Additional file 1 of A statistical model for the analysis of beta values in DNA methylation studies

Contains distributional results and proofs of propositions. (PDF 54.1 kb

Inhibition of BMP signaling is crucial for initiation of reprogramming.

<p>(A) IF /IHC staining of pSMAD 1 /5 in parental and xenografted TCam-2 at indicated time points. Scale bar: 100 μm. (B, C) qRT-PCR analysis of indicated genes after NOGGIN treatment of TCam-2 cells for 1–8 days. (D) Western blotting of indicated proteins 8 days after NOGGIN treatment of TCam-2 cells.</p

BMP Inhibition in Seminomas Initiates Acquisition of Pluripotency via NODAL Signaling Resulting in Reprogramming to an Embryonal Carcinoma

<div><p>Type II germ cell cancers (GCC) can be subdivided into seminomas and non-seminomas. Seminomas are similar to carcinoma in situ (CIS) cells, the common precursor of type II GCCs, with regard to epigenetics and expression, while embryonal carcinomas (EC) are totipotent and differentiate into teratomas, yolk-sac tumors and choriocarcinomas. GCCs can present as seminomas with a non-seminoma component, raising the question if a CIS gives rise to seminomas and ECs at the same time or whether seminomas can be reprogrammed to ECs. In this study, we utilized the seminoma cell line TCam-2 that acquires an EC-like status after xenografting into the murine flank as a model for a seminoma to EC transition and screened for factors initiating and driving this process. Analysis of expression and DNA methylation dynamics during transition of TCam-2 revealed that many pluripotency- and reprogramming-associated genes were upregulated while seminoma-markers were downregulated. Changes in expression level of 53 genes inversely correlated to changes in DNA methylation. Interestingly, after xenotransplantation 6 genes (<i>GDF3</i>, <i>NODAL</i>, <i>DNMT3B</i>, <i>DPPA3</i>, <i>GAL</i>, <i>AK3L1</i>) were rapidly induced, followed by demethylation of their genomic loci, suggesting that these 6 genes are poised for expression driving the reprogramming. We demonstrate that inhibition of BMP signaling is the initial event in reprogramming, resulting in activation of the pluripotency-associated genes and NODAL signaling. We propose that reprogramming of seminomas to ECs is a multi-step process. Initially, the microenvironment causes inhibition of BMP signaling, leading to induction of NODAL signaling. During a maturation phase, a fast acting NODAL loop stimulates its own activity and temporarily inhibits BMP signaling. During the stabilization phase, a slow acting NODAL loop, involving WNTs re-establishes BMP signaling and the pluripotency circuitry. In parallel, DNMT3B-driven de novo methylation silences seminoma-associated genes and epigenetically fixes the EC state.</p></div

Inhibition of BMP signaling is crucial for initiation of reprogramming.

<p>(A) IF /IHC staining of pSMAD 1 /5 in parental and xenografted TCam-2 at indicated time points. Scale bar: 100 μm. (B, C) qRT-PCR analysis of indicated genes after NOGGIN treatment of TCam-2 cells for 1–8 days. (D) Western blotting of indicated proteins 8 days after NOGGIN treatment of TCam-2 cells.</p

Model of the dynamics and molecular mechanisms during the SET.

<p>(A) Model summarizing the dynamics and events driving acquisition of pluripotency and epigenetic reprogramming of TCam-2 cells to an EC-like cell fate. (B) Models of the fast and slow acting NODAL feedback loop. Arrows indicate 'activation', T-shaped arrows indicate 'inhibition'.</p

BMP, NODAL and WNT signaling in GCC tissues and cell lines.

<p>(A) cDNA microarray expression data of BMP, NODAL and WNT signaling-associated genes in GCC tissues. <i>SOX2</i> and <i>SOX17</i> were used to determine zero level of expression intensity (black line). (B) Pie diagrams summarizing ID1 IHC data of GCC-TMAs. Stainings were classified as ID1 positive (+), negative (-), mixed with much more positive than negative cells (+ >-) and mixed with much more negative than positive cells (+ <-). (C) ZIC3 protein levels in indicated GCC cell lines and tissues. (D) Pie diagrams summarizing beta-CATENIN IHC data of GCC-TMAs. Beta-CATENIN staining was classified as membraneous (m), cytoplasmic (c) and nuclear (n). (E) IF /IHC staining of beta-CATENIN in in parental and xenografted TCam-2 cells (1, 2 and 6 weeks). Scale bar: 100 μm.</p

Global dynamics of Gex and 5mC during reprogramming of TCam-2 cells.

<p>(A—B) Unsupervised hierarchical clustering illustrates genome-wide changes in Gex (A) and 5mC (B) over time in indicated samples. Dendrograms indicate that during SET TCam-2 cells become more similar to the EC control cells.</p

Consistently enriched categories of the GWAS on BC survival.

<p>Only categories enriched in all four gene lists and by more than one tool were considered consistently enriched. * Genes present in the 0.01 gene list (allelic SNP <i>p</i>-value cut-off 0.01).</p

Flow chart of the pathway enrichment analysis of the GWAS on BC survival.

<p>Flow chart of the pathway enrichment analysis of the GWAS on BC survival.</p

Distribution of the seven generic terms among the 50 top pathways in the enrichment analyses of the 13 gene lists.

<p>Distribution of the seven generic terms among the 50 top pathways in the enrichment analyses of the 13 gene lists.</p