Specification of germ cell fate is fundamental in development and heredity. Recent evidence indicates that in mice, specification of primordial germ cells (PGCs), the common source of both oocytes and spermatozoa, occurs through the integration of three key events: repression of the somatic program, reacquisition of potential pluripotency and ensuing genome-wide epigenetic reprogramming. Here we provide genetic evidence that Prdm14, a PR domain-containing transcriptional regulator with exclusive expression in the germ cell lineage and pluripotent cell lines, is critical in two of these events, the reacquisition of potential pluripotency and successful epigenetic reprogramming. In Prdm14 mutants, the failure of these two events manifests even in the presence of Prdm1 (also known as Blimp1), a key transcriptional regulator for PGC specification. Our combined evidence demonstrates that Prdm14 defines a previously unknown genetic pathway, initiating independently from Prdm1, for ensuring the launching of the mammalian germ cell lineage.

We previously reported that primordial germ cells (PGCs) in mice erase genome-wide DNA methylation and histone H3 lysine9 dimethylation (H3K9me2), and instead acquire high levels of tri-methylation of H3K27 (H3K27me3) during their migration, a process that might be crucial for the re-establishment of potential totipotency in the germline. We here explored a cellular dynamics associated with this epigenetic reprogramming. We found that PGCs undergo erasure of H3K9me2 and upregulation of H3K27me3 in a progressive, cell-by-cell manner, presumably depending on their developmental maturation. Before or concomitant with the onset of H3K9 demethylation, PGCs entered the G2 arrest of the cell cycle, which apparently persisted until they acquired high H3K27me3 levels. Interestingly, PGCs exhibited repression of RNA polymerase II-dependent transcription, which began after the onset of H3K9me2 reduction in the G2 phase and tapered off after the acquisition of high-level H3K27me3. The epigenetic reprogramming and transcriptional quiescence were independent from the function of Nanos3. We found that before H3K9 demethylation, PGCs exclusively repress an essential histone methyltransferase, GLP, without specifically upregulating histone demethylases. We suggest the possibility that active repression of an essential enzyme and subsequent unique cellular dynamics ensures successful implementation of genome-wide epigenetic reprogramming in migrating PGCs.

Persistent infection of hepatitis C virus (HCV) is a major cause of liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Searching for a substance with anti-HCV potential, we examined the effects of a variety of compounds on HCV replication using a HCV subgenomic replicon cell culture system. Consequently, the immunosuppressant cyclosporin A (CsA) was found to have a suppressive effect on the HCV replicon RNA level and HCV protein expression in these cells. CsA also inhibited multiplication of the HCV genome in a cultured human hepatocyte cell line infected with HCV using HCV-positive plasma. This anti-HCV activity of CsA appeared to be independent of its immunosuppressive function. In conclusion, our results suggest that CsA may represent a new approach for the development of anti-HCV therapy. (HEPATOLOGY 2003;38:1282-1288 P ersistent infection with the hepatitis C virus (HCV), identified as the major causative agent of non-A, non-B hepatitis, 1,2 has been closely related to liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. 3 The development of these liver diseases from HCV carriers, an estimated 170 million people throughout the world, is a major public health problem. Effective anti-HCV therapy has been restricted mainly to therapy with interferon (IFN) and a combination of IFN and ribavirin. However, because the virus is not eliminated from approximately one half of HCVinfected patients treated with these agents, 4 alternative approaches to the treatment of HCV infection are needed.Recently, an HCV subgenomic replicon cell culture system has been established in which an HCV subgenomic replicon autonomously replicated in Huh-7 cells (HCV replicon cells). 5 This replicon is composed of the HCV 5Ј-untranslated region containing an internal ribosomal entry site, the neomycin phosphotransferase gene, the encephalomyocarditis virus internal ribosomal entry site, HCV nonstructural proteins (NS) 3 through NS5B; and the HCV 3Ј-untranslated region (Fig. 1A). This system provides a unique tool for studying the mechanisms of HCV replication and screening as well as evaluating anti-HCV compounds. Taking advantage of this feature, we examined the effects of various types of compounds on the replication of HCV using HCV replicon cells established in our laboratory 6 (Miyanari et al., manuscript accepted for publication). Consequently, we found that a well-known immunosuppressant, cyclosporin A (CsA), 7 had a strong suppressive effect on HCV replication in these cells. Moreover, we found suppressive activity of CsA for multiplication of the HCV genome in cultured human hepatocytes infected with HCV. The mechanism of the anti-HCV activity of CsA was also studied. Materials and MethodsCell Culture. Huh-7 and MH-14 cells, HCV replicon cells, were cultured in Dulbecco's modified Eagle medium with 10% fetal bovine serum. PH5CH8 cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle medium and F12 medium supplemented with 100 ng/mL epidermal gro...

In serum, mouse embryonic stem cells (mESCs) fluctuate between a naive inner cell mass (ICM)-like state and a primed epiblast-like state, but when cultured with inhibitors of the mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 pathways (2i), they are harnessed exclusively in a distinct naive pluropotent state, the ground state, that more faithfully recapitulates the ICM. Understanding the mechanism underlying this naive pluripotent state will be critical for realizing the full potential of ESCs. We show here that PRDM14, a PR-domain-containing transcriptional regulator, ensures naive pluripotency through a dual mechanism: antagonizing activation of the fibroblast growth factor receptor (FGFR) signaling by the core pluripotency transcriptional circuitry, and repressing expression of de novo DNA methyltransferases that modify the epigenome to a primed epiblast-like state. PRDM14 exerts these effects by recruiting polycomb repressive complex 2 (PRC2) specifically to key targets and repressing their expression.

SUMMARYGerm cell development creates totipotency through genetic as well as epigenetic regulation of the genome function. Primordial germ cells (PGCs) are the first germ cell population established during development and are immediate precursors for both the oocytes and spermatogonia. We here summarize recent findings regarding the mechanism of PGC development in mice. We focus on the transcriptional and signaling mechanism for PGC specification, potential pluripotency, and epigenetic reprogramming in PGCs and strategies for the reconstitution of germ cell development using pluripotent stem cells in culture. Continued studies on germ cell development may lead to the generation of totipotency in vitro, which should have a profound influence on biological science as well as on medicine.