SummaryThe AP-2 family of transcription factors consists of five different proteins in humans and mice: AP-2␣, AP-2␤, AP-2␥, AP-2␦ and AP-2⑀. Frogs and fish have known orthologs of some but not all of these proteins, and homologs of the family are also found in protochordates, insects and nematodes. The proteins have a characteristic helix-span-helix motif at the carboxyl terminus, which, together with a central basic region, mediates dimerization and DNA binding. The amino terminus contains the transactivation domain. AP-2 proteins are first expressed in primitive ectoderm of invertebrates and vertebrates; in vertebrates, they are also expressed in the emerging neural-crest cells, and AP-2␣ -/-animals have impairments in neural-crest-derived facial structures. AP-2␤ is indispensable for kidney development and AP-2␥ is necessary for the formation of trophectoderm cells shortly after implantation; AP-2␣ and AP-2␥ levels are elevated in human mammary carcinoma and seminoma. The general functions of the family appear to be the cell-type-specific stimulation of proliferation and the suppression of terminal differentiation during embryonic development. Gene organization and evolutionary historyThe AP-2 family of transcription factors (Ensembl Family ENSF00000001105) consists in humans and mice of five members, AP-2␣, AP-2␤, AP-2␥, AP-2␦ and AP-2⑀; frogs and fish have some of these proteins, and homologs are also known in invertebrates. The chromosomal locations and accession numbers of the family are given in Tables 1 and 2, respectively. All mammalian AP-2 proteins except AP-2␦ are encoded by seven exons and share a characteristic domain structure (reviewed in [1]; for AP-2␦ see [2] and for AP-2⑀ see [3,4]). Orthologs show a similarity between 60 and 99% at the amino-acid level, whereas paralogs show a similarity between 56 and 78%.Analysis of the phylogenetic tree (Figure 1) reveals that the vertebrate AP-2 proteins are grouped together and are divided into five groups. The single Xenopus AP-2 is most closely related to mammalian AP-2␣ proteins. As the genes AP-2␤ and AP-2␦ are found on the same chromosome in chickens, rodents and humans (Table 1), it is likely that they are the result of an internal duplication. According to the phylogenetic tree, AP-2␦ genes appear to have separated from the rest of the family early in the vertebrate clade and to have evolved separately (Figure 1). A BLAST search of the puffer fish Fugu rubripes fourth genome assembly database [5] suggests that there are orthologs of AP-2␣, AP-2␤, AP-2␥ and AP-2⑀ but not AP-2␦ genes in bony fish, although only orthologs of AP-2␣ and AP-2␤ have been found in zebrafish.In the genome of the protochordate Ciona intestinalis a single AP-2 gene has been predicted; the phylogenetic tree shows that the protein evolved before the split of the AP-2␣, AP-2␤, AP-2␥ and AP-2⑀ proteins, with the highest sequence similarity with the AP-2␣ group, suggesting that AP-2␣ might be most similar to the ancestor of AP-2 proteins. This hypothesis is further supported b...

Formation of the germ cell lineage involves multiple processes, including repression of somatic differentiation and reacquisition of pluripotency as well as a unique epigenetic constitution. The transcriptional regulator Prdm1 has been identified as a main coordinator of this process, controlling epigenetic modification and gene expression. Here we report on the expression pattern of the transcription factor Tcfap2c, a putative downstream target of Prdm1, during normal mouse embryogenesis and the consequences of its specific loss in primordial germ cells (PGCs) and their derivatives. Tcfap2c is expressed in PGCs from Embryonic Day 7.25 (E 7.25) up to E 12.5, and targeted disruption resulted in sterile animals, both male and female. In the mutant animals, PGCs were specified but were lost around E 8.0. PGCs generated in vitro from embryonic stem cells lacking TCFAP2C displayed induction of Prdm1 and Dppa3. Upregulation of Hoxa1, Hoxb1, and T together with lack of expression of germ cell markers such Nanos3, Dazl, and Mutyh suggested that the somatic gene program is induced in TCFAP2C-deficient PGCs. Repression of TCFAP2C in TCam-2, a human PGC-resembling seminoma cell line, resulted in specific upregulation of HOXA1, HOXB1, MYOD1, and HAND1, indicative of mesodermal differentiation. Expression of genes indicative of ectodermal, endodermal, or extraembryonic differentiation, as well as the finding of no change to epigenetic modifications, suggested control by other factors. Our results implicate Tcfap2c as an important effector of Prdm1 activity that is required for PGC maintenance, most likely mediating Prdm1-induced suppression of mesodermal differentiation.

Of all malignancies diagnosed in men between 17 and 45 years of age, 60% are germ cell tumors (GCT). GCT arise from carcinoma in situ cells, which are thought to originate from a transformed fetal germ cell, the gonocyte. Seminoma together with embryonal carcinoma represent the most frequent subtypes of GCT. However, the nature of the molecular pathways involved in seminoma formation remains elusive. Therefore, analysis of appropriate cell culture systems is an important prerequisite for further understanding of the etiology of this tumor entity. Although several cell lines for embryonal carcinoma have been established and analyzed, so far only two cell lines from seminoma patients have been reported. In the present study, we have analyzed these seminoma cell lines (TCam-2 and JKT-1) and compared the gene-expression profiles with those of normal tissue and of seminoma and embryonal carcinoma by using DNA Array technology. We have found that TCam-2 clusters with the group of classical seminoma, whereas JKT-1 clusters with the group of embryonal carcinoma. Using reverse transcription/polymerase chain reaction, Western blot, and immunohistochemistry, we have confirmed the seminoma-like nature of TCam-2, whereas JKT-1 lacks expression for most of the genes detectable in GCTs, thus making doubtful the germ cell nature of this cell line. The data represent the first genome-wide expression analysis of the two cell lines and comparison/clustering with subgroups of germ cell tumors. Only TCam-2 seems to represent a suitable in vitro model for seminoma.

Background: Most testicular germ cell tumors arise from intratubular germ cell neoplasia unclassified (IGCNU, also referred to as carcinoma in situ), which is thought to originate from a transformed primordial germ cell (PGC)/gonocyte, the fetal germ cell. Analyses of the molecular profile of IGCNU and seminoma show similarities to the expression profile of fetal germ cells/ gonocytes. In murine PGCs, expression and interaction of Blimp1 and Prmt5 results in arginine 3 dimethylation of histone H2A and H4. This imposes epigenetic modifications leading to transcriptional repression in mouse PGCs enabling them to escape the somatic differentiation program during migration, while expressing markers of pluripotency.

Testicular germ cell tumours (TGCTs) are the most frequent cancer type in young men; 5% of these patients develop a second TGCT in the contralateral testis. The pathogenesis of TGCT is closely linked to primordial germ cells (PGCs) or gonocytes. The receptor tyrosine kinase (c-KIT) is necessary for migration and survival of PGCs and is expressed in intratubular neoplastic germ cells (IGCNUs) and seminomas. We studied the frequency of c-KIT exon 11 and 17 mutations in 155 unilateral (108 seminomas and 47 non-seminomas) and 22 bilateral (18 seminomas, two embryonal carcinomas, two IGCNU) cases. While no mutations were detected in exon 11, the mutation frequency in exon 17 was significantly higher in bilateral (14/22, 63.6%) compared to unilateral TGCT (10/155, 6.4%) (p < 0.001). Different activating mutations (Y823D, D816V, D816H and N822K) were detected in bilateral TGCT. Y823D mutation was identical in both testes in three cases and quantitative pyrosequencing showed that up to 76% of the cells analysed in tumour samples carried this mutation. One bilateral synchronous seminoma revealed a S821F mutation in one testis and a Y823D mutation contralaterally. To study the role of c-KIT in TGCT progression, we compared its expression in 41 seminomas and adjacent IGCNUs. Immunohistochemical analysis revealed that c-KIT expression was significantly reduced in seminomas compared to IGCNUs (p < 0.006) and that there were no significant changes in c-KIT mRNA copy numbers in progressed compared to low-stage seminomas. In summary, our study shows that patients with c-KIT mutations are more prone to develop a bilateral TGCT and suggests that in a portion of bilateral TGCTs, c-KIT mutations occur early during embryonal development, prior to the arrival of PGCs at the genital ridge. Furthermore, our findings show that c-KIT down-regulation occurs during the progression of IGCNU to seminoma.