Gene expression within the amplified region.

<p>A) Expression histogram indicating the relative expression of genes mapped within the amplified region in a series of SE and EC. The stars indicate genes significantly differentially expressed between SE and EC (according to Mann Whitney U test). Most of the genes are represented by multiple specific probes; B) DNA-FISH result for SOX2 in NCCIT cells. A centromere 12 (C12) specific probe is used as control. Red dye (Cye3) shows SOX2 probe. For C12 probe green dye (FITC) is used. The blue background color is DAPi. Multiple red spots for SOX2 are detectable in each cell containing two green spots for C12, indicating SOX2 amplification. Magnification used was 630x. DNA-FISH was performed on cut tissue section with a thickness of 4 micron. This results in possible heterogeneity of the probe sizes detected. This issue was not a limitation as the purpose of this experiment was to determine the copy numbers and the size or intensity of the region. The FISH (BAC) probes were ordered at BACPAC Resources Center (BPRC) online:bacpac.chori.org. They were verified and confirmed at the department of genetics.</p

OCT3/4 and SOX2 knockdown in NCCIT.

<p>A) Percentage of positive cells for OCT3/4 and SOX2 in cells with reduced OCT3/4 and SOX2 levels compared to cells transfected with control siRNA in the NCCIT cells in three different time-points post transfection based on immunohistochemistry; The controls are set to 100 in all cases. B) Relative expression pattern of 32 genes representing targets for pluripotency and differentiation (ectoderm, mesoderm and endoderm). The expression levels are normalized based on the housekeeping gene <i>HPRT</i>; C) Percentage of living NCCIT cells with reduced level of OCT3/4 and SOX2 compared to cells transfected with control siRNA at each time point based on Trypan blue measurement; D) Percentage of positive NCCIT cells for Ki67 in untreated cells, cells transfected with control siRNA and cells with reduced levels of OCT3/4 and SOX2 at 48+12 h post transfection; E) Percentage of positive cells for Caspase 3 in untreated NCCIT cells, Cells transfected with control siRNA show a reduced level of OCT3/4 and SOX2 at all three time points after the transfection; F) FACS analysis with Propidium Iodide staining in cells transfected with control siRNA, cells transfected with SOX2 and OCT3/4 siRNA at 60 h post transfection. NCCIT cells transfected with control siRNA show the presence of 78% living cells, while cells transfected with SOX2 siRNA show 51% living cells, NCCIT cells with OCT3/4kd show 62% live cells. G) Annexin V assay results in cells with siRNA control and cells with reduced SOX2 and OCT3/4 at 60 h after transfection. In the control, percentages of living-annexin negative cells are 89.6%, while dead-annexin positive cells are 12.2%. In SOX2kd cells, the amount of living-annexin negative cells is 57.5% while the amount of dead-annexin positive cells is 17.2%. In cells transfected with OCT3/4 siRNA, living-annexin negative cells are 68.3% while dead-annexin positive cells are 12.8%. The differences between the percentages of live and dead cells in all experiments are due to using independent and various methods in order to prove apoptosis and different sensitivity of the materials and methods used. As it is demonstrated, OCT3/4 knock-down also induces apoptosis, however, this effect is not as dramatic as SOX2 knock-down. The FACS analysis were performed in triplicate.</p

Schematic representation of the hypothetical indirect regulation of SOX2 by p53 status.

<p>Wild type p53 results in induction of expression of miR-145 and miR-34a. Subsequently, miR-34a and mir-145 down-regulates pluripotency genes, including SOX2, the latter via Wnt signaling.</p

Methylation profile at GCT subtype specific differentially methylated regions (DMRs).

<p>Visualization of the methylation percentage at specific loci is used to zoom in on a predefined region and investigate local methylation differences between GCT subtypes. <b>(A) <i>DMRT3</i>, (B) <i>SOX2</i>, (C) <i>POU5F1</i> (<i>OCT3/4</i>), (D) <i>TEX14</i>. (Visualizations)</b> From top to bottom the following is depicted: (1) Four-color heat map indicating methylation % for each individual probe in the depicted region. For the sample groups specified on the left the median methylation % is shown. (2) Position of all probes in the region of interest (ROI) is annotated as black rectangles. (3) HMM segments are displayed as grey boxes spanning the segment’s width and grouped per state. Numbers indicate the state of each (group of) segment(s). (5) GC% was obtained from the UCSC genome browser database (gc5Base table). (6) Transcripts overlapping with the ROI are plotted at the bottom. Plot generated using the Gviz package. Abbreviations of histological subtypes are explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">Fig 1A</a>. Please note that the TE group is subdivided based on gender and localization: I = type I; II = type II/formally part of the mNS group, s = sacrum, t = testis, o = ovary, m = male, f = female. CL indicates cell lines.</p

Methylation of imprinting control regions and the X chromosome.

<p>Analogous to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g002" target="_blank">Fig 2</a> the differences in methylation status between histological GCT subtypes is illustrated by two methods. Firstly, the methylation pattern is visualized using the distribution of the methylation percentage β. Next, the discriminatory power of the methylation pattern for each individual sample is shown using principal component analysis. <b>(A)</b> All probes associated with paternally expressed genes (ICR_P). <b>(B)</b> All probes associated with maternally expressed genes (ICR_M). <b>(C)</b> All probes located on the X chromosome. <b>(D)</b> Distribution of methylation in individual TE samples ordered by sex and localization. To compare type I and II TE the n = 3 type II pure TEs from the mNS were included in this visualization. Methylation levels of all probes, and probes associated with ICRs (P/M) and probes on the X chromosome are subsequently shown. <b>(Distribution plots of methylation percentage.)</b> Violin plots: grey areas indicate a kernel density plot of the methylation percentage (β) of all probes in all samples in a certain category. The boxplot indicates the interquartile range (black bars) and median (white squares). X-axis labels indicate histological subgroup according to Fig <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">1A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">1B</a>. TE indicates type I TE only. (<b>Principal Component Analysis.)</b> The first two principal components (PC) are plotted to evaluate the discriminative power of the methylation pattern between the subtypes. Abbreviations of histological subtypes are explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">Fig 1A</a>. CL indicates cell lines. Please note that in the legend of the PCA the TE group is subdivided based on gender and localization: I = type I; II = type II/formally part of the mNS group, s = sacrum, t = testis, o = ovary, m = male, f = female.</p

GCT methylation status in context of methylation during germ cell development.

<p>The top and bottom line charts depict normal germ cell development in female and male respectively (stages specified in the middle black bar). Methylation status during normal germ cell development is depicted for the global genome, ICRs and chromosome X (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#sec010" target="_blank">Discussion</a>). Putative cells of origin of the various types of GCTs are indicated in the brown boxes. ICR_P/M = ICR regulating paternally/maternally expressed genes. Bimodal indicates a methylation pattern peaking 0 and 100% with the exception of SE/DG (between 0 and ≈50). The table (bottom) provides a summary of the results, mainly Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g006" target="_blank">6</a>. Abbreviations: pf = primordial follicle. Type I tumors are indicated with their type (I), sex (m = male, f = female) and location (s = sacral, t = testis, o = ovary). Other GCT subtypes are indicated with their type (I, II, IV) and the abbreviation of each histological class, which are explained in the main text. Gradient bars indicate percentages of methylation (0→100%, green-white-grey-red) analogous to the gradient used in the other figures.</p

Functional enrichment of DMPs.

<p>DMPs were classified according to their functional genomic location (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">Fig 1C</a>). Statistical over- and underrepresentation of probes in certain categories provides clues to differences between GCT subtypes in regarding function of methylation. Enrichment was assessed by comparing the number of probes in a functional category in a subset of DMPs with the that in the total dataset (Fisher’s Exact test, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#sec011" target="_blank">Materials & Methods</a> section). Results are shown for four pairwise (A vs B) comparisons of histological subtypes: <b>(A)</b> SE/DG versus EC/mNS; <b>(B)</b> SE/DG vs type I TE; <b>(C)</b> EC/MNS vs type I TE and <b>(D)</b> SE/DG vs SS. <b>(LEFT)</b> The number (n) of DMPs identified in either the DMP[<u><b>A</b></u>-B] (hypermethylated in A, green) or DMP[A-<u><b>B</b></u>] (hypermethylated in B, red) group. <b>(MIDDLE/RIGHT)</b> Functional enrichment in the DMP[<u><b>A</b></u>-B] and DMP[A-<u><b>B</b></u>] group respectively. X-axis: positive numbers indicate a significant overrepresentation of DMPs in a functional category compared to non-DMPs while negative numbers indicate a significant underrepresentation. Depicted is the log2 ratio of (1) the % of either DMP group assigned to a category and (2) the % of non-DMPs assigned to that category. Only significant enrichments are depicted (2-sided Fisher’s Exact test, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#sec011" target="_blank">Methods</a> section for Bonferroni corrected α threshold). DMPs[se/dgvs<u><b>SS</b></u>].IMPR_P1500 showed significant underrepresentation, but could not be plotted on log scale (0 probes in DMP group). Details of calculations and raw counts and percentages are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.s008" target="_blank">S2 Table</a>. Y-axis: functional categories as specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">Fig 1C</a>.</p

Methylation patterns in GCT subtypes and cell lines.

<p>To illustrate differences in methylation status between histological GCT subtypes two (visualization) methods were applied. Firstly, the methylation pattern over the whole genome and specific functional categories (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">Fig 1C</a>) is visualized using the distribution of the methylation percentage β in all samples of a certain GCT subtype. Next, the discriminatory power of the methylation pattern for each individual sample is shown using principal component analysis. <b>(A) Distribution of methylation percentage.</b> Violin plots: grey areas indicate a kernel density plot of the methylation percentage (β) of all probes in all samples in a certain category. The boxplot indicates the interquartile range (black bars) and median (white squares). X-axis labels indicate histological subgroup according to Fig <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">1A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">1B</a>. TE indicates type I TE only. <b>(B) Principal Component Analysis.</b> The first two principal components (PC) are plotted to evaluate the discriminative power of the methylation pattern between the subtypes. Abbreviations of histological subtypes are explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">Fig 1A</a>. CL indicates cell lines. Please note that in the legend of the PCA the TE group is subdivided based on gender and localization: I = type I; II = type II/formally part of the mNS group, s = sacrum, t = testis, o = ovary, m = male, f = female. A more detailed visualization of the TE classes is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.s002" target="_blank">S2 Fig</a>, which also includes the full series of 18 functional categories, bootstrap validation of the PCA and an estimation of the variance explained by the first two principal components.</p

Tumor types/samples and cell lines analyzed and schematic visualization of genomic functional categories of interest.

<p><b>(A)</b> GCT subtypes in the hypothesized context of normal germ cell development as proposed in earlier studies (grey box). Developmental schemes are indicated in blue (male), red (female) or when possible in both sexes (white). DG does not originate from CIS but is indicated together with SE for reasons of consistency. <b>(B)</b> Samples included in this study. Abbreviations match Fig 1A and roman numbers indicate the GCT type to which the histological subtypes belongs. n indicates the number of tumor samples per group. All samples are from male patients except the DGs, DCs and a subset of the type I TEs. Please note that when only TE is denoted, this indicates the group of all type I TEs together. Otherwise II.TE (type II pure TE) or the abbreviations for specific localizations are used as indicated in this figure. Four GCT cell lines were included; tumor of origin between brackets. <b>(C)</b> Reference to (abbreviations of) the functional genomic regions as mentioned in the rest of the manuscript. Probes were classified according to their relation to gene coding regions, micro-RNA (MIR) coding regions, CpG islands and/or transposon elements (LINE/SINE). The distance to the transcription start site (TSS) was used in accordance with the Illumina manifest: 200 or 1500 bp. Of note, the TSSAssociated category contains all probes with a distance < 1500 bp to the TSS in contrast to the TSS1500 category from Illumina which is only contains probes 200-1500bp from the TSS. Probes within imprinting associated regions were classified as (1) mapped inside a known imprinting control region (ICR) or (2) either mapped inside an ICR or mapped close to the TSS of a transcript of an imprinted gene (200/1500bp upstream, not mutually exclusive). P/M indicates the expressed allele, i.e. paternal/maternal respectively. Numbers between brackets indicate the number of valid probes within each specific category (total number of valid probes: 437,881). *The visualization did not permit including the probe count for all categories. The counts for the empty categories are: 5’UTR = 59,338; ISLAND = 136,339; IMPR_P200 = 638; IMPR_P1500 = 1,659; IMPR_M200 = 610; IMPR_M1500 = 2,265.</p

Methylation profile of GCT specific genes and regions of interest (ROIs).

<p>Visualization of the methylation percentage at specific loci is used to zoom in on a predefined region and investigate local methylation differences between GCT subtypes. The genes are reviewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.t002" target="_blank">Table 2</a>. <b>(A) <i>AR</i>, (B) <i>miR-371-2-3</i>, (C) <i>NANOG</i>, (D) <i>SOX17</i>. (Visualizations)</b> From top to bottom the following is depicted: (1) Four-color heat map indicating methylation % for each individual probe in the depicted region. For the sample groups specified on the left the median methylation % is shown. (2) Position of all probes in the region of interest (ROI) is annotated as black rectangles. (3) HMM segments are displayed as grey boxes spanning the segment’s width and grouped per state. Numbers indicate the state of each (group of) segment(s). (5) GC% was obtained from the UCSC genome browser database (gc5Base table). (6) Transcripts overlapping with the ROI are plotted at the bottom. Plot generated using the Gviz package. Abbreviations of histological subtypes are explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122146#pone.0122146.g001" target="_blank">Fig 1A</a>. Please note that the TE group is subdivided based on gender and localization: I = type I; II = type II/formally part of the mNS group, s = sacrum, t = testis, o = ovary, m = male, f = female. CL indicates cell lines.</p