REVIEWS H3K4 unmodified/ CpG spacing DNMT3A DNMIBL D|[ piRNA Figure 3 Cross-talk between DNA methylation, histone marks and the pirNa pathway in male germ cells In prospermatogonia of the fetal testis, de novo dNa methylation of paternally imprinted loci and retrotransposons occurs. The DNMT3A (DNA methyltransferase 3A)-DNMT3L complex recognizes its targets, such as imprinted loci, by sensing unmodified histone 3 lysine 4(H3K4 )and CpG spacing at 10-nucleotide intervals. Both DNMT3A- DNMT3L and DNMT3B-DNMT3L complexes methylate retrotransposons but how these sequences are recognized is unknown. MILL, a member of the Argonaute family of proteins, is involved in the piwi-interacting RNA(piRNA) pathway, but recent studies have shown that it also has a role in DNA methylation of retrotransposons. Whether MILI functions in de novo methylation or maintenance methylation is currently unknown. Sme C, 5-methylcytosine (the product of DNA methylation). derepressed, which is consistent with the fact that a large Epigenetic regulation of meiosis proportion of piRNAs from pre-pachytene spermato- Germ cells undergo several types of change in their cytes and prospermatogonia(Kuramochi-Miyagawa, S, epigenetic profile during the various stages of meiosis Watanabe, T, H.S. and Nakano, T, unpublished data) For example, in premeiotic PGCs and spermatogonia, were retrotransposon-derived. As RIWI, a member of unique patterns of histone modifications such as low the rat Piwi family, co-purifies with an RNA-cleavage H3K9me2 levels are observed,, but these patterns ctivity", it is likely that the suppression involves RNA- are dynamically changed upon the initiation of meiosis, guided cleavage of target RNAs. Interestingly, however, especially in male germ cells"(FIG 4) the Mili mutants also showed decreased DNA methyla- tion at LINEl retrotransposons and meiotic defects", The role of histone variants. Male germ cells also express similar to the phenotype of Dnmt3L mutants. At present, an unusually high number of histone variants, including how the MILl-PiRNA complex leads to DNA methyla- TH2A, TH2B, TH3, H3. 3A, H33B and HTl, which tion is an open question. Recent studies in Drosophila are incorporated in the nucleosomes of spermatogonia suggest a link between H3K9me3 and H3K9me2, het- and/or spermatocytes In oocytes, somatic histone Hl erochromatin protein 1(HPI)and Piwi proteinss, and is replaced by an oocyte-specific variant HIFoO dur- it is interesting to ask whether such a link exists in the ing meiotic prophase I (REF 5). The changes in histone mammalian germline. composition and modification might contribute to a Dnmt3L-mutant females can produce fully mature chromatin state that is required for meiosis to take place oocytes(although embryos derived from them die correctly, and for the further maturation of the gametes in utero because of the imprinting defects). However, (see REF. 5 for a detailed review of the role of histone a recent study showed that loss of LSH (lymphoid- variants during meiosis atin specific helicase), a member of the SNF2-helicase fam This is the highly compacted ily of chromatin remodelling proteins, leads to DNA Crucial functions of histone methyltransferases in uxtaposed to centromeres and demethylation and derepression of IAP retrotrans- prophase. The functional significance of histone modifi- contains large blocks of tandem posons in pachytene oocytes". This suggests a role cations during the process of meiosis is particularly clear repeats. It is irreversib for DNA methylation in retrotransposon suppression from gene-knockout studies(TABLE 1). Double mutations silenced and remains so throughout the cell cycle. in oocytes as well. Furthermore, the mutant oocytes of the H3K9 trimethyltransferase genes Suv39h1 and showed demethylation of repeats at pericentric hetero- Suy39h2 cause abnormal meiotic prophase in the testis chromatin as well as incomplete chromosome synapsis, The mutant spermatocytes lack pericentric H3K9me3 ructurally distinct, non- leading to a severe loss of oocytes at an early postnatal just before and during early meiotic prophase, and mei pical versions of the histone stage. In addition to DNA methylation, oocytes also otic chromosomes in these mutant cells undergo non- proteins. They are encoded use small-RNA pathways to silence retrotransposon omologous interactions, predominantly at centromeres (REF. 77 and Watanabe, T and H.S., unpublished data). and delayed synapsis. Meiosis is arrested at the pachytene that is distinct from that of So, both male and female germ cells use multiple stage in these cells, which triggers pronounced apopto the canonical histones. mechanisms for defence against retrotransposons sis of spermatocytes. The results suggest that specific @2008 Nature Publishing GroupPericentric heterochromatin This is the highly compacted chromatin region that is juxtaposed to centromeres and contains large blocks of tandem repeats. It is irreversibly silenced and remains so throughout the cell cycle. Histone variants Structurally distinct, non￾typical versions of the histone proteins. They are encoded by independent genes and often subject to regulation that is distinct from that of the canonical histones. derepressed69, which is consistent with the fact that a large proportion of piRNAs from pre-pachytene spermato￾cytes69 and prospermatogonia (Kuramochi-Miyagawa, S., Watanabe, T., H.S. and Nakano, T., unpublished data) were retrotransposon-derived. As RIWI, a member of the rat Piwi family, co-purifies with an RNA-cleavage activity72, it is likely that the suppression involves RNA￾guided cleavage of target RNAs. Interestingly, however, the Mili mutants also showed decreased DNA methyla￾tion at LINE1 retrotransposons69 and meiotic defects73, similar to the phenotype of Dnmt3L mutants. At present, how the MILI–piRNA complex leads to DNA methyla￾tion is an open question. Recent studies in Drosophila suggest a link between H3K9me3 and H3K9me2, het￾erochromatin protein 1 (HP1) and Piwi proteins74,75, and it is interesting to ask whether such a link exists in the mammalian germline. Dnmt3L-mutant females can produce fully mature oocytes (although embryos derived from them die in utero because of the imprinting defects)58,59. However, a recent study showed that loss of LSH (lymphoid￾specific helicase), a member of the SNF2-helicase fam￾ily of chromatin remodelling proteins, leads to DNA demethylation and derepression of IAP retrotrans￾posons in pachytene oocytes76. This suggests a role for DNA methylation in retrotransposon suppression in oocytes as well. Furthermore, the mutant oocytes showed demethylation of repeats at pericentric hetero‑ chromatin as well as incomplete chromosome synapsis, leading to a severe loss of oocytes at an early postnatal stage. In addition to DNA methylation, oocytes also use small-RNA pathways to silence retrotransposons (REF. 77 and Watanabe, T. and H.S., unpublished data). So, both male and female germ cells use multiple mechanisms for defence against retrotransposons. Epigenetic regulation of meiosis Germ cells undergo several types of change in their epigenetic profile during the various stages of meiosis. For example, in premeiotic PGCs and spermatogonia, unique patterns of histone modifications such as low H3K9me2 levels are observed27,78, but these patterns are dynamically changed upon the initiation of meiosis, especially in male germ cells79 (FIG. 4). The role of histone variants. Male germ cells also express an unusually high number of histone variants, including TH2A, TH2B, TH3, H3.3A, H3.3B and HT1, which are incorporated in the nucleosomes of spermatogonia and/or spermatocytes5 . In oocytes, somatic histone H1 is replaced by an oocyte-specific variant H1FOO dur￾ing meiotic prophase I (Ref. 5). The changes in histone composition and modification might contribute to a chromatin state that is required for meiosis to take place correctly, and for the further maturation of the gametes (see REF. 5 for a detailed review of the role of histone variants during meiosis). Crucial functions of histone methyltransferases in meiotic prophase. The functional significance of histone modifi￾cations during the process of meiosis is particularly clear from gene-knockout studies (table 1).Double mutations of the H3K9 trimethyltransferase genes Suv39h1 and Suv39h2 cause abnormal meiotic prophase in the testis79. The mutant spermatocytes lack pericentric H3K9me3 just before and during early meiotic prophase, and mei￾otic chromosomes in these mutant cells undergo non￾homologous interactions, predominantly at centromeres, and delayed synapsis. Meiosis is arrested at the pachytene stage in these cells, which triggers pronounced apopto￾sis of spermatocytes. The results suggest that specific Nature Reviews | Genetics Imprinted locus Retrotransposon H3K4 unmodified/ CpG spacing Target-RNA degradation DNMT3A DNMT3L DNMT3L De novo DNA methylation DNMT3A/B Effect on DNA methylation MILI piRNA piRNA biogenesis 5meC 5meC Figure 3 | Cross-talk between DNA methylation, histone marks and the piRNA pathway in male germ cells. In prospermatogonia of the fetal testis, de novo DNA methylation of paternally imprinted loci and retrotransposons occurs. The DNMT3A (DNA methyltransferase 3A)–DNMT3L complex recognizes its targets, such as imprinted loci, by sensing unmodified histone 3 lysine 4 (H3K4) and CpG spacing at 10-nucleotide intervals. Both DNMT3A– DNMT3L and DNMT3B–DNMT3L complexes methylate retrotransposons but how these sequences are recognized is unknown. MILI, a member of the Argonaute family of proteins, is involved in the piwi-interacting RNA (piRNA) pathway, but recent studies have shown that it also has a role in DNA methylation of retrotransposons. Whether MILI functions in de novo methylation or maintenance methylation is currently unknown. 5meC, 5-methylcytosine (the product of DNA methylation). R E V I E W S 134 | february 2008 | volume 9 www.nature.com/reviews/genetics © 2008 Nature Publishing Group