Magnúsdóttir et al 2012 combinatorial control of cell fate and reprogramming in the mammalian germli by magnusdottir

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Combinatorial control of cell fate and reprogramming in the mammalian germline Erna MagnuÂ´sdoÂ´ttir*, Astrid Gillich*, Nils Grabole and M Azim Surani Development of mammalian primordial germ cells (PGCs) presents a unique example of a cell fate specification event that is intimately linked with epigenetic reprogramming. Cell fate commitment is governed by transcription factors which, together with epigenetic regulators, instruct lineage choice in response to signalling cues. Similarly, the reversal of epigenetic silencing is driven by the combinatorial action of transcriptional regulators, resulting in an increase in cellular plasticity. PGCs constitute a paradox, since their development as a unipotent specialised lineage is coupled with extensive reprogramming, which eventually leads to an increase in cellular potency. In this review we discuss the role of key factors in the specification of the germ cell lineage that are also important for the comprehensive erasure of epigenetic modifications, which provides the foundation for regeneration of totipotency. We further discuss current concepts of transcriptional and epigenetic control of cell fate decisions, with a particular focus on emerging principles of enhancer activity and their potential implications for the transcriptional control of PGC specification. Address Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, United Kingdom Corresponding author: Surani, M Azim (a.surani@gurdon.cam.ac.uk, azim.surani@gurdon.cam.ac.uk) * These authors contributed equally to this work.

Current Opinion in Genetics & Development 2012, 22:1â&#x20AC;&#x201C;9 This review comes from a themed issue on Cell reprogramming Edited by Kathrin Plath and Martin Pera

in any given cell type and an even smaller fraction of those expressed are capable of driving lineage choice by orchestrating the specificity of the transcriptional output in a cell. A hierarchy of transcription factors acts to restrict lineage choice during development that culminates in terminal differentiation into all the lineages of an organism [2,3]. Similarly, reprogramming of a differentiated cell back to a pluripotent state in vitro is governed by the combinatorial and hierarchical action of transcriptional regulators that collaborate to induce a gradual increase in cellular plasticity [4,5]. In this review, we discuss current concepts of combinatorial transcriptional and epigenetic regulation by illustrating the complex process of specification and reprogramming of primordial germ cells (PGCs) in the early mammalian embryo. Although PGCs are unipotent, they show some characteristics of pluripotent cells [6,7], representing a unique system to study the balance between differentiation and lineage restriction on one hand and an underlying pluripotency on the other. At the same time, PGCs also show some unique features of reprogramming such as extensive and global DNA demethylation that includes erasure of all parental imprints [8]. Therefore, the development of the germ cell lineage is a fascinating example of temporal and spatial coupling of cell fate determination and epigenetic reprogramming. Notably, mouse post-implantation epiblast and its cognate epiblast stem cells (EpiSCs) [9,10] represent a distinct intermediate cellular state between ground state pluripotency and specification, which can give rise to PGCs or embryonic stem cells (ESCs) in response to appropriate signalling cues [11,12 ,13]. The alternative cell states that can be generated from such epiblast cells constitute a suitable model for the study of transcriptional regulation of embryonic lineage choice.

http://dx.doi.org/10.1016/j.gde.2012.06.002

Introduction The complex process of cell fate determination in metazoan organisms is orchestrated by the combinatorial action of specific transcription factors that govern the cell type specific interpretation of the genotype, which results in a multitude of different cellular phenotypes. The mouse genome encodes between 2000 and 3300 predicted transcription factors, out of which roughly 1200 have a confirmed transcription factor activity [1]. However, only a fraction of these factors are co-expressed www.sciencedirect.com

We present how two principal interconnected and interdependent networks can govern cell fate specification and reprogramming in which Prdm14, a member of the Prdm (PRDI-BFI-RIZ-domain containing) family of transcription factors [14 ], plays an important role. During PGC specification, Prdm14 acts together with the transcriptional repressor Blimp1/Prdm1 and AP2g, encoded by Tcfap2c [14 ,15â&#x20AC;&#x201C;17], while reversion to ground state pluripotency is enhanced by Prdm14 in conjunction with Klf2 [18 ]. We further propose that the appropriate priming of a repertoire of enhancers in the post-implantation epiblast is critical for providing the competence for the interpretation of signalling cues that can either direct Current Opinion in Genetics & Development 2012, 22:1â&#x20AC;&#x201C;9

Please cite this article in press as: MagnuÂ´sdoÂ´ttir E, et al.: Combinatorial control of cell fate and reprogramming in the mammalian germline, Curr Opin Genet Dev (2012), http://dx.doi.org/10.1016/ j.gde.2012.06.002

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2 Cell reprogramming

the induction of Blimp1 concurrently with Prdm14 leading to PGC specification [13,14,16,17], or enable reprogramming to an ESC-like state in the absence of Blimp1 [18 ,19 ].

Lineage acquisition and epigenetic reprogramming in primordial germ cells PGCs are specified in the proximal epiblast in response to BMP signalling emanating primarily from extraembryonic ectoderm cells on day 6.25 of gestation (Figure 1) [20â&#x20AC;&#x201C; 22]. This results in the induction of Blimp1, a transcriptional repressor critical for the induction of the germ cell gene expression programme as well as for the repression of a somatic cell fate [16,17]. AP2g is induced downstream of Blimp1 and is also critical for PGC specification as AP2g knockout embryos show an early loss of PGCs similar to that of Blimp1 deficient embryos (MagnuÂ´sdoÂ´ttir and Surani, unpublished data) [15,23]. Before PGC specification and during the transition from pre-implantation to post-implantation development, epiblast cells undergo major transcriptional and epigenetic

changes, including DNA methylation, chromatin compaction, random X-chromosome inactivation, silencing of the distal Oct4 enhancer and a switch from monoallelic to biallelic Nanog expression [24 ,25 ,26 ,27,28]. Reversal of many of these changes occurs exclusively in PGCs following their specification from post-implantation epiblast and is a prerequisite for acquisition of totipotency [14 ,29]. Prdm14 is a key factor in the early phase of germ cell reprogramming, which shows specific expression in PGCs and pluripotent stem cells [14 ]. Prdm14 deficient PGCs fail to undergo characteristic chromatin changes, as they maintain high levels of histone 3 lysine 9 dimethylation (H3K9me2), which could be attributed to their inability to repress the histone lysine methyltransferase Glp (Ehmt1), and they fail to induce global H3K27me3 [14 ,30â&#x20AC;&#x201C;32]. Moreover, negative regulation of Dnmt3b and Uhrf1, a cofactor of the maintenance DNA methyltransferase Dnmt1, could influence early loss of DNA methylation and might be further enhanced by the action of Tet enzymes that promote conversion of 5-methylcytosine

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Transcriptional and epigenetic changes upon specification of PGCs. In mammals, germ cell fate is induced in the posterior proximal epiblast by BMP signalling from the extraembryonic ectoderm at E6.25 resulting in the transcription of Blimp1, Prdm14 and Tcfap2c, which are crucial for PGC specification. Specified PGCs migrate to the developing gonads and induce pluripotency genes, such as Klf2, Sox2 and Nanog. Migrating PGCs undergo epigenetic reprogramming, including activation of the distal Oct4 enhancer and X-chromosome reactivation, which is followed by genomewide DNA demethylation and imprint erasure in the genital ridges. Current Opinion in Genetics & Development 2012, 22:1â&#x20AC;&#x201C;9

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Cell fate and reprogramming in the mammalian germline MagnuÂ´sdoÂ´ttir et al.

(5mC) to hydroxymethylcytosine (5hmC) [8,14 ,31,33, 34,35 ]. Indeed, Prdm14 is crucial for germ cell development as evidenced by a reduction of PGC numbers as early as embryonic day (E) 7.5, and their complete loss by E11.5 [14 ], indicating a defect preceding the epigenetic changes. Thus, three genes, Blimp1, Prdm14 and AP2g, establish a critical transcriptional network for PGC specification leading to extensive epigenetic programming of this lineage (Figure 1).

Cell-based assays reveal combinatorial roles of germline genes in epigenetic reprogramming

Although Prdm14 has been identified as a key germ cell determinant, it also has a general effect in epigenetic reprogramming. Notably, the human PRDM14 homologue enhances the efficiency of reprogramming of human fibroblasts to induced pluripotent stem cells (iPSCs), when combined with the other key reprogramming factors [36 ]. Prdm14 can also synergize with Klf2 to accelerate reversion of mouse EpiSCs to ESC-like cells [18 ]. Prdm14 knockout PGCs are apparently unable to convert to pluripotent embryonic germ cells (EGCs) in medium containing serum and leukemia inhibitory factor (LIF) [14 ], although their reprogramming in 2i/LIF conditions [37,38] remains to be tested. PRDM14 is essential for self-renewal of human ESCs, but loss of function in mouse ESCs has a subtle phenotype [35,36 ]. Prdm14 knockout embryos certainly show no defect at the blastocyst stage, indicating that the generation of pluripotent cells in the inner cell mass is not affected; thus the phenotype of Prdm14 mutant mice is restricted to the germline [14 ]. Therefore, Prdm14 might not be essential for the acquisition of pluripotency, but could facilitate the maintenance of ESCs and a naÄąÂ¨ve pluripotent state, in part by inhibition of differentiation [35 ]. Alternatively, functional redundancy of Prdm14 with other Prdm family members, such as Prdm5, and epigenetic modifiers may mask the phenotype in mouse ESCs and the inner cell mass [36 ].

Although genetic studies have provided crucial insight into PGC specification, the molecular mechanism of germ cell development and reprogramming is not well understood. This is because there are limited numbers of PGCs and currently there are no suitable methods that can sustain PGCs in vitro [7]. It is therefore important to consider cell-based systems to complement genetic studies. EpiSC-based assays can be used to investigate the role of germline factors in epigenetic reprogramming [18 ]. EpiSCs self-renew continuously, can be expanded to large quantities and are easy to manipulate [9,10,42]. They revert to ESC-like cells at a low rate and efficiency by exposure to LIF-Stat3 signalling on feeder cells, and undergo specification to unipotent PGCs upon exposure to BMP4 (Figure 2) [11,12 ]. Loss of DNA methylation, activation of the distal Oct4 enhancer, X-chromosome reactivation and re-expression of Stella and Rex1 occur during both EpiSC reversion and PGC specification [12 ]. Thus, reversion of EpiSCs to ESCs represents a tractable and an objective model for testing the effects of germline factors on reprogramming. This system was used to uncover a synergistic effect of Prdm14 and Klf2 in EpiSC reversion that includes rapid and efficient X-chromosome reactivation and loss of DNA methylation [18 ]. Notably, however, Prdm14 does not act as reprogramming factor on its own, but appears to enhance the competence for reprogramming, and promotes recruitment of Klf2 to specific loci, including the distal enhancer of Oct4 and Nr5a2 [18 ]. Klf2 is a target of Prdm14 that is induced in PGCs [31,35 ], at the time when epigenetic reprogramming commences, which could indicate a potential cooperation of the two factors in early germ cells.

Shortly after the induction of Blimp1, Prdm14 and AP2g, several pluripotency and reprogramming factors, such as Klf2, Sox2 and Nanog, are specifically induced in PGCs (Figure 1) [31] and may benefit from the concurrent activity of Prdm14. Oct4 and Nanog are required in migrating PGCs [29,39,40], but the precise molecular functions of these and other pluripotency factors in germ cells remains to be elucidated. Expression of some pluripotency genes in PGCs may simply reflect the change to a pluripotency-like programme, whereas others could be critical for PGC development or function [29], such as for silencing of transposons or the erasure of imprints. The role of germline genes could be tested by gain-of-function and loss-of-function studies in an in vitro system for PGC specification, such as the induction of epiblast-like cells (EpiLCs) from mouse ESCs and subsequent stimulation with BMP4 [41 ]. A similar system, if developed for other mammals including human ESCs, could allow wider investigations on the mechanism of PGC specification and epigenetic reprogramming.

Notably, EpiSC reversion by Prdm14 and Klf2 occurs efficiently in the absence of Blimp1, indicating that a progression through a germ cell-like intermediate state may not be required for successful reprogramming [18 ]. Indeed, Blimp1 is neither required for the derivation of mouse ESCs and EpiSCs, nor for the reversion of EpiSCs to ESCs upon exposure to LIF-Stat3 signalling [19], demonstrating that Blimp1 has a unique function in the germline and plays no role in pluripotent cells such as ESCs. Unlike ESCs, PGCs are unipotent cells that eventually generate the totipotent state through sperm and oocytes. Indeed, PGCs do not contribute to chimeras upon blastocyst injection [43], yet they can undergo efficient conversion to pluripotent EGCs in medium containing 2i and LIF [38]. The repression of Blimp1 appears to be a prerequisite for conversion of PGCs to EGCs [44], and may be required for complete activation of the pluripotency network. Thus, Blimp1 may be a key factor that initiates and sustains unipotency of PGCs.

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EpiSCs can undergo reversion to ESCs upon exposure to LIF-Stat3 signalling and specification to PGCs in response to BMP4. EpiSC reversion is accelerated by Prdm14 and Klf2, which are also induced in early PGCs. The epigenetic reprogramming events during EpiSC reversion and upon specification to PGCs share similarities and include X-chromosome reactivation, DNA demethylation and activation of the distal enhancer (DE) of Oct4. A notable exception in this assay is the erasure of imprints that occurs in PGCs, but not in reverting EpiSCs, which may require additional germline factors.

The priming of enhancer repertoire provides competence for cell fate commitment in response to signalling cues Although the general requirements for transcription factors during PGC specification and reprogramming are being discovered, further insights on signalling events and transcriptional regulation could provide valuable clues as to how these early embryonic events are regulated. Consistent with PGC specification occurring downstream of BMP signalling, embryos null for BMP4 or BMP8b fail to properly specify PGCs [21,22], as do epiblast cells lacking the BMP responsive transcription factors Smad1 or Smad5 [45,46]. Although only a small cluster of cells most proximal to the extraembryonic ectoderm in the Current Opinion in Genetics & Development 2012, 22:1–9

post-implantation epiblast is subject to these signalling cues, when tested in vitro, the majority of epiblast cells are competent for PGC induction in response to BMP signalling [13]. Interestingly, mouse ESCs, like post-implantation epiblast, also respond to BMP4 signalling but in this instance it can promote self-renewal and maintenance of the ESC state [47]. By contrast, BMP4 is required for PGC specification from the post-implantation epiblast [13,22]. How the same signalling molecules can drive different transcriptional outputs depending on the cellular context is of considerable interest. Recent studies into the properties of tissue specific enhancers have uncovered a dynamic interplay between signal responsive transcription factors, lineage determining transcription factors and enhancer dynamics. www.sciencedirect.com

Please cite this article in press as: Magnu´sdo´ttir E, et al.: Combinatorial control of cell fate and reprogramming in the mammalian germline, Curr Opin Genet Dev (2012), http://dx.doi.org/10.1016/ j.gde.2012.06.002

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Cell fate and reprogramming in the mammalian germline MagnuÂ´sdoÂ´ttir et al.

Genome-wide profiling of histone modifications and histone modifying enzymes has revealed that enhancers are characterised by specific epigenetic signatures, and that the marking of enhancer chromatin is highly cell type specific [48]. The majority of active and poised enhancers are enriched for H3K4me1 and depleted for H3K4me3, a modification that is highly enriched on promoters [49]. In addition, active and poised enhancers can be distinguished through the modification of H3K27, that is unmodified or trimethylated on poised enhancers of mouse and human ESCs, respectively [50 ]. In contrast, active enhancers are marked by H3K27 acetylation in both species [51 ]. Analysis of p300 occupancy in early embryos and subsequent transgenic reporter analysis further identified several tissue specific enhancers [49,52,53 ]. Interestingly, the chromatin state of promoters does not correlate with cell type specific activity, whereas chromatin modification profile and transcription factor occupancy on enhancers does [48,49,54 ]. The chromatin state of enhancers is established by the occupancy of key lineage determining transcription factors that bring in chromatin modifiers to covalently modify histones, resulting in enhancer licensing [53 ,54 ,55 ,56]. Thus the epigenetic marking of appropriate enhancers precedes and anticipates cell fate decisions. Additionally, the enhancers rather than promoters are critical for providing cell type specificity to transcriptional regulation [50 ,52]. The process of enhancer licensing is initiated by pioneer factors that occupy enhancers and restrict cells to relatively broad lineages that then become continuously more restricted as cells differentiate and express further lineage specific factors [56]. It is therefore likely that enhancers directing PGC specific gene expression would be in a primed state, anticipating the appropriate signalling cues, most notably BMP4, in cells of the post-implantation epiblast. The haematopoietic system has provided additional insights on how enhancers act to mediate tissue specific gene expression. In common lymphoid progenitors, the entire set of enhancers of both B-cells and macrophages is occupied by PU.1 and marked by H3K4me1 [54 ]. Upon differentiation to either of the two respective lineages, PU.1 becomes restricted to enhancers that lie proximal to genes expressed in the relevant lineage. This restriction directs signal responsive transcription factors to these enhancers and provides cell type specificity to signalling cues. Importantly, the presence of the B-cell lineage determining factors EBF and E2A has an effect on PU.1 occupancy at distinct stages of B-cell development, as is the case for C/EBPb in macrophages. However, the absence of signal responsive factors LXRa or LXRb did not alter the occupancy of PU.1, revealing the hierarchical nature of these transcription factors [54 ]. www.sciencedirect.com

More recently, a study by Troumpouki et al. [55 ] showed that the signal dependent factors Smad1 and Tcf7l1 that are downstream effectors of BMP and Wnt signalling, respectively, are targeted to enhancers occupied by Gata factors in haematopoietic stem cells and erythroblasts. Whereas Gata2 directs the signal responsive factors to a broad set of enhancers in haematopoietic progenitors, the set of enhancers to which the factors are targeted is subsequently restricted to sites of high Gata1 occupancy in the more differentiated erythroblasts [55 ]. Transcriptional regulation in mouse ESCs is governed by the combinatorial action of the major pluripotency factors Oct4, Sox2 and Nanog that are critical for inducing and maintaining the pluripotent properties of ESCs and iPSCs [4], whereas a distinct set of factors including Myc and Zfx promote stem cell self-renewal [57,58]. In fact, Oct4, Sox2 and Nanog form a core set of factors that bind to a subset of mouse ESC enhancers where multiple other transcription factors have been shown to co-localise [58]. The binding of Oct4 to these enhancers is critical since depletion of Oct4 leads to the reduction in occupancy of co-bound factors [59,60]. Interestingly, and perhaps more instructive of potential mechanisms of PGC specification and epiblast reprogramming, Mullen et al. [61 ] recently found that analogous to the findings in haematopoiesis, the response to signal induced factors, in this case Smad3 downstream of TGFb signalling was governed by Oct4. Thus, Smad3 binding downstream of TGFb was targeted to Oct4 sites, whereas overexpression of MyoD in mouse ESCs redirected the Smad3 response to MyoD occupied enhancers, which are characteristically active during myogenesis. A previous study had mapped the Smad1 binding events that occur downstream of BMP4 signalling in mouse ESCs to the target regions of Oct4, Sox2 and Nanog [59]. Thus, BMP signalling response in mouse ESCs is likely directed to enhancers by the pluripotency factors. The picture that emerges reflects a sequential refinement of enhancer activity during development, where transcription factors governing lineage choice dictate the repertoire of active enhancers in the respective cell lineages. This activity subsequently directs the targeting of signal responsive transcription factors to the appropriate genetic elements, generating cell type specific responses to broadly expressed signal responsive factors.

Transcriptional competence of primordial germ cell specification It is interesting to think about PGC specification in the context of the accumulating data revealing the different nature and role of cis-acting enhancers and trans-acting lineage specific transcription factors that occupy them during lineage choice decisions. It is important to Current Opinion in Genetics & Development 2012, 22:1â&#x20AC;&#x201C;9

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A model depicting Oct4 enhancers, chromatin modifications and transcription factor occupancy. Transcription of Oct4/Pou5f1 in mouse ESCs is driven by the distal enhancer (DE), which is active and highly occupied by transcription factors that regulate pluripotency and self-renewal. The histone modification mark H3K4me1 marks both the DE and the poised proximal enhancer (PE), but the active DE is further marked by H3K27 acetylation that characterises active enhancers. Signal responsive transcription factors, including Stat3 and Smad1/5/8, are correspondingly recruited to the DE. In post-implantation epiblast and EpiSCs, Oct4 expression is driven from the PE, that is correspondingly occupied by pluripotency factors and signal responsive transcription factors such as Smads are recruited to the PE. The activity of Oct4 reverts to being driven from the DE in PGCs, which resumes its function as a binding hub for major pluripotency and germ cell regulators. However, unlike mouse ESCs, only Klf2 and Klf5, but not Klf4, occupy the DE in PGCs. H3K4me3 marks the active Oct4 promoter in ESCs, EpiSCs and PGCs.

emphasise that the regulatory logic of gene expression in the post-implantation epiblast is likely to be different from that of the inner cell mass and pre-implantation epiblast, which is in part reflected in Oct4 expression itself that is driven from its proximal enhancer (PE) rather than the distal one (DE) employed in the inner cell mass and ESCs (Figure 3) [28]. At the Current Opinion in Genetics & Development 2012, 22:1–9

onset of PGC specification, the post-implantation epiblast retains the expression of Oct4, but Nanog and Sox2 are repressed in the region where PGCs get specified [31]. This suggests that an altered epigenetic environment and enhancer repertoire in the post-implantation epiblast leads to a different interpretation of the BMP4 signal www.sciencedirect.com

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Cell fate and reprogramming in the mammalian germline MagnuÂ´sdoÂ´ttir et al.

compared to inner cell mass and ESCs, thus providing the basis for competence towards PGC specification. Interestingly, mouse EpiSCs that express Oct4, Sox2 and Nanog have an inactive Oct4 DE [12 ,18 ], indicating that the binding of these factors to the DE in EpiSCs is either insufficient for its activation or that the factors are not properly recruited to the locus. This could suggest that recruitment of Prdm14, Klf2, Klf4 and/or Klf5, that are highly expressed and bind to Oct4 DE in mouse ESCs, but are expressed only at low levels in post-implantation epiblast and EpiSCs [18 ,35 ,42], may be crucial for the activation of DE. Indeed, activation of Oct4 DE is highly accelerated during EpiSC reversion by Prdm14 and Klf2 [18 ]. These observations on activation of Oct4 DE provide a potential paradigm for the engagement of distinct set of enhancers during reprogramming to ground state pluripotency, compared to those that are required during PGC specification. How enhancer switching occurs and which of them are specifically activated could generally illuminate our understanding of mechanisms of cell fate determination. One of the unique effects of BMP4 in PGC specification is the induction of Blimp1/Prdm1 [13,16]. Blimp1 mediates lineage restriction by binding to and repressing the promoters of genes encoding transcriptional activators of somatic cell fate commitment (MagnuÂ´sdoÂ´ttir and Surani, unpublished) [16]. The superimposition of such a repressive activity on gene induction mediated by BMP4 presumably acts to restrict the transcriptional response and to limit the lineage choice to the unipotent germ cell lineage. Downstream of BMP4 signalling, there is also independent induction of Prdm14 soon thereafter, and subsequently, expression of Klf2, Sox2 and Nanog is reinstated [14,31]. Representing a change in enhancer activity in PGCs, Oct4 DE that is bound by Prdm14 in mouse ESCs becomes reactivated [28,35 ]. Since Prdm14 predominantly binds to distal regulatory elements [35 ], it could function as a priming factor by inducing a permissive chromatin state on enhancers, including Oct4 DE, and facilitating the switch in enhancer occupancy also of other key transcriptional regulators apart from Oct4. During reprogramming of EpiSCs to ESCs, ectopic expression of Prdm14 might re-direct the signal responsive transcription factors such as Stat3 and Smad1 to their typical mouse ESC targets, thus resulting in an accelerated response to LIF and BMP4 [18 ]. In PGCs, Prdm14 might have a similar function of switching the enhancer repertoire of post-implantation epiblast cells to a more nai#ve state (Figure 3). Furthermore, one could envision the direct cooperativity of binding by Prdm14 and Klf2 to regulatory elements, leading to the facilitation of signalling cue interpretation. Other factors may similarly also play a role in the establishment and the key properties of the germ cell lineage. Furthermore, expression of www.sciencedirect.com

Blimp1 probably alters the topology of the transcriptional network by restricting the response to signalling cues and influencing which genes are transcribed downstream of Prdm14 and Klf2-occupied enhancers, explaining the difference in cellular response during EpiSC reversion on the one hand and upon PGC specification on the other.

Perspectives Traditionally, research on PGCs has been hampered by the limited amount of material in the early embryo and the lack of suitable in vitro systems to recapitulate PGC development. Recent progress in methods, however, will accelerate progress in germ cell biology and reprogramming, through analysis of a small number of cells or even single cells, live-cell imaging and in vitro systems for PGC induction using continuously growing stem cell lines. A combination of these approaches should permit largescale screens for the combinatorial action of Blimp1, Prdm14 and AP2g, elucidation of their downstream effectors and analysis of the complex gene regulatory networks in the mammalian germline. This unipotent lineage undergoes extensive epigenetic reprogramming to ensure establishment of totipotency and Blimp1 could well be the key factor controlling the balance between pluripotency and unipotent germ cell fate.

Acknowledgments We thank members of our group for discussions and apologise to all the authors whose work could not be cited due to space constraints. E.M. was supported by a Marie Curie Intra-European Fellowship for Career Development. A.G. and N.G. were recipients of a Wellcome Trust PhD Studentship (RG44593). This work was supported by grants from the Wellcome Trust to M.A.S.

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8 Cell reprogramming

Brons IG, Smithers LE, Trotter MW, Rugg-Gunn P, Sun B, Chuva de Sousa Lopes SM, Howlett SK, Clarkson A, Ahrlund-Richter L, Pedersen RA et al.: Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 2007, 448:191-195.

10. Tesar PJ, Chenoweth JG, Brook FA, Davies TJ, Evans EP, Mack DL, Gardner RL, McKay RD: New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 2007, 448:196-199. 11. Hayashi K, Surani MA: Self-renewing epiblast stem cells exhibit continual delineation of germ cells with epigenetic reprogramming in vitro. Development 2009, 136:3549-3556. 12. Bao S, Tang F, Li X, Hayashi K, Gillich A, Lao K, Surani MA: Epigenetic reversion of post-implantation epiblast to pluripotent embryonic stem cells. Nature 2009, 461:1292-1295. This study shows that mouse post-implantation epiblast and EpiSCs can revert to pluripotent ESC-like cells upon exposure to LIF-Stat3 signalling on feeder cells and without the introduction of transcription factors. 13. Ohinata Y, Ohta H, Shigeta M, Yamanaka K, Wakayama T, Saitou M: A signaling principle for the specification of the germ cell lineage in mice. Cell 2009, 137:571-584. 14. Yamaji M, Seki Y, Kurimoto K, Yabuta Y, Yuasa M, Shigeta M, Yamanaka K, Ohinata Y, Saitou M: Critical function of Prdm14 for the establishment of the germ cell lineage in mice. Nat Genet 2008, 40:1016-1022. Genetic experiments demonstrate that Prdm14 is required for PGC specification and reveal a loss of germ cells as early as E7.5. Prdm14deficient PGCs fail to induce Sox2 and do not show a global increase in H3K9me2 levels, indicating impaired epigenetic reprogramming. 15. Weber S, Eckert D, Nettersheim D, Gillis AJ, Schafer S, Kuckenberg P, Ehlermann J, Werling U, Biermann K, Looijenga LH et al.: Critical function of AP-2 gamma/TCFAP2C in mouse embryonic germ cell maintenance. Biol Reprod 2010, 82:214-223. 16. Ohinata Y, Payer B, Oâ&#x20AC;&#x2122;Carroll D, Ancelin K, Ono Y, Sano M, Barton SC, Obukhanych T, Nussenzweig M, Tarakhovsky A et al.: Blimp1 is a critical determinant of the germ cell lineage in mice. Nature 2005, 436:207-213. 17. Vincent SD, Dunn NR, Sciammas R, Shapiro-Shalef M, Davis MM, Calame K, Bikoff EK, Robertson EJ: The zinc finger transcriptional repressor Blimp1/Prdm1 is dispensable for early axis formation but is required for specification of primordial germ cells in the mouse. Development 2005, 132:1315-1325. 18. Gillich A, Bao S, Grabole N, Hayashi K, Trotter MWB, Pasque V, MagnuÂ´sdoÂ´ttir E, Surani MA: Epiblast stem cell-based system reveals reprogramming synergy of germline factors. Cell Stem Cell 2012, 10:425-439. This study reveals a synergy of the germline factor Prdm14 with Klf2 to accelerate and enhance reversion of EpiSCs to a naÄąÂ¨ve ESC-like pluripotent state that can occur in the absence of Blimp1. This indicates parallels and differences between reprogramming of EpiSCs to ESC-like cells and germline reprogramming. 19. Bao S, Leitch HG, Gillich A, Nichols J, Tang F, Kim S, Lee C, Zwaka T, Li X, Surani MA: The germ cell determinant blimp1 is not required for derivation of pluripotent stem cells [Internet]. Cell Stem Cell 2012, 11:110-117. This study demonstrates that Blimp1 is not required for either the derivation of mouse ESCs and EpiSCs, nor for the reversion of EpiSCs to ESC-like cells, indicating that a transition through a germ cell-like state may not be required for the acquisition of a naÄąÂ¨ve pluripotent state. 20. Okamura D, Hayashi K, Matsui Y: Mouse epiblasts change responsiveness to BMP4 signal required for PGC formation through functions of extraembryonic ectoderm. Mol Reprod Dev 2005, 70:20-29. 21. Ying Y, Liu XM, Marble A, Lawson KA, Zhao GQ: Requirement of Bmp8b for the generation of primordial germ cells in the mouse. Mol Endocrinol 2000, 14:1053-1063. 22. Lawson KA, Dunn NR, Roelen BA, Zeinstra LM, Davis AM, Wright CV, Korving JP, Hogan BL: Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev 1999, 13:424-436. 23. Schafer S, Anschlag J, Nettersheim D, Haas N, Pawig L, Schorle H: The role of BLIMP1 and its putative downstream target Current Opinion in Genetics & Development 2012, 22:1â&#x20AC;&#x201C;9

TFAP2C in germ cell development and germ cell tumours. Int J Androl 2011, 34:e152-e158 [discussion e158â&#x20AC;&#x201C;e159]. 24. Ahmed K, Dehghani H, Rugg-Gunn P, Fussner E, Rossant J, Bazett-Jones DP: Global chromatin architecture reflects pluripotency and lineage commitment in the early mouse embryo. PLoS One 2010, 5:e10531. Electron spectroscopic imaging to examine global chromatin organisation in mouse pre-implantation and post-implantation embryos shows that the chromatin is highly dispersed in the epiblast of E3.5 blastocysts and more compact in the epiblast of E5.5 post-implantation embryos. 25. Borgel J, Guibert S, Li Y, Chiba H, Schubeler D, Sasaki H, Forne T, Weber M: Targets and dynamics of promoter DNA methylation during early mouse development. Nat Genet 2010, 42:1093-1100. Using methylated DNA immunoprecipitation (MeDIP) to analyse the genome-wide pattern of DNA methylation in mouse pre-implantation and post-implantation embryos, this study shows that a major wave of de novo DNA methylation of promoters, particularly of germline genes, takes place at around implantation, and is primarily mediated by the DNA methyltransferase Dnmt3b. 26. Miyanari Y, Torres-Padilla M-E: Control of ground-state pluripotency by allelic regulation of Nanog. [Internet]. Nature 2012, 483:470-473. This study reveals that Nanog expression is monoallelic in 4-cell and 8cell stage mouse embryos and switches to biallelic expression in the pluripotent epiblast of E3.5 blastocysts. Nanog expression is also biallelic in ESCs cultured in medium containing 2i and LIF. Notably, a gradual reversal to monoallelic Nanog expression takes place upon implantation. 27. Monk M, Harper MI: Sequential X chromosome inactivation coupled with cellular differentiation in early mouse embryos. Nature 1979, 281:311-313. 28. Yeom YI, Fuhrmann G, Ovitt CE, Brehm A, Ohbo K, Gross M, Hubner K, Scholer HR: Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 1996, 122:881-894. 29. Chambers I, Silva J, Colby D, Nichols J, Nijmeijer B, Robertson M, Vrana J, Jones K, Grotewold L, Smith A: Nanog safeguards pluripotency and mediates germline development. Nature 2007, 450:1230-1234. 30. Hajkova P, Ancelin K, Waldmann T, Lacoste N, Lange UC, Cesari F, Lee C, Almouzni G, Schneider R, Surani MA: Chromatin dynamics during epigenetic reprogramming in the mouse germ line. Nature 2008, 452:877-881. 31. Kurimoto K, Yabuta Y, Ohinata Y, Shigeta M, Yamanaka K, Saitou M: Complex genome-wide transcription dynamics orchestrated by Blimp1 for the specification of the germ cell lineage in mice. Genes Dev 2008, 22:1617-1635. 32. Seki Y, Hayashi K, Itoh K, Mizugaki M, Saitou M, Matsui Y: Extensive and orderly reprogramming of genome-wide chromatin modifications associated with specification and early development of germ cells in mice. Dev Biol 2005, 278:440-458. 33. Ficz G, Branco MR, Seisenberger S, Santos F, Krueger F, Hore TA, Marques CJ, Andrews S, Reik W: Dynamic regulation of 5hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 2011, 473:398-402. 34. Hajkova P, Jeffries SJ, Lee C, Miller N, Jackson SP, Surani MA: Genome-wide reprogramming in the mouse germ line entails the base excision repair pathway. Science 2010, 329:78-82. 35. Ma Z, Swigut T, Valouev A, Rada-Iglesias A, Wysocka J: Sequence-specific regulator Prdm14 safeguards mouse ESCs from entering extraembryonic endoderm fates. Nat Struct Mol Biol 2011, 18:120-127. The first global analysis of Prdm14 targets in mouse ESCs by ChIP-Seq reveals its localisation to distal regulatory elements including the distal enhancer of Oct4 and an overlap with enhancers occupied by pluripotency factors such as Nanog. The study also suggests that Prdm14 represses differentiation of ESCs into extraembryonic endoderm. 36. Chia NY, Chan YS, Feng B, Lu X, Orlov YL, Moreau D, Kumar P, Yang L, Jiang J, Lau MS et al.: A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity. Nature 2010, 468:316-320. www.sciencedirect.com

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Cell fate and reprogramming in the mammalian germline MagnuÂ´sdoÂ´ttir et al.

This study presents a global analysis of PRDM14 targets in human ESCs by ChIP-Seq and reveals that PRDM14 enhances the efficiency of reprogramming of human fibroblasts to iPSCs, when overexpressed together with OCT4, SOX2, KLF4 and c-MYC. 37. Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A: The ground state of embryonic stem cell self-renewal. Nature 2008, 453:519-523. 38. Leitch HG, Blair K, Mansfield W, Ayetey H, Humphreys P, Nichols J, Surani MA, Smith A: Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state. Development 2010, 137:2279-2287. 39. Yamaguchi S, Kurimoto K, Yabuta Y, Sasaki H, Nakatsuji N, Saitou M, Tada T: Conditional knockdown of Nanog induces apoptotic cell death in mouse migrating primordial germ cells. Development 2009, 136:4011-4020. 40. Kehler J, Tolkunova E, Koschorz B, Pesce M, Gentile L, Boiani M, Lomeli H, Nagy A, McLaughlin KJ, Scholer HR et al.: Oct4 is required for primordial germ cell survival. EMBO Rep 2004, 5:1078-1083. 41. Hayashi K, Ohta H, Kurimoto K, Aramaki S, Saitou M: Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 2011, 146:519-532. This study reports efficient generation of PGC-like cells by induction of epiblast-like cells (EpiLCs) from mouse ESCs cultured in 2i and LIF, and subsequent stimulation with BMP4, providing the first attempt at inducing PGCs in bulk culture potentially amenable to biochemical characterisation. 42. Guo G, Yang J, Nichols J, Hall JS, Eyres I, Mansfield W, Smith A: Klf4 reverts developmentally programmed restriction of ground state pluripotency. Development 2009, 136:1063-1069. 43. Durcova-Hills G, Adams IR, Barton SC, Surani MA, McLaren A: The role of exogenous fibroblast growth factor-2 on the reprogramming of primordial germ cells into pluripotent stem cells. Stem Cells 2006, 24:1441-1449. 44. Durcova-Hills G, Tang F, Doody G, Tooze R, Surani MA: Reprogramming primordial germ cells into pluripotent stem cells. PLoS One 2008, 3:e3531. 45. Hayashi K, Kobayashi T, Umino T, Goitsuka R, Matsui Y, Kitamura D: SMAD1 signaling is critical for initial commitment of germ cell lineage from mouse epiblast. Mech Dev 2002, 118:99-109. 46. Chang H, Matzuk MM: Smad5 is required for mouse primordial germ cell development. Mech Dev 2001, 104:61-67. 47. Ying QL, Nichols J, Chambers I, Smith A: BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003, 115:281-292.

Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc Natl Acad Sci U S A 2010, 107:21931-21936. Different classes of enhancers in mouse ESCs are characterised, where a distinction between active and poised enhancers can be made by the presence or absence of the H3K27 acetylation mark, respectively. The H3K27 acetylation mark is restricted to active enhancers in B-cells, neural progenitors and liver cells. Poised enhancers, characterised by H3K4me1, but unmodified H3K27, acquire H3K27 acetylation upon developmental progression. 52. Visel A, Blow MJ, Li Z, Zhang T, Akiyama JA, Holt A, Plajzer-Frick I, Shoukry M, Wright C, Chen F et al.: ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 2009, 457:854-858. 53. Ghisletti S, Barozzi I, Mietton F, Polletti S, De Santa F, Venturini E, Gregory L, Lonie L, Chew A, Wei C-L et al.: Identification and characterization of enhancers controlling the inflammatory gene expression program in macrophages. Immunity 2010, 32:317-328. This study characterises the inducible recruitment of the histone acetyltransferase p300 to enhancers governing the inflammatory response in macrophages and to sites occupied by the haematopoietic regulator PU.1 downstream of lipopolysaccharide (LPS) binding to toll-like receptors (TLRs). 54. Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, Cheng JX, Murre C, Singh H, Glass CK: Simple combinations of lineage-determining transcription factors prime cisregulatory elements required for macrophage and B cell identities. Mol Cell 2010, 38:576-589. This is one of the first papers addressing the relationship between the occupancy of signal responsive and lineage determining transcription factors. There is a step-wise refinement of PU.1 enhancer binding sites, from a broad repertoire of enhancers in haematopoietic stem cells to subsequent restriction to subsets of enhancers depending on the coexpression of small sets of either macrophage or B-cell lineage factors. The sequential restriction of enhancer repertoires culminates in the direction of ligand responsive transcription factors to PU.1 occupied enhancers, driving transcriptional activation from enhancer proximal promoters. 55. Trompouki E, Bowman TV, Lawton LN, Fan ZP, Wu DC, DiBiase A, Martin CS, Cech JN, Sessa AK, Leblanc JL et al.: Lineage regulators direct BMP and Wnt pathways to cell-specific programs during differentiation and regeneration. Cell 2011, 147:577-589. This paper addresses the relationship between lineage determining transcription factors and the signal responsive Smad and Tcf factors during differentiation and regeneration in haematopoiesis. Smad1 and Tcf7l2 co-occupy enhancers with Runx1 and ETS transcription factors in haematopoietic stem cells, but subsequently become restricted to enhancers occupied by GATA factors during erythropoiesis, revealing a refinement in enhancer occupancy and transcriptional output upon differentiation.

48. Heintzman ND, Hon GC, Hawkins RD, Kheradpour P, Stark A, Harp LF, Ye Z, Lee LK, Stuart RK, Ching CW et al.: Histone modifications at human enhancers reflect global cell-typespecific gene expression. Nature 2009, 459:108-112.

56. Smale ST: Pioneer factors in embryonic stem cells and differentiation. Curr Opin Genet Dev 2010, 20:519-526.

49. Heintzman ND, Stuart RK, Hon G, Fu Y, Ching CW, Hawkins RD, Barrera LO, Van Calcar S, Qu C, Ching K et al.: Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat Genet 2007, 39:311318.

58. Ng HH, Surani MA: The transcriptional and signalling networks of pluripotency. Nat Cell Biol 2011, 13:490-496.

50. Rada-Iglesias A, Bajpai R, Swigut T, Brugmann SA, Flynn RA, Wysocka J: A unique chromatin signature uncovers early developmental enhancers in humans. Nature 2011, 470:279-283. The epigenetic signature of two classes of enhancers in human ESCs demonstrates the distinction between active enhancers that are marked by H3K27 acetylation, and poised enhancers marked by H3K27me3. Active enhancers are associated with genes actively transcribed in human ESCs, whereas the poised enhancers lie proximal to genes that undergo activation during early embryonic differentiation. A reporter assay in zebrafish shows that the enhancers alone are sufficient to activate appropriate gene expression in a context dependent manner during development. 51. Creyghton MP, Cheng AW, Welstead GG, Kooistra T, Carey BW, Steine EJ, Hanna J, Lodato Ma, Frampton GM, Sharp Pa et al.: www.sciencedirect.com

57. Young RA: Control of the embryonic stem cell state. Cell 2011, 144:940-954.

59. Chen X, Xu H, Yuan P, Fang F, Huss M, Vega VB, Wong E, Orlov YL, Zhang W, Jiang J et al.: Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 2008, 133:1106-1117. 60. van den Berg DL, Snoek T, Mullin NP, Yates A, Bezstarosti K, Demmers J, Chambers I, Poot RA: An Oct4-centered protein interaction network in embryonic stem cells. Cell Stem Cell 2010, 6:369-381. 61. Mullen Alan C, Orlando David A, Newman Jamie J, LoveÂ´n J, Kumar Roshan M, Bilodeau S, Reddy J, Guenther Matthew G, DeKoter RP, Young Richard A: Master transcription factors determine cell-type-specific responses to TGF-b signaling. Cell 2011, 147:565-576. This paper reveals TGFb responsive Smad3 targeting to enhancers by the pluripotency factor Oct4, and the myeloid-lineage transcription factor MyoD1. Oct4/MyoD1 binding to enhancers directs Smad3 binding and subsequent transcriptional activation of proximal genes. Current Opinion in Genetics & Development 2012, 22:1â&#x20AC;&#x201C;9