RESEARCH REPORT 3881
Development 136, 3881-3887 (2009) doi:10.1242/dev.034637
xol-1, the master sex-switch gene in C. elegans, is a
transcriptional target of the terminal sex-determining factor
TRA-1
Balázs Hargitai1, Vera Kutnyánszky1, Timothy A. Blauwkamp2, Attila Steták3, Györgyi Csankovszki2,
Krisztina Takács-Vellai1 and Tibor Vellai1,*
In the nematode Caenorhabditis elegans, sex is determined by the ratio of X chromosomes to sets of autosomes: XX animals
(2X:2A1.0) develop as hermaphrodites and XO animals (1X:2A0.5) develop as males. TRA-1, the worm ortholog of Drosophila
Cubitus interruptus and mammalian Gli (Glioma-associated homolog) proteins, is the terminal transcription factor of the C. elegans
sex-determination pathway, which specifies hermaphrodite fate by repressing male-specific genes. Here we identify a consensus
TRA-1 binding site in the regulatory region of xol-1, the master switch gene controlling sex determination and dosage
compensation. xol-1 is normally expressed in males, where it promotes male development and prevents dosage compensation. We
show that TRA-1 binds to the consensus site in the xol-1 promoter in vitro and inhibits the expression of xol-1 in XX animals in vivo.
Furthermore, inactivation of tra-1 enhances, whereas hyperactivation of tra-1 suppresses, lethality in animals with elevated xol-1
activity. These data imply the existence of a regulatory feedback loop within the C. elegans sex-determination and dosagecompensation cascade that ensures the accurate dose of X-linked genes in cells destined to adopt hermaphrodite fate.
INTRODUCTION
The primary signal that determines sexual differentiation in C.
elegans is the ratio of sex chromosomes to sets of autosomes: diploid
animals with two X chromosomes (XX) are hermaphrodites and
animals with one X chromosome (XO) are males (Meyer, 2005;
Zarkower, 2006). All aspects of somatic sexual fate in this organism
are controlled by the sex-determination and dosage-compensation
pathway, which comprises a cascade of negative regulatory
interactions (Fig. 1A). Upstream in this pathway, the Xchromosome-counting mechanism culminates in switching xol-1
(XO lethal) on in males and off in hermaphrodites (Meyer, 2005;
Zarkower, 2006). The activity of xol-1 is determined by dosesensitive signals from both the X chromosome and autosomes. Xsignal elements (XSEs), including fox-1 (feminizing gene on X), sex1 (signal element on X) and sex-2, repress xol-1. By contrast,
autosomal signal elements (ASEs), including sea-1 (signal element
on autosome), sea-2 and sea-3, promote xol-1 activity. In XO
animals, the inhibitory effects of XSEs on xol-1 are not sufficient to
overcome the stimulatory effects of ASEs, thereby rendering xol-1
active. In XX animals, the combined dose of XSEs is able to repress
xol-1.
XOL-1 inhibits the sdc (sex-determination and dosagecompensation defect) genes sdc-1, sdc-2 and sdc-3, which in turn
regulate both somatic sex determination and X-chromosome dosage
compensation (Meyer, 2005). In the sex-determination pathway,
the SDC proteins downregulate the autosomal gene her-1
1
Department of Genetics, Eötvös Loránd University, Budapest H-1117, Hungary.
Department of Molecular, Cellular, and Developmental Biology, University of
Michigan, Ann Arbor, MI 48109, USA. 3Division of Molecular Psychology, Life
Sciences Training Facility, Biozentrum, University of Basel, Basel CH-4056,
Switzerland.
2
*Author for correspondence ([email protected])
Accepted 22 September 2009
(hermaphroditization of XO animals) (Trent et al., 1991; Dawes et
al., 1999; Chu et al., 2002). her-1 encodes a secreted protein that
binds to and inhibits the transmembrane receptor product of tra-2
(sexual transformer) (Hodgkin, 1980). When TRA-2 function is off,
the FEM (feminization of XO and XX animals) proteins FEM-1,
FEM-2 and FEM-3, together with CUL-2 (Cullin-2-like ubiquitin
ligase), form a complex to target the zinc-finger transcription factor
TRA-1A for proteasome-mediated degradation (Starostina et al.,
2007). In the absence of functional TRA-1A, male-specific genes
can be transcribed. When the SDCs are inactive, HER-1 is able to
block TRA-2. As a result, the inactive FEM-1/2/3–CUL-2 complex
allows TRA-1A to be cleaved C-terminally, and to thereby become
resistant to proteasomal degradation. In turn, TRA-1A represses
male-specific genes (Zarkower, 2006). Thus, tra-1 functions as the
terminal control gene of the nematode sex-determination pathway.
The SDC proteins also constitute components of the worm
dosage-compensation complex (DCC), which ensures that X-linked
genes are expressed at similar levels in both sexes (Chuang et al.,
1996; Lieb et al., 1996; Lieb et al., 1998; Meyer, 2005). In addition
to the SDCs, this complex contains other proteins, including MIX1 (mitosis and X-associated), DPY-21 (Dumpy), DPY-26, DPY-27,
DPY-28, CAPG-1 (CAP-G subunit of condensin I) and DPY-30
(Meyer, 2005; Csankovszki et al., 2009) (see Fig. 1A). Interestingly,
dosage-compensation proteins establish both gene-specific (her-1)
and chromosome-wide (i.e. X-specific) transcriptional repression
(Chu et al., 2002). The DCC is recruited to specific recognition
elements on hermaphrodite X chromosomes, then spreads to the rest
of the chromosome to reduce gene expression by half (Csankovszki
et al., 2004; McDonel et al., 2006; Jans et al., 2009).
In this study, we report a novel feedback mechanism that controls
dosage compensation through repressing xol-1. We show that xol-1
is a direct molecular target of TRA-1A repression. Inactivation of
tra-1 causes ectopic expression of xol-1 in XX embryos. This
suggests that the XSEs alone are not sufficient to fully repress xol1. Moreover, decreased activity of tra-1 enhances, whereas its
DEVELOPMENT
KEY WORDS: Sex determination, Dosage compensation, Caenorhabditis elegans, TRA-1/Gli/Ci, xol-1, Chromatin
increased activity suppresses, lethality in animals with elevated xol1 activity. Thus, tra-1 contributes to the maintenance of xol-1
repression, and indirectly to halving the expression of X-linked
genes in cells destined to adopt hermaphrodite fate.
MATERIALS AND METHODS
Nematode strains and alleles
The wild-type C. elegans strain corresponds to var. Bristol (N2). The
following mutant strains were used: CB2823 tra-1(e1488)III; eDp6(III;f);
CB2590 tra-1(e1099)/dpy-18(e1096)III; CB3844 fem-3(e2006)IV; NG41
sex-1(gm41)X; TY2384 sex-1(y263)X; CB428 dpy-21(e428)V; TY2431
him-8(e1487)IV; xol-1::gfp(yIs34)V; CB1489 him-8(e1489)IV; CB4088
him-5(e1490)V; BU099 hbEx2[mut pxol-1::gfp + unc-119(+) + rol6(su1006)]; TY1807 xol-1(y9)X; CB3769 tra-1(e1575gf)/+III; tra3(e1767)IV; DH1033 sqt(sc103)II; bIs1[VIT-2::GFP + rol-6(su1006)]X; and
CB678 lon-2(e678)X.
Gel mobility shift assay
TRA-1A protein was generated by in vitro transcription and translation of
full-length tra-1 cDNA from pDZ118 (kindly provided by David Zarkower,
University of Minnesota, Minneapolis, MN, USA), using a T7-based
coupled reticulocyte lysate system (TNT Coupled Reticulocyte Lysate
System, Promega). After in vitro translation, ZnSO4 was added to 50 M.
For preparing DNA probes, single-stranded oligonucleotides were annealed
in TE (10 mM Tris-Cl, 1 mM EDTA, pH 7.6) and labeled by filling in the
single-stranded termini with Klenow polymerase in the presence of
[32P]dCTP according to standard procedures. The following
oligonucleotides were used:
wt xol-1 L 5⬘-GGGGCCCCTGTAAGACCACACACGACGAAAACCTCTTGTT-3⬘and R 5⬘-GGGGAACAAGAGGTTTTCGTCGTGTGTGGTCTTACAGGGG-3⬘;
mut xol-1 L 5⬘-GGGGGCCCCTGTAACGGTACACACGACGAAAACCTCTTGTT-3⬘ and R 5⬘-GGGGGAACAAGAGGTTTTCGTCGTGTGTACCGTTACAGGGG-3⬘;
wt mab-3 L 5⬘-GGGGGCGTTCTCTAATTATCGTCGTGTGAGGTCTTCTAT-3⬘ and R 5⬘-GGGGGATAGAAGACCTCACACGACGATAATTAGAGAACG-3⬘.
Experiments were performed essentially as described previously (Yi et
al., 2000). Briefly, probes of 40,000 cpm (~1 ng DNA) were incubated for
20 minutes on ice in the presence of 3 l protein before electrophoresis on
4% polyacrylamide 0.5⫻ TBE gels at 160 V at room temperature. The gels
were dried and exposed to Kodak XAR film.
RNA interference and transgene construction
Total RNA was isolated from mix-staged wild-type worms, and specific
cDNA fragments were amplified by RT-PCR. The following forward and
reverse primers were used:
tra-1, 5⬘-CTAGCTAGCTAGACAATCCGGAGCATCTCAAG-3⬘ and 5⬘GGGGTACCCCTGATGATGTTGAGCCAGAGC-3⬘;
xol-1, 5⬘-CATGCCATGGCATGGCGCGAAAACAGTCCAGTC-3⬘ and
5⬘-CATGCCATGGCATGGCCGTCGTCGAAAAATGAG-3⬘;
sea-2, 5⬘-CATGCCATGGCATGCGGAAAGCTCCTCAACTCTG-3⬘ and
5⬘-CATGCCATGGCATGACCGTCACGAATGAGGTTTC-3⬘.
The amplified fragments were cloned into pGEM-T Easy (Promega) and
subcloned into pPD129.36. The resulting constructs were transformed into
E. coli HT115(DE3). RNA interference (RNAi) experiments were performed
at 25°C. To generate a xol-1::gfp reporter lacking the putative TRA-1A
binding site (pmutxol-1::gfp), two genomic fragments were amplified using
the following forward and reverse primer pairs:
Forw1 5⬘-AAGGCGCGCCTTGCAGAACAGCTTTGATCG-3⬘ and Rev1
5⬘-TGTTGACCGTTACAGGGGTATTCGCGGGCGTTTGAAAATAGTG3⬘;
Forw2 5⬘-CGAATACCCCTGTAACGGTCAACACGACGAAAACCTCTTGTTCC-3⬘ and Rev2 5⬘-TTGCGGCCGCAAGGACCCTGCAACAAAACG-3⬘.
A single fragment was generated by fusion PCR, digested with AscI and
NotI, and cloned into pRH21. Extrachromosomal array-bearing animals
were examined in wild-type and him-5 mutant backgrounds.
Development 136 (23)
RESULTS AND DISCUSSION
TRA-1A binds to the regulatory region of xol-1
The transcriptional regulator tra-1 encodes two proteins: TRA-1A
with five zinc-finger motifs and TRA-1B with two zinc fingers
(Zarkower and Hodgkin, 1992). TRA-1A has a DNA-binding
ability, whereas TRA-1B does not (Zarkower and Hodgkin, 1993).
Until now, only a few direct targets of TRA-1A have been identified,
including egl-1 (egg-laying defective), mab-3 (male abnormal), ceh30 (C. elegans homeobox), fog-1 (feminization of germline), fog-3
and dmd-3 (DM-domain family) (Conradt and Horvitz, 1999; Yi et
al., 2000; Schwartz and Horvitz, 2007; Peden et al., 2007; Mason et
al., 2008). These TRA-1A targets, all of which are repressed in XX
animals, determine different aspects of sexual fate determination.
The availability of sequence information of TRA-1 binding sites in
the regulatory regions of known targets prompted us to deduce a
consensus binding site from the nucleotides conserved within these
sites (Fig. 1B). We found that nine of the 18 bases in TRA-1A binding
sites are strictly conserved, and three bases change only once. We then
searched the C. elegans genome for consensus TRA-1A binding sites,
considering the highly conserved nucleotides at their appropriate
positions (TTATTCNNNNTGTGGATGGTC). Our analysis identified
35 novel putative TRA-1A binding sites within upstream regulatory
or intronic sequences. One of these sites is located within the xol-1
promoter, 154 bp upstream of the ATG translational initiation site (Fig.
1C). This consensus site, which is almost identical to that found in the
mab-3 regulatory region, is highly conserved in Caenorhabditis
species, and, interestingly, is repeated three times in the C. remanei
xol-1 genomic environment (Fig. 1D).
We next assessed whether in-vitro-translated full-length TRA-1A
is able to bind to an oligonucleotide corresponding to this consensus
site in the xol-1 promoter. Using gel electrophoretic mobility shift
assays, we detected efficient binding of TRA-1A to this element
(Fig. 1E). By contrast, TRA-1A was not able to bind to the
oligonucleotide when the putative TRA-1A binding site was
mutated in four crucial positions (see Materials and methods). In
addition, unlabeled wild-type, but not mutant, xol-1 oligonucleotide
was able to compete with the labeled oligonucleotide in a
concentration-dependent manner (Fig. 1E). We conclude that TRA1A is able to bind to the consensus site in the xol-1 promoter in vitro.
TRA-1 represses xol-1 in XX animals
xol-1 promotes the male mode of dosage compensation and sexual
fate determination. Normally, xol-1 is expressed in XO embryos to
repress the SDC proteins. Therefore, XOL-1 prevents the DCC from
undergoing assembly, causing the her-1 autosomal locus and the
single male X chromosome to remain transcriptionally fully active.
In XX animals, xol-1 is repressed and unable to inhibit the SDCs. To
monitor xol-1 expression, we analyzed the expression of an
integrated xol-1::gfp transcriptional fusion reporter, yIs34 (Dawes
et al., 1999). Consistent with previous results (Carmi et al., 1998),
xol-1::gfp was inactive in a wild-type background and was
expressed in XO, but repressed in XX, embryos in the him-8(e1489)
(high incidence of males) mutant background (Fig. 2A,B). The
nuclear hormone receptor SEX-1 acts as an X-chromosome-specific
signal to downregulate xol-1 transcription (Carmi et al., 1998).
Consistently, yIs34 was ectopically expressed in sex-1(gm41)
mutant XX embryos (Fig. 2C). A significant proportion of sex-1
mutant embryos was arrested in development, probably owing to
xol-1 misregulation (see below).
We monitored xol-1::gfp expression in tra-1(e1488) mutant
animals. e1488 is a reduction-of-function mutation that confers an
intersex phenotype to animals of XX karyotype. Homozygous tra-
DEVELOPMENT
3882 RESEARCH REPORT
TRA-1 represses xol-1
RESEARCH REPORT 3883
1(e1488) mutants have a hermaphrodite gonad and intestine, but the
rest of their body (e.g. the tail region, musculature and nervous
system) is masculinized (Hodgkin, 1993). We found that at certain
developmental stages xol-1::gfp was expressed in tra-1(e1488)
mutant XX embryos (Fig. 2D). This implies that TRA-1A represses
xol-1 in animals of hermaphrodite karyotype. To confirm these
results, we also examined xol-1::gfp expression in tra-1(e1099)
mutant animals. e1099 is a strong loss-of-function allele that
transforms XX animals into low-fertility males. Similar to what we
observed in the hypomorph tra-1 mutant background, the xol-1::gfp
reporter was ectopically active in a proportion of the progeny of tra1(+/e1099) heterozygous hermaphrodites, which we infer to be
homozygous for tra-1(e1099) (Fig. 2E). Furthermore, we depleted
TRA-1 by RNA interference (RNAi) and examined the effect of this
treatment on xol-1 expression. tra-1 RNAi (see Szabó et al., 2009)
strongly phenocopied tra-1(e1488): tra-1(RNAi) animals were
DEVELOPMENT
Fig. 1. xol-1 is a transcriptional target of TRA-1A. (A)The C. elegans sex-determination and dosage-compensation pathway. xol-1 is controlled
by X-chromosome and autosomal signal elements (XSEs and ASEs). tra-1 is the terminal regulator of somatic sexual fate. The genetic mechanisms
of sex determination and dosage compensation are known to be decoupled downstream of the SDC proteins. For details, see text. T-bars indicate
negative regulatory interactions, arrows indicate activation. (B)Determination of the consensus sequence ‘logo’ for TRA-1A binding sites. (C)The
xol-1 promoter contains a consensus TRA-1A binding site. (D)This site is highly conserved in Caenorhabditis species, and is repeated three times in
the C. remanei xol-1 genomic environment. (E)TRA-1A is able to bind to an oligonucleotide that contains the xol-1 TRA-1 binding site. Competition
assays were performed using unlabeled wild-type or mutant competitor oligonucleotides. The relative concentrations of competitors are indicated
above the lanes. Unlabeled wild-type oligonucleotide is able, but mutant oligonucleotide fails, to abolish the specific binding of TRA-1A to the wildtype xol-1 oligonucleotide. Lysate, reticulocyte lysate; wt, wild-type; oligo, oligonucleotide.
3884 RESEARCH REPORT
Development 136 (23)
Fig. 2. tra-1 contributes to xol-1 repression in XX animals. (A)xol1 is not active in wild-type XX animals. An integrated xol-1::gfp
reporter (yIs34) is not expressed in wild-type embryos of XX karyotype.
(B)Expression of yIs34 in a him-8(e1489) mutant genetic background.
him-8(e1489) mutant hermaphrodites generate both XX (~63%) and
XO (~37%) progeny. The green embryo is inferred to be of XO
karyotype (see Nicoll et al., 1997). (C-F)Expression of yIs34 in sex1(gm41) mutant (C), tra-1(e1488) mutant (D), tra-1(e1099) mutant (E)
and tra-1(RNAi) embryos (F). The strains shown are of XX karyotype.
Thus, inactivation of tra-1 (similarly to insufficient sex-1 activity) causes
ectopic expression of yIs34 in XX embryos. Some green embryos
produced by tra-1(e1099/+) hermaphrodites were transferred onto
NGM agar plates, allowed to develop into adulthood, and indeed
identified as males. (G)Expression of an extrachromosomal xol-1::gfp
reporter (hbEx2) in an otherwise wild-type background (XX karyotype).
To generate this reporter, the putative TRA-1A binding site was
mutated in the xol-1 regulatory region of yIs34 (see Materials and
methods). The corresponding wild-type promoter (hbEx1) failed to drive
the expression of the gfp transgene in XX embryos (data not shown).
Overlay of Nomarski and fluorescence images, except F, where only the
fluorescence image is shown. Scale bars: 50m.
[Ex(pmutxol-1::gfp)], in which the putative TRA-1A binding site is
mutated. A proportion of the progeny of these worms, assumed to
be the array-bearing XX embryos, were green (Fig. 2G). Together,
these results indicate that TRA-1A binds to the consensus site in the
xol-1 promoter and inhibits expression.
Fig. 3. Lethality in tra-1-deficient animals
depends on increased xol-1 activity.
(A)Lethality in tra-1 mutants is suppressed by
depletion of XOL-1. Control animals were fed
E. coli HT115 expressing the empty vector
alone and were maintained under inducing
conditions. For mutant backgrounds, P<0.001
(unpaired Student’s t-test). (B)xol-1(y9)
mutation also markedly suppresses lethality in
tra-1(–) mutant animals. Lethality of xol-1; tra1 double mutants was compared with the
corresponding tra-1 single mutants (P<0.001,
unpaired Student’s t-test). Depletion of SEA-2
(which normally activates xol-1) also suppresses
lethality in tra-1(–) mutants (P<0.001, unpaired
Student’s t-test). Control RNAi: animals were
fed bacteria expressing the empty vector. (C)A
model for the control of xol-1 expression by
TRA-1A (red bar). In XX animals, tra-1
maintains the activity of the SDC proteins by
repressing xol-1. In turn, the SDC and DPY
proteins reduce the transcriptional activity of
the X chromosomes by half.
DEVELOPMENT
intersexes, i.e. they developed both vulval structure and male tail at
almost full penetrance. xol-1 activity was obvious in embryos
produced by XX animals treated with tra-1 double-stranded RNA
(Fig. 2F). Finally, we generated worms carrying extrachromosomal
arrays containing a modified form of the xol-1::gfp reporter
Inactivation of tra-1 enhances, whereas
hyperactivation of tra-1 suppresses, lethality in
animals with elevated xol-1 activity
Inactivation of the XSE sex-1 causes greater than 30% embryonic
lethality in XX animals, presumably owing to ectopic xol-1
expression (Nicoll et al., 1997; Carmi et al., 1998) (Table 1).
Inactivating another xol-1 repressor, such as tra-1, would be
expected to enhance this lethality. To test this, we first quantified
lethality in mutant animals defective for tra-1. At 25°C, 41.4% and
47.9% of tra-1(e1488) and tra-1(e1099) loss-of-function mutant
embryos failed to develop to adulthood, respectively (Table 1).
These values are comparable to, or even higher than, those obtained
TRA-1 represses xol-1
RESEARCH REPORT 3885
Table 1. tra-1 activity influences the penetrance of lethality in sex-1 and dpy-21 loss-of-function mutants
Maternal genotype
Wild type
Wild type
tra-1(e1488/+)
tra-1(e1488/+)
tra-1(e1099/+)
tra-1(e1099/+)
tra-1(e1575gf)/+
tra-1(e1575gf)/+
fem-3(e2006)
fem-3(e2006)
sex-1(gm41)
sex-1(gm41)
sex-1(gm41); tra-1(e1488)/+
sex-1(gm41); tra-1(e1488)/+
sex-1(gm41); tra-1(e1099)/+
sex-1(gm41); tra-1(e1099)/+
sex-1(gm41); fem-3(e2006)
sex-1(gm41); fem-3(e2006)
sex-1(gm41); tra-1(e1575gf/+)
sex-1(gm41); tra-1(e1575gf/+)
sex-1(y263)
sex-1(y263)
sex-1(y263); tra-1(e1488)/+
sex-1(y263); tra-1(e1488)/+
sex-1(y263); fem-3(e2006)
sex-1(y263); fem-3(e2006)
dpy-21(e428)
dpy-21(e428)
dpy-21(e428); tra-1(e1488)/+
dpy-21(e428); tra-1(e1488)/+
dpy-21(e428); tra-1(e1099)/+
dpy-21(e428); tra-1(e1099)/+
Temp.
(°C)
Embryos
(n)
Expected
homozygous
mutant embryos
(n)
20
25
20
25
20
25
20
25
20
25
20
25
20
25
20
25
20
25
20
25
20
25
20
25
20
25
20
25
20
25
20
25
97
91
1634
629
160
566
334
117
376
97
419
325
871
699
59
283
878
133
135
135
372
535
ND
394
575
160
136
147
402
212
207
560
97*
91*
409
157
40
142
251†
88†
376
97
419
325
218
175
15
71
878
100
101
101
372
535
ND
99
575
160
136
147
101
53
52
140
Adults
(n)
Homozygous
mutant adults
(n)
Lethality of
homozygous
embryos (%)
P<
97
90
1449
490
153
481
324
110
326
77
202
179
317
396
17
159
683
96
101
110
227
367
ND
301
385
119
72
101
231
90
72
303
97*
90*
300
92
29
74
242
83
326
77
202
179
46
40
2
6
683
96
91
95
227
367
ND
49
385
119
72
101
52
21
6
57
0
0.9
26.7
41.4
27.5
47.9
3.6‡
5.7‡
13.3
20.6
51.8
44.9
78.9
77.1
86.7
91.5
22.2
27.8
10.1
6.2
39.0
31.4
ND
50.5
33.0
25.6
47.1
31.3
48.5
60.4
88.5
59.3
–
–
0.001
0.001
0.001
0.001
0.03
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
–
0.001
0.001
0.001
0.001
0.001
NS
0.001
0.001
0.001
*Homozygous wild-type animals.
†
Expected homozygous and heterozygous animals (e1575gf behaves as a dominant mutation).
‡
Lethality of embryos bearing at least one tra-1(e1751gf) allele. Unpaired Student’s t-test was used to calculate the P-value. For statistics, single mutants were compared with
the wild type, double mutants were compared with the corresponding single mutants.
ND, not determined; NS, not significant; gf, gain-of-function mutation.
function that is required for viability and independent of sex
determination. Because inhibiting other components of the sexdetermination pathway similarly caused developmental arrest with
moderate penetrance [at 20°C: fem-2(b245) mutants, 4.8% lethality
(n126); her-1(e1518) mutants, 5.1% (n147)], the observed lethality
might be a general property of the pathway.
XOL-1 functions to inhibit dosage compensation (Meyer, 2005).
Consistent with the potential role of tra-1 in controlling xol-1,
insufficient TRA-1 levels enhanced lethality in dpy-21(e428) mutant
nematodes, in which the DCC is already compromised (Table 1).
Together, these data indicate that tra-1 modulates the activity of xol1, which regulates both sex determination and dosage compensation.
Lethality of tra-1 mutant embryos depends on
xol-1 activity
As shown above, a significant proportion of tra-1-deficient, but not
of tra-1(e1575gf) mutant, XX animals die at different stages of
development (Table 1). Therefore, we tested whether the lethality of
tra-1 mutant embryos was a result of xol-1 overexpression. If it
were, repressing xol-1 should enhance the viability of tra-1 loss-offunction mutants. Embryonic lethality was compared for tra-1
mutants treated with xol-1 versus control double-stranded RNA (Fig.
3A). Although xol-1 RNAi treatment is likely to deplete XOL-1 only
partially [as most xol-1(RNAi) XO embryos were able to develop
DEVELOPMENT
in sex-1(gm41) and sex-1(y263) mutant animals maintained under
identical conditions. Then, we generated sex-1(–); tra-1(–) double
mutants. Lethality detected in either single mutant appeared to be
additive in the corresponding sex-1(–); tra-1(–) double-mutant
animals. This suggests that sex-1 and tra-1 repress xol-1 via parallel
mechanisms.
To test whether an increase in tra-1 activity can suppress sex-1
lethality, we also generated sex-1(–); fem-3(–) double-mutant
nematodes and scored them for viability. fem-3 inhibits the activity of
tra-1 in the sex-determination pathway (Fig. 1A). Loss-of-function
mutations in fem-3, similarly to tra-1 gain-of-function mutations,
feminize both XX and XO animals (i.e. fem-3-deficient and tra-1hyperactive animals produce only oocytes). The fem-3(e2006)
mutation significantly increased viability in the sex-1 mutant
background (Table 1). Thus, hyperactivation of tra-1 is able to
suppress lethality in XX animals with derepressed xol-1. Consistent
with these data, the tra-1 gain-of-function mutation e1575 markedly
suppressed embryonic lethality in sex-1(gm41) mutant animals (Table
1). It is worth noting that fem-3(e2006) single-mutant animals also
exhibited a significant degree of embryonic lethality (Table 1). This
was unexpected because xol-1 is normally repressed in XX animals,
and thereby downregulation of fem-3, if it acts through tra-1, should
not influence xol-1 activity in the soma. Since tra-1(e1575gf) led to
only modest embryonic lethality (Table 1), fem-3 probably has a
into adults; data not shown], it did significantly decrease the
percentage of dead embryos in both sex-1 (control) and tra-1 mutant
backgrounds. For example, embryonic lethality in the tra-1(e1099)
mutant background was reduced by half with xol-1 RNAi (Fig. 3A).
Similarly, the xol-1(y9) mutation markedly enhanced viability in tra1(–) mutant embryos (Fig. 3B). Depletion of the ASE SEA-2 (which
normally activates xol-1) also caused a significant suppression of
lethality in tra-1(e1099) mutant animals (Fig. 3B). These results
indicate that lethality caused by tra-1 deficiency is, at least partially,
xol-1 dependent.
To address whether lethality caused by tra-1 deficiency is XX
specific, we scored the ratio of males in him-8(e1489) mutant
animals depleted for tra-1. Indeed, the percentage of males was
increased in him-8(e1489); tra-1(RNAi) animals (46.9% males,
n168), as compared with him-8(e1489) mutants treated with
control RNAi (35.7% males, n145). To directly determine the sex
of arrested embryos, we used an integrated X-linked GFP reporter,
bIs1 (Grant and Hirsh, 1999). lon-2(e678)X hermaphrodites were
treated with tra-1 double-stranded RNA from the L2 larval stage,
then mated with males carrying bIs1. The arrested F1 progeny were
tested for the presence of gfp by PCR. Sixteen out of the 20 embryos
tested were gfp positive (XX karyotype), indicating that XX
embryos are overrepresented in the arrested population.
TRA-1A is similar to Drosophila Cubitus interruptus and to
vertebrate Glioma-associated homolog proteins (Zarkower and
Hodgkin, 1992). Members of this protein family act as the terminal
transcription factors of the Hedgehog (Hh) signaling pathway and
control several key developmental processes in both invertebrates
and vertebrates. In humans, dysregulated Hh signaling has been
implicated in cancer, skeletal malformation and defective neuronal
patterning. Studies of tra-1 in nematodes might therefore shed light
on the role of Hh signaling in human development and disease.
tra-1 has been shown to interact with the class B synMuv
(synthetic Multivulva) pathway: it cooperates with the synMuv B
gene tra-4 to promote female development (Grote and Conradt,
2006), represses vulval induction in a synMuv A mutant background
(Szabó et al., 2009), and appears to directly regulate the expression
of the Hox gene lin-39 (Szabó et al., 2009), a central regulator of
vulval development (Takács-Vellai et al., 2007). The synMuv B
pathway includes chromatin remodeling factors, such as members
of the NuRD nucleosome remodeling and histone deacetylase
complex and the Rb (Retinoblastoma)/E2F complex (Harrison et al.,
2006). It is therefore likely that xol-1 repression by TRA-1A
involves changes in chromatin structure.
In C. elegans, both dosage compensation and sex determination
are thought to be irreversibly determined early in life by the X:A
ratio, which renders the master sex-switch gene xol-1 to be active or
inactive (Meyer, 2005; Zarkower, 2006). In XX animals, the
combined dose of XSEs represses xol-1, allowing the DCC to
assemble and repress the autosomal gene her-1 and halve the
expression from both X chromosomes. However, following Xchromosome repression, the expression of XSEs is also reduced by
half (Gladden et al., 2007). It is possible that repression of the XSEs
necessitates an additional mechanism to maintain xol-1 repression.
In this study, we have shown that TRA-1A, the terminal
transcription factor of the C. elegans sex-determination pathway,
also represses xol-1, the upstream regulator of the pathway (Fig. 3C).
Since the other known autosomal regulators of xol-1, the ASEs,
promote xol-1 expression, tra-1 is the first autosomal gene identified
as inhibiting xol-1. Our study shows that the role of TRA-1A in
hermaphrodites is to repress male-specific genes at two levels. TRA1A not only represses the terminal male sexual differentiation genes,
Development 136 (23)
but also xol-1, the male-specific upstream regulator of the pathway.
By repressing xol-1 in XX animals, TRA-1A indirectly contributes
to the maintenance of X-chromosome repression during dosage
compensation. Understanding the mechanism by which Xchromosome repression is maintained throughout the lifetime of
hermaphrodites is an important area of research (Meyer, 2005). Our
results might help to better understand the molecular mechanism
underlying X-linked gene dosage equalization between
hermaphrodites and males.
Acknowledgements
We thank David Zarkower for the tra-1 cDNA clone; the Caenorhabditis
Genetics Center funded by the NIH for nematode strains; and Martha Snyder
for critical reading of the manuscript. This work was supported by grants from
the OTKA Hungarian Scientific Research Funds K68372 to T.V. and PD75477 to
K.T-V, from the National Office for Research and Technology (TECH_08_A1/22008-0106) to T.V, and by the NIH grant (NIH RO1 GM079533) to G.C. T.V.
and K.T.-V. are grantees of the János Bolyai Scholarship. Deposited in PMC for
release after 12 months.
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TRA-1 represses xol-1