Abstract 4240: Transcription factor networks that regulate pluripotency and lineage differentiation in adult human male germ cell tumors
Authors: Nirmala Jagadish, Manjunath Kustagi, Geetu Mendiratta, Ritu Kushwaha, James E. Korkola, Jane Houldsworth, Agnes Viale, Kim R. Hyunjae, Pavel Sumazin, George J. Bosl, Andrea Califano, Raju S K Chaganti
Journal: Cancer Research (2010)

Abstract Recent interest and effort have been focused on identification of transcription factors (TFs) and TF networks that regulate pluripotency and lineage differentiation in human and murine cells. Yet, a complete repertoire of regulatory interactions supporting these processes remains elusive. Embryonic stem (ES) cells, induced pluripotent (iPS) cells, and embryonal carcinoma (EC) cells derived from germ cell tumors (GCTs) comprise the three classic pluripotent cell types. EC cells exhibit, in vivo and in vitro, many properties in common with ES cells, making them an excellent system to study pluripotency and lineage differentiation. We previously characterized the gene expression profile (GEP) of a panel of 135 GCT biopsies comprising all described in vivo differentiation lineages and 6 normal testes as controls, using the Affymetrix U133A+B arrays (Korkola et al. Cancer Res., 66: 820-7, 2006 and J. Clin. Oncol., 27: 5240-7, 2009). These GEP data were analyzed using the ARACNe reverse engineering algorithm (Basso et al., Nat. Genet., 37: 382-90, 2005) to reconstruct a global transcriptional interaction network regulating pluripotency and lineage differentiation in these tumors. The ensuing interactome, comprising 1312 TFs, displayed three strongly intra-connected modules: Module A, including 187 genes associated with regulation of development and differentiation, Module B, including 89 genes associated with regulation of immune response and B-cell development, and Module C, including 80 genes associated with regulation of transcription. Furthermore, the interactome recapitulated 830 regulatory targets of the core pluripotency TFs, POU5F1, NANOG, and SOX2 (369 of POU5F1, 375 of NANOG, and 306 of SOX2). Validation of these targets by ChIP-PCR and ChIP-seq in NT2/D1 (EC) and H9 (ES) cells revealed that all of the top 10 target genes of POU5F1 (NANOG, FAM46B, L1TD1, DND1, ZYG11, FRAT2, DPPA4, ARGEF1, KLRG2 and CBR3) and SOX2 (SEPHS1, PLCB2, ZNF518, ECE2, SLC26A3, GPM6B, PHLDA2, PATZ1, ADRB3 and MAP1A) could specifically bind the corresponding TF in their promoter regions, within both cell types. For NANOG, 9 of the top 10 targets (POU5F1, FAM46B, DPPA4, L1TD1, UTF1, FRAT2, RAB15, TEAD4 and ZYG11A) showed specific promoter binding of the TF in both cell types, while one target (DND1) showed specific binding only in H9 cells. ChIP-seq analysis was consistent with the results of ChIP-PCR. We sought functional validation of the targets by performing lentivirus-mediated shRNA silencing of POU5F1 in NT2/D1 cells. Results showed that the ARACNe-inferred targets were both positively and negatively regulated, consistently with regulation of pluripotency and/or differentiation. Taken together, these results define a novel and highly accurate regulatory model for the systems biology study of TFs involved in regulation of pluripotency and lineage differentiation in mammalian cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4240.