Department of Biology, Yonsei University, 134 Shinchon-dong,
Seodaemun-ku, Seoul 120-749, Korea
*
Author for correspondence (e-mail:
Leej{at}yonsei.ac.kr
)
Accepted 29 May 2001
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SUMMARY |
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Key words: C. elegans, Transcriptional mediator, med-6, RNAi, rde-1, Ras, Wnt
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INTRODUCTION |
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To date, many mediator complexes have been identified in various
multicellular species. These complexes include the human Srb/Med-containing
co-factor complex (SMCC) (Gu et al.,
1999), the negative regulator
of activated transcription (NAT) complex (Sun et al.,
1998
), mouse and human
Mediator complexes (Boyer et al.,
1999
; Jiang et al.,
1998
), and the Mediator
complex in the nematode C. elegans (Kwon et al.,
1999
).
Although the biological significance of the mediators at the organismic
level is largely unknown in higher eukaryotes, some experimental evidence of a
physiological role for these Mediator complexes in development has come from
studies in the nematode Caenorhabditis elegans. The med-6,
med-7 and med-10 mediator genes were initially identified from a
genome search for homologs of the yeast mediators (Lee et al.,
1997), and we have previously
reported that these mediators are required for regulated transcription of
tissue- and stage-specific developmental genes, and that loss of function in
any of these genes causes embryonic lethality, confirming their essential
roles in development (Kwon et al.,
1999
). Another mediator gene
characterized in the nematode was sur-2. sur-2 was originally
isolated as a suppressor of a let-60 Ras gain-of-function mutation
(Singh and Han, 1995
).
Loss-of-function mutations in sur-2 alone resulted in pleiotropic,
incompletely penetrant phenotypes, including a vulvaless phenotype in
hermaphrodites, defects in development of the male tail, gonadal abnormalities
and larval lethality. When sur-2 was cloned, it was found to have no
homology to any other gene, but later biochemical studies on human mediator
complexes revealed that human SUR-2 was a component of the mediator complex.
It was also shown that the adenovirus E1A protein binds to SUR-2, activating
transcription in vitro (Boyer et al.,
1999
). A third C.
elegans gene, sop-1, which encodes a transcriptional
mediator-related protein, was isolated as a suppressor of a pal-1
mutation. pal-1 is a gene that encodes the ortholog of the
transcription factor caudal (Waring and Kenyon,
1991
), and its gene activity
is required for both early and late embryogenesis. During late embryogenesis,
pal-1 is required for specifying the fate of V6 cells. Expression of
pal-1 in V6 neuroblasts activates expression of mab-5, a
gene encoding a homeobox protein, which in turn activates expression of
egl-5 and lin-32, thus defining male ray-specific properties
(Costa et al., 1988
; Ferreira
et al., 1999
; Hunter et al.,
1999
; Wrischnik and Kenyon,
1997
). One of the
pal-1 mutations, pal-1 (e2091), is a tissue-and
stage-specific mutation in that it does not perturb any early embryonic
development but causes defects in the fate specification of V6 cells.
Mutations in the sop-1 gene were isolated in a screen for suppressors
of pal-1(e2091), and the sop-1 gene turned out to encode a
homolog of TRAP230, which is a component of the human mediator-related protein
complex (Zhang and Emmons,
2000
).
One common feature of the two mediator-related proteins, SUR-2 and SOP-1, found in the nematode, is that these proteins do not have homologs in yeast, but are conserved in the various metazoan species studied so far, including humans, indicating that these mediator-related proteins may relay signals from metazoan-specific transcriptional regulators. As MED-6, MED-7, MED-10 and SRB-7 are conserved not only in the metazoa but also in yeast, it is conceivable that these universally conserved mediators may be the point of convergence at which diverse transcriptional signaling mediated by metazoan-specific transcription factors and mediator-related proteins converge, at least in metazoa. Based on these inferences, we became interested in resolving the issue of the relationship between the metazoan-specific mediator-related proteins and the conserved mediator components. We pursued this by studying the roles of a universally conserved mediator med-6, using Caenorhabditis elegans.
In this study, we have isolated and characterized the genetic mutation in the med-6 gene, and have examined the in vivo functions of this mediator gene in the development of C. elegans by establishing biological assay systems involving the Ras and Wnt pathway, in which metazoan-specific mediator-related proteins are known to be involved. Based on our observation that the med-6 mutant animals had a vulval defect, which is due to hypo-induction of the vulval precursor cells (VPCs), we hypothesized that med-6 is involved in transcriptional regulation of genes involved in the Ras pathway. Accordingly we examined the effect of med-6 RNAi on the lin-3, let-23, and let-60 genes, which act in the Ras pathway. We also examined the effect of med-6 RNAi on male ray development, in which pathway sop-1, a mediator-related gene, is involved. Another interesting finding was that RNAi of med-6 in wild-type background caused more severe phenotypic changes than the putative null mutation of med-6. We examined the possibility that this phenotypic discrepancy was due to maternal rescue by employing a genetic experiment involving the rde-1 mutation.
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MATERIALS AND METHODS |
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Identification of let-425 as the med-6 gene
The physical location of med-6 was determined to be in the YAC
Y57E12 (Y.-J. Kim, personal communication), on chromosome V. However, no
positive cosmids were detected by Southern hybridization using med-6 cDNA as a
probe. We reasoned that this was because the med-6 gene resided in a
cosmid gap that is located between the cosmid K11C4, encoding unc-68,
and VC5, encoding odr-2. As the region was saturated with lethal
mutations, and sDf20 and sDf30 delete both unc-68
and odr-2 while nDf32 does not, we searched for lethal
mutations mapped between the break points of the deficiencies. According to
the genetic map available (http://elegans.swmed.edu), let-404, let-438,
let-468, let-442, let-339, let-343, let-425, let-346 and let-332
reside in this region. We examined the phenotypes of animals containing each
mutation by DIC microscopy, and decided that let-425 and
let-332 could be candidates for the med-6 mutation. A
let-425 mutation had been isolated as a lethal mutation with a
sterile phenotype, and a let-332 mutation isolated with embryonic
lethality (Johnsen and Baillie,
1991). We amplified the
genomic region containing the entire coding sequence of the med-6
gene from either heterozygotes of let-332 or animals homozygous for
let-425. The primers used for amplifying the genomic region from
single larvae were CM6F (5'-GGACTAGTATGGGACCTCCAGCAGCTGCAC-3') and
CM6R (5'-TAATTTAATGTTCATTTTTAGTG-3'). The amplified fragments were
cloned into pGEM-T easy vector (Promega). We determined the sequences from six
independent colonies with T7 sequenase 2.0 DNA sequencing kits (Amersham). To
confirm the identity of the mutation, we directly determined the sequence of
the PCR-amplified fragments from eight let-425 homozygous animals,
eight heterozygotes and eight N2 animals. In an effort to rescue the mutation
of let-425 with the wild-type med-6 gene, we amplified a
full-length med-6 gene containing the upstream 2 kb region and the
entire 3'UTR as well as the coding region. The PCR primers used for this
purpose were CM6PF (5'-CGTCGACCACATCCTTCGCCGGAAGC-3') and CM6PR
(5'-CTCTAGACTCATAAACCACAAAGAGGAGC-3'). We microinjected the linear
PCR product into wild-type animals with genomic DNA digested with
EcoRI, which cuts the coding region of med-6. Heterozygous
males of the genotype dpy-18/eT1; unc-46 let-425/eT1 were mated with
transgenic animals containing the med-6 transgene, and F1
hermaphrodites that segregated Dpy animals in the next generation were
individually selected. Among the siblings of the Dpy animals, rolling UNC-46
animals, whose genotype should be unc-46 let-425; [Ex pRF4 + med-6 +
fragmented genomic DNA] were examined for their phenotypes. These UNC-46
animals were normal in vulval development and were able to lay eggs, a
phenomenon never seen in the let-425 homozygous animals. This result
indicates that the linear PCR fragment containing the med-6 gene
complemented the let-425 mutation.
Characterization of let-425 homozygous animals
The let-425 mutation is maintained in the heterozygote form in the
genotype dpy-18(e364)/eT1 III; unc-46(e177) let-425(s385)/eT1 V. In
order to measure the lethality and sterility of the let-425
homozygotes, we removed the eT1 balancer by mating dpy-18 homozygote
hermaphrodites with N2 males to produce males of the genotype dpy-18/
+, and the dpy-18/+ heterozygous males were mated with the
let-425 heterozygotes. In the next generation, Dpy virgins of the
genotype dpy-18(e364)/dpy-18(e364) III; unc-46(e177) let-425(s385)/+
V were selected, and the phenotypes of their progeny were examined. In
order to determine whether the existing allele of let-425 is null, we
mated males of the genotype dpy-18(e364)/eT1 III; unc-46(e177)
let-425(s385)/eT1 V with hermaphrodites of the genotype
dpy-18(e364)/eT1 III; unc-46(e177) sDf20/eT1 V. The number of non-Dpy
male progeny and that of Dpy Unc male and hermaphrodite progeny were compared,
and the phenotypes of the hemizygotes were examined. The expected ratio of the
non-Dpy males to Dpy Unc animals (hermaphrodites and males) is 4:2, if there
had been no embryonic lethality (Rosenbluth and Baillie,
1981). The observation was
that the ratio of the non-Dpy males to Dpy Unc animals was close to 2.45:1
(n=913). Among the hemizygous hermaphrodites containing a single copy of the
let-425 mutation, 78% arrested at the L4 stage and the remaining 22%
reach adulthood. This number is comparable with that of the homozygous
let-425 mutants. These results indicate that the let-425
mutation is a severe reduction-of-function mutation, if not a complete
null.
RNAi and microscopy
The med-6 dsRNA used in this study was identical to the dsRNA used
in the previous study (Kwon et al.,
1999). The size of the RNA
used in RNAi was about 800 nucleotides, and contained the full
med-6-coding region. The concentration of the med-6 RNA used
in the experiments was 100 µg/ml. In order to observe the effect of
med-6 RNAi on vulval development in various backgrounds, vulval
invagination at the L4 stage of F1 progeny laid at 6-24 hours after
microinjection was observed. We confirmed the effectiveness of the RNAi by
observing that the embryos laid after that time were 100% embryonic lethal.
For the RNAi experiments in the rde-1 background, med-6
dsRNA was injected into rde-1 mutant animals, and individual animals
were mated with three N2 males. After each 12 hour period, we moved the P0
individuals to new plates and provided new males. Individual F1
progeny were transferred to new plates and were examined for their phenotypes
after four days of culture. The phenotype `small brood size' was used to
describe F1 animals with fewer than 20 F2 progeny. Thus,
animals classified as normal had fewer progeny than wild-type animals in many
cases. Embryonic lethality was calculated by subtracting the number of viable
animals from the total number of eggs. In the rde-1 background
experiments, we attempted to see if microinjection of dsRNA into the gonad,
body cavity or intestine gave different ranges of phenotypes, but we did not
see any correlation between the location of injection and severity of the
resulting phenotypes. To characterize the role of med-6 in ray
development, we likewise microinjected med-6 dsRNA into animals of
various mutant backgrounds. When using the rde-1 mutant background,
we mated the injected animals with N2 males and examined the phenotypes of the
male progeny. When using pal-1(e2091) III; him-5(e1490) V and
pal-1(e2091) III; him-5(e1490) V; sop-1(bx92) X backgrounds, we
examined the males from the mothers directly. To examine the change in
pal-1 transcription by med-6 RNAi, we microinjected
med-6 dsRNA into the pal-1::GFP integration line, and
observed F1 embryos inside the PO animals. A Zeiss Axioplan2
microscope was used for Nomarski images, and a Zeiss AxioCam digital camera
and MC200 camera (Carl Zeiss) were used for taking photographs.
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RESULTS |
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Phenotypes associated with a putative null mutation of let-425
We first determined whether the let-425 mutation was null. As
hemizygous animals containing a single copy of let-425 mutation
displayed comparable phenotypes with those of the let-425 homozygotes
(see Materials and Methods), the existing let-425 mutation is a
severe reduction-of-function mutation. The phenotypes associated with the
let-425 mutation were then examined in order to define the biological
significance of the med-6 gene in development. We examined the
let-425 homozygous animals laid by heterozygous mothers because
homozygous animals are completely infertile. Two phenotypes were apparent in
the let-425 homozygous progeny; late larval arrest and adult
sterility, but they exhibited no embryonic lethality. In order to check if
there was any embryonic lethality associated with the mutation, we examined
the progeny from heterozygous mothers with the genotype dpy-18/dpy-18;
unc-46 let-425/+. Among the 2145 progeny laid from the heterozygous
mothers, we found that 25.7% were let-425 homozygous animals. This
percentage is comparable with the expected number of homozygous
let-425 progeny if there had not been any embryonic lethality
associated with the let-425 mutation. In another experiment, we let
the animals of the genotype dpy-18/dpy-18; unc-46 let-425/+ lay eggs
and then transferred all the progeny individually to single plates and allowed
them to grow. Among the 194 progeny examined, 25.8% were Dpy Unc larvae or
sterile adults, while 47.9% were heterozygotes and 26.3% were dpy-18
homozygotes, confirming that there was no embryonic lethality and that the
let-425 mutation caused 100% larval arrest or adult sterility.
Next, we examined the phenotypes of the larvae and adults of the
let-425 homozygous animals in more detail. Among the 132 animals
observed under Nomarski optics, 72% were arrested in the L4 larval stage and
28% were adults with 100% sterility. The sterile adults were defective both in
oogenesis and spermatogenesis: when the male progeny from RNAi-affected
him-5 hermaphrodites were mated with unc-101 hermaphrodites,
they could not produce any cross progeny (n=20). Additionally, in a
reciprocal mating, in which RNA-affected hermaphrodites were mated with N2
males, no progeny were produced (data not shown). Because male tail defects
and vulval defects, which may interfere with mating ability by themselves, are
not 100% penetrant (see the following sections), enough numbers of matings
should have resulted in production of cross progeny if there was no defect in
spermatogenesis or oogenesis, respectively. Interestingly, the sterile adult
animals showed an additional phenotype of protruding vulvae
(Fig. 2F). Accordingly, we
examined the let-425 homozygous animals at the L3 molt stage, which
is when the vulval precursor cells usually divide to form the vulval tissue
(Sternberg and Horvitz, 1986;
Sternberg and Horvitz, 1989
),
in order to explain the cellular defects associated with this phenotype. Out
of 15 L3 molt animals, eight had less than wild-type vulval induction, in
which we commonly observed induction of P6.p but not P5.p and P7.p (for
example, see Fig. 2B).
RNAi-affected animals displayed an identical hypo-induction phenotype
(Fig. 2C). It is known that
hypo-induced VPCs later form protruding vulval tissue, and in our study, we
found that the animals confirmed to have hypo-induced VPCs later either
arrested as L4 larvae or grew to adulthood with protruding vulvae (data not
shown).
MED-6 is involved in regulating genes involved in a Ras pathway
A possible explanation for the hypo-induction of VPCs in the
let-425 homozygous animals is that the med-6 mutation causes
transcriptional downregulation of genes involved in vulval induction. To test
our hypothesis, we examined the effect of reduction of med-6 gene
function on lin-3, let-23 and let-60, which encode an
epidermal growth factor (EGF), the EGF receptor and Ras, respectively (see
Table 1; Fig. 3; Aroian et al.,
1990; Han and Sternberg,
1990
; Hill and Sternberg,
1992
). Either gain-of-function
or reduction-of-function alleles of these genes were used. Gain-of-function
mutations in any one of these genes result in a multivulva phenotype in which
more than three VPCs are induced to make additional vulval tissue in addition
to a functional vulva. On the other hand, reduction-of-function mutations in
these genes cause hypo-induction of VPCs. We reasoned that reduction in
med-6 activity would result in reduction of the transcription levels
of these genes, which would suppress the multivulva phenotype of the
gain-of-function mutations and enhance the hypo-induction phenotype of
reduction-of-function mutations. The alleles of lin-3 that we used
were syIs1, an allele generated by integration of multicopies of the
wild-type lin-3 gene, and lin-3 (e1417), a
reduction-of-function mutation. Reduction of med-6 activity by RNAi
did not suppress the multivulva phenotype resulting from overexpression of
lin-3 driven by multicopies of this gene
(Fig. 3C,D). On the contrary,
the hypo-induction phenotype of lin-3 (e1417) was significantly
enhanced (Fig. 3A,B). Furthermore, gain-of-function mutations in let-23 or let-60
were partially suppressed to the wild-type form by reduction of med-6
activity (Fig. 3E-H). From
these results, we concluded that reduction of med-6 activity could
cause the genes involved in the Ras signaling pathway to be transcribed at
lower levels.
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MED-6 is involved in regulating genes involved in ray
development
A recent study has shown that a tissue-specific mediator is involved in
regulating transcription of gene(s) involved in ray development (Zhang and
Emmons, 2000). Transcription
of pal-1, a gene encoding a transcription factor required for proper
ray development, is regulated by two separate circuits, one of which is
normally silent, owing to sop-1 activity, which represses the
transcription of pal-1 from the 5' upstream regulatory element.
This transcriptional regulation is mediated by the Wnt pathway. We wished to
examine whether med-6 was also involved in the regulation of genes
involved in ray development. We first examined the male tails of a few
let-425 homozygous animals and found that the male structures were
defective (data not shown). We then used RNAi to examine the effect of
med-6 on male ray development. Because we reasoned that genes
required for proper ray development should be functional at the zygotic level,
we examined the male tail phenotypes associated with males produced by the
mating of N2 males with rde-1 homozygous hermaphrodites that had been
microinjected with med-6 dsRNA (see Materials and Methods; for
rde-1 experiments, see the next Result sections). We found that 17
out of 45 males observed under Nomarski optics displayed abnormal ray defects
(Fig. 4B). A typical phenotype
was that the V6-derived rays, 2-6, were partially or completely missing. We
observed essentially identical phenotypes when we examined the tail phenotypes
of the males produced from N2 hermaphrodites that had first been mated with N2
males, and then subjected to med-6 RNAi (data not shown). It is known
that the development of the V6-derived ray structure is dependent on the
expression of the caudal homolog pal-1 in the V6 progeny. Because we
did not see significant defects in ray number 1, nor in 7-9 in our
experimental conditions, it is probable that med-6 may be involved in
ray development at the level of pal-1 transcription, although we
cannot rule out the possibility that genes downstream of pal-1 were
affected. We next examined the ray phenotypes of animals of the genotype
sop-1; pal-1 in order to determine whether med-6 is involved
in the transcriptional regulation mediated by the Wnt pathway in ray
development. In wild-type animals, pal-1 is transcriptionally
activated through an intronic regulatory element and the transcriptional
activation by the Wnt pathway is repressed by the activity of sop-1.
As in the pal-1; sop-1 mutants pal-1 is transcriptionally
activated by the Wnt pathway (Zhang and Emmons,
2000
), it is possible to
examine if med-6 is involved in the Wnt-mediated transcription of
pal-1 by using these mutants. If med-6 is involved in the
transcriptional activation of pal-1 both by the original intronic
regulatory element and by the Wnt pathway, then med-6 RNAi should
reduce the development of the V6-derived rays in the double mutant animals. We
observed ray defects in the pal-1; sop-1 double mutant animals that
were almost identical to those caused by med-6 RNAi in the wild-type
background (Fig. 4C-F),
indicating that Wnt signal-mediated transcription may also require MED-6 as
its transcriptional mediator.
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Examination of maternal and zygotic functions of med-6 using
an rde-1 mutation
Our previous RNAi result showed that RNAi using a high concentration of
med-6 double-stranded RNA in the wild-type background caused almost
100% embryonic lethality and that at a lower concentration of RNA, the animals
showed both embryonic lethality and adult sterility (Kwon et al.,
1999). When we examined the
phenotype of the let-425 mutation, we found no evidence of embryonic
lethality (see the previous Results section). One possible explanation is that
maternal med-6 activity required for embryogenesis may still be
provided by the heterozygous mother. We examined this possibility by using the
rde-1 mutation (Tabara et al.,
1999
). To test our hypothesis,
we designed a genetic experiment in which we could selectively reduce the
zygotic med-6 gene function. Fig.
5 shows the mating schemes that we employed. As rde-1
homozygous animals are resistant to RNAi both maternally and zygotically, and
the rde-1 phenotype is fully recessive (Tabara et al.,
1999
), we performed RNAi of
med-6 at a high concentration in the rde-1 homozygous
animals and mated them with N2 males. The progeny from this mating should have
a zygotic RNAi effect, but not a maternal RNAi effect, because rde-1
is now in the heterozygote form. This mating allowed for selective reduction
of the zygotic med-6 gene function
(Table 2). Unlike wild-type
animals, rde-1 mutant animals were indeed 100% resistant to RNAi of
med-6: we did not see any effect of med-6 RNAi in the
rde-1 mutant animals. However, after mating the RNA-injected
rde-1 hermaphrodites with N2 males, the progeny showed various
degrees of severity in their phenotypes. About 10% of the eggs counted
(n=1693) were arrested at the embryonic stage, 6% grew up to
adulthood and became sterile, and 28% of the eggs grew to adulthood with no
obvious phenotype. About 7% of the adults had limited brood sizes. In
addition, males produced in this experiment displayed ray defects and
hermaphrodites showed defective vulval induction (data not shown). In control
experiments, wild-type animals injected with the dsRNA, whether mated or not,
produced nonviable embryos. In summary, we observed phenotypes in
rde-1 mutant RNAi that were comparable with, although less severe
than, let-425 homozygous mutant phenotypes. From these data, we
propose that the maternal med-6 is required for early embryogenesis,
and that the zygotic function of med-6 is mostly required for
postembryonic development such as vulval development and ray development.
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DISCUSSION |
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Role of transcription mediators in development
Mediators were shown to be required for regulated gene transcription in
yeast and in the nematode (Kwon et al.,
1999; Lee and Kim,
1998
). In yeast,
med-6 mediates transcription of about 10% of the whole genome
(Holstege et al., 1998
). It is
therefore conceivable that med-6 also is involved in the regulated
transcription of many genes in the nematode C. elegans. In order to
establish biological assay systems to study the role of transcription
mediators in development, we examined genes acting in two signaling pathways
of the nematode: the Ras and the Wnt pathways. The Ras signaling pathway is
one major signaling pathway in hermaphrodite vulval development, and the Wnt
pathway is involved in ray development. Because vulval and ray development
should occur at the appropriate time in development at the correct place, the
genes involved in those pathways should be regulated temporally and spatially.
Therefore, it is conceivable that mediators may be involved in regulated
transcription of the genes in these pathways. In this report, we showed that
nematode med-6 is involved in the regulation of genes associated with
the Ras pathway and the Wnt pathway.
We first examined vulval development because we observed abnormal vulval
morphogenesis in the let-425 homozygous mutant animals. From the
animals produced at early time points after injection of med-6 dsRNA,
we were able to show that med-6 is indeed involved in the Ras
pathway: reduction of med-6 activity lowered the gene activity of
both the gain-of-function mutant genes and a reduction-of-function mutant
gene. The fact that embryos laid at later time points after RNAi were arrested
at the embryonic stage indicates that the animals produced earlier might have
had residual med-6 activity, sufficient to enable the animals to
survive up to adulthood. We were unable to obtain direct evidence showing that
the transcription levels of the genes were reduced. However, from the
phenotypes of the RNAi-affected animals, we could see the effects caused by
the reduction of med-6 activity. It was not surprising that reduction
of med-6 activity did not suppress the multivulva phenotype caused by
multicopies of lin-3. We interpret this as follows: the level of
lin-3 activity is known to be crucial in determining how many VPCs
are induced (Katz et al.,
1995), and it is probable that
reduction of med-6 activity by RNAi was not effective enough to
reduce the transcription level of lin-3 to a point where fewer VPCs
were induced. However, it is obvious that lin-3 is directly or
indirectly activated by med-6, because the reduction-of-function
mutation of lin-3 was enhanced by the med-6 RNAi. We do not
know at this point whether transcription of all three of the genes we
examined, lin-3, let-23, and let-60, is directly regulated
by med-6 activity. We cannot rule out the possibility that other
genes downstream of these are affected by the med-6 RNAi, resulting
in the effects we saw in the experiments. It would be necessary to establish
an in vitro transcription assay system to test this directly.
It was recently reported that components of the nucleosome remodeling and
histone deacetylase (NURD) complex in C. elegans antagonize
Ras-induced vulval development by deacetylating specific target genes to
repress vulval development (Solari and Ahringer,
2000). Now that it is evident
that both the mediators and the chromatin remodeling complex are involved in
the strict temporal and spatial regulation of sets of genes for proper
development, the C. elegans vulval development system can serve as a
model in which one can study the biological significance of the mediators and
chromatin remodeling complexes.
We examined ray development because it was reported that another mediator
protein was involved in repression of the Wnt pathway in ray precursor cells
(Zhang and Emmons, 2000), and
we subsequently found that med-6 was also involved in regulation of
the gene(s) involved in ray development. Reduction of med-6 activity
caused V6 progeny to undergo abnormal development, in that ray structures
produced by these cells were missing or reduced in number. We also
demonstrated that med-6 is required for proper regulation of the
genes in the Wnt signaling pathway by examining the RNAi effect in the
pal-1; sop-1 mutant animals. The data presented in this report
suggest that med-6 is involved in the development of V6-derived ray
structures by regulation of pal-1 transcription. Consistent with
this, we found that med-6 RNAi lowered pal-1 transcription
during embryogenesis (data not shown). However, it is formally possible that
med-6 is involved in regulated transcription of genes downstream of
pal-1, such as mab-5.
The fact that SUR-2 and SOP-1 proteins do not have homologs in yeast, but are conserved only in metazoa, while MED-6 is conserved from yeast to humans, raises the possibility that MED-6 is a more general mediator of transcriptional regulatory signaling. Consistent with this, SUR-2 has been shown to have roles in the Ras pathway, but not in ray development, while SOP-1 has been shown to be involved in the Wnt pathway, but not in the Ras pathway. On the contrary, med-6 has been shown to be involved both in the Ras pathway and in the Wnt pathway. Our model for the action of the mediator-related proteins in the nematode is shown in Fig. 6. We propose that the metazoan-specific mediator-related proteins interact with metazoan-specific transcription regulators to generate signals that converge at a more conserved mediator complex containing MED-6, which in turn transmits the signal(s) to the basal transcriptional machinery, leading to developmental specific gene expression.
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Using the rde-1 mutation background as a tool to dissect
maternal and zygotic gene function
The finding that the let-425 mutant phenotypes were less severe
than the effects of RNAi prompted us to examine the differences between the
maternal and zygotic gene functions of med-6 in development. If
mutations are introduced in essential genes that are required both maternally
and zygotically, the phenotype of homozygous mutant animals from heterozygous
mothers would be masked because of maternal rescue. We were able to show by
using the rde-1 mutant background that one reason why the phenotypes
of the let-425 mutation are less severe than the effects of RNAi is
due to the maternal contribution of wild-type med-6 gene activity. As
RNAi in the wild-type background does not distinguish between the reduction in
maternal or zygotic functions of genes that are expressed both maternally and
zygotically, we employed the rde-1 mutant background. By using the
rde-1 mutant background, we were able to examine the phenotypes
caused by the reduction in zygotic gene expression only, leaving the
maternally expressed genes intact. From the data we presented above, we could
infer that the maternal contribution of med-6 is required for normal
embryogenesis and that its zygotic gene activity is mostly required for normal
larval development and fertility, as well as normal ray development.
Additionally, we were able to confirm the limitations of RNAi, one of the main ones being that the effect of RNAi is not as severe as the null mutation of the gene, particularly for genes acting late in development. The rde-1 RNAi experiment we described above is an example of this. Sterility and larval arrest are late phenotypes compared with embryonic lethality, and quite a few progeny from the RNAi-affected rde-1 animals were able to escape the RNAi effect, while the effect of the let-425 genetic mutation was persistent through adulthood.
In summary, we have shown in this report that MED-6 is encoded by let-425, that the maternal and zygotic let-425 have distinct roles in development. Maternal let-425 has been shown to be required for early embryogenesis and zygotic let-425 has been shown to be required for late development, including hermaphrodite vulval development and male ray development. As for the action mechanism of the mediators, we suggested that MED-6, a universally conserved transcription mediator, is required for actions of metazoan-specific mediator-related proteins, such as SOP-1, for appropriate transcriptional regulation of development-specific genes. The next step will be to examine whether and how med-6 is involved in regulated transcription of other genes in development. One way to pursue this issue on a large scale would be to perform a microarray experiment, as has been done in the yeast system.
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ACKNOWLEDGMENTS |
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