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RESEARCH ARTICLE
A Differentiation Transcription Factor
Establishes Muscle-Specific Proteostasis in
Caenorhabditis elegans
Yael Bar-Lavan
1
, Netta Shemesh
1
, Shiran Dror
1
, Rivka Ofir
2
, Esti Yeger-Lotem
3
, Anat Ben-
Zvi
1
*
1Department of Life Sciences and The National Institute for Biotechnology in the Negev, Ben-Gurion
University of the Negev, Beer Sheva, Israel, 2Regenerative Medicine and Stem Cell Research Center, Ben-
Gurion University of the Negev, Beer Sheva, Israel, 3Department of Clinical Biochemistry and Pharmacology
and The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer Sheva,
Israel
*anatbz@bgu.ac.il
Abstract
Safeguarding the proteome is central to the health of the cell. In multi-cellular organisms,
the composition of the proteome, and by extension, protein-folding requirements, varies
between cells. In agreement, chaperone network composition differs between tissues.
Here, we ask how chaperone expression is regulated in a cell type-specific manner and
whether cellular differentiation affects chaperone expression. Our bioinformatics analyses
show that the myogenic transcription factor HLH-1 (MyoD) can bind to the promoters of
chaperone genes expressed or required for the folding of muscle proteins. To test this
experimentally, we employed HLH-1 myogenic potential to genetically modulate cellular dif-
ferentiation of Caenorhabditis elegans embryonic cells by ectopically expressing HLH-1 in
all cells of the embryo and monitoring chaperone expression. We found that HLH-1-depen-
dent myogenic conversion specifically induced the expression of putative HLH-1-regulated
chaperones in differentiating muscle cells. Moreover, disrupting the putative HLH-1-binding
sites on ubiquitously expressed daf-21(Hsp90) and muscle-enriched hsp-12.2(sHsp) pro-
moters abolished their myogenic-dependent expression. Disrupting HLH-1 function in mus-
cle cells reduced the expression of putative HLH-1-regulated chaperones and compromised
muscle proteostasis during and after embryogenesis. In turn, we found that modulating the
expression of muscle chaperones disrupted the folding and assembly of muscle proteins
and thus, myogenesis. Moreover, muscle-specific over-expression of the DNAJB6 homolog
DNJ-24, a limb-girdle muscular dystrophy-associated chaperone, disrupted the muscle
chaperone network and exposed synthetic motility defects. We propose that cellular differ-
entiation could establish a proteostasis network dedicated to the folding and maintenance of
the muscle proteome. Such cell-specific proteostasis networks can explain the selective vul-
nerability that many diseases of protein misfolding exhibit even when the misfolded protein
is ubiquitously expressed.
PLOS Genetics | DOI:10.1371/journal.pgen.1006531 December 30, 2016 1 / 27
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OPEN ACCESS
Citation: Bar-Lavan Y, Shemesh N, Dror S, Ofir R,
Yeger-Lotem E, Ben-Zvi A (2016) A Differentiation
Transcription Factor Establishes Muscle-Specific
Proteostasis in Caenorhabditis elegans. PLoS
Genet 12(12): e1006531. doi:10.1371/journal.
pgen.1006531
Editor: Gregory P. Copenhaver, The University of
North Carolina at Chapel Hill, UNITED STATES
Received: July 24, 2016
Accepted: December 8, 2016
Published: December 30, 2016
Copyright: ©2016 Bar-Lavan et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: This research was supported by a grant
from the Israel Science Foundation (ABZ, grant No.
91/11; https://www.isf.org.il/#/) and by the Legacy
Heritage Biomedical Science Partnership Program
of the Israel Science Foundation (ABZ, grant No.
804/13; https://www.isf.org.il/#/). EYL was
supported by a grant from the Israel Science
Foundation (EYL, grant No. 860/13; https://www.
Author Summary
Molecular chaperones protect proteins from misfolding and aggregation. In multi-cellular
organisms, the composition and expression levels of chaperones vary between tissues.
However, little is known of how such differential expression is regulated. We hypothesized
that the cellular differentiation that regulates the cell-type specific expression program
may be involved in establishing a cell-type specific chaperone network. To test this possi-
bility, we addressed the myogenic commitment transcription factor HLH-1 (CeMyoD)
that converts embryonic cells to muscle cells in Caenorhabditis elegans. We demonstrated
that HLH-1 regulates the expression of muscle chaperones during muscle differentiation.
Moreover, we showed that HLH-1-dependent expression of chaperones is required for
embryonic development and muscle function. We propose that cellular differentiation
results in cell-specific differences in the chaperone network that may be detrimental in
terms of the susceptibility of neurons and muscle cells to protein misfolding diseases.
Introduction
Molecular chaperones are a diverse group of highly conserved proteins that evolved to cope
with protein quality control challenges [1–3]. The cellular chaperone machinery is involved in
a multitude of cellular functions, including de novo folding, assembly and disassembly of pro-
tein complexes, protein translocation across membranes, assisting proteolytic degradation and
unfolding and reactivation of stress-denatured proteins [1,3,4]. The function and specificity
of a chaperone-based reaction can be mediated by co-chaperones that choose the substrate,
present it to the chaperone, and then coordinate cycles of binding and release by the chaperone
in a manner that facilitates polypeptide unfolding [5–7]. Acute stress or chronic expression of
metastable proteins leads to the accumulation of misfolded proteins that disrupts cellular pro-
tein homeostasis (proteostasis). Misfolded proteins continually occupy the chaperone machin-
ery, such that overwhelming this machinery results in a shortage of chaperones for other
cellular functions [8–12]. Activation of stress responses, such as the heat shock response, can
induce chaperone genes, (chaperone and co-chaperone) expression and restore proteostasis
[13]. However, this activation is also regulated by cell non-autonomous signals that can inhibit
or induce a heat shock response regardless of protein damage [14]. Although chaperone over-
expression often alleviates misfolded protein-associated toxicity [2,15], accumulation of chap-
erones and activation of the heat shock response can also be detrimental to organismal health
[12,16–22], possibly by disrupting sub-networks of chaperones and co-chaperones [23–25].
The chaperone network in unicellular eukaryotes consists of two separately regulated chap-
erone sets, where one is co-regulated with the translational apparatus and one is stress-induced
[26]. In multi-cellular eukaryotes, however, the complexity of the chaperone network is
increased, with expression of components of the proteostasis network being highly heteroge-
neous between tissues, as well as dependent on age [2,27]. Thus, the chaperone network may
parallel the diverse composition of the proteome and its cellular folding requirements. How-
ever, it remains unknown how the expression of cell type-specific or ubiquitously expressed
chaperones is regulated in different tissues. We reasoned that if chaperones expression is regu-
lated in a cell-specific manner then differentiation transcription factors could play a role in
defining the proteostatic network.
Muscle differentiation in Caenorhabditis elegans provides a well-studied case of highly regu-
lated changes in cellular proteome composition within a specific time window [28–31], as well
as information on molecular chaperones associated with muscle function [32]. C.elegans
Differentiation Can Determine Cellular Proteostasis
PLOS Genetics | DOI:10.1371/journal.pgen.1006531 December 30, 2016 2 / 27
isf.org.il/#/). YBL was supported by Kreitman
short-term post-doctoral scholarship. NS was
supported by Kreitman Negev scholarship. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
development is determined by the essentially invariant somatic cell lineage, so that the 81
embryonic muscle cells of the organism arise in a deterministic manner [33]. Muscle gene
expression starts ~300 min after the first division. By ~350 min, dorsal and ventral muscle
quadrants are formed, followed by the organization of muscle components into sarcomeres,
and then by contraction of myofilaments at ~420–450 min [34]. Failure to properly fold and
assemble the myofilaments disrupts myogenesis (arrest at two-fold phenotype) and can result
in embryonic lethality [34]. C.elegans body-wall muscle differentiation is dependent on the
core myogenic transcription factor modules HLH-1 (MyoD), UNC-120 and HND-1. Ectopic
expression of each of these transcription factors can convert early blastomeres into muscle-like
cells. However, in their absence only morphogenesis is disrupted and muscle differentiation
can still occur [28,35–37]. These transcription factors regulate the expression of many muscle
proteins, such as myosin and actin [28,30].
Many sarcomeric proteins require chaperones for their folding and assembly [32]. For
instance, myosin folding and assembly requires the coordinated functions of the Hsp90 chap-
erone machinery (Hsp90 and its co-chaperones STI1-AHA1-P23) and the myosin-specific
chaperone UNC-45 [25,32,38]. Moreover, there are examples of muscle-specific diseases that
are associated with mutations in a ubiquitously expressed chaperone, such as DNAJB6 associ-
ated with the limb-girdle muscular dystropy [18,39]. Here, we examined whether muscle
chaperone expression is regulated by HLH-1 during C.elegans myogenesis. We found that
the expression of chaperone genes with putative HLH-1-binding sites is induced by HLH-1-
dependent myogenic conversion. We then demonstrated that disrupting the putative E-box
motifs at the promoters of such chaperones inhibited HLH-1-dependent expression. More-
over, reduced HLH-1 expression resulted in a limited muscle proteostasis capacity during
embryogenesis, larval development and adulthood. Finally, we showed that modulating the
levels of muscle chaperones impacted the folding environment of muscle cells, disrupting mus-
cle function and embryogenesis. We thus concluded that the myogenic transcription factor
HLH-1 can regulate the expression of chaperones required for the folding and assembly of
muscle proteins, establishing a cell-specific proteostasis network to fit cellular needs. We pro-
pose that cell-specific differences in the proteostatic network may contribute to tissue-specific
vulnerability to protein misfolding diseases.
Results
Putative HLH-1 occupancy sites are associated with muscle chaperones
HLH-1 is the main myogenic transcription factor in C.elegans. To test whether chaperone
expression is associated with cellular differentiation, we first assessed the potential of HLH-1
to regulate chaperone expression during muscle differentiation. Using chromatin immunopre-
cipitation and next-generation sequencing (ChIP-seq), two independent studies mapped the
occupancy sites for this factor. One study used myogenic conversion, while the second used
animals expressing HLH-1::GFP to increase HLH-1 detection [29,30]. We used a set of 97 C.
elegans chaperone genes [25] to ask whether there are putative HLH-1-binding sites associated
with chaperone genes. Chaperone genes identified in at least one ChIP-seq experiment as
being bound by HLH-1 were defined as chaperones with a HLH-1 occupancy site. This analy-
sis resulted in a set of 62 chaperone genes (Fig 1A and S1 Table). The occupancy sites for these
genes were found mainly in the promoter region, similar to other genes possessing HLH-1
occupancy sites [29,30] (Fig 1B).
We ranked the 97 chaperone genes according to the number of independent ChIP-Seq
experiments in which they were identified. Strong candidate genes, such as unc-45 and daf-21
(Hsp90), were found to bind HLH-1 in all three ChIP-Seq experiments. Unlikely candidates
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Differentiation Can Determine Cellular Proteostasis
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included hsp-17(sHsp) and fkb-6(FKBP)for which an HLH-1-binding site was not identified
(Fig 1A).
We then asked whether chaperones with HLH-1 occupancy sites are expressed in muscle
cells. To define muscle-expressed chaperones, we considered three independent datasets of
muscle-enriched genes: (1) An RNA-sequencing dataset of genes expressed in myogenic-con-
verted embryos [30]; (2) a microarray dataset of genes expressed in muscle cells isolated by
sorting cells from dissociated embryos expressing green fluorescence protein-tagged myosin
(MYO-3::GFP) [31]; and (3) an mRNA dataset isolated from muscle cells at the first larval
stage (L1) using mRNA-binding proteins expressed specifically in body-wall muscles [40].
This last dataset represents proteins that were expressed in functional muscle cells during
post-embryonic development. Here, too, chaperones were ranked according to the number of
datasets in which they were identified (Fig 1A). Combining these datasets, we identified 46
chaperones that were muscle-enriched (S1 Table).
Next, we used manual curation to identify muscle-required chaperones. The literature was
scanned for reports of: (1) Chaperones shown in vivo to function in the folding of abundant
muscle proteins, such as CCT/TRiC that is required for actin folding; (2) chaperones known to
cause myopathies in humans, such as DNAJB6 (DNJ-24), as well as chaperones that affect
C.elegans motility, such as UNC-23; and (3) chaperones that are localized to the sarcomere,
such as HSP-12.1 [18,25,38,39,41–54] (S1 Table). This yielded 24 genes that were ranked
according to the number of these criteria they matched (Fig 1A). Supporting a role for these
chaperones in the folding and assembly of muscle proteins in vivo, the muscle-required set sig-
nificantly overlapped with the muscle-enriched set (17 of 24, P = 0.008, Fisher exact test; Fig
1C). Most of the chaperone genes with HLH-1 occupancy sites were associated with muscle
chaperones (enriched or required) (39 of 62, P = 0.025, Fisher exact test; Fig 1D), while chaper-
ones with no identifiable HLH-1 occupancy site were not significantly associated with muscle
chaperones (14 out of 35, P = 0.99, Fisher exact test). Thus, many muscle-enriched or -required
chaperones have HLH-1 occupancy sites and can potentially be regulated by HLH-1.
Expression of well-established HLH-1-depndent muscle genes, such as myosins, is first
observed ~300 min after the first division [34]. If HLH-1 occupancy sites are functional, chap-
erone genes that are bound by HLH-1 are expected to show a similar pattern of expression.
While changes in muscle expression of ubiquitously expressed chaperones could be masked by
their expression in other tissues [56], muscle specific or muscle-enriched chaperones are
expected to show this pattern. We utilized the C.elegans developmental gene expression time
course to characterize the myogenic-induced (MI) expression of genes during embryogenesis.
This dataset, derived from whole embryos, records the expression of over 19,000 genes at ten
different developmental stages over the course of embryogenesis [55]. Using this dataset, we
first examined the expression dynamics of a set of known muscle genes that are also enriched
in embryos showing increased muscle content upon myogenic conversion [30]. Of the 35
genes examined, the expression of 21 muscle-specific genes clustered into a single distinct
Fig 1. Promoter occupancy and transcriptional analysis of muscle chaperones reveals potential HLH-
1-dependent regulation of chaperones. (A) A list of 97 C.elegans chaperones genes ranked according to
potential for HLH-1 binding [29,30] (HLH-1 occupancy), muscle-enrichment information [30,31,40] (Muscle-
enriched) and literature-curated information [18,25,38,39,41–54] (Muscle-required) (see Methods). (B)
HLH-1 occupancy sites associated with the promoter region of unc-54(myosin heavy chain B),unc-45,daf-21
(Hsp90) and hsp-12.2(sHsp) [29]. (C) Overlap between muscle-required and muscle-enriched chaperone
sets. (D) Overlap between muscle-chaperones and chaperones with HLH-1 occupancy site sets. (E)
Hierarchical clustering of the relative expression of 62 chaperone genes with HLH-1 occupancy sites across
10 developmental stages (at 4-cells, E cell division, 4
th
-7
th
AB cell divisions, ventral enclosure (VE), comma
stage (cs), first movement, and L1) [55]. MI marks the myogenesis-induced subset.
doi:10.1371/journal.pgen.1006531.g001
Differentiation Can Determine Cellular Proteostasis
PLOS Genetics | DOI:10.1371/journal.pgen.1006531 December 30, 2016 5 / 27
developmental expression pattern (S1 Fig). The pattern showed little change in mRNA levels
during early embryogenesis (<200 min) but a strong increase at the ventral enclosure stage
(~290 min). We then asked whether the expression of some chaperone genes with HLH-1
occupancy site also follows this pattern of muscle protein expression. Of the 62 chaperone
genes in this set, eight genes clustered into the MI expression pattern, of which seven were
muscle-enriched and all eight were muscle-required (Fig 1A and 1E). As expected, ubiqui-
tously expressed genes were not detected in this analysis. Thus, we were able to find a myo-
genic-induced expression pattern for a subset of HLH-1-associated chaperones also linked to
muscle expression and function, supporting our hypothesis that muscle chaperones could be
regulated by HLH-1 during muscle differentiation.
Myogenic conversion modulates chaperone expression
To experimentally test whether HLH-1 regulates chaperone expression during muscle differ-
entiation, we first examined whether the ectopic expression of HLH-1 that induced myogenic
conversion could also induce the expression of muscle chaperones in non-muscle cells. As
such, we utilized animals expressing HLH-1 under the control of the inducible hsp-16.41(sHsp)
promoter, HLH-1(ec) [28]. When such animals were exposed to a short heat shock (30 min at
34˚C) during early embryogenesis, HLH-1 was ectopically expressed in all embryonic cells.
Because heat shock induced the expression of heat shock genes, some of which are chaperones,
we examined the expression of each gene in both HLH-1(ec) and wild type embryos with or
without exposure to heat shock (Fig 2A). To control for heat shock-induced activation, ani-
mals expressing GFP under the control of the inducible chaperone promoter hsp-16.2(sHsp)
were crossed with HLH-1(ec) animals and GFP expression was monitored. Upon heat shock,
robust GFP expression was detected in most cells of the HLH-1(ec) embryos (Fig 2B), similar
to wild type animals (S2A Fig). Likewise, heat shock genes, such as hsp-16.2(sHsp) and F44E5.4
(Hsp70), were similarly induced in both HLH-1(ec) and wild type embryos (Fig 2C). When we
examined the expression of known HLH-1-regulated genes, such as myosin, by immuno-stain-
ing, heat shock-treated HLH-1(ec) embryos showed ectopic expression of myosin heavy chain
A (MYO-3) in most cells of the embryo (Fig 2B) but not in wild type embryos (S2A Fig). In
agreement, levels of actin (act-4) and myosin heavy chain B (unc-54) were induced in heat
shock-treated HLH-1(ec) but not in wild type embryos (Fig 2D).
We then asked whether ectopic expression of HLH-1 and altered cellular fate affected the
pattern and levels of expression of chaperone genes. We divided the chaperone list into four
groups: (1) Chaperones with HLH-1 occupancy site identified in at least one experiment and
that are muscle-associated (39 genes), or (2) that are not associated with muscle (21 genes); (3)
chaperones with no identified HLH-1 occupancy site that are associated with muscle (14
genes), or (4) that are not associated with muscle (22 genes) (S1 Table). We then tested candi-
date genes (Fig 1A, gray shaded) from each group for myogenesis-dependent changes in
expression induced by ectopic induction of HLH-1 (Fig 2E–2H). As expected, the expression
of UNC-45, considered a HLH-1-specific substrate [30], was ectopically induced in most of
the cells of the heat shocked HLH-1(ec) embryos (Fig 2B) but not of wild type animals (S2A
Fig). To test for changes in expression of ubiquitously expressed chaperones, animals express-
ing GFP under the control of the cct-2(Hsp60) or cct-7(Hsp60) promoter were crossed with
HLH-1(ec) animals and GFP fluorescence was monitored. Similar to the HLH-1 muscle genes
tested, cct-2(Hsp60)- and cct-7(Hsp60)-dependent GFP expression was detected in most cells of
the HLH-1(ec) embryos upon heat shock (Fig 2B) but not in wild type embryos (S2A Fig).
Thus, myogenic-converted cells, differentiating into muscle cells, began to express chaperone
genes. Indeed, mRNA of 14 muscle-associated chaperone genes with an HLH-1 occupancy site
Differentiation Can Determine Cellular Proteostasis
PLOS Genetics | DOI:10.1371/journal.pgen.1006531 December 30, 2016 6 / 27
Fig 2. Myogenic conversion induced the expression of muscle chaperones. (A) Schematic representation
of the experimental setup. Wild type (wt) or HLH-1(ec) embryos were untreated or subjected to heat shock
(34˚C, 30 min) and chaperone expression was examined. (B) Representative images (>90%) of the expression
pattern of the indicated chaperones in untreated or heat shock embryos expressing HLH-1(ec) after a 6 h
recovery. Scale bar is 25 μm. (C-H) Relative chaperone mRNA levels of heat shock-treated wild type (gray) or
HLH-1(ec) (red) embryos (normalized to T07A9.15). Data are normalized to values obtained with untreated
embryos and are presented as means ±SEM of at least 5 independent experiments. Gene groups were defined
Differentiation Can Determine Cellular Proteostasis
PLOS Genetics | DOI:10.1371/journal.pgen.1006531 December 30, 2016 7 / 27
(group 1) were all induced (10–80 folds) in HLH-1(ec) embryos upon heat shock. This group
included all MI chaperones tested (5 out of 8, Fig 1E), as well as ubiquitously expressed chaper-
ones. In wild type embryos, in contrast, these chaperones expression levels (apart from sip-1
(sHsp)) did not increase and indeed, some decreased following heat shock (Fig 2E and S2B
Fig). Although sip-1(sHsp) levels increased in wild type embryos, its induction in HLH-1(ec)
embryos was 10-fold higher (Fig 2E and S2B Fig). Chaperone genes with HLH-1 occupancy
sites that were not associated with muscle (group 2) also showed increased levels in HLH-1(ec)
embryos upon heat shock (3 of the 4 genes tested), albeit to a modest extent (1.5–3.5 fold).
Thus, of the 18 chaperone genes with an identified HLH-1 occupancy site, 17 were signifi-
cantly induced by ectopic expression of HLH-1 (Fig 2E and 2F and S2B and S2C Fig). In con-
trast, when we examined chaperones for which HLH-1 occupancy sites was not identified,
regardless of their muscle association (groups 3 and 4), only one gene, C01G10.8(Aha1),
showed increased expression in HLH-1(ec) embryos upon heat shock (Fig 2G and 2H and
S2D and S2E Fig). Thus, under conditions of induced myogenic conversion, when HLH-
1-dependent muscle differentiation is activated, chaperones genes that were shown to bind
HLH-1 are induced. This indicates that the majority of HLH-1 occupancy sites identified for
chaperone genes are functional (24 out of 26 genes tested, i.e. 92%) and, similar to other mus-
cle genes, are up-regulated when cells differentiate into muscle cells.
To verify that chaperone expression was due to HLH-1, HLH-1(ec) embryos from animals
treated with control or hlh-1 RNAi were heat shocked and changes in mRNA levels following
heat shock were assessed. While expression of the inducible heat shock gene hsp-70(Hsp70)
was unaffected by hlh-1(RNAi), the induced expression of the muscle genes act-4 and unc-54
and the muscle chaperone genes unc-45,cct-2(Hsp60) and cct-5(Hsp60) was strongly reduced
in hlh-1(RNAi)-treated HLH-1(ec) embryos, as compared to control RNAi-treated embryos
(Fig 2I). Likewise, the expression of muscle and chaperone genes was not significantly induced
when the transcription factor CHE-1 was ectopically expressed upon heat shock in embryos
expressing hsp-16.2::che-1, although expression of hsp-70(Hsp70) and che-1 was induced (Fig
2J). Thus, ectopic expression of HLH-1 that led to myogenic conversion, resulted in HLH-
1-dependent induced expression of muscle chaperones in differentiating muscle cells.
Mutations in putative HLH-1-binding motifs disrupt chaperone
expression
A previous attempt to validate HLH-1 function using a HLH-1-binding site upstream of a
minimal promoter was very limited in its ability to induce muscle expression, even of known
muscle genes [29]. We, therefore, took a different approach to examine whether HLH-1 is
required for chaperone expression during muscle differentiation. Accordingly, we asked how
disruption of the HLH-1 E-box-binding motif at chaperone promoters would affect their
expression in myogenic-converted embryos. Because the muscle-specific chaperone UNC-45
is considered one of the “gold standard” muscle genes regulated by HLH-1 [30], we examined
two ubiquitously expressed chaperones. Specifically, DAF-21(Hsp90), a well-established myo-
sin chaperone and HSP-12.2, a small HSP (sHsp) that showed a myogenic expression pattern
during embryogenesis (Fig 1E). The DAF-21(Hsp90) HLH-1 occupancy site was identified in
in S1 Table.(I) Relative mRNA levels of heat shocked HLH-1(ec) embryos grown on control (gray stripes) or hlh-
1(red stripes) RNAi (normalized to T07A9.15). Data are relative to values obtained with untreated embryos and
are presented as means ±SEM of at least 3 independent experiments. (J) Relative mRNA levels of untreated
(gray stripes) or heat shocked (red stripes) CHE-1(ec) embryos (normalized to T07A9.15). Data are relative
to values obtained with wild type embryos and are presented as means ±SEM of at least 3 independent
experiments.
doi:10.1371/journal.pgen.1006531.g002
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three independent ChIP-seq experiments and its binding peak at the promoter showed a clear
E-Box consensus motif. The HSP-12.2(sHsp) HLH-1 occupancy site was identified in two
independent ChIP-seq experiments and its binding peak at the promoter has two
E-Box consensus motifs (S1 Table) [29,30].
We constructed a transcription reporter containing the promoter region of daf-21(Hsp90)
or hsp-12.2(sHsp) upstream of GFP (daf-21::gfpor hsp12.2::gfp) and mutated the E-box se-
quences (Fig 3A). These constructs were injected into HLH-1(ec) animals and stable trans-
genic animals were established. The expression of GFP in myogenic-converted embryos was
then monitored following heat shock. In 82.6±0.4% of the daf-21(Hsp90) and 57.7±4.8% of the
hsp12.2(sHsp) embryos carrying the wild type transcription reporters, GFP was ectopically
expressed in most cells of the embryos upon heat shock. In contrast, GFP expression was
Fig 3. Mutation in the putative HLH-1-binding motifs of daf-21(Hsp90) and hsp-12.2(sHsp) promoters
abolished their HLH-1-dependent expression. (A) Wild type or mutated promoter reporter constructs for daf-21
(Hsp90)- or hsp-12.2(sHsp)-regulated GFP expression. (B) Representative images of HLH-1(ec) embryos
expressing GFP under the regulation of the wild type or mutant daf-21(hsp90) (top) or hsp-12.2(sHsp) (bottom)
promoter following heat shock (34˚C, 30 min). Scale bar is 25 μm.
doi:10.1371/journal.pgen.1006531.g003
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undetected (less than 5 cells) in all the heat shock embryos carrying the mutated transcription
reporters (P<0.05, Fig 3B). When daf-21::GFP embryos were allowed to develop, expression of
both wild type and mutated constructs was observed in various tissues of the adult animals,
including intestine and neurons (S3 Fig). However, we could not detect GFP expression in
muscle cells of adult animals carrying the mutated transcription reporters. For example, no
muscle expression was detected in animals carrying the mutated daf-21::GFP transcription
reporter (n = 120), although wild type daf-21::GFP was expressed in muscle cells (S3 Fig).
Thus, disrupting putative E-box sequences abolished the HLH-1-dependent regulation of daf-
21::GFP and hsp-12.2::GFP in embryonic muscle cells, suggesting that HLH-1 occupancy sites
at these promoters are transcriptionally functional and can drive muscle expression.
Reduced HLH-1 levels limit muscle proteostasis capacity
To complement the approach taken above and to determine the contribution of HLH-1 to
muscle proteostasis, we examined the effects of down-regulating hlh-1 on chaperone expres-
sion during embryogenesis, using a truncation allele, hlh-1(cc561). This nonsense (Glu222-
Stop) mutation does not affect HLH-1 function but results in temperature-dependent hlh-1
mRNA clearance by the nonsense mRNA decay pathway and, therefore, temperature-depen-
dent knockdown of HLH-1 levels [28,57]. We thus asked whether the expression of chaperone
genes with HLH-1 occupancy sites was affected in hlh-1(cc561) animals grown at 25˚C, as com-
pared to animals grown at 15˚C. Wild type or hlh-1(cc561) embryos laid at 25˚C were allowed
to develop for 6 h. Protein expression and mRNA levels of muscle genes were then compared
with those values obtained in embryos maintained at 15˚C (Fig 4A). Some muscle proteins,
including the major myosins and actins, were unaffected by hlh-1(cc561) because UNC-120
serves a partially overlapping function and can compensate for a loss of HLH-1 [30]. In agree-
ment, immuno-staining of hlh-1(cc561) embryos with anti-MYO-3 antibodies showed a typical
organization pattern in body-wall muscle cells in embryos grown at 15˚C and 25˚C (Figs 4B
and S4A). The relative mRNA levels (25˚C/15˚C) of act-4 and unc-54 were also similar in hlh-1
Fig 4. Reduced HLH-1 levels result in a decline in chaperone expression. (A) Schematic representation of the experimental
setup. Wild type or hlh-1(cc561) embryos were grown at 15 or 25˚C for 6 h and chaperone expression was examined. (B)
Representative images (>90%) of the expression pattern of the indicated chaperones in hlh-1(cc561) embryos grown at 15 or 25˚C.
Scale bar is 25 μm. (C-E) Relative mRNA levels (25/15˚C) of wild type (gray) or hlh-1(cc561) (green) embryos (normalized to
T07A9.15). Data are presented as means ±SEM of 5 independent experiments.
doi:10.1371/journal.pgen.1006531.g004
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(cc561) and wild type animals (Fig 4C). In contrast, the localization of myosin chaperone
UNC-45 was lost in hlh-1(cc561) embryos grown at 25˚C and relative unc-45 mRNA levels
were reduced in hlh-1(cc561), as compared to wild type embryos (Fig 4B and 4C and S4A Fig).
Likewise, the expression of GFP under the control of the cct-2(Hsp60) or cct-7(Hsp60) pro-
moter in hlh-1(cc561) embryos grown at 25˚C was lost and the relative mRNA levels of differ-
ent muscle chaperones shown to be regulated by HLH-1(ec) (group 1 and 2) were significantly
reduced in hlh-1(cc561) embryos, as compared to wild type embryos (Fig 4B and 4D,S4A and
S4B Fig). While the expression of C01G10.8(Aha1) that was induced in heat-shocked and
treated HLH-1(ec) embryos was significant reduced (S4C Fig), chaperones, such as dnj-2
(Hsp40) and fkb-6(FKBP), for which no HLH-1 occupancy site or HLH-1(ec)-induced expres-
sion were identified, were unaffected by hlh-1 knockdown (Fig 4E). Thus, the expression of
ubiquitously expressed and muscle-enriched chaperones associated with muscle protein fold-
ing and assembly was strongly reduced in hlh-1(cc561) embryos.
We next considered the consequences of disrupting HLH-1-dependent chaperone expres-
sion for muscle proteostasis during embryogenesis. To challenge muscle proteostasis, we
crossed hlh-1(cc561) with animals expressing yellow fluorescent protein (YFP) fused to 35 glu-
tamine repeats (Q35) or YFP alone (Q0) expressed under the muscle-specific unc-54 myosin
promoter (Q35;hlh-1(cc561)and Q0;hlh-1(cc561), respectively). As noted above, hlh-1(cc561) is
a knockdown mutant. The nonsense allele occurs at a position coding 13 amino acids after the
bHLH domain, resulting in a functional protein. Indeed, the hlh-1(cc561) phenotype under
restrictive conditions was fully rescued by over-expression of the cc561 allele or by inhibiting
the nonsense mRNA decay pathway [57]. Under permissive conditions, <10% of the animals
expressing Q0;hlh-1(cc561)exhibited embryonic arrest and typical myofilaments were formed
(>90%) (Fig 5A and 5B). Likewise, embryonic development was unaffected by Q0- or Q35-
expression and myofilament organization, examined by UNC-54 immuno-staining, was nor-
mal (Fig 5A and 5B) [11]. In contrast, 45.5±6% of the Q35;hlh-1(cc561)embryos were arrested
at the two-fold stage and assumed deformed shapes when grown at 15˚C. Q35;hlh-1(cc561)
embryos showed severe mislocalization of UNC-54 and myofilaments were not formed in
many of the embryos (>60%, Fig 5A and 5B). This phenotype was partially rescued by inhibit-
ing the nonsense mRNA decay pathway. RNAi knockdown of smg-2 or smg-7 did not affect
Q35 embryos, yet rescued 30–50% of Q35;hlh-1(cc561)-arrested embryos, as compared to
those treated with the empty vector control (S5A Fig). Thus, expression of aggregation-prone
Q35 in a hlh-1(cc561) background resulted in severe disruption of muscle protein folding.
These data suggest that muscle proteostasis capacity is limited in hlh-1(cc561) embryos, sup-
porting a role for hlh-1 in establishing muscle proteostasis.
To examine whether reduced HLH-1 levels also impacted muscle proteostasis capacity later
in life, i.e., after muscle development has completed, we monitored Q35;hlh-1(cc561) young
adults for muscle function and myosin organization. Although we excluded deformed or para-
lyzed animals, motility of Q35;hlh-1(cc561)young adults was reduced 2.5-3-fold, as compared
to Q0;hlh-1,Q0 or Q35 young adults (Fig 5C). In agreement, Q35;hlh-1(cc561)young adults
exhibited severe UNC-54 disorganization, while Q0;hlh-1(cc561)myofilaments maintained
their striated structures and were only mildly disorganized (Fig 5D and S5B Fig). Myofilament
organization was normal in Q0 or Q35 young adults (Fig 5D) [11]. Thus, the disruption of
muscle protein folding observed for Q35;hlh-1(cc561)embryos was not mitigated in adult
animals.
Disruption of cellular proteostasis was previously shown to increase Q35 foci formation [11].
Foci formation in Q35-expressing animals begins at the transition to reproductive adulthood
[58]. As such, no foci were observed in Q35 animals at the first larval stage (L1) (n = 430). Fol-
lowing the onset of reproduction, (day 5) Q35 animals had an average of ~4 foci per animal. In
Differentiation Can Determine Cellular Proteostasis
PLOS Genetics | DOI:10.1371/journal.pgen.1006531 December 30, 2016 11 / 27
Fig 5. HLH-1 is required for establishing muscle proteostasis. (A) Q0, Q35, Q0;hlh-1(cc561) or Q35;hlh-1
(cc561) embryos laid at 15˚C were scored for embryonic arrest. Data are presented as means ±SEM of at least 6
Differentiation Can Determine Cellular Proteostasis
PLOS Genetics | DOI:10.1371/journal.pgen.1006531 December 30, 2016 12 / 27
contrast, foci were observed in ~10% of the Q35;hlh-1(cc561) animals (n = 425) even by the L1
stage, while by day 5, Q35;hlh-1(cc561)animals had an average of ~50 foci per animal (Fig 5E
and 5F). Still, Q35 protein levels in Q35;hlh-1(cc561)animals were ~50% lower than in Q35 ani-
mals (S5C and S5D Fig). Thus, reduced HLH-1 levels also resulted in limited muscle proteosta-
sis in adulthood.
The disruption in muscle function and increased aggregation of Q35;hlh-1(cc561) later in
life could be due to HLH-1 function after embryogenesis but could also stem from defects
acquired during myogenesis. Indeed, Q35;hlh-1(cc561)L1 animals were already affected at
15˚C (Fig 5E). To test the impact of hlh-1 on proteostasis past embryogenesis, we treated Q35;
hlh-1(cc561) animals with smg-2(RNAi) at the L1 stage to rescue hlh-1 expression levels after
embryogenesis was completed. We found that motility and aggregation of Q35;hlh-1(cc561)
young adults treated with smg-2(RNAi) from L1 were partially rescued as compared to those
treated with the empty vector control (S5E and S5F Fig). In contrast, shifting hlh-1(cc561) to
25˚C at the L1 stage to reduced hlh-1 expression levels past embryogenesis, did not signifi-
cantly affect its motility as compared to wild type (S5G Fig). These data suggest that HLH-1 is
not required but can contribute to muscle proteostasis in adulthood. Taken together, our data
support a role for HLH-1 in establishing muscle proteostasis, as well as impacting proteostasis
capacity in adulthood.
Modulating muscle chaperone expression can disrupt myogenesis
The correct folding and assembly of myosin thick filaments and thus, myogenesis, requires
UNC-45. Myofilaments are assembled and begin to contract some ~420 min after the first divi-
sion (1.5-fold stage), thereby facilitating embryo elongation (3-fold stage) [34]. In contrast,
proper myofilament assembly is disrupted in unc-45 null mutants, leading to muscle-depen-
dent embryonic arrest at the two-fold stage and lethality. Given that DAF-21(Hsp90) and
UNC-45 were shown to compete for myosin binding in vitro [59], we postulated that the regu-
lation of ubiquitously expressed chaperone genes by the myogenic transcription factor HLH-1
should also be adjusted to muscle proteomic needs. To directly test whether specifically chang-
ing the levels of ubiquitously expressed chaperone in body-wall muscle cells disrupted myo-
genesis, we asked how over-expression of muscle DAF-21(Hsp90) affected the folding of
UNC-54, a known Hsp90 substrate, and hence, myogenesis. A temperature-sensitive mutation
in myosin, unc-54(e1301ts)(unc-54(ts)), shows temperature-dependent misfolding [11] but
only mildly induced the arrest at two-fold phenotype [34]. We crossed unc-54(ts) with animals
that specifically over-express DAF-21(Hsp90) in body-wall muscle cells (strain AM780). These
animals express daf-21(Hsp90) tagged with GFP (daf-21::GFP) under the muscle specific unc-
54 promoter (HSP90M). We then monitored embryonic arrest and UNC-54 localization in
wild type, HSP90M,unc-54(ts) and HSP90M;unc-54(ts) embryos laid at 20 or 25˚C. HSP90M
did not induce arrest at the two-fold stage when the animals were grown at 20 or 25˚C (2.1
±0.6% and 3.9±0.5%, respectively). unc-54(ts) embryos showed a mild arrest at 20 and 25˚C
(5.2±0.6% and 13.6±1.2%, respectively). In contrast, HSP90M;unc-54(ts) embryos were
severely delayed (S6A Fig), with the percentage of embryo arrested at the two-fold stage at
independent experiments. (B) Representative confocal images of Q0, Q35, Q0;hlh-1(cc561) or Q35;hlh-1(cc561)
embryos laid at 15˚C. Scale bar is 25 μm. (C) The number of body movements per minute scored in age-synchronized
Q0, Q35, Q0;hlh-1(cc561) or Q35;hlh-1(cc561) animals on the first day of adulthood. (D) Representative confocal
images of myofilaments. Age-synchronized Q0, Q35, Q0;hlh-1(cc561) or Q35;hlh-1(cc561) animals expressing GFP
(green) and stained with anti-UNC-54 antibodies (red). Scale bar is 10 μm. (E) The average number of visible foci
scored in age-synchronized Q35 or Q35;hlh-1(cc561) animals. (F) Images of representative Q35 or Q35;hlh-1(cc561)
animals 5 days after hatching.
doi:10.1371/journal.pgen.1006531.g005
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both 20 and 25˚C being increased (12.7±1.7% and 40.6±3.3%, respectively, Fig 6A). HSP90M;
unc-54(ts) embryos showed defective myofilament and muscle elongation. Immuno-staining
with anti-UNC-54 antibodies of HSP90M;unc-54(ts)embryos grown at 20˚C exhibited strongly
reduced UNC-54 staining (Fig 6B). Although the embryos examined were arrested at the two-
fold stage, most eventually hatched (Fig 6A). Similar UNC-54 immuno-staining was observed
for HSP90M;unc-54(ts) embryos grown at 25˚C but only about half of these embryos hatched
(Fig 6A and S6B Fig). In contrast, UNC-54 myofilament assembled correctly in most wild
type, HSP90M,unc-54(ts) embryos grown at 20˚C (Fig 6B). Our data suggest that DAF-21
(Hsp90) levels are adjusted for proper myosin folding to support muscle elongation and
embryo development. Thus, changes in chaperone expression can disrupt proteostasis and
abrogate myogenesis.
Modulating chaperone expression disrupts the chaperone network
The expression of aggregation-prone proteins was suggested to disrupt proteostasis by engag-
ing chaperones and competing for their substrates [9,11]. Differences in chaperones expres-
sion levels and composition could also alter chaperone and co-chaperone interactions. Thus,
modulating chaperone expression in a given tissue could transform the network of that chap-
erone. To ask how changing chaperone levels modulate chaperone interactions, we focused on
dnj-24(Hsp40), encoding the C.elegans homolog of DNAJB6. DNAJB6 is a ubiquitously
expressed chaperone linked to limb-girdle muscular dystropy type 1D (LGMD1D) [18].
LGMD1D mutations were shown to result in stabilization and, therefore, increased levels of
DNAJB6. While the amino acids associated with LGMD1D are not conserved in DNJ-24
(Hsp40), DNJ-24(Hsp40) is enriched in muscle and shows the expected muscle and nuclear
distribution pattern [49]. To address whether increased levels of this chaperones disrupted
chaperone interactions in muscle cells, we examined the effects of muscle over-expression of
dnj-24(Hsp40) (DNJ-24M) on synthetic motility defects induced by chaperone knock-down.
We reasoned that if DNJ-24M perturbed chaperone interactions in muscle cells, then this
might exacerbate the effects of knocking-down the levels of other muscle chaperones [25]. If
so, then RNAi of chaperones that do not affect motility in wild type animals should induce
motility defects in DNJ-24M-expressing animals. Consistent with previous work in a zebrafish
model [18], over-expression of wild type dnj-24(Hsp40) in body-wall muscle of C.elegans did
Fig 6. Muscle proteostasis and myogenesis are disrupted in HSP90M;unc-54(ts) embryos. (A) Wild type, unc-54(ts),
HSP90M and HSP90M;unc-54(ts) embryos laid at the indicated temperature were scored for embryo arrest. Data are
presented as means ±SEM of at least 5 independent experiments. (B) Representative confocal images (>90%) of wild type,
unc-54(ts),HSP90M and HSP90M;unc-54(ts) embryos laid at 20˚C and stained with anti-UNC-54 antibodies. Scale bar is
25 μm.
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not result in notable motility defects (Fig 7). However, when age-synchronized DNJ-24M-
expressing animals were treated with RNAi for different Hsp70 chaperones and co-chaper-
ones, three genes (of 48 examined), namely hsp-1,rme-8, and dnj-8, specifically affected the
motility of DNJ-24M-expressing but not wild type or HSP90M-expressing animals (Fig 7A
and 7B). RNAi knock-down of the hsp-1(Hsc70) induced a strong larval arrest in wild type,
HSP90M and DNJ-24M animals, yet only in the DNJ-24M animals did such treatment induce
100% paralysis (Fig 7A and 7B). Of note, DNAJB6 interacts with several chaperones associated
with chaperones-assisted selective autophagy, one of which is HSPA8, a Hsp-1(Hsc70) homo-
log [18]. Knocking-down the expression of rme-8(Hsp40) and dnj-8(Hsp40) resulted in no
motility phenotype in wild type or HSP90M animals, while knocking-down the expression
of these genes in a DNJ-24M background resulted in motility defects (72±10.7 and 61±2.7,
p<0.0002, Fig 7B) and disrupted myosin organization (S7 Fig). Thus, over-expression of DNJ-
24(Hsp40) in body-wall muscle cells disrupted muscle proteostasis such that muscle cells were
more susceptible to hsp-1(Hsc70),rme-8(Hsp40) and dnj-8(Hsp40) knock-down. Cell type-spe-
cific regulation of chaperone expression could, therefore, impact tissue-specific chaperone
networks.
Discussion
Differentiation can establish cellular proteostasis
In the present study, we asked whether the cellular chaperone network is regulated in a cell
type-specific manner. Specifically, we asked whether muscle chaperones are regulated by the
myogenic transcription factor HLH-1 during C.elegans myogenesis. We found that muscle
chaperones that have HLH-1 occupancy sites in their promoter are induced in myogenic-con-
verted embryos. This muscle-specific induction was fully dependent on HLH-1, as no induc-
tion was observed for most chaperones without HLH-1 occupancy sites or when HLH-1
expression was down-regulated. Moreover, we showed that disrupting the putative HLH-1
binding sites in two different chaperone promoters inhibited their myogenic-induced expres-
sion and muscle expression later in life. Thus, HLH-1 is required for the expression of muscle
chaperones with HLH-1 occupancy sites in cells undergoing differentiation into body-wall
muscle cells. While a HLH-1 differentiation-independent function in embryonic muscle cells
is possible, we instead propose that muscle chaperone genes are regulated by HLH-1 together
with other muscle genes during myogenesis. Linking the regulation of chaperone expression
to the differentiation program could result in a distinct chaperone network, ensuring that
chaperones are expressed at the required levels and with proper timing. Indeed, we found that
down-regulation of HLH-1 strongly restricted proteostasis capacity, leading to misfolding of
muscle protein and myogenesis arrest.
Tissue-specific differences in the expression levels of chaperones can explain why down-
regulation of ubiquitously expressed chaperones led to a tissue-selective activation of the heat
shock response [60] and why the cellular folding environment is sensitive to chronic expres-
sion of aggregation-prone proteins and expression of stress-induced chaperones [9,11,19,61].
The importance of regulating chaperone levels in a tissue-specific manner is supported by
prior findings and our data showing that both down-regulation and over-expression of the
myosin-specific chaperone UNC-45 and the ubiquitously expressed Hsp90 were detrimental
to myosin assembly and muscle elongation [45,59]. Given that DAF-21(Hsp90) and UNC-45
were shown to compete for myosin binding in vitro, their relative levels are critical for myosin
folding and can abrogate myogenesis. We, therefore, suggest that physiological tissue-specific
chaperone networks can enable cells to respond to the folding requirement of their unique
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Differentiation Can Determine Cellular Proteostasis
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proteomes, leading to distinct responses to folding challenges, such as acute stress or chronic
expression of misfolded proteins.
We found that muscle proteostasis can also be critical for muscle differentiation and can, as
in the case of UNC-45 and Hsp90, lead to embryonic arrest and lethality. In support of this
claim, a recent study showed that activation of Janus kinase 2 (JAK2) signaling associated with
myeloproliferative neoplasms (MPNs) resulted in reduced expression of proteostasis compo-
nent AIRAPL (arsenite-inducible RNA-associated protein-like) and led to increased insulin/
insulin-like growth factor 1 (IGF1R) stability and disrupted hematopoietic differentiation [62].
Thus, changes in expression of proteostasis components can result in modulated folding or
degradation of cellular factors, such as signaling proteins that, in turn, can lead to alterations
in differentiation.
A general role for differentiation-related transcription factors in regulating
cell type-specific proteostasis
Our data demonstrate that the myogenic factor HLH-1 regulates the expression of chaperones
in muscle cells. By extension, chaperones can be differentially regulated in different cell types
to meet the needs of a specific proteome. The large available ChIP-seq dataset (ModEncode)
[63] shows that transcription factors involved in development and differentiation can bind the
promoter region of chaperone genes, supporting our proposal of tissue-specific developmental
regulation of chaperone expression, and raises the possibility that tissue differentiation pro-
motes the expression of required chaperones. In agreement, PHA-4 is required for the devel-
opment of the pharynx and foregut and also regulates the expression of autophagy-required
genes [64]. ChIP-seq analysis of the occupancy sites of PHA-4 [65] showed significant overlap
between PHA-4 binding to chaperone promoters under starvation stress conditions and chap-
erone promoters occupied during embryogenesis (33 out of 38 overlapped between L1 stress
and embryos, p = 0.0001). This overlap suggests, in turn, a possible role for transcription fac-
tors involved in development and differentiation in tissue maintenance later in life. Indeed,
the pha-4 occupancy site in the daf-21(hsp90) promoter, as identified by modEncode, was
shown to be functional in cell non-autonomous expression of daf-21(Hsp90) in adult C.elegans
muscle, intestinal and neuronal cells [12]. For HLH-1, we observed disrupted proteostasis in
adulthood in a Q35;hlh-1(cc561) background that could be alleviated by blocking the nonsense
mRNA decay pathway and thus, hlh-1 mRNA clearance. Indeed, hlh-1(cc561) exacerbated the
effect of a dystrophin mutation associated with Duchenne’s muscular dystrophy, leading to
muscle degeneration in adulthood [66,67]. We, therefore, propose that similar to the speciali-
zation of the chaperone networks in unicellular eukaryotes into two separate sets, one dedi-
cated to coping with stress-induced misfolding and the other to newly translated proteins [26],
the regulation of chaperone expression in multi-cellular organisms is specialized to establish
chaperone networks dedicated to the folding and maintenance of cell type-specific proteomes
in development and possibility in adulthood as well.
Myogenic-dependent regulation of chaperones suggests that the proteostatic requirements
of the muscle proteome might dictate the expression of other quality control machineries to fit
functional and folding characteristics of that proteome. Careful analysis of HLH-1 targets
Fig 7. Muscle over-expression of dnj-24 disrupts chaperone interactions, exposing sensitivity to
specific chaperone down-regulation. (A) Age-synchronized L1 wild type, DNJ-24M or HSP90M animals
grown at 15˚C were transferred to plates containing control, hsp-1,rme-8, or dnj-8 RNAi-expressing bacteria,
and images were taken on day 1 of adulthood. (B) Age-synchronized wild type, DNJ-24M or HSP90M animals
treated as in (A) were scored for motility on day 1 of adulthood. Data are presented as means ±SEM of 3
independent experiments.
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revealed that most were not muscle-enriched [30]. One interpretation of this analysis is that
the expression of general factors required for muscle differentiation is specifically regulated to
meet the needs of muscle cells. Indeed, SKN-1, required for specification of the EMS blasto-
meres that give rise to pharyngeal, muscle and intestinal cells, regulates the expression of the
oxidative stress response [68]. As noted above, PHA-4 is involved in both development and
autophagy [64]. Moreover, autophagy is activated in different tissues of zebrafish during
embryogenesis and is required for vertebrate cardiac morphogenesis [69]. Likewise, efficient
differentiation of human embryonic stem cells required increased expression of the 19S sub-
unit PSMD11 [70]. We, therefore, propose that rather than relying on a generic proteostatic
machinery, each cell and tissue type with a defined folding capacity possesses a specific compo-
sition of the quality control machinery and perhaps even cell type-specific heat shock response
and unfolded proteins responses to deal with the highly specialized challenges of each cell type.
Implications of cell type-specific regulation for misfolding diseases
Although the expression of many disease-associated proteins is not tissue-specific, many protein
misfolding diseases exhibit tissue-specific vulnerability [71,72]. For example, mutations in the
ubiquitously expressed co-chaperone DNAJB6 cause a tissue-specific disease, limb-girdle mus-
cular dystropy [18]. The mechanism for this selective vulnerability in certain tissues is unknown,
although differences in folding and clearance capacities were suggested to affect the onset of sev-
eral tissue-specific diseases and stress activation [60,73–75]. Here, we showed that over-expres-
sion of the DNAJB6 homolog DNA-24(Hsp40) in muscle cells affected the muscle function of
HSP-1(Hsc70) and two other DnaJ co-chaperones, RME-8(Hsp40) and DNJ-8(Hsp40). HSP-1
(Hsc70) and RME-8(Hsp40) are required for receptor-mediated and fluid-phase endocytosis
[76,77]. This suggests that the balance between co-chaperones may affect Hsc70 function, simi-
lar to protein misfolding [9,11]. Indeed, a lack of RME-8(Hsp40) resulted in mislocalization
and clearance of endosomal proteins to the lysosome, associated with autophagic function [78].
Given the link of DNAJB6 to autophagy [18], it is possible that DNJ-24M disrupts HSP-1
(Hsc70) and RME-8(Hsp40) interactions, in turn affecting endocytic trafficking in LGMD1D.
Thus, the observed stabilization of DNAJB6 might play a role in LGMD1D muscle etiology by
competing for RME-8(Hsp40) or other Hsp40s function. Differential susceptibility to misfolding
or stress may, therefore, spring from cell-specific differences in the composition and expression
levels of components of the proteostatic network. We propose that differences in chaperone lev-
els and composition between tissues could impact tissue-specific vulnerability to protein mis-
folding diseases that are globally expressed yet which are manifested in a specific tissue.
Methods
Bioinformatics and statistics
The chaperone list was complied based on the work of Brehme et al. [79], focusing on the
main chaperone families and their co-chaperones (97 genes), including Hsp60 and Hsp10,
Hsp70, Hsp40 and NEF, Hsp90 and Hsp90 co-chaperones and sHSP [25]. Three curated lists
of HLH-1 occupancy sites were used, i.e. ChiP-seq (e
-6
) peak call data, were provided in the
manuscript as supporting information [29] and two curated lists, namely a union set of genes
that were identified in experiment (mex-3 or mex-3;skn-1;elt-1 RNAi) and an overlap set iden-
tified in both [30]. The later curated lists (Union and overlap) were kindly provided by Dr. Ste-
ven Kuntz and Dr. Paul Sternberg (S1 Table). Three curated lists of genes enriched in muscle
were used: (1) Myogenic-converted embryos [30], kindly provided by Dr. Steven Kuntz and
Dr. Paul Sternberg; (2) Muscle cells from dissociated embryos and (3) L1 body-wall muscles
[31,40]. These later curated lists (2–3) were provided in the manuscripts as supporting
Differentiation Can Determine Cellular Proteostasis
PLOS Genetics | DOI:10.1371/journal.pgen.1006531 December 30, 2016 18 / 27
information. Chaperone genes occupancy sites and muscle enrichment were ranked according
to the number of independent experiments in which they were identified, giving equal weight
to each experiment. Muscle-required chaperone genes were ranked according to the number
of criteria (function, phenotype or sarcomeric localization) they fulfilled. The list was sorted by
HLH-1 occupancy ranking (Fig 1A). The flowchart outlining the bioinformatics analyses and
all the data included in these analyses are summarized in S1 Table.
HLH-1 binds to E-Box motif (CANNTG) [29,80]. For each of the 97 chaperone genes, we
downloaded upstream sequences (1000bp) from the ensambel biomart webserver and
searched using the FIMO tool from MEME suite 4.11.2 with a p-value<0.001 [81]. Putative
HLH-1 E-box-binding motifs were found at the promoters of 39 of the 62 chaperones with
HLH-1 occupancy sites but were not enriched in these promoters (S1 Table).
Venn diagrams were plotted using the BioVenn diagram generator http://www.cmbi.ru.nl/
cdd/biovenn/ (BioVenn) [82]. Microarray-normalized data for C.elegans embryonic develop-
ment gene expression was provided by Dr. Itai Yanai [55]. Data were complied and clustered
using the EXPANDER (6.5.1) program [83]. The probability of overlap between chaperone
sets was calculated using the Fisher exact test. The Pvalues in Figs 2–7were calculated using
the Mann-Whitney test, where () denotes P<0.05 and () denotes P<0.01.
Nematode strains and maintenance
The list of strains used in this work is provided in S2 Table. Nematodes were grown on NGM
plates seeded with the Escherichia coli OP50-1 strain at 15˚C, unless indicated otherwise.
Cross-strains were generated using standard C.elegans procedures.
To generate the promoter reporter constructs for daf-21(Hsp90) and hsp-12.2(sHsp), a 2492 bp
fragment for daf-21(Hsp90) and a 921 bp fragment for the Hsp-12.2(sHsp) promoter were ampli-
fied from N2 genomic DNA and assembled into plasmid pNU106 to create plasmids pNU314
and pNU374, respectively, using Gibson ligation. Mutated promoter reporters for daf-21(Hsp90)
and hsp-12.2(sHsp),pNU315::Pdaf-21(mut)::gfpand pNU375::hsp-12.2p(mut)::gfp, respectively,
were generated by site-directed mutagenesis of the putative HLH-1-binding motifs at -432 bp
and at -921 bp and -266 bp to ACGCGT. Plasmids were validated by DNA sequencing and
injected into animals expressing unc-119(ed3);hsp-16.41::hlh-1. Promoter reporter constructs
were generated and injected by Knudra Transgenics. Stable transgenic lines are listed in S2 Table.
Embryo synchronization and treatment
Synchronized animals were grown at 15˚C for five days or transferred to 20 or 25˚C at the L2
stage for 24–48 h to reach adulthood. These synchronized gravid adults (first day of egg laying)
were allowed to lay eggs for 45 min and then removed. Synchronized embryos were heat
shock-treated, allowed to grow for 6 h to pass the comma stage or allowed to grow for 24–48 h
and complete embryogenesis.
Heat shock treatment
Embryos laid at 15˚C were moved to new plates and were untreated or subjected to heat shock
at 34˚C for 30 min. To determine RNA levels, embryos were frozen immediately following
heat shock. To examine expression patterns, embryos were collected after a 6 h recovery.
Temperature shift treatment
Wild type and hlh-1(cc561) embryos laid at 15˚C or 25˚C for 45 min were allowed to grow for
6 h. The embryonic developmental stage was examined to determine whether embryos grown
Differentiation Can Determine Cellular Proteostasis
PLOS Genetics | DOI:10.1371/journal.pgen.1006531 December 30, 2016 19 / 27
at 15˚C had passed the comma stage. To determine the effect of hlh-1(cc561) on gene expres-
sion, we compared the relative mRNA levels (25˚C/15˚C) of the genes examined. In S5G Fig,
the temperature shift was carried out at L1.
RNA interference experiments
RNAi knockdown treatments were performed as previously described [84]. RNAi constructs
were obtained from the “RNAi chaperone library” kindly provided by Prof. Richard Mori-
moto, Northwestern University [79] or from the Julie Ahringer library. To collect RNAi-
treated embryos, animals were grown on E.coli strain HT115(DE3) transformed with specified
RNAi or empty (pL4440) vectors and allowed to lay eggs, as above. Otherwise, synchronized
L1 larvae stage worms were washed from regular NGM plates, transferred to RNAi plates and
grown at 15˚C for five days until day 1 of adulthood.
RNA levels
RNA extraction from synchronized embryos, cDNA synthesis and quantitative real-time PCR
were performed as previously described [25]. Samples were normalized (2
-ΔΔCT
method) to
T07A9.15 or tbc-10, determined to be stably expressed during embryogenesis [55]. The list of
primers used in this work is provided in S3 Table.
Embryo arrest
To determine embryo arrest, synchronized embryos were grown at 15–25˚C. Embryos that
did not hatch until their siblings reached L2 (24–48 hours) or hatched arrested at the two-fold
state were counted as arrested embryos.
Motility assays
Age-synchronized young adults were moved to a new plate and their movement was moni-
tored after 10 min. Animals that did not move one body length were scored as paralyzed. Oth-
erwise, age-synchronized young adults were placed in wells containing M9 and allowed to
acclimate for 5–10 min. Each animal was monitored for 15 sec and thrashes (changes in direc-
tion of bending at mid-body) were counted.
Immuno-staining and fluorescence reporters
Embryos were fixed in methanol and 2% paraformaldehyde and permeabilized by freeze-thaw-
ing and then immuo-stained as described [85]. Adult animals were fixed with 4% paraformal-
dehyde and permeabilized with β-mercaptoethanol and collagenase IV treatment as described
[86]. Antibodies used in this work included anti-MYO-3 (5–6), anti-UNC-45 (gift from Dr.
Thorsten Hoppe) and anti-UNC-54 (28.2, gift from Dr. Jose Barral) [87,88] and secondary
DyLight 488, DyLight 549 or DyLight 633 anti-mouse or anti-rabbit antibodies (Jackson
Immuno Research). Embryos or adult animals expressing fluorescence reporters or tagged
proteins were fixed with 4% paraformaldehyde. Treated samples were imaged using an Olym-
pus Fluoview FV1000 or an LEICA DM5500 confocal microscope with 488 or 549 or 633 nm
laser lines for excitation or with LEICA DFC360FX camera. Otherwise, treated samples were
imaged using an LEICA M165FC stereomicroscope with QIMAGINE Exi blue camera.
Aggregation quantification
The number of bright foci of age-synchronized animals expressing Q35::YFP was counted
using an LEICA M165FC stereomicroscope.
Differentiation Can Determine Cellular Proteostasis
PLOS Genetics | DOI:10.1371/journal.pgen.1006531 December 30, 2016 20 / 27
Protein levels
Age-synchronized animals were collected and lyzed in SDS sample buffer (95˚C for 10 min).
Samples were separated by SDS-PAGE and analyzed by western blot (LF PVDF membrane),
using primary anti-tubulin (Sigma) and anti-GFP (Enco Scientific) and secondary DyLight
488 and DyLight 549, anti-mouse and anti-rabbit antibodies, respectively (Jackson Immuno
Research). Membranes were imaged using the ChemiDoc MP Imaging System (BioRad).
Supporting Information
S1 Fig. Transcriptional analysis of muscle gene expression. Hierarchical clustering of the rel-
ative expression of 35 muscle-specific genes across 10 developmental stages (at 4-cells, E cell
division, 4
th
-7
th
AB cell divisions, ventral enclosure (VE), comma stage (cs), first movement,
and L1) [55]. MI marks the myogenesis-induced subset.
(TIF)
S2 Fig. Heat shock-induced changes in chaperone expression. (A) Representative images
(>90%) of the expression pattern of chaperones in wild type embryos untreated or subjected
to heat shock after a 6 h recovery. Scale bar is 25 μm. (B-E) Relative chaperone mRNA levels in
heat shock-treated wild type (gray) or HLH-1(ec) (red) embryos. Data are relative to values
obtained with untreated embryos (normalized to tbc-10) and are presented as means ±SEM of
at least 5 independent experiments.
(TIF)
S3 Fig. A mutation in the putative HLH-1-binding motif of daf-21(Hsp90) promoter
affected its expression pattern in adult animals. Representative images of HLH-1(ec) ani-
mals expressing GFP under the regulation of the wild type or a mutant daf-21(Hsp90) pro-
moter, without myogenic induction. Arrows indicate body-wall muscle cells.
(TIF)
S4 Fig. Reduced HLH-1 levels modulate the expression of some chaperones. (A) Represen-
tative images (>90%) of the expression pattern of the indicated chaperones in wild type
embryos grown at 25˚C. (B-C) Relative mRNA levels (25/15˚C) of wild type (gray) or hlh-1
(cc561) (green) embryos (normalize to T07A9.15). Data are presented as means ±SEM of 5
independent experiments.
(TIF)
S5 Fig. Disruption of muscle proteostasis results in embryo arrest. (A) Embryonic arrest scored
for Q35;hlh-1(cc561) or Q35 embryos treated with smg-2,smg-7 or empty vector control RNAi.
Data are presented as means ±SEM of at least 3 independent experiments. (B) Representative con-
focal images of Q35;hlh-1(cc561) muscles. Scale bar is 10 μm. (C-D) Extracts of age-synchronized
(day 4) Q35 or Q35;hlh-1(cc561) animals were separated on a SDS-PAGE gel and probed with
anti-GFP (top) and anti-tubulin (bottom) antibodies. Relative levels were determined by quantifi-
cation of Q35::YFP protein bands. Data are presented as means ±SEM of at least 3 independent
experiments. (E) The number of body movements per minute scored on the first day of adulthood
in age-synchronized Q35;hlh-1(cc561) animals treated with smg-2 or empty vector control RNAi
from L1. (F) The average number of visible foci scored in age-synchronized Q35;hlh-1(cc561)
young adults treated with smg-2 or empty vector control RNAi from L1. (G) The number of body
movements per minute scored in wild type or hlh-1(cc561) young adults shifted to 25˚C at L1.
(TIF)
S6 Fig. Modulating HSP90 levels results in embryo arrest. (A) Images of a population of
wild type, unc-54(ts), HSP90M or HSP90M;unc-54(ts) embryos laid at 20˚C. (B) Representative
Differentiation Can Determine Cellular Proteostasis
PLOS Genetics | DOI:10.1371/journal.pgen.1006531 December 30, 2016 21 / 27
confocal images (>90%) of unc-54(ts), and HSP90M;unc-54(ts) embryos laid at 25˚C and
stained with anti-UNC-54 antibodies. The scale bar is 25 μm.
(TIF)
S7 Fig. Down-regulation of hsp-1(Hsc70),rme-8(Hsp40) and dnj-8(Hsp40) in DNJ-24M ani-
mals disrupts myosin organization. Representative confocal images of age-synchronized
DNJ-24M animals treated with control, hsp-1(Hsc70),rme-8(Hsp40) or dnj-8(Hsp40) RNAi
and stained with anti-MYO-3 antibodies. Scale bar is 25 μm.
(TIF)
S1 Table. Chaperone association with muscle and HLH-1 binding. (A) Flowchart outlining
the filtering analyses of the chaperone list. (B) Summarized data from bioinformatics analyses.
(XLSX)
S2 Table. List of strains used in this study.
(PDF)
S3 Table. List of quantitative PCR primers used in this study.
(PDF)
Acknowledgments
Some nematode strains used in this work were provided by the Caenorhabditis Genetics Cen-
ter, which is funded by the NIH National Center for Research Resources (NCRR). The mono-
clonal antibody 5–6 developed by H.F. Epstein was obtained from the Developmental Studies
Hybridoma Bank developed under the auspices of the NICHD and maintained by the Depart-
ment of Biology, University of Iowa.
Author Contributions
Conceptualization: YBL ABZ.
Formal analysis: YBL NS EYL ABZ.
Funding acquisition: ABZ.
Investigation: YBL NS SD ABZ.
Methodology: YBL NS SD RO EYL ABZ.
Project administration: ABZ.
Resources: YBL NS SD RO EYL ABZ.
Supervision: RO EYL ABZ.
Validation: YBL NS SD ABZ.
Visualization: YBL NS SD ABZ.
Writing – original draft: YBL NS SD RO EYL ABZ.
Writing – review & editing: YBL NS SD RO EYL ABZ.
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