《遗传学》课程教学资源（学科前沿）遗传与表观遗传 Epigenetic events in mammalian germ-cell development

REVIEWS Epigenetic events in mammalian germ-cell development reprogramming and beyond Hiroyuki Sasaki* and Yasuhisa Matsui* Abstract The epigenetic profile of germ cells, which is defined by modifications of DNA and chromatin, changes dynamically during their development. Many of the changes are associated with the acquisition of the capacity to support post-fertilization development. Our knowledge of this aspect has greatly increased-for example insights into how the re-establishment of parental imprints is regulated. In addition, an emerging theme from recent studies is that epigenetic modifiers have key roles in germ-cell development itself-for example, epigenetics contributes to the gene- and genomic integrity. Understanding epigenetic regulation in germ cells har of meiosis expression programme that is required for germ-cell development, regulation implications for reproductive engineering technologies and human health Epigenetics refers to a collection of mechanisms and The role of epigenetics in germ cells can be viewed phenomena that define the phenotype of a cell without differently from that in somatic cells During somatic cell affecting the genotype. In molecular terms, it repre- differentiation, cells start in a pluripotent state and make a nts a range of chromatin modifications including series of decisions about their fates, thereby giving rise to DNA methylation, histone modifications, remodelling of a range of cell types. Their gene-expression programme nucleosomes and higher order chromatin reorganiza- become more restricted and potentially locked in by tion. These epigenetic modifications constitute a changes in epigenetic modifications. However, germ cells unique profile in each cell and define cellular identity are different in that, once their fate has been determined by regulating gene expression. Epigenetic profiles are during early development, there is no need for develop- Division of Human Genetics. modifiable during cellular differentiation, but herit- mental decisions to be made. Instead, germ cells have a Department of Integrated ability is an important aspect of epigenetics: it ensures specific fate and go through a series of epigenetic events Genetics, National Institute that daughter cells have the same phenotype as the that are unique to this cell type. The aspects of germ-cell Organization of Information parental cell development that are relevant to these epigenetic events The process of germ-cell development is regulated are the need for a unique gene-expression programme of Genetics. School of Life by both genetic and epigenetic mechanisms2-. Among that is different from somatic cells, the fact that germ Science The Graduate the various cell types that constitute an animal body, cells undergo meiosis and the particular importance of niversity for Advanced udies, 1111 yata germ cells are unique in that they can give rise to a new maintaining genomic integrity in these cells organism. On fertilization, the products of germ-cell In this Review, we discuss dynamic epigenetic Cell Resource Center for development, the oocyte and sperm cell, fuse to form changes that occur during mammalian germ-cell devel a zygote, which is totipotent- it can develop a whole opment. Recent studies have identified a number of epi stitute of Development, new organism. For the zygote to acquire this totipo- genetic modifiers, including DNA methyltransferase ging and Cancer, Tohoku niversity, Seiryo-machi4-I, tency, germ cells and the zygote undergo extensive ep histone-modification enzymes and their regulatory genetic reprogramming. In mammalian germ cells, factors, that have crucial influences on germ-cell devel Correspondence to HSor Y.M. reprogramming also strips existing parental imprints opment. There is also an increasing understanding of epigenetic marks that ensure parental-origin- the mechanisms of the epigenetic reprogramming that hisasakiglab nig acip: pecific monoallelic expression of about a hundred takes place during germ-cell development-for exam do:10.1038/nrg229 mammalian imprinted genes in the next generation ple, how imprints are re-established in the male and Published online and establishes new ones that are different in male female germ cells. Our discussion follows the temporal and female gametes progression of events during germ-cell development, NATURE REVIEWS GENETICS VOLUME 9 FEBRUARY 2008 129 @2008 Nature Publishing Group

Epigenetics refers to a collection of mechanisms and phenomena that define the phenotype of a cell without affecting the genotype1 . In molecular terms, it repre￾sents a range of chromatin modifications including DNA methylation, histone modifications, remodelling of nucleosomes and higher order chromatin reorganiza￾tion. These epigenetic modifications constitute a unique profile in each cell and define cellular identity by regulating gene expression. Epigenetic profiles are modifiable during cellular differentiation, but herit￾ability is an important aspect of epigenetics: it ensures that daughter cells have the same phenotype as the parental cell. The process of germ-cell development is regulated by both genetic and epigenetic mechanisms2–5. Among the various cell types that constitute an animal body, germ cells are unique in that they can give rise to a new organism. On fertilization, the products of germ-cell development, the oocyte and sperm cell, fuse to form a zygote, which is totipotent — it can develop a whole new organism2 . For the zygote to acquire this totipo￾tency, germ cells and the zygote undergo extensive epi￾genetic reprogramming2,3. In mammalian germ cells, reprogramming also strips existing parental imprints — epigenetic marks that ensure parental-origin￾specific monoallelic expression of about a hundred mammalian imprinted genes in the next generation — and establishes new ones that are different in male and female gametes. The role of epigenetics in germ cells can be viewed differently from that in somatic cells. During somatic cell differentiation, cells start in a pluripotent state and make a series of decisions about their fates, thereby giving rise to a range of cell types6 . Their gene-expression programmes become more restricted and potentially locked in by changes in epigenetic modifications. However, germ cells are different in that, once their fate has been determined during early development, there is no need for develop￾mental decisions to be made. Instead, germ cells have a specific fate and go through a series of epigenetic events that are unique to this cell type. The aspects of germ-cell development that are relevant to these epigenetic events are the need for a unique gene-expression programme that is different from somatic cells, the fact that germ cells undergo meiosis and the particular importance of maintaining genomic integrity in these cells. In this Review, we discuss dynamic epigenetic changes that occur during mammalian germ-cell devel￾opment. Recent studies have identified a number of epi￾genetic modifiers, including DNA methyltransferases, histone-modification enzymes and their regulatory factors, that have crucial influences on germ-cell devel￾opment. There is also an increasing understanding of the mechanisms of the epigenetic reprogramming that takes place during germ-cell development — for exam￾ple, how imprints are re-established in the male and female germ cells. Our discussion follows the temporal progression of events during germ-cell development, *Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Research Organization of Information and Systems & Department of Genetics, School of Life Science, The Graduate University for Advanced Studies, 1111 Yata, Mishima 411‑8540, Japan. ‡Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4‑1, Sendai 980‑8575, Japan. Correspondence to H.S. or Y.M. e-mails: hisasaki@lab.nig.ac.jp; ymatsui@idac.tohoku.ac.jp doi:10.1038/nrg2295 Published online 16 January 2008 Epigenetic events in mammalian germ-cell development: reprogramming and beyond Hiroyuki Sasaki* and Yasuhisa Matsui‡ Abstract | The epigenetic profile of germ cells, which is defined by modifications of DNA and chromatin, changes dynamically during their development. Many of the changes are associated with the acquisition of the capacity to support post-fertilization development. Our knowledge of this aspect has greatly increased— for example, insights into how the re-establishment of parental imprints is regulated. In addition, an emerging theme from recent studies is that epigenetic modifiers have key roles in germ-cell development itself — for example, epigenetics contributes to the gene￾expression programme that is required for germ-cell development, regulation of meiosis and genomic integrity. Understanding epigenetic regulation in germ cells has implications for reproductive engineering technologies and human health. R E V I E W S nature reviews | genetics volume 9 | february 2008 | 129 © 2008 Nature Publishing Group

REVIEWS Paternal trol of meiosis Imprinting Germ-cell specification Birth/prepuberty/adult ression and activation Suppression of somatic genes Transposon repression PGC founder population PGCs zygote MIll oocyte/egg PGCs settled in gonad Onset of Sex differentiation ully grown oocyte E6.0 Ovulation reprogramming Imprint erasure E17.5 Birth/ prepuberty/adult X-chromosome reactivation Figure 1 Germ cell development and associated epigenetic events in mice. Chronology of mouse germ cell development and the main epigenetic events that occur. PGCs(primordial germ cells) first emerge at embryonic day 7. 25(E7. 25)as a cluster of about 20 cells. Subsequently, they rapidly proliferate with an average doubling time of approximately 16 hours Before they stop dividing at E13.5, their number reaches up to about 26,000 MSCl, meiotic sex-chromosome inactivation DNA methylation A covalent modification that epigenetic changes ar might be important suppression. PGC-like cel dinucelotides in the vertebrate contributions to germ-cell-specific functions at each in Blimpl-null embryos have aberrant expression of the nome.It is catalysed by stage. Understanding the epigenetic changes that take Hox genes6, which are normally repressed in PGCs. This DNA methyltransferases place during germ-cell development has important suggests that BLIMPI is crucial for suppression of the transcription directly by resses implications for animal cloning, assisted reproductive somatic programme, which might ensure that the PGC echnologies and human health precursors and nascent PGCs are restricted to the rganisms such as Caenorhabditis ele specific transcription factors, Germ-cell specification and differentiation and Drosophila melanogaster, this repression involves nd indirectly by recruiting Determination and maintenance of the germ-cell fate. global inhibition of RNA polymerase II (RNAPII) In post-implantation mammalian embryos, a popula- dependent transcription 7-19. In D. melanogaster, the and their associated repressive tion of pluripotent cells in the epiblast gives rise to pri- pole cells that develop into PGCs also have reduced chromatin-remodelling ctivities mondial germ cells(PGCs), the fate of which is specified levels of histone H3 lysine 4 methylation(H3K4me), by tissue interaction during gastrulation. In mice, PGCs a mark that is associated with the permissive(active) Histone modifications first emerge inside the extra-embryonic mesoderm at state, and are enriched in H3K9me, a mark that is translational modifications the posterior end of the primitive streak as a cluster of associated with repression, suggesting a role for epi that alter their interactions cells at embryonic day 7.25(E7. 25)(REFS 7-9)(FIG. 1). genetic modifications in suppressing the somatic pro Before the final specification of PGCs, their precursors gramme. Here, maternally inherited molecules such roteins. In particular, the tails are induced within the proximal epiblast cell popula- as the products of gcl (germ-cell-less)212, pgc(polar of histones H3 and H4 can b tion by signals from the adjacent extra-embryonic ecto- granule component)2.2and nanos u are involved in esidues. Modifications of dermo-ls. A transcriptional regulator, B-lymphocyte the transcriptional quiescence. Therefore, suppres- the tail include methylation, maturation-induced protein 1(BLIMPI, also known as sion of somatic differentiation through transcriptional etylation, phosphorylation PR-domain-containing 1), is expressed specifically in regulation might be an evolutionarily conserved theme the precursor cells as early as E6. 25(REF. 16), and this for germ-cell specification. However, as RNAPII is processes, including gene molecule is essential for PGC specification learly active in nascent PGCs in mice, the molecules The PGCprecursors need to suppress the somaticgene- and mechanisms that regulate the process might differ pression programme, and epigenetic modifications between species www.nature.com/reviews/genetics @2008 Nature Publishing Group

DNA methylation A covalent modification that occurs predominantly at CpG dinucelotides in the vertebrate genome. It is catalysed by DNA methyltransferases and converts cytosines to 5‑methylcytosines. It represses transcription directly by inhibiting the binding of specific transcription factors, and indirectly by recruiting methyl-CpG-binding proteins and their associated repressive chromatin-remodelling activities. Histone modifications Histones undergo post￾translational modifications that alter their interactions with DNA and nuclear proteins. In particular, the tails of histones H3 and H4 can be covalently modified at several residues. Modifications of the tail include methylation, acetylation, phosphorylation and ubiquitylation, and influence several biological processes, including gene expression, DNA repair and chromosome condensation. and we describe the epigenetic changes and their contributions to germ-cell-specific functions at each stage. Understanding the epigenetic changes that take place during germ-cell development has important implications for animal cloning, assisted reproductive technologies and human health. Germ-cell specification and differentiation Determination and maintenance of the germ-cell fate. In post-implantation mammalian embryos, a popula￾tion of pluripotent cells in the epiblast gives rise to pri￾mordial germ cells (PGCs), the fate of which is specified by tissue interaction during gastrulation. In mice, PGCs first emerge inside the extra-embryonic mesoderm at the posterior end of the primitive streak as a cluster of cells at embryonic day 7.25 (E7.25) (REFS 7–9) (FIG. 1). Before the final specification of PGCs, their precursors are induced within the proximal epiblast cell popula￾tion by signals from the adjacent extra-embryonic ecto￾derm10–15. A transcriptional regulator, B‑lymphocyte maturation-induced protein 1 (BLIMP1, also known as PR-domain-containing 1), is expressed specifically in the precursor cells as early as E6.25 (REF. 16), and this molecule is essential for PGC specification. The PGC precursors need to suppress the somatic gene￾expression programme, and epigenetic modifications might be important for this suppression. PGC-like cells in Blimp1-null embryos have aberrant expression of the Hox genes16, which are normally repressed in PGCs. This suggests that BLIMP1 is crucial for suppression of the somatic programme, which might ensure that the PGC precursors and nascent PGCs are restricted to the germ￾cell fate. In organisms such as Caenorhabditis elegans and Drosophila melanogaster, this repression involves global inhibition of RNA polymerase II (RNAPII)- dependent transcription17–19. In D. melanogaster, the pole cells that develop into PGCs also have reduced levels of histone H3 lysine 4 methylation (H3K4me), a mark that is associated with the permissive (active) state, and are enriched in H3K9me, a mark that is associated with repression, suggesting a role for epi￾genetic modifications in suppressing the somatic pro￾gramme20. Here, maternally inherited molecules such as the products of gcl (germ-cell-less)21,22, pgc (polar granule component)23,24 and nanos20,25 are involved in the transcriptional quiescence. Therefore, suppres￾sion of somatic differentiation through transcriptional regulation might be an evolutionarily conserved theme for germ-cell specification. However, as RNAPII is clearly active in nascent PGCs in mice, the molecules and mechanisms that regulate the process might differ between species. Nature Reviews | Genetics Transposon repression Germ-cell specification Suppression of somatic genes E3.5 E6.0 E7.25 E10.5 E12.5 Meiosis Sperm cell Fully grown oocyte MII oocyte/egg Zygote PGC precursors PGC founder population Migration Repression and activation of germ-cell-specific genes Imprint erasure PGCs settled in gonad Sex differentiation X-chromosome reactivation E13.5 E17.5 Birth/prepuberty/adult Onset of meiosis Maternal imprinting Genome-wide deacetylation Spermatocyte E13.5 Birth/prepuberty/adult Paternal imprinting Control of meiosis MSCI Histone– protamine exchange Spermatogonium PGCs Oocyte growth Maturation Ovulation Genome-wide reprogramming Non-growing oocyte Prospermatogonium Figure 1 | Germ cell development and associated epigenetic events in mice. Chronology of mouse germ cell development and the main epigenetic events that occur. PGCs (primordial germ cells) first emerge at embryonic day 7.25 (E7.25) as a cluster of about 20 cells. Subsequently, they rapidly proliferate with an average doubling time of approximately 16 hours. Before they stop dividing at E13.5, their number reaches up to about 26,000. MSCI, meiotic sex-chromosome inactivation. R E V I E W S 130 | february 2008 | volume 9 www.nature.com/reviews/genetics © 2008 Nature Publishing Group

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 Group

Nucleosome 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

REVIEWS Erasure and establishment of parental imprints Imprinting in the male and female germline. Once the An early stage of mammalian Imprint erasure in PGCs. When they arrive at the parental imprints have been erased, new imprints must mbryonic development at genital ridge, which occurs by E11.5, PGCs undergo be re-established according to the gender of the animal. which the first cell lineage extensive epigenetic reprogramming, including the This re-establishment occurs only after sex determina- erasure of parental imprints(FIG. 1). The erasure of tion has been initiated, and male and female germ-cell imprints is reflected by demethylation of the imprinted development diverges to give rise to sperm or oocytes, Spherical structure formed loci, which occurs concomitantly with demethylation respectively. In mice, the gonads of males and females by diferentiating ES cells in of other regions. The erasure occurs at different become morphologically distinguishable by E12.5.In culture, which resembles the imprinted loci at different times between E10.5 and female embryos, germ cells arrest in meiotic prophase E12.5, as shown in a study of cloned embryos that were at around E13. 0, whereas male germ cells enter into produced from PGC nuclei". Since the imprint eras- Gl-phase mitotic arrest at a similar time(FIGS 1,2).A ure at each locus is a rapid process that is completed number of environmental cues, including retinoic acid within one day of development, this might be an active signals from the mesonephroi, determine the timing demethylation process of entry into meiosis by germ cells In somatic cells of female mammals, one of the two G1-arrested male germ cells are called prosper X chromosomes is inactivated so that the dosage of the matogonia or gonocytes(FIG. 1). Paternal methylation genes on this chromosome is equalized between males imprints, which have been identified at just three loci, and females. The inactive X chromosome is reactivated are progressively established in these cells between during female germ-cell development. It had been E14.5 and the newborn stage7-st A germline-specific thought that this reactivation occurs around the time gene-knockout study indicated that the de novo dNA of imprint erasure 39. However, more recent studies methyltransferase, DNMT3A, has a central role in the e novo methylation process of all known paternally at an even earlier stagei. So, X-chromosome reactiva- methylated loci, and another de novo methyltrans tion occurs progressively over a prolonged period and ferase, DNMT3B, is involved only at the Raserfl(ras is completed in post-migratory PGCs. The initiation protein-specific guanine nucleotide-releasing factor 1) of reactivation in migrating PGCs is reminiscent of locus 0. 2. The reason why RasgrfI requires an addi the X reactivation in inner-cell-mass cells of female tional enzyme is unknown, but this could be related to blastocysts, but the mechanisms of these processes are the presence of several retrotransposon sequences at yet to be clarified. this locus(see below ). The establishment of paternal Not all sequences show DNA demethylation in methylation imprints at all loci requires another mem post-migratory PGCs For example, DNA methylation ber of the Dnmt3 family, DNMT3L, which is highly of the intra-cisternal A particle(IAP)retrotransposon expressed in prospermatogonia052-4. This protein has family is only partially reprogrammed. Incomplete no DNA-methyltransferase activity but forms a complex removal of epigenetic marks in the germ line can lead with DNMT3A and/or DNMT3B and stimulates their to epigenetic inheritance from one generation to the activities. next, evidence of which is now accumulating in both The established methylation imprints are then main- factors, it has been suggested that this phenomenon established from neonatal testes, can be maintained could provide a basis for adaptive evolution and/or stably in culture and can give rise to sperm when heritable disease susceptibility without changes in transplanted into testes- possess paternal methyla DNA sequence-[BOX 2). tion imprints, whereas their multipotent derivatives, mGS cells, show partial demethylation at these sites, similar to ES cells. GS cells and mGS cells provide Box 1 Derivation of germ cells from embryonic stem cells invaluable tools for germ-cell study and reproductive Various types of somatic cell, including blood cells and neural cells, have been In the female germline, the initiation of dna obtained from embryonic stem(ES)cells in culture dishes. Recent studies have methylation imprinting occurs after birth, during the revealed that it is also possible to generate gametes from ES cells. Gametes or gamete-like cells were derived when mouse ES cells were cultured under various oocyte growth?. The growing oocytes are at the diplo tene stage of meiotic prophase I, and the de novo methyl differentiation conditions including simple monolayer culture(oocyte), embryoid- ation process is complete by the fully-grown oocyte body formation (sperm)o, embryoid-body formation followed by treatment with retinoic acid (sperm)and retinoic acid induction alone (sperm). In the most ge(FIG. 1). Both DNMT3A and DNMT3L also have successful case, ES-derived sperm cells were able to fertilize oocytes after essential roles in this process 2.9, but DNMT3B seems and survived only up to five months, indicating that reprogramming of the o. K intracytoplasmic injection and support embryonic development to term1.The resultant pups, however, had abnormalities in DNA methylation at imprinted loci Recent studies have started to provide some clues on the mechanism by which the DNMT3A-DNMT3L germ-cell genome was not properly accomplished. When we fully understand the complex recognizes the imprinted loci(and some ret- mechanisms of germ-cell reprogramming, we might be able to derive appropriately rotransposons, see below). A crystallographic analysis reprogrammed, functional gametes from cultured cells, which will allow new of the complexed C-terminal domains of DNMT3A approaches to reproductive engineering, although ethical and safety issues must be and DNMT3L revealed a tetrameric structure with two carefully considered active sites. This structure suggests that DNA regions @2008 Nature Publishing Group

Blastocyst An early stage of mammalian embryonic development at which the first cell lineages become established. Embryoid body Spherical structure formed by differentiating ES cells in culture, which resembles the early embryo. Erasure and establishment of parental imprints Imprint erasure in PGCs. When they arrive at the genital ridge, which occurs by E11.5, PGCs undergo extensive epigenetic reprogramming, including the erasure of parental imprints (FIG. 1). The erasure of imprints is reflected by demethylation of the imprinted loci, which occurs concomitantly with demethylation of other regions32. The erasure occurs at different imprinted loci at different times between E10.5 and E12.5, as shown in a study of cloned embryos that were produced from PGC nuclei37. Since the imprint eras￾ure at each locus is a rapid process that is completed within one day of development, this might be an active demethylation process32. In somatic cells of female mammals, one of the two X chromosomes is inactivated so that the dosage of the genes on this chromosome is equalized between males and females. The inactive X chromosome is reactivated during female germ-cell development. It had been thought that this reactivation occurs around the time of imprint erasure38,39. However, more recent studies showed that it is initiated in the migratory stage40 or at an even earlier stage41. So, X-chromosome reactiva￾tion occurs progressively over a prolonged period and is completed in post-migratory PGCs. The initiation of reactivation in migrating PGCs is reminiscent of the X reactivation in inner-cell-mass cells of female blastocysts, but the mechanisms of these processes are yet to be clarified. Not all sequences show DNA demethylation in post-migratory PGCs. For example, DNA methylation of the intra-cisternal A particle (IAP) retrotransposon family is only partially reprogrammed42. Incomplete removal of epigenetic marks in the germ line can lead to epigenetic inheritance from one generation to the next, evidence of which is now accumulating in both mice and humans43–45. Together with the fact that epigenetic marks can be influenced by environmental factors, it has been suggested that this phenomenon could provide a basis for adaptive evolution and/or heritable disease susceptibility without changes in DNA sequence43–45 (BOX 2). Imprinting in the male and female germline. Once the parental imprints have been erased, new imprints must be re-established according to the gender of the animal. This re-establishment occurs only after sex determina￾tion has been initiated, and male and female germ-cell development diverges to give rise to sperm or oocytes, respectively. In mice, the gonads of males and females become morphologically distinguishable by E12.5. In female embryos, germ cells arrest in meiotic prophase at around E13.0, whereas male germ cells enter into G1-phase mitotic arrest at a similar time (FIGS 1,2). A number of environmental cues, including retinoic acid signals from the mesonephroi46, determine the timing of entry into meiosis by germ cells. G1-arrested male germ cells are called prosper￾matogonia or gonocytes (FIG. 1). Paternal methylation imprints, which have been identified at just three loci, are progressively established in these cells between E14.5 and the newborn stage47–51. A germline-specific gene-knockout study indicated that the de novo DNA methyltransferase, DNMT3A, has a central role in the de novo methylation process of all known paternally methylated loci, and another de novo methyltrans￾ferase, DNMT3B, is involved only at the Rasgrf1 (RAS protein-specific guanine nucleotide-releasing factor 1) locus50,52. The reason why Rasgrf1 requires an addi￾tional enzyme is unknown, but this could be related to the presence of several retrotransposon sequences at this locus (see below). The establishment of paternal methylation imprints at all loci requires another mem￾ber of the Dnmt3 family, DNMT3L, which is highly expressed in prospermatogonia50,52–54. This protein has no DNA-methyltransferase activity but forms a complex with DNMT3A and/or DNMT3B and stimulates their activities. The established methylation imprints are then main￾tained throughout the rest of male germ-cell develop￾ment. Notably, germline stem (GS) cells — which are established from neonatal testes, can be maintained stably in culture and can give rise to sperm when transplanted into testes — possess paternal methyla￾tion imprints, whereas their multipotent derivatives, mGS cells, show partial demethylation at these sites, similar to ES cells55. GS cells and mGS cells provide invaluable tools for germ-cell study and reproductive engineering. In the female germline, the initiation of DNA￾methylation imprinting occurs after birth, during the oocyte growth56,57. The growing oocytes are at the diplo￾tene stage of meiotic prophase I, and the de novo methyl￾ation process is complete by the fully-grown oocyte stage (FIG. 1). Both DNMT3A and DNMT3L also have essential roles in this process52,58,59, but DNMT3B seems dispensable52. Recent studies have started to provide some clues on the mechanism by which the DNMT3A–DNMT3L complex recognizes the imprinted loci (and some ret￾rotransposons, see below). A crystallographic analysis of the complexed C-terminal domains of DNMT3A and DNMT3L revealed a tetrameric structure with two active sites60. This structure suggests that DNA regions Box 1 | Derivation of germ cells from embryonic stem cells Various types of somatic cell, including blood cells and neural cells, have been obtained from embryonic stem (ES) cells in culture dishes. Recent studies have revealed that it is also possible to generate gametes from ES cells108–111. Gametes or gamete-like cells were derived when mouse ES cells were cultured under various differentiation conditions including simple monolayer culture (oocyte)108, embryoid￾body formation (sperm)109, embryoid-body formation followed by treatment with retinoic acid (sperm)110 and retinoic acid induction alone (sperm)111. In the most successful case, ES-derived sperm cells were able to fertilize oocytes after intracytoplasmic injection and support embryonic development to term111. The resultant pups, however, had abnormalities in DNA methylation at imprinted loci and survived only up to five months, indicating that reprogramming of the germ-cell genome was not properly accomplished. When we fully understand the mechanisms of germ-cell reprogramming, we might be able to derive appropriately reprogrammed, functional gametes from cultured cells, which will allow new approaches to reproductive engineering, although ethical and safety issues must be carefully considered. R E V I E W S 132 | february 2008 | volume 9 www.nature.com/reviews/genetics © 2008 Nature Publishing Group

REVIEWS Box 2 I Transgenerational influence of epigenetic alterations in germ cells in mammals because expression levels of the imprinted genes, which include ny important developmental Recent studies have suggested that exposure to chemicals and malnutrition conditions can affect not only the children of the affected individuals, but also genes, are unbalanced in such embryos. When the their grandchildren. This might be attributable to epigenetic alterations that occur imprinted genes are appropriately modified by genetic engineering and developmental manipulation, however, disruptors, the number of spermatogenic cells decreased in the F1 generation. This it was possible to derive adult femalemice with two mater effect was transmittable through the male germ line to subsequent generations, nal genomes and no paternal complement 66(BOX 3) and this was correlated with altered DNA-methylation patterns. In another As the method involves genetically engineered animals example, exposure to methyl-donor supplementation during midgestation and highly complex nuclear-transfer technologies, affected the epigenetic s of fetal germ cells. The mouse A y gene, which its direct application to livestock seems difficult. influences coat colour, is regulated by the DNA methylation status of an intra- cisternal A particle(lAP)retrotransposon inserted at pseudoexon 1A of the gene The methyl-donor supplementation shifted the coat colour of the F2 generation to - Pigenetic silencing of retrotransposons darker one. This suggests that the methyl donor directly or indirectly affected the Only germ cells can transmit genetic information to epigenetic status of Ay in fetal germ cells. Finally, epidemiological studies have the next generation. Therefore, transposons, which indicated that grandchildren of malnourished women show low birth weight4, mobilize in the genome and might cause insertional that grandchildren of men who were well-fed before adolescence have a greater nutations, have to be strictly controlled in these cells. Approximately 40-50% of the mammalian genome is have low cardiovascular mortality In these cases, it is possible that the nutrition occupied by retrotransposons, which mobilize through status caused epigenetic alterations in germ cells, but further studies are needed an RNA intermediate, although many of them are to confirm this possibility. truncated or have accumulated mutations Mammalian retrotransposons include short interspersed nuclear elements(SINEs), long interspersed nuclear elements with a 10-nucleotide CpG interval are a preferred sub- (LINEs) and endogenous retroviruses(long terminal strate,and these are found in many imprinted loci. repeat(LTR)-type retrotransposons However, there are many other regions in the genome One way to control transposable elements is through with the same CpG spacing. Another study showed that epigenetic mechanisms?. In the male germline, al DNMT3L interacts with unmodified H3K4 (REF. 61), retrotransposon sequences undergo de which might restrict targets to regions without H3K4me. methylation during the fetal prospermatogonium Together, both nucleosome modification and CpG spac- stage, concomitant with the de novo methylation ing might provide the basis for the recognition of the of the imprinted loci(FIG. 1). Gene-knockout studies imprinted loci by DNMT3A-DNMT3L(REF. 60)(FIG. 3). in mice showed both common and differential target The differential methylation of the imprinted loci in the specificities of DNMT3A and DNMT3B with respect male and female germlines might require additional to these sequences: SINEBl is mainly methylated factors. In the case of paternally imprinted H19, pro- by DNMT3A, whereas LINEl and IAP are methyl X-chromosome inactivation tection of this locus from de novo DNA methylation in ated by both DNMT3A and DNMT3B (. 50). By The process that occurs in oocytes requires CCCTC-binding factor(CTCE), which contrast, DNMT3L is required for methylation of is known to bind to an unmethylated H19(H19 fetal all these sequences, indicating the crucial function liver mRNA)regulatory region and broad specificity of this factor in de novo dNA the pair of x chromosomes is X-chromosome inactivation in female mice is imprinted methylation((FIG 3) ownregulated to match the in pre-implantation embryos and the extra-embryonic The functional importance of DNA methylation in tissues of post-implantation embryos, and in both cases retrotransposon silencing and germ-cell development that is present in males. The the paternal X chromosome is preferentially inactivated. was first seen in Dnmt3L knockout mice. The LINE and inactivation process involves This imprinted X inactivation depends on both an acti- AP retrotransposons, of which de novo methylation a range of epigenetic vating imprint on the maternal X chromosome and an was prevented by Dnmt3L mutations, were highly tran mechanisms on the inactivated inactivating imprint on the paternal X chromosome. scribed in spermatogonia and spermatocytes' hanges in dNa methylation As mentioned above, the inactive X chromosome is mutations also caused meiotic failure with widespread histone modifications. reactivated in female PGCs, but maintenance of the non-homologous chromosome synapsis and progressive active state of the maternal X chromosome beyond fer- loss of germ cells by the mid-pachytene stage[TABLE 1) Chromosome synapsis tilization requires an imprint. Nuclear transplantation This resulted in complete azoospermia in older ani wo pairs of sister chromatids experiments showed that this maternal imprint is set on mals. The non-homologous synapsis could arise from the X chromosomes during the growth of the oocyte, illegitimate interactions between dispersed retrotrans- chromosomes)that occurs at as with the imprints at autosomal loci. As the maternal poson sequences that were unmasked by demethyla X chromosome from Dnmt3a/Dnmt3b double-mutant tion or from single-or double-strand breaks that were oocytes seems to have normal imprints, the mecha- produced during replicative retrotransposition Argonaute proteins are the nism of this imprinting might be different from that of Recently, a link between a small-RNA pathway and central components of RNA. autosomal imprinting DNA methylation of retrotransposons was discovered silencing mechanisms. They k Parthenogenesis, which is a successful development of (FIG 3).MILL, a member of the Piwi subfamily of Argonaute roteins, Is and, if it were possible in mammals, would provide a way early as E12.5(REF. 70) and interacts with a class of small and the catalytic activity for to produce clones of livestock animals. However, imprint- RNAs called piwi-interacting RNAs(piRNAs) In ing is a major barrier to parthenogenetic development Mili-mutant testis, LINEl and IAP retrotransposons were NATURE REVIEWS GENETICS @2008 Nature Publishing Group

X-chromosome inactivation The process that occurs in female mammals by which gene expression from one of the pair of X chromosomes is downregulated to match the levels of gene expression from the single X chromosome that is present in males. The inactivation process involves a range of epigenetic mechanisms on the inactivated chromosome, including changes in DNA methylation and histone modifications. Chromosome synapsis The association or pairing of the two pairs of sister chromatids (representing homologous chromosomes) that occurs at the start of meiosis. Argonaute proteins Argonaute proteins are the central components of RNA￾silencing mechanisms. They provide the platform for target-mRNA recognition by short guide RNA strands and the catalytic activity for mRNA cleavage. with a 10-nucleotide CpG interval are a preferred sub￾strate, and these are found in many imprinted loci60. However, there are many other regions in the genome with the same CpG spacing. Another study showed that DNMT3L interacts with unmodified H3K4 (Ref. 61), which might restrict targets to regions without H3K4me. Together, both nucleosome modification and CpG spac￾ing might provide the basis for the recognition of the imprinted loci by DNMT3A–DNMT3L (Ref. 60) (FIG. 3). The differential methylation of the imprinted loci in the male and female germlines might require additional factors. In the case of paternally imprinted H19, pro￾tection of this locus from de novo DNA methylation in oocytes requires CCCTC-binding factor (CTCF), which is known to bind to an unmethylated H19 (H19 fetal liver mRNA) regulatory region62. X-chromosome inactivation in female mice is imprinted in pre-implantation embryos and the extra-embryonic tissues of post-implantation embryos, and in both cases the paternal X chromosome is preferentially inactivated. This imprinted X inactivation depends on both an acti￾vating imprint on the maternal X chromosome and an inactivating imprint on the paternal X chromosome. As mentioned above, the inactive X chromosome is reactivated in female PGCs, but maintenance of the active state of the maternal X chromosome beyond fer￾tilization requires an imprint. Nuclear transplantation experiments showed that this maternal imprint is set on the X chromosomes during the growth of the oocyte63, as with the imprints at autosomal loci. As the maternal X chromosome from Dnmt3a/Dnmt3b double-mutant oocytes seems to have normal imprints64, the mecha￾nism of this imprinting might be different from that of autosomal imprinting. Parthenogenesis, which is a successful development of unfertilized eggs, is observed in many vertebrate species and, if it were possible in mammals, would provide a way to produce clones of livestock animals. However, imprint￾ing is a major barrier to parthenogenetic development in mammals because expression levels of the imprinted genes, which include many important developmental genes, are unbalanced in such embryos. When the imprinted genes are appropriately modified by genetic engineering and developmental manipulation, however, it was possible to derive adult female mice with two mater￾nal genomes and no paternal complement65,66 (BOX 3). As the method involves genetically engineered animals and highly complex nuclear-transfer technologies, its direct application to livestock seems difficult. Epigenetic silencing of retrotransposons Only germ cells can transmit genetic information to the next generation. Therefore, transposons, which mobilize in the genome and might cause insertional mutations, have to be strictly controlled in these cells. Approximately 40–50% of the mammalian genome is occupied by retrotransposons, which mobilize through an RNA intermediate, although many of them are truncated or have accumulated mutations. Mammalian retrotransposons include short interspersed nuclear elements (SINEs), long interspersed nuclear elements (LINEs) and endogenous retroviruses (long terminal repeat (LTR)-type retrotransposons). One way to control transposable elements is through epigenetic mechanisms67. In the male germline, all retrotransposon sequences undergo de novo DNA methylation during the fetal prospermatogonium stage50, concomitant with the de novo methylation of the imprinted loci (FIG. 1). Gene-knockout studies in mice showed both common and differential target specificities of DNMT3A and DNMT3B with respect to these sequences: SINEB1 is mainly methylated by DNMT3A, whereas LINE1 and IAP are methyl￾ated by both DNMT3A and DNMT3B (REF. 50). By contrast, DNMT3L is required for methylation of all these sequences50, indicating the crucial function and broad specificity of this factor in de novo DNA methylation (FIG. 3). The functional importance of DNA methylation in retrotransposon silencing and germ-cell development was first seen in Dnmt3L knockout mice. The LINE and IAP retrotransposons, of which de novo methylation was prevented by Dnmt3L mutations, were highly tran￾scribed in spermatogonia and spermatocytes53,54,68. The mutations also caused meiotic failure with widespread non-homologous chromosome synapsis and progressive loss of germ cells by the mid-pachytene stage (TABLE 1). This resulted in complete azoospermia in older ani￾mals. The non-homologous synapsis could arise from illegitimate interactions between dispersed retrotrans￾poson sequences that were unmasked by demethyla￾tion or from single- or double-strand breaks that were produced during replicative retrotransposition53. Recently, a link between a small-RNA pathway and DNA methylation of retrotransposons was discovered69 (FIG. 3). MILI, a member of the Piwi subfamily of Argonaute proteins, is expressed in the male and female gonads as early as E12.5 (REF. 70) and interacts with a class of small RNAs called piwi-interacting RNAs (piRNAs)69,71. In Mili-mutant testis, LINE1 and IAP retrotransposons were Box 2 | Transgenerational influence of epigenetic alterations in germ cells Recent studies have suggested that exposure to chemicals and malnutrition conditions can affect not only the children of the affected individuals, but also their grandchildren. This might be attributable to epigenetic alterations that occur in fetal germ cells. When gestating female rats were exposed to some endocrine disruptors, the number of spermatogenic cells decreased in the F1 generation. This effect was transmittable through the male germ line to subsequent generations, and this was correlated with altered DNA-methylation patterns112. In another example, exposure to methyl-donor supplementation during midgestation affected the epigenetic status of fetal germ cells113. The mouse Avy gene, which influences coat colour, is regulated by the DNA methylation status of an intra￾cisternal A particle (IAP) retrotransposon inserted at pseudoexon 1A of the gene. The methyl-donor supplementation shifted the coat colour of the F2 generation to a darker one. This suggests that the methyl donor directly or indirectly affected the epigenetic status of Avy in fetal germ cells. Finally, epidemiological studies have indicated that grandchildren of malnourished women show low birth weight114, that grandchildren of men who were well-fed before adolescence have a greater risk of mortality from diabetes, and that descendants of men who suffered famine have low cardiovascular mortality115. In these cases, it is possible that the nutrition status caused epigenetic alterations in germ cells, but further studies are needed to confirm this possibility. R E V I E W S nature reviews | genetics volume 9 | february 2008 | 133 © 2008 Nature Publishing Group