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First published online 14 June 2006
doi: 10.1242/dev.02444
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Howard Hughes Medical Institute, Department of Biology, Room 68-425, MIT, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Author for correspondence (e-mail:
horvitz{at}mit.edu)
Accepted 15 May 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: ISWI, NURF, Rb, Ras, C. elegans
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). These
two mechanisms for chromatin remodeling have been characterized extensively in
vitro (Roth et al., 2001;
Smith and Peterson, 2005), but
efforts to understand how each functions in the development of metazoa have
just begun.
Studies of vulval development in the nematode Caenorhabditis
elegans could help establish the roles of chromatin remodeling during
development. The vulva of the C. elegans hermaphrodite is formed by
the 22 descendants of three ectodermal blast cells (P5.p, P6.p and P7.p)
located along the ventral surface of the animal
(Sulston and Horvitz, 1977).
P(5-7).p, three of a set of six equipotent cells P(3-8).p called the vulval
equivalence group, are induced to generate vulval cells by an epidermal growth
factor (EGF)-like signal from the gonad
(Sulston and White, 1980;
Hill and Sternberg, 1992). The
inductive signal is received and transduced by a conserved receptor tyrosine
kinase (RTK)/Ras pathway (Kornfeld,
1997). Unlike the other cells of the vulval equivalence group
(P3.p, P4.p and P8.p), which divide once and fuse with the nearby hypodermal
syncytium (hyp7), P(5-7).p divide three times to generate the cells that form
the adult vulva (Sulston and Horvitz,
1977). Mutations that reduce or eliminate the function of the
let-23 RTK/let-60 Ras pathway can result in a vulvaless
(Vul) animal in which no cells of the vulval equivalence group express vulval
fates; by contrast, mutations that increase the function of this pathway can
cause the ectopic adoption of vulval cell fates by P3.p, P4.p and P8.p, and
result in a multivulva (Muv) animal (Beitel
et al., 1990; Han et al.,
1990; Katz et al.,
1996).
Loss-of-function mutations in the synthetic multivulva (synMuv) genes also
can cause a Muv phenotype (Horvitz and
Sulston, 1980; Ferguson and
Horvitz, 1989). These genes have been grouped into three classes:
A, B and C (Ferguson and Horvitz,
1989; Ceol and Horvitz,
2004). Loss-of-function mutations within any one class do not
cause a Muv phenotype, whereas mutations in any two genes within two different
classes cause a Muv phenotype (Ferguson
and Horvitz, 1989; Ceol and
Horvitz, 2004). The class A synMuv genes encode novel, nuclear
components (Clark et al.,
1994; Huang et al.,
1994; Davison et al.,
2005). Many class B synMuv genes encode homologs of
transcriptional repressors and factors that remodel chromatin, including
LIN-35 Rb (Lu and Horvitz,
1998), the EFL-1/DPL-1 E2F heterodimeric transcription factor
(Ceol and Horvitz, 2001), the
HDA-1 HDAC1, LET-418 Mi2, LIN-53 RbAp48 NuRD complex
(Lu and Horvitz, 1998;
Tong et al., 1998;
Xue et al., 1998;
von Zelewsky et al., 2000;
Unhavaithaya et al., 2002) and
HPL-2 Heterochromatin Protein 1 (HP1)
(Couteau et al., 2002). The
Drosophila melanogaster homologs of some class B synMuv proteins form
a complex, identified by two different groups and called Myb-MuvB or dREAM,
that is likely to repress the transcription of genes through chromatin
remodeling (Korenjak et al.,
2004; Lewis et al.,
2004). Class C synMuv genes encode homologs of a putative
Tip60/NuA4 histone acetyltransferase complex
(Ceol and Horvitz, 2004).
Because of these homologies, the synMuv genes, which negatively regulate the
vulval cell fate, probably act by repressing the transcription of genes that
promote the expression of vulval cell fates.
In this study, we describe the identification of a C. elegans
ortholog of Drosophila ISWI, called isw-1, as a suppressor
of the synMuv phenotype. ISWI is an ATP-dependent chromatin-remodeling enzyme
identified by homology to S. cerevisiae Snf2/Swi2
(Elfring et al., 1994). We
show that ISW-1 probably acts as a component of a Nucleosome Remodeling Factor
(NURF)-like complex with the Drosophila NURF301 ortholog NURF-1 to
antagonize the synMuv genes. Our observations reveal the antagonistic
functions of a NURF-like chromatin remodeling complex and complexes similar to
Myb-MuvB/dREAM, NuRD and TIP60/NuA4 in the determination of multiple cell
fates, including the antagonistic regulation of at least one putative target
of synMuv transcriptional repression.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). N2 (Bristol)
was the wild-type strain. The following mutations and integrants were
used:
LGI: dpy-5(e61), lin-35(n745), lin-53(n833, n3368)
(n3368, this study), ccIs4251
(Hsieh et al., 1999).
LGII: unc-4(e120), nurf-1(n4293, n4295) (this study),
dpl-1(n3316) (Ceol and Horvitz,
2001), lin-8(n111, n2731)
(Thomas et al., 2003),
lin-56(n2728) (Thomas et al.,
2003), lin-38(n751), trr-1(n3712)
(Ceol and Horvitz, 2004),
lin-31(n301), let-23(sa62) (Katz
et al., 1996), rrf-3(pk1426)
(Simmer et al., 2002).
LGIII: dpy-17(e164), isw-1(n3294, n3297, n4066) (this study),
lin-9(n112, n942) (Beitel et al.,
2000), lin-36(n766)
(Thomas and Horvitz, 1999),
lin-37(n758, n2234) (Ferguson and
Horvitz, 1989; Thomas et al.,
2003), lin-52(n3718)
(Thomas et al., 2003),
lin-13(n387) (Melendez and
Greenwald, 2000), hpl-2(tm1489), flt-1(tm235).
LGIV: unc-30(e191), pyp-1(n4599) (this study),
let-60(n1046) (Beitel et al.,
1990; Han et al.,
1990), lin-1(n304, e1275).
LGV: unc-46(e177), let-418(n3536, n3719), mep-1(q660), lin-54(n3423),
hda-1(e1795) (Dufourcq et al.,
2002), tam-1(cc567)
(Hsieh et al., 1999),
lin-45(ku112) (Sundaram and Han,
1995), him-5(e1490).
LGX: lin-15A(n767, n433), lin-15B(n744), lin-15AB(n765, e1763), lin-2(n768).
The following reciprocal translocations containing GFP-expressing
transgenes integrated at or near the translocation breakpoints were used:
hT2 [qIs48] LGI; LGIII and nT1 [qIs51] LGIV; LGV. mIn1
[mIs14 dpy-10(e128)] is a balancer chromosome that expresses GFP. The
following mutations were provided by C. Ceol, F. Stegmeier and M. Harrison:
let-418(n3536, n3719), lin-54(n3423), mep-1(n3703). flt-1(tm235) and
hpl-2(tm1489) were provided by S. Mitani. Those mutant alleles for
which no citation is given are described by Riddle
(Riddle, 1997).
RNAi analyses
RNAi by injection was performed as described by Ceol and Horvitz
(Ceol and Horvitz, 2004),
except single-stranded RNA molecules were annealed by incubation at 85°C
for 15 minutes, then at 37°C for 30 minutes and slowly cooled to room
temperature for 1 hour. RNAi of F37A4.6, the gene predicted to be
within an intron of isw-1, did not suppress the synMuv phenotype of
lin-53(n833); lin-15A(n767) mutants (data not shown). The following
constructs were used to make dsRNA: for isw-1, yk593a10 and yk617c10;
for nurf-1, yk273g2, yk1151c6, pEA30 (a cDNA encoding the 3'
end of nurf-1b, c, d and e) and pEA147 (a RT-PCR product
corresponding to only nurf-1a); for pyp-1, yk169c6; for
rba-1, yk117c9; for H20J04.2, yk230c2 and yk323f2; for
T26A5.8, yk471d3; and forY53F4B.3, yk393b2 and yk1412b12.
Yuji Kohara kindly provided all yk clones.
Determination of gene structures
For isw-1, the sequences of two independent cDNAs, yk593a10 and
yk617c10, were determined. 5' rapid amplification of cDNA ends (5'
RACE, Invitrogen) was used to determine the 5' end of isw-1,
and an SL1 splice-leader sequence was detected. For nurf-1, the
sequences of 15 independent cDNAs were determined: yk62e9 (pEA30), yk98g1,
yk172b9, yk374b9, yk381c2, yk480b9, yk565d9, yk752a4, yk765d8, yk879b11,
yk1030g7, yk1151c6, yk1288b1, yk1456g11, yk1691d5. Gene-specific primers were
used to amplify reverse-transcribed products by PCR to confirm several
nurf-1 transcripts, and a Stratagene C. elegans cDNA library
also was used for PCR analyses of nurf-1 gene structures. The
5' ends of nurf-1b and nurf-1c were identified by SL1
RT-PCR, and the 5' ends of nurf-1a, d and e are from
GeneFinder (Liang et al.,
2001) predictions.
Isolation of deletion alleles
Genomic DNA pools from EMS-mutagenized animals were screened for deletions
using PCR as described by Ceol and Horvitz
(Ceol and Horvitz, 2001).
Deletion mutant animals were isolated from frozen stocks and backcrossed to
the wild type at least twice. isw-1(n4066) removes nucleotides 20629
to 21932 of cosmid F37A4. nurf-1(n4293) removes 3058 to 3782, and
nurf-1(n4295) removes 18656 to 19733 of cosmid F26H11.
pyp-1(n4599) removes nucleotides 26777 to 28936 of cosmid C47E12.
lin-53(n3368) removes nucleotides 38104 to 38857 of cosmid K07A1.
Scoring of vulval cell fates
For trr-1(n3712) and trr-1(n3712); lin-15B(n744), vulval
induction was scored during the L4 larval stage using Nomarski optics. If more
than three out of the six Pn.p cells were induced, the animals were counted as
Muv.
Antibody staining
We cloned a full-length isw-1 cDNA into a vector containing the
coding sequence for the maltose-binding protein (MBP). Antisera recognizing
ISW-1 were generated by injecting MBP:ISW-1 into two rabbits (Covance).
Anti-ISW-1 antibodies were affinity purified using GST (glutathione
Stransferase)-tagged ISW-1 as described by Koelle and Horvitz
(Koelle and Horvitz, 1996).
Embryos, larvae and adults were fixed as described by Finney and Ruvkun
(Finney and Ruvkun, 1990).
Affinity-purified antibodies were used at a 1:10 dilution for whole-mount
staining and at 1:1000 for western blots. Horseradish peroxidase-conjugated
secondary antibodies (Jackson ImmunoResearch) were used at 1:3000 for western
blots, and Alexfluor 488 (Invitrogen) was used at a 1:200 dilution for
detection by whole-mount immunocytochemistry.
Determination of mutant sequences
We used PCR-amplified regions of genomic DNA to determine mutant sequences.
For both isw-1 alleles, all exons and splice junction sequences were
determined. All mutations were confirmed using independently derived PCR
products. Sequences were determined using an ABI Prism 3100 Genetic
Analyzer.
Germline transformation experiments
Germline transformation experiments were performed as described by Mello et
al. (Mello et al., 1991). For
rescue of the lin-53(n833); isw-1(n3294); lin-15A(n767) synMuv
suppression phenotype, we injected cosmid C28G2 (30 ng/µl). 100 µg/µl
of 1 kb DNA ladder (Invitrogen) was used to increase the complexity of the
extrachromosomal arrays, and pTG96 (sur-5::gfp)
(Yochem et al., 1998) was used
as the co-injection marker at 20 ng/µl.
Suppression of non-vulval defects caused by class B synMuv mutations
Using the same exposure time, GFP expression (Tam phenotype) was quantified
for each micrograph within the linear range for signal detection using the
Profiler function of the OpenLab software package (Improvision). PGL-1
staining and RNAi sensitivity were scored as described by Wang et al.
(Wang et al., 2005). The L1
larval arrest phenotypes of let-418(n3536) and mep-1(n3701)
were scored at 25°C.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) rescued the synMuv suppression phenotype of n3294
animals (Table 1). We
determined that n3294 and n3297 harbor two distinct missense
mutations in F37A4.8 (Fig.
1B; see Fig. S1 in the supplementary material).
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)
(Fig. 1B). F37A4.8
encodes the C. elegans homolog of Drosophila ISWI, a
regulator of transcription and chromatin
(Elfring et al., 1994;
Deuring et al., 2000). We
named F37A4.8 isw-1 (isw, ISWI-like).
ISW-1 is 60% identical to Drosophila ISWI
(Elfring et al., 1994) and 69%
identical to human SNF2H (Okabe et al.,
1992). ISW-1 contains an AT-hook
(Reeves and Nissen, 1990) and
two SANT (Aasland et al., 1996)
domains; each of these domains can bind DNA. Additionally, ISW-1 contains a
domain similar to many helicases (the DEXD/H box) and an ATPase domain. Each
are required for chromatin-remodeling activity
(Corona et al., 1999).
isw-1(n3294) is predicted to cause a proline-to-leucine substitution
within the DEXD/H domain, implicating this domain in isw-1 function.
isw-1(n3297) is predicted to cause a leucine-to-phenylalanine
substitution within a non-conserved region
(Fig. 1C; see Fig. S1 in the
supplementary material).
isw-1(n3294) and isw-1(n3297) each conferred a fully penetrant recessive synMuv suppression and incompletely penetrant sterile phenotype (Table 1; see Table S1 in the supplementary material). By contrast, RNAi of isw-1 caused a fully penetrant synMuv suppression and sterile phenotype (Table 1; see Table S1 in the supplementary material). We isolated a deletion allele of isw-1, n4066, which removes part of the ATPase domain and most of the DEXD/H domain, causing a presumptive null phenotype. The isw-1(n4066) deletion allele caused a fully penetrant recessive sterile phenotype (see Table S1 in the supplementary material) but failed to cause strong suppression of the synMuv phenotype (Table 1). Because isw-1(n4066) homozygotes are sterile, the animals we studied were descended from isw-1(n4066) heterozygotes. Homozygous missense mutants descended from heterozygous mothers also did not have a strong synMuv suppression phenotype (Table 1), so the lack of synMuv suppression observed in isw-1 homozygotes derived from heterozygous mothers was probably caused by maternally inherited wild-type isw-1 gene product. isw-1(n4066) did perturb the synMuv suppressor function of isw-1, because this mutation failed to complement the synMuv suppression phenotype caused by each of the missense alleles (Table 1). isw-1(n3294) and isw-1(n3297) cause a decrease of isw-1 function, because each resulted in recessive suppression of the synMuv phenotype and failed to complement the phenotype conferred by a deletion of isw-1. Additionally, RNAi of isw-1 caused a synMuv suppression and sterile phenotype, so the two isw-1 missense alleles probably cause a partial loss of isw-1 gene function.
ISW-1 is nuclear, ubiquitously expressed and associated with chromatin
We generated specific antibodies that recognized a protein of the predicted
ISW-1 size 
115 kDa) by western blot analysis of wild-type but not
isw-1(n4066) adults (see Fig. S2A in the supplementary material).
ISW-1 was present in the nuclei of most, if not all, cells during every stage
of C. elegans development (see Fig. S2B-D in the supplementary
material). ISW-1 was associated with chromatin, as indicated by colocalization
of anti-ISW-1 immunoreactivity with DAPI-stained condensed chromosomes (see
Fig. S2E in the supplementary material).
Decreased function of the C. elegans homolog of Drosophila nurf301 suppresses the synMuv phenotype
In Drosophila, ISWI acts as the ATPase subunit of several
ATP-dependent chromatin-remodeling complexes, including ACF
(Ito et al., 1997), CHRAC
(Varga-Weisz et al., 1997) and
NURF (Tsukiyama and Wu, 1995).
We determined whether any of the C. elegans genes encoding
presumptive components of homologous complexes act similarly to isw-1
to promote the synMuv phenotype.
Using BLAST (Altschul et al.,
1990) and SMART (Sonnhammer et
al., 1997) searches, we identified C. elegans orthologs
of the ACF, CHRAC and NURF complex members. The ACF and CHRAC complexes share
one component: ACF1. Deletion of one of the two genes encoding a C.
elegans ACF1 ortholog (flt-1), RNAi of the other ortholog
(H20J04.2), or both deletion and RNAi together failed to suppress the
synMuv phenotype. Furthermore, RNAi of each of the remaining genes encoding
CHRAC complex orthologs failed to suppress the synMuv phenotype
(Table 2). In these RNAi
experiments in which a failure to suppress the synMuv phenotype was observed,
it remains possible that the gene plays a role in the antagonism of the synMuv
genes, but this role was not seen because the gene was not inactivated
sufficiently.
|
). RNAi
of the sole C. elegans NURF38 ortholog, pyp-1, or of one of
the two NURF55 orthologs, rba-1, caused embryonic lethality (data not
shown), and thus precluded the scoring of the synMuv suppression phenotype.
The other NURF55 ortholog is a class B synMuv gene, lin-53
(Lu and Horvitz, 1998), and
was not tested for synMuv suppression for this reason. Additionally, a
deletion allele of pyp-1 caused larval lethality before the third
larval stage (data not shown), which precluded scoring of the synMuv
suppression phenotype. Using the sequences determined from 15 independent cDNA clones (see Materials and methods), RT-PCR products and 5' RACE products, we identified five distinct transcripts generated from the nurf-1 locus (Fig. 2A). Each transcript is predicted to encode a protein with domains similar to some of the domains of Drosophila NURF301. However, none of the identified transcripts is predicted to encode a protein with all of the domains contained in full-length NURF301 (Fig. 2B).
nurf-1a encodes a protein most similar to the N terminus of
NURF301 and contains domains implicated in binding DNA, including an HMGY/I
domain (Reeves and Beckerbauer,
2001), a DDT domain (Doerks et
al., 2001) and a PHD finger
(Schindler et al., 1993;
Aasland et al., 1995).
nurf-1b and nurf-1c share two exons with nurf-1a
and encode proteins with similarity to the C-terminus of NURF301. Unlike
nurf-1b, which encodes a protein with only a Q-rich domain,
nurf-1c encodes a protein with two PHD fingers and a bromodomain. The
nurf-1d and nurf-1e transcripts are initiated at different
sites but encode the same predicted protein, which shares a C terminus with
NURF-1C. RNAi of nurf-1a, but not of the other nurf-1
transcripts, suppressed the synMuv phenotype of lin-15AB(n765)
mutants and caused sterility (Fig.
2C and data not shown).
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The C. elegans NURF-like genes isw-1 and nurf-1 promote the synMuv phenotypes of all synMuv mutant combinations
The C. elegans NURF-like genes might promote the ectopic vulval
fates of only specific synMuv mutant combinations, e.g. the lin-53;
lin-15A double mutant. To address this issue, we used RNAi to reduce the
function of isw-1 or nurf-1 in a variety of synMuv double
mutants. Inactivation of isw-1 or nurf-1 suppressed not only
the synMuv phenotype of lin-53(n833); lin-15A(n767) animals but also
the synMuv phenotype of the null double mutant combination lin-53(n3368);
lin-15A(n767) (Table 3).
Additionally, RNAi of isw-1 or nurf-1 suppressed other class
AB, BC and AC synMuv double mutant combinations
(Table 3). Reduction of
isw-1 function suppressed the synMuv phenotype of
lin-53(n833) in combination with putative null mutations in each of
the class A synMuv genes. Additionally, reduction of isw-1 function
suppressed the synMuv phenotype of lin-15A(n767) in combination with
putative null alleles of all identified class B synMuv genes
(Table 3; see Table S2 in the
supplementary material).
|
;
Beitel et al., 1990;
Han and Sternberg, 1990).
Therefore, reduction of isw-1 function might suppress the synMuv
phenotype by reducing the activity of the Ras pathway. If so, isw-1
might act by promoting the activity of the Ras pathway. However unlike many
mutants defective in the Ras pathway, isw-1 mutants did not have
abnormalities in the expression of vulval cell fates.
We tested for a subtle role of isw-1 in the specification of
vulval cells by asking if a weak Vul phenotype conferred by a decrease in Ras
pathway activity could be enhanced by an isw-1 mutation. A
lin-2 partial loss-of-function mutation, e1453, causes an
incompletely penetrant Vul phenotype
(Ferguson and Horvitz, 1985)
and a weak mutation in lin-45 Raf, ku112, does not cause a
vulval cell-fate defect (Sundaram and Han,
1995). lin-45(ku112) has been used to identify mutations
that as single mutants do not cause a vulval-fate defect but in combination
with lin-45(ku112) cause a synthetic vulvaless (synVul) phenotype,
implicating the genes defined by such mutations in the generation of vulval
cell fates (Rocheleau et al.,
2002). We scored vulval induction in isw-1; lin-2 and
isw-1; lin-45 double mutants and observed that isw-1 did not
enhance the Vul phenotype caused by lin-2(e1453) (42% versus 38%) and
did not cause a synVul phenotype in combination with lin-45(ku112)
(data not shown). These data suggest that if isw-1 promotes the
activity of the Ras pathway, it might act redundantly with other factors.
Gain-of-function mutations in let-23 RTK or let-60 Ras
cause a Muv phenotype (Beitel et al.,
1990; Han et al.,
1990; Katz et al.,
1996). Reduction of isw-1 and nurf-1 function
partially suppressed the Muv phenotype caused by increased let-23 and
let-60 gain-of-function mutations
(Table 4). The Ras pathway
terminates with the transcription factors LIN-1 ETS and LIN-31 HNF/Forkhead.
lin-1 has a primary role in inhibiting vulval cell fates, such that
lin-1 null mutants have a Muv phenotype
(Beitel et al., 1995;
Tiensuu et al., 2005). LIN-31
when bound to LIN-1 inhibits vulval cell fates, but after phosphorylation by
MPK-1 MAPK, LIN-31 promotes vulval cell fates
(Tan et al., 1998;
Miller et al., 2000).
lin-31 mutants can have either a Muv or Vul phenotype, because the
vulval cells are unregulated and stochastically adopt a vulval or non-vulval
cell fate (Miller et al.,
2000). Reduction of isw-1 and nurf-1 function
suppressed the Muv phenotype caused by a partial loss-of-function
lin-1 mutation and a null lin-31 mutation but failed to
suppress the null lin-1 mutant phenotype
(Table 4). The failure to
enhance a sensitized abnormal vulval phenotype and to suppress completely the
Muv phenotype caused by an increase in Ras pathway activity suggests either
that a greater inhibition of the functions of isw-1 and
nurf-1 is required to observe complete effects or that other factors
act redundantly with the NURF-like genes to promote Ras pathway activity.
|
); by a germline-like appearance of somatic cells in
mep-1 and let-418 arrested larvae
(Unhavaithaya et al., 2002);
by the tandemarray-modifier (Tam) phenotype of increased somatic silencing of
expression from repetitive transgenes by a process similar to that which
occurs in the germline (Kelly et al.,
1997; Kelly and Fire,
1998; Hsieh et al.,
1999); and by enhanced RNAi sensitivity, perhaps caused by ectopic
expression of a germline RNA polymerase EGO-1 in the soma producing an
increased RNAi effect (Wang et al.,
2005). Reduction of isw-1 and nurf-1 function suppressed abnormalities associated with defects in the germline-versus-soma cell-fate decision caused by lin-15B(n744) and lin-35(n745), including the ectopic somatic expression of the germlineexpressed protein PGL-1, the Tam phenotype (see Fig. S4 in the supplementary material) and the RNAi hypersensitivity phenotype (Fig. 3A-C; data not shown). However, the reduction of isw-1 or nurf-1 function did not cause a germline desilencing of expression from repetitive transgenes (data not shown), so both genes might not be required for mechanisms of transcriptional repression in the germline. Additionally, reduction of isw-1 and nurf-1 function suppressed the mep-1 and let-418 larval-arrest phenotypes (Fig. 3D), and somatic cells did not have a germline-like appearance in isw-1; mep-1 or in nurf-1; mep-1 double mutants (data not shown). These data indicate that not only are isw-1 and nurf-1 required for the synMuv vulval phenotype but also for the ectopic adoption of germline fates in the soma caused by loss of class B synMuv gene function. Therefore, the putative synMuv complexes and the NURF-like complex might antagonize the transcription of similar sets of target genes.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Ferguson and Horvitz, 1989).
The loss of isw-1 or nurf-1 function must antagonize a
deficit in one or both of the synMuv genes in each suppressed strain to
counteract the synMuv phenotype. Because the synMuv phenotypes of class AB, BC
and AC double mutants were each suppressed by loss of isw-1 or
nurf-1 function, the C. elegans NURF-like genes must
antagonize the functions of at least two classes of synMuv genes. Defects
caused by single class B mutants were suppressed by reduction of
isw-1 function (Fig.
3), and the vulval phenotype of the class C synMuv mutant
trr-1(n3712) was suppressed (see Table S2 in the supplementary
material). Thus, isw-1 and nurf-1 likely antagonize the
activities of at least the class B and C synMuv genes. Because a functional
Ras pathway is required for the synMuv phenotype, it is possible that the
antagonism of the class B and C gene functions caused by reduction of
isw-1 or nurf-1 function involves a downregulation of the
Ras pathway.
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ISW-1 and NURF-1 might be components of a NURF-like chromatin-remodeling complex involved in the C. elegans vulval cell-fate decision
C. elegans ISW-1 probably acts as part of a NURF-like complex and
not as part of a CHRAC-like or ACF-like complex to antagonize the actions of
the synMuv proteins, because inhibition of isw-1 or nurf-1
but not inhibition of ACF or CHRAC ortholog gene functions suppressed the
synMuv phenotype (Table 2). The
Drosophila NURF complex is composed of four subunits: ISWI, NURF38,
NURF55 and NURF301 (Tsukiyama and Wu,
1995). Because loss of the C. elegans homolog of NURF38,
PYP-1, caused embryonic lethality (data not shown), we have not determined if
it functions as a NURF-like complex component to antagonize the actions of the
synMuv proteins. Drosophila NURF55 is similar to two proteins in
C. elegans, LIN-53 and RBA-1 (72% and 53% identical, respectively).
LIN-53 is a class B synMuv protein and 54% identical to its neighbor RBA-1.
Strong reduction-of-function mutations in lin-53 and isw-1
cause opposite mutant phenotypes. Therefore, it is unlikely that LIN-53 and
ISW-1 always act in the same complex. The LIN-53 homolog NURF55/RbAp48/CAF-1
is present in many chromatin-regulatory complexes, and it is possible that
LIN-53 similarly acts in a number of C. elegans complexes, possibly
both preventing and promoting the synMuv phenotype. If so, the role of LIN-53
in preventing the synMuv phenotype must be predominant, because
loss-of-function mutations in lin-53 cause a synMuv phenotype in
combination with mutations in class A genes
(Lu and Horvitz, 1998).
Alternatively, RBA-1 might act with ISW-1 as part of a NURF-like complex.
Because rba-1(RNAi) caused embryonic lethality (data not
shown), we have not tested this possibility.
nurf-1, the C. elegans homolog of Drosophila
nurf301, is predicted to encode at least five different proteins, each of
which has some similarity to NURF301. Using deletion alleles and RNAi, we
found that nurf-1a but not nurf-1b, nurf-1c, nurf-1d or
nurf-1e was required to promote the synMuv phenotype. The region of
NURF301 between the DDT domain (Doerks et
al., 2001) and the C-terminal PHD fingers
(Aasland et al., 1995)
interacts with transcription factors required for recruitment of the NURF
complex to target gene promoters (Xiao et
al., 2001). The corresponding regions of NURF-1A, NURF-1B and
NURF-1C differ in length and could mediate interactions with distinct sets of
transcription factors to direct recruitment of the complex to different
promoters. The NURF-1A region that presumably interacts with transcription
factors might be responsible for recruitment of the NURF-like complex to
promoters of genes required for the vulval cell-fate decision.
The functions of ISW-1 and NURF-1 might be involved in the generation of normal vulval cell fates
It is possible that the function of the putative C. elegans
NURF-like complex is required only for the generation of ectopic vulval cell
fates, e.g. in synMuv mutants, but is not involved in normal vulval
development. For example, when the inhibitory actions of the synMuv proteins
are absent or when the activity of the Ras pathway is increased, the NURF-like
complex might promote the specification of ectopic vulval cell fates. However,
we observed that isw-1 is required not only for the Tam phenotype of
class B synMuv mutants (Fig.
3B) but also for a basal level of repression of expression from
the ccIs4251 GFP reporter (see Fig. S4 in the supplementary
material). This observation suggests that isw-1 is required not only
in the absence of synMuv activity but also in a wild-type synMuv background to
promote expression of genes repressed by the synMuv proteins. By analogy, we
propose that the putative NURF-like complex helps promote the normal
generation of vulval cell fates in a wild-type synMuv background.
The C. elegans NURF-like complex acts antagonistically to complexes similar to Myb-MuvB/dREAM, NuRD and Tip60/NuA4 to control transcription
The Drosophila NURF complex slides nucleosomes along the DNA to
allow access for transcription factors to bind target sequences in vitro
(Hamiche et al., 1999). Both
ISWI and NURF301 are required for the transcription of Hox and heat-shock
genes in vivo (Deuring et al.,
2000; Badenhorst et al.,
2002). Therefore, the NURF complex has been hypothesized to be
involved in transcriptional activation. The homologs of many class B synMuv
proteins are components of at least two complexes, Myb-MuvB/dREAM and NuRD,
involved in transcriptional repression
(Tong et al., 1998;
Xue et al., 1998;
Korenjak et al., 2004;
Lewis et al., 2004). Studies
of Drosophila and mammalian cells argue that the NURF complex and the
Myb-MuvB/dREAM and NuRD complexes have opposite effects on transcription.
The vulval cell-fate decision in C. elegans demonstrates the
biological consequences of the opposing effects of the Myb-MuvB/dREAM and NuRD
and the NURF chromatin-remodeling activities. We propose that a complex
containing both ISW-1 and NURF-1 antagonizes one or more synMuv protein
complexes in the transcriptional control of the vulval cell-fate decision. One
hypothesis is that loss of transcriptional repression by the synMuv proteins
causes a Muv phenotype, as a result of the increased transcription of genes
that promote the vulval cell-fate decision. The NURF-like complex might be
required for this ectopic expression of synMuv target genes. Alternatively,
the NURF-like complex might act at targets distinct from those that are
misregulated in synMuv mutants, and transcription of these genes would
antagonize the activities of the synMuv proteins. The identification of the
transcriptional targets of the synMuv proteins and of the NURF-like complex
should help differentiate between these two hypotheses. The
Drosophila Myb-MuvB complex copurified with sub-stoichiometric
amounts of NURF complex members. The actions of and requirements for the NURF
complex components for Myb-MuvB function were not investigated
(Lewis et al., 2004). Perhaps
NURF-like complexes bind Myb-MuvB-like complexes to directly inhibit
activities of these complexes.
The antagonism of the lin-35 Rb and let-60 Ras mutant phenotypes by partial loss of isw-1 ISWI function suggests a possible approach to cancer therapy
The functional antagonism between a NURF-like complex and synMuv repressive
complexes and/or activation of the Ras pathway could be conserved in humans
and be important for human cancer. The loss of Rb function is associated with
the majority of human carcinomas (Adams and
Kaelin, 1998), and methods to inhibit the defects of Rb-deficient
cells should be beneficial as cancer treatment strategies. Additionally
oncogenic forms of human Ras are involved in many cancers, especially cancers
of the lung (Minamoto et al.,
2000). Because a reduction of isw-1 ISWI function can
suppress defects associated with a complete loss of lin-35 Rb or
activation of let-60 Ras in C. elegans, inhibition of the
human ISW-1 homolog (SNF2H; SMARCA5 - Human Gene Nomenclature Database) might
suppress the effects of Rb loss or of oncogenic Ras in human cells and hence
reduce or eliminate the consequences of oncogenic mutations. SNF2H is a
chromatin-remodeling enzyme (Okabe et al.,
1992; Aihara et al.,
1998) and might be a reasonable target for therapeutic
intervention.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/14/2695/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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