Highly specific activation of Core and Myc modules and repression of the PRC module in pluripotent cells.

<p>Publicly available DNA microarray data for 20 different tissue/somatic cell and stem cell types were obtained from the NCBI GEO database. To compare the same sets of genes used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#pone-0083769-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#pone-0083769-g002" target="_blank">2</a>, data obtained using the same DNA microarray platform (Mouse Expression Array 430 platform, Affymetrix) by Hayashi et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#B29" target="_blank">29</a>] were selected from the database. Average gene expression values (log₂) of Core (upper panel), Myc (meddle panel), and PRC (lower panel) modules in each sample were calculated using those in ESCs as references. The data were aligned in an ordered fashion based on the value of average Myc module gene expression in which a sample showing the highest score, i.e., gPSC, was put at the left end of graph. The accession numbers of the obtained DNA microarray data are listed in the Materials and Methods. Data from germline stem cells and their derivatives, somatic stem cells, tissues, terminally differentiated hematopoietic cells and EpiSCs/EpiLCs are indicated by pink, blue, green, red, and gray bars, respectively, in the graph.</p

Comparison of the expression of Core and Myc module genes in EpiSCs and ESCs.

<div><p>(A) Average gene expression values (log₂) of Core, Myc, and PRC module genes in EpiSCs using values from ESCs as references. Data from 99 Core, 426 Myc, and 474 PRC module genes deposited in GEO under GSE30056 were used for the analyses. Data from 12 Core (111 genes), 77 Myc (503 genes), and 86 PRC (560) module genes are not available in the deposited data sets.</p> <p>(B) Comparison of the expression of individual Core, Myc, and PRC module genes between ESCs and EpiSCs. Left, middle, and right scatter plots show the expression values of individual Core, Myc, and PRC module genes, respectively, in ESCs and EpiSCs. Red and blue spots indicate genes with expression levels that are higher or lower by more than 2-fold in EpiSCs compared with those in ESCs, respectively. Gene symbols corresponding to red and blue are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#pone.0083769.s011" target="_blank">Table S2</a>. The variance value was calculated and is shown for each scatter plot.</p> <p>(C) Left, middle, and right scatter plots show the expression values of the selected Core, Myc, and Core module genes (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#pone.0083769.s012" target="_blank">Table S3</a>), respectively, in ESCs and EpiSCs. Red and blue spots indicate as described in B. The variance value was calculated and is shown for each scatter plot.</p></div

Striking Similarity in the Gene Expression Levels of Individual Myc Module Members among ESCs, EpiSCs, and Partial iPSCs

<div><p>Predominant transcriptional subnetworks called Core, Myc, and PRC modules have been shown to participate in preservation of the pluripotency and self-renewality of embryonic stem cells (ESCs). Epiblast stem cells (EpiSCs) are another cell type that possesses pluripotency and self-renewality. However, the roles of these modules in EpiSCs have not been systematically examined to date. Here, we compared the average expression levels of Core, Myc, and PRC module genes between ESCs and EpiSCs. EpiSCs showed substantially higher and lower expression levels of PRC and Core module genes, respectively, compared with those in ESCs, while Myc module members showed almost equivalent levels of average gene expression. Subsequent analyses revealed that the similarity in gene expression levels of the Myc module between these two cell types was not just overall, but striking similarities were evident even when comparing the expression of individual genes. We also observed equivalent levels of similarity in the expression of individual Myc module genes between induced pluripotent stem cells (iPSCs) and partial iPSCs that are an unwanted byproduct generated during iPSC induction. Moreover, our data demonstrate that partial iPSCs depend on a high level of c-Myc expression for their self-renewal properties.</p> </div

Most Myc module members maintain constant levels of expression in naïve and primed human iPSCs.

<div><p>(A) Average gene expression values (log₂) of Core, Myc, and PRC module genes in primed human iPSCs using those in human iPSCs converted to a naïve state as references <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#B33" target="_blank">33</a>. Data from 69 Core, 321 Myc, and 423 PRC module genes deposited in GEO under GSE21222 were used for the analyses. Data from six Core, 34 Myc, and 28 PRC module genes are not available in the deposited data sets.</p> <p>(B) Comparison of the expression of individual Core, Myc, and PRC module genes between naïve and primed human iPSCs. Left, middle, and right scatter plots show the expression values of individual Core, Myc, and PRC module genes, respectively, in naïve and primed human iPSCs. Red and blue spots indicate genes with expression levels that are higher or lower by more than 2-fold in primed human iPSCs compared with those in naïve human iPSCs, respectively. Gene symbols corresponding to red and blue are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#pone.0083769.s014" target="_blank">Table S5</a>. The variance value was calculated and is shown for each scatter plot.</p> <p>(C) Scatter plot analyses of the selected genes from Core (left), Myc (middle), and PRC (right) modules. The same sets of genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#pone.0083769.s012" target="_blank">Table S3</a>) used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083769#pone-0083769-g001" target="_blank">Figure 1C</a> were used for the analyses. The data lacked information for 15, 33, and 13 genes of the selected Core (50), Myc (98), and PRC (115) module genes, respectively. Red and blue spots indicate as described in B.</p></div

<i>In Vivo</i> Function and Evolution of the Eutherian-Specific Pluripotency Marker UTF1

<div><p>Embryogenesis in placental mammals is sustained by exquisite interplay between the embryo proper and placenta. <i>UTF1</i> is a developmentally regulated gene expressed in both cell lineages. Here, we analyzed the consequence of loss of the <i>UTF1</i> gene during mouse development. We found that homozygous <i>UTF1</i> mutant newborn mice were significantly smaller than wild-type or heterozygous mutant mice, suggesting that placental insufficiency caused by the loss of <i>UTF1</i> expression in extra-embryonic ectodermal cells at least in part contributed to this phenotype. We also found that the effects of loss of <i>UTF1</i> expression in embryonic stem cells on their pluripotency were very subtle. Genome structure and sequence comparisons revealed that the <i>UTF1</i> gene exists only in placental mammals. Our analyses of a family of genes with homology to UTF1 revealed a possible mechanism by which placental mammals have evolved the <i>UTF1</i> genes.</p></div

The <i>UTF1</i> gene is present only in the genomes of eutherian (placental) mammals.

<p>(A) Genomic organizations surrounding the <i>UTF1</i> locus in mammals and their corresponding genomic regions in other organisms. Black boxes indicate regions without available genomic sequences. (B) VISTA Browser (VGB2.0) plot of the 65.5 kb interval (ch10∶134,506,934-134,572,432) containing <i>KNDC1</i>, <i>UTF1</i> and <i>VENTX</i> genes in the human genome. Conservation plots for elephant (top panel) mouse (second panel), opossum (third panel) and wallaby (bottom panel), with respect to human, are shown in the coordinates of the human sequence (horizontal axis). Dark blue boxes indicate portions with unavailable DNA sequences in the database.</p

Isolation of UTF1 homozygous mutant ESCs from blastocysts.

<p>(A) Western blot analyses of pluripotency marker proteins in ESCs generated by outgrowth of <i>UTF1</i> homozygous and heterozygous mutant blastocysts. (B) Comparison of the differentiation propensity between <i>UTF1</i> homozygous and heterozygous mutant ESCs. <i>UTF1</i> homozygous and heterozygous mutant ESCs were induced to differentiate by embryoid body formation. RNAs were prepared at the indicated days. cDNAs generated by reverse transcription were used to examine the levels of differentiation marker gene expression by real-time PCR. Data represent the mean with SD (n = 3). (C) Western blot analyses of Arf tumor suppressor protein in <i>UTF1</i> heterozygous and homozygous mutant ESCs. (D) Chimeric mouse analyses of <i>UTF1</i> homozygous mutant ESCs. Fluorescent Kusabira orange-labeled <i>UTF1</i> homozygous mutant ESCs generated from a blastocyst were injected into blastocysts. Embryos were allowed to develop in a recipient female mouse. Left and right panels are bright-field and fluorescence images of a 9.5 dpc embryo recovered from the recipient mouse, respectively.</p

Implication of derivation of <i>UTF1</i> from <i>ZCSAN20-like</i> in an ancestor of placental mammals.

<p>(A) Diagram showing the domain structure of ZCSAN20-like protein with the amino acid sequence (1–867). Triangles with numbers indicate positions corresponding to exon-intron boundaries of the gene encoding ZCSAN20-like protein. Brown and black bars indicate positions with significant similarity to UTF1, which are shown in B and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068119#pone.0068119.s006" target="_blank">Figure S6</a>, respectively. (B) Alignment of the amino acid sequence of the SANT domain of human UTF1 with that of ZSCAN20-like protein. Amino acid identity and similarity between UTF1 and ZSCAN20-like are marked by double and single dots, respectively. (C) A neighbor-joining tree analysis was constructed based on the number of nucleotide differences per site. The bootstrap value supporting each internal branch is indicated at the node. A cluster containing UTF1s and opossum ZSCAN20-like is boxed with a red line. (D) Model of <i>UTF1</i> evolution in a common ancestor of eutherian (placental) mammals after divergence which separates from marsupials. Data in B, C, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068119#pone.0068119.s006" target="_blank">Figure S6</a> suggest that the <i>UTF1</i> gene was derived from a portion of the <i>ZCAN20-like</i> gene. After this transition, extensive nucleotide sequence changes occurred to evolve the <i>UTF1</i> gene, which eliminated the trace of homology between UTF1 and ZSCAN20-like except for two regions including their SANT domains. However, the depicted panel is just one possibility. For example, regions without similarity to the <i>ZCAN20-like</i> gene (depicted with a red rectangle) could have arisen <i>de novo</i> by modification of the sequences that had been present before translocation of the <i>ZCAN20-like</i> gene. The second SANT domain of ZSCAN20-like and its possible derivative in UTF1 are indicated by a brown bold line, while regions corresponding to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068119#pone.0068119.s006" target="_blank">Figure S6</a> are marked by a black line.</p

Phenotypes of <i>UTF1</i> homozygous mutant mice with a mixed genetic background of C57BL/6J (25%) and ICR (75%).

<p>(A) Weight of viable <i>UTF1</i> homozygous mutant mice generated by intercrossing heterozygous mutant mice that had been backcrossed twice with wild-type ICR mice. These analyses revealed that adult <i>UTF1</i> homozygous mutant mice (4-weeks-old) were viable, but significantly smaller than corresponding wild-type and heterozygous mutant mice. Data represent the mean with SD (n = 4). **, p<0.01. (B) Intercrosses between <i>UTF1</i> homozygous and heterozygous mutant mice. These intercrosses showed that the <i>UTF1</i> homozygous mutation with a mixed mouse genetic background did not affect fertility, but led to the generation of small mice. Mouse weights were examined at 4 weeks after birth. Data represent the mean with SD (n = 4). *, p<0.05;</p

Acquisition of pluripotency by <i>UTF1</i>-knockout homozygotes.

<p>(A) Comparison of the frequency between knockouts of first and second alleles in serial <i>UTF1</i> gene targeting. A <i>UTF1-</i>targeting vector carrying the puromycin resistance gene was introduced into wild-type and <i>UTF1</i> heterozygous mutant ESCs bearing the blasticidin resistance gene in one of the <i>UTF1</i> loci by electroporation, and were then selected with medium containing puromycin only or puromycin and blasticidin, respectively. Genomic DNAs were prepared from drug-resistant clones and used as templates for PCR to distinguish between homologous recombination and random integration of the vector. Frequencies of targeted disruption in wild-type and <i>UTF1</i> heterozygous mutant ESCs were 2.08% and 0.88%, respectively. Because the probability of targeted disruption in wild-type ESCs is considered to be twice as high as that in heterozygous mutant ESCs, we concluded that the second allele knockout in serial gene targeting of the <i>UTF1</i> loci is not a rare event compared with the first allele knockout. (B) Western blot analyses of UTF1 and other pluripotency marker proteins in <i>UTF1</i> homozygous mutant ESCs (UTF1 KO ES) generated in A and iPSCs generated from <i>UTF1</i> homozygous mutant MEFs (UTF1 KO iPS). Wild-type ESCs were used as references. (C) H&E-staining of sections of a teratoma containing differentiated cells of all three germ layers generated by injection of <i>UTF1</i>-null iPSCs into nude mice. Representative portions of the three germ layers are marked by brackets.</p