Promoter DNA methylation couples genome-defence mechanisms to epigenetic reprogramming in the mouse germline

Mouse primordial germ cells (PGCs) erase global DNA methylation (5mC) as part of the comprehensive epigenetic reprogramming that occurs during PGC development. 5mC plays an important role in maintaining stable gene silencing and repression of transposable elements (TE) but it is not clear how the extensive loss of DNA methylation impacts on gene expression and TE repression in developing PGCs. Using a novel epigenetic disruption and recovery screen and genetic analyses, we identified a core set of germline-specific genes that are dependent exclusively on promoter DNA methylation for initiation and maintenance of developmental silencing. These gene promoters appear to possess a specialised chromatin environment that does not acquire any of the repressive H3K27me3, H3K9me2, H3K9me3 or H4K20me3 histone modifications when silenced by DNA methylation. Intriguingly, this methylation-dependent subset is highly enriched in genes with roles in suppressing TE activity in germ cells. We show that the mechanism for developmental regulation of the germline genome-defence genes involves DNMT3B-dependent de novo DNA methylation. These genes are then activated by lineage-specific promoter demethylation during distinct global epigenetic reprogramming events in migratory (～E8.5) and post-migratory (E10.5-11.5) PGCs. We propose that genes involved in genome defence are developmentally regulated primarily by promoter DNA methylation as a sensory mechanism that is coupled to the potential for TE activation during global 5mC erasure, thereby acting as a failsafe to ensure TE suppression and maintain genomic integrity in the germline.     

Expression patterns and miRNA regulation of DNA methyltransferases in chicken primordial germ cells

DNA methylation is widespread in most species, from bacteria to mammals, and is crucial for genomic imprinting, gene expression, and embryogenesis. DNA methylation occurs via two major classes of enzymatic reactions: maintenance-type methylation catalyzed by DNA (cytosine-5-)-methyltransferase (DNMT) 1, and de novo methylation catalyzed by DNMT 3 alpha (DNMT3A) and -beta (DNMT3B). The expression pattern and regulation of DNMT genes in primordial germ cells (PGCs) and germ line cells has not been sufficiently established in birds. Therefore, we employed bioinformatics, RT-PCR, real-time PCR, and in situ hybridization analyses to examine the structural conservation and conserved expression patterns of chicken DNMT family genes. We further examined the regulation of a candidate de novo DNA methyltransferase gene, cDNMT3B by cotransfection of cDNMT3B 3'UTR- and cDNMT3B 3'UTR-specific miRNAs through a dual fluorescence reporter assay. All cDNMT family members were differentially detected during early embryonic development. Of interest, cDNMT3B expression was highly detected in early embryos and in PGCs. During germ line development and sexual maturation, cDNMT3B expression was reestablished in a female germ cell-specific manner. In the dual fluorescence reporter assay, cDNMT3B expression was significantly downregulated by four miRNAs: gga-miR-15c (25.82%), gga-miR-29b (30.01%), gga-miR-383 (30.0%), and gga-miR-222 (31.28%). Our data highlight the structural conservation and conserved expression patterns of chicken DNMTs. The miRNAs investigated in this study may induce downregulation of gene expression in chicken PGCs and germ cells.     

Variant Histone H2afv reprograms DNA methylation during early zebrafish development

The DNA methylome is re-patterned during discrete phases of vertebrate development. In zebrafish, there are 2 waves of global DNA demethylation and re-methylation: the first occurs before gastrulation when the parental methylome is changed to the zygotic pattern and the second occurs after formation of the embryonic body axis, during organ specification. The occupancy of the histone variant H2A.Z and regions of DNA methylation are generally anti-correlated, and it has been proposed that H2A.Z restricts the boundaries of highly methylated regions. While many studies have described the dynamics of methylome changes during early zebrafish development, the factors involved in establishing the DNA methylation landscape in zebrafish embryos have not been identified. We test the hypothesis that the zebrafish ortholog of H2A.Z (H2afv) restricts DNA methylation during development. We find that, in control embryos, bulk genome methylation decreases after gastrulation, with a nadir at the bud stage, and peaks during mid-somitogenesis; by 24 hours post -fertilization, total DNA methylation levels return to those detected in gastrula. Early zebrafish embryos depleted of H2afv have significantly more bulk DNA methylation during somitogenesis, suggesting that H2afv limits methylation during this stage of development. H2afv deficient embryos are small, with multisystemic abnormalities. Genetic interaction experiments demonstrate that these phenotypes are suppressed by depletion of DNA methyltransferase 1 (Dnmt1). This work demonstrates that H2afv is essential for global DNA methylation reprogramming during early vertebrate development and that embryonic development requires crosstalk between H2afv and Dnmt1.     

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.     

综述: DNA甲基化在动物胚胎和生殖细胞发育过程中的重编程

表观遗传信息DNA甲基化在动物的发育、细胞分化和器官形成过程中,起着至关重要的作用．近期,关于DNA甲基化在脊椎动物胚胎发育和生殖细胞发育过程重编程的研究取得了重要的进展．发现斑马鱼的早期胚胎完整地继承了精子的DNA甲基化图谱,而哺乳动物的早期胚胎和原始生殖细胞发育过程则经历了整体去甲基化并重新建立甲基化图谱的过程,但胚胎发育过程中基因的印迹区未发生DNA去甲基化,而生殖细胞发育过程中印迹区的甲基化修饰被消除．     

Epigenetic reprogramming in mouse primordial germ cells

Genome-wide epigenetic reprogramming in mammalian germ cells, zygote and early embryos, plays a crucial role in regulating genome functions at critical stages of development. We show here that mouse primordial germ cells (PGCs) exhibit dynamic changes in epigenetic modifications between days 10.5 and 12.5 post coitum (dpc). First, contrary to previous suggestions, we show that PGCs do indeed acquire genome-wide de novo methylation during early development and migration into the genital ridge. However, following their entry into the genital ridge, there is rapid erasure of DNA methylation of regions within imprinted and non-imprinted loci. For most genes, the erasure commences simultaneously in PGCs in both male and female embryos, which is completed within 1 day of development. Based on the kinetics of this process, we suggest that this is an active demethylation process initiated upon the entry of PGCs into the gonadal anlagen. The timing of reprogramming in PGCs is crucial since it ensures that germ cells of both sexes acquire an equivalent epigenetic state prior to the differentiation of the definitive male and female germ cells in which new parental imprints are established subsequently. Some repetitive elements, however, show incomplete erasure, which may be essential for chromosome stability and for preventing activation of transposons to reduce the risk of germline mutations. Aberrant epigenetic reprogramming in the germ line would cause the inheritance of epimutations that may have consequences for human diseases as suggested by studies on mouse models.     

生殖细胞及早期胚胎基因组印记的表观调控

基因组印记是一种区别父母等位基因的表观遗传过程,可导致父源和母源基因特异性表达。印记是在配子发生过程中全基因组表观重编程时获得的,且在早期胚胎发育过程中得以维持。因此,在全基因组重编程过程中,对印记的识别和维持十分重要。本文概述了原始生殖细胞的印记清除、双亲原始生殖细胞的印记获得以及早期胚胎发育过程中印记维持的相关过程,并对在印记区域内保护印记基因免受全基因组DNA去甲基化的表观遗传因子的相关作用机制进行了讨论。     

哺乳动物原始生殖细胞体内特化和体外培养

原始生殖细胞(primordial germ cells, PGCs)是胚胎中最先出现的生殖细胞。PGCs来源于上胚层,最早出现在后肠,随后向生殖嵴迁移。这一过程伴随一系列复杂的分子调控机制,以及DNA甲基化重编程和组蛋白修饰等表观遗传过程。PGCs经过不断的分裂、发育及分化,最终形成配子。为了更好地研究PGCs发育与分化的调控和表观遗传过程,体外培养的研究变得越来越重要。本文以小鼠和人为例,介绍了哺乳动物PGCs的特化过程、PGCs特化过程中的表观遗传过程和PGCs的体外培养研究进展。     

Functions of DNA methyltransferase 3-like in germ cells and beyond

DNA methyltransferase 3-like (DNMT3L) is one of the key players in de novo DNA methylation of imprinting control elements and retrotransposons, which occurs after genome-wide epigenetic erasure during germ cell development. In this review, we summarise the biochemical properties of DNMT3L and discuss the possible mechanisms behind DNMT3L-mediated imprinting establishment and retrotransposon silencing in germ cells. We also discuss possible connections between DNMT3L and non-coding RNA-mediated epigenetic remodelling, the roles of DNMT3L in germ cell development and the implications in stem cell and cancer research.     

Development of a pheasant interspecies primordial germ cell transfer to chicken embryo: Effect of donor cell sex on chimeric semen production

This study was conducted to evaluate whether the sex of donor primordial germ cells (PGCs) influences production of chimeric semen from recipient hatchlings produced by interspecies transfer between pheasant (Phasianus colchicus) and chicken (Gallus gallus). Pheasant PGCs were retrieved from 7-d-old embryos and subsequently transferred into circulatory blood of 2.5-d-old (Stage 17) embryos. The sex of embryos was discerned 3 to 6 days after laying, and in preliminary study, overall rate of embryo survival after sexing was 74.6% with male-to-female ratio of 0.49 to 0.51. In Experiment 1, magnetic-activated cell sorting (MACS) using QCR1 antibody was effective for enriching the population of male and female PGCs in gonadal cells (9.2- to 12.5-fold and 10.8- to 19.5-fold increase, respectively). In Experiment 2, an increase in the number of hatchlings producing chimeric semen was detected after the homosexual transfer of male-to-male compared with that after the heterosexual transfer of female-to-male (68% to 88%). Significant increase was found in the frequency of chimeric semen production (0.96 to 1.68 times); production of pheasant progenies by artificial insemination using chimeric semen was also increased in the homosexual transfer (0 to 3 cases). In conclusion, the homosexual PGC transfer of male-to-male yielded better rate of generating pheasant progenies after test cross-reproduction than that of the heterosexual transfer of female-to-male, which could improve the efficiency of interspecies germ cell transfer system.     

Functions of DNA methyltransferase 3‐like in germ cells and beyond

DNA methyltransferase 3‐like (DNMT3L) is one of the key players in de novo DNA methylation of imprinting control elements and retrotransposons, which occurs after genome‐wide epigenetic erasure during germ cell development. In this review, we summarise the biochemical properties of DNMT3L and discuss the possible mechanisms behind DNMT3L‐mediated imprinting establishment and retrotransposon silencing in germ cells. We also discuss possible connections between DNMT3L and non‐coding RNA‐mediated epigenetic remodelling, the roles of DNMT3L in germ cell development and the implications in stem cell and cancer research.     

Genome wide abnormal DNA methylome of human blastocyst in assisted reproductive technology

Proper reprogramming of parental DNA methylomes is essential for mammalian embryonic development.However,it is unknown whether abnormal methylome reprogramming occurs and is associated with the failure of embryonic development.Here we analyzed the DNA methylomes of 57 blastocysts and 29 trophectoderm samples with different morphological grades during assisted reproductive technology(ART) practices.Our data reveal that the global methylation levels of high-quality blastocysts are similar(0.30 ± 0.02,mean ± SD).while the methylation levels of low-quality blastocysts are divergent and away from those of high-quality blastocysts.The proportion of blastocysts with a methylation level falling within the range of 0.30 ± 0.02 in different grades correlates with the live birth rate for that grade.Moreover,abnormal methylated regions are associated with the failure of embryonic development.Furthermore,we can use the methylation data of cells biopsied from trophectoderm to predict the blastocyst methylation level as well as to detect the aneuploidy of the blastocysts.Our data indicate that global abnormal methylome reprogramming often occurs in human embryos,and suggest that DNA methylome is a potential biomarker in blastocyst selection in ART.     

Changes of DNA methylation level and spatial arrangement of primordial germ cells in embryonic day 15 to embryonic day 28 pig embryos

The mammalian germline is generally assumed to undergo extensive epigenetic reprogramming during embryonic development, including a nearly complete erasure of DNA methylation. This assumption does, however, to large degree rely on data from mouse, and despite a well-grounded picture the general nature of these data needs to be validated by investigations of other mammalian species. This study represents such a contribution in the examination of the germline in the domestic pig (Sus scrofa). Semiquantitative immunohistochemistry was used to investigate the level of DNA methylation in the POU5F1-positive primordial germ cells (PGCs) compared with neighboring somatic cells in porcine embryos at Embryonic Day 15 (E15), E17, E20, E21, and E28. We show that, in agreement with the mouse model, a significantly lower level of DNA methylation was observed in the early migrating PGCs. This level was decreasing until a stage coinciding with the entrance of the PGCs to the genital ridge. After this, the methylation level increased. Using whole-mount immunostaining, we determined the spatial arrangement of the porcine PGCs in the period between E15 and E28, allowing some comparison with the migration of the murine germline. The overall conclusion from the obtained data is that the DNA methylation changes in porcine PGCs, as well as the migration of these cells, parallels the picture reported for the mouse.     

Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells.

Genomic reprogramming of primordial germ cells (PGCs), which includes genome-wide demethylation, prevents aberrant epigenetic modifications from being transmitted to subsequent generations. This process also ensures that homologous chromosomes first acquire an identical epigenetic status before an appropriate switch in the imprintable loci in the female and male germ lines. Embryonic germ (EG) cells have a similar epigenotype to PGCs from which they are derived. We used EG cells to investigate the mechanism of epigenetic modifications in the germ line by analysing the effects on a somatic nucleus in the EG-thymic lymphocyte hybrid cells. There were striking changes in methylation of the somatic nucleus, resulting in demethylation of several imprinted and non-imprinted genes. These epigenetic modifications were heritable and affected gene expression as judged by re-activation of the silent maternal allele of Peg1/Mest imprinted gene in the somatic nucleus. This remarkable change in the epigenotype of the somatic nucleus is consistent with the observed pluripotency of the EG-somatic hybrid cells as they differentiated into a variety of tissues in chimeric embryos. The epigenetic modifications observed in EG-somatic cell hybrids in vitro are comparable to the reprogramming events that occur during germ cell development.     

黄河鲤性别决定时间和相关基因表达研究

鱼类性别决定和分化机制极为复杂,通过性腺组织切片鉴定得出黄河鲤从未分化性腺发育为Ⅱ期精巢、卵巢的时间为受精后第40天到第80天。选取一些可能参与黄河鲤性别决定分化相关的基因（amh、ar、cyp19a、cyp19b、dax1、dmrt1、er、foxl2、nobox、sox9a、sox9b、zp2）进行实时荧光定量PCR分析各个基因在受精后40d、45d、50d、55d、65d和80d的表达情况。结果显示性别决定相关基因在50d都有高表达,推测45-50 d为性别决定的关键时间。ar、amh、dax1、dmrt1、sox9a、sox9b六个基因在80d雄性表达量升高,且雄性明显高于雌性,推测这些基因参与精巢分化发育过程。cyp19a、cyp19b、foxl2、nobox、zp2五个基因在80d雌性表达升高,且高于雄性,推测其可能参与卵巢分化发育。