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Single Cell Sequencing Identifies Key Epigenetic Regulators in Nuclear Transfer Mediated Reprogramming [RNA-seq]

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE70605 | GEO | 2016/05/17

SECONDARY ACCESSION(S): PRJNA289146

REPOSITORIES: GEO

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Single Cell Sequencing Identifies Key Epigenetic Regulators in Nuclear Transfer Mediated Reprogramming [ChIP-seq]

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.

2016-05-17 | E-GEOD-70606 | biostudies-arrayexpress

Single Cell Sequencing Identifies Key Epigenetic Regulators in Nuclear Transfer Mediated Reprogramming [RRBS]

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. For SCNT embryos 4-8 replicates were performed for each stage . As the control, 3 replicates were performed for each stage of wild type samples

2016-05-17 | E-GEOD-70607 | biostudies-arrayexpress

Single Cell Sequencing Identifies Key Epigenetic Regulators in Nuclear Transfer Mediated Reprogramming [RNA-seq]

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. For SCNT embryos or injected SCNT embryos 3-8 replicates were performed for each stage . As the control, 3-6 replicates were performed for each stage of wild type samples

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Single Cell Sequencing Identifies Key Epigenetic Regulators in Nuclear Transfer Mediated Reprogramming [RRBS]

2016-05-17 | GSE70607 | GEO

Single Cell Sequencing Identifies Key Epigenetic Regulators in Nuclear Transfer Mediated Reprogramming [ChIP-seq]

2016-05-17 | GSE70606 | GEO

Inhibition of aberrant DNA re-methylation improves development of cloned embryos (WGBS-Seq)

Project description:Somatic cell nuclear transfer (SCNT) enables the genome of a differentiated somatic cell to be reprogrammed to totipotency. However, this process is extremely inefficient, and the underlying mechanism of the epigenetic rearrangements following SCNT remains largely unknown. Here, we generated a genome-wide DNA methylome of mouse SCNT preimplantation embryos. Surprisingly, we identified widespread re-methylated regions (rDMRs) in 2- to 4-cell stage cloned embryos, which caused mis-expression of genes and retrotransposons important for zygotic genome activation and embryo development. Knocking-down DNA methyltransferases can specifically rescue the re-methylation defects of SCNT embryos and evidently improve the poor developmental capacity of cloned embryos. In addition, inactivation of DNA methyltransferases combined with overexpression of histone demethylases led to a more significant reduction in DNA methylation as well as exhibited a synergistic enhancement effect on the full-term development of nuclear transfer embryos. Our study therefore reveals that aberrant re-methylation functions as an unavoidable barrier for SCNT embryo development, and that the removal of multiple epigenetic barriers would be a promising approach to achieve the highest cloning efficiency.

2018-08-14 | GSE116103 | GEO

Inhibition of aberrant DNA re-methylation improves development of cloned embryos (RNA-Seq data set 2)

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Inhibition of aberrant DNA re-methylation improves development of cloned embryos (RNA-Seq data set 1)

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Inhibition of aberrant DNA re-methylation improves development of cloned embryos (RRBS-Seq)

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RNA sequencing revealed abnormal gene expression and benefits of vitamin C treatment in cloned bovine embryos

Project description:Somatic cell nuclear transfer (SCNT) in mammals has been mostly unsuccessful. In this study, we measured and compared transcription profiles of cloned and fertilized embryos using RNA sequencing. Before zygotic genome activation (ZGA), fewer genes were activated in the cloned embryos than in vitro fertilization (IVF), leading to insufficient activation of protein transport and degradation functions. In blastocysts, terms of “embryo development” and “adherens junction” represented major differences for repressed genes in SCNT compared to IVF. Vitamin C (Vc) was found to relax donor cell chromatin and promote cloned embryo development. RNA sequencing indicated that Vc treatment restored some abnormal genes and processes in the cloned embryos. Processes of autophagy, RNA-editing and cell junction were not fully established, but they were improved by Vc treatment. Overexpression of ZNF641, one of the completely restored genes by Vc treatment, improved the cloned embryos’ potential. Finally, Vc treatment rescued some long non-coding RNAs that were aberrantly expressed in the cloned embryos. Collectively, many important genes and biological processes were abnormal in the cloned embryos, some of which can be improved by Vc treatment. This investigation provides several practical aspects that could be useful for improving cloning efficiency and its future applications.

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