CTCF-dependent chromatin bias constitutes transient epigenetic memory of the mother at the H19-Igf2 imprinting control region in prospermatogonia

Genomic imprints-parental allele-specific DNA methylation marks at the differentially methylated regions (DMRs) of imprinted genes-are erased and reestablished in germ cells according to the individual's sex. Imprint establishment at paternally methylated germ line DMRs occurs in fetal male germ cells. In prospermatogonia, the two unmethylated alleles exhibit different rates of de novo methylation at the H19/Igf2 imprinting control region (ICR) depending on parental origin. We investigated the nature of this epigenetic memory using bisulfite sequencing and allele-specific ChIP-SNuPE assays. We found that the chromatin composition in fetal germ cells was biased at the ICR between the two alleles with the maternally inherited allele exhibiting more H3K4me3 and less H3K9me3 than the paternally inherited allele. We determined genetically that the chromatin bias, and also the delayed methylation establishment in the maternal allele, depended on functional CTCF insulator binding sites in the ICR. Our data suggest that, in primordial germ cells, maternally inherited allele-specific CTCF binding sets up allele-specific chromatin differences at the ICR. The erasure of these allele-specific chromatin marks is not complete before the process of de novo methylation imprint establishment begins. CTCF-dependent allele-specific chromatin composition imposes a maternal allele-specific delay on de novo methylation imprint establishment at the H19/Igf2 ICR in prospermatogonia.     

Sex-specific dynamics of global chromatin changes in fetal mouse germ cells

Mammalian germ cells undergo global reprogramming of DNA methylation during their development. Global DNA demethylation occurs around the time when the primordial germ cells colonize the embryonic gonads and this coincides with dynamic changes in chromatin composition. Global de novo DNA methylation takes place with remarkably different dynamics between the two sexes, prospermatogonia attaining methylation during fetal stages and oocytes attaining methylation postnatally. Our hypothesis was that dynamic changes in chromatin composition may precede or accompany the wave of global DNA de novo methylation as well. We used immunocytochemistry to measure global DNA methylation and chromatin components in male and female mouse fetal germ cells compared to control somatic cells of the gonad. We found that global DNA methylation levels sharply increased in male germ cells at 17.5 days post coitum, but remained low in female germ cells at all fetal stages. Global changes in chromatin composition: i, preceded global DNA methylation in fetal germ cells; ii, sex specifically occurred in male but not in female germ cells; iii, affected active and repressive histone marks and iv, included histone tail and histone globular domain modifications. Our data suggest that dynamic changes of chromatin composition may provide a framework for the pattern of male-specific de novo DNA methylation in prospermatogonia.     

Developmental acquisition of genome-wide DNA methylation occurs prior to meiosis in male germ cells

The development of germ cells is a highly ordered process that begins during fetal growth and is completed in the adult. Epigenetic modifications that occur in germ cells are important for germ cell function and for post-fertilization embryonic development. We have previously shown that male germ cells in the adult mouse have a highly distinct epigenetic state, as revealed by a unique genome-wide pattern of DNA methylation. Although it is known that these patterns begin to be established during fetal life, it is not known to what extent DNA methylation is modified during spermatogenesis. We have used restriction landmark genomic scanning (RLGS) and other techniques to examine DNA methylation at multiple sites across the genome during postnatal germ cell development in the mouse. Although a significant proportion of the distinct germ cell pattern is acquired prior to the type A spermatogonial stage, we find that both de novo methylation and demethylation occur during spermatogenesis, mainly in spermatogonia and spermatocytes in early meiotic prophase I. Alterations include predominantly non-CpG island sequences from both unique loci and repetitive elements. These modifications are progressive and are almost exclusively completed by the end of the pachytene spermatocyte stage. These studies better define the developmental timing of genome-wide DNA methylation pattern acquisition during male germ cell development.     

Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers

In mammalian development, epigenetic modifications, including DNA methylation patterns, play a crucial role in defining cell fate but also represent epigenetic barriers that restrict developmental potential. At two points in the life cycle, DNA methylation marks are reprogrammed on a global scale, concomitant with restoration of developmental potency. DNA methylation patterns are subsequently re-established with the commitment towards a distinct cell fate. This reprogramming of DNA methylation takes place firstly on fertilization in the zygote, and secondly in primordial germ cells (PGCs), which are the direct progenitors of sperm or oocyte. In each reprogramming window, a unique set of mechanisms regulates DNA methylation erasure and re-establishment. Recent advances have uncovered roles for the TET3 hydroxylase and passive demethylation, together with base excision repair (BER) and the elongator complex, in methylation erasure from the zygote. Deamination by AID, BER and passive demethylation have been implicated in reprogramming in PGCs, but the process in its entirety is still poorly understood. In this review, we discuss the dynamics of DNA methylation reprogramming in PGCs and the zygote, the mechanisms involved and the biological significance of these events. Advances in our understanding of such natural epigenetic reprogramming are beginning to aid enhancement of experimental reprogramming in which the role of potential mechanisms can be investigated in vitro. Conversely, insights into in vitro reprogramming techniques may aid our understanding of epigenetic reprogramming in the germline and supply important clues in reprogramming for therapies in regenerative medicine.     

Distinct dynamics of HISTONE3 variants between the two fertilization products in plants

Sexual reproduction involves epigenetic reprogramming comprising DNA methylation and histone modifications. In addition, dynamics of HISTONE3 (H3) variant H3.3 upon fertilization are conserved in animals, suggesting an essential role. In contrast to H3, H3.3 marks actively transcribed regions of the genome and can be deposited in a replication-independent manner. Although H3 variants are conserved in plants, their dynamics during fertilization have remained unexplored. We overcame technical limitations to live imaging of the fertilization process in Arabidopsis thaliana and studied dynamics of the male-gamete-specific H3.3 and the centromeric Histone Three Related 12 (HTR12). The double-fertilization process in plants produces the zygote and the embryo-nourishing endosperm. We show that the zygote is characterized by replication-independent removal of paternal H3.3 and homogeneous incorporation of parental chromatin complements. In the endosperm, the paternal H3.3 is passively diluted by replication while the paternal chromatin remains segregated apart from the maternal chromatin (gonomery). Hence epigenetic regulations distinguish the two products of fertilization in plants. H3.3-replication-independent dynamics and gonomery also mark the first zygotic divisions in animal species. We thus propose the convergent selection of parental epigenetic imbalance involving H3 variants in sexually reproducing organisms.     

The Role of DNA Methylation Reprogramming During Sex Determination and Transition in Zebrafish

DNA methylation is a prevalent epigenetic modification in vertebrates, and it has been shown to be involved the regulation of gene expression and embryo development. However, it remains unclear how DNA methylation regulates sexual development, especially in species without sex chromosomes. To determine this, we utilized zebrafish to investigate DNA methylation reprogramming during juvenile germ cell development and adult female-to-male sex transition.We reveal that primordial germ cells(PGCs) undergo significant DNA methylation reprogramming during germ cell development, and the methylome of PGCs is reset to an oocyte/ovary-like pattern at 9 days post fertilization(9 dpf). When DNA methyltransferase(DNMT) activity in juveniles was blocked after 9 dpf, the zebrafish developed into females. We also show that Tet3 is involved in PGC development. Notably, we find that DNA methylome reprogramming during adult zebrafish sex transition is similar to the reprogramming during the sex differentiation from 9 dpf PGCs to sperm. Furthermore, inhibiting DNMT activity can prevent the female-to-male sex transition, suggesting that methylation reprogramming is required for zebrafish sex transition. In summary, DNA methylation plays important roles in zebrafish germ cell development and sexual plasticity.     

Methylation changes in porcine primordial germ cells

Epigenetic re-programming is an important event in the development of primordial germ cells (PGC) into functional gametes, characterized by genome-wide erasure of DNA methylation and re-establishment of epigenetic marks, a process essential for restoration of the potential for totipotency. In this study changes in the methylation status of centromeric repeats and two IGF2-H19 differentially methylated domain (DMD) sequences were examined in porcine PGC between Days 24 and 31 of pregnancy. The methylation levels of centromeric repeats and IGF2-H19 DMD sequences decreased rapidly from Days 24 to 28 in both male and female PGC. At Days 30 and 31 of pregnancy centromeric repeats and IGF2-H19 DMD sequences acquired new methylation in male PGC, while in female PGC these sequences were completely demethylated by Day 30 and remained hypomethylated at Day 31. To characterize methylation changes that PGC undergo in culture, the methylation status of embryonic germ cells (EGCs) derived from PGC at Day 26 of pregnancy was examined. Centromeric repeats and IGF2-H19 DMD sequences were similarly methylated in both male and female EGC and hypermethylated in female EGC compared with female PGC at the same embryonic age. Our results show that, similar to murine PGC, porcine PGC undergo genome-wide DNA demethylation shortly after arrival in the genital ridges. When placed in culture porcine PGC terminate their demethylation program and may acquire new DNA methylation marks. To our knowledge, this is the first report regarding epigenetic re-programming of genital ridge PGC in the pig.     

The essential role of histone H3 Lys9 di-methylation and MeCP2 binding in MGMT silencing with poor DNA methylation of the promoter CpG island

Silencing of the O (6)-methylguanine-DNA methyltransferase (MGMT) gene, a key to DNA repair, is involved in carcinogenesis. Recent studies have focused on DNA hypermethylation of the promoter CpG island. However, cases showing silencing with DNA hypomethylation certainly exist, and the mechanism involved is not elucidated. To clarify this mechanism, we examined the dynamics of DNA methylation, histone acetylation, histone methylation, and binding of methyl-CpG binding proteins at the MGMT promoter region using four MGMT negative cell lines with various extents of DNA methylation. Histone H3K9 di-methylation (H3me2K9), not tri-methylation, and MeCP2 binding were commonly seen in all MGMT negative cell lines regardless of DNA methylation status. 5Aza-dC, but not TSA, restored gene expression, accompanied by a decrease in H3me2K9 and MeCP2 binding. In SaOS2 cells with the most hypomethylated CpG island, 5Aza-dC decreased H3me2K9 and MeCP2 binding with no effect on DNA methylation or histone acetylation. H3me2K9 and DNA methylation were restricted to in and around the island, indicating that epigenetic modification at the promoter CpG island is critical. We conclude that H3me2K9 and MeCP2 binding are common and more essential for MGMT silencing than DNA hypermethylation or histone deacetylation. The epigenetic mechanism leading to silent heterochromatin at the promoter CpG island may be the same in different types of cancer irrespective of the extent of DNA methylation.