Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations.

Human pluripotent stem cells (hPS cells) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with the acquisition of large copy number variants that provide mutated cells with a growth advantage in culture. The nature, extent and functional effects of other acquired genome sequence mutations in cultured hPS cells are not known. Here we sequence the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hES cell) lines, including 26 lines prepared for potential clinical use. We then apply computational strategies for identifying mutations present in a subset of cells in each hES cell line. Although such mosaic mutations were generally rare, we identified five unrelated hES cell lines that carried six mutations in the TP53 gene that encodes the tumour suppressor P53. The TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We found that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that the P53 mutations confer selective advantage. We then mined published RNA sequencing data from 117 hPS cell lines, and observed another nine TP53 mutations, all resulting in coding changes in the DNA-binding domain of P53. In three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from the loss of heterozygosity at the TP53 locus. As the acquisition and expansion of cancer-associated mutations in hPS cells may go unnoticed during most applications, we suggest that careful genetic characterization of hPS cells and their differentiated derivatives be carried out before clinical use.NB is the Herbert Cohn Chair in Cancer Research and was partially supported by The Rosetrees Trust and The Azrieli Foundation. Costs associated with acquiring and sequencing hESC lines were supported by HHMI and the Stanley Center for Psychiatric Research. FTM, SAM, and KE were supported by grants from the NIH (HL109525, 5P01GM099117, 5K99NS08371). KE was supported by the Miller consortium of the HSCI and FTM is currently supported by funds from the Wellcome Trust, the Medical Research Council (MR/P501967/1), and the Academy of Medical Sciences (SBF001\1016)

Global Characterization of X Chromosome Inactivation in Human Pluripotent Stem Cells

Summary: Dosage compensation of sex-chromosome gene expression between male and female mammals is achieved via X chromosome inactivation (XCI) by employing epigenetic modifications to randomly silence one X chromosome during early embryogenesis. Human pluripotent stem cells (hPSCs) were reported to present various states of XCI that differ according to the expression of the long non-coding RNA XIST and the degree of X chromosome silencing. To obtain a comprehensive perspective on XCI in female hPSCs, we performed a large-scale analysis characterizing different XCI parameters in more than 700 RNA high-throughput sequencing samples. Our findings suggest differences in XCI status between most published samples of embryonic stem cells (ESCs) and induced PSCs (iPSCs). While the majority of iPSC lines maintain an inactive X chromosome, ESC lines tend to silence the expression of XIST and upregulate distal chromosomal regions. Our study highlights significant epigenetic heterogeneity within hPSCs, which may bear implications for their use in research and regenerative therapy. : Bar et al. perform a large-scale analysis of X chromosome inactivation (XCI) in over 700 samples of human pluripotent stem cells (PSCs). Erosion of XCI involves stable silencing of XIST and partial overexpression of distal X-linked genes and is prevalent in embryonic stem cells, but not in most induced PSCs. Keywords: X inactivation, human embryonic stem cells, human induced pluripotent stem cells, XIS

Differentiation of Human Parthenogenetic Pluripotent Stem Cells Reveals Multiple Tissue- and Isoform-Specific Imprinted Transcripts

Parental imprinting results in monoallelic parent-of-origin-dependent gene expression. However, many imprinted genes identified by differential methylation do not exhibit complete monoallelic expression. Previous studies demonstrated complex tissue-dependent expression patterns for some imprinted genes. Still, the complete magnitude of this phenomenon remains largely unknown. By differentiating human parthenogenetic induced pluripotent stem cells into different cell types and combining DNA methylation with a 5′ RNA sequencing methodology, we were able to identify tissue- and isoform-dependent imprinted genes in a genome-wide manner. We demonstrate that nearly half of all imprinted genes express both biallelic and monoallelic isoforms that are controlled by tissue-specific alternative promoters. This study provides a global analysis of tissue-specific imprinting in humans and suggests that alternative promoters are central in the regulation of imprinted genes

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

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

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