Bourc'his D, et al reported on Dnmt3L and the establishment of maternal genomic imprints.
Complementary sets of genes are epigenetically silenced in male and female gametes in a process
termed genomic imprinting. The Dnmt3L gene is expressed during gametogenesis at stages where
genomic imprints are established. Targeted disruption of Dnmt3L caused azoospermia in homozygous
males, and heterozygous progeny of homozygous females died before midgestation. Bisulfite genomic
sequencing of DNA from oocytes and embryos showed that removal of Dnmt3L prevented
methylation of sequences that are normally maternally methylated. The defect was specific to
imprinted regions, and global genome methylation levels were not affected. Lack of maternal
methylation imprints in heterozygous embryos derived from homozygous mutant oocytes caused
biallelic expression of genes that are normally expressed only from the allele of paternal origin. The
key catalytic motifs characteristic of DNA cytosine methyltransferases have been lost from Dnmt3L,
and the protein is more likely to act as a regulator of imprint establishment than as a DNA
methyltransferase.
NCBI Summary:
CpG methylation is an epigenetic modification that is important for embryonic development, imprinting, and X-chromosome inactivation. Studies in mice have demonstrated that DNA methylation is required for mammalian development. This gene encodes a nuclear protein with similarity to DNA methyltransferases. This protein is not thought to function as a DNA methyltransferase as it does not contain the amino acid residues necessary for methyltransferase activity. However, this protein does stimulate de novo methylation by DNA cytosine methyltransferase 3 alpha and it is thought to be required for the establishment of maternal genomic imprints. This protein also mediates transcriptional repression through interaction with histone deacetylase 1. Alternative splicing results in two transcript variants. An additional splice variant has been described but its biological validity has not been determined.
General function
Oncogenesis
, Epigenetic modifications
Comment
This protein is not thought to function as a DNA methyltransferase as it does not contain the amino acid residues necessary for methyltransferase activity. However, this protein does stimulate de novo methylation by DNA cytosine methyltransferase 3 alpha and it is thought to be required for the establishment of maternal genomic imprints.
Dynamic CpG island methylation landscape in oocytes and preimplantation embryos. Smallwood SA et al. Elucidating how and to what extent CpG islands (CGIs) are methylated in germ cells is essential to understand genomic imprinting and epigenetic reprogramming. Here we present, to our knowledge, the first integrated epigenomic analysis of mammalian oocytes, identifying over a thousand CGIs methylated in mature oocytes. We show that these CGIs depend on DNMT3A and DNMT3L but are not distinct at the sequence level, including in CpG periodicity. They are preferentially located within active transcription units and are relatively depleted in H3K4me3, supporting a general transcription-dependent mechanism of methylation. Very few methylated CGIs are fully protected from post-fertilization reprogramming but, notably, the majority show incomplete demethylation in embryonic day (E) 3.5 blastocysts. Our study shows that CGI methylation in gametes is not entirely related to genomic imprinting but is a strong factor in determining methylation status in preimplantation embryos, suggesting a need to reassess mechanisms of post-fertilization demethylation.
Bovine DNA Methylation Imprints Are Established in an Oocyte Size-Specific Manner, Which Are Coordinated with the Expression of the DNMT3 Family Proteins. O'Doherty AM et al. A subset of genes, known as imprinted genes, is present in the mammalian genome. Genomic imprinting governs the monoallelic expression of these genes depending on whether the gene was inherited from the sperm or the egg. This parent-of-origin specific gene expression is generally dependent on the epigenetic modification, DNA methylation, and the DNA methylation status of CpG dinucleotides residing in loci known as differentially methylated regions (DMRs). The enzymatic machinery responsible for the addition of methyl (-CH(3)) groups to the cytosine residue in the CpG dinucleotides are known as the DNA methyltransferases (DNMTs). Correct establishment and maintenance of methylation patterns at imprinted genes has been associated with placental function and regulation of embryonic/fetal development. Much work has been carried out on imprinted genes in mouse and human, however little is known about the methylation dynamics in the bovine oocyte. The primary objective of the present study was to characterize the establishment of methylation at maternally imprinted genes in bovine growing oocytes and to determine if the expression of the bovine DNA methyltransferases, DNMT3A, DNMT3B, and DNMT3L, is coordinated with DNA methylation during oocyte development. To this end a panel of maternally imprinted genes was selected (SNRPN, MEST, IGF2R, PEG10, and PLAGL1), putative DMRs for MEST, IGF2R, PEG10, and PLAGL1 were identified within the 5' regions for each gene, the SNRPN DMR has been reported previously. Conventional bisulfite sequencing (BiS) revealed that methylation marks were acquired, at all five DMRs investigated, in an oocyte size dependent fashion. This was confirmed for a selection of genes using pyrosequencing analysis (PyS). Furthermore, mRNA expression and protein analysis revealed that the DNA methyltransferases, DNMT3A, DNMT3B, and DNMT3L, are also present in the bovine oocyte during its growth phase. This study demonstrates for the first time that an increase in bovine imprinted gene DMR methylation occurs during oocyte growth, as is observed in mouse.
Cellular localization
Nuclear
Comment
Ovarian function
Early embryo development
Comment
Genetic evidence for Dnmt3a-dependent imprinting during oocyte growth obtained by conditional knockout with Zp3-Cre and complete exclusion of Dnmt3b by chimera formation. Kaneda M et al. 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.
Expression regulated by
Comment
DNA methylation plays an important role in promoter choice and protein production at the mouse Dnmt3L locus. O'Doherty AM et al. The DNA methyltransferase 3-like (Dnmt3L) protein is a crucial cofactor in the germ line for the de novo methyltransferase Dnmt3a, which establishes imprints and represses transposable elements. We have previously shown that Dnmt3L transcription is regulated via three different promoters in mice, producing transcripts we term Dnmt3L(s) (stem cell), Dnmt3L(o) (oocyte) and Dnmt3L(at) (adult testis). Here we show that both Dnmt3L(s) and Dnmt3L(o) produce full-length proteins but that the Dnmt3L(at) transcripts are not translated. Although not a canonical CpG island, the Dnmt3L(s) promoter is silenced by methylation during somatic differentiation in parallel with germ-cell-specific genes. During oocyte growth, Dnmt3L(s) also becomes heavily methylated and silenced and this requires its own gene product, since there is complete loss of methylation and derepression of transcription from this promoter in oocytes derived from Dnmt3L(-/-) mice. Methylation of the Dnmt3L(s) promoter is established prior to the completion of imprinting and explains the requirement in mouse oocytes for the Dnmt3L(o) promoter, located in an intron of the neighboring unmethylated Aire gene. Overall these results give insight into how and why promoter switching at the mouse Dnmt3L locus occurs and provide one of the first examples of a non-imprinted locus where methylation plays a role in promoter choice. The derepression of the Dnmt3L(s) promoter in the knockout oocytes also suggests that other non-imprinted loci may be dysregulated in these cells and contribute to the phenotype of the resultant mice.
Ovarian localization
Primordial Germ Cell, Oocyte
Comment
Sex-specific promoters regulate Dnmt3L expression in mouse germ cells. Shovlin TC et al. BACKGROUND: Dnmt3L, a member of the DNA methyltransferase 3 family, lacks enzymatic activity but is required for de-novo methylation of imprinted genes in oocytes and for transposon repression in male germ cells. METHODS: We used northern blots, RT-PCR, 5' rapid amplification of complementary DNA (cDNA) ends (RACE), RNase H mapping, real-time/quantitative RT-PCR and in situ hybridization to identify and characterize Dnmt3L transcripts produced during germ cell development. RESULTS: Mouse Dnmt3L uses three sex-specific promoters, not the single promoter previously thought. A promoter active in prospermatogonia drives transcription of an mRNA encoding the full-length protein in perinatal testis, where de-novo methylation occurs. Late pachytene spermatocytes activate a second promoter in intron 9 of the Dnmt3L gene. After this stage, the predominant transcripts are three truncated mRNAs, which appear to be non-coding. We could also detect similar adult testis transcripts in humans. In the mouse ovary, an oocyte-specific promoter located in an intron of the neighbouring autoimmune regulator (Aire) gene produces a transcript with the full open reading frame (ORF). This is the only Dnmt3L transcript found in growing oocytes and is absent in the oocytes of Dnmt3L-/- females. CONCLUSIONS: Sex-specific promoters control Dnmt3L expression in the mouse germ line, mirroring the situation at the Dnmt1 and Dnmt3A loci.