Stem cell transcription factor networks in adult male germ cell tumors

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The transcription factors (TFs) SOX2, NANOG, and POU5F1 play essential roles in the initiation and maintenance of pluripotency in embryonic stem (ES) cells. Identification of target genes of these TFs would be useful in understanding the processes involved in pluripotency, but to date, only one such study has been performed, using chromatin immunoprecipitation (ChIP). We identified targets of SOX2, NANOG, and POU5F1 in pluripotent adult male germ cell tumors (GCTs) using ARACNE (Algorithm for the Reconstruction of Accurate Cellular Networks), an information theoretic algorithm that uses expression profiles to generate target gene lists with a high degree of accuracy, although it does not identify all target genes. GCTs are useful for such studies, since the GCT subtype embryonal carcinoma (EC) expresses many stem cell-specific markers and displays pluripotency both in vivo and in vitro. ARACNE identified 738 probe sets (714 genes) that were regulated by at least one of the TFs in a panel of 141 GCT profiles. SOX2 regulated 395, NANOG regulated 209, and POU5F1 regulated 208 genes respectively. There were 11 genes that were co-regulated by all three TFs, comprised of two uncharacterized EST, A4GALT, ADD2, DPPA4, FAM9C, HLA-DPB2, JARID2, NALP7, PHACTR1, and TERF1. Hierarchical clustering of the 738 probe sets separated the GCTs on the basis of histology, with three main gene clusters showing expression in subsets of either the pluripotent or differentiated tumors. These results are consistent with previous studies which suggest that the TFs not only activate pro-pluripotency genes, but also repress differentiation specific genes. In comparison to the previously published ChIP data, there was only partial overlap of the target genes. For example, of 395 SOX2 targets identified by ARACNE, 48 were also found to be targets of one of the three TFs in the ChIP data (29 genes overlapped between the SOX2-specific lists). Similar results were seen for NANOG and POU5F1. Differences between the data sets may reflect non-functional interactions and culture artifacts (ChIP data), or differentiation processes including methylation changes and incomplete identification of all target genes (ARACNE data). Expression of target genes in independent data sets indicated that most of the genes expressed in EC tumors were also highly expressed in ES cells, and that induction of differentiation led to their down-regulation. Finally, SOX2 was found to be the major TF controlling differential expression between pluripotent EC tumors and non-pluripotent Seminomas, consistent with our prediction that SOX2 expression differences play an essential role in the observed differentiation potential of these GCT subtypes. In conclusion, this study has identified a large panel of novel genes that are targets of SOX2, NANOG, and POU5F1, which should complement the previously generated ChIP gene list to expand our understanding of the genes important in pluripotency.