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Intrinsic de novo DNA methylation in early embryos enables the generation of semi-cloned mice from methylation-deficient androgenetic haploid stem cells

ABSTRACT: This SuperSeries is composed of the SubSeries listed below. Abstract Among all mammalian cell types, sperm cells exhibit one of the highest levels of DNA methylation, with ~80% of CpG sites being methylated. The role of this sperm hypermethylation in offspring development, aside from the methylation at the well-characterized H19-DMR and Dlk1-Dio3 intergenic germline DMR (IG-DMR) loci, remains largely unexplored. In mouse germ cells, removal of DNA methylation by deleting methyltransferase (Dnmt) genes causes meiotic catastrophe and infertility. To circumvent this limitation, we inactivated them instead in androgenetic haploid embryonic stem cells (AG-haESCs) lacking H19-DMR and IG-DMR to remove methylation prior to oocyte injection, and subsequently reactivated them in the resulting embryos during cleavage, to ensure Dnmt sufficiency. This strategy enabled the generation of viable offspring from unmethylated paternally-derived haploid cells. In the resulting embryos, the paternal genome rapidly reacquired methylation and was comparable to wild-type in post-implantation embryos. These semi-cloned embryos could develop to term and survived to adulthood, exhibiting near-normal morphological and physiological parameters. These findings indicate that the essential hereditary function of the paternal genome methylation is mainly restricted to the imprinted loci Igf2-H19 and Dlk1-Dio3, while methylation elsewhere appears dispensable for subsequent development, as de novo methylation activity intrinsic to the early embryo ensures epigenetic reprogramming for offspring development.

ORGANISM(S): Mus musculus

PROVIDER: GSE292524 | GEO | 2025/07/31

REPOSITORIES: GEO

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Viable semi-cloned mice derived from androgenetic haploid stem cells lacking DNA methylation [RNA-seq]

Project description:Among all mammalian cell types, sperm exhibit the highest level of DNA methylation, with approximately 70–80% of CpG sites being methylated. The role of this paternal hypermethylation in embryonic developmental competence, aside from the well-characterized H19-DMR and Dlk1-Dio3 intergenic germline DMR (IG-DMR) loci, remains largely unexplored. In mouse male germ cells, the lack of DNA methyltransferase (Dnmt) causes a loss of methylation, leading to meiotic catastrophe and infertility. To circumvent this limitation, we used sperm-like androgenetic haploid embryonic stem cells (AG-haESCs) for oocyte injection to produce offspring. Using CRISPR/Cas9, we effectively inactivated Dnmt1, Dnmt3a and Dnmt3b in AG-haESCs and later reactivated them in the resulting embryos, overcoming Dnmt haploinsufficiency and enabling the generation of viable offspring from methylation-deficient haploid cells. In the offspring embryos, the paternal genome derived from Dnmt-inactivated AG-haESCs rapidly reacquired methylation and restored a methylome in post-implantation embryos comparable to that of wild-type AG-haESC counterparts. These semi-cloned embryos could develop to term and survived to adulthood, exhibiting near-normal morphological and physiological parameters. These findings indicate that the functional significance of paternal genome methylation is mainly restricted to the imprinted loci Igf2-H19 and Dlk1-Dio3, while methylation of the paternal genome elsewhere appears dispensable for normal development.

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Viable semi-cloned mice derived from androgenetic haploid stem cells lacking DNA methylation [TAPS]

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Imprinted gene expression at the Dlk1-Dio3 cluster is controlled by both maternal and paternal IG-DMRs in a tissue-specific fashion.

Project description:Imprinting at the Dlk1-Dio3 cluster is controlled by the IG-DMR, an imprinting control region differentially methylated between maternal and paternal chromosomes. The maternal IG-DMR is essential for imprinting control, functioning as a cis enhancer element. Meanwhile, DNA methylation at the paternal IG-DMR is thought to prevent enhancer activity. To explore whether suppression of enhancer activity at the methylated IG-DMR requires the transcriptional repressor TRIM28, we analyzed Trim28chatwo embryos and performed epistatic experiments with IG-DMR deletion mutants. We found that while TRIM28 regulates the enhancer properties of the paternal IG-DMR, it also controls imprinting through other mechanisms. Additionally, we found that the paternal IG-DMR, previously deemed dispensable for imprinting, is required in certain tissues, demonstrating that imprinting is regulated in a tissue-specific manner. Using ChRO-seq to analyze nascent transcription, we show that different tissues have a distinctive regulatory landscape at the Dlk1-Dio3 cluster, providing insight into potential mechanisms of tissue-specific imprinting control. ChRO-seq identified 30 novel transcribed regulatory elements, including a candidate regulatory region that depends on the paternal IG-DMR. Together, our findings challenge the model that Dlk1-Dio3 imprinting is regulated through a single mechanism and demonstrate that different tissues use distinct strategies for imprinting control.

2019-02-28 | GSE119882 | GEO

RNA Seq analyzed the transcriptional difference between the ICSI and ICAHCI embryo in blastocyst and placenta

Project description:Our lab first derived mouse androgenetic haploid embryonic stem cells (AG-haESCs) and demonstrated that AG-haESCs can be used as an “artificial spermatids” to generate gene-edited semi-cloned (SC) mice through intracytoplasmic injection (ICAHCI) into mature oocyte, even though the birth efficiency is very low. Further we proved that H19-DMR and IG-DMR were the main barrier to generate viable mice through androgenetic and parthenogenetic haESCs. More importantly, AG-haESCs mediated SC technology combined with CRISPR-Cas9 is a powerful tool to generate gene-modified mouse models and carry out genetic screening at organismal level. However, it is still not clear how the H19-DMR and IG-DMR coordinately regulate SC embryo development. Here, we found that the H19-DMR and IG-DMR regulate the development of SC embryos in spatio-temporal scales. Firstly, we found that the H19-DMR and IG-DMR are not indispensable for the development of preimplantation of SC embryos. Secondly, H19-DMR is essential for the development of SC embryos in mid-gestation and IG-DMR takes effect in late-gestation. Further, the maintenance of paternal H19-DMR methylation status and deletion of paternal H19 transcription unit play a key role in the structures and transport functions of SC embryo placenta. Importantly, AG-haESCs carrying triple deletions, including H19, H19-DMR and IG-DMR, can further improve the efficiency in generation of viable, normal-size, and fertile mice.

2019-09-17 | GSE132254 | GEO

A bipartite element with allele-specific functions safeguards DNA methylation imprints at the Dlk1-Dio3 locus [4C-seq]

Project description:Dysregulation of imprinted gene loci also referred to as loss of imprinting (LOI) can result in severe developmental defects and other diseases, but the molecular mechanisms that ensure imprint stability remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) and the mechanism by which they ensure imprinting maintenance. Using pluripotent stem cells carrying an allele-specific reporter system, we demonstrate that the IG-DMR consists of two antagonistic regulatory elements: a paternally methylated CpG-island that prevents the activity of Tet dioxygenases and a maternally unmethylated regulatory element, which serves as a non-canonical enhancer and maintains expression of the maternal Gtl2 lncRNA by precluding de novo DNA methyltransferase function. Targeted genetic or epigenetic editing of these elements leads to LOI with either bi-paternal or bi-maternal expression patterns and respective allelic changes in DNA methylation and 3D chromatin topology of the entire Dlk1-Dio3 locus. Although the targeted repression of either IG-DMR or Gtl2 promoter is sufficient to cause LOI, the stability of LOI phenotype depends on the IG-DMR status, suggesting a functional hierarchy. These findings establish the IG-DMR as a novel type of bipartite control element and provide mechanistic insights into the control of Dlk1-Dio3 imprinting by allele-specific restriction of the active DNA (de)methylation machinery.

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Loss of imprinting at the CTCF binding sites in the DLK1-DIO3 domain reactivates microRNA expression in acute promyelocytic leukaemia

Project description:We report the DNA-methylation profiling of 10 regions selected from the DLK1-DIO3 domain on chromosome 14q32 in BM/PB samples from patients with acute promyelocytic leukaemia (APL), other subclasses of acute myeloid leukaemia and healthy donors, using high-throughput amplicon bisulfite sequencing with Roche 454 technology. We identify monoallelic-hypermethylation in APL only at the differentially methylated region (DMR) located upstream from the MEG3 gene (MEG3-DMR), whereas no changes in the DNA methylation profile were detected at the imprinting control region of the domain (IG-DMR) among the samples analysed. We show that the expression profile of 6 miRNAs clustered downstream from the MEG3-DMR correlates with the methylation profile at both DMRs. We demonstrate that miRNAs expression negatively correlates with DNA-methylation at the IG-DMR and MEG3 gene-body, whereas the correlation was positive for the CpGs located in the promoter of MEG3, including the binding sites for the insulator CTCF. We propose a loss of imprinting at the CTCF binding sites in patients with APL. These results are consistent with the previously reported DLK1-DIO3 miRNAs overexpression in APL, indicating a possible involvement of these ncRNAs in the pathogenesis of the disease. Investigation of the epigenetic regulation of the miRNAs clustered in 14q32 by next-generation sequencing

2014-02-13 | E-GEOD-42543 | biostudies-arrayexpress

Project description:IG-DMR hypomethylation in Dlk1-Dio3 imprint locus mediates offspring hippocampal neurogenesis

| PRJNA1045103 | ENA

Loss of imprinting at the CTCF binding sites in the DLK1-DIO3 domain reactivates microRNA expression in acute promyelocytic leukaemia

2014-02-13 | GSE42543 | GEO

Silencing of maternally expressed RNAs in Dlk1-Dio3 domain is critical for embryonic development

Project description:The mammalian imprinted Dlk1-Dio3 domain contains multiple lncRNAs, mRNAs, the largest miRNA cluster in the genome and four differentially methylated regions (DMRs), and deletion of maternal RNA within this locus results in embryonic lethality, but the mechanism by which this occurs is not clear. Here, we optimized the model of maternally expressed RNAs transcription termination in the domain and found that the cause of embryonic death was apoptosis in the embryo, particularly in the liver. We generated a mouse model of maternally expressed RNAs silencing in the Dlk1-Dio3 domain by inserting a 3×polyA termination sequence in Gtl2 locus. By analyzing mouse embryos RNA-Seq data combined with histological analysis, we found that silence of maternally expressed RNAs in the domain activated apoptosis, causing vascular rupture of fetal liver, resulting hemorrhage and injury. Mechanistically, termination of Gtl2 transcription results in the silencing of the maternally expressed RNAs and activation of the paternally expressed genes in the interval, and it is the gene itself rather than the IG-DMR and Gtl2-DMR that causes the above phenotypes. In conclusion, these findings illuminate a novel mechanism by which silencing of the maternally expressed RNAs within Dlk1-Dio3 domain leads to hepatic hemorrhage and embryonic death through activation of the apoptosis.

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