Dataset Information

Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR.

ABSTRACT: BACKGROUND:Zrsr1 is a paternally expressed imprinted gene located in the first intron of Commd1, and the Zrsr1 promoter resides in a differentially methylated region (DMR) that is maternally methylated in the oocyte. However, a mechanism for the establishment of the methylation has remained obscure. Commd1 is transcribed in the opposite direction to Zrsr1 with predominant maternal expression, especially in the adult brain. RESULTS:We found Commed1 transcribed through the DMR in the growing oocyte. Zrsr1-DMR methylation was abolished by the prevention of Commd1 transcription. Furthermore, methylation did not occur at the artificially unmethylated maternal Zrsr1-DMR during embryonic development when transcription through the DMR was restored in the zygote. Loss of methylation at the maternal Zrsr1-DMR resulted in biallelic Zrsr1 expression and reduced the extent of the predominant maternal expression of Commd1. CONCLUSIONS:These results indicate that the establishment of methylation at Zrsr1-DMR occurs in a transcription-dependent and oocyte-specific manner and caused Zrsr1 imprinting by repressing maternal expression. The predominant maternal expression of Commd1 is likely caused by transcriptional interference by paternal Zrsr1 expression.

SUBMITTER: Joh K

PROVIDER: S-EPMC5989421 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR.

Joh Keiichiro K Matsuhisa Fumikazu F Kitajima Shuji S Nishioka Kenichi K Higashimoto Ken K Yatsuki Hitomi H Kono Tomohiro T Koseki Haruhiko H Soejima Hidenobu H

Epigenetics & chromatin 20180606 1

<h4>Background</h4>Zrsr1 is a paternally expressed imprinted gene located in the first intron of Commd1, and the Zrsr1 promoter resides in a differentially methylated region (DMR) that is maternally methylated in the oocyte. However, a mechanism for the establishment of the methylation has remained obscure. Commd1 is transcribed in the opposite direction to Zrsr1 with predominant maternal expression, especially in the adult brain.<h4>Results</h4>We found Commed1 transcribed through the DMR in th ...[more]

PMID: 29875017

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Project description:BackgroundGametogenesis in mammals entails profound re-patterning of the epigenome. In the female germline, DNA methylation is acquired late in oogenesis from an essentially unmethylated baseline and is established largely as a consequence of transcription events. Molecular and functional studies have shown that imprinted genes become methylated at different times during oocyte growth; however, little is known about the kinetics of methylation gain genome wide and the reasons for asynchrony in methylation at imprinted loci.ResultsGiven the predominant role of transcription, we sought to investigate whether transcription timing is rate limiting for de novo methylation and determines the asynchrony of methylation events. Therefore, we generated genome-wide methylation and transcriptome maps of size-selected, growing oocytes to capture the onset and progression of methylation. We find that most sequence elements, including most classes of transposable elements, acquire methylation at similar rates overall. However, methylation of CpG islands (CGIs) is delayed compared with the genome average and there are reproducible differences amongst CGIs in onset of methylation. Although more highly transcribed genes acquire methylation earlier, the major transitions in the oocyte transcriptome occur well before the de novo methylation phase, indicating that transcription is generally not rate limiting in conferring permissiveness to DNA methylation. Instead, CGI methylation timing negatively correlates with enrichment for histone 3 lysine 4 (H3K4) methylation and dependence on the H3K4 demethylases KDM1A and KDM1B, implicating chromatin remodelling as a major determinant of methylation timing. We also identified differential enrichment of transcription factor binding motifs in CGIs acquiring methylation early or late in oocyte growth. By combining these parameters into multiple regression models, we were able to account for about a fifth of the variation in methylation timing of CGIs. Finally, we show that establishment of non-CpG methylation, which is prevalent in fully grown oocytes, and methylation over non-transcribed regions, are later events in oogenesis.ConclusionsThese results do not support a major role for transcriptional transitions in the time of onset of DNA methylation in the oocyte, but suggest a model in which sequences least dependent on chromatin remodelling are the earliest to become permissive for methylation.

Project description:BackgroundReproductive biology methods rely on in vitro follicle cultures from mature follicles obtained by hormonal stimulation for generating metaphase II oocytes to be fertilised and developed into a healthy embryo. Such techniques are used routinely in both rodent and human species. DNA methylation is a dynamic process that plays a role in epigenetic regulation of gametogenesis and development. In mammalian oocytes, DNA methylation establishment regulates gene expression in the embryos. This regulation is particularly important for a class of genes, imprinted genes, whose expression patterns are crucial for the next generation. The aim of this work was to establish an in vitro culture system for immature mouse oocytes that will allow manipulation of specific factors for a deeper analysis of regulatory mechanisms for establishing transcription regulation-associated methylation patterns.ResultsAn in vitro culture system was developed from immature mouse oocytes that were grown to germinal vesicles (GV) under two different conditions: normoxia (20% oxygen, 20% O2) and hypoxia (5% oxygen, 5% O2). The cultured oocytes were sorted based on their sizes. Reduced representative bisulphite sequencing (RRBS) and RNA-seq libraries were generated from cultured and compared to in vivo-grown oocytes. In the in vitro cultured oocytes, global and CpG-island (CGI) methylation increased gradually along with oocyte growth, and methylation of the imprinted genes was similar to in vivo-grown oocytes. Transcriptomes of the oocytes grown in normoxia revealed chromatin reorganisation and enriched expression of female reproductive genes, whereas in the 5% O2 condition, transcripts were biased towards cellular stress responses. To further confirm the results, we developed a functional assay based on our model for characterising oocyte methylation using drugs that reduce methylation and transcription. When histone methylation and transcription processes were reduced, DNA methylation at CGIs from gene bodies of grown oocytes presented a lower methylation profile.ConclusionsOur observations reveal changes in DNA methylation and transcripts between oocytes cultured in vitro with different oxygen concentrations and in vivo-grown murine oocytes. Oocytes grown under 20% O2 had a higher correlation with in vivo oocytes for DNA methylation and transcription demonstrating that higher oxygen concentration is beneficial for the oocyte maturation in ex vivo culture condition. Our results shed light on epigenetic mechanisms for the development of oocytes from an immature to GV oocyte in an in vitro culture model.

Project description:BackgroundDuring early mammalian development, DNA methylation undergoes two waves of reprogramming, enabling transitions between somatic cells, oocyte and embryo. The first wave of de novo DNA methylation establishment occurs in oocytes. Its molecular mechanisms have been studied in mouse, a classical mammalian model. Current model describes DNA methyltransferase 3A (DNMT3A) and its cofactor DNMT3L as two essential factors for oocyte DNA methylation-the ablation of either leads to nearly complete abrogation of DNA methylation. However, DNMT3L is not expressed in human oocytes, suggesting that the mechanism uncovered in mouse is not universal across mammals.ResultsWe analysed available RNA-seq data sets from oocytes of multiple mammals, including our novel data sets of several rodent species, and revealed that Dnmt3l is expressed only in the oocytes of mouse, rat and golden hamster, and at a low level in guinea pigs. We identified a specific promoter sequence recognised by an oocyte transcription factor complex associated with strong Dnmt3l activity and demonstrated that it emerged in the rodent clade Eumuroida, comprising the families Muridae (mice, rats, gerbils) and Cricetidae (hamsters). In addition, an evolutionarily novel promoter emerged in the guinea pig, driving weak Dnmt3l expression, likely without functional relevance. Therefore, Dnmt3l is expressed and consequently plays a role in oocyte de novo DNA methylation only in a small number of rodent species, instead of being an essential pan-mammalian factor. In contrast to somatic cells, where catalytically inactive DNMT3B interacts with DNMT3A, forming a heterotetramer, we did not find evidence for the expression of such inactive Dnmt3b isoforms in the oocytes of the tested species.ConclusionsThe analysis of RNA-seq data and genomic sequences revealed that DNMT3L is likely to play a role in oocytes de novo DNA methylation only in mice, rats, gerbils and hamsters. The mechanism governing de novo DNA methylation in the oocytes of most mammalian species, including humans, occurs through a yet unknown mechanism that differs from the current model discovered in mouse.

Project description:Genomic imprinting is the epigenetic marking of genes that results in parent-of-origin monoallelic expression. Most imprinted domains are associated with differentially DNA methylated regions (DMRs) that originate in the gametes, and are maintained in somatic tissues after fertilization. This allelic methylation profile is associated with a plethora of histone tail modifications that orchestrates higher order chromatin interactions. The mouse chromosome 15 imprinted cluster contains multiple brain-specific maternally expressed transcripts including Ago2, Chrac1, Trappc9 and Kcnk9 and a paternally expressed gene, Peg13. The promoter of Peg13 is methylated on the maternal allele and is the sole DMR within the locus. To determine the extent of imprinting within the human orthologous region on chromosome 8q24, a region associated with autosomal recessive intellectual disability, Birk-Barel mental retardation and dysmorphism syndrome, we have undertaken a systematic analysis of allelic expression and DNA methylation of genes mapping within an approximately 2 Mb region around TRAPPC9.Utilizing allele-specific RT-PCR, bisulphite sequencing, chromatin immunoprecipitation and chromosome conformation capture (3C) we show the reciprocal expression of the novel, paternally expressed, PEG13 non-coding RNA and maternally expressed KCNK9 genes in brain, and the biallelic expression of flanking transcripts in a range of tissues. We identify a tandem-repeat region overlapping the PEG13 transcript that is methylated on the maternal allele, which binds CTCF-cohesin in chromatin immunoprecipitation experiments and possesses enhancer-blocker activity. Using 3C, we identify mutually exclusive approximately 58 and 500 kb chromatin loops in adult frontal cortex between a novel brain-specific enhancer, marked by H3K4me1 and H3K27ac, with the KCNK9 and PEG13 promoters which we propose regulates brain-specific expression.We have characterised the molecular mechanism responsible for reciprocal allelic expression of the PEG13 and KCNK9 transcripts. Therefore, our observations may have important implications for identifying the cause of intellectual disabilities associated with the 8q24 locus.

Dataset Information

Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR.

Publications

Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR.

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