Hatsune Chiba

Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes.

RNA interference (RNAi) is a mechanism by which double-stranded RNAs (dsRNAs) suppress specific transcripts in a sequence-dependent manner. dsRNAs are processed by Dicer to 21–24-nucleotide small interfering RNAs (siRNAs) and then incorporated into the argonaute (Ago) proteins. Gene regulation by endogenous siRNAs has been observed only in organisms possessing RNA-dependent RNA polymerase (RdRP). In mammals, where no RdRP activity has been found, biogenesis and function of endogenous siRNAs remain largely unknown. Here we show, using mouse oocytes, that endogenous siRNAs are derived from naturally occurring dsRNAs and have roles in the regulation of gene expression. By means of deep sequencing, we identify a large number of both ∼25–27-nucleotide Piwi-interacting RNAs (piRNAs) and ∼21-nucleotide siRNAs corresponding to messenger RNAs or retrotransposons in growing oocytes. piRNAs are bound to Mili and have a role in the regulation of retrotransposons. siRNAs are exclusively mapped to retrotransposons or other genomic regions that produce transcripts capable of forming dsRNA structures. Inverted repeat structures, bidirectional transcription and antisense transcripts from various loci are sources of the dsRNAs. Some precursor transcripts of siRNAs are derived from expressed pseudogenes, indicating that one role of pseudogenes is to adjust the level of the founding source mRNA through RNAi. Loss of Dicer or Ago2 results in decreased levels of siRNAs and increased levels of retrotransposon and protein-coding transcripts complementary to the siRNAs. Thus, the RNAi pathway regulates both protein-coding transcripts and retrotransposons in mouse oocytes. Our results reveal a role for endogenous siRNAs in mammalian oocytes and show that organisms lacking RdRP activity can produce functional endogenous siRNAs from naturally occurring dsRNAs.

Targets and dynamics of promoter DNA methylation during early mouse development

DNA methylation is extensively reprogrammed during the early phases of mammalian development, yet genomic targets of this process are largely unknown. We optimized methylated DNA immunoprecipitation for low numbers of cells and profiled DNA methylation during early development of the mouse embryonic lineage in vivo. We observed a major epigenetic switch during implantation at the transition from the blastocyst to the postimplantation epiblast. During this period, DNA methylation is primarily targeted to repress the germline expression program. DNA methylation in the epiblast is also targeted to promoters of lineage-specific genes such as hematopoietic genes, which are subsequently demethylated during terminal differentiation. De novo methylation during early embryogenesis is catalyzed by Dnmt3b, and absence of DNA methylation leads to ectopic gene activation in the embryo. Finally, we identify nonimprinted genes that inherit promoter DNA methylation from parental gametes, suggesting that escape of post-fertilization DNA methylation reprogramming is prevalent in the mouse genome.

Maternal and zygotic Dnmt1 are necessary and sufficient for the maintenance of DNA methylation imprints during preimplantation development

Parental origin-specific DNA methylation regulates the monoallelic expression of the mammalian imprinted genes. The methylation marks or imprints are established in the parental germline and maintained throughout embryonic development. However, it is unclear how the methylation imprints are maintained through extensive demethylation in cleavage-stage preimplantation embryos. Previous reports suggested that DNA methyltransferase(s) other than Dnmt1 is involved in the maintenance of the imprints during cleavage. Here we demonstrate, by using conditional knockout mice, that the other known DNA methyltransferases Dnmt3a and Dnmt3b are dispensable for the maintenance of the methylation marks at most imprinted loci. We further demonstrate that a lack of both maternal and zygotic Dnmt1 results in complete demethylation of all imprinted loci examined in blastocysts. Consistent with these results we find that zygotic Dnmt1 is expressed in the preimplantation embryo. Thus, contrary to the previous reports, Dnmt1 alone is sufficient to maintain the methylation marks of the imprinted genes.

Mouse Oocyte Methylomes at Base Resolution Reveal Genome-Wide Accumulation of Non-CpG Methylation and Role of DNA Methyltransferases

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.

Genome-Wide Analysis of DNA Methylation Dynamics during Early Human Development

DNA methylation is globally reprogrammed during mammalian preimplantation development, which is critical for normal development. Recent reduced representation bisulfite sequencing (RRBS) studies suggest that the methylome dynamics are essentially conserved between human and mouse early embryos. RRBS is known to cover 5–10% of all genomic CpGs, favoring those contained within CpG-rich regions. To obtain an unbiased and more complete representation of the methylome during early human development, we performed whole genome bisulfite sequencing of human gametes and blastocysts that covered>70% of all genomic CpGs. We found that the maternal genome was demethylated to a much lesser extent in human blastocysts than in mouse blastocysts, which could contribute to an increased number of imprinted differentially methylated regions in the human genome. Global demethylation of the paternal genome was confirmed, but SINE-VNTR-Alu elements and some other tandem repeat-containing regions were found to be specifically protected from this global demethylation. Furthermore, centromeric satellite repeats were hypermethylated in human oocytes but not in mouse oocytes, which might be explained by differential expression of de novo DNA methyltransferases. These data highlight both conserved and species-specific regulation of DNA methylation during early mammalian development. Our work provides further information critical for understanding the epigenetic processes underlying differentiation and pluripotency during early human development.

Genetic evidence for Dnmt3a-dependent imprinting during oocyte growth obtained by conditional knockout with Zp3-Cre and complete exclusion of Dnmt3b by chimera formation.

In the male and female germ‐lines of mice, both of the two de novo DNA methyltransferases Dnmt3a and Dnmt3b are expressed. By the conditional knockout experiments using the Tnap‐Cre gene, we previously showed that deletion of Dnmt3a in primordial germ cells disrupts paternal and maternal imprinting, however, Dnmt3b mutants did not show any defect. Here, we have knocked out Dnmt3a after birth in growing oocytes by using the Zp3‐Cre gene and obtained genetic evidence that de novo methylation by Dnmt3a during the oocyte growth stage is indispensable for maternal imprinting. We also carried out DNA methylation analysis in the mutant oocytes and embryos and found that hypomethylation of imprinted genes in Dnmt3a‐deficient oocytes was directly inherited to the embryos, but repetitive elements were re‐methylated during development. Furthermore, we show that Dnmt3b‐deficient cells can contribute to the male and female germ‐lines in chimeric mice and can produce normal progeny, establishing that Dnmt3b is dispensable for mouse gametogenesis and imprinting. Finally, Dnmt3‐related protein Dnmt3L is not only essential for methylation of imprinted genes but also enhances de novo methylation of repetitive elements in growing oocytes.

Re-investigation and RNA sequencing-based identification of genes with placenta-specific imprinted expression

Within the vertebrate groups, only mammals are subject to a specialized epigenetic process termed genomic imprinting in which genes are preferentially expressed from one parental allele. Imprinted expression has been reported for >100 mouse genes and, for approximately one-quarter of these genes, the imprinted expression is specific to the placenta (or extraembryonic tissues). This seemingly placenta-specific imprinted expression has garnered much attention, as has the apparent lack of conserved imprinting between the human and mouse placenta. In this study, we used a novel approach to re-investigate the placenta-specific expression using embryo transfer and trophoblast stem cells. We analyzed 20 genes previously reported to show maternal allele-specific expression in the placenta, and only 8 genes were confirmed to be imprinted. Other genes were likely to be falsely identified as imprinted due to their relatively high expression in contaminating maternal cells. Next, we performed a genome-wide transcriptome assay and identified 133 and 955 candidate imprinted genes with paternal allele- and maternal allele-specific expression. Of those we analyzed in detail, 1/6 (Gab1) of the candidates for paternal allele-specific expression and only 1/269 (Ano1) candidates for maternal allele-specific expression were authentically imprinted genes. Imprinting of Ano1 and Gab1 was specific to the placenta and neither gene displayed allele-specific promoter DNA methylation. Imprinting of ANO1, but not GAB1, was conserved in the human placenta. Our findings impose a considerable revision of the current views of placental imprinting.

Imprinting methylation errors in ART

There has been an increase in incidence reports of rare imprinting disorders associated with assisted reproductive technology (ART). ART, including in vitro fertilization and intracytoplasmic sperm injections, is an important treatment for infertile people of reproductive age and increasingly produces children. The identification of epigenetic changes at imprinted loci in ART infants has led to the suggestion that ART techniques themselves may predispose embryos to acquire imprinting errors and diseases. In this review, we note that the particular steps of ART may be prone to induction of imprinting methylation errors during gametogenesis, fertilization and early embryonic development. In addition, we explain imprint-associated diseases and their causes. Moreover, from a Japanese nationwide epidemiological study of imprint-associated diseases, we determine their associations with ART. Epigenetic studies will be required to understand the pathogenesis, ART-related risk factor(s) and what precautions can be taken to prevent the occurrence of input methylation errors. We hope that the constitution of children born after each ART procedure will reveal the safest and most ethical approach to use, which will be invaluable for the future development of standard ART.

De novo DNA methylation independent establishment of maternal imprint on X chromosome in mouse oocytes

In female mouse embryos, the paternal X chromosome (Xp) is preferentially inactivated during preimplantation development and trophoblast differentiation. This imprinted X‐chromosome inactivation (XCI) is partly due to an activating imprint on the maternal X chromosome (Xm), which is set during oocyte growth. However, the nature of this imprint is unknown. DNA methylation is one candidate, and therefore we examined whether disruptions of the two de novo DNA methyltransferases in growing oocytes affect imprinted XCI. We found that accumulation of histone H3 lysine‐27 trimethylation, a hallmark of XCI, occurs normally on the Xp, and not on the Xm, in female blastocysts developed from the mutant oocytes. Furthermore, the allelic expression patterns of X‐linked genes including Xist and Tsix were unchanged in preimplantation embryos and also in the trophoblast. These results show that a maternal disruption of the DNA methyltransferases has no effect on imprinted XCI and argue that de novo DNA methylation is dispensable for Xm imprinting. This underscores the difference between imprinted XCI and autosomal imprinting. genesis 46:768–774, 2008.

RNA sequencing-based identification of aberrant imprinting in cloned mice

Animals cloned by somatic cell nuclear transfer (SCNT) provide a unique model for understanding the mechanisms of nuclear epigenetic reprogramming to a state of totipotency. Though many phenotypic abnormalities have been demonstrated in cloned animals, the underlying mechanisms are not well understood. In this study, we performed transcriptome-wide allelic expression analyses in brain and placental tissues of cloned mice. We found that Gab1, Sfmbt2 and Slc38a4 showed loss of imprinting in all cloned mice analyzed, which might be involved in placentomegaly of cloned mice. These three genes did not require de novo DNA methylation in growing oocytes for the establishment of imprinting, implying the involvement of a de novo DNA methylation-independent mechanism. Loss of Dlk1-Dio3 imprinting was also observed in nearly half of cloned mouse embryos and showed a strong correlation with embryonic lethality. Our findings are essential to understand the underlying mechanisms of developmental abnormalities of cloned animals. We also emphasize that particular attention should be paid to specific imprinted genes for therapeutic and agricultural applications of SCNT.