br Experimental Procedures br Author Contributions

Experimental Procedures
Author Contributions
Acknowledgments
Introduction Nuclear reprogramming of somatic ras gtpase using genetic modification and overexpression of pluripotent transcription factors is essential for creating induced pluripotent stem cells (iPSCs) in vitro (Takahashi and Yamanaka, 2006). An alternate approach that does not entail genetic modification involves primordial germ cells (PGCs) from the embryo, followed by culture-induced reversion into embryonic germ cell (EGC) lines. Both EGCs and iPSCs transcriptionally resemble undifferentiated embryonic stem cells (ESCs) in vitro, and, similar to ESCs, EGCs derived by PGC reversion in vitro have the capacity to contribute to chimeras with germline transmission (Labosky et al., 1994; Leitch et al., 2013b; Matsui et al., 1992; Resnick et al., 1992; Sharova et al., 2007; Stewart et al., 1994; Takahashi and Yamanaka, 2006). Despite these similarities, one of the major epigenetic differences with EGCs and iPSCs is variable methylation at imprinting control centers (ICCs). Competency for PGC reversion to EGCs in the mouse embryo is found during a 6-day window starting from the time of PGC specification at embryonic day 7.5 (E7.5) through E13.5 after PGCs have settled in the genital ridge (Labosky et al., 1994; Leitch et al., 2013b, 2013c; Matsui et al., 1992; Resnick et al., 1992; Shim et al., 2008; Stewart et al., 1994; Tada et al., 1998). During this time, the germline undergoes a unique two-stage DNA demethylation event. In the first stage, which is completed soon after specification (at around E8.0), cytosine methylation is removed genome-wide from more than 50% of cytosines in a CG sequence context (Seisenberger et al., 2012). Methylated regions that are protected in the first stage include ICCs, some gonadal stage germline genes, particularly those involved in meiosis, as well as endogenous retroviruses (Guibert et al., 2012; Hajkova et al., 2002; Seisenberger et al., 2012; Vincent et al., 2013). In the second stage, which extends from E9.5 to E13.5, cytosine methylation in PGCs is removed in a time and locus-specific manner, with E13.5 PGCs considered the most hypomethylated germline epigenetic ground state. This includes removing (also referred to as erasing) methylation from ICCs in preparation for establishing new methylated marks on DNA in a sex-specific manner (Guibert et al., 2012; Hajkova et al., 2002; Kagiwada et al., 2013; Seisenberger et al., 2012). Cytosine methylation in EGC lines has been studied extensively and the prevailing hypothesis is that ICCs are generally hypomethylated. However, close analysis reveals that ICC cytosine methylation is extremely variable, not only between lines but also between ICC sites within a given line (Labosky et al., 1994; Leitch et al., 2013a; McLaren and Durcova-Hills, 2001; Shim et al., 2008; Shovlin et al., 2008; Tada et al., 1998). This variability has led to three major hypotheses for cytosine methylation dynamics during PGC reversion to EGCs. The first hypothesis is that EGC lines reflect the epigenetic status of the PGCs in the embryo from which they were originally derived. The second hypothesis is that PGCs undergo incomplete cytosine methylation erasure, and thus the variable status in established EGC lines is due to heterogeneously terminated demethylation. The third hypothesis is that PGCs undergo demethylation as well as de novo methylation during the process of reverting from PGCs to EGCs.
Results
Discussion In this study we have addressed a long-standing question in the field regarding methylation erasure at ICCs during reversion of PGCs to EGCs in vitro. We discovered that initiating reprogramming from E9.5 PGCs results in erasure of cytosine methylation from ICCs within the first 4 days of in vitro culture, therefore rejecting the notion that established EGC lines represent the epigenetic state of PGCs from which they were derived. Furthermore, our data suggest that the first 4 days of PGC culture resemble the dynamics of ICC methylation erasure normally observed in the mouse embryo (Hackett et al., 2013; Yamaguchi et al., 2012, 2013). Therefore, we propose that the 7F PGC culture system is an important new model for understanding mechanisms of locus-specific cytosine demethylation in the germline. In particular in future studies, this model could be used to address the locus-specific removal of cytosine methylation downstream of oxidation (Hackett et al., 2013; Yamaguchi et al., 2012, 2013), activation-induced cytidine deaminase (Popp et al., 2010), or base excision repair (Ciccarone et al., 2012; Kawasaki et al., 2014), the outcomes of which will be important to stem cell biology and reprogramming in general.