, which is normally achieved only by the VPC
colsest to the anchor cell, the source of LIN-3. Consistent with this model,
P6.p is positioned closest to the anchor cell and invariantly responds to
LIN-3.
Genetic studies of negative regulation of vulval development indicate that
additional mechanisms operate to ensure the selective response of P6.p to
LIN-3. Although ectopically activated LET-23 induces vulval fate
transformations at high frequency in P3.p and P4.p, the posteriorly expressed
mab-5 homeobox gene inhibits this response in the most posterior Pn.p
cell, P8.p (Clandinin et al.,
1997). Another mechanism of restricting the response to LIN-3
involves two classes of genes that function redundantly to inhibit vulval
fates. Animals that harbor loss-of-function mutations both in a class A and
class B `synmuv' gene display a synthetic multivulva phenotype because of the
adoption of vulval fates by most of the VPCs
(Ferguson and Horvitz, 1989
).
This vulval induction is independent of the LIN-3-producing anchor cell, but
dependent on LET-23 and its downstream effectors
(Ferguson et al., 1987
;
Huang et al., 1994
;
Lu and Horvitz, 1998
), raising
the possibility that this pathway exists to repress low, but functional,
levels of ligand-independent activity by the LET-23 pathway. Molecular
identification of some of the synmuv genes and RNA interference experiments
suggest that this pathway comprises components of a histone deacetylase
complex, which represses LET-23-dependent gene expression
(Chen and Han, 2001
;
Lu and Horvitz, 1998
;
Solari and Ahringer,
2000
).
Several genes have been identified whose properties do not fully resemble
the synmuv genes, but nevertheless, function as negative regulators of vulval
induction. These include unc-101(AP47 medium chain of trans-Golgi
AP-1 complexes) (Lee et al.,
1994), sli-1(c-CBL)
(Yoon et al., 1995
),
gap-1 (Hajnal et al.,
1997
), ark-1 (ACK-related tyrosine kinase)
(Hopper et al., 2000
) and
lip-1 (MAP kinase phosphatase)
(Berset et al., 2001
).
Mutations in these genes suppress loss-of-function mutations in the
let-23 pathway that cause vulvaless phenotypes, and in different
genetic backgrounds, they enhance the frequency of multivulva phenotypes,
indicating they affect all six VPCs, similar to the synmuv genes. These
negative regulators may function to raise the requirement for the amount of
LET-23 pathway activity necessary to drive a functional response.
To address whether these are the only mechanisms and points of negative
regulation of the response to LIN-3, we performed a genetic screen for
mutations that enhance the frequency of ectopic vulval fate transformations in
the presence of gain-of-function let-23(sa62). We isolated two new
alleles of sop-1 (Zhang and
Emmons, 2000), which has recently been found to be allelic to the
older locus, dpy-22 (Meneely and
Wood, 1987
) (H. Sawa, personal communication). DPY-22 is most
closely related to human TRAP230 (Ito et
al., 1999
; Nagase et al.,
1996
; Philibert et al.,
1998
), a component of the transcriptional mediator complex
(Ito et al., 1999
), and has
been shown to be an inhibitor of WNT-dependent ray formation in the C.
elegans male tail (Zhang and Emmons,
2000
). We describe some of the phenotypes of our new
dpy-22 alleles, and present evidence that DPY-22 also is an inhibitor
of RAS-dependent vulval fate specification, independent of its role in
regulating WNT signaling.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
bar-1(mu63) was previously reported to have a mutation causing a
L130F change (Maloof et al.,
1999), which we did not detect in the extant bar-1(mu63)
strains. In order to determine the molecular lesion in bar-1(mu63),
we amplified 1 kb regions of bar-1 genomic DNA from
bar-1(mu63) animals by PCR, and directly sequenced the products.
Primer sets used included BAR1-6 5'-ttc agt tct act tgt cta ttg gtg
tgc-3' and BAR1-7 5'-cac atg gta gtc cgc gac ttg tac-3';
BAR1-8 5'-cga gaa ttg acc agc tcc aga aga g-3' and BAR1-9
5'-gc tgc tta ctg atg aag ccg gtg-3'; and BAR1-10 5'-gc ttt
gtg cac aac ctc ctg taa g-3' and BAR1-11 5'-ct ctt cat ccg gca gac
aaa tcg-3. After sequencing 36% of the bar-1 genomic locus, we
identified a C to T mutation at position 39108 of C54D1 in
bar-1(mu63) animals. This change was detected on both strands, and
not in N2 animals. This mis-sense mutation is predicted to cause a G524D
change in the BAR-1 protein. Linkage of bar-1(mu63) to
dpy-22(sy622) was confirmed by DNA sequencing. Linkage of
bar-1(ga80) to dpy-22(sy622) was confirmed by PCR and
digestion with MseI, which detects the MseI restriction site
created by the ga80 C to T point mutation.
let-23(sa62); him-5(e1490) animals were subjected to standard
mutagenesis with ethylmethanesulfonate
(Brenner, 1974). One thousand
F1 offspring were picked to individual plates, and those segregating adult
males with ventral protrusions were saved.
Molecular biology
Full-length dpy-22 used for PCR-based rescue was amplified by PCR
(Expand Long, Roche) from positions 21115 to 6068 of cosmid F47A4 with the
primers 5'-gtc ccg tta tga taa cgt atc tcc aag-3' and 5'-caa
gcg tta tct tga tga cgc ggt c-3'. The PCR fragment was injected at 10
ng/µl with pPD118.33 (myo-2::gfp) (10 ng/µl) and pBSSK
(Stratagene) (160 ng/µl) into dpy-6(e14) dpy-22(sy622); stDp2
animals. Rescuing arrays were subsequently crossed into dpy-22(sy622)
single mutants.
A full-length dpy-22 gene spanning 20759 to 6464 of cosmid F47A4
was reconstructed in pBR322 as follows. First, an XhoI fragment from
F47A4 (20759-10290) was cloned into the SalI site of pBR322 to yield
pBRF47A4Xh. This plasmid was digested with AgeI, and an AgeI
fragment from F47A4 (10406-6464) was introduced into this vector, to generate
pBRsop1FL, which harbors a full-length dpy-22 gene. dpy-22::gfp,
dpy-22 2548::gfp and dpy-22 2141::gfp transgenes were
constructed using overlap extension PCR (High Fidelity, Roche) to make
C-terminal in-frame translational fusions between appropriately truncated
dpy-22 fragments and gfp. In all cases, codons encoding two
glycine residues were placed between the two genes, and the fusions were made
to the codon encoding the first Ser residue of GFP. dpy-22 fragments
were amplified by PCR from F47A4 using the 5' primer sop1-17 5'-ct
tat gtt cca cgg tat cat caa tcc-3' and the 3' primer sop1-28
5'-c ttc tcc ttt act tcc tcc gta ctg att tgg tgg ttg ttg gtt g-3';
sop1-26 5'-c ttc tcc ttt act tcc tcc ttt ctg ctg ctc cac aag ttg ttg atg
g-3'; or sop1-35 5'- c ttc tcc ttt act tcc tcc gaa cat tct gaa ctt
cca tcc gcc-3', for dpy-22::gfp, dpy-22 2548::gfp and
dpy-22 2141::gfp, respectively. gfp fragments included the
unc-54 3' untranslated region, and were amplified by PCR from
pPD95.79 with the 5' primers sop1-34 5'-aat cag tac gga gga agt
aaa gga gaa gaa ctt ttc act gg-3', sop1-33 5'-cag cag aaa gga gga
agt aaa gga gaa gaa ctt ttc act gg-3' or sop1-32 5'-aga atg ttc
gga gga agt aaa gga gaa gaa ctt ttc act gg-3', for dpy-22::gfp,
dpy-22 2548::gfp, and dpy-22 2141::gfp, respectively, and the
3' primer unc54-5 5'-a taa gaa tgc ggc cgc aaa cag tta tgt ttg gta
tat tgg gaa tg-3'. The individual fragments were purified by agarose gel
electrophoresis, mixed in the appropriate combinations, and then subjected to
PCR in the presence of the 5' primer sop1-17, and the 3' primer
unc54-5. The dpy-22::gfp fusion was digested with NotI and
cloned into NotI-digested pBRsop1FL. dpy-22 2548::gfp and
dpy-22 2141::gfp truncations were digested with BstEII and
NotI, and cloned into BstEII/NotI-digested
pBRsop1FL. dpy-22::gfp and dpy-22 2548::gfp transgenes were
injected at 10 ng/µl with pPD118.33 (myo-2::gfp) (10 ng/µl) and
160 ng/µl of pBSSK into dpy-6(e14) dpy-22(bx93)/dpy-22(sy622)
animals. dpy-22 2141::GFP was injected at 12.5 ng/µl with pBX-1
(Granato et al., 1994) (100
ng/µl) and pBSSK (37.5 ng/µl) into pha-1(e2123ts) animals.
Identification of sy622 and sy665 as alleles of
dpy-22/sop-1
sy622 and sy665 were placed on LGX by crossing N2 males
into mutant hermaphrodites and observing that 100% of F1 males were
small/dumpy (Dpy) and had abnormal tails (Mab). Three-factor mapping was
carried out following the small/Dpy and egg-laying-defective (Egl) phenotypes
of sy622. sy622 was placed between lon-2 and unc-9
as 6/11 Lon non-Unc and 4/11 Unc non-Lon recombinants picked up the mutation.
sy622 was placed between dpy-6 and unc-9 as 6/13
Dpy non-Unc and 8/10 Unc non-Dpy recombinants picked up sy622. sy622
was placed between lon-2 and egl-15 as 23/26 Lon-non-Egl and
4/33 Egl non-Lon recombinants picked up sy622. Using sy622
egl-15 double mutants and CB4856, a Hawaiian isolate of C.
elegans, Egl-non sy622 recombinants were generated that allowed
us to analyze the positions of crossovers by the absence or presence of single
nucleotide polymorphisms (SNPs) from CB4856
(Wicks et al., 2001).
sy622 was mapped to the left of the SNP at 36555 in F47A4. Using
dpy-6 sy622 double mutants and CB4856, and picking Dpy
non-sy622 and sy622 non-Dpy recombinants, sy622 was
mapped to the right of the SNP at 19169 of F15G9. Owing to the unhealthiness
of sy622 animals, cosmids from this region initially were coinjected
with pPD118.33 (myo-2::gfp) and pBX-1 into pha-1(e2123ts)
animals, and stable extrachromosomal arrays were crossed into sy622
animals to test for rescue. Initial attempts at rescue failed using this
strategy. Later, cosmid F47A4 (30 ng/µl) was co-injected with pPD118.33
(myo-2::gfp)(10 ng/µl) and pBSSK (140 ng/µl) into
pal-1(e2091); him-5(e1490); dpy-22(bx93) animals. Arrays conferring
functional rescue in this background were crossed into sy622 animals
and found to rescue all of the sy622 phenotypes. dpy-22
genomic DNA was amplified in 1 kb pieces from sy622 and
sy665 worms, and the products directly sequenced. Mutations were
confirmed by sequencing both strands, and comparing the sequencing data from
N2, sy622 and sy665 animals in the appropriate regions.
RNAi
Exon 17 from dpy-22 was amplified by PCR from the cosmid F47A4
with the primers 5'-tta ata cga ctc act ata ggg aga cat tcg aac tag ctc
cag aga aac-3' and 5'-tta ata cga ctc act ata ggg aga atc aaa tgg
gta ctt ccc agc ttc-3', which introduce a T7 bacteriophage promoter at
both ends of the fragment. An intronless GFP gene was amplified from the
plasmid pPD79.44, with the primers 5'-tta ata cga ctc act ata ggg aga
tga gta aag gag aag aac ttt tca c-3' and 5'-tta ata cga ctc act
ata ggg aga cta ttt gta tag ttc atc cat gcc atg-3', which also add a T7
promoter to both ends of the PCR product. dsRNA was synthesized in vitro using
the MEGAscript T7 kit (Ambion). The presence of dsRNA was confirmed by agarose
gel electrophoresis, and quantified by spectrophotometry. L1 stage
hermaphrodites were incubated in 12 µl M9 buffer containing 1.5 mg/ml total
RNA and OP50 (A600 nm=1.0) for 24 hours at 20°C. After incubation, worms
were recovered and placed on standard NG plates with OP50, and allowed to
develop to the mid-L4 stage, at which time they were examined by Nomarski.
Vulval induction assay and gonad ablations
Vulval development was scored during the L4 stage under Nomarski optics
(Sternberg and Horvitz, 1986).
Nuclei in the ventral region of the worm that were not of hypodermal, neuronal
or muscle descent were counted. In wild-type animals, 22 nuclei arise from
vulval fates. The number of vulval nuclei is used to extrapolate how many of
the Pn.p cells were induced to adopt vulval fates. A vulval precursor cell
(VPC) in which both daughter cells divide one more time, and both
granddaughters divide to generate seven or eight great granddaughters and no
hypodermal tissue, is scored as 1.0 cell induction. A VPC in which one
daughter fuses with the hypodermis, and one daughter continues to divide over
the next two generations, resulting in four great granddaughter cells is
scored as 0.5 cell induction. In wild-type animals, P5.p, P6.p and P7.p each
undergo the equivalent of 1.0 cell induction, whereas the other Pn.p cells do
not adopt vulval fates, resulting in an overall cell induction of 3.0. Animals
displaying a cell induction of more than 3.0 are multivulva, and animals with
a cell induction less than 3.0 are vulvaless. Laser ablations were conducted
using a standard protocol (Bargmann and
Avery, 1995
). Gonadal cells (Z1, Z2, Z3 and Z4) were ablated
during the L1 stage.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
To determine whether sy622 and sy665 affected vulval fate specification only in male Pn.p cells, we introduced these mutations into a sensitized hermaphrodite background. Hermaphrodites heterozygous for let-23(sa62) have mostly wild-type vulvae, but occasionally some animals display ectopic vulval tissue (Table 1). sy622 and sy665 enhance the frequency of ectopic vulval fate specification in let-23(sa62)/+ animals (Table 1), indicating that the mutations also affect the response of hermaphrodite Pn.p cells to activated LET-23, and that some regulators of LET-23 signaling are shared between male and hermaphrodite Pn.p cells.
We tested whether the multivulva-enhancing effect of sy622 was
unique to let-23(sa62), or reflected an interaction with activated
RAS. let-60(n1046) is a gain-of-function let-60 allele that
encodes a RAS protein with a G13E change
(Beitel et al., 1990).
Seventy-three percent of let-60(n1046) homozygotes display ectopic
vulval cell fate differentiation (Table
1). However, similar to let-23(sa62)/+ animals,
let-60(n1046)/+ heterozygotes have mostly wild-type vulvae
(Table 1). We therefore used
let-60(n1046)/+ as a sensitized background to examine specifically
the interaction of sy622 with activated RAS. sy622 enhanced
ectopic vulval fate specification in a let-60(n1046)/+ background
(Table 1), consistent with the
sy622 mutation increasing the response to RAS activation.
We also tested if sy622 and sy665 could affect vulval fate specification in the central VPCs, P5.p-P7.p, which are normally specified to form vulval tissue. We found that both sy622 and sy665 could restore the ability of P6.p to adopt a vulval fate in the presence of the lin-3(n378) or let-23(sy1) reduction-of-function mutations, which reduce RAS signaling (Table 1). However, many of these double mutants still do not display wild-type vulval induction, indicating sy622 and sy665 do not bypass the requirements for RAS signaling.
sy622 and sy665 have additional phenotypes
In addition to enhancing vulval fate specification in the presence of
activated LET-23, the sy622 and sy665 mutations cause other
phenotypes. sy622 and sy665 hermaphrodites have a 90%
penetrant egg-laying defect (Table
2), and, as young adults, are dumpyish and only about 70% of the
size of wild-type animals (Fig.
1A-C; Table 2). In
addition, 95% of sy622 and sy665 adult males have abnormal
ray development in the tail (Fig.
1D-F; Table 2).
Given the shortened body length of sy622 and sy665 animals,
we asked whether the effect on ectopic vulval fate specification might
indirectly result from a reduced distance between the anchor cell and P3.p,
P4.p and P8.p. We, therefore, introduced the dpy-4(e1166) mutation,
which comparably reduces body length (Table
2), into let-23(sa62)/+ animals. Unlike sy622
and sy665, dpy-4(e1166) did not significantly enhance ectopic vulval
fate specification in this background, indicating that the effect of
sy622 and sy665 on vulval induction is not due to a general
reduction in body size (Table
1).
|
|
sy622 and sy665 are strong loss-of-function alleles of
dpy-22/sop-1
Based on the similarity in phenotypes between sy622 and
sy665, and their common linkage to the X chromosome, we tested
whether they were allelic. These recessive alleles failed to complement each
other for egg-laying and body size defects, suggesting that they define a
single locus (data not shown). We three-factor mapped sy622 between
dpy-6 and egl-15 on the genetic map, and using single
nucleotide polymorphisms in CB4856, a Hawaiian isolate of C. elegans,
we placed sy622 between the cosmids F15G9 and F47A4 on the physical
map. Cosmid F47A4 rescued the egg-laying, body size and male tail defects
(Table 2). A PCR fragment
encompassing the complete predicted coding region of only F47A4.2,
sop-1 (Zhang and Emmons,
2000) (Fig. 2A),
also rescued the egg-laying, body size and male tail defects
(Table 2).
F47A4.2/sop-1 has recently been found to be allelic to an older
genetic locus, dpy-22 (Meneely
and Wood, 1987
) (H. Sawa, personal communication). When crossed
into a let-23(sa62)/+; sy622 background, extra-chromosomal
arrays containing either F47A4 or the dpy-22 PCR product suppressed
the ectopic vulval fate specification observed in these animals
(Table 3). These results
suggest that all of the sy622 phenotypes result from mutation of a
single gene, dpy-22. We then sequenced dpy-22 genomic DNA
from sy622 and sy665 animals. We found that relative to
wild-type animals, sy622 animals harbored a C to T mutation at
position 13,100 of F47A4, which changes a CAG glutamine codon in exon 12 to an
Amber STOP codon. sy665 animals harbored a C to T mutation at
position 11,718 of F47A4, which changes a CAA glutamine codon in exon 13 to a
TAA Ochre STOP codon. The mutant DNA in sy622 and sy665
animals is predicted to truncate the DPY-22 protein after amino acids 1697 and
2141, respectively (Fig.
2B).
|
|
dpy-22 was cloned as sop-1, by virtue of the isolation of
non-Dpy alleles that suppress the ray loss phenotype conferred by a regulatory
region mutation in the homeobox gene pal-1
(Zhang and Emmons, 2000). The
dpy-22 alleles that suppress pal-1(e2091) consist of one
splicing mutation and three nonsense mutations, with the strength of the
allele correlated with the extent of the predicted C-terminal truncation.
dpy-22(bx93) and dpy-22(bx92) are the strongest alleles, and
they are expected to make proteins that are truncated after amino acids 2548
and 3165, respectively. RNAi experiments indicate that all of these
dpy-22 alleles are reduction-of-function mutations, raising the
possibility that dpy-22(sy622) and dpy-22(sy665) are more
severe loss-of-function alleles, because they would cause even earlier
truncations than would dpy-22(bx93). Consistent with this notion,
recent RNAi experiments against dpy-22, and double mutant analyses
using the dpy-22 alleles that suppress pal-1(e2091) and
either unc-37 or sur-2 also have revealed a ray loss
phenotype (Zhang and Emmons,
2002
), similar to that observed in dpy-22(sy622) and
dpy-22(sy665) single mutants (Fig.
1). This observation suggests that ray development is ultimately
compromised when DPY-22 pathway activity is reduced below a certain threshold.
In addition, we find that the loss-of-function allele dpy-22(bx93)
weakly promotes ectopic vulval fate transformations in a
let-23(sa62)/+ background (Table
3). Moreover, dpy-22(bx93) fails to complement
dpy-22(sy622) for enhancing ectopic vulval fate specification in the
presence of let-23(sa62)/+ (Table
3), providing further evidence that DPY-22 negatively regulates
vulval development.
To examine directly the effects of reducing DPY-22 levels on vulval development, we used RNAi against dpy-22. In an otherwise wild-type background, dpy-22 RNAi did not affect vulval development (data not shown). However, in a sensitized background consisting of the let-23(sy1) loss-of-function mutation, dpy-22 RNAi could partially suppress the vulvaless phenotype of let-23(sy1), similar to the dpy-22 alleles sy622 and sy665 (Table 3). By contrast, control gfp dsRNA did not suppress let-23(sy1) (Table 3). Together, these data indicate that sy622 and sy665 are stronger loss-of-function alleles of dpy-22.
dpy-22 is expressed in vulval precursor cells and is
mislocalized in dpy-22(sy665) animals
DPY-22 is most closely related to human TRAP230
(Ito et al., 1999;
Nagase et al., 1996
;
Philibert et al., 1998
), a
component of human mediator complexes, and KOHTALO
(Treisman, 2001
), a regulator
of cell fate in the Drosophila eye. The fly and human proteins have
similar length, and display 35% overall identity. DPY-22 is larger than both
proteins by
1000 amino acids, and BLAST analysis indicates that its
homology with TRAP230 and KOHTALO is spread over three regions
(Fig. 2B). These include small
regions of identity at the N and C termini, and a larger region of identity in
the middle of the protein. In all three proteins, the C terminus is rich in
glutamine. Thirty-three percent of the terminal 781 and 589 amino acids are
glutamine in DPY-22 and KOHTALO, respectively, and 41% of the 278 terminal
amino acids in TRAP230 are glutamine. Of the previously isolated nonsense
mutations in dpy-22, bx93 and bx92 are the strongest.
bx93 results in the greatest truncation, removing all the amino acids
C-terminal to 2548, including the entire glutamine-rich region. As
dpy-22(sy622) and dpy-22(sy665) cause a number of highly
penetrant phenotypes (Fig. 1;
Tables 1,
2) not observed in the other
nonsense mutations, domains N-terminal to the glutamine-rich region must be
important for DPY-22 function. The nonsense mutation in dpy-22(sy622)
would be predicted to truncate DPY-22 in the largest central region of
identity; however, dpy-22(sy665) is predicted to truncate DPY-22
after amino acid 2141, close to the end of this region
(Fig. 2B). This observation
suggests that another important functional domain exists between amino acids
2141 and 2548 that does not have identity with TRAP230 or KOHTALO.
PROSITE analysis of DPY-22 indicated the presence of two putative nuclear
localization signals in this region. To test whether the stronger phenotypes
observed in dpy-22(sy622) and dpy-22(sy665) animals are
correlated with a mislocalized, non-nuclear form of DPY-22, we constructed
transgenes in which dpy-22 DNA was fused in-frame to the green
fluorescent protein (GFP) open reading frame
(Fig. 2C). dpy-22 has
been reported to be expressed in vulval cells, during the later stages of
vulval development (Zhang and Emmons,
2000). We found that a rescuing (see last section of the Results)
fusion of wild-type DPY-22 to GFP was expressed in the VPCs, the anchor cell,
and hyp7 nuclei at the time of vulval fate specification, and continued to be
expressed in these cells throughout vulval development
(Fig. 3A-F). Wild-type DPY-22
and DPY-22 truncated after amino acid 2548, as generated in
dpy-22(bx93) mutants, directed nuclear expression of GFP
(Fig. 3A-H). By contrast,
DPY-22 truncated after amino acid 2141 could not direct GFP into the nucleus,
and instead, caused GFP to accumulate in the cytoplasm
(Fig. 3I,J). These data
indicate that the predicted nuclear localization sequences (NLSs) in this
region are functional, and suggest that the severity of the
dpy-22(sy622) and dpy-22(sy665) phenotypes may result from
mislocalization of the DPY-22 protein, and/or loss of other functional regions
of the protein.
|
DPY-22 is a gonad-independent inhibitor of vulval fate specification
of multiple Pn.p cells
let-23(sa62)/+ and let-60(n1046) animals are responsive
to LIN-3 (Chang et al., 2000;
Katz et al., 1996
;
Sundaram and Han, 1995
)
(Table 3), and suppression of
the lin-3(n378) and let-23(sy1) hypomorphic mutations could
in principle occur through elevated production of LIN-3. As DPY-22::GFP is
expressed in the anchor cell and the VPCs
(Fig. 3), we tested whether
dpy-22 affects vulval fate specification by modulating the production
of LIN-3 from the gonad. We ablated the gonadal primordium in
let-23(sa62)/+ and let-23(sa62)/+; dpy-22(sy622)
early L1 larvae. Gonad-ablated let-23(sa62)/+ animals displayed very
little vulval fate specification in any of the Pn.p cells
(Table 4). By contrast,
dpy-22(sy622) increased the frequency of vulval fate specification in
all six VPCs in this background (Table
4), indicating that DPY-22 does not act primarily by regulating
LIN-3 production from the gonad, and that the fates of all the VPCs are
regulated by DPY-22.
DPY-22-mediated inhibition of vulval-fate specification involves
transcription factors other than BAR-1 (ß-catenin)
In males, a bar-1(ß-catenin) null allele, ga80, does
not affect ray development on its own
(Zhang and Emmons, 2000).
However, it reduces the degree to which dpy-22(bx92) can restore
wild-type ray development in a pal-1(e2091) mutant background
(Zhang and Emmons, 2000
). This
property has led to the proposal that in the presence of the
pal-1(e2091) regulatory region mutation, DPY-22 inhibits
BAR-1-dependent regulation of the pal-1 gene
(Zhang and Emmons, 2000
).
WNT/ß-catenin signaling also promotes vulval fate specification, parallel
to the LET-23-LET-60 pathway. A null mutation in bar-1 results in an
incompletely penetrant vulvaless phenotype
(Table 5)
(Eisenmann et al., 1998
), and
a loss-of-function mutation in the axin-like inhibitor of WNT signaling,
pry-1 (Korswagen et al.,
2002
; Maloof et al.,
1999
), causes ectopic vulval fate specification
(Table 5)
(Gleason et al., 2002
).
Furthermore, hyperactivated WNT signaling can bypass loss-of-function
mutations in the let-23 pathway
(Gleason et al., 2002
). We
therefore tested whether relief of inhibition of BAR-1 might account for the
interactions between our dpy-22 alleles and mutations in the LET-23
pathway during vulval development.
|
We linked two bar-1 mutations to dpy-22(sy622).
bar-1(mu63) encodes a weak loss-of-function mutation
(Maloof et al., 1999) that
does not confer defects in vulval development on its own
(Table 5). The mis-sense
mutation in this allele results in a protein with a G524D change (see
Materials and Methods). We hypothesized that under conditions where a
phenotype was dependent on elevated BAR-1 activity, bar-1(mu63)
should quantitatively reduce it. Consistent with this notion,
bar-1(mu63) suppresses the ectopic mab-5 expression and poly
ray phenotypes observed in pry-1(mu38) animals
(Maloof et al., 1999
). As a
control, we tested whether bar-1(mu63) also could reduce vulval
induction in a background where WNT signaling was specifically hyperactivated.
We built a pry-1(mu38); let-23(sy1) double mutant and, as recently
reported (Gleason et al.,
2002
), found that excess WNT signaling could strongly suppress
this let-23 reduction-of-function allele
(Table 5). bar-1(mu63)
reduced the ability of pry-1(mu38) to suppress let-23(sy1)
(Table 5), indicating that
bar-1(mu63) can be used to define conditions under which WNT
signaling is hyperactivated during vulval development. Although
dpy-22(sy622) is a weaker suppressor of let-23(sy1) than is
pry-1(mu38), its ability to suppress the let-23 mutation was
not reduced in the presence of the bar-1(mu63) mutation
(Table 5). These data suggest
that DPY-22 does not interfere with vulval development through inhibition of
BAR-1. We also examined the body size, egg-laying behavior and male rays in
bar-1(mu63) dpy-22(sy622) animals, and found none of
dpy-22(sy622) phenotypes to be suppressed by the bar-1
mutation (Table 2).
As our data suggested that DPY-22 might primarily inhibit the output of the RAS pathway, rather than the WNT pathway, during vulval development, we tested whether the dpy-22(sy622) mutation was strong enough to compensate for the absence of BAR-1 in bar-1(ga80) null mutants. bar-1(ga80) dpy-22(sy622) double mutants were sick, but still displayed the bar-1(ga80) vulvaless phenotype (Table 5). Because the gain-of-function let-60(n1046) allele, which is far stronger than dpy-22(sy622) [(Table 1) 73% Muv and 4.0 cell induction versus 3% Muv and 3.0 cell induction, respectively] only suppresses bar-1(ga80) to wild-type levels, we hypothesized that failure of dpy-22(sy622) to suppress bar-1(ga80) might result from insufficient activation of the RAS pathway. Therefore, we used a more sensitive assay to test further whether DPY-22 acts independently of BAR-1 during vulval development. let-60(n1046gf); bar-1(ga80) double mutants are mostly wild type and are mutually suppressed with regards to the let-60(n1046) multivulva phenotype and the bar-1(ga80) vulvaless phenotype (Table 5). As dpy-22(sy622) is a strong enhancer of the gain-of-function let-60(n1046) allele (Table 1), we asked whether dpy-22(sy622) could still enhance activated RAS even in the absence of BAR-1 protein. let-60(n1046); bar-1(ga80) dpy-22(sy622) triple mutants were very sick, but survived to L4 and displayed much more vulval induction than did let-60(n1046); bar-1(ga80) double mutants (Table 5). The enhanced vulval induction in the triple mutants was abrogated by the presence of the dpy-22-rescuing cosmid, F47A4, demonstrating that enhancement in the triple mutant was indeed due to loss of DPY-22 activity (Table 5). These data indicate that DPY-22 can regulate vulval induction independently of any effects on BAR-1 activity.
The C-terminal glutamine-rich region is dispensable for most of the
activity of DPY-22
The absence of body size, egg-laying, and ray defects, and the presence of
only a weak vulval phenotype in dpy-22(bx93) mutants, suggest that
the glutamine-rich region is dispensable for the function of DPY-22 in these
processes. Because it is possible that small amounts of wild-type DPY-22 might
be produced through translational readthrough in dpy-22(bx93)
mutants, we directly examined the functional properties of a DPY-22 protein
that completely lacks the glutamine-rich region. We injected a construct
consisting of dpy-22 DNA ending with the codon for amino acid 2548,
fused in-frame to the GFP open reading frame, into dpy-22(sy622)
animals (Fig. 2C). This
transgene should produce a protein similar to that made in
dpy-22(bx93) animals, which ends after amino acid 2548, before the
beginning of the glutamine-rich region. Wild-type and glutamine-deleted DPY-22
fully rescued the body length, egg-laying and male tail defects observed in
dpy-22(sy622) mutants (Fig.
1G,H; Table 2).
Furthermore, glutamine-deleted DPY-22 also rescued the ectopic vulval fate
specification observed in a let-23(sa62)/+; dpy-22(sy622)
background, comparable with wild-type DPY-22
(Table 3). These results
demonstrate that the glutamine-rich region is not essential for the majority
of functions performed by DPY-22.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Besides the RAS pathway, LIN-12/NOTCH and WNT promote vulval fates.
However, LIN-12 and RAS antagonize each other with respect to the type of
vulval fate that is induced (Berset et al.,
2001; Wang and Sternberg,
1999
). High levels of LIN-12 signaling promote secondary vulval
fates (Sternberg and Horvitz,
1989
), and high levels of RAS signaling promote primary vulval
fates (Katz et al., 1995
). In
our double mutants consisting of dpy-22 and let-23 pathway
mutations, P6.p induction is restored
(Table 1), and all animals
displaying full vulval induction have correctly patterned vulvae consisting of
2°-1°-2° fates for P5.p-P7.p. These data indicate that
dpy-22 mutations cooperate with RAS to induce the primary fate in
P6.p, rather than antagonize it, and suggest that DPY-22 does not act
primarily by inhibiting the LIN-12 pathway.
DPY-22 has previously been described as an inhibitor of BAR-1-dependent WNT
signaling (Zhang and Emmons,
2000). In contrast to the LIN-12-RAS relationship, RAS and WNT
signaling are known to converge on at least one common target, necessary for
vulval development, lin-39
(Eisenmann et al., 1998
;
Maloof and Kenyon, 1998
).
Thus, DPY-22 inhibition likely occurs through interference with RAS and/or WNT
signaling. However, our triple mutant analysis with a bar-1 null
mutation indicates that DPY-22 can inhibit activated RAS even in the absence
of BAR-1 protein (Table 5).
Together with our molecular data that the glutamine-rich region, which is
necessary for inhibition of BAR-1-dependent ray development
(Zhang and Emmons, 2000
), is
dispensable for inhibition of vulval development
(Table 3), we propose that
DPY-22 largely acts on the RAS pathway, rather than the WNT pathway, to
inhibit vulval development. The strong gain-of-function let-60(n1046)
allele only suppresses the bar-1(ga80) null mutation to wild type,
and is itself suppressed for its multivulva phenotype by the
bar-1(ga80) mutation (Table
5). Furthermore, our dpy-22(sy622) mutation which does
not cause a strong multivulva phenotype, partially suppresses the moderate
vulvaless phenotype of let-23 pathway mutations
(Table 1), but not the weak
vulvaless phenotype of bar-1(ga80)
(Table 5). These results
suggest that shared targets between RAS and WNT signaling are unequally
activated by the two pathways. In addition, it is likely that the WNT pathway
has targets that are not shared with the RAS pathway.
Most inhibitors of vulval development affect all six VPCs. This observation
is consistent with a model in which negative regulators of the RAS pathway
raise the requirement for the amount of pathway activation needed to generate
a biological response. Although the LIN-3 growth factor might diffuse outside
of the source anchor cell, only P6.p, because of its proximity to the anchor
cell, achieves sufficient pathway activation to adopt a primary vulval fate. A
surprising number of points in the pathway must be negatively regulated,
presumably to achieve invariant positional specificity for the response to
LIN-3. SLI-1(c-CBL) (Jongeward et al.,
1995; Yoon et al.,
1995
), GAP-1 (Hajnal et al.,
1997
) and LIP-1 (MAPK phosphatase)
(Berset et al., 2001
) appear to
directly regulate LET-23 (EGFR), LET-60 (RAS) and SUR-1/MPK-1(MAP kinase),
respectively, while ARK-1 might control some aspect of SEM-5(GRB2)-dependent
RAS activation (Hopper et al.,
2000
).
Ultimately, the end point for growth factor signaling can be considered to
be a change in RNA Pol II activity on specific promoters. Thus, in principle,
sequence-specific and global regulators of transcription also might play
important roles in regulating the output of the RAS pathway. Genetic studies
have identified LIN-1, an Ets-domain sequence-specific DNA-binding protein as
an inhibitor of vulval fate specification
(Beitel et al., 1995). Global
regulators of transcription broadly include chromatin remodeling proteins, RNA
Pol II and the general transcription factors, and components of the mediator
complex, which promote the activity of sequence-specific activators and
repressors. Mutations in the synmuv genes lin-35(RB) and
lin-53(RBAP48) have suggested that a histone deacetylase complex is
inhibitory towards vulval fate specification
(Lu and Horvitz, 1998
). In
support of this, experiments that directly examine HDAC-1 and components of
the NURD complex have provided some evidence for their inhibitory roles
(Chen and Han, 2001
;
Lu and Horvitz, 1998
;
Solari and Ahringer, 2000
).
Our work with dpy-22, which is most related to humanTRAP230
(Ito et al., 1999
;
Nagase et al., 1996
;
Philibert et al., 1998
), now
indicates that a specific component of the mediator also can act as an
inhibitor of a RAS-dependent response.
At least 20 components of the mediator complex have been identified in
yeast. Ten are essential for viability and seven of these appear to have
related counterparts in higher species including C. elegans (for
reviews, see Boube et al.,
2002; Gustafsson and
Samuelsson, 2001
; Myers and
Kornberg, 2000
; Woychik and
Hampsey, 2002
). By contrast, the remaining yeast mediator
components do not have clear orthologs in higher species. This has led to the
proposal that these components and their counterparts in other species might
not peform core mediator functions but, instead, specifically integrate
information from rapidly evolving transcription factor families. Consistent
with this model, mutation or RNA interference against the conserved components
in C. elegans result in early lethality, or multiple phenotypes in
the rare animals escaping lethality (Kwon
et al., 1999
; Kwon et al.,
2001
; Kwon and Lee,
2001
). By contrast, the metazoan-specific mediator, sur-2
(Boyer et al., 1999
;
Singh and Han, 1995
), is not
essential for viability, and single mutants have a strong vulvaless phenotype,
resembling a RAS pathway mutation (Singh
and Han, 1995
). Similarly, prior to the discovery that
sop-1 was allelic to dpy-22, it was thought that mutations
in C. elegans TRAP230, another metazoan-specific mediator component,
do not affect viability and cause one major phenotype, which is the relief of
inhibition on BAR-1-dependent regulation of pal-1 expression in males
(Zhang and Emmons, 2000
).
However, our alleles of dpy-22, which cause more severe
truncations of the protein and mislocalize it
(Fig. 3I), result in multiple
developmental and behavioral phenotypes
(Fig. 1,
Table 2) that do not appear to
be restricted to one specific signaling pathway. Some of these include an
overall reduction in body length by 30%, a Dpy appearance, partial sterility
(data not shown), abnormal ray development in the male tail and an egg-laying
defect that does not result from the absence of vulval tissue or sex muscles,
or a functional uterine-vulval connection (data not shown). We have also found
that DPY-22 antagonizes vulval fates specified by activated RAS, and that the
major target(s) of this inhibition is distinct from BAR-1
(Table 5). These findings are
consistent with the original description of dpy-22(e652) phenotypes,
and the observation that dpy-22(e652) enhances the multivulva
phenotypes of lin-53(n833); lin-15(n767) and lin-15(n765)
males (Meneely and Wood,
1987). Because we observe effects of dpy-22 mutations on
vulval development with multiple non-X-linked alleles in the RAS pathway, we
do not think that DPY-22 acts on vulval development through regulation of
dosage compensation from the X-chromosome, as originally proposed
(Meneely and Wood, 1987
).
Neither loss-of-function nor gain-of-function mutants in the RAS pathway
result in the body size or ray defects observed in our mutants, and
bar-1(mu63) and bar-1(ga80) do not suppress at least the
body size phenotype of our dpy-22 mutants
(Table 2, data not shown).
These results suggest that transcription factors downstream of other signaling
pathway also must be regulated by DPY-22. Similarly, recent data with
dpy-22(bx92) and sur-2 double mutants have shown a synthetic
loss of rays (Zhang and Emmons,
2002
), indicating SUR-2 also can function in pathways distinct
from those using RAS and BAR-1. Thus, it is likely that DPY-22 and SUR-2 relay
information from multiple activator/promoter contexts to RNA Pol II, but how
they do it, and their relative contribution at a given promoter may be
different.
The molecular identification of our new dpy-22 alleles provides
some insight into how a mediator component such as DPY-22 can have general and
specific functions, depending on the promoter context. Although
dpy-22(bx93), which deletes the entire glutamine-rich domain of
DPY-22, has a weak ability to promote ectopic vulval fate specification in the
presence of activated LET-23, it has a profound ability to alleviate
inhibition on BAR-1-dependent regulation of the pal-1 gene
(Zhang and Emmons, 2000).
Furthermore, whereas a transgene harboring nonsense mutations affecting the
glutamine-rich domain cannot restore inhibition to the pal-1 gene as
well as a wild-type dpy-22 transgene
(Zhang and Emmons, 2000
), we
find that a transgene completely lacking the glutamine-rich domain has the
same activity as a wild-type transgene in rescuing vulval fate specification
(Table 3), ray development
(Fig. 1), body size and
egg-laying defects (Table 2).
The glutamine-rich region thus defines a domain that probably distinguishes
promoter/activator-dependent activity for DPY-22. Inhibition of
BAR-1-dependent pal-1 expression is critically dependent on this
domain, but not the activity of DPY-22 in the other pathways studied in this
work. BLAST analysis (Boube et al.,
2002
; Gustafsson and
Samuelsson, 2001
) indicates the N-terminal region of TRAP230 is
similar to both DPY-22 and SRB8
(Hengartner et al., 1995
), a
yeast mediator component without a clear ortholog in metazoans. SRB8 is
responsible for recruiting (Myer and
Young, 1998
) the SRB10/SRB11 cyclin/cdk complex
(Liao et al., 1995
) that
represses Pol II-dependent transcription on many promoters
(Holstege et al., 1998
). In
cases where DPY-22/TRAP230 represses transcription, this function might be
dependent on the SRB8-like domain, but modulated by the metazoan-specific
domains. TRAP230 and PCQAP (Berti et al.,
2001
), two glutamine-rich components of human mediator complexes
have been implicated in human disease. TRAP230 has been reported to undergo a
12 bp expansion in a region encoding the glutamine-rich domain in a subset of
mentally retarded individuals (Philibert
et al., 1998
), and PCQAP is in the 22q11 deletion associated with
DiGeorge syndrome (Berti et al.,
2001
). If TRAP230 and PCQAP contribute to these conditions, one
would expect them to arise out of loss of specific and general functions,
respectively.
Although compared with sequence-specific DNA-binding proteins, proteins
involved in nucleosome displacement, histone acetylation and deacetylation,
and components of the mediator complex appear to be global regulators of
transcription, these global regulators may not act equally at all promoters.
In particular, multiple chromatin remodeling and histone acetylation and
deacetylation complexes with distinct subunit composition, substrate
specificity and activator preferences have been described (reviewed by
Emerson, 2002;
Narlikar et al., 2002
).
Although it is still controversial whether distinct mediator complexes are
associated with the RNA Pol II holoenzyme at a given promoter, mutations in
different components have different effects on gene expression in yeast
(Holstege et al., 1998
;
Myers et al., 1999
). Some
mediator components may perform unique functions for distinct activators or
repressors, while others may be used to varying degrees or redundantly with
other components. Finally, as in the case of DPY-22, a single component may
use distinct domains to regulate transcription on different promoters. Genetic
studies thus demonstrate that a specific component of the mediator provides an
important point for regulating the output of the RAS pathway, and that
specific mutations can be generated with distinct properties, which might
contribute to disease in humans.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aroian, R. and Sternberg, P. (1991). Multiple
functions of let-23, a Caenorhabditis elegans receptor tyrosine
kinase gene required for vulval induction. Genetics
128,251
-267.
Aroian, R. V., Koga, M., Mendel, J. E., Ohshima, Y. and Sternberg, P. W. (1990). The let-23 gene necessary for Caenorhabditis elegans vulval induction encodes a tyrosine kinase of the EGF receptor subfamily. Nature 348,693 -699.[CrossRef][Medline]
Avery, L., Bargmann, C. and Horvitz, H. (1993).
The Caenorhabditis elegans unc-31 gene affects multiple nervous
system-controlled functions. Genetics
134,455
-464.
Bargmann, C. and Avery, L. (1995). Laser killing of cells in Caenorhabditis elegans. Methods in Cell Biology. Caenorhabditis elegans: Modern Biological Analysis of an Organism. Vol. 48 (ed. H. F. Epstein and D. C. Shakes), pp. 225-250. San Diego: Academic Press.
Beitel, G., Clark, S. and Horvitz, H. (1990). Caenorhabditis elegans ras gene let-60 acts as a switch in the pathway of vulval induction. Nature 348,503 -509.[CrossRef][Medline]
Beitel, G., Tuck, S., Greenwald, I. and Horvitz, H. (1995). The Caenorhabditis elegans gene lin-1 encodes an ETS-domain protein and defines a branch of the vulval induction pathway. Genes Dev. 9,3149 -3162.[Abstract]
Berset, T., Hoier, E. F., Battu, G., Canevascini, S. and Hajnal,
A. (2001). Notch inhibition of RAS signaling through MAP
kinase phosphatase LIP-1 during C. elegans vulval development.
Science 291,1055
-1058.
Berti, L., Mittler, G., Przemeck, G. K., Stelzer, G., Gunzler, B., Amati, F., Conti, E., Dallapiccola, B., Hrabe de Angelis, M., Novelli, G. et al. (2001). Isolation and characterization of a novel gene from the DiGeorge chromosomal region that encodes for a mediator subunit. Genomics 74,320 -332.[CrossRef][Medline]
Boube, M., Joulia, L., Cribbs, D. L. and Bourbon, H. M. (2002). Evidence for a mediator of RNA polymerase II transcriptional regulation conserved from yeast to man. Cell 110,143 -151.[Medline]
Boyer, T. G., Martin, M. E., Lees, E., Ricciardi, R. P. and Berk, A. J. (1999). Mammalian Srb/Mediator complex is targeted by adenovirus E1A protein. Nature 399,276 -279.[CrossRef][Medline]
Brenner, S. (1974). The genetics of
Caenorhabditis elegans. Genetics
77, 71-94.
Chang, C., Hopper, N. and Sternberg, P. (2000).
Caenorhabditis elegans SOS-1 is necessary for multiple Ras-mediated
developmental signals. EMBO J.
19,3283
-3294.
Chen, Z. and Han, M. (2001). C. elegans Rb, NuRD, and Ras regulate lin-39-mediated cell fusion during vulval fate specification. Curr. Biol. 11,1874 -1879.[CrossRef][Medline]
Clandinin, T., Katz, W. and Sternberg, P. (1997). Caenorhabditis elegans HOM-C genes regulate the response of vulval precursor cells to inductive signal. Dev. Biol. 182,150 -161.[CrossRef][Medline]
Cox, G., Laufer, J., Kusch, M. and Edgar, R.
(1980). Genetic and phenotypic characterization of roller mutants
of C. elegans. Genetics
95,317
-339.
Eisenmann, D., Maloof, J., Simske, J., Kenyon, C. and Kim,
S. (1998). The ß-catenin homolog BAR-1 and LET-60 Ras
coordinately regulate the Hox gene lin-39 during Caenorhabditis
elegans vulval development. Development
125,3667
-3680.
Emerson, B. M. (2002). Specificity of gene regulation. Cell 109,267 -270.[Medline]
Ferguson, E. and Horvitz, H. (1985).
Identification and characterization of 22 genes that affect the vulval cell
lineages of the nematode C. elegans. Genetics
110, 17-72.
Ferguson, E. and Horvitz, H. (1989). The
multivulva phenotype of certain Caenorhabditis elegans mutants
results from defects in two functionally redundant pathways.
Genetics 123,109
-121.
Ferguson, E., Sternberg, P. and Horvitz, H. (1987). A genetic pathway for the specification of the vulval cell lineages of C. elegans. Nature 326,259 -267.[CrossRef][Medline]
Gleason, J. E., Korswagen, H. C. and Eisenmann, D. M.
(2002). Activation of Wnt signaling bypasses the requirement for
RTK/Ras signaling during C. elegans vulval induction.
Genes Dev. 16,1281
-1290.
Granato, M., Schnabel, H. and Schnabel, R. (1994). pha-1, a selectable marker for gene-transfer in C. elegans. Nucleic Acids Res. 22,1762 -1763.[Medline]
Greenwald, I. (1997). Development of the vulva. In C. Elegans II (ed. D. L. Riddle, T. Blumenthal, B. J. Meyer and J. R. Priess), pp. 519-541. Cold Spring Harbor, NY: Cold Spring Habor Laboratory Press.
Gustafsson, C. M. and Samuelsson, T. (2001). Mediator a universal complex in transcriptional regulation. Mol. Microbiol. 41,1 -8.[CrossRef][Medline]
Hajnal, A., Whitfield, C. and Kim, S. (1997).
Inhibition of Caenorhabditis elegans vulval induction by gap-1 and by
let-23 receptor tyrosine kinase. Genes Dev.
11,2715
-2728.
Han, M. and Sternberg, P. (1990). let-60, a gene that specifies cell fates during C. elegans vulval induction, encodes a ras protein. Cell 63,921 -931.[Medline]
Hengartner, C. J., Thompson, C. M., Zhang, J., Chao, D. M., Liao, S. M., Koleske, A. J., Okamura, S. and Young, R. A. (1995). Association of an activator with an RNA polymerase II holoenzyme. Genes Dev. 9, 897-910.[Abstract]
Herman, R. (1978). Crossover suppressors and
balanced recessive lethals in C. elegans. Genetics
88, 49-65.
Hill, R. and Sternberg, P. (1992). The gene lin-3 encodes an inductive signal for vulval development in C. elegans. Nature 358,470 -476.[CrossRef][Medline]
Hodgkin, J., Horvitz, H. and Brenner, S.
(1979). Nondisjunction mutants of the nematode C. elegans.Genetics 91,67
-94.
Holstege, F. C., Jennings, E. G., Wyrick, J. J., Lee, T. I., Hengartner, C. J., Green, M. R., Golub, T. R., Lander, E. S. and Young, R. A. (1998). Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95,717 -728.[Medline]
Hopper, N., Lee, J. and Sternberg, P. (2000). ARK-1 inhibits EGFR signaling in C. elegans. Mol. Cell 6, 65-75.[Medline]
Huang, L., Tzou, P. and Sternberg, P. (1994). The lin-15 locus encodes two negative regulators of Caenorhabditis elegans vulval development. Mol. Biol. Cell 5,395 -411.[Abstract]
Ito, M., Yuan, C. X., Malik, S., Gu, W., Fondell, J. D., Yamamura, S., Fu, Z. Y., Zhang, X., Qin, J. and Roeder, R. G. (1999). Identity between TRAP and SMCC complexes indicates novel pathways for the function of nuclear receptors and diverse mammalian activators. Mol. Cell 3,361 -370.[Medline]
Jongeward, G., Clandinin, T. and Sternberg, P.
(1995). sli-1, a negative regulator of let-23-mediated signaling
in C. elegans. Genetics
139,1553
-1566.
Kaech, S., Whitfield, C. and Kim, S. (1998). The LIN-2/LIN-7/LIN-10 complex mediates basolateral membrane localization of the C. elegans EGF receptor LET-23 in vulval epithelial cells. Cell 94,761 -771.[Medline]
Katz, W., Hill, R., Clandinin, T. and Sternberg, P. (1995). Different levels of the C. elegans growth factor LIN-3 promote distinct vulval precursor fates. Cell 82,297 -307.[Medline]
Katz, W., Lesa, G., Yannoukakos, D., Clandinin, T., Schlessinger, J. and Sternberg, P. (1996). A point mutation in the extracellular domain activated LET-23, the Caenorhabditis elegans epidermal growth factor receptor homolog. Mol. Cell. Biol. 16,529 -537.[Abstract]
Korswagen, H. C., Coudreuse, D. Y., Betist, M. C., van de Water,
S., Zivkovic, D. and Clevers, H. C. (2002). The Axin-like
protein PRY-1 is a negative regulator of a canonical Wnt pathway in C.
elegans. Genes Dev. 16,1291
-1302.
Kwon, J., Park, J., Gim, B., Han, S., Lee, J. and Kim, Y.
(1999). Caenorhabditis elegans mediator complexes are
required for developmental-specific transcriptional activation.
Proc. Natl. Acad. Sci. USA
96,14990
-14995.
Kwon, J. Y., Kim-Ha, J., Lee, B. J. and Lee, J. (2001). The MED-7 transcriptional mediator encoded by let-49 is required for gonad and germ cell development in Caenorhabditis elegans. FEBS Lett. 508,305 -308.[CrossRef][Medline]
Kwon, J. Y. and Lee, J. (2001). Biological
significance of a universally conserved transcription mediator in metazoan
developmental signaling pathways. Development
128,3095
-3104.
Lackner, M., Kornfeld, K., Miller, L., Horvitz, H. and Kim, S. (1994). A MAP kinase homolog, mpk-1, is involved in ras-mediated induction of vulval cell fates in Caenorhabditis elegans.Genes Dev. 8,160 -173.[Abstract]
Lee, J., Jongeward, G. and Sternberg, P. (1994). unc-101, a gene required for many aspects of Caenorhabditis elegans development and behavior, encodes a clathrin-associated protein. Genes Dev. 8, 60-73.[Abstract]
Liao, S. M., Zhang, J., Jeffery, D. A., Koleske, A. J., Thompson, C. M., Chao, D. M., Viljoen, M., van Vuuren, H. J. and Young, R. A. (1995). A kinase-cyclin pair in the RNA polymerase II holoenzyme. Nature 374,193 -196.[CrossRef][Medline]
Lu, X. and Horvitz, H. (1998). lin-35 and lin-53, two genes that antagonize a C. elegans Ras pathway, encode proteins similar to Rb and its binding protein RbAp48. Cell 95,981 -991.[Medline]
Maloof, J. and Kenyon, C. (1998). The Hox gene
lin-39 is required during C. elegans vulval induction to
select the outcome of Ras signaling. Development
125,181
-190.
Maloof, J., Whangbo, J., Harris, J., Jongeward, G. and Kenyon,
C. (1999). A Wnt signaling pathway controls Hox gene
expression and neuroblast migration in C. elegans.Development 126,37
-49.
Meneely, P. and Wood, W. (1987). Genetic
analysis of X-chromosome dosage compensation in C. elegans.Genetics 117,25
-41.
Myer, V. E. and Young, R. A. (1998). RNA
polymerase II holoenzymes and subcomplexes. J. Biol.
Chem. 273,27757
-27760.
Myers, L. C., Gustafsson, C. M., Hayashibara, K. C., Brown, P.
O. and Kornberg, R. D. (1999). Mediator protein mutations
that selectively abolish activated transcription. Proc. Natl. Acad.
Sci. USA 96,67
-72.
Myers, L. C. and Kornberg, R. D. (2000). Mediator of transcriptional regulation. Annu. Rev. Biochem. 69,729 -749.[CrossRef][Medline]
Nagase, T., Seki, N., Ishikawa, K., Tanaka, A. and Nomura, N. (1996). Prediction of the coding sequences of unidentified human genes. V. The coding sequences of 40 new genes (KIAA0161-KIAA0200) deduced by analysis of cDNA clones from human cell line KG-1. DNA Res. 3,17 -24.[Medline]
Narlikar, G. J., Fan, H. Y. and Kingston, R. E. (2002). Cooperation between complexes that regulate chromatin structure and transcription. Cell 108,475 -487.[Medline]
Philibert, R. A., King, B. H., Winfield, S., Cook, E. H., Lee, Y. H., Stubblefield, B., Damschroder-Williams, P., Dea, C., Palotie, A., Tengstrom, C. et al. (1998). Association of an X-chromosome dodecamer insertional variant allele with mental retardation. Mol. Psychiatry 3,303 -309.[CrossRef][Medline]
Rogalski, T. and Riddle, D. (1988). A C.
elegans RNA polymerase II gene, ama-1 IV, and nearby essential
genes. Genetics 118,61
-74.
Sigurdson, D., Spanier, G. and Herman, R.
(1984). Caenorhabditis elegans deficiency mapping.
Genetics 108,331
-345.
Simske, J. and Kim, S. (1995). Sequential signaling during Caenorhabditis elegans vulval induction. Nature 375,142 -146.[CrossRef][Medline]
Simske, J., Kaech, S., Harp, S. and Kim, S. (1996). LET-23 receptor localization by the cell junction protein LIN-7 during C. elegans vulval induction. Cell 85,195 -204.[Medline]
Singh, N. and Han, M. (1995). sur-2, a novel gene, functions late in the let-60 ras-mediated signaling pathway during Caenorhabditis elegans vulval induction. Genes Dev. 9,2251 -2265.[Abstract]
Solari, F. and Ahringer, J. (2000). NURD-complex genes antagonise Ras-induced vulval development in Caenorhabditis elegans. Curr. Biol. 10,223 -226.[CrossRef][Medline]
Sternberg, P. and Han, M. (1998). Genetics of RAS signaling in C. elegans. Trends Genet. 14,466 -472.[CrossRef][Medline]
Sternberg, P. and Horvitz, H. (1986). Pattern formation during vulval development in C. elegans.Cell 44,761 -772.[Medline]
Sternberg, P. and Horvitz, H. (1989). The combined action of two intercellular signaling pathways specifies three cell fates during vulval induction in C. elegans. Cell 58,679 -693.[Medline]
Sundaram, M. and Han, M. (1995). The C. elegans ksr-1 gene encodes a novel Raf-related kinase involved in Ras-mediated signal transduction. Cell 83,889 -901.[Medline]
Treisman, J. (2001). Drosophila
homologues of the transcriptional coactivation complex subunits TRAP240 and
TRAP230 are required for identical processes in eye-antennal disc development.
Development 128,603
-615.
Trent, C., Tsung, N. and Horvitz, H. (1983).
Egg-laying defective mutants of the nematode C. elegans.Genetics 104,619
-647.
Wang, M. and Sternberg, P. (1999). Competence and commitment of Caenorhabditis elegans vulval precursor cells. Dev. Biol. 212,12 -24.[CrossRef][Medline]
White, J., Southgate, E. and Thomson, J. (1992). Mutations in the Caenorhabditis elegans unc-4 gene alter the synaptic input to ventral cord motor neurons. Nature 355,838 -841.[CrossRef][Medline]
Wicks, S. R., Yeh, R. T., Gish, W. R., Waterston, R. H. and Plasterk, R. H. (2001). Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map. Nat. Genet. 28,160 -164.[CrossRef][Medline]
Woychik, N. A. and Hampsey, M. (2002). The RNA polymerase II machinery: structure illuminates function. Cell 108,453 -463.[Medline]
Wu, Y. and Han, M. (1994). Suppression of activated Let-60 Ras protein defines a role of Caenorhabditis elegans Sur-1 MAP kinase in vulval differentiation. Genes Dev. 8, 147-159.[Abstract]
Yochem, J., Weston, K. and Greenwald, I. (1988). The Caenorhabditis elegans lin-12 gene encodes a transmembrane protein with overall similarity to Drosophila Notch. Nature 335,547 -550.[CrossRef][Medline]
Yoon, C., Lee, J., Jongeward, G. and Sternberg, P. (1995). Similarity of sli-1, a regulator of vulval development in C. elegans, to the mammalian protooncogene c-cbl. Science 269,1102 -1105.[Medline]
Zhang, H. and Emmons, S. (2000). A C.
elegans mediator protein confers regulatory selectivity on
lineage-specific expression of a transcription factor gene. Genes
Dev. 14,2161
-2172.
Zhang, H. and Emmons, S. W. (2002).
Caenorhabditis elegans unc-37/groucho interacts genetically with
components of the transcriptional mediator complex.
Genetics 160,799
-803.