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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Single Cell Sequencing Identifies Key Epigenetic Regulators in Nuclear Transfer Mediated Reprogramming [ChIP-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. Examination of histone H3K9me3 modifications in 2-cell embryos and cumulus cells.

ORGANISM(S): Mus musculus

SUBMITTER: Shaorong Gao

PROVIDER: E-GEOD-70606 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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

2016-05-17 | E-GEOD-70605 | biostudies-arrayexpress

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

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

2016-05-17 | GSE70605 | GEO

Identification of somatic memory genes during bovine nuclear reprogramming by transcriptome sequencing

Project description:We hypothesized aberrant transcriptional silence was existed in bovine clone reprogramming as well as abnormal transcriptional activation and the result of RNA-seq confirmed this hypothesis. On the one hand, SCNT (somatic cell nuclear transfer) embryos exhibited excessive RNA processing and translation while these two biological processes were deficient in human and mouse; on the other hand, SCNT embryos exhibited the transcriptional defects of reproduction-related genes. These results proved the existences of active- and silent-memory genes which inherited from donor cells in bovine early SCNT embryos. Then, H3K4me3-specific demethylase 5B (KDM5B) mRNAs were injected into cloned embryos to erase active or silent memory respectively. KDM5B overexpression not only reduced the transcription level of active-memory genes but also promoted the expression of silent-memory genes, especially rescued multiple development-related genes.

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Expression profiling reveals differential expressed genes between bovine IVF and SCNT embryos

Project description:Global hypermethylations of histone H3 lysine 9 (H3K9) tri- and di-methylation (H3K9me3/2) have been identified in bovine cloned embryos at the embryonic genome activation (EGA) stage (eight-cell stage), but the intrinsic reason for these anomalies remains elusive. To ascertain key factors responsible for aberrant H3K9 methylation, we performed RNA sequencing of transcripts in eight-cell bovine in vitro fertilized (IVF) and SCNT embryo. From the differentially expressed genes (DEGs) between IVF and SCNT embryos, we identified that the unsuccessful reactivation of two histone demethylases, KDM4D and KDM4E, is responsible for the incomplete H3K9me3/2 demethylation in SCNT embryos at the EGA stage. By mRNA injection, ectopic expression of either KDM4D or KDM4E could erase H3K9me3/2 barriers, improve blastocyst formation, and elevate cloning efficiency of bovine SCNT. To examine the detailed genes responsive to KDM4E overexpression, we also performed RNA sequencing of bovine eight-cell SCNT embryos with KDM4E compensation and found an obvious restoration of global transcriptional patterns in SCNT embryos. Our study first provides the transcriptome data set of bovine IVF and SCNT embryos during EGA with or without KDM4E overexpression, which advance the understanding of incomplete nuclear reprogramming, and contribute to the practical implications for genetically modified livestock breeding using SCNT.

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Histone Demethylase-Assisted Somatic Cell Nuclear Transfer Facilitates Derivation of Human Pluripotent Stem Cells

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2016-03-01 | E-GEOD-73362 | biostudies-arrayexpress

Unreprogrammed H3K9me3 prevents minor zygotic genome activation and lineage commitment in SCNT embryos

Project description:Somatic cell nuclear transfer (SCNT) can be used to reprogram differentiated somatic cells to a totipotent state but has poor efficiency in supporting full-term development. H3K9me3 is considered to be an epigenetic barrier to zygotic genomic activation in 2-cell SCNT embryos. However, the mechanism underlying the failure of H3K9me3 reprogramming during SCNT embryo development remains elusive. Here, we perform genome-wide profiling of H3K9me3 in cumulus cell-derived SCNT embryos. We find redundant H3K9me3 marks are closely related to defective minor zygotic genome activation. Moreover, SCNT blastocysts show severely indistinct lineage-specific H3K9me3 deposition. We identify MAX and MCRS1 as potential H3K9me3-related transcription factors and are essential for early embryogenesis. Overexpression of Max and Mcrs1 significantly benefits SCNT embryo development. Notably, MCRS1 partially rescues lineage-specific H3K9me3 allocation, and further improves the efficiency of full-term development. Importantly, our data confirm the conservation of deficient H3K9me3 differentiation in Sertoli cell-derived SCNT embryos, which may be regulated by alternative mechanisms.

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Unreprogrammed H3K9me3 prevents minor zygotic genome activation and lineage commitment in SCNT embryos

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