REVIEWS 1In11111111111111111111111 5mec The global changes in repressive marks in migrat aIlllIll11 H3K9me12 ing PGCs might reflect the reprogramming of the PGC genome, which is eventually necessary for the zygote to acquire totipotency. Recent studies of chromatin modi I Transcriptional quiescence fications in embryonic stem cells(ES cells)showed that their pluripotency might be ensured by bivalent chro- E]05 En.5 E125 EB3.5 matin-that is, coincidence of H3K27me and H3K4me Arrival in at genes that encode key developmental transcrip- Entry into meiosis(female) tion factors2.30.Such modifications might temporarily keep the developmental genes poised for activation Figure 2 Epigenetic reprogramming in primordial germ cells(PGCs). Changes in undifferentiated ES cells. The increased level of in epigenetic modifications that occur during the genome-wide reprogramming that H3K27me3 and loss of other repressive marks in PGCs akes place during mouse PGC development Dashed lines indicate that the level of seem to make the PCG genome partly resemble such a the epigenetic modification is lower during these periods than that during the periods chromatin state. Understanding the epigenetic profile shown by solid lines. Sme C, 5-methylcytosine(the product of DNA methylation). of ES cells and germ cells should facilitate research on e exciting possibility of deriving functional gametes from pluripotent cells in culture(BOX 1) How BLIMPI regulates germ-cell specification and suppresses the somatic genes is currently obscure. Regulation of post-migratory PGC-specific genes by Although BLIMPI has a histone-methyltransferase epigenetic mechanisms. Recent studies have shown Nucleosome motif, such an activity has not been detected for this that changes in epigenetic modifications also have he basic structural subunit protein. As discussed below, BLIMPI binds to a histone- important roles in the regulation of post-migratory part for the compactness of a arginine methyltransferase, PRMT5 (protein argini GC-specific genes. Genes such as Ddx4 (DEAD chromosome. Each nucleosome N-methyltransferase 5), to repress premature expression box polypeptide 4, also known as Mvh), Sycp3(syn e of DNA of some germ-cell-specific genes in more advanced aptonemal complex protein 3)and Dazl(deleted in rapped around a histone core, PGCs, and it is possible that epigenetic modification azoospermia-like)are induced after migrating PGCs containing two copies of each might also contribute to the somatic repression role of have entered the genital ridge between E10. 5 and of the core histones: H2A, H2B. BLIMPI Ell.5(FIG. 1). DNA-methylation analysis revealed that, despite the genome-wide decrease in DNA methyl Genome-wide epigenetic changes during early PGC dif- ation after E8.0, the flanking regions of these genes ferentiation. An important recent insight into germ-cell remain methylated at E10.5, but become hypomethyl development comes from findings that unique epige- ated by E13.5 when they are expressed. Furthermore, eages usually all netic and transcriptional changes are seen in further these genes are derepressed in E9.5 embryos that lack lineages and a subset of differentiating, migrating PGCs2 2 When the germ- the maintenance DNA methyltransferase, DNMTI ctraembryonic lineages cell fate is established at E7. 25, levels of genome-wide (REF. 31). The results suggest that DNA methylation Epiblast DNA methylation, H3K9 dimethylation(H3K9me2) regulates the timing of activation of these genes n embryonic lineage that is and H3K27 trimethylation(H3K27 me3)-all marks This demethylation might be part of the second wave derived from the inner cell mass that are associated with transcriptional repression of demethylation that occurs around El1.5(REF. 32) of the blastocyst, which gives are similar to those in surrounding somatic cells. (see below). of the fetus. Subsequently, H3K9me2 starts to be erased at E7.5 A recent study has shown that histone methylation Gastrulation and DNA methylation decreases after about E8. 0, with that is mediated by BLIMPI and its associated histone the level of the former being clearly lower than in the arginine methyltransferase PRMT5 also regulates PGC- movements whereby the cells neighbouring somatic cells by E8.75(REFS 27, 28(FIG. 2). specific genes in post-migratory PGCs. In migrating of the blastula are rearranged Following this initial decrease in these two repressive PGCs, a nuclear-localized BLIMPI-PRMT5 com plan, which consists of the outer marks, the level of H3K27me3, another repressive mark, plex mediates dimethylation of histone H2AR3 and toderm. inner ectoderm and starts to be upregulated after E8. 25 and most PGCs show H4R3. After PGCs have settled in the genital ridge, terstitial mesoderm significantly higher levels of this mark at E9.5(REF 28) the BLIMP1-PRMT5 complex translocates to the FIG. 2). It is likely that this upregulation of H3K27me3 cytoplasm and the levels of H2AR3me2 and H4R3me2 Primitive streak complements the erasure of H3K9me2 to maintain a are diminished. Subsequently, Dhx38(DEAH box structure, which is present as proper repressive chromatin state of the PGC genome. polypeptide 38), a putative target of BLIMPI-PRMT5 relatively free of repressive chromatin between E7.5 and prevents premature expression of this gene Dhx38 E8. 25, which could result in deregulated transcription. encodes a protein that contains a DEAD box, which is gastrulation embryonic cells However, global RNAPII-dependent transcription is an RNA-helicase motif, and its homologue in C. elegans progress through the streak transiently repressed during this period, as demonstrated is involved in germ-cell development. It is interest m ce by the absence of both 5-bromouridine 5'triphosphate ing that H4R3me is associated with activation ofother A type of pluripotent stem cell(BrUTP) incorporation and RNAPII C-terminal- genes 4.35 and H3R8me, which is also mediated by that is derived from the inner domain phosphorylation. As RNAPII is active in PRMT5, can repress transcription6.Together,these cell mass of the early embryo nascent PGCs, as mentioned above, this transcrip- findings suggest that H2AR3me2 and H4R3me2 that of generating virtually all cell tional quiescence seems indifferent to the suppression are mediated by BLIMPI-PRMT5 might have a novel types of the organism. of the somatic program repressive role in PGCs @2008 Nature Publishing GroupNucleosome The basic structural subunit of chromatin, responsible in part for the compactness of a chromosome. Each nucleosome consists of a sequence of DNA wrapped around a histone core, which is a histone octamer containing two copies of each of the core histones: H2A, H2B, H3 and H4. Pluripotent Able to give rise to a wide range of, but not all, cell lineages (usually all fetal lineages and a subset of extraembryonic lineages). Epiblast An embryonic lineage that is derived from the inner cell mass of the blastocyst, which gives rise to the body of the fetus. Gastrulation A process of cell and tissue movements whereby the cells of the blastula are rearranged to form a three-layered body plan, which consists of the outer ectoderm, inner ectoderm and interstitial mesoderm. Primitive streak A transitory embryonic structure, which is present as a strip of cells, that pre-figures the anterior–posterior axis of the embryo. During gastrulation embryonic cells progress through the streak. Embryonic stem cell A type of pluripotent stem cell that is derived from the inner cell mass of the early embryo. Pluripotent cells are capable of generating virtually all cell types of the organism. How BLIMP1 regulates germ-cell specification and suppresses the somatic genes is currently obscure. Although BLIMP1 has a histone-methyltransferase motif, such an activity has not been detected for this protein. As discussed below, BLIMP1 binds to a histone￾arginine methyltransferase, PRMT5 (protein arginine N-methyltransferase 5), to repress premature expression of some germ-cell-specific genes in more advanced PGCs26, and it is possible that epigenetic modification might also contribute to the somatic repression role of BLIMP1. Genome-wide epigenetic changes during early PGC dif￾ferentiation. An important recent insight into germ-cell development comes from findings that unique epige￾netic and transcriptional changes are seen in further differentiating, migrating PGCs27,28. When the germ￾cell fate is established at E7.25, levels of genome-wide DNA methylation, H3K9 dimethylation (H3K9me2) and H3K27 trimethylation (H3K27me3) — all marks that are associated with transcriptional repression — are similar to those in surrounding somatic cells. Subsequently, H3K9me2 starts to be erased at E7.5 and DNA methylation decreases after about E8.0, with the level of the former being clearly lower than in the neighbouring somatic cells by E8.75 (REFs 27,28) (FIG. 2). Following this initial decrease in these two repressive marks, the level of H3K27me3, another repressive mark, starts to be upregulated after E8.25 and most PGCs show significantly higher levels of this mark at E9.5 (REF. 28) (FIG. 2). It is likely that this upregulation of H3K27me3 complements the erasure of H3K9me2 to maintain a proper repressive chromatin state of the PGC genome. The observation indicates that the PGC genome is relatively free of repressive chromatin between E7.5 and E8.25, which could result in deregulated transcription. However, global RNAPII-dependent transcription is transiently repressed during this period, as demonstrated by the absence of both 5-bromouridine 5′ triphosphate (BrUTP) incorporation and RNAPII C-terminal￾domain phosphorylation28. As RNAPII is active in nascent PGCs, as mentioned above, this transcrip￾tional quiescence seems indifferent to the suppression of the somatic programme. The global changes in repressive marks in migrat￾ing PGCs might reflect the reprogramming of the PGC genome, which is eventually necessary for the zygote to acquire totipotency. Recent studies of chromatin modi￾fications in embryonic stem cells (ES cells) showed that their pluripotency might be ensured by bivalent chro￾matin — that is, coincidence of H3K27me and H3K4me — at genes that encode key developmental transcrip￾tion factors29,30. Such modifications might temporarily keep the developmental genes poised for activation in undifferentiated ES cells. The increased level of H3K27me3 and loss of other repressive marks in PGCs seem to make the PCG genome partly resemble such a chromatin state. Understanding the epigenetic profiles of ES cells and germ cells should facilitate research on the exciting possibility of deriving functional gametes from pluripotent cells in culture (BOX 1). Regulation of post-migratory PGC-specific genes by epigenetic mechanisms. Recent studies have shown that changes in epigenetic modifications also have important roles in the regulation of post-migratory PGC-specific genes. Genes such as Ddx4 (DEAD box polypeptide 4, also known as Mvh), Sycp3 (syn￾aptonemal complex protein 3) and Dazl (deleted in azoospermia-like) are induced after migrating PGCs have entered the genital ridge between E10.5 and E11.5 (FIG. 1). DNA-methylation analysis revealed that, despite the genome-wide decrease in DNA methyl￾ation after E8.0, the flanking regions of these genes remain methylated at E10.5, but become hypomethyl￾ated by E13.5 when they are expressed31. Furthermore, these genes are derepressed in E9.5 embryos that lack the maintenance DNA methyltransferase, DNMT1 (REF. 31). The results suggest that DNA methylation regulates the timing of activation of these genes. This demethylation might be part of the second wave of demethylation that occurs around E11.5 (REF. 32) (see below). A recent study has shown that histone methylation that is mediated by BLIMP1 and its associated histone￾arginine methyltransferase PRMT5 also regulates PGC￾specific genes in post-migratory PGCs26. In migrating PGCs, a nuclear-localized BLIMP1–PRMT5 com￾plex mediates dimethylation of histone H2AR3 and H4R3. After PGCs have settled in the genital ridge, the BLIMP1–PRMT5 complex translocates to the cytoplasm and the levels of H2AR3me2 and H4R3me2 are diminished. Subsequently, Dhx38 (DEAH box polypeptide 38), a putative target of BLIMP1–PRMT5, is upregulated, suggesting that arginine methylation prevents premature expression of this gene26. Dhx38 encodes a protein that contains a DEAD box, which is an RNA-helicase motif, and its homologue in C. elegans is involved in germ-cell development33. It is interest￾ing that H4R3me is associated with activation of other genes34,35 and H3R8me, which is also mediated by PRMT5, can repress transcription36. Together, these findings suggest that H2AR3me2 and H4R3me2 that are mediated by BLIMP1–PRMT5 might have a novel repressive role in PGCs. Nature Reviews | Genetics Migration Arrival in genital ridge Mitotic arrest at G1 (male) Entry into meiosis (female) 5meC H3K9me1/2 H3K27me3 Transcriptional quiescence E7.5 E8.5 E9.5 E10.5 E11.5 E12.5 E13.5 Figure 2 | Epigenetic reprogramming in primordial germ cells (PGCs). Changes in epigenetic modifications that occur during the genome-wide reprogramming that takes place during mouse PGC development. Dashed lines indicate that the level of the epigenetic modification is lower during these periods than that during the periods shown by solid lines. 5meC, 5-methylcytosine (the product of DNA methylation). R E V I E W S nature reviews | genetics volume 9 | february 2008 | 131 © 2008 Nature Publishing Group