A Differentiation Transcription Factor Establishes Muscle-Specific Proteostasis in <i>Caenorhabditis elegans</i>

<div><p>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 differentiation of <i>Caenorhabditis elegans</i> embryonic cells by ectopically expressing HLH-1 in all cells of the embryo and monitoring chaperone expression. We found that HLH-1-dependent 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 <i>daf-21(Hsp90)</i> and muscle-enriched <i>hsp-12</i>.<i>2(sHsp)</i> promoters abolished their myogenic-dependent expression. Disrupting HLH-1 function in muscle 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 differentiation could establish a proteostasis network dedicated to the folding and maintenance of the muscle proteome. Such cell-specific proteostasis networks can explain the selective vulnerability that many diseases of protein misfolding exhibit even when the misfolded protein is ubiquitously expressed.</p></div

Mutation in the putative HLH-1-binding motifs of <i>daf-21(Hsp90)</i> and <i>hsp-12</i>.<i>2(sHsp)</i> promoters abolished their HLH-1-dependent expression.

<p><b>(A)</b> Wild type or mutated promoter reporter constructs for <i>daf-21(Hsp90)</i>- or <i>hsp-12</i>.<i>2(sHsp)</i>-regulated GFP expression. (<b>B</b>) Representative images of HLH-1(ec) embryos expressing GFP under the regulation of the wild type or mutant <i>daf-21(hsp90)</i> (top) or <i>hsp-12</i>.<i>2(sHsp)</i> (bottom) promoter following heat shock (34°C, 30 min). Scale bar is 25 μm.</p

Myogenic conversion induced the expression of muscle chaperones.

<p><b>(A)</b> 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>(B)</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. <b>(C-H)</b> Relative chaperone mRNA levels of heat shock-treated wild type (gray) or HLH-1(ec) (red) embryos (normalized to <i>T07A9</i>.<i>15</i>). 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 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006531#pgen.1006531.s008" target="_blank">S1 Table</a>. <b>(I)</b> Relative mRNA levels of heat shocked HLH-1(ec) embryos grown on control (gray stripes) or <i>hlh-1</i> (red stripes) RNAi (normalized to <i>T07A9</i>.<i>15</i>). Data are relative to values obtained with untreated embryos and are presented as means ± SEM of at least 3 independent experiments. <b>(J)</b> Relative mRNA levels of untreated (gray stripes) or heat shocked (red stripes) CHE-1(ec) embryos (normalized to <i>T07A9</i>.<i>15</i>). Data are relative to values obtained with wild type embryos and are presented as means ± SEM of at least 3 independent experiments.</p

Reduced HLH-1 levels result in a decline in chaperone expression.

<p><b>(A)</b> Schematic representation of the experimental setup. Wild type or <i>hlh-1(cc561)</i> embryos were grown at 15 or 25°C for 6 h and chaperone expression was examined. <b>(B)</b> Representative images (>90%) of the expression pattern of the indicated chaperones in <i>hlh-1(cc561)</i> embryos grown at 15 or 25°C. Scale bar is 25 μm. <b>(C-E)</b> Relative mRNA levels (25/15°C) of wild type (gray) or <i>hlh-1(cc561)</i> (green) embryos (normalized to <i>T07A9</i>.<i>15</i>). Data are presented as means ± SEM of 5 independent experiments.</p

Muscle over-expression of <i>dnj-24</i> disrupts chaperone interactions, exposing sensitivity to specific chaperone down-regulation.

<p><b>(A)</b> Age-synchronized L1 wild type, DNJ-24M or HSP90M animals grown at 15°C were transferred to plates containing control, <i>hsp-1</i>, <i>rme-8</i>, or <i>dnj-8</i> RNAi-expressing bacteria, and images were taken on day 1 of adulthood. <b>(B)</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.</p

HLH-1 is required for establishing muscle proteostasis.

<p><b>(A)</b> Q0, Q35, Q0;<i>hlh-1(cc561)</i> or Q35;<i>hlh-1(cc561)</i> embryos laid at 15°C were scored for embryonic arrest. Data are presented as means ± SEM of at least 6 independent experiments. <b>(B)</b> Representative confocal images of Q0, Q35, Q0;<i>hlh-1(cc561)</i> or Q35;<i>hlh-1(cc561)</i> embryos laid at 15°C. Scale bar is 25 μm. <b>(C)</b> The number of body movements per minute scored in age-synchronized Q0, Q35, Q0;<i>hlh-1(cc561)</i> or Q35;<i>hlh-1(cc561)</i> animals on the first day of adulthood. <b>(D)</b> Representative confocal images of myofilaments. Age-synchronized Q0, Q35, Q0;<i>hlh-1(cc561)</i> or Q35;<i>hlh-1(cc561)</i> animals expressing GFP (green) and stained with anti-UNC-54 antibodies (red). Scale bar is 10 μm. <b>(E)</b> The average number of visible foci scored in age-synchronized Q35 or Q35;<i>hlh-1(cc561)</i> animals. <b>(F)</b> Images of representative Q35 or Q35;<i>hlh-1(cc561)</i> animals 5 days after hatching.</p

Muscle proteostasis and myogenesis are disrupted in <i>HSP90M;unc-54(ts)</i> embryos.

<p><b>(A)</b> Wild type, <i>unc-54(ts)</i>, <i>HSP90M</i> and <i>HSP90M;unc-54(ts)</i> embryos laid at the indicated temperature were scored for embryo arrest. Data are presented as means ± SEM of at least 5 independent experiments. <b>(B)</b> Representative confocal images (>90%) of wild type, <i>unc-54(ts)</i>, <i>HSP90M</i> and <i>HSP90M;unc-54(ts)</i> embryos laid at 20°C and stained with anti-UNC-54 antibodies. Scale bar is 25 μm.</p

Promoter occupancy and transcriptional analysis of muscle chaperones reveals potential HLH-1-dependent regulation of chaperones.

<p><b>(A)</b> A list of 97 <i>C</i>. <i>elegans</i> chaperones genes ranked according to potential for HLH-1 binding [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006531#pgen.1006531.ref029" target="_blank">29</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006531#pgen.1006531.ref030" target="_blank">30</a>] (HLH-1 occupancy), muscle-enrichment information [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006531#pgen.1006531.ref030" target="_blank">30</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006531#pgen.1006531.ref031" target="_blank">31</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006531#pgen.1006531.ref040" target="_blank">40</a>] (Muscle-enriched) and literature-curated information [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006531#pgen.1006531.ref018" target="_blank">18</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006531#pgen.1006531.ref025" target="_blank">25</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006531#pgen.1006531.ref038" target="_blank">38</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006531#pgen.1006531.ref039" target="_blank">39</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006531#pgen.1006531.ref041" target="_blank">41</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006531#pgen.1006531.ref054" target="_blank">54</a>] (Muscle-required) (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006531#sec013" target="_blank">Methods</a>). <b>(B)</b> HLH-1 occupancy sites associated with the promoter region of <i>unc-54(myosin heavy chain B)</i>, <i>unc-45</i>, <i>daf-21(Hsp90)</i> and <i>hsp-12</i>.<i>2(sHsp)</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006531#pgen.1006531.ref029" target="_blank">29</a>]. <b>(C)</b> Overlap between muscle-required and muscle-enriched chaperone sets. <b>(D)</b> Overlap between muscle-chaperones and chaperones with HLH-1 occupancy site sets. <b>(E)</b> 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) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006531#pgen.1006531.ref055" target="_blank">55</a>]. MI marks the myogenesis-induced subset.</p