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Resolving multiple epigenetic barriers to pig SCNT-mediated reprogramming during embryonic genome activation

ABSTRACT: Pig cloning by somatic cell nuclear transfer (SCNT) frequently undergoes incomplete epigenetic remodeling during the maternal-to-zygotic transition, which leads to a significant embryonic loss before implantation. Here, we generated the first genome-wide landscapes of histone methylation in pig SCNT embryos. Excessive H3K9me3 and H3K27me3, but not H3K4me3, were observed in the genomic regions with unfaithful embryonic genome activation and donor cell-specific gene silencing. A combination of H3K9 demethylase KDM4A and GSK126, an inhibitor of H3K27me3 writer, could remove these epigenetic barriers and restore the global transcriptome in SCNT embryos. More importantly, TDG was defined as a pig-specific epigenetic regulator for nuclear reprogramming, which was not reactivated by H3K9me3 and H3K27me3 removal. Both combined treatment and transient TDG overexpression could promote DNA demethylation and enhance the blastocyst forming rates of SCNT embryos, which offers valuable methods to increase the cloning efficiency of genome-edited pigs for agricultural and biomedical purposes. This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Sus scrofa

PROVIDER: GSE161527 | GEO | 2021/09/21

REPOSITORIES: GEO

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Resolving multiple epigenetic barriers to pig SCNT-mediated reprogramming during embryonic genome activation [ChIP-Seq]

Project description:Pig cloning by somatic cell nuclear transfer (SCNT) frequently undergoes incomplete epigenetic remodeling during the maternal-to-zygotic transition, which leads to a significant embryonic loss before implantation. Here, we generated the first genome-wide landscapes of histone methylation in pig SCNT embryos. Excessive H3K9me3 and H3K27me3, but not H3K4me3, were observed in the genomic regions with unfaithful embryonic genome activation and donor cell-specific gene silencing. A combination of H3K9 demethylase KDM4A and GSK126, an inhibitor of H3K27me3 writer, could remove these epigenetic barriers and restore the global transcriptome in SCNT embryos. More importantly, TDG was defined as a pig-specific epigenetic regulator for nuclear reprogramming, which was not reactivated by H3K9me3 and H3K27me3 removal. Both combined treatment and transient TDG overexpression could promote DNA demethylation and enhance the blastocyst forming rates of SCNT embryos, which offers valuable methods to increase the cloning efficiency of genome-edited pigs for agricultural and biomedical purposes.

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SCNT embryos are defective for H3K27me3 imprinting [ChIP-seq]

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SCNT embryos are defective for H3K27me3 imprinting [RNA-seq]

Project description:Animal cloning can be achieved through somatic cell nuclear transfer (SCNT), yet the success rate remains very low. Recent studies have revealed two epigenetic barriers, H3K9me3 in donor cells and abnormal Xist activation, that impede SCNT reprogramming. Here we overcome both barriers by combining the use of Xist knockout donor cells and overexpressing Kdm4d, which allowed us to achieve the highest mouse cloning efficiency. However, SCNT-associated developmental defects and abnormal placenta were still observed, suggesting the existence of additional epigenetic defects in these SCNT embryos. Comparative DNA methylome analysis of IVF and SCNT blastocysts identified many abnormally methylated regions in SCNT embryos, despite successful global methylome reprogramming. Strikingly, allelic transcriptome analyses of SCNT blastocysts revealed a complete loss-of-imprinting at the H3K27me3-dependent imprinted genes, which may account for postimplantation developmental defects of SCNT embryos. This study thus not only provides the most efficient method for mouse cloning but also points the way for further improve SCNT cloning.

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Project description:Differentiated cell can be reprogrammed into totipotent embryo through somatic cell nuclear transfer (SCNT). However, this process is highly inefficient and most cloned embryos arrest at certain developmental stages. Through single cell sequencing combined with embryo biopsy, here we generate a global map of DNA methylome and RNA transcriptome for SCNT embryos with distinct developmental fates. We subsequently demonstrate that the unfaithful reactivation of two histone demethylases, Kdm4b and Kdm5b, accounts for the arrest of cloned embryos at 2-cell and 4-cell stage, respectively. Ectopic expression of Kdm4b and Kdm5b in SCNT can remove H3K9me3 barrier, restore the transcription profile and facilitate the blastocyst developmental efficiency over 95%. Moreover, these cloned embryos can further support full-term development and the derivation of SCNT-embryonic stem cells with greater efficiency. Our study reveals that histone methylation reset is crucial for the development of SCNT embryos, which provides a clue to further improve therapeutic cloning. Examination of histone H3K9me3 modifications in 2-cell embryos and cumulus cells.

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2018-01-16 | GSE99294 | GEO

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