Coordinated inhibition of C/EBP by Tribbles in multiple tissues is essential for Caenorhabditis elegans development
Kim et al. BMC Biology
Coordinated inhibition of C/EBP by Tribbles in multiple tissues is essential for Caenorhabditis elegans development
Kyung Won Kim 1
Nishant Thakur 0 3
Christopher A. Piggott 1
Shizue Omi 0 3
Jolanta Polanowska 0 3
Yishi Jin 1 2
Nathalie Pujol 0 3
0 Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université, Inserm, CNRS , Marseille , France
1 Section of Neurobiology, Division of Biological Sciences, University of California San Diego , La Jolla, CA 92093 , USA
2 Howard Hughes Medical Institute, University of California San Diego , La Jolla, CA 92093 , USA
3 Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université, Inserm, CNRS , Marseille , France
Background: Tribbles proteins are conserved pseudokinases that function to control kinase signalling and transcription in diverse biological processes. Abnormal function in human Tribbles has been implicated in a number of diseases including leukaemia, metabolic syndromes and cardiovascular diseases. Caenorhabditis elegans Tribbles NIPI-3 was previously shown to activate host defense upon infection by promoting the conserved PMK-1/p38 mitogen-activated protein kinase (MAPK) signalling pathway. Despite the prominent role of Tribbles proteins in many species, our knowledge of their mechanism of action is fragmented, and the in vivo functional relevance of their interactions with other proteins remains largely unknown. Results: Here, by characterizing nipi-3 null mutants, we show that nipi-3 is essential for larval development and viability. Through analyses of genetic suppressors of nipi-3 null mutant lethality, we show that NIPI-3 negatively controls PMK-1/p38 signalling via transcriptional repression of the C/EBP transcription factor CEBP-1. We identified CEBP-1's transcriptional targets by ChIP-seq analyses and found them to be enriched in genes involved in development and stress responses. Unlike its cell-autonomous role in innate immunity, NIPI-3 is required in multiple tissues to control organismal development. See also companion paper by McEwan et al http://bmcbiol.biomedcentral.com/articles/10.1186/s12915-016-0334-6#Bib1.
Tribbles; C/EBP transcription factor; p38 MAP kinase; Genetic suppression; Transcriptional regulation; Signal transduction; Development; Stress responses
The Tribbles genes encode a family of highly conserved
pseudokinases which lack key catalytic amino acids in the
kinase domain [1–3]. Functional studies in multiple
organisms have shown that these pseudokinases play diverse
roles in innate immunity, cell signalling, energy homeostasis
and cell division [2, 4]. The Drosophila tribbles gene is
required for cell proliferation and migration in embryogenesis
and oogenesis [5–8]. The mammalian Tribbles family
includes three genes, Trib1, Trib2 and Trib3, each of which
plays unique roles in signalling networks regulating adipose
tissue, metabolic homeostasis and the immune system [2, 4].
Abnormal function in human Tribbles has been implicated
in a number of diseases including leukaemia, metabolic
syndromes and cardiovascular disease .
Mammalian Tribbles can interact with components of
the mitogen-activated protein (MAP) kinase pathway and
act as adaptor proteins to modulate the strength and
output of kinase signalling cascades [10–12]. Like fly Tribbles
[7, 13], mammalian Tribbles bind to the basic leucine
zipper (bZIP) transcription factors. Trib1 and Trib2 can
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induce degradation of several CCAAT/enhancer-binding
protein (C/EBP) members in a context-dependent manner
[1, 14, 15]. Trib3 binds to and inhibits the transcriptional
activity of both activating transcription factor 4 (ATF4)
and C/EBP homologous protein (CHOP) in cultured cell
lines [16–19], although the in vivo functional relevance of
such protein interactions remains unknown.
Given its extensive connections to different cellular
processes, many questions remain to be answered regarding
the role of Tribbles at the organismal level. No induction of
peptide after Drechmeria infection 3 (NIPI-3) is the single
Tribbles protein in Caenorhabditis elegans . A role for
nipi-3 in the innate immune response was previously
uncovered through the isolation of a partial loss-of-function
mutation, which contains a missense mutation in the
pseudokinase domain . NIPI-3 is required for the
upregulation of antimicrobial peptide (AMP) gene
expression following infection by the fungus Drechmeria
coniospora . It acts upstream of a p38 MAP kinase
(MAPK) pathway consisting of NSY-1/MAPKKK,
SEK1/MAPKK and PMK-1/MAPK [20, 21]. Both NIPI-3
and all components of the MAPK cascade are required
cell autonomously in the epidermis during the immune
In this study, we generated null mutations of nipi-3
and uncovered a novel role in animal development and
viability. The lethality of nipi-3 null animals is
completely suppressed by loss of function in CEBP-1, a C.
elegans member of the C/EBP family, previously known
to be required for adult sensory axon regeneration and
neuronal stress responses [22, 23]. Unexpectedly, loss of
function in components of the PMK-1/p38 MAPK cascade
also suppresses the lethality of nipi-3 null animals. In nipi-3
mutants, the levels of activated PMK-1 are increased, in a
cebp-1-dependent manner. Through chromatin
immunoprecipitation and deep sequencing (ChIP-seq) analyses and
identification of target genes, we found that CEBP-1 binds
to a conserved DNA motif. Our analyses of candidate target
genes of CEBP-1 suggest a functional enrichment in
development and stress responses. Importantly, in contrast to its
role in innate immunity, we show that NIPI-3 acts in
multiple tissues to negatively regulate transcriptional expression
of cebp-1. This inhibition of CEBP-1 by NIPI-3 is also
required across multiple tissues to enable larval development
and maintain fecundity. The coordinated inhibition of
CEBP-1 by NIPI-3 in multiple tissues reveals novel
requirements for systemic regulation of signalling pathways
in organism development.
repeats (CRISPR)-Cas9 genome editing [24–27] to
generate two deletion alleles of nipi-3 (fr148 and ju1293) and a
green fluorescent protein (GFP) knock-in (KI) (fr152)
(Fig. 1a; see Methods and below). The fr148 and ju1293
deletion alleles remove 1.6 kb and 0.6 kb of the 5′ region
of the gene, respectively (Fig. 1a), resulting in molecular
nulls for nipi-3. The phenotypes of homozygous mutants
of either nipi-3 deletion allele, designated as null (0), were
indistinguishable (Fig. 1c, d). Mutants arrested
development at the second to third larval stages (L2–L3) (see
below) and eventually died between 5–10 days after
hatching. When compared to wild-type larvae at the same stage
(2 days post-hatching), nipi-3(0) arrested larvae displayed
a small and dumpy body morphology. At 3 days
posthatching, wild-type animals reached the adult stage, as
evidenced by fusion of seam cells (lateral epidermal cells),
formation of adult alae and the vulva (Fig. 1b, f ). By
contrast, all age-matched nipi-3(0) animals were arrested at
L2–L3, as the seam cells did not fuse, adult alae were not
observed and the vulval invagination did not occur (Fig. 1c,
d, f ). In these mutant animals, the germline also appeared
to be arrested, generally at L3 based on the size of the
gonad and the number of germ cells (Fig. 1c, d, g).
Occasionally, in nipi-3(0) animals with longer bodies, we
observed some sperm or a few unfertilized oocytes. The
nipi-3(0) animals also exhibited an abnormal pharyngeal
morphology (Additional file 1: Figure S1). We rescued the
larval lethality and sterility of nipi-3(0) by expressing the
wild-type nipi-3 genomic DNA as high-copy-number
extrachromosomal arrays (Fig. 1a, e, h; Methods). As
expression from such transgenes is silenced in the
germline , this result indicates that the larval lethality
and germline development defects of nipi-3(0) are both
primarily due to its function in somatic tissues. Thus,
the analyses of nipi-3 null animals indicate an essential
somatic role of nipi-3 in organism development.
Knocking-in gfp to the nipi-3 locus, which produced a
protein tagged at its N-terminus (GFP::NIPI-3), had no
adverse effect; KI animals (fr152) were fully viable and
indistinguishable from wild type in growth and movement.
We observed GFP expression in the epidermis, intestine
and in neurons (Fig. 1i), consistent with the previously
reported expression pattern obtained using transgenic
transcriptional reporters . Interestingly, although NIPI-3
does not have a clearly identifiable nuclear-localization
signal, GFP::NIPI-3 expression was observed
predominantly in the nuclei (Fig. 1i), with an overall intensity
peaking at the L2–L3 stages. This nuclear localization suggests
a role for NIPI-3 in regulation of gene expression.
Loss of cebp-1 suppresses the lethality, but not the innate
immune response defect, of nipi-3 null animals
To dissect the molecular mechanisms involving NIPI-3, we
undertook a yeast two-hybrid screen using the full-length
Fig. 1 C. elegans Tribbles nipi-3 is required for larval development and viability. a The nipi-3 locus. Top, nipi-3 encodes a pseudokinase of the
Tribbles family. Middle, nipi-3 deletions generated using CRISPR-Cas9 genome editing. Bottom, the extent of the nipi-3 genomic region used to
rescue the deletion mutants. b–e Bright-field images of worms at 3 days post-hatching; wild type (b), nipi-3 null mutants (c,d) and a transgenic
animal (Tg) expressing the wild-type nipi-3 genomic DNA in a nipi-3(0) background (e). f Fluorescence images of worms expressing AJM-1::GFP
reporter in the epithelial cells to allow visualization of seam cells. g Overlaid differential interference contrast (DIC) and fluorescence image of a
nipi3(fr148) mutant at 3 days post-hatching expressing lag-2p::GFP reporter. This image corresponds to the boxed region in d. Two distal tip cells (DTC) are
shown in green (arrow), and the germline of arrested nipi-3 null larva is denoted by a dotted red line. h Body length (μm) of worms at 3 days
posthatching. Each dot represents a single animal measured as shown; each red line represents the mean value. ***P < 0.001; ns, not significant (one-way
ANOVA with Tukey’s post hoc tests). i Fluorescence images of endogenous NIPI-3 expression visualized in the GFP KI strain (fr152). Expression is
observed in the nuclei (yellow arrows) of the epidermis (left panel), the intestine (upper right panel) and the head neurons (lower right panel)
NIPI-3 protein as bait (Cypowyj S. et al., manuscript in
preparation). One prominent candidate interacting partner
was CEBP-1, a member of the C/EBP family of
transcription factors . The CEBP-1 protein consists of 319 amino
acids, with a bZIP domain at its C-terminus. In further
analyses using the yeast two-hybrid assay, we found that the
N-terminal region (amino acids 1–115) of CEBP-1 was
sufficient for binding NIPI-3 (Additional file 2: Figure S2). This
interaction is reminiscent of those observed for fly and
vertebrate Tribbles proteins, which bind and degrade C/EBP
family proteins [1, 7, 13–15, 29], with human Trib1 binding
the N-terminus of C/EBPα . Thus, the ability of Tribbles
and C/EBP proteins to interact directly is likely conserved
from C. elegans to humans.
To understand the functional significance of the
observed protein interaction, we next performed genetic
analysis using null mutations of nipi-3 and cebp-1. Null
mutants of cebp-1 show normal development and body
appearance. Remarkably, cebp-1(0) completely suppressed
the growth and fertility defects of nipi-3(0) mutants
(Fig. 2b, h; Methods). cebp-1(0) also completely suppressed
the body size defect and the developmental delay observed
at 25 °C of the partial loss-of-function nipi-3(fr4) mutants
(Additional file 3: Figure S3). NIPI-3 is also known to be
Fig. 2 nipi-3(0) lethality is suppressed by loss of cebp-1 or components of a PMK-1/p38 MAPK cascade. a Schematic overview of the forward
genetic screen designed to identify the suppressors of nipi-3(0) larval arrest and lethality. b–g The mutations isolated from the nipi-3(0) suppressor
screen and the deletion null mutations tested for nipi-3(0) suppression assay are shown in the left column and bright-field images of worms at
3 days post-hatching are shown in the right column. h Body length (μm) of worms at 3 days post-hatching. Each dot represents a single animal
measured as shown; each red line represents the mean value; some data are replicated from Fig. 1 as shown with darker grey dots. ***P < 0.001;
ns, not significant (one-way ANOVA with Tukey’s post hoc tests)
necessary for the epidermal innate immune response upon
fungal infection . The expression of AMP genes after
fungal infection is highly induced in wild-type animals and
abrogated in nipi-3(fr4) mutants . However, cebp-1(0)
nipi-3(0) animals did not exhibit an induction of the AMP
gene nlp-34 upon infection (Additional file 4: Figure S4a).
Further, cebp-1(0) nipi-3(0) animals expressing wild-type
cebp-1 in a tissue-specific manner in either the
epidermis or neurons also showed no AMP gene induction
(Additional file 4: Figure S4a). These results indicate
that loss of cebp-1 does not suppress the immune
response defect in nipi-3(0). As cebp-1(0) strongly impairs
sensory axon regeneration after laser axotomy , we
also tested whether nipi-3 affected posterior lateral
microtubule (PLM) axon regeneration. Neither nipi-3(0) nor
nipi-3(fr4) showed significant effects in axon regeneration,
and cebp-1(0) nipi-3(0) double mutants showed impaired
axon regeneration similar to cebp-1(0) (Additional file 4:
Figure S4b). These results show that the genetic
interaction between nipi-3 and cebp-1 is highly specific for
larval development and organism fecundity.
Loss of the PMK-1/p38 MAPK pathway also suppresses
To gain further insight into the mechanism underlying
nipi-3’s role in animal development, we performed a
forward genetic screen for suppressors of nipi-3(0)
lethality. We mutagenized nipi-3(ju1293); Tg[nipi-3(+);
myo-2p::mCherry] animals (Methods). Among their F2
progeny, we isolated fertile animals that had lost the
rescuing transgene (Fig. 2a) and established multiple
suppressor lines (genotypes designated as nipi-3(0);
suppressor). We screened ~16,000 haploid genomes and
identified 7 independent suppressor alleles. All the
identified suppressors in this study were associated with a full
reversion of the nipi-3(0) lethality; fertile adults could be
propagated without the nipi-3(+) rescuing transgene.
We characterized several suppressor mutations using
candidate gene analyses in combination with whole
genome sequencing. One suppressor caused a missense
mutation in the bZIP domain of cebp-1, and behaved
like cebp-1(0) (Fig. 2b, h). Among the other suppressors
of nipi-3(0), we found missense alterations in TIR-1/the
sterile alpha (SAM) and Toll-interleukin receptor (TIR)
motif-containing protein SARM), NSY-1/MAPKKK,
SEK1/MAPKK and MAK-2/MAPK-activated protein kinase
(MAPKAPK) (Fig. 2c–f). These mutations are located in
known functional domains, including the TIR domain for
TIR-1, the kinase domains for NSY-1, SEK-1 and MAK-2,
and the DUF4071 domain, commonly found at the
Nterminus of many serine-threonine kinase-like proteins, for
NSY-1 (Fig. 2c–f). We then tested known null mutants in
each of these genes and found them to suppress nipi-3(0)
to the same degree as our suppressor mutations, as shown
by quantification of the body length (Fig. 2b–f, h;
Additional file 5: Figure S5a). Thus, loss of function in tir-1 and
these three kinase genes causes strong suppression of
NIPI-3 has a specific role in the regulation of
epidermal defense genes. It acts together with TIR-1, NSY-1
and SEK-1, as well as several other genes including tpa-1,
pmk-1 and sta-2 [20, 21, 30]. We therefore tested mutants
for these three latter genes for their genetic interaction
with nipi-3(0). We found that loss of pmk-1, but not tpa-1
or sta-2, suppressed the lethality of nipi-3(0) (Fig. 2 g, h
for pmk-1; Additional file 5: Figure S5b for tpa-1 and
sta-2). Loss of pmk-1 resulted in suppression of nipi-3(0)
phenotypes similar to the other suppressor mutants such
that nipi-3(0); pmk-1(0) double mutants developed into
fertile adults with adult alae and vulva. The rescue of body
size, however, was not complete (Fig. 2 g, h). As cebp-1 is
involved in two other MAPK cascades known for their
roles in adult axon regeneration [22, 31, 32], we tested
mutations in several other candidate genes and found that
loss of function in dlk-1, pmk-3, mlk-1 or kgb-1 did not
suppress nipi-3(0) defects (Additional file 5: Figure S5b).
Together, our analyses from both the forward genetic
screening and test of candidate mutants reveal a
previously unknown role of CEBP-1 in larval development
mediated by NIPI-3, and a novel genetic interaction between
NIPI-3 and the PMK-1/p38 MAPK cascade.
NIPI-3 inhibits PMK-1 phosphorylation via CEBP-1
To dissect the mechanism underlying the interaction
between PMK-1/p38 MAPK and NIPI-3, we first asked how
the levels of active PMK-1 might be altered in nipi-3(0)
suppressor animals. We performed a Western blot analysis
using an anti-phospho-p38 MAPK antibody that
specifically recognizes phosphorylated, active PMK-1 . We
made protein lysates from animals at 1 day post-hatching
(L2) because nipi-3(0) animals at this stage were as healthy
as the wild type. We observed that levels of active PMK-1
were significantly increased in nipi-3(0), but remained
similar to wild type in cebp-1(0) and cebp-1(0) nipi-3(0)
animals (Fig. 3a, b). Phosphorylated PMK-1 was
undetectable in nipi-3(0) sek-1(0) animals, consistent with PMK-1
being activated by SEK-1 (Fig. 3a, b). The total PMK-1
levels were likely unchanged in nipi-3(0), as the mRNA
levels of pmk-1 were comparable between nipi-3(0) and
wild type when assessed by quantitative RT-PCR (Fig. 3c).
We note that mak-2(0) did not affect phosphorylated
PMK-1 (Additional file 6: Figure S6), suggesting that
MAK-2 likely acts downstream of, or in parallel to, PMK-1.
Together, these results suggest that the abnormally high
levels of phosphorylated PMK-1 in nipi-3(0) are dependent
Fig. 3 NIPI-3 represses PMK-1 phosphorylation via cebp-1 and represses cebp-1 transcription. a Western blot analysis on total protein lysate from
various animal strains using the indicated antibodies, α-phospho-p38 MAPK antibody to detect a phosphorylated form of PMK-1 proteins (p-PMK-1)
or α-actin antibody as a loading control. b Densitometric quantifications of immunoblot signals normalized to actin. n = 3; error bars represent standard
error of the mean (SEM);
*** P < 0.001 (one-way ANOVA with Tukey’s post hoc tests). c Quantitative RT-PCR (qRT-PCR) analysis of pmk-1. Relative abundance of pmk-1
mRNA normalized to actin mRNA. n = 3; error bars represent SEM; ns, not significant (one-way ANOVA with Tukey’s post hoc tests). d–j
Fluorescence images of cebp-1p::GFP reporter animals at L2. k qRT-PCR analysis of cebp-1 in WT, nipi-3(fr4), nipi-3(0) animals. Relative abundance of
cebp-1 mRNA normalized to actin mRNA. n = 3; error bars represent SEM; *P < 0.05; **P < 0.01; ns, not significant (one-way ANOVA with Tukey’s
post hoc tests). l, m Confocal fluorescence images (z-stack) of col-154p(epidermis)::CEBP-1::GFP reporter animals at L2. n Quantification of GFP
intensity measured in each epidermal nucleus (10 per animal). n = 6; error bars represent SEM; ns, not significant (Student’s unpaired t test). Primary
data for panels (b, c, and k) are provided in Additional file 14
NIPI-3 represses the transcription of cebp-1 in multiple
To dissect how NIPI-3 inhibits CEBP-1, we examined
whether cebp-1 levels were altered in nipi-3(0) and in
each nipi-3(0) suppressor. Transcriptional reporters of
cebp-1 (Tg[cebp-1p::GFP]) were broadly expressed in most
post-embryonic tissues, including epidermis, muscles,
pharynx, intestine and neurons (Fig. 3d–j). Strikingly, the
expression of the cebp-1 transcriptional reporter was
highly and significantly increased in both nipi-3(fr4) and
nipi-3(0) mutants, compared with wild-type animals
(Fig. 3d–f ). Quantitative RT-PCR analysis also showed
significantly increased expression of cebp-1 mRNAs in nipi-3
mutant animals (Fig. 3 k). Consistent with the observed
transcriptional regulation, we found that a translational
CEBP-1::GFP reporter driven by a heterologous epidermal
promoter showed no detectable differences in GFP
expression in a nipi-3(0) background (Fig. 3 l–n). The increased
expression of cebp-1p::GFP in nipi-3(0) was reduced to
normal levels when a nipi-3(+) transgene was introduced
(Fig. 3 g), indicating that NIPI-3 represses the
transcription of cebp-1. Additionally, the transcriptional repression
of cebp-1 by NIPI-3 was largely independent of the PMK-1
pathway since cebp-1p::GFP expression in nipi-3(0)
remained high in animals that also carried a null mutation
of nsy-1, pmk-1 or mak-2 (Fig. 3 h–j). Together, the results
show that NIPI-3 negatively regulates expression of cebp-1
at the transcriptional level and that CEBP-1 acts upstream
of the PMK-1 pathway.
The tight regulation of cebp-1’s expression level is
critical for animal viability. We found that suppression of
nipi-3(0) by cebp-1(0) was semi-dominant, as cebp-1(0)/+
caused partial but significant suppression of the short body
length of nipi-3(0) mutants (Additional file 7: Figure S7).
Moreover, in a wild-type background, the transgene
eft3p::CEBP-1::GFP, which drives strong and ubiquitous
expression of CEBP-1, caused dose-dependent lethality
(see Methods). In addition, expression of a full-length
functional translational reporter of cebp-1
(cebp-1p::CEBP1::GFP) in nipi-3(0) mutant animals exacerbated
developmental defects and accelerated larval lethality, while the
same transgene showed no such effects in a wild-type
background. Together, these results support the
conclusion that the lethality observed in nipi-3(0) mutants is a
direct consequence of cebp-1 overexpression.
Overexpression of truncated forms of CEBP-1 suppresses
To dissect the molecular basis of CEBP-1’s role in animal
development, we expressed truncated forms of CEBP-1
lacking the bZIP domain (amino acids 1–230 or 1–115),
or lacking the N-terminus (amino acids 237–319) in
nipi3(0) mutants. Surprisingly, in stark contrast to the strong
lethality caused by overexpressing full-length CEBP-1 in
nipi-3(0) mutants, we found that expression of either the
N-terminal fragment or the bZIP domain of CEBP-1 alone
resulted in significant suppression of nipi-3(0) defects
(Fig. 4a–d). Overexpression of either the N- or C-terminal
truncated protein caused no defects either in the wild-type
or cebp-1(0) backgrounds. Among the three CEBP-1
fragments, the expression of C-terminal CEBP-1 (amino acids
237–319) showed the most effective rescue, judged by the
fecundity of the transgenic lines and quantitative
comparisons of body length (Fig. 4c, d). The expression
levels of CEBP-1(amino acids 1–230)::GFP were markedly
increased in nipi-3(0), presumably reflecting the
transcriptional regulation of cebp-1 described above. We noticed
the fluorescence intensity was most strongly increased in
the epidermis and neurons, throughout the head region
and in the ventral nerve cords (Fig. 4e–g). These
observations suggest that the truncated forms of CEBP-1 act in a
dominant negative manner to inhibit the activity of the
CEBP-1 binds conserved DNA motifs in genes regulating
development and stress response
To gain further insight into CEBP-1’s function in animal
development, we next sought candidate target genes of
CEBP-1 by performing ChIP-seq analysis on transgenic
animals expressing a functional FLAG-tagged CEBP-1
protein in a cebp-1(0) background (Methods). We found
209 CEBP-1 ChIP-seq peaks in the genome that were
associated with 212 coding genes (Additional file 8: Table
S1). CEBP-1 peaks were preferentially located within the
promoter regions of the target genes (169 genes, 79 %),
less frequently within introns (43 genes, 21 %) and never
We then performed motif analysis of the genomic
regions bound by CEBP-1 using the motif discovery tools
Multiple Em for Motif Elicitation (MEME)  and
Regulatory Sequence Analysis Tools (RSAT) . The
most over-represented motif, NTTDYGAAAH, was found
in 139 out of 209 CEBP-1 ChIP-seq peak regions (Fig. 5a).
We then compared this motif with published motifs using
the motif comparison tool Tomtom  and found the
most statistically significant similarities to vertebrate C/
EBP binding motifs . The conservation of CEBP-1
binding motif further reinforces the functional parallels
between C. elegans CEBP-1 and vertebrate C/EBPs.
As CEBP-1 likely acts upstream of the PMK-1 pathway,
we searched among the targets of CEBP-1 for components
of the PMK-1 pathway and found CEBP-1 ChIP-seq peaks
present in the promoter of sek-1 (Additional file 9: Figure
S8). When we examined the mRNA levels of sek-1 by
quantitative RT-PCR in nipi-3(0) animals where cebp-1 is
overexpressed, we observed increased sek-1 mRNA levels
(Fig. 5b). In contrast, in nipi-3(0) cebp-1(0) animals the
levels of sek-1 mRNAs were similar to those of the wild
Fig. 4 Overexpression of truncated forms of CEBP-1 protein suppresses nipi-3(0) lethality. a–c Bright-field images of worms at 3 days post-hatching
expressing truncated forms of CEBP-1 proteins. d Body length (μm) of worms at 3 days post-hatching. Each dot represents a single animal measured
as shown; each red line represents the mean value; some data are replicated from Fig. 1 as shown with darker grey dots. ***P < 0.001; ns, not significant
(one-way ANOVA with Tukey’s post hoc tests). e, f Confocal fluorescence images (z-stack) of cebp-1p::CEBP-1(aa 1-230)::GFP reporter animals at L2.
g Quantification of GFP intensity measured in the region of head neurons. Error bars represent SEM; ***P < 0.001 (Student’s unpaired t test)
Fig. 5 CEBP-1 binds conserved DNA motifs in genes regulating development and stress response. a Motif logo of the most over-represented
motif among CEBP-1 ChIP-seq peaks. b qRT-PCR analysis of sek-1. Relative abundance of pmk-1 mRNA normalized to actin mRNA. n = 3; error bars
represent SEM; *P < 0.05; ns, not significant (one-way ANOVA with Tukey’s post hoc tests). Primary data are provided in Additional file 14. c Hierarchical
clustering of genes and functional classes (see Additional file 10: Figure S9 and Additional file 8: Table S1 for class labels and full data); the presence of
a gene in a class is represented by a red rectangle, its absence by blue
type. These results suggest that the abnormally high levels
of cebp-1 in nipi-3(0) can promote the expression of sek-1,
which in turn promotes the phosphorylation of PMK-1
, leading to abnormal larval development and lethality.
We then asked whether the list of potential CEBP-1
targets was enriched for genes with specific functions.
To this end, we searched for enriched categories through
an Expression Analysis Systematic Explorer (EASE)
analysis [37, 38], using our in-house database of functional
annotations as described . This includes 4600 datasets
automatically updated from multiple sources including
WormBase, FlyBase, the Kyoto Encyclopedia of Genes and
Genomes (KEGG) and relevant RNAi databases. Most
(80 %) of CEBP-1 target genes were associated with at
least one of 33 enriched functional classes (P < 10-5;
Additional file 8: Table S1). Hierarchical clustering of
the genes in each of the enriched classes identified two
main groups (Fig. 5c; Additional file 10: Figure S9;
Additional file 8: Table S1). One group is related to
development with phenotypic classes such as 'larval lethal’
or 'slow growth’ and includes genes involved in basic
cellular processes, transcription, translation or endocytosis.
The second group is related to the response to biotic or
abiotic stress, including response to cadmium, hygromycin
or bacterial toxins. Interestingly, McEwan et al. have found
that most of the genes upregulated in nipi-3(fr4) mutants
are also induced by the translational inhibitory toxin
ToxA. Out of the 14 stress-related classes associated with
the cebp-1 targets, 10 are shared with those found for
genes upregulated in nipi-3(fr4) (D.L. McEwan, http://
0334-6#Bib1; P < 10-10; Additional file 8: Table S1). On the
other hand, consistent with the fact that the nipi-3(fr4)
allele does not provoke larval lethality, only 1 out of the 10
classes in the development cluster is shared between the
cebp-1 targets and the genes upregulated in nipi-3(fr4).
These analyses suggest that CEBP-1 regulates genes
functioning in development and in stress responses, and might
have a particularly important impact on organismal
physiology when overexpressed in a nipi-3 mutant context.
NIPI-3 is required in multiple tissues to ensure proper
We next asked in which tissue the expression of nipi-3(+)
is required for animal viability. We expressed nipi-3(+) in
a tissue-specific manner, using intestinal, epidermal and
pan-neuronal promoters, in the nipi-3(0) background
(Fig. 6a–f ). In contrast to the complete rescue of body size
and lethality in nipi-3(0) mutants expressing nipi-3(+)
under its own promoter (Fig. 1e, h), expressing nipi-3(+)
in individual tissues failed to rescue the developmental
arrest (Fig. 6a–c, f ). Pan-neuronal expression of nipi-3(+)
resulted in slightly increased body length of nipi-3(0)
3 days post-hatching (Fig. 6c, f ). Since nipi-3 activity
strongly inhibits cebp-1 transcription in the epidermis and
neurons (Fig. 4e–g), we expressed nipi-3(+) in both of
these tissues together, and found that these transgenic
animals showed an increased body size (Fig. 6e, f ), compared
to those expressing nipi-3(+) in each tissue alone, or in the
intestine and epidermis simultaneously (Fig. 6a–d, f ).
Some of the transgenic animals with a body length closer
to that of wild-type animals showed improved somatic
and germline development, with formation of vulva and
adult alae, and produced a few viable but infertile progeny.
When we expressed nipi-3(+) in all three tissues together,
the transgenic animals showed no further improvement of
body size compared to those expressing nipi-3(+) in both
the epidermis and neurons (Fig. 6f), and did not
recapitulate the rescue of lethality associated with the expression
of nipi-3 under its own promoter. Thus, we conclude that
NIPI-3 is required in multiple tissues, particularly
epidermis and neurons, for animal growth and development.
Suppression of nipi-3(0) by cebp-1(0) also requires a block
of CEBP-1 activity in multiple tissues
Conversely, we asked whether expression of cebp-1(+) in
a single tissue might cause the viable nipi-3(0) cebp-1(0)
animals to die. We expressed cebp-1(+) using the intestinal,
epidermal and pan-neuronal promoters in cebp-1(0)
nipi3(0) double mutants (Fig. 6 g). Expression of cebp-1(+) in
individual tissues was insufficient to produce larval lethal
phenotypes in cebp-1(0) nipi-3(0) animals. Interestingly,
epidermal or neuronal expression of cebp-1(+) in cebp-1(0)
nipi-3(0) double mutants caused short body length, but not
in cebp-1(0) mutants (Fig. 6 g). Co-expression of cebp-1(+)
in the epidermis and neurons resulted in further reductions
in body length, although these were not as severe as those
in nipi-3(0) animals (Fig. 6 g). In addition, we noticed that
the same transgenes expressing cebp-1(+) in both epidermis
and neurons caused an abnormal pharyngeal morphology
in cebp-1(0) nipi-3(0) animals, similar to that seen in
nipi3(0) mutants (Additional file 1: Figure S1). Together, our
data suggest that the tight regulation of both NIPI-3 and
CEBP-1 in multiple tissues is required in a systemic manner
for normal animal growth and development.
C. elegans Tribbles NIPI-3 was identified on the basis of
its roles in host defense [20, 21]. Here, through generation
and analyses of null alleles, we find nipi-3 to be essential
for animal development and viability. Remarkably, the
larval arrest and lethality caused by complete loss of nipi-3 is
fully suppressed by loss of cebp-1, a C/EBP bZIP
transcription factor, or by loss of function in the PMK-1/p38
MAPK cascade including tir-1/SARM, nsy-1/MAPKKK,
sek-1/MAPKK and pmk-1/MAPK. Our data show that
complete elimination of the function of nipi-3 causes
abnormally high expression of CEBP-1, and activation
Fig. 6 Tight regulation of both NIPI-3 and CEBP-1 is required in multiple tissues for proper organism development. a–e Bright-field images and (f)
the body length of worms expressing tissue-specific nipi-3(+) driven by the intestinal (mtl-2), epidermal (col-12) or pan-neuronal (rgef-1) promoters
in a nipi-3(0) background. g The body length of worms expressing tissue-specific cebp-1(+) driven by the intestinal (ges-1), epidermal (col-154) or
pan-neuronal (rgef-1) promoters in a nipi-3(0) cebp-1(0) background. f, g Each dot represents a single animal measured as shown; each red line
represents the mean value; some data are replicated from Figs. 1 and 2 as shown with darker grey dots. **P < 0.01; ***P < 0.001; ns, not significant
(one-way ANOVA with Tukey’s post hoc tests). h Working model for NIPI-3 function in C. elegans development. In wild type, presence of NIPI-3
keeps cebp-1 expression level optimal for coordinated tissue development. In nipi-3(0), however, cebp-1 and sek-1 are overexpressed and in turn
PMK-1 is hyperactivated
of PMK-1 MAPK. This then disrupts development and
leads to death. The level of sek-1 mRNA is increased in
nipi-3(0) mutants but not in cebp-1(0) or in nipi-3 cebp-1
animals. The level of phosphorylated (active) PMK-1
follows the same trend. Coupled with our ChIP-seq analyses
and genetic epistasis data, this suggests that CEBP-1 acts
as a direct positive regulator of sek-1. The PMK-1 pathway
is therefore activated when CEBP-1 expression is high in
nipi-3(0). On the other hand, cebp-1 expression levels
remain high in nipi-3(0); pmk-1(0) animals, confirming that
CEBP-1 does not act downstream of the PMK-1 pathway.
Together, these results suggest that NIPI-3 negatively
regulates the PMK-1 MAPK cascade, via CEBP-1, to promote
animal viability and development (Fig. 6 h).
In innate immunity, however, nipi-3 cell-autonomously
promotes or enhances the same p38 kinase cascade to
activate host defense in the epidermis . It has been
shown that overexpression of sek-1 in the epidermis
rescues the block of AMP induction in nipi-3 mutants upon
fungal infection , and an overexpression of nipi-3
provokes an increase in the constitutive expression of AMP
which is dependent on the p38 cascade . It is
intriguing that NIPI-3 appears to be capable of activating
or inhibiting PMK-1/p38 in the epidermis at different
times or under different conditions (infection versus
development). How might NIPI-3 achieve this dual role
under different stresses and in altered cellular contexts?
As Tribbles proteins are well known to act as adaptors,
NIPI-3 might be regulated via binding with other
cofactors only present under specific circumstances. Indeed,
we find that other upstream and downstream components
of the epidermal immune response cascade are not
involved in the developmental regulation described here.
Thus, the core PMK-1/p38 MAPK cassette has evolved
context-specific functions depending on different
upstream regulators or co-factors [40, 41]. Members of the
Tribbles family in other species have been mostly studied
in the context of cell proliferation, adipocyte tissue
differentiation, energy metabolism and immunity, where they
function in a cell-autonomous manner. Our discovery of
the opposing roles of NIPI-3 in development and in the
immune response illustrates how cellular context can alter
the function of highly conserved signalling molecules.
Negative regulation of C/EBP by Tribbles has been
observed throughout the animal kingdom. Drosophila and
mammalian Tribbles bind and degrade C/EBP proteins
[1, 7, 13–15]. We find that C. elegans NIPI-3 represses
the transcription of cebp-1, which has important
functional consequences in vivo. This form of regulation has
not been reported in other organisms. Given its nuclear
localization, NIPI-3 may inhibit the transcription of
cebp-1 by interfering with other transcription factor(s).
The promoter of cebp-1 contains putative CEBP-1
binding consensus motifs, raising the possibility that NIPI-3,
by binding to CEBP-1, may also alter the transcriptional
activity of CEBP-1.
NIPI-3 is required to control CEBP-1 levels in multiple
tissues for animal development and viability. Consistent
with the inhibition of CEBP-1 expression by NIPI-3 in
the epidermis and neurons, simultaneous expression of
nipi-3(+) in both tissues makes a noticeable contribution
to animal development in nipi-3(0) mutants, compared
with nipi-3(+) expression in single tissues. Conversely,
simultaneous expression of cebp-1(+) in both epidermis
and neurons causes noticeable defects in animal
development in nipi-3(0) cebp-1(0) mutants, compared with
cebp1(+) expression in single tissues. Thus, a tightly regulated
coordination of these two genes’ interactions in multiple
tissues is required to ensure proper development.
A key conclusion from our study is that the precise
control of CEBP-1 and PMK-1/p38 MAPK pathways in
multiple tissues is critical for organismal development. NIPI-3
acts as a master regulator to prevent improper activation
of CEBP-1 and PMK-1, whose hyperactivation during
development has deleterious consequences. Interestingly,
hyperactivation of PMK-1/p38 was previously shown to
block larval development when the endoplasmic reticulum
unfolded protein response was altered . Moreover,
innate immune activation with a xenobiotic that provides
protection from bacterial infection in the adult has been
shown to provoke a growth delay during development
. Subsequently, an elegant genetic suppressor screen
revealed that mutations in the PMK-1/p38 MAPK
pathway suppressed this developmental phenotype . Thus,
the NIPI-3/CEBP-1 axis is a key mechanism by which
immune effector expression is held in check during
During normal development, both CEBP-1 and PMK-1
are maintained at a basal level by NIPI-3. The levels of
inducible signalling from these pathways are, however,
important for animals to protect themselves or to promote
repair. For instance, following fungal infection, NIPI-3
promotes the PMK-1/p38 MAPK signalling pathway in
the epidermis . Thus, animals can successfully defend
themselves from fungal infection with activated PMK-1
locally in the epidermis, while survival is not affected
as PMK-1 remains inactive in other tissues. Similarly,
CEBP-1 is known to play a key role in neuronal stress
responses [22, 23, 45], and we identified potential CEBP-1
target genes that are involved in different stress responses.
Moreover, a concomitant study has identified NIPI-3 as a
negative regulator of CEBP-1 in intestinal defense against
the bacterial toxin ToxA (McEwan et al.,
http://bmcbiol.biomedcentral.com/articles/10.1186/s12915-016-03346#Bib1). An important challenge for the future will be to
understand how NIPI-3 regulates its downstream pathways
and how NIPI-3 itself is regulated depending on
developmental and environmental conditions. Understanding the
molecular mechanism of this systemic, coordinated
regulation should advance our knowledge of how animal
development can be maintained in the face of environmental
We showed a novel essential role for the C. elegans Tribbles
homolog NIPI-3 in animal development and viability,
which requires NIPI-3’s function in multiple tissues. NIPI-3
acts as a master regulator to prevent improper activation of
a C/EBP transcription factor and a conserved PMK-1/p38
MAPK signalling cascade known to control innate
immunity. These findings suggest that innate immune responses
are tightly controlled for proper organismal development.
Strains, transgenes and plasmids
C. elegans strains were maintained under standard
conditions at 20 °C unless otherwise mentioned. The wild
type was the N2 Bristol strain . New strains were
constructed using standard procedures, and all
genotypes were confirmed by PCR or sequencing. All strains
and their genotypes used in this study are described in
Additional file 11: Table S2. Extrachromosomal array
transgenic lines were generated as described previously
. Expression constructs, transgenes and strain
genotypes are also summarized in Additional file 11: Table
S2. For all experiments, at least two independent
transgenic lines were examined and quantitative data are
shown for one. In our studies of cebp-1 dosage effect,
we could not generate transgenic animals when
injecting 10 ng/μl of eft-3p::CEBP-1::GFP transgenes into
wild-type animals, while many transgenic lines were
obtained when cebp-1::gfp DNA was injected with lower
concentrations (i.e. 1 ng/μl or 0.01 ng/μl). All plasmids
used in this study are described in Additional file 12:
CRISPR-Cas9-mediated deletion and GFP KI
We generated the nipi-3(ju1293) deletion allele using the
co-CRISPR method [25, 48]. We used four single guide
RNAs (sgRNAs) targeting the N-terminus of the nipi-3
gene (Additional file 12: Table S3a). U6p::nipi-3 sgRNAs
were generated by Gibson assembly and injected into
cebp-1(tm2807) worms, using standard methods, in
mixtures composed of 30 ng/μl of each nipi-3 sgRNA, 50 ng/
μl of eft-3p::Cas9-SV40NLS::tbb-2 3′UTR, 50 ng/μl of
U6p::unc-22 sgRNA and 1.5 ng/μl of myo-2p::mCherry.
For the nipi-3(fr148) deletion allele, a single sgRNA
targeting the N-terminus of the nipi-3 gene (Additional file 12:
Table S3a) was injected into cebp-1(tm2807) worms, in
mixtures composed of 50 ng/μl of nipi-3 sgRNA, 30 ng/μl
of eft-3p::Cas9-SV40NLS::tbb-2 3′UTR and 30 ng/μl of
col-12p::dsRed . Note that we also injected many
wildtype worms with various combinations of nipi-3 sgRNAs,
but failed to isolate any deletion alleles.
GFP KI in the nipi-3 locus, nipi-3(fr152) was generated
with the same mixture as for nipi-3(fr148), providing
30 ng/μl of the nipi-3 repair template pSO1. This template
was generated by Gibson assembly in the self-excising
(SEC) cassette containing vector pDD282  with 716 bp
and 518 bp homology arms upstream and downstream of
the nipi-3 start codon, respectively (Additional file 12:
Genetic screen for nipi-3(0) lethality suppressors
nipi-3(ju1293) mutant animals carrying juEx6807[nipi-3
genomic DNA; myo-2p::mCherry] were mutagenized using
45 mM ethyl methane sulphonate (EMS) following
standard procedures as described . Animals were
distributed onto nematode growth media (NGM) plates seeded
with Escherichia coli OP50 and screened in the F2
generation for normal animal growth reaching adulthood
without expressing the transgene (no pharyngeal mCherry)
under a fluorescence dissecting microscope.
Mapping and cloning of nipi-3 suppressor alleles
We first performed a conventional Sanger sequencing
analysis for all suppressor alleles for cebp-1 and determined
that the ju1367 allele affected cebp-1. We next sequenced
mak-2, which was previously known to act in the same
pathway as cebp-1 in neurons , and found ju1349 and
ju1352 alleles to affect mak-2. All other suppressors were
analysed by whole genome sequencing analysis and
singlenucleotide polymorphism (SNP) mapping following
established methods . Briefly, genomic DNA was prepared
using a Puregene Cell and Tissue Kit (Qiagen) according to
the manufacturer’s instructions, and 20X coverage of
sequences was obtained using a 90 bp paired-end Illumina
HiSeq 2000 at Beijing Genomics Institute (BGI Americas).
The raw sequences were mapped to the C. elegans
reference genome (WS220/ce10) using Burrows-Wheeler
Aligner (BWA)  in the Galaxy platform
(http://usegalaxy.org) . Following subtraction of the
nucleotide variants in the original strains, we generated a list
of candidate genes containing unique homozygous
nucleotide variants that were predicated to alter the
function of the gene. We then confirmed the causality of
the candidate genes by testing the known null alleles
on the suppression of nipi-3(0).
Body length analysis
To examine the body length of animals during
development, we obtained synchronized animals. Briefly, 5–15
gravid adults were placed on a seeded NGM plate to
allow egg-laying for 3 h. Eggs laid during this period
were incubated at 20 °C for 72 h, and the animals were
then mounted on 2 % agarose pads containing a drop of
2.5 mM levamisole and photographed with a Zeiss
Axioplan compound microscope, using Nomarski-DIC optics
and an attached AxioCam digital camera. ImageJ software
(National Institutes of Health (NIH), Bethesda, MD, USA)
was used to measure body length by drawing a freehand
midline from the tip of the nose to the tip of the tail of
Western blot analysis
Worms of each genotype (80–100 individuals of L2 stage
worms) were collected and washed with M9 buffer and
boiled in sodium dodecyl sulphate (SDS) sample buffer
for 10 min and loaded onto SDS-polyacrylamide gel
electrophoresis (PAGE) gel (Bio-Rad). A 1:500 dilution of
rabbit anti-phospho-p38 MAPK (Cell Signaling, #9211)
and a 1:10,000 dilution of mouse anti-actin (MPbio,
#08691001) were used as primary antibodies. ImageJ was
used to quantify the intensity of the immunoblot bands.
Quantitative real-time PCR was performed as
previously described . Sequences of primers are given
in Additional file 12: Table S3b. To collect
synchronized nipi-3(0) homozygous animals, we maintained
nipi-3(0) animals on cebp-1 RNAi plates. Gravid adults
were then treated by bleaching solution to collect
nipi-3(0) embryos, which were placed directly on
regular NGM plates for 2 days in parallel to other strains.
Fluorescence microscopy and axon regeneration by laser
Animals were mounted on 2 % agarose pads and
immobilized with 2.5 mM levamisole. For transcriptional
cebp-1p::GFP, GFP expression was imaged with a Zeiss
Axioplan compound microscope, using Nomarski optics
and an attached AxioCam digital camera. Translational
CEBP-1::GFP expression was imaged with a Zeiss LSM710
confocal microscope for quantitative analyses. For confocal
images, z stacks were obtained and maximum projection
images were created using Zeiss Zen 2012 software. ImageJ
was used to measure the GFP intensity at the nerve ring
area for cebp-1p::CEBP-1::GFP and at each nucleus (10 per
animal) for col-154p::CEBP-1::GFP. GFP::NIPI-3 KI
expression was imaged with a spinning disk confocal microscope
as described  to improve the signal-to-noise ratio.
We cut PLM axons and quantified the length of
regrown axons as described .
CEBP-1 ChIP-seq analysis
We generated transgenic animals expressing a functional
FLAG-tagged CEBP-1 protein in a cebp-1(tm2807)
mutant background (cebp-1(0); juIs418
[cebp-1p::FLAG::CEBP-1::cebp-1 3′UTR]) (Additional file 13: Figure S10)
and then immunoprecipitated FLAG-CEBP-1-associated
DNA fragments using anti-FLAG antibodies (M2
antiFLAG magnetic beads; Sigma). We collected mixed stage
worms grown at 20 °C on NGM plates followed by 2 %
formaldehyde and sonicated the samples as described
. We next generated ChIP-seq DNA libraries via
ligating DNA to specific adaptors and amplification with
barcode primers, then sequenced them on the Illumina
HiSeq 2000 platform. We performed two independent
ChIP-seq experiments with parallel genomic DNA
controls prepared from the same strain. We conducted peak
calling using a CLC Genomics Workbench 6.0 (CLC
bio). To define genes associated with the peaks, we used
the annotation of transcription start site (TSS) and
transcription end site (TES) from Wormbase WS220 (http://
www.wormbase.org) and annotated the peak if it
overlapped with the gene or the 3 kb upstream of the TSS.
We then manually confirmed the peaks and associated
genes using the University of California, Santa Cruz
(UCSC) browser and an update to WS252. If the peak
was found within the promoter for one isoform and
introns for other isoforms, we categorized it as a peak
within a promoter. The ChIP-seq data are available at
the Gene Expression Omnibus [GEO:GSE83330].
MEME  and RSAT  were used to identify
overrepresented motifs from 209 CEBP-1 ChIP-seq peak
sequences, and then Tomtom  was used to compare
the most over-represented motif against a database of
known motifs in vertebrates. All informatics tools can be
found at http://meme-suite.org and http://www.rsat.eu.
Enrichment analyses were run on a newly developed
database of functional annotations including 4600
datasets  updated to Wormbase WS252 (http://
www.wormbase.org). Classes considered for enrichment
had a maximum size of 2000 genes, and the P value for
enrichment was lower than 10-5.
Yeast two-hybrid assay
Full-length or fragments of complementary DNAs (cDNAs)
were cloned into the pACT2 (Gal4 activation domain) or
pBTM116 (LexA DNA-binding domain) vectors (Clontech)
and constructs were co-transformed into yeast strain L40.
We grew transformed yeasts on agar plates with synthetic
defined (SD) minimal medium lacking leucine and
tryptophan; interactions were examined on plates with SD
medium lacking leucine, tryptophan and histidine.
Statistical analysis was performed using GraphPad Prism
5. Significance was determined using unpaired t tests for
two samples, one-way ANOVA followed by Tukey’s
multiple comparison tests for multiple samples. For two
nominal variables, Fisher’s exact test was used to evaluate the
statistical significance. P < 0.05 (*) was considered
statistically significant (* P < 0.05; ** P < 0.01; *** P < 0.001).
Additional file 1: Figure S1. CEBP-1 expression in multiple tissues causes
an abnormal pharyngeal morphology in nipi-3(0) cebp-1(0) animals. (a)
Differential interference contrast (DIC) images of worms at 3 days
posthatching. Pharynx and intestine are denoted by a dotted red and yellow
line, respectively. (b) Co-expression of cebp-1(+) in the epidermis and
neurons in nipi-3(0) cebp-1(0) animals caused pharyngeal morphology
defect. *P < 0.05; **P < 0.01; ns, not significant (two-tailed Fisher’s exact
test). (PDF 3776 kb)
Additional file 2: Figure S2. NIPI-3 interacts with CEBP-1 in yeast
two-hybrid assay. CEBP-1 variants were fused to the Gal4 activation domain
and tested for their interaction with full-length NIPI-3 fused to the LexA
DNA-binding domain. A large CEBP-1 fragment (Δ2, amino acids 1–115) was
sufficient for the NIPI-3 interaction. – refers to no growth and + refers to
growth in the absence of histidine. (PDF 433 kb)
Additional file 3: Figure S3. Loss of cebp-1 rescues the nipi-3(fr4)
phenotype. (a) Bright-field images and (b) body length of worms at 3 days
post-hatching grown at 25 °C. (b) Each dot represents a single animal
measured as shown; each red line represents the mean value. ***P < 0.001; ns,
not significant (one-way ANOVA with Tukey’s post hoc tests). (PDF 1253 kb)
Additional file 4: Figure S4. cebp-1 and nipi-3 are dispensable in
immune response and axon regeneration, respectively. (a) qRT-PCR analysis
of AMP gene, nlp-34. Relative abundance of nlp-34 mRNA normalized to
actin mRNA. n = 3. Error bars represent SEM. ***P < 0.001; ns, not significant
(one-way ANOVA with Tukey’s post hoc tests). Primary data are provided in
Additional file 14. (b) Axotomy and axon regeneration analysis. Normalized
PLM axon regrowth is shown in the bar graph. Error bars represent
SEM. ***P < 0.001; ns, not significant (one-way ANOVA with Tukey’s post
hoc tests). (PDF 472 kb)
Additional file 5: Figure S5. Quantification of the body length of
suppressor alleles identified from the screen and loss-of-function mutants
of tpa-1, pmk-1, sta-2, dlk-1, pmk-3, mlk-1 and kgb-1. (a, b) Body length of
worms at 3 days post-hatching. Each dot represents a single animal
measured as shown; each red line represents the mean value; some
data are replicated from Fig. 1 as shown with darker grey dots. ***P < 0.001
(one-way ANOVA with Tukey’s post hoc tests). (PDF 569 kb)
Additional file 6: Figure S6. Phosphorylated PMK-1 levels are unchanged
in nipi-3(0); mak-2(0) animals. (a) Western blot analysis on total protein lysate
from various animal strains using the indicated antibodies, α-phospho-p38
MAPK antibody to detect a phosphorylated form of PMK-1 proteins (p-PMK-1)
or α-actin antibody as a loading control. (b) Densitometric quantifications of
immunoblot signals normalized to actin. n = 4; error bars represent SEM; ns,
not significant (Student’s paired t test). Primary data are provided in Additional
file 14. (PDF 595 kb)
Additional file 7: Figure S7. cebp-1 shows a dosage sensitive effect in
nipi-3(0) mutants. (a) Bright-field images and (b) body length of worms at
3 days post-hatching. (b) Each dot represents a single animal measured
as shown; each red line represents the mean value. ***P < 0.001 (one-way
ANOVA with Tukey’s post hoc tests). (PDF 1501 kb)
Additional file 8: Table S1. List of CEBP-1 target genes and hierarchical
clustering of genes and functional classes (XLSX 64 kb)
Additional file 9: Figure S8. The promoter of sek-1 contains a ChIP-seq
peak of CEBP-1. The promoter region of sek-1 contains two consensus
DNA-binding motifs for CEBP-1 (black triangles). Top, the sek-1 locus. Middle,
sequencing reads from CEBP-1-IP. Bottom, sequencing reads from genomic
DNA input. (PDF 253 kb)
Additional file 10: Figure S9. Hierarchical clustering of genes and
functional classes. The presence of a gene in a class is represented by a
red rectangle, its absence in blue. See Additional file 8: Table S1 for class
labels and full data. (PDF 680 kb)
Additional file 11: Table S2. List of strains and alleles. (XLSX 18 kb)
Additional file 13: Figure S10. FLAG-tagged CEBP-1 protein rescues
PLM axon regeneration defects of cebp-1(0). Axotomy and axon
regeneration analysis. Normalized PLM axon regrowth is shown in the bar graph.
Error bars represent SEM. ***P < 0.001; ns, not significant (one-way ANOVA
with Tukey’s post hoc tests). (PDF 432 kb)
AMP: Antimicrobial peptide; bZIP: Basic leucine zipper; C/EBP: CCAAT/
enhancer-binding protein; ChIP-seq: Chromatin immunoprecipitation and
deep sequencing; CRISPR: Clustered regularly interspaced short palindromic
repeats; EASE: Expression Analysis Systematic Explorer; GO: Gene ontology;
KEGG: Kyoto Encyclopedia of Genes and Genomes; MAPK: Mitogen-activated
protein kinase; MEME: Multiple Em for Motif Elicitation; NIPI: No induction of
peptide after Drechmeria infection; RSAT: Regulatory Sequence Analysis Tools
We thank J. Ewbank for support on the project, A.D. Chisholm and members
of the Jin and Chisholm laboratories for valuable discussions and critical
comments on the manuscript and D.L. McEwan and F.M. Ausubel for
discussions and sharing of unpublished data. We thank S. Cypowyj for the
initial findings on cebp-1, J. Ruiz for assistance in strain construction, Z. Wu
for help in axotomy analyses, M. Bulle and A. Boned for technical assistance,
D.J. Dickinson and Z. Wang for CRISPR plasmids, H-R. Li and Y. Zhou in X-D
Fu’s lab at UC San Diego for help in Illumina sequencing and advice on
mapping ChIP-seq reads, and WormBase as an information resource. YJ
gratefully acknowledges the support from IMéRA, Aix-Marseille University.
Some strains were provided by the Caenorhabditis Genetics Center (CGC),
which is funded by the NIH Office of Research Infrastructure Programs
(P40 OD010440), and by the National Bioresource Project of Japan (NBRP).
This work was supported by institutional grants from AMU, INSERM and CNRS, the
ANR (ANR-12-BSV3-0001-01, ANR-11-LABX-0054 (Investissements d’Avenir–Labex
INFORM) and ANR-11-IDEX-0001-02 (Investissements d’Avenir–A*MIDEX) and
ANR-10-INBS-04-01 (France Bio Imaging)), NIH grant R01 NS035546 to YJ
and an American Heart Association postdoctoral fellowship to KWK. YJ is
an investigator of the Howard Hughes Medical Institute.
Availability of data and material
All data generated or analysed during this study are included in this published
article (and its additional files). The ChIP-seq data have been deposited in the
Gene Expression Omnibus database [GEO:GSE83330] (http://www.ncbi.nlm.nih.
gov/geo/query/acc.cgi?token=avylwwgwvtmrlmz&acc=GSE83330). Requests for
material should be made to the corresponding authors.
KWK and YJ designed the genetic suppressor screen, and KWK and CAP
performed the screen. KWK performed all experiments on nipi-3 suppressors
and CEBP-1 ChIP-seq analyses. SO and JP performed experiments, and NT
performed the bioinformatic analyses. YJ and NP supervised the experiments.
KWK, YJ and NP analysed the data and wrote the manuscript. All authors
read and approved the final manuscript.
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