1. science
2. biology
3. breeding

Copyright Ó 2007 by the Genetics Society of America
DOI: 10.1534/genetics.107.076653
Genetic Suppressors of Caenorhabditis elegans pha-4/FoxA Identify
the Predicted AAA Helicase ruvb-1/RuvB
Dustin L. Updike and Susan E. Mango1
Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah, 84112
Manuscript received May 25, 2007
Accepted for publication July 31, 2007
ABSTRACT
FoxA transcription factors are critical regulators of gut development and function. FoxA proteins specify
gut fate during early embryogenesis, drive gut differentiation and morphogenesis at later stages, and affect
gut function to mediate nutritional responses. The level of FoxA is critical for these roles, yet we know
relatively little about regulators for this family of proteins. To address this issue, we conducted a genetic
screen for mutants that suppress a partial loss of pha-4, the sole FoxA factor of Caenorhabditis elegans. We
identified 55 mutants using either chemical or insertional mutagenesis. Forty-two of these were informational suppressors that affected nonsense-mediated decay, while the remaining 13 were pha-4 suppressors.
These 13 alleles defined at least six different loci. On the basis of mutational frequencies for C. elegans and the
genetic dominance of four of the suppressors, we predict that many of the suppressors are either unusual
loss-of-function mutations in negative regulators or rare gain-of-function mutations in positive regulators.
We characterized one dominant suppressor molecularly and discovered the mutation alters a likely cisregulatory region within pha-4 itself. A second suppressor defined a new locus, the predicted AAA1 helicase
ruvb-1. These results indicate that our screen successfully found cis- or trans-acting regulators of pha-4.
D
URING metazoan development, cells acquire specialized identities such as cell type, position, organ,
or tissue. The developmental programs that establish
these identities often depend on the activity of a subset
of transcription factors called selector genes (Mann
and Carroll 2002). Here we focus on the selector
gene responsible for identity of cells of the foregut,
which is the winged-helix transcription factor FoxA
(Mango et al. 1994; Horner et al. 1998; Kalb et al.
1998).
FoxA, previously known as HNF3b (Kaestner et al.
2000), was identified as a protein enriched in liver
nuclear extracts (Lai et al. 1990; Lai et al. 1991) and the
gene defined by Drosophila fork head ( Jurgens et al. 1984;
Jurgens and Weigel 1988; Weigel et al. 1989). More
recently, FoxA factors have been implicated in transcriptional control of breast cancer genes (Carroll
et al. 2005; Laganiere et al. 2005), metabolic processes
(Friedman and Kaestner 2006) and aging (Panowski
et al. 2007). Loss of FoxA activity results in severe defects
in foregut-derived organs such as liver and pancreas
(Weigel et al. 1989; Ang and Rossant 1994; Mango
et al. 1994; Weinstein et al. 1994; Dufort et al. 1998;
reviewed in Zaret 2002; Lee et al. 2005). Conversely,
ubiquitous expression of pha-4/FoxA in Caenorhabditis
elegans is sufficient to induce ectopic foregut cells
1
Corresponding author: Room 4345, 2000 Circle of Hope Huntsman
Cancer Institute, Salt Lake City, UT 84112.
E-mail: susan.mango@hci.utah.edu
Genetics 177: 819–833 (October 2007)
(Horner et al. 1998). These data reveal that appropriately regulated expression of pha-4/FoxA is critical for
its normal functions.
Microarray studies have identified .300 potential
targets of PHA-4 in C. elegans, and promoter analysis has
revealed that many of these genes are direct PHA-4
targets (Gaudet and Mango 2002). The targets are
expressed at varying times during the development, differentiation, or functioning of the pharynx. The appropriate timing of expression depends on combinatorial
mechanisms among different cis-regulatory sites (Kalb
et al. 2002; Ao et al. 2004; Gaudet et al. 2004; Vilimas
et al. 2004; Raharjo and Gaudet 2007). In addition, the
affinity of PHA-4 for its binding sites is an important
contributor to temporal control (Gaudet and Mango
2002; Ao et al. 2004; Gaudet et al. 2004). FoxA proteins
recognize the consensus TRTTKRY (R ¼ A/G, K ¼ T/G,
and Y ¼ T/C) in the cis-regulatory regions of gut-specific
targets (Overdier et al. 1994; Kalb et al. 1998; Gaudet
and Mango 2002). Target genes with high affinity sites
are competent to fire early in embryogenesis, whereas
genes with lower affinity sites are typically expressed late
(Gaudet and Mango 2002; Gaudet et al. 2004). This
regulatory configuration implies that the level and activity of PHA-4 are carefully controlled to achieve proper
timing for pharyngeally expressed genes. This raises the
question of what mechanisms control PHA-4 expression
and activity. Here, we take advantage of our pha-4 alleles
to identify mutants that can suppress a partial loss of
pha-4 function. We chose a genetic approach over other
820
D. L. Updike and S. E. Mango
possible schemes (e.g., yeast two-hybrid screen, coimmunoprecipitation) because this strategy could potentially identify proteins that do not physically touch
PHA-4, such as components of signaling pathways, as
well as those that do. Here we present the results and
discuss the implications of this screen for regulators of
PHA-4.
MATERIALS AND METHODS
Worm growth: C. elegans strains were maintained as described in Brenner (1974). Wild type (WT) was N2 (Bristol).
pha-4(ts) strains, SM190 smg-1(cc546ts)I; pha-4(zu225)V and
SM568 smg-1(cc546ts)I; pha-4(q500) rol-9(sc148)V, KK822 par1(zu310ts)V, SM279 smg-1(cc546ts)I five times outcrossed,
SM141 or PGP is pxIs1(PHA-4TGFP 1 rol-6) were described
previously (Gaudet and Mango 2002; Alder et al. 2003;
Kaltenbach et al. 2005; Updike and Mango 2006). Strains
obtained from the CGC include TR1331 smg-1(r861)I, BC2511
dpy-18(e364); eT1III; unc-60(e677) dpy-11(e224) sDf35/eT1V, and
CB4856 Hawaiian isolate. Strain TM2786 ruvb-1(tm2786) was
obtained from the National Bioresource Project, Tokyo
Women’s Medical College, Tokyo (Gengyo-Ando and Mitani
2000).
Strains created for this study include the Mos1 mutagenesis
strain SM948 smg-1(cc546ts)I; pha-4(zu225); oxIs30(Mos1 Transposase) X; oxEx229(Mos1 Transposon). SM948 was created from
SM190 and EG2412, which carries the Mos1 transposon and
transposase (Bessereau et al. 2001). SM1547 was used for singlenucleotide polymorphism (SNP) mapping and was generated
by crossing SM190 10 times with Hawaiian CB4856, using
pxEx259(sur-5TGFP 1 unc-54TGFP) to mark cross progeny.
SM1153 was smg-1(cc546ts); pha-4(zu225) 1 (coelomocyteTGFP).
SM1261 was smg-1(cc546ts)I; pha-4(px63 zu225)/fog-2(q71) rol9(sc148)V. SM1237 was dpy-11(e224) unc-42(e270) ruvb-1(px34)/
evl-1(ar115) V. SM1238 was ruvb-1(px34)/unc-42(e270) sqt-3(sc63)
V. SM1239 is ruvb-1(px34) sqt-3(sc63)/unc-42(e270) sqt-3(sc63) V.
SM1262 was dpy-11(e224) unc-42(e270) ruvb-1(px34)/evl-1(ar115)V;
pxEx210a(R05D5 1 sur-5TGFP). SM1263 was dpy-11(e224) unc42(e270) ruvb-1(px34)/evl-1(ar115)V; pxEx210b(T10A5 1 sur5TGFP). SM1276 was pxEx211(ruvb-1pTGFPTHis2B 1 rol-6).
SM1415 is smg-1(cc546ts)I; (px23)II; pha-4(zu225)V 1 (coelomocyteTGFP). SM1261 was pha-4(px63 zu225)/fog-2(q71) rol9(sc148). SM1592 was ruvb-1(tm2786)/unc-42(e270) sqt-3(sc63).
Suppressor strains in the pha-4(ts) background are shown in
Table 1.
Isolation of pha-4(ts) suppressors: pha-4(ts) strains were
mutagenized with 50 mm ethylmethanesulfonate (EMS) for 4
hr at 24°. P0’s were rinsed with M9 to remove EMS, and fourth
larval stage (L4) worms were picked to new plates and
incubated at 24°. F1 progeny were shifted to the intermediate
restrictive temperature of 20° at the L3–L4. Viable F2 progeny
were selected, and these strains were maintained at 20°, with
no more than one F2 suppressor line isolated from one F1.
Mos1 transposon mutagenesis was performed with SM948.
This strain carries pha-4(ts) and an extrachromosomal array
with the Mos1 transposon (oxEx229) and an integrated
HSTMos1 transposase (oxIs30) (Bessereau et al. 2001). Mutagenesis was performed as described in (Bessereau et al.
2001). To synchronize populations, several plates of SM948
worms were treated with bleach to isolate embryos. Embryos
were grown to the adult stage to provide the P0 population.
Synchronized P0 hermaphrodites were incubated at 33° for
1 hr to induce the transposase and then dispensed to several
10-cm plates at a density of 100 P0 animals per plate. F1
progeny were shifted to 20° at the L3–L4 stage. Viable F2
progeny were selected, and their progeny maintained at 20°,
with no more than one F2 suppressor line isolated from each
plate.
PCR amplification of a Mos1 sequence was used to detect an
insertion event after the Mos1 extrachromosomal array was
lost from the strain (Bessereau et al. 2001). No Mos1 insertion
events were detected in the pha-4(ts) suppressor strains. A
Mos1 insertion in unc-74 provided by Dan Williams and Erik
Jorgensen was used as a positive control in these experiments
and was always detected (Williams et al. 2005; D.W. and E.J.L.,
personal communication).
Genetic analysis: To test for suppression in heterozygous
cross progeny, 10 SM1153 males were crossed to five to eight
sup; pha-4(ts) hermaphrodites at 20°. GFP-positive F1 cross
progeny were observed for all suppressors. SM1153 hermaphrodite controls had no viable F1 progeny. To determine
whether suppressors were dominant, five GFP-positive F1 cross
progeny were picked to new plates in L4 and examined for
viable F2 progeny at 20°.
To test for dominant zygotic suppression, males were
generated for each of the 13 pha-4 suppressor strains. Twenty
suppressor males were crossed to 10 pha-4(ts) worms at 20°. Ten
pha-4(ts) males were used for a control. After two dates of
mating, P0 mothers were picked from the plates. Two days
later, the percentage of progeny larger than the L1 stage were
scored (n ¼ 100 progeny).
To examine the gonad, pha-4(ts) animals were shifted to 20°
at the L1 or the pretzel stage of embryogenesis. Worms were
examined for gonad defects under the light microscope two
days later.
RT–PCR of rpl-7a: For total RNA extraction, 100 ml of
frozen worm pellets were crushed and resuspended in Trizol
Reagent (Gibco BRL, Grand Island, NY), followed by chloroform extraction, according to the manufacturer’s specifications. RNA was precipitated with isopropanol and washed with
70% ethanol. Resuspended RNA was treated with DNase,
extracted with phenol:chloroform and precipitated with
ethanol. A FirstStrand reaction kit (Gibco BRL) was used to
perform the reverse transcriptase reaction. This reaction was
followed by phenol:chloroform extraction and ethanol precipitation. Amplification of rpl-7a from the cDNA was performed as described in (Mitrovich and Anderson 2000),
and PCR products were analyzed on a 1% agarose gel.
Antibody staining: To detect possible nonsense-codon,
readthrough suppression, embryos were stained as described
previously with an antibody that recognizes the carboxyl
terminus of PHA-4 (Horner et al. 1998; Kaltenbach et al.
2005). Embryos from pha-4 suppressing strains were stained
with a-carboxyl PHA-4 at a 1:20 dilution and costained with a-P
granule (OIC1D4) at 1:10 ½received from the Developmental
Studies Hybridoma Bank, University of Iowa (Beanan and
Strome 1992). par-1 mutant embryos (Kemphues et al. 1988)
were placed on the same slide as the suppressors as a positive
control for PHA-4 staining. Their altered morphology allowed
them to be distinguished from pha-4(ts) embryos.
To assess early pharyngeal development, embryos were stained
with pharyngeal muscle antibody 3NB12 (Priess and Thomson
1987) and costained with a-LIN-26 antibody (Labouesse et al.
1996), as described in (Horner et al. 1998).
To count pharyngeal cells, embryos were stained with a
1:2000 dilution of affinity purified pan-PHA-4 antibody as
described previously (Kaltenbach et al. 2005). Confocal sections were taken every 0.25 mm and the number of PHA-4
positive nuclei counted in 6–12 embryos for each strain.
Single nucleotide polymorphism mapping of suppressors:
SM1547, which is pha-4(ts) outcrossed 10 times in the Hawaiian
CB4856 background, males were crossed to sup; pha-4(ts)
C. elegans pha-4 Suppressors
hermaphrodites (Bristol background) at 20° (px17, px33, px34,
and px63) or 24° (px8, px11, px12, px16, px23, px28, px31, px70,
and px71). GFP fusions with sur-5 (Yochem et al. 1998) or unc54 (Sha and Fire 2005) in SM1547 males were used to identify
F1 cross progeny. Hermaphrodite F1 cross progeny were
cloned to individual plates at 20°. Viable F2 progeny were
picked to individual plates at 20°. Individual F2 animals that
gave rise to viable F3 progeny at 20° were picked into 20 ml of
worm lysis buffer with proteinase K and frozen for later
analysis.
Worms in lysis buffer (50 mm KCl, 10 mm Tris pH 8.3, 2.5 mm
MgCl2, 0.45% IGEPAL CA-630, 0.45% Tween 20, 0.01%
gelatin, 60 mg/ml proteinase K) were incubated at 65° for 1
hr, followed by 15 min at 95°. This lysate was used as a template
in PCR reactions to amplify SNPs spanning all chromosomes.
After PCR, the appropriate restriction enzymes and buffers
were added to detect SNPs between the Hawaiian and N2
isolates (Wicks et al. 2001; Davis et al. 2005). Digested
amplicons were analyzed by 2% agarose gel electrophoresis.
SNP mapping data: F2 segregation ratio of N2:N2/Hi:Hi is
given for suppressors linked to chromosomes. Map positions
and physical locations in this article correspond to WormBase
release WS174.
px11: Chromosome III. snp_Y71H2B½2 at 12.05 cM 28:1:2.
pkP3101 at 0.92 cM 29:1:1. snp_F56C9½1 at 0.75 cM
26:4:1. snp_Y39A1½9 at 14.13 cM 29:0:2.
px12: Chromosome IV. snp_Y41D4B½2 at 17.51 cM 6:1:0.
px16: Chromosome II. pkP2101 at 15.91 cM 45:6:1. pkP2103
at 6.21 cM 45:6:1. snp_T24B8½1 at 10.98 cM 43:6:2.
pkP2116 at 115.77 cM 45:3:4. snp_Y39G8B½1 at 121.19
cM 45:3:4.
px17: Chromosome III. snp_Y71H2B½2 at 12.05 cM 142:210:28.
Of the 28 Hi/Hi at 12.05 cM, only 1 was still Hi/Hi at
pkP3076 at 0.85 cM, and it was N2/Hi by snp_F56C9½1 at
0.75 cM. snp_Y39A1½9 at 14.13 cM 145:209:11. Of the 11
Hi/Hi at 14.13 cM, only 2 were still Hi/Hi at pkP3105
at 0.59 cM, and both were N2/Hi by snp_F56C9½1 at
0.75 cM.
px23: Chromosome II. pkP2116 at 115.77 cM 89:4:0. Of the
four N2/Hi at 115.77 cM, two were N2 homozygotes by
pkP2118 at 120.71 cM. snp_Y39G8B½1 at 121.19 cM 92:0:0.
px28: Chromosome II. pkP2116 at 115.77 cM 6:1:0.
snp_Y39G8B½1 at 121.19 cM 7:0:0
px33: Chromosome III. snp_F56C9½1 at 0.75 cM 5:10:0.
px34: Chromosome V. nP118 at 18.81 cM 3:6:1.
snp_K12B6½7 at 0.18 cM 10:13:1. pkP5117 at 12.00 cM
10:12:1. snp_F46F3½1 at 13.25 cM 12:12:0. pkP5125 at
15.83 cM 9:15:1. Next, F2 arrested L3 worms were picked
for finer mapping instead of suppressors. snp_K12B6½7 at
0.18 cM 12:9:0. pkP5117 at 12.00 cM 68:1:0. Remaining
N2/Hi is N2/N2 by snp_F58E6½1 at 12.14 cM. pkP5064 at
12.51 cM 47:1:0. Remaining N2/Hi is still N2/Hi by
snp_C12D8½2 at 12.40 cM, but is N2/N2 by snp_C08B6½1
at 2.28 cM. snp_F29F11½1 at 12.72 67:2:0. snp_F46F3½1 at
13.25 cM 18:3:0. Three-point mapping demonstrated that
px34 mapped to the right of unc-42, which is at 12.16 cM.
For finer mapping from the left, non-L3 arrested Unc
recombinants were mapped from px34 animals marked with
unc-42. From 14 non-L3 arrested Unc recombinants, 2 were
still N2/N2 by pkP5119 at 12.20 cM, but N2/Hi by pkP5064
at 12.51 cM. For finer mapping from the right, non-L3
arrested Sqt recombinants were mapped from px34 animals
marked with sqt-3. From 48 non-L3 arrested Sqt recombinants, 10 were still N2/N2 by snp_F29F11½1 at 12.72 cM,
only 1 was N2/N2 by snp_C08B6½1 at 2.28 cM, but N2/Hi by
pkP5119 at 12.20 cM.
px63: Chromosome V. pkP5117 at 12.00 cM 4:6:0.
821
Southern blot analysis: Genomic DNA was prepared from
frozen 0.5-ml pellets of N2 or smg-1(cc546ts); pha-4(zu225)/pha4(px63 zu225) worms by standard proteinase K lysis, phenol:chloroform extraction, and ethanol precipitation (Sulston
and Hodgkin 1988). A total of 5 mg of genomic DNA from
each strain was digested overnight with EcoRI, HindIII, XbaI, or
HindIII and XbaI. Digested DNA was run in a 0.8% agarose gel,
blotted, and cross linked to a nylon membrane. 32P-labeled
PCR amplicons were used as probes and hybridized to the blot.
Primers flanking HindIII internal amplicon: (left-CAGG
TCCCCTGACAAG) and (right-GGTGTAGTCAATTCCTGGA
TAACTGC). Primers flanking XbaI internal amplicon: (leftCAGGTTCAGAGGTCAAACGGGAC) and (right-CGGTGGT
GCCAGTGGTAAAAC). These probes placed the px63 rearrangement between XbaI sites in intron 2 and exon 5.
px23 and px28 complementation: SM1415 (1coelomocyteT
GFP) males were crossed to SM1430 hermaphrodites at 20°.
As complementation controls, SM1153(1coelomocyteTGFP)
males were crossed to SM1415 (coelomocyteTGFP) and
SM1430 hermaphrodites at 20°. From each of the three crosses
10 L4 cross progeny (1coelomocyteTGFP) were cloned to
individual plates at 20°. As noncomplementation controls,
10 SM1415 and SM1430 L4 animals were cloned to individual plates at 20°. 10/10 plates from SM1415/SM1430
(1coelomocyteTGFP) cross progeny had viable F2 progeny, as
did 10/10 plates from SM1415 and SM1430. 0/10 SM1415/1
and 0/10 SM1430/1 cross progeny had viable F2 offspring.
Quantitation of nuclear PHA-4: In situ antibody staining was
performed as described in Horner et al. (1998). Single layer
confocal images were taken using a Ziess LSM510. Intensity
of PHA-4 staining was quantified in four nuclei for each of
10 1.5-fold embryos using ImageJ. The intensity of PHA-4,
after background subtraction, was normalized to the intensity
of PHA-4 in 10 par-1 mutant embryos on the same slide
(Kaltenbach et al. 2005) ½P # 0.0072, t-test; significance of
PHA-4 levels in suppressors from pha-4(ts) at 20°. For this
experiment, all embryos were in the smg-1(cc546ts)I; pha4(zu225)V background, with the exception of px12 and px17,
which were in smg-1(cc546ts)I; pha-4(q500) rol-9(sc148)V.
ruvb-1 expression and rescue: DNA was prepared from
cosmids R05D5 and T10A5 with QIAGEN (Valencia, CA) spin
miniprep columns. A total of 30 ng/ml of T10A5 was injected
with 2 ng/ml of a sur-5TGFP reporter (Yochem et al. 1998) to
rescue ruvb-1 lethality in SM1237. Of the four lines carrying
T10A5 in SM1237, two lines produced Dpy Unc progeny that
suppressed L3 lethality of ruvb-1 (60%, n ¼ 5; and 33%, n ¼ 9;
rescue). A total of 11 ng/ml of R05D5 was injected with 2 ng/ml
of sur-5TGFP to rescue ruvb-1-associated lethality of SM1237
and SM1238. Of the five lines carrying R05D5 in SM1237, two
lines produced Dpy Unc progeny that suppressed L3 lethality
(33%, n ¼ 15; and 69%, n ¼ 16; rescue). Of the seven lines
carrying R05D5 in SM1238, one produced non-Unc non-Dpy
non-Rol progeny that suppressed L3 lethality (28% rescue at
25°, n ¼ 36). A total of 0.2 ng/ml of a 3777-bp amplicon
containing ruvb-1 (677 to 13100 relative to the ATG) was
injected with 2 ng of an amplicon containing the sur-5TGFP
reporter and 98 ng/ml herring sperm carrier DNA to rescue
ruvb-1 lethality in SM1238, and three non-Unc non-Dpy non-Rol
sur-5TGFP positive transgenic animals suppressed L3 lethality.
The ruvb-1 promoter was amplified from R05D5, encompassing nucleotide positions 677 to 1 relative to the ruvb-1
start codon. This promoter fragment was engineered with 59
SphI and 39 KpnI restriction sites to facilitate cloning into the
GFPTHIS2B reporter vector pAP.10 (Gaudet and Mango
2002). GFP expression was examined on a Zeiss LSM510
confocal microscope.
Suppression of pha-4 using RNAi: For pha-4(ts) suppression,
HT115 bacteria expressing GFP, ruvb-1 or ruvb-2 double-stranded
822
D. L. Updike and S. E. Mango
Figure 1.—Strategy for pha-4 suppressor screen EMS or
Mos1 transposon mutagenesis was performed on pha-4(ts) animals at 24°. F1 progeny were shifted to 20°. F2 progeny died in
L1 at 20° unless a suppressing mutation had been introduced.
RNA were grown overnight in 60 mg/ml carbenicillin at 37°,
spotted to unseeded plates containing 1 mm IPTG and 60 mg/
ml carbenicillin and incubated at 37° overnight (Kamath et al.
2003). Five L4 stage pha-4(ts) or smg-1; ruvb-1 pha-4/1 pha-4
worms were picked to five HT115 (RNAi) or OP50 (control)
plates and incubated at 20°. P0 animals were allowed to lay eggs
for 1 day and then removed. Two days later, the percentage of
animals older than L1 (as determined by size) were counted
(40 F1 animals/plate).
For pha-4(RNAi) suppression, HT115 bacteria expressing
GFP or pha-4 double-stranded RNA were grown overnight in
60 mg/ml carbenicillin at 37°. Three parts of a GFP dsRNAexpressing culture were mixed with one part pha-4 dsRNAexpressing culture to weaken the potency of pha-4(RNAi). The
mixture was spotted to unseeded plates containing no IPTG or
antibiotic and incubated at 37° overnight. Five L4 stage WT N2
or ruvb-1(px34)/unc-42(e270) sqt-3(sc63) worms were picked to
six HT115 (RNAi) plates and incubated at 25°. P0 animals were
allowed to lay eggs for 36 hr and then removed. Two days later
the percentage of animals older than L1 were counted (100
F1 animals/plate). P-value ¼ 5.9 3 105 for significance
between N2 and ruvb-1 suppression (t-test).
RESULTS
Identification of suppressors of pha-4(ts) lethality:
To screen for pha-4 suppressors, we used two temperature-sensitive (ts) configurations of pha-4 that allowed us
to regulate PHA-4 accumulation. pha-4(zu225) and pha4(q500) each carry a premature stop codon predicted
to truncate PHA-4 after the DNA binding domain
and render pha-4 mRNA subject to degradation by the
nonsense-mediated decay (NMD) pathway (Mango 2001;
Gaudet and Mango 2002; Kaltenbach et al. 2005;
Updike and Mango 2006). Our previous analyses revealed that animals homozygous for these alleles were
viable provided the NMD pathway was also inactivated
(Kaltenbach et al. 2005). We took advantage of a ts
allele of an NMD component smg-1(cc546ts), which ex-
hibits robust NMD activity at 15° but compromised
activity at higher temperatures (Pulak and Anderson
1993; Mango 2001; Grimson et al. 2004; A. Fire, personal communication). At 15°, SMG-1 was active and
pha-4 mRNA from either pha-4(zu225) or pha-4(q500) was
degraded. We call this ‘‘restrictive temperature’’ because
it results in Pha-4 lethality at L1 (Kaltenbach et al.
2005). At the ‘‘permissive temperature’’ of 24°, SMG-1
was compromised and pha-4 mRNA was stabilized. Truncated PHA-4 protein accumulated, and worms survived
(Kaltenbach et al. 2005). At 20°, an intermediate level
of truncated PHA-4 accrued, producing a phenotype
less severe than the null phenotype, but lethal nonetheless (Kaltenbach et al. 2005). We define pha-4(ts) strains
as those strains carrying one of the two pha-4 nonsense
alleles and smg-1(cc546ts).
To identify pha-4 suppressors, we mutagenized pha4(ts) worms at the permissive temperature of 24° and
shifted the first generation progeny (F1) to 20° at the L4
(Figure 1). The second generation (F2), which could be
homozygous or heterozygous for any induced mutation,
was selected for suppression of pha-4 lethality at the
intermediate temperature of 20°. We selected suppressors at the intermediate temperature because we were
most interested in proteins within the pha-4 pathway
(not bypass mutations), which would likely require some
pha-4 activity. We predicted that this screen would find
regulators or cofactors of pha-4, but not downstream
targets. PHA-4 has many downstream targets (Gaudet
and Mango 2002), and it is unlikely that mutation of a
single target could restore viability to pha-4(ts) mutants.
We will determine if this prediction holds true in the
future, as the suppressors are analyzed at the molecular
and mechanistic levels.
Twenty-six suppressors from 15,000 haploid genomes
were obtained using the alkylating agent EMS as a mutagen (Tables 1 and 2). Twenty-nine more suppressors
from 1,160,000 haploid genomes were obtained using
the Mos1 transposon as a mutagen (Bessereau et al.
2001). Surprisingly, none of our transposon-induced
alleles carried a Mos1 insertion, despite robust positive
controls (see materials and methods). These alleles
likely represent either spontaneous or ‘‘hit-and-run’’
mutations by Mos1 (Williams et al. 2005).
We predicted we would isolate two classes of pha-4
suppressors. First, we expected informational suppressors, including mutations in NMD components. Second, we expected ‘‘true’’ pha-4 suppressors, including
regulators of pha-4 expression or modifiers of PHA-4
activity. We performed three tests to distinguish informational suppressors from true pha-4 suppressors. First,
we examined a natural target of NMD rpl-7a (Mitrovich
and Anderson 2000). This gene produces two mRNAs by
alternative splicing (Figure 2A). The larger isoform is
created by alternative splicing of exon 3, which introduces a stop codon and makes this mRNA an NMD target.
The smaller mRNA isoform, which lacks the entire third
C. elegans pha-4 Suppressors
823
TABLE 1
List of non-NMD pha-4 suppressors
Allele
px8
px11
px12
px16
px17
px23
px28
px31
px33
px34
px63
px70
px71
Strain
name
M/ Z/1 M1/1 Z/1 M/1 Z/1 Dominant/
Back
Suppress
alive1
alive1
alivea (%)
recessive
cross Mutagen lethalitya (%)
SM1150 43
SM1114 43
SM1591 53
SM1118 43
SM1569 43
SM1415 73
SM1430 43
SM1570 43
SM1108 43
SM1149 63
SM1134 103
SM1135 43
SM1133 43
EMS
EMS
EMS
EMS
EMS
EMS
EMS
EMS
EMS
EMS
Mos1
Mos1
Mos1
7
58
29
55
12
62
18
43
32
86d
10d
78
28
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
d
d
Yes
Yes
0
0
0
0
0
0
0
0
0
0
6
0
0
No
No
No
No
Yes
No
No
No
Yes
Yes
Yes
No
No
b
R
Rb
Rb
Rb
MatDc
Rb
Rb
Rb
MatDc
MatDc
ZygDe
Rb
Rb
Map
position
Locus
ND
IIIM
IVL
II
IIIM
IIR
IIR
ND
IIIM
V: 12.22 ruvb-1
V: 125.11 pha-4
ND
ND
None of these suppressors were components of the nonsense-mediated decay (NMD) pathway, according to rpl-7a mRNA analysis. None grew at 15° in combination with pha-4(ts), and none stained with an antibody that recognizes the carboxyl terminus of
PHA-4. , suppressor allele (note that it is unknown if a given allele is a loss of function). 1, scored as ‘‘yes’’ if there were at least
five viable progeny from #100 total. ND, no data.
a
Percentage viable at 20°, n ¼ 100. pha-4(ts) is 0% viable at 20°.
b
Maternal absence effect, recessive.
c
Maternal dominant.
d
px34 and px63 are homozygous lethal, so analysis was performed on heterozygotes.
e
Zygotic dominant.
intron, is not targeted by NMD. RT–PCR of rpl-7a from
WT animals amplifies primarily the smaller mRNA. In
animals defective for NMD, RT–PCR also amplifies the
larger transcript since this mRNA is now stable. rpl-7a RT–
PCR of the suppressors revealed that 16/26 EMS and
26/29 transposon-mutagenized suppressors were components of NMD, leaving 13 potential regulators of PHA-4
activity or expression (Figure 2B; Tables 1 and 2).
As a second test that these 13 suppressors were not
mutations in the NMD pathway or bypass mutations that
no longer required PHA-4, we examined suppression at
the more restrictive temperature of 15°. We reasoned
that mutations that completely inactivated the NMD
pathway would suppress pha-4(ts) at any temperature
(Kaltenbach et al. 2005). For each suppressor, we shifted
10 worms at L4 from 20° to 15°, and searched for progeny that were viable past L1. None of the 13 suppressors
were viable when grown at 15° (Table 1). These data
suggest that either we have isolated cold-sensitive lethal
alleles, which is unlikely, or none of the 13 suppressors
can bypass the requirement for some pha-4 activity.
These data also demonstrate that none of the 13 suppressors are null alleles of NMD components.
Third, we wanted to determine whether suppression
could be explained by readthrough of the pha-4(zu225)
or pha-4(q500) nonsense mutations. We used antibodies
that recognize an epitope located in the carboxyl terminus of WT PHA-4, but are absent from the truncated proteins made by pha-4(zu225) or pha-4(q500) (Kaltenbach
et al. 2005). We detected no staining with the PHA-4
carboxyl terminus antibody for any of the 13 pha-4 suppressors (Figure 2C). PHA-4 staining of par-1 mutant embryos (Kemphues et al. 1988) placed on the same slides
served as a positive control for staining (Figure 2C).
These results indicate that suppression of Pha-4 lethality cannot be explained by translational readthrough
of pha-4(zu225) or pha-4(q500) nonsense mutations. We
conclude that 13 of 55 pha-4 suppressors are not informational suppressors. We focus on the 13 pha-4 suppressors here; the NMD suppressors will be described
elsewhere.
pha-4 suppressors increase PHA-4 levels: We wanted
to investigate the effect of the suppressors on pha-4
expression. We compared levels of endogenous PHA-4
from pha-4(ts) and suppressed pha-4(ts) embryos at the
restrictive temperature of 20°. We measured the nuclear
accumulation of truncated PHA-4 protein in individual
nuclei, using a pan-PHA-4 antibody, and compared it to
PHA-4 in par-1 mutant embryos placed on the same slide
(our positive control). Levels were expressed as the ratio
between the query strain and par-1. In previous experiments, this approach was the most sensitive measure of
PHA-4 accumulation (Kaltenbach et al. 2005). Our
analysis revealed that PHA-4 accumulated to higher levels in sup; pha-4(ts) embryos compared to pha-4(ts) embryos (Figure 3). We observed a range of 1.6- to 2.2-fold
higher expression, which resembled the level of PHA-4
in pha-4(ts) embryos at permissive temperature (24°).
Surprisingly, however, we did not observe a strong correlation between PHA-4 levels and suppressor strength
824
D. L. Updike and S. E. Mango
TABLE 2
List of NMD suppressors
Allele
Strain name
Mutagen
Growth at 15°
px9
px10
px13
px14
px15
px19
px20
px21
px22
px24
px25
px26
px27
px29
px30
px32
px52
px53
px54
px55
px56
px57
px58
px59
px60
px61
px62
px64
px65
px66
px67
px68
px69
px72
px73
px74
px75
px76
px77
px78
px79
px80
SM579
SM580
SM583
SM584
SM585
SM600
SM601
SM604
SM610
SM612
SM613
SM621
SM622
SM624
SM625
SM635
SM701
SM702
SM703
SM704
SM705
SM706
SM780
SM781
SM785
SM786
SM787
SM827
SM828
SM866
SM867
SM876
SM949
SM956
SM957
SM958
SM959
SM961
SM964
SM965
SM966
SM967
EMS
EMS
EMS
EMS
EMS
EMS
EMS
EMS
EMS
EMS
EMS
EMS
EMS
EMS
EMS
EMS
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Mos1
Yes
No
Yes
No
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
No
No
NMD was established by rpl-7a RT–PCR.
(Table 1), which may reflect regulation of PHA-4 activity
as well as levels for suppression. For example, px8 embryos had high levels of PHA-4 but poor suppression,
whereas px34 mutants had relatively lower levels of PHA4 but strong suppression (Figure 3; Table 1). These data
reveal that the 13 suppressors lead to a modest increase
in PHA-4 accumulation at restrictive temperature. We
note that this experiment did not address whether the
increase in PHA-4 was direct or indirect.
Four dominant pha-4 suppressors: All 13 suppressors
½i.e., sup; pha-4(ts) or sup/1;pha-4(ts) had viable progeny
when crossed with pha-4(ts) males at 20°, indicating that
pha-4 suppression was either dominant (gain-of-function
or haploinsufficient) or subject to a maternal-absence
effect. To distinguish between these possibilities, heterozygous sup/1;pha-4(ts) hermaphrodites were tested
for their ability to produce viable F2 progeny at 20°. Four
heterozygous suppressors (px17, px33, px34, and px63)
generated viable F2 progeny at 20°, indicating these four
alleles were dominant (Table 1). Five of the remaining
suppressors (px11, px16, px31, px70, and px71) had dead
F2 progeny, indicating that suppression required both
maternal copies of the suppressing gene be mutated
(Table 1). F1 heterozygotes for the final four suppressors
(px8, px12, px23, and px28) were sterile and gave no F2
progeny. The sterility of the heterozygotes may reflect
poor feeding, due to altered pharyngeal development.
Alternatively, gonad formation may require pha-4 activity directly during postembryonic development (Figure
4). First stage larvae do not express PHA-4TGFP in their
gonads (data not shown), and L1 animals with pha-4(null)
mutations are born with a normal gonad primordium
(Mango et al. 1994). However, we observed that as larval
development progressed, PHA-4TGFP (Alder et al. 2003)
was activated in the developing gonad (Figure 4A; Kalb
et al. 1998; Gaudet et al. 2004) and was required for its
proper formation (Figure 4B; Ao et al. 2004). A shift of
pha-4(ts) larvae to nonpermissive temperature (20°) at
the L1 stage produced sterile adults (Figure 4B). A shift
to nonpermissive temperature (15°) at the pretzel stage
of embryogenesis generated larvae that arrested at the
L2 or L3 stage, with appropriately undeveloped gonads
(data not shown). These data indicate that pha-4 is required to form the gonad, and that px8, px12, px23, and
px28 heterozygotes cannot rescue the gonad defects of
pha-4(ts).
To determine whether the suppressors could suppress pha-4 lethality when contributed paternally, 20
males from each suppressor ½sup; pha-4(ts) or for px34
and px63, sup/1;pha-4(ts) were crossed to 10 pha-4(ts) L4
hermaphrodites at the restrictive temperature of 20°.
Only px63/1 males gave rise to viable F1 progeny, suggesting that suppression by px63 was contributed zygotically, whereas the remaining three dominant suppressors
(px17, px33, and px34) required a maternal contribution. We note that zygotic suppression did not track with
overall strength of suppression. px63 M1/ Z1/ gave
one of the lowest suppression rates (10%), yet this allele
could still suppress as M1/1 Z1/. px34 M1/ Z1/,
on the other hand, was one of the strongest suppressors,
yet M1/1 Z1/ did not suppress pha-4(ts). These
behaviors indicate that suppression reflected maternal
vs. zygotic contributions rather than overall strength of
suppression.
Location of pha-4 suppressors: We were able to assign
a linkage group (LG) to 9 of the 13 suppressors using
single nucleotide polymorphisms (SNPs). Mapping
data from the 9 suppressors revealed that at least six
different loci were involved.
C. elegans pha-4 Suppressors
825
Figure 2.—Identification of informational suppressors. (A) rpl-7a encodes two transcripts, the
longer of which contains a premature nonsense codon and is degraded by NMD (Mitrovich and
Anderson, 2000). (B) PCR amplification of rpl-7a
cDNA from mutant strains identifies when NMD
is compromised and the longer mRNA accumulates ½smg-1(null) and suppressors px21, px26, px27,
and px29. WTstrains exhibit normal NMD activity.
NMD activity is partially restored in the remaining
strains grown at 20° ½smg-1(ts) and suppressors px33
and px34 (see also Kaltenbach et al. 2005). (C)
pha-4 suppression is not caused by readthrough of
carboxyl-terminal nonsense alleles. Red, a-PHA-4
carboxy-terminal antibody (Horner et al. 1998);
green, P-granule costain (arrowhead) (Strome and
Wood 1982). par-1 embryos have excess pharyngeal
cells (Kemphues et al. 1988) and were used as an
on-slide control for PHA-4 staining. The aberrant
morphology of par-1 embryos allowed us to distinguish control embryos from sup; pha-4(ts) embryos.
LG II: px23 and px28 mapped to the same location on
the far right arm of chromosome II (see materials
and methods). When grown at the restrictive temperature of 20°, px23/px28; pha-4(ts) heterozygotes
produced viable progeny, unlike px23/1; pha-4(ts) or
px28/1; pha-4(ts) mothers ½progeny scored from 10
px23/px28, px23/1 or px28/1 animals in the pha-4(ts)
background. Thus, px23 and px28 fail to complement
each other for suppression of pha-4(ts) lethality and
are likely alleles of the same gene. A third suppressor,
px16, also mapped to chromosome II with a recessive
segregation ratio, but was not linked to the far right
arm, indicating it was distinct from px23 and px28.
None of the three alleles had an obvious phenotype
when the pha-4 and smg-1 mutations were removed
(data not shown).
LG III: The alleles px11, px17, and px33 mapped to the
center of chromosome III. Both px17 and px33 were
maternally dominant, whereas px11 was recessive. The
dominant attributes of px17 and px33 prevented
further complementation analysis. These three genes
may comprise an allelic series for one locus or represent individual genes located near each other. None
of the three genes had an obvious phenotype when
the pha-4 and smg-1 mutations were removed (data
not shown).
LG IV: px12 was the only suppressor that mapped to the
left end of chromosome IV. px12 exhibited a recessive
segregation ratio, where homozygous but not heterozygous px12 animals gave rise to viable F3 progeny.
px12 did not reveal an obvious phenotype when the
pha-4 and smg-1 mutations were removed (data not
shown).
LG V: SNP mapping was used to position px63 to the
right arm of chromosome V. Homozygous px63 pha4(ts) animals from balanced smg-1; pha-4(px63 zu225)/
fog-2 rol-9 heterozygotes arrested as late-stage embryos
or L1 larvae with a pharynx unattached (Pun) phenotype (Figure 5A).
px34 mapped to the center of chromosome V. Suppression was vigorous, as 90% of progeny from smg-1;
ruvb-1(px34) pha-4/1 pha-4 mothers survived at 20°,
compared to 0% in the absence of px34 (Table 3). Our
data suggest that in addition to being dominant, px34
suppression is contributed maternally since .90% of
progeny from a px34 heterozygous mother survived.
Intriguingly, homozygous px34 animals arrested as
larvae even in the absence of smg-1 or pha-4, suggesting the px34 mutation disrupts an essential gene (data
not shown).
Additional alleles: To date, we have not assigned a
linkage group for px8, px31, px70, or px71. Our difficulty
may reflect where these genes are located. To map
suppression, the mutant alleles smg-1(cc546ts) and pha4(zu225) from C. elegans Bristol were introduced into the
Hawaiian CB4856 strain. The resulting Bristol/Hawaiian
chimera was crossed with Hawaiian C. elegans 10 times to
remove as much Bristol DNA as possible. Nevertheless,
SNPs at I: 6 and I: 1, which are near the smg-1 locus
on chromosome I, still harbored DNA from the Bristol
isolate (data not shown). Thus, suppressors that map to
the central region of chromosome I or to the far right
end of chromosome V (near pha-4), would elude SNP mapping using our pha-4(ts) Hawaiian mapping strain.
px63 is an allele of pha-4: The location of px63 on the
right arm of chromosome V, near pha-4 itself, suggested
that px63 might be an allele of pha-4. As a first test of this
idea, we attempted to exchange the pha-4(zu225) allele
of smg-1; pha-4(zu225) px63/pha-4(zu225) with pha-4(q500),
another pha-4 nonsense allele targeted by NMD. We
observed that the pha-4(zu225) allele could not be
exchanged with pha-4(q500) rol-9 (n ¼ 0/96 non-Rol F2
offpsring). This result suggested that either suppression
826
D. L. Updike and S. E. Mango
Figure 3.—PHA-4 restoration in sup; pha-4(ts) strains. (A)
px34 increases endogenous PHA-4 levels in the pha-4(ts) background at 20°. pan-PHA-4 antibody (red) (Kaltenbach et al.
2005). (B) PHA-4 levels increase in pha-4(ts) strains at 20° in
the presence of each of the pha-4 suppressors. PHA-4 was
quantified in individual nuclei as described in materials
and methods ½P # 0.0072 for suppressor strain compared
to pha-4(ts) alone. The ratio of PHA-4 in the suppressed strain
vs. a par-1 control is shown.
was specific for the pha-4(zu225) allele or px63 was very
closely linked to the pha-4 locus.
To determine whether px63 was an allele of pha-4, we
sequenced the pha-4 locus from smg-1(cc546ts); pha4(px63 zu225)/pha-4(zu225) animals (Figure 5C). We
verified that the zu225 stop codon had not reverted to
enable suppression, and we also found a 156-bp deletion
in the second intron of pha-4 (Figure 5, B and C). In
addition to the deletion, Southern blotting revealed an
insertion of $5.3 kb between the 39 end of intron 2 and
the middle of exon 5 (Figure 5, B and D). Taken together, these results suggest that px63 is an allele of pha-4
that contains a complex rearrangement in the 39 end of
intron 2 and possibly other regions as well.
The pha-4 gene produces three transcripts that initiate at exons 1, 2, and 3 and that generate three inframe proteins (Figure 5B) (Azzaria et al. 1996). The
rearrangement associated with px63 affected the second
intron, which could possibly function as a promoter for
the third, most abundant mRNA (Azzaria et al. 1996).
We predict that the rearrangement associated with pha4(px63) either disrupts a negative regulatory element
or introduces a positive element within the third pha-4
promoter. The identification of a pha-4 cis-acting mutation validated our suppressor screen as an effective
method for finding regulators of pha-4.
px34 is an allele of the predicted AAA1 helicase ruvb-1:
px34 was positioned to within 220 kb on chromosome
V (V: 9906715 . . . 10124252) using standard SNP mapping (Figure 6A) (Wicks et al. 2001). Each of 10 cosmids
spanning the 220-kb region were injected into dpy-11
unc-42 px34/evl-1 hermaphrodites and scored for rescue
of lethality. Of the 10 cosmids, R05D5 and T10A5 could
each rescue px34, implicating one of the five genes
common to both of these cosmids (see materials and
methods). One of these five genes, the predicted helicase ruvb-1 (C27H6.2) was associated with a larval arrest
phenotype, suggesting ruvb-1 was a good candidate for
px34 (Piano et al. 2002; Simmer et al. 2003).
Two lines of evidence revealed that px34 was an allele
of ruvb-1. First, a 3777-bp fragment, which contained the
ruvb-1 locus with 677 bp of 59 upstream sequences, could
rescue the lethality associated with px34 larvae (Figure
6A; materials and methods). Second, we detected a
C-to-T transition within ruvb-1 that was predicted to
change the conserved glutamate at position 371 to a
lysine (Figure 6B). This mutation was located after the
conserved Walker A and Walker B motifs required for
ATP binding of the helicase and four residues before
a conserved Arginine finger motif thought to regulate
ATP hydrolysis and DNA binding (Putnam et al. 2001;
Ohnishi et al. 2005; Matias et al. 2006). These data
demonstrate that px34 is an allele of ruvb-1.
Four experiments suggested that ruvb-1(px34) was a
loss-of-function mutation. First, RNAi against ruvb-1
could suppress the lethality of pha-4(ts) animals reared
at 20°, demonstrating that reduced ruvb-1 was responsible for pha-4 suppression (Table 3). Similarly, RNAi
against ruvb-2 could suppress pha-4(ts) lethality (Table
3). In other organisms, orthologs of RUVB-1 and RUVB2 bind each other and function as a hexameric helicase
(Ikura et al. 2000; Shen et al. 2000; Wood et al. 2000;
Fuchs et al. 2001; Krogan et al. 2003; Doyon et al. 2004;
Mizuguchi et al. 2004; Jin et al. 2005; Bakshi et al. 2006).
Second, some ruvb-1(RNAi) animals arrested as larvae
and resembled ruvb-1(px34) homozygotes (Simmer et al.
2003; Piano et al. 2002). Third, when ruvb-1(px34)/unc42(e270) sqt-3(sc63) males were crossed to sDf35/1 heterozygotes, 24% (17/71) of the progeny arrested as
C. elegans pha-4 Suppressors
827
Figure 4.—PHA-4 is required in the developing gonad. (A) PHA-4TGFP is broadly expressed
in the somatic gonad of a third stage larval worm
(asterisk, left). Arrows denote the distal tip cells.
PHA-4TGFP is low or undetectable in the gonads
of young adult animals (right) and older L4 larvae (not shown). Some epifluorescence derives
from the intestine. (B) Somatic gonad development after 2 days of growth at 20° from the L1.
Somatic gonad of a WT worm (top). A sterile
pha-4(ts) adult with abnormal somatic gonad development (bottom). Arrowhead denotes the
vulva.
larvae, with a phenotype resembling that of ruvb-1(px34).
sDf35 removes the ruvb-1 region (Mckim et al. 1988).
Fourth, we observed an identical larval arrest for a
second ruvb-1 allele, tm2786, which contained a deletion
in exons 4 and 5 (Figure 6A). A total of 23.5% of progeny from ruvb-1(tm2786)/unc-42(e270) sqt-3(sc63) mothers arrested, indicating a zygotic larval arrest phenotype
(n ¼ 251). These data indicate that the ruvb-1 E371K
mutation causes a loss-of-function in ruvb-1 (see also
discussion).
Restoration of pharyngeal cells by ruvb-1(px34): PHA-4
is required during embryogenesis to form the pharynx
(Mango et al. 1994; Kiefer et al. 2007), suggesting that the
pha-4 suppressors function embryonically to restore pharynx development. To explore this idea, we examined one
suppressor ½ruvb-1(px34) in greater detail. At 20°, 82%
(n ¼ 50) of pha-4(ts) embryos had an unattached (Pun)
pharynx, compared to 0% (n ¼ 50) for embryos from a
smg-1; ruvb-1 pha-4/1 pha-4 mother (Figure 7, A and B).
We reasoned that the pharyngeal phenotype associated
with pha-4(ts) could reflect either fewer pharyngeal cells
or defective pharyngeal morphology (Mango 2007). To
distinguish between these possibilities, embryos were
stained with a pan-a-PHA-4 antibody, and pharyngeal
cells counted at the 1.5-fold stage of embryogenesis. The
number of pharyngeal cells was reduced for pha-4(ts)
worms incubated at 20° (76 6 10.21, n ¼ 12) compared to
24° (90.5 6 2.35, n ¼ 6) (Figure 7C). In embryos from
smg-1; ruvb-1 pha-4/1 pha-4 mothers, the number of pharyngeal cells at 20° was restored to the WT level (91.8 6
2.40, n ¼ 11). These data indicate that suppression of
Pha-4 lethality by ruvb-1(px34) is embryonic and affects
accumulation of PHA-4. We note that we did not observe
ectopic expression of the ectodermal factor LIN-26
(Labouesse et al. 1996) within presumptive pharyngeal
cells in either pha-4(ts) or smg-1; ruvb-1 pha-4/1 pha-4
embryos (data not shown). This result differs from pha-4
null embryos, which express ectopic LIN-26 within cells
that would normally become pharyngeal (Horner et al.
1998; Kiefer et al. 2007).
ruvb-1 is expressed in the pharynx during embryogenesis: To monitor zygotic expression of ruvb-1, we
designed a transcriptional, nuclear GFP reporter driven
by 677 bp of ruvb-1 promoter (Figure 8A). This construct included all bases between the ruvb-1 ATG and
the upstream gene C27H6.3, which corresponds to the
sequences used in the ruvb-1 rescuing construct. We
observed expression in most cells of the early embryo,
but as development progressed to the twofold stage,
expression became restricted to pharyngeal cells (Figure 8B). We also observed GFP in two intestinal cells
near the center of larval stage worms, which could
828
D. L. Updike and S. E. Mango
Figure 5.—px63 is a complex rearrangement
in pha-4. (A) In px63 homozygotes, the pharynx
is differentiated, but unattached (arrowheads).
(B) Overview of the pha-4 locus, the 156-bp deletion in the second intron (red), and the probe
used for Southern blotting (green). (C) 156-bp
deletion (red) in intron 2. Sequence numbers
correspond to the Z92633 GeneBank entry. (D)
Southern blot comparing WT and px63/1 genomic DNA. A probe internal to the XbaI sites revealed an insertion of at least 5.3 kb
(arrowheads).
reflect artifactual intestinal expression (Figure 8C)
(Reece-Hoyes et al. 2007). The expression of ruvb-1 in
pharyngeal cells supports its role as a regulator of
pharyngeal development.
DISCUSSION
Selector genes specify regional, tissue, organ, and cell
identity in processes that require the precise temporal
and spatial expression of target genes. In this study, we
used a forward genetic screen to identify potential regulators or cofactors for the selector gene pha-4/FoxA. We
isolated 55 mutants, of which 13 were true pha-4 suppressors and the remainder were likely NMD compoTABLE 3
ruvb-1(px34) suppression of pha-4
Genotype of parent
pha-4(ts) 20°
smg-1; ruvb-1 pha-4/1 pha-4 20°
GFP(RNAi); pha-4(ts) 20°
ruvb-1(RNAi) pha-4(ts) 20°
ruvb-2(RNAi) pha-4(ts) 20°
pha-4(RNAi)a
ruvb-1(px34)/1; pha-4(RNAi)a
Viable . L1 SD
(%)
(%) Replicates
0.0
90.0
0.9
48.8
47.6
14.7
56.4
0.0
4.3
2.1
9.0
4.8
6.5
14.0
5
5
5
5
5
6
6
SD, standard deviation.
a
1:4 ratio of pha-4(RNAi) to GFP(RNAi) for weak pha4(RNAi).
nents. The 13 suppressors represented at least six different
loci and included rare dominant alleles, as well as recessive alleles that exhibited a maternal-absence effect.
Previous studies have identified genes that functioned
upstream of pha-4 in either the ABa or EMS cell lineage
(Mango et al. 1994). The screen described here complements these previous approaches since it likely required
activation of pha-4-dependent processes in both ABaand EMS-derived cells.
The recessive suppressors px23 and px28 failed to
complement each other and likely define the same
gene. Supporting this conclusion, both map to the same
area on the far right arm of chromosome II. px16 also
maps to chromosome II, but shows no preference for
the right arm of the chromosome. A potential allelic
series mapped to the center of chromosome III and
consisted of px11, px17, and px33. px12 was the only
suppressor to map to the far left arm of chromosome IV.
We were unable to assign a linkage group to px8, px31,
px70, or px71, which may reflect the SNP mapping
strain. With the exception of ruvb-1(px34) and pha4(px63), we did not observe obvious phenotypes other
than suppression of pha-4(ts) lethality.
PHA-4 is first expressed at the 28–50 cell stage of
embryogenesis (Horner et al. 1998). Given this early
expression, we were not surprised to uncover a maternal contribution for the nine recessive suppressors
and three of the four dominant suppressors (px17,
px33, and px34). Genes affecting the activity or expression of PHA-4 might need to be present during early
C. elegans pha-4 Suppressors
829
Figure 6.—Identification of
px34 within the ruvb-1 locus.
(A) px34 was mapped to the center of LG V. Rescuing cosmids
R05D5 and T10A5 overlap ruvb-1.
A PCR fragment (green) containing ruvb-1 was used to rescue the
larval arrest phenotype. (B) Alignment of ruvb-1 with its orthologs.
The E371K mutation associated
with px34 (arrow) is located just
upstream of the Arginine finger.
(C) The E371K mutation is positioned at the predicted interface
between RuvB molecules. The position of the C. elegans ruvb-1 E371K
has been superimposed on the
human RuvbL1 structure adapted
from Matias et al. (2006).
embryogenesis, which could be accomplished by a
maternal contribution. The only suppressor that clearly
did not have a maternal requirement was px63, which
was a mutation in the pha-4 locus. pha-4 loss-of-function
mutations are also zygotic (Mango et al. 1994).
The px63 mutation was a complex rearrangement in a
likely regulatory region of pha-4. There are three transcriptional initiation sites for pha-4, which produce three
mRNAs and three PHA-4 proteins (Azzaria et al. 1996).
The location of the px63 rearrangement is predicted to
affect the third and strongest initiation site, but could
also affect the two upstream promoters. We note that all
sequence conservation between FoxA proteins and
PHA-4 is located within the third open reading frame
(Azzaria et al. 1996). Thus, the transcript and protein
derived from the third promoter, and affected by px63,
appear to be critical for PHA-4 function.
px63 was generated by Mos1 transposon mutagenesis
(Bessereau et al. 2001), yet we were unable to detect
Mos1 sequences within pha-4. Mos1-mediated mutagenesis can create deletions where the transposon inserts
into a region of DNA and then excises, leaving a nucleotide footprint (Williams et al. 2005). It is also possible
that repair of a double-strand break created by Mos1
830
D. L. Updike and S. E. Mango
Figure 7.—ruvb-1(px34) suppression of pha-4(ts) restores
pharyngeal cells. (A) Suppression of the pha-4(ts) Pun phenotype by ruvb-1(px34). Green, 3NB12 antibody, which stains
pharynx muscle (Priess and Thomson 1987). (B) Severity
of pha-4(ts) pharyngeal defects is suppressed by ruvb-1(px34)
(n ¼ 50 threefold embryos for each strain; Pun, pharynx unattached). (C) Reduction of pharyngeal cell number (PHA-41
cells within the head) in pha-4(ts) at 20° is suppressed by
ruvb-1(px34).
transposase or insertion of a non-Mos1 substrate could
be responsible for creating a complex rearrangement.
We note that the strain we used harbored an integrated
copy of the transposase but an extrachromosomal array of the transposase substrate (i.e., the transposon).
Transposase in worms, including worms lacking the
extrachromosomal array, might target genomic DNA
at low frequency, and our selection scheme would
have enabled these rare events to be detected.
The frequencies at which we obtained suppressors
by EMS mutagenesis suggest that many of our suppressors represent unusual loss-of-function mutations or
rare gain-of-function mutations. Typically, null mutations
Figure 8.—ruvb-1 expression. (A) Design of ruvb-1 transcriptional GFP reporter. (B) ruvb-1 transcriptional GFP reporter is expressed in most cells of the early embryo, but
becomes restricted to pharyngeal cells during late embryogenesis. (C) ruvb-1 transcriptional GFP reporter is frequently
expressed in two intestinal cells during larval development.
are generated at a frequency of 1/2000 haploid genomes (Jorgensen and Mango 2002). If a typical lossof-function mutation were able to suppress pha-4(ts)
lethality, we would have obtained approximately seven
alleles/gene from EMS mutagenesis of 15,000 haploid
genomes. One explanation for our low frequency is that
our screen selected for viability. If regulators of pha-4
are essential, we would not be able to isolate null alleles. This phenomenon may explain the ratio of NMD
mutations vs. pha-4 suppressors in EMS vs. Mos1-mediated mutagenesis. Only 3 of 29 suppressors (10%)
from the Mos1 transposon mutagenesis were pha-4 suppressors, compared to 10 of 26 (40%) from the EMS
mutagenesis (P , 0.02, Fisher’s exact test). Unlike EMS
mutagenesis, which typically creates point mutations,
Mos1 transposon creates insertions or deletions, which often have stronger loss-of-function phenotypes (Williams
et al. 2005). For example, smg-4 was identified at a low
C. elegans pha-4 Suppressors
frequency by traditional genetic screens using EMS
(Hodgkin et al. 1989), and smg-4(r1169) is a large deletion (Aronoff et al. 2001). An alternative explanation
for the high frequency of NMD alleles by Mos mutagenesis is that Mos1, but not EMS, could inactivate pairs of
redundant, linked NMD genes, such as those found in
operons.
Role of RuvB proteins in development: A second
dominant suppressor was associated with the predicted
helicase ruvb-1. RUVB-1 belongs to the AAA1 ATPase
class of helicases represented by RuvB in bacteria and
RUVBL1 in mammals (e.g., TIP49a, TAP54a, Pontin52)
(Kanemaki et al. 1997; Bauer et al. 1998; Ikura et al.
2000). We isolated a single allele of ruvb-1, px34, which
carried a lysine substitution for glutamate at position
371; we obtained no alleles of ruvb-2. A comparison of
C. elegans ruvb-1 with the crystal structure for human
RuvbL1 (Matias et al. 2006) suggests that the px34 mutation lies at the interface between RUVB molecules
within the hexameric complex (see Figure 6C). Intriguingly, multiple dominant-negative alleles of RuvB have
been generated in bacteria, and these map to the interface between two RuvB molecules in the hexameric
ring (Iwasaki et al. 2000; Putnam et al. 2001). These observations suggest that the lysine substitution of RUVB-1
may disrupt the formation or stability of the hexameric
ring, despite the presence of WT RUVB-1 and RUVB-2
proteins. Thus, this mutation may be a weak dominant
negative and not a simple loss of function. Consistent
with this hypothesis, we observed dominant suppression
of pha-4 lethality by ruvb-1(px34).
The role of RuvB in bacteria is DNA repair and recombination, where RuvB is required to translocate the
Holliday junction along DNA (West 1996). Eukaryotes
posses a RuvB homolog but not RuvA or RuvC, raising
the question of what RuvB proteins do in higher organisms. Expression of ruvb-1 orthologs in Drosophila,
Xenopus, zebrafish, and mice suggests a possible role
in gut development. Strong expression of Pontin/ruvb-1
and Reptin/ruvb-2 is found in the anterior and posterior
midgut of Drosophila (Bauer et al. 2000). In Xenopus,
xPontin/ruvb-1 is expressed in different gastrointestinal
organs (Etard et al. 2000). Zebrafish express higher
levels of zReptin/ruvb-2 in the heart, brachial arches,
liver, exocrine pancreas, and intestine (Rottbauer et al.
2002). In mice, robust expression of mPontin/ruvb-1 and
mReptin/ruvb-2 is found in the gut and pancreas (Chauvet
et al. 2005). The pharyngeal expression pattern we observed in C. elegans, together with the expression of RuvB
genes in Drosophila, Xenopus, zebrafish, and mice, suggest an evolutionarily conserved role for RuvB in gut
development.
It has been proposed that the paralogs ruvb-1/Pontin/
Tip49 and ruvb-2/Reptin/Tip48 have antagonistic roles
for wingless signaling in Drosophila and zebrafish
(Bauer et al. 2000; Rottbauer et al. 2002). In contrast,
Xenopus Xpontin and Xreptin appear synergistic (Etard
831
et al. 2005). Our results resemble those of Xenopus,
since both ruvb-1 and ruvb-2 negatively regulate pha-4
and pharyngeal development (Table 3). Biochemical
assays suggest that RUVB-1 and RUVB-2 are present
together in multiprotein complexes in other organisms
(Ikura et al. 2000; Shen et al. 2000; Wood et al. 2000;
Fuchs et al. 2001; Krogan et al. 2003; Doyon et al. 2004;
Mizuguchi et al. 2004; Jin et al. 2005; Bakshi et al. 2006).
Direct interaction between these paralogs has been
reported in multiple systems, most likely in the form
of a dodecameric RUVB-1/2 ring (Puri et al. 2007). This
configuration may help explain why ruvb-1 and ruvb-2
have synergistic roles for Xenopus and C. elegans development (Gohshi et al. 1999; Kanemaki et al. 1999;
Bauer et al. 2000; Ikura et al. 2000; Wood et al. 2000;
Walhout et al. 2002; Puri et al. 2007).
Biochemical studies have identified RuvB orthologs
as members of several multiprotein complexes including those that control DNA repair (RuvABC complex,
TIP60/SRCAP, and URI-1), transcription ½c-Myc, TIP60/
SRCAP, and Polycomb repressive complex 1 (PRC1),
and ribosome biogenesis (snoRNP assembly complex)
(Ikura et al. 2000; Shen et al. 2000; Fuchs et al. 2001;
Jonsson et al. 2001; Frank et al. 2003; Krogan et al. 2003;
Doyon et al. 2004; Kobor et al. 2004; Jonsson et al. 2004;
Taubert et al. 2004). How might these interactions
explain a role for ruvb-1/2 during pharyngeal development? Vertebrate FoxA binds chromatin and modulates
compaction in vitro, indicating FoxA factors function
by modifying the chromatin environment (Cirillo et al.
2002). Previous work from our lab has shown that homologs of the TIP60/SRCAP histone acetyltransferase
and nucleosome-remodeling complex cooperate with
pha-4 to achieve the proper timing and expression of
pharyngeal genes (Updike and Mango 2006). Therefore, one possibility is that ruvb-1/2 modulates the
activity of the TIP60/SRCAP complex. PRC1 is another
attractive candidate, however C. elegans lacks an obvious
correlate to this complex. We favor the notion that ruvb-1
and ruvb-2 modulate pha-4 activity through interactions
with the chromatin environment.
We thank Dan Williams, Wayne Davis, Marc Hammarlund, and Erik
Jorgensen for sharing unpublished information; Jeb Gaudet and
numerous rotation students for help with the genetics; and Michel
Labouesse and the National Bioresource Project for reagents. Some
strains used in this work were obtained by the Caenorhabditis Genetics
Center. This work was supported by T32 HD007491 to D.L.U. S.E.M.
was supported by National Institutes of Health R01 GM056264, the
Huntsman Cancer Institute, and the Department of Oncological
Sciences.
LITERATURE CITED
Alder, M. N., S. Dames, J. Gaudet and S. E. Mango, 2003 Gene
silencing in Caenorhabditis elegans by transitive RNA interference.
RNA 9: 25–32.
Ang, S. L., and J. Rossant, 1994 HNF-3 beta is essential for node
and notochord formation in mouse development. Cell 78:
561–574.
832
D. L. Updike and S. E. Mango
Ao, W., J. Gaudet, W. J. Kent, S. Muttumu and S. E. Mango,
2004 Environmentally induced foregut remodeling by PHA-4/
FoxA and DAF-12/NHR. Science 305: 1743–1746.
Aronoff, R., R. Baran and J. Hodgkin, 2001 Molecular identification of smg-4, required for mRNA surveillance in C. elegans. Gene
268: 153–164.
Azzaria, M., B. Goszczynski, M. A. Chung, J. M. Kalb and J. D.
McGhee, 1996 A fork head/HNF-3 homolog expressed in
the pharynx and intestine of the Caenorhabditis elegans embryo.
Dev. Biol. 178: 289–303.
Bakshi, R., A. K. Mehta, R. Sharma, S. Maiti, S. Pasha et al.,
2006 Characterization of a human SWI2/SNF2 like protein
hINO80: demonstration of catalytic and DNA binding activity.
Biochem. Biophys. Res. Commun. 339: 313–320.
Bauer, A., S. Chauvet, O. Huber, F. Usseglio, U. Rothbacher
et al., 2000 Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity. EMBO J 19: 6121–6130.
Bauer, A., O. Huber and R. Kemler, 1998 Pontin52, an interaction
partner of beta-catenin, binds to the TATA box binding protein.
Proc. Natl. Acad. Sci. USA 95: 14787–14792.
Beanan, M. J., and S. Strome, 1992 Characterization of a germ-line
proliferation mutation in C. elegans. Development 116: 755–766.
Bessereau, J. L., A. Wright, D. C. Williams, K. Schuske, M. W. Davis
et al., 2001 Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line. Nature 413: 70–74.
Brenner, S., 1974 The genetics of Caenorhabditis elegans. Genetics
77: 71–94.
Carroll, J. S., X. S. Liu, A. S. Brodsky, W. Li, C. A. Meyer et al.,
2005 Chromosome-wide mapping of estrogen receptor binding
reveals long-range regulation requiring the forkhead protein
FoxA1. Cell 122: 33–43.
Chauvet, S., F. Usseglio, D. Aragnol and J. Pradel, 2005 Analysis
of paralogous pontin and reptin gene expression during mouse
development. Dev. Genes Evol. 215: 575–579.
Cirillo, L. A., F. R. Lin, I. Cuesta, D. Friedman, M. Jarnik et al.,
2002 Opening of compacted chromatin by early developmental
transcription factors HNF3 (FoxA) and GATA-4. Mol. Cell 9: 279–
289.
Davis, M. W., M. Hammarlund, T. Harrach, P. Hullett, S. Olsen
et al., 2005 Rapid single nucleotide polymorphism mapping in
C. elegans. BMC Genomics 6: 118.
Doyon, Y., W. Selleck, W. S. Lane, S. Tan and J. Cote, 2004 Structural and functional conservation of the NuA4 histone acetyltransferase complex from yeast to humans. Mol. Cell Biol. 24:
1884–1896.
Dufort, D., L. Schwartz, K. Harpal and J. Rossant, 1998 The
transcription factor HNF3beta is required in visceral endoderm
for normal primitive streak morphogenesis. Development 125:
3015–3025.
Etard, C., D. Gradl, M. Kunz, M. Eilers and D. Wedlich,
2005 Pontin and Reptin regulate cell proliferation in early Xenopus embryos in collaboration with c-Myc and Miz-1. Mech. Dev.
122: 545–556.
Etard, C., D. Wedlich, A. Bauer, O. Huber and M. Kuhl, 2000 Expression of Xenopus homologs of the beta-catenin binding protein pontin52. Mech. Dev. 94: 219–222.
Frank, S. R., T. Parisi, S. Taubert, P. Fernandez, M. Fuchs et al.,
2003 MYC recruits the TIP60 histone acetyltransferase complex
to chromatin. EMBO Rep. 4: 575–580.
Friedman, J. R., and K. H. Kaestner, 2006 The Foxa family of transcription factors in development and metabolism. Cell. Mol. Life
Sci. 63: 2317–2328.
Fuchs, M., J. Gerber, R. Drapkin, S. Sif, T. Ikura et al., 2001 The
p400 complex is an essential E1A transformation target. Cell 106:
297–307.
Gaudet, J., and S. E. Mango, 2002 Regulation of organogenesis by
the Caenorhabditis elegans FoxA protein PHA-4. Science 295:
821–825.
Gaudet, J., S. Muttumu, M. Horner and S. E. Mango, 2004 Wholegenome analysis of temporal gene expression during foregut development. PLoS Biol. 2: e352.
Gengyo-Ando, K., and S. Mitani, 2000 Characterization of mutations induced by ethyl methanesulfonate, UV, and trimethylpsoralen in the nematode Caenorhabditis elegans. Biochem. Biophys.
Res. Commun. 269: 64–69.
Gohshi, T., M. Shimada, S. Kawahire, N. Imai, T. Ichimura et al.,
1999 Molecular cloning of mouse p47, a second group mammalian RuvB DNA helicase-like protein: homology with those
from human and Saccharomyces cerevisiae. J. Biochem. 125: 939–
946.
Grimson, A., S. O’Connor, C. L. Newman and P. Anderson,
2004 SMG-1 is a phosphatidylinositol kinase-related protein kinase required for nonsense-mediated mRNA Decay in Caenorhabditis elegans. Mol. Cell. Biol. 24: 7483–7490.
Hodgkin, J., A. Papp, R. Pulak, V. Ambros and P. Anderson, 1989 A
new kind of informational suppression in the nematode Caenorhabditis elegans. Genetics 123: 301–313.
Horner, M. A., S. Quintin, M. E. Domeier, J. Kimble, M. Labouesse
et al., 1998 pha-4, an HNF-3 homolog, specifies pharyngeal
organ identity in Caenorhabditis elegans. Genes Dev. 12: 1947–
1952.
Ikura, T., V. V. Ogryzko, M. Grigoriev, R. Groisman, J. Wang et al.,
2000 Involvement of the TIP60 histone acetylase complex in
DNA repair and apoptosis. Cell 102: 463–473.
Iwasaki, H., Y. W. Han, T. Okamoto, T. Ohnishi, M. Yoshikawa,
et al., 2000 Mutational analysis of the functional motifs of RuvB,
an AAA1 class helicase and motor protein for holliday junction
branch migration. Mol. Microbiol. 36: 528–538.
Jin, J., Y. Cai, T. Yao, A. J. Gottschalk, L. Florens et al., 2005 A
mammalian chromatin remodeling complex with similarities to
the yeast INO80 complex. J. Biol. Chem. 280: 41207–41212.
Jonsson, Z. O., S. K. Dhar, G. J. Narlikar, R. Auty, N. Wagle et al.,
2001 Rvb1p and Rvb2p are essential components of a chromatin remodeling complex that regulates transcription of over 5%
of yeast genes. J. Biol. Chem. 276: 16279–16288.
Jonsson, Z. O., S. Jha, J. A. Wohlschlegel and A. Dutta,
2004 Rvb1p/Rvb2p recruit Arp5p and assemble a functional
Ino80 chromatin remodeling complex. Mol. Cell. 16: 465–477.
Jorgensen, E. M., and S. E. Mango, 2002 The art and design of genetic screens: caenorhabditis elegans. Nat. Rev. Genet. 3: 356–369.
Jurgens, G., and D. Weigel, 1988 Terminal versus segmental development in the Drosophila embryo: the role of the homeotic gene
fork head. Rouxs Arch. Dev. Biol. 197: 345–354.
Jurgens, G., E. Wieschaus, C. Nusslein-Volhard and H. Kluding,
1984 Mutations affecting the pattern of the larval cuticle in
Drosophila melanogaster. II: Zygotic loci on the third chromosome.
Rouxs Arch. Dev. Biol. 193: 283–295.
Kaestner, K. H., W. Knochel and D. E. Martinez, 2000 Unified
nomenclature for the winged helix/forkhead transcription factors. Genes Dev. 14: 142–146.
Kalb, J. M., L. Beaster-Jones, A. P. Fernandez, P. G. Okkema, B.
Goszczynski et al., 2002 Interference between the PHA-4
and PEB-1 transcription factors in formation of the Caenorhabditis
elegans pharynx. J. Mol. Biol. 320: 697–704.
Kalb, J. M., K. K. Lau, B. Goszczynski, T. Fukushige, D. Moons et al.,
1998 pha-4 is Ce-fkh-1, a fork head/HNF-3alpha,beta,gamma
homolog that functions in organogenesis of the C. elegans pharynx. Development 125: 2171–2180.
Kaltenbach, L. S., D. L. Updike and S. E. Mango, 2005 Contribution of the amino and carboxyl termini for PHA-4/FoxA
function in Caenorhabditis elegans. Dev. Dyn. 234: 346–354.
Kamath, R. S., A. G. Fraser, Y. Dong, G. Poulin, R. Durbin et al.,
2003 Systematic functional analysis of the Caenorhabditis elegans
genome using RNAi. Nature 421: 231–237.
Kanemaki, M., Y. Kurokawa, T. Matsu-ura, Y. Makino, A. Masani
et al., 1999 TIP49b, a new RuvB-like DNA helicase, is included
in a complex together with another RuvB-like DNA helicase, TIP49a. J. Biol. Chem. 274: 22437–22444.
Kanemaki, M., Y. Makino, T. Yoshida, T. Kishimoto, A. Koga et al.,
1997 Molecular cloning of a rat 49-kDa TBP-interacting protein
(TIP49) that is highly homologous to the bacterial RuvB. Biochem. Biophys. Res. Commun. 235: 64–68.
Kemphues, K. J., J. R. Priess, D. G. Morton and N. S. Cheng,
1988 Identification of genes required for cytoplasmic localization in early C. elegans embryos. Cell 52: 311–320.
Kiefer, J. C., P. A. Smith and S. E. Mango, 2007 PHA-4/FoxA cooperates with TAM-1/TRIM to regulate cell fate restriction in
the C. elegans foregut. Dev. Biol. 303:611–624.
Kobor, M. S., S. Venkatasubrahmanyam, M. D. Meneghini, J. W.
Gin, J. L. Jennings et al., 2004 A protein complex containing
C. elegans pha-4 Suppressors
the conserved Swi2/Snf2-related ATPase Swr1p deposits histone
variant H2A.Z into euchromatin. PLoS Biol. 2: E131.
Krogan, N. J., M. C. Keogh, N. Datta, C. Sawa, O. W. Ryan et al.,
2003 A Snf2 family ATPase complex required for recruitment
of the histone H2A variant Htz1. Mol. Cell 12: 1565–1576.
Labouesse, M., E. Hartwieg and H. R. Horvitz, 1996 The Caenorhabditis elegans LIN-26 protein is required to specify and/or
maintain all non-neuronal ectodermal cell fates. Development
122: 2579–2588.
Laganiere, J., G. Deblois, C. Lefebvre, A. R. Bataille, F. Robert
et al., 2005 From the Cover: Location analysis of estrogen receptor alpha target promoters reveals that FOXA1 defines a domain
of the estrogen response. Proc. Natl. Acad. Sci. USA 102: 11651–
11656.
Lai, E., V. R. Prezioso, E. Smith, O. Litvin, R. H. Costa et al.,
1990 HNF-3A, a hepatocyte-enriched transcription factor of novel
structure is regulated transcriptionally. Genes Dev. 4: 1427–1436.
Lai, E., V. R. Prezioso, W. F. Tao, W. S. Chen and J. E. Darnell, Jr.,
1991 Hepatocyte nuclear factor 3 alpha belongs to a gene family in mammals that is homologous to the Drosophila homeotic
gene fork head. Genes Dev. 5: 416–427.
Lee, C. S., J. R. Friedman, J. T. Fulmer and K. H. Kaestner,
2005 The initiation of liver development is dependent on Foxa
transcription factors. Nature 435: 944–947.
Mango, S. E., 2001 Stop making nonSense: the C. elegans smg genes.
Trends Genet. 17: 646–653.
Mango, S. E., 2007 The C. elegans pharynx: a model for organogenesis, in Wormbook, edited by The C. elegans Research Community.
Wormbook (http://www.wormbook.org).
Mango, S. E., E. J. Lambie and J. Kimble, 1994 The pha-4 gene is
required to generate the pharyngeal primordium of Caenorhabditis
elegans. Development 120: 3019–3031.
Mann, R. S., and S. B. Carroll, 2002 Molecular mechanisms of selector gene function and evolution. Curr. Opin. Genet. Dev. 12:
592–600.
Matias, P. M., S. Gorynia, P. Donner and M. A. Carrondo,
2006 Crystal structure of the human AAA1 protein RuvBL1.
J. Biol. Chem. 281: 38918–38929.
McKim, K. S., M. F. Heschl, R. E. Rosenbluth and D. L. Baillie,
1988 Genetic organization of the unc-60 region in Caenorhabditis elegans. Genetics 118: 49–59.
Mitrovich, Q. M., and P. Anderson, 2000 Unproductively spliced
ribosomal protein mRNAs are natural targets of mRNA surveillance in C. elegans. Genes Dev. 14: 2173–2184.
Mizuguchi, G., X. Shen, J. Landry, W. H. Wu, S. Sen et al., 2004 ATPdriven exchange of histone H2AZ variant catalyzed by SWR1
chromatin remodeling complex. Science 303: 343–348.
Ohnishi, T., T. Hishida, Y. Harada, H. Iwasaki and H. Shinagawa,
2005 Structure-function analysis of the three domains of RuvB
DNA motor protein. J. Biol. Chem. 280: 30504–30510.
Overdier, D. G., A. Porcella and R. H. Costa, 1994 The DNAbinding specificity of the hepatocyte nuclear factor 3/forkhead
domain is influenced by amino-acid residues adjacent to the recognition helix. Mol. Cell. Biol. 14: 2755–2766.
Panowski, S. H., S. Wolff, H. Aguilaniu, J. Durieux and A. Dillin,
2007 PHA-4/Foxa mediates diet-restriction-induced longevity
of C. elegans. Nature 447: 550–555.
Piano, F., A. J. Schetter, D. G. Morton, K. C. Gunsalus, V. Reinke
et al., 2002 Gene clustering based on RNAi phenotypes of ovaryenriched genes in C. elegans. Curr. Biol. 12: 1959–1964.
Priess, J. R., and J. N. Thomson, 1987 Cellular interactions in early
C. elegans embryos. Cell 48: 241–250.
Pulak, R., and P. Anderson, 1993 mRNA surveillance by the Caenorhabditis elegans smg genes. Genes Dev. 7: 1885–1897.
Puri, T., P. Wendler, B. Sigala, H. Saibil and I. R. Tsaneva,
2007 Dodecameric structure and ATPase activity of the human
TIP48/TIP49 Complex. J. Mol. Biol. 366: 179–192.
Putnam, C. D., S. B. Clancy, H. Tsuruta, S. Gonzalez, J. G. Wetmur
et al., 2001 Structure and mechanism of the RuvB Holliday junction branch migration motor. J. Mol. Biol. 311: 297–310.
833
Raharjo, I., and J. Gaudet, 2007 Gland-specific expression of C. elegans hlh-6 requires the combinatorial action of three distinct
promoter elements. Dev. Biol. 302: 295–308.
Reece-Hoyes, J. S., J. Shingles, D. Dupuy, C. A. Grove, A. J. Walhout
et al., 2007 Insight into transcription factor gene duplication
from Caenorhabditis elegans Promoterome-driven expression patterns. BMC Genomics 8: 27.
Rottbauer, W., A. J. Saurin, H. Lickert, X. Shen, C. G. Burns et al.,
2002 Reptin and pontin antagonistically regulate heart growth
in zebrafish embryos. Cell 111: 661–672.
Sha, K., and A. Fire, 2005 Imprinting capacity of gamete lineages in
Caenorhabditis elegans. Genetics 170: 1633–1652.
Shen, X., G. Mizuguchi, A. Hamiche and C. Wu, 2000 A chromatin
remodelling complex involved in transcription and DNA processing. Nature 406: 541–544.
Simmer, F., C. Moorman, A. M. van der Linden, E. Kuijk, P. V. van
den Berghe et al., 2003 Genome-wide RNAi of C. elegans using
the hypersensitive rrf-3 strain reveals novel gene functions. PLoS
Biol. 1: e12.
Strome, S., and W. B. Wood, 1982 Immunofluorescence visualization of germ-line-specific cytoplasmic granules in embryos, larvae, and adults of Caenorhabditis elegans. Proc. Natl. Acad. Sci.
USA 79: 1558–1562.
Sulston, J. E., and J. Hodgkin, 1988 Methods: In the Nematode C. elegans. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY.
Taubert, S., C. Gorrini, S. R. Frank, T. Parisi, M. Fuchs et al.,
2004 E2F-dependent histone acetylation and recruitment of
the Tip60 acetyltransferase complex to chromatin in late G1.
Mol. Cell. Biol. 24: 4546–4556.
Updike, D. L., and S. E. Mango, 2006 Temporal regulation of foregut development by HTZ-1/H2A.Z and PHA-4/FoxA. PLoS
Genet 2: e161.
Vilimas, T., A. Abraham and P. G. Okkema, 2004 An early pharyngeal muscle enhancer from the Caenorhabditis elegans ceh-22 gene
is targeted by the Forkhead factor PHA-4. Dev. Biol. 266: 388–
398.
Walhout, A. J., J. Reboul, O. Shtanko, N. Bertin, P. Vaglio et al.,
2002 Integrating interactome, phenome, and transcriptome
mapping data for the C. elegans germline. Curr. Biol. 12: 1952–
1958.
Weigel, D., G. Jurgens, F. Kuttner, E. Seifert and H. Jackle,
1989 The homeotic gene fork head encodes a nuclear protein
and is expressed in the terminal regions of the Drosophila embryo. Cell 57: 645–658.
Weinstein, D. C., A. Ruiz i Altaba, W. S. Chen, P. Hoodless, V. R.
Prezioso et al., 1994 The winged-helix transcription factor
HNF-3 beta is required for notochord development in the mouse
embryo. Cell 78: 575–588.
West, S. C., 1996 DNA helicases: new breeds of translocating motors
and molecular pumps. Cell 86: 177–180.
Wicks, S. R., R. T. Yeh, W. R. Gish, R. H. Waterston and R. H.
Plasterk, 2001 Rapid gene mapping in Caenorhabditis elegans
using a high density polymorphism map. Nat. Genet. 28: 160–
164.
Williams, D. C., T. Boulin, A. F. Ruaud, E. M. Jorgensen and J. L.
Bessereau, 2005 Characterization of Mos1-mediated mutagenesis in Caenorhabditis elegans: a method for the rapid identification
of mutated genes. Genetics 169: 1779–1785.
Wood, M. A., S. B. McMahon and M. D. Cole, 2000 An ATPase/
helicase complex is an essential cofactor for oncogenic transformation by c-Myc. Mol. Cell 5: 321–330.
Yochem, J., T. Gu and M. Han, 1998 A new marker for mosaic analysis in Caenorhabditis elegans indicates a fusion between hyp6 and
hyp7, two major components of the hypodermis. Genetics 149:
1323–1334.
Zaret, K. S., 2002 Regulatory phases of early liver development:
paradigms of organogenesis. Nat. Rev. Genet. 3: 499–512.
Communicating editor: K. Kemphues