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  • br Experimental Procedures br Author


    Experimental Procedures
    Author Contributions
    Acknowledgments The authors thank Chyuan-Sheng Lin from the Transgenic Mouse Facility at the Herbert Irving Comprehensive Cancer Center at Columbia University for development of the Zfyve27-CreERT2 mice; Theresa Swayne from the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University for support for the confocal imaging and 2-photon microscopy (she is supported by NIH grant #P30 CA013696, National Cancer Institute, and the Nikon A1RMP confocal microscope was purchased with NIH grant #S10 RR025686); Kristie Gordon from the Flow Cytometry Shared Resource Facility of the Herbert Irving Comprehensive Cancer Center at Columbia University for support with FCAS; Mathew Zimmer from the New York State Foundation for support with flow cytometry; and Roseann Zott and Serge Cremers from the Irving Institute for Clinical and Translational Research for the LC/MS measurements.
    Introduction Human male germ cell tumors (GCTs) are thought to originate in primordial germ cells (PGCs) most likely by a mechanism similar to that recently described for the origin of teratocarcinomas in strain 128 family mice (Heaney et al., 2012). The key driver for this process is suggested to be upregulation of genes in the pathways controlling pluripotency and proliferation, such as NANOG, CCND2, and RASK2 that map to chromosome 12p (Chaganti and Houldsworth, 2000; Korkola et al., 2006). GCTs comprise two main subsets, seminoma (SEM) and nonseminoma (NS), with a common precursor, germ cell neoplasia in situ (GCNIS). SEM is unipotent whereas the NS subset embryonal carcinoma (EC) is pluripotent, analogous to the lxr agonist (Andrews et al., 2005), and has a gene-expression profile (GEP) similar to that of embryonic stem cells (ESCs) (Sperger et al., 2003; Josephson et al., 2007). EC differentiates to extraembryonic (choriocarcinoma, yolk sac tumor) and embryonic (teratoma) lineages (Chaganti and Houldsworth, 2000). Comparison of GEPs of human PGC (hPCG)-like cells derived in vitro from ESCs, gonadal GCs, and the SEM cell line TCam-2 suggested that SEM arises in PGCs and hence is a good model system to investigate hPGC biology (Irie et al., 2015). SOX17 was shown to be the key specifier of hPGC fate, with the downstream PRDM1 repressing mesendodermal genes (Irie et al., 2015). The core pluripotency regulatory master transcription factors (TFs) POU5F1 and NANOG are expressed in both EC and SEM, whereas SOX2 is repressed in hPGCs (Perrett et al., 2008; Irie et al., 2015), GCNIS, and SEM (Korkola et al., 2006). The molecular mechanism of SOX2 repression in the hPGC-GCNIS-SEM lineage has so far not been characterized. We show here that SOX2 repression in TCam-2 cells is due to the co-occupation by the Polycomb group (PcG) proteins and the repressive chromatin mark H3K27me3 near its transcription start site (TSS). We further show that the occupancy of H3K27me3 decreases when UTX, a H3K27-specific demethylase, is recruited to the SOX2 promoter in response to retinoid signaling, leading to SOX2 transcriptional derepression and induction of neuronal genes, consistent with its function as a neuroectodermal effector (Thomson et al., 2011; Zhang and Cui, 2014). Thus, SOX2 repression in TCam-2/SEM is imposed by PcG and its derepression is regulated by UTX. These data are consistent with a model of hPGC development initiated by SOX17, with PRDM1 repressing mesodermal genes and SOX2 repression inhibiting neuroectodermal genes. Although murine and human PGCs re-express pluripotency genes following specification, pluripotency remains latent and becomes functional only when PGCs are cultured in vitro as embryonic germ cells or transform in vivo as GCTs (Leitch et al., 2013). By analysis of GEPs of SEM and EC, we show here that the functional pathways of SEM reflect their derivation from PGCs, while those of EC, also derived from PGCs, reflect re-establishment of pluripotency in the transformed PGCs. These data are of value in understanding the biology of hPGCs and regulation of the pluripotency state in the unique GCT system.