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A CRISPR/Cas9 screen in embryonic stem cells reveals that Mdm2 regulates totipotency exit [RNA-Seq]

ABSTRACT: During early embryonic development, the transition from totipotency to pluripotency is a fundamental and critical process for proper development. However, the regulatory mechanisms governing this transition remain elusive. In this study, we conducted a comprehensive genome-wide CRISPR/Cas9 screen to investigate the 2-cell-like cells (2CLCs) phenotype in mouse embryonic stem cells (mESCs). This effort led to the identification of ten regulators that play a pivotal role in determining cell fate during this transition. Notably, our study revealed Mdm2 as a significant negative regulator of 2CLCs, as perturbation of Mdm2 resulted in a higher proportion of 2CLCs. Mdm2 appears to influence cell fate through its impact on cell cycle progression and H3K27me3 epigenetic modifications. In summary, the results of our CRISPR/Cas9 screen have uncovered several genes with distinct functions in regulating totipotency and pluripotency at various levels, offering a valuable resource for potential targets in future molecular studies.

ORGANISM(S): Mus musculus

PROVIDER: GSE269168 | GEO | 2024/07/01

REPOSITORIES: GEO

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A CRISPR/Cas9 screen in embryonic stem cells reveals that Mdm2 regulates totipotency exit [CUT&Tag]

Project description:During early embryonic development, the transition from totipotency to pluripotency is a fundamental and critical process for proper development. However, the regulatory mechanisms governing this transition remain elusive. In this study, we conducted a comprehensive genome-wide CRISPR/Cas9 screen to investigate the 2-cell-like cells (2CLCs) phenotype in mouse embryonic stem cells (mESCs). This effort led to the identification of ten regulators that play a pivotal role in determining cell fate during this transition. Notably, our study revealed Mdm2 as a significant negative regulator of 2CLCs, as perturbation of Mdm2 resulted in a higher proportion of 2CLCs. Mdm2 appears to influence cell fate through its impact on cell cycle progression and H3K27me3 epigenetic modifications. In summary, the results of our CRISPR/Cas9 screen have uncovered several genes with distinct functions in regulating totipotency and pluripotency at various levels, offering a valuable resource for potential targets in future molecular studies.

2024-07-01 | GSE269169 | GEO

A genome-wide screen reveals new regulators of the 2-cell-like cell state

Project description:In mammals, only the zygote and blastomeres of the early embryo are totipotent. This totipotency is mirrored in vitro by mouse '2-cell-like cells' (2CLCs), which appear at low frequency in cultures of embryonic stem cells (ESCs). Because totipotency is not completely understood, we carried out a genome-wide CRISPR knockout screen in mouse ESCs, searching for mutants that reactivate the expression of Dazl, a gene expressed in 2CLCs. Here we report the identification of four mutants that reactivate Dazl and a broader 2-cell-like signature: the E3 ubiquitin ligase adaptor SPOP, the Zinc-Finger transcription factor ZBTB14, MCM3AP, a component of the RNA processing complex TREX-2, and the lysine demethylase KDM5C. All four factors function upstream of DPPA2 and DUX, but not via p53. In addition, SPOP binds DPPA2, and KDM5C interacts with ncPRC1.6 and inhibits 2CLC gene expression in a catalytic-independent manner. These results extend our knowledge of totipotency, a key phase of organismal life.

2023-03-10 | GSE221710 | GEO

Myc and Dnmt1 impede the pluripotent to totipotent state transition in embryonic stem cell [CRISPR-Cas9 screen]

Project description:Totipotency is defined as the ability of a cell to generate all the cell types of an organism, including those of the extraembryonic tissues. Unlike pluripotency, the molecular features and the establishment of totipotency are poorly understood. In mouse embryonic stem cell (ESC) culture, a small percentage of cells transits into a totipotent state by expressing a group of genes that are only expressed in 2-cell-stage embryos. To understand how this transition takes place, we performed single cell RNA-seq analysis which revealed a two-step transcriptional reprogramming process characterized by downregulation of pluripotent genes in the first step and upregulation of the 2-cell embryo-specific genes in the second step. To identify factors controlling the transition process, we performed a CRISPR/Cas9-mediated genetic screen which revealed Myc and Dnmt1 as two factors preventing the transition. Mechanistic studies demonstrate that Myc prevents down-regulation of the genes in the first step, while Dnmt1 impedes gene activation in the second step. Collectively, our study reveals insights into the mechanism underlying establishment and regulation of totipotent state in ESCs.

2019-04-12 | GSE128307 | GEO

Myc and Dnmt1 impede the pluripotent to totipotent state transition in embryonic stem cell [RNA-Seq]

2019-04-12 | GSE121451 | GEO

Myc and Dnmt1 impede the pluripotent to totipotent state transition in embryonic stem cell [RRBS]

2019-04-12 | GSE121458 | GEO

Myc and Dnmt1 impede the pluripotent to totipotent state transition in embryonic stem cell [Drop-seq]

2019-04-12 | GSE121457 | GEO

DUX-miR-344-ZMYM2-mediated activation of MERVL LTRs induces a totipotent 2C-like state [LSD1 ChIP-seq]

Project description:Mouse embryonic stem cells (ESCs) sporadically express preimplantation two-cell-stage (2C) transcripts, including MERVL endogenous retrovirus and Zscan4 cluster genes. Such 2C-like cells (2CLCs) can contribute to both embryonic and extraembryonic tissues when reintroduced into early embryos. We examined global nucleosome occupancy and gene expression in 2CLCs and identified miR-344 as the noncoding molecule that positively controls 2CLC potency. We found that activation of endogenous MERVL or miR-344-2 alone is sufficient to induce 2CLCs with induction of 2C genes and an expanded potency. Mechanistically, miR-344 is activated by the 2C-state driver DUX and post-transcriptionally represses ZMYM2 and LSD1, which recruit the HDAC corepressor to MERVL LTR for transcriptional repression. Consistently, zygotic depletion of Zmym2 compromises the totipotency-to-pluripotency transition during early development. Our studies establish the novel DUXmiR-344--|Zmym2/LsdSD1 axis that controls MERVL for expanded stem cell potency.

2020-02-06 | GSE142280 | GEO

DUX-miR-344-ZMYM2-mediated activation of MERVL LTRs induces a totipotent 2C-like state [ZMYM2 ChIP-seq]

Project description:Mouse embryonic stem cells (ESCs) sporadically express preimplantation two-cell-stage (2C) transcripts, including MERVL endogenous retrovirus and Zscan4 cluster genes. Such 2C-like cells (2CLCs) can contribute to both embryonic and extraembryonic tissues when reintroduced into early embryos. We examined global nucleosome occupancy and gene expression in 2CLCs and identified miR-344 as the noncoding molecule that positively controls 2CLC potency. We found that activation of endogenous MERVL or miR-344-2 alone is sufficient to induce 2CLCs with induction of 2C genes and an expanded potency. Mechanistically, miR-344 is activated by the 2C-state driver DUX and post-transcriptionally represses ZMYM2 and LSD1, which recruit the HDAC corepressor to MERVL LTR for transcriptional repression. Consistently, zygotic depletion of Zmym2 compromises the totipotency-to-pluripotency transition during early development. Our studies establish the novel DUX->miR-344--|Zmym2/Lsd1 axis that controls MERVL for expanded stem cell potency.

2020-02-06 | GSE119818 | GEO

DUX-miR-344-ZMYM2-mediated activation of MERVL LTRs induces a totipotent 2C-like state [ATAC-seq]

Project description:Mouse embryonic stem cells (ESCs) sporadically express preimplantation two-cell-stage (2C) transcripts, including MERVL endogenous retrovirus and Zscan4 cluster genes. Such 2C-like cells (2CLCs) can contribute to both embryonic and extraembryonic tissues when reintroduced into early embryos. We examined global nucleosome occupancy and gene expression in 2CLCs and identified miR-344 as the noncoding molecule that positively controls 2CLC potency. We found that activation of endogenous MERVL or miR-344-2 alone is sufficient to induce 2CLCs with induction of 2C genes and an expanded potency. Mechanistically, miR-344 is activated by the 2C-state driver DUX and post-transcriptionally represses ZMYM2 and LSD1, which recruit the HDAC corepressor to MERVL LTR for transcriptional repression. Consistently, zygotic depletion of Zmym2 compromises the totipotency-to-pluripotency transition during early development. Our studies establish the novel DUX->miR-344--|Zmym2/Lsd1 axis that controls MERVL for expanded stem cell potency.

2020-02-06 | GSE119817 | GEO

DUX-miR-344-ZMYM2-mediated activation of MERVL LTRs induces a totipotent 2C-like state [RNA-seq]

Project description:Mouse embryonic stem cells (ESCs) sporadically express preimplantation two-cell-stage (2C) transcripts, including MERVL endogenous retrovirus and Zscan4 cluster genes. Such 2C-like cells (2CLCs) can contribute to both embryonic and extraembryonic tissues when reintroduced into early embryos. We examined global nucleosome occupancy and gene expression in 2CLCs and identified miR-344 as the noncoding molecule that positively controls 2CLC potency. We found that activation of endogenous MERVL or miR-344-2 alone is sufficient to induce 2CLCs with induction of 2C genes and an expanded potency. Mechanistically, miR-344 is activated by the 2C-state driver DUX and post-transcriptionally represses ZMYM2 and LSD1, which recruit the HDAC corepressor to MERVL LTR for transcriptional repression. Consistently, zygotic depletion of Zmym2 compromises the totipotency-to-pluripotency transition during early development. Our studies establish the novel DUX->miR-344--|Zmym2/Lsd1 axis that controls MERVL for expanded stem cell potency.

2020-02-06 | GSE119819 | GEO

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