[Show abstract][Hide abstract] ABSTRACT: In mice, primordial germ cells migrate into the genital ridges by embryonic day 13.5 (E13.5), where they are then subjected to a sex-specific fate with female and male primordial germ cells undergoing mitotic arrest and meiosis, respectively. However, the sex-specific basis of primordial germ cell differentiation is poorly understood. The aim of this study was to investigate the sex-specific features of mouse primordial germ cells. We performed RNA-sequencing (seq) of E13.5 female and male mouse primordial germ cells using next-generation sequencing. We identified 651 and 428 differentially expressed transcripts (>2-fold, P < 0.05) in female and male primordial germ cells, respectively. Of these, many transcription factors were identified. Gene ontology and network analysis revealed differing functions of the identified female- and male-specific genes that were associated with primordial germ cell acquisition of sex-specific properties required for differentiation into germ cells. Furthermore, DNA methylation and ChIP-seq analysis of histone modifications showed that hypomethylated gene promoter regions were bound with H3K4me3 and H3K27me3. Our global transcriptome data showed that in mice, primordial germ cells are decisively assigned to a sex-specific differentiation program by E13.5, which is necessary for the development of vital germ cells.
[Show abstract][Hide abstract] ABSTRACT: In the male germline, neonatal prospermatogonia give rise to spermatogonia, which include stem cell population (undifferentiated spermatogonia) that supports continuous spermatogenesis in adults. Although the levels of DNA methyltransferases change dynamically in the neonatal and early postnatal male germ cells, detailed genome-wide DNA methylation profiles of these cells during the stem cell formation and differentiation have not been reported.
To understand the regulation of spermatogonial stem cell formation and differentiation, we examined the DNA methylation and gene expression dynamics of male mouse germ cells at the critical stages: neonatal prospermatogonia, and early postntal (day 7) undifferentiated and differentiating spermatogonia. We found large partially methylated domains similar to those found in cancer cells and placenta in all these germ cells, and high levels of non-CG methylation and 5-hydroxymethylcytosines in neonatal prospermatogonia. Although the global CG methylation levels were stable in early postnatal male germ cells, and despite the reported scarcity of differential methylation in the adult spermatogonial stem cells, we identified many regions showing stage-specific differential methylation in and around genes important for stem cell function and spermatogenesis. These regions contained binding sites for specific transcription factors including the SOX family members.
Our findings show a distinctive and dynamic regulation of DNA methylation during spermatogonial stem cell formation and differentiation in the neonatal and early postnatal testes. Furthermore, we revealed a unique accumulation and distribution of non-CG methylation and 5hmC marks in neonatal prospermatogonia. These findings contrast with the reported scarcity of differential methylation in adult spermatogonial stem cell differentiation and represent a unique phase of male germ cell development.
[Show abstract][Hide abstract] ABSTRACT: Recent studies of the demethylation process in murine zygotes have shown that 5-methylcytosine (5mC) is first converted into 5-hydroxymethylcytosine (5hmC) or further-oxidized cytosines in the paternal genome by the maternal ten–eleven translocation 3 (TET3) enzyme. This process is crucial for normal embryogenesis, and our aim was to elucidate the effect of Tet3 on the maternal genome during female germ-line development. Immunofluorescence analysis showed that 5hmC was clearly present in fully grown oocytes but not in nongrowing and early growth-stage oocytes. The 5hmC in the maternal genome was clearly detectable in DNA methyltransferase 3-like enzyme (Dnmt3L)-null oocytes and their fertilized zygotes, although Dnmt3L is essential for DNA methylation in oocytes. An analysis using an enzyme digestion-based method showed that 5hmC was present in LTR retrotransposons from the late growth period of oocytes. Quantitative RT-PCR analysis showed that Tet3 expression was enhanced during oocyte growth and exhibited an approximately 40-fold increase between nongrowing and fully grown oocytes. Our results show that 5hmC is generated since the oocyte growth stage, accompanied by up-regulation of Tet3; 5hmC is located mainly in LTR retrotransposons, indicating that 5hmC generated in growth-stage oocytes is responsible for genomewide demethylation after fertilization.
[Show abstract][Hide abstract] ABSTRACT: Long non-coding RNAs (lncRNAs), transcribed from the intergenic regions of animal genomes, play important roles in key biological processes. In mice, Zdbf2linc was recently identified as an lncRNA isoform of the paternally expressed imprinted Zdbf2 gene. The functional role of Zdbf2linc remains undefined, but it may control parent-of-origin-specific expression of protein-coding neighbors through epigenetic modification in cis, similar to imprinted Nespas, Kcnq1ot1 and Airn lncRNAs. Here, we identified a novel imprinted long-range non-coding RNA, termed GPR1AS, in the human GPR1-ZDBF2 intergenic region. Although GPR1AS contains no human ZDBF2 exons, this lncRNA is transcribed in the antisense orientation from the GPR1 intron to a secondary, differentially methylated region upstream of the ZDBF2 gene (ZDBF2 DMR), similar to mouse Zdbf2linc. Interestingly, GPR1AS/Zdbf2linc is exclusively expressed in human/mouse placenta with paternal-allele-specific expression and maternal-allele-specific promoter methylation (GPR1/Gpr1 DMR). The paternal-allele specific methylation of the secondary ZDBF2 DMR was established in human placentas as well as somatic lineage. Meanwhile, the ZDBF2 gene showed stochastic paternal-allele-specific expression, possibly methylation-independent, in placental tissues. Overall, we demonstrated that epigenetic regulation mechanisms in the imprinted GPR1-GPR1AS-ZDBF2 region were well-conserved between human and mouse genomes without the high sequence conservation of the intergenic lncRNAs. Our findings also suggest that lncRNAs with highly conserved epigenetic and transcriptional regulation across species arose by divergent evolution from a common ancestor, if they do not have identical exon structures.
Preview · Article · May 2013 · Epigenetics: official journal of the DNA Methylation Society
[Show abstract][Hide abstract] ABSTRACT: DNA methylation is an epigenetic modification that plays a crucial role in normal mammalian development, retrotransposon silencing, and cellular reprogramming. Although methylation mainly occurs on the cytosine in a CG site, non-CG methylation is prevalent in pluripotent stem cells, brain, and oocytes. We previously identified non-CG methylation in several CG-rich regions in mouse germinal vesicle oocytes (GVOs), but the overall distribution of non-CG methylation and the enzymes responsible for this modification are unknown. Using amplification-free whole-genome bisulfite sequencing, which can be used with minute amounts of DNA, we constructed the base-resolution methylome maps of GVOs, non-growing oocytes (NGOs), and mutant GVOs lacking the DNA methyltransferase Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3L. We found that nearly two-thirds of all methylcytosines occur in a non-CG context in GVOs. The distribution of non-CG methylation closely resembled that of CG methylation throughout the genome and showed clear enrichment in gene bodies. Compared to NGOs, GVOs were over four times more methylated at non-CG sites, indicating that non-CG methylation accumulates during oocyte growth. Lack of Dnmt3a or Dnmt3L resulted in a global reduction in both CG and non-CG methylation, showing that non-CG methylation depends on the Dnmt3a-Dnmt3L complex. Dnmt3b was dispensable. Of note, lack of Dnmt1 resulted in a slight decrease in CG methylation, suggesting that this maintenance enzyme plays a role in non-dividing oocytes. Dnmt1 may act on CG sites that remain hemimethylated in the de novo methylation process. Our results provide a basis for understanding the mechanisms and significance of non-CG methylation in mammalian oocytes.
[Show abstract][Hide abstract] ABSTRACT: Dynamic epigenetic reprogramming occurs during mammalian germ cell development, although the targets of this process, including DNA demethylation and de novo methylation, remain poorly understood. We performed genome-wide DNA methylation analysis in male and female mouse primordial germ cells at embryonic days 10.5, 13.5, and 16.5 by whole-genome shotgun bisulfite sequencing. Our high-resolution DNA methylome maps demonstrated gender-specific differences in CpG methylation at genome-wide and gene-specific levels during fetal germline progression. There was extensive intra- and intergenic hypomethylation with erasure of methylation marks at imprinted, X-linked, or germline-specific genes during gonadal sex determination and partial methylation at particular retrotransposons. Following global demethylation and sex determination, CpG sites switched to de novo methylation in males, but the X-linked genes appeared resistant to the wave of de novo methylation. Significant differential methylation at a subset of imprinted loci was identified in both genders, and non-CpG methylation occurred only in male gonocytes. Our data establish the basis for future studies on the role of epigenetic modifications in germline development and other biological processes.
[Show abstract][Hide abstract] ABSTRACT: The paternally-expressed imprinted genes Gpr1 and Zdbf2 form a gene cluster wherein the imprinted-methylated regions of these two genes differ. We identified a novel, paternally expressed, long intergenic non-coding Zdbf2 variant (Zdbf2linc) transcribed from maternally methylated Gpr1 DMR during early embryogenesis in the mouse. While the Gpr1 DMR displayed biallelic hypermethylation, Zdbf2linc expression was rarely observed in the post-gastrulation, despite a positive correlation between the methylation of Zdbf2 DMRs and the mono-allelic transcription of the original Zdbf2 coding variant. Furthermore, lack of the maternal methylation imprint resulted in the biallelic expression of both coding and non-coding Zdbf2 transcripts as well as complete methylation of Zdbf2 DMRs. Globally, our findings suggest the role of Zdbf2linc in the establishment of secondary epigenetic modifications after implantation.
[Show abstract][Hide abstract] ABSTRACT: Genome-wide dynamic changes in DNA methylation are indispensable for germline development and genomic imprinting in mammals. Here, we report single-base resolution DNA methylome and transcriptome maps of mouse germ cells, generated using whole-genome shotgun bisulfite sequencing and cDNA sequencing (mRNA-seq). Oocyte genomes showed a significant positive correlation between mRNA transcript levels and methylation of the transcribed region. Sperm genomes had nearly complete coverage of methylation, except in the CpG-rich regions, and showed a significant negative correlation between gene expression and promoter methylation. Thus, these methylome maps revealed that oocytes and sperms are widely different in the extent and distribution of DNA methylation. Furthermore, a comparison of oocyte and sperm methylomes identified more than 1,600 CpG islands differentially methylated in oocytes and sperm (germline differentially methylated regions, gDMRs), in addition to the known imprinting control regions (ICRs). About half of these differentially methylated DNA sequences appear to be at least partially resistant to the global DNA demethylation that occurs during preimplantation development. In the absence of Dnmt3L, neither methylation of most oocyte-methylated gDMRs nor intragenic methylation was observed. There was also genome-wide hypomethylation, and partial methylation at particular retrotransposons, while maintaining global gene expression, in oocytes. Along with the identification of the many Dnmt3L-dependent gDMRs at intragenic regions, the present results suggest that oocyte methylation can be divided into 2 types: Dnmt3L-dependent methylation, which is required for maternal methylation imprinting, and Dnmt3L-independent methylation, which might be essential for endogenous retroviral DNA silencing. The present data provide entirely new perspectives on the evaluation of epigenetic markers in germline cells.
[Show abstract][Hide abstract] ABSTRACT: Dynamic changes in DNA methylation at the gene-specific and genome-wide level occur during mammalian germ-cell development. However, the details of how the methylation profiles change remain largely unknown. Bisulfite sequencing analysis is a powerful technique to determine the methylation status of DNA at individual cytosine-guanine dinucleotide (CpG) sites and requires only a small amount of DNA for analysis. Here, we introduce two methods for bisulfite-based DNA methylation analyses using small samples such as germ cells: bisulfite Sanger sequencing at a specific locus and high-throughput bisulfite sequencing at the whole genome level.
No preview · Article · Jan 2012 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Mammalian imprinted genes are associated with differentially methylated regions (DMRs) that are CpG methylated on one of the two parental chromosomes. In mice, at least 21 DMRs acquire differential methylation in the germline and many of them act as imprint centres. We previously reported the physical extents of differential methylation at 15 DMRs in mouse embryos at 12.5 days postcoitum. To reveal the ontogeny of differential methylation, we determined and compared methylation patterns of the corresponding regions in sperm and oocytes. We found that the extent of the gametic DMRs differs significantly from that of the embryonic DMRs, especially in the case of paternal gametic DMRs. These results suggest that the gametic DMR sequences should be used to extract the features specifying methylation imprint establishment in the germline: from this analysis, we noted that the maternal gametic DMRs appear as unmethylated islands in male germ cells, which suggests a novel component in the mechanism of gamete-specific marking. Analysis of selected DMRs in blastocysts revealed dynamic changes in allelic methylation in early development, indicating that DMRs are not fully protected from the major epigenetic reprogramming events occurring during preimplantation development. Furthermore, we observed non-CpG methylation in oocytes, but not in sperm, which disappeared by the blastocyst stage. Non-CpG methylation was frequently found at maternally methylated DMRs as well as non-DMR regions, suggesting its prevalence in the oocyte genome. These results provide evidence for a unique methylation profile in oocytes and reveal the surprisingly dynamic nature of DMRs in the early embryo.
[Show abstract][Hide abstract] ABSTRACT: There is an increased prevalence of imprinting disorders, such as Beckwith-Wiedemann syndrome, associated with human assisted reproductive technologies (ART). Work on animal models suggests that in vitro culture may be the source of these imprinting errors. However, in this study we report that, in some cases, the errors are inherited from the father. We analyzed DNA methylation at seven autosomal imprinted loci and the XIST locus in 78 paired DNA samples. In seven out of seventeen cases where there was abnormal DNA methylation in the ART sample (41%), the identical alterations were present in the parental sperm. Furthermore, we also identified DNA sequence variations in the gene encoding DNMT3L, which were associated with the abnormal paternal DNA methylation. Both the imprinting errors and the DNA sequence variants were more prevalent in patients with oligospermia. Our data suggest that the increase in the incidence of imprinting disorders in individuals born by ART may be due, in some cases, to the use of sperm with intrinsic imprinting mutations.
Full-text · Article · Jun 2009 · European journal of human genetics: EJHG
[Show abstract][Hide abstract] ABSTRACT: In mammals, both the maternal and paternal genomes are necessary for normal embryogenesis due to parent-specific epigenetic modification of the genome during gametogenesis, which leads to non-equivalent expression of imprinted genes from the maternal and paternal alleles. In this study, we identified a paternally expressed imprinted gene, Zdbf2, by microarray-based screening using parthenogenetic and normal embryos. Expression analyses showed that Zdbf2 was paternally expressed in various embryonic and adult tissues, except for the placenta and adult testis, which showed biallelic expression of the gene. We also identified a differentially methylated region (DMR) at 10 kb upstream of exon 1 of the Zdbf2 gene and this differential methylation was derived from the germline. Furthermore, we also identified that the human homolog (ZDBF2) of the mouse Zdbf2 gene showed paternal allele-specific expression in human lymphocytes but not in the human placenta. Thus, our findings defined mouse chromosome 1 and human chromosome 2 as the loci for imprinted genes.
[Show abstract][Hide abstract] ABSTRACT: The Xenopus adult limb has very limited regeneration ability, and only a simple cartilaginous spike structure without digits is formed after limb amputation. We found that expression of Shh and its downstream genes is absent from the regenerating blastema of the Xenopus froglet limb. Moreover, we found that a limb enhancer region of the Shh gene is highly methylated in the froglet, although the sequence is hypomethylated in the Xenopus tadpole, which has complete limb regeneration ability. These findings, together with the fact that the promoter region of Shh is hardly methylated in Xenopus, suggest that regenerative failure (deficiency in repatterning) in the Xenopus adult limb is associated with methylation status of the enhancer region of Shh and that a target-specific epigenetic regulation is involved in gene re-activation for repatterning during the Xenopus limb regeneration process. Because the methylation level of the enhancer region was low in other amphibians that have Shh expression in the blastemas, a low methylation status may be the basic condition under which transcriptional regulation of Shh expression can progress during the limb regeneration process. These findings provide the first evidence for a relationship between epigenetic regulation and pattern formation during organ regeneration in vertebrates.
Full-text · Article · Jan 2008 · Developmental Biology
[Show abstract][Hide abstract] ABSTRACT: Recent studies suggest that assisted reproductive technologies (ART), which involve the isolation, handling and culture of gametes and early embryos, are associated with an increased incidence of rare imprinting disorders. Major epigenetic events take place during this time and the process of ART may expose the epigenome to external influences, preventing the proper establishment and maintenance of genomic imprints. However, the risks of ART cannot be simply evaluated because the patients who receive ART may differ both demographically and genetically from the general population at reproductive age. In this study, we examined the DNA methylation status of seven imprinted genes using a combined bisulphite-PCR restriction analysis and sequencing technique on sperm DNA obtained from 97 infertile men. We found an abnormal paternal methylation imprint in 14 patients (14.4%) and abnormal maternal imprint in 20 patients (20.6%). The majority of these doubly defective samples were in men with moderate or severe oligospermia. These abnormalities were specific to imprinted loci as we found that global DNA methylation was normal in these samples. The outcome of ART with sperm shown to have an abnormal DNA methylation pattern was generally poor. However, one sample of sperm with both paternal and maternal methylation errors used in ICSI produced a child of normal appearance without any abnormalities in their imprinted methylation pattern. Our data suggest that sperm from infertile patients, especially those with oligospermia, may carry a higher risk of transmitting incorrect primary imprints to their offspring, highlighting the need for more research into ART.
Full-text · Article · Dec 2007 · Human Molecular Genetics