Cell cycle heterogeneity directs spontaneous 2C state entry and exit in mouse embryonic stem cells (ChIP-Seq)
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ABSTRACT: Mouse embryonic stem cells (ESCs) show cell-to-cell heterogeneity. A small fraction of 2-cell-like cells (2CLCs) marked by endogenous retrovirus activation emerge spontaneously. The 2CLCs are instable and they transit back to the pluripotent state without extrinsic stimulus. To understand how this bidirectional transition takes place, we performed single-cell RNA-seq on isolated 2CLCs which underwent 2C-like state exit and re-entry, and revealed a novel circular transitional process between 2C-like and pluripotent states. Mechanistically, we found that the cell cycle played an important role in mediating these transitions by regulating assembly of nucleolus and peri-nucleolar heterochromatin to influence 2C gene Dux expression. Collectively, our findings provide a roadmap of the 2C-like state entry and exit in ESCs and also a causal role of the cell cycle in promoting these transitions.
Project description:Mouse embryonic stem cells (ESCs) show cell-to-cell heterogeneity. A small fraction of 2-cell-like cells (2CLCs) marked by endogenous retrovirus activation emerge spontaneously. The 2CLCs are instable and they transit back to the pluripotent state without extrinsic stimulus. To understand how this bidirectional transition takes place, we performed single-cell RNA-seq on isolated 2CLCs which underwent 2C-like state exit and re-entry, and revealed a novel circular transitional process between 2C-like and pluripotent states. Mechanistically, we found that the cell cycle played an important role in mediating these transitions by regulating assembly of nucleolus and peri-nucleolar heterochromatin to influence 2C gene Dux expression. Collectively, our findings provide a roadmap of the 2C-like state entry and exit in ESCs and also a causal role of the cell cycle in promoting these transitions.
Project description:In mouse embryonic stem cell (ESC) culture, a small proportion of cells display totipotent features by expressing a set of genes that are only active in 2-cell-stage embryos. These 2-cell-like (2C-like) cells spontaneously transit back into pluripotent state. We previously dissected the transcriptional dynamics of pluripotent to 2C-like transition and identified factors that modulate the transition. However, how 2C-like cells transits back to the pluripotent state and what factors drive this process remains largely unknown. To address these questions, we examined the transcriptional dynamics during the reverse transition from the 2C-like state to ESCs and identified an intermediate state involved in the transition. Interestingly, we found that mESCs exit from the 2C-like state through a molecular path characterized by a two-wave upregulation of pluripotent genes different from the one observed during the 2C-like entry transition. We also showed that nonsense-mediated mRNA decay (NMD) targets Dux mRNA and affects 2C-like state maintenance, suggesting that Dux degradation contributes to the reversal of 2C-like state.
Project description:In mouse embryonic stem cell (ESC) culture, a small proportion of cells display totipotent features by expressing a set of genes that are only active in 2-cell-stage embryos. These 2-cell-like (2C-like) cells spontaneously transit back into pluripotent state. We previously dissected the transcriptional dynamics of pluripotent to 2C-like transition and identified factors that modulate the transition. However, how 2C-like cells transits back to the pluripotent state and what factors drive this process remains largely unknown. To address these questions, we examined the transcriptional dynamics during the reverse transition from the 2C-like state to ESCs and identified an intermediate state involved in the transition. Interestingly, we found that mESCs exit from the 2C-like state through a molecular path characterized by a two-wave upregulation of pluripotent genes different from the one observed during the 2C-like entry transition. We also showed that nonsense-mediated mRNA decay (NMD) targets Dux mRNA and affects 2C-like state maintenance, suggesting that Dux degradation contributes to the reversal of 2C-like state.
Project description:Mouse embryonic stem cells (ESCs) consist of a rare population of heterogeneous cells which express 2-cell-stage-specific transcripts and are referred to as 2-cell-like cells (2CLCs). Accumulating evidence has demonstrated that transcription factors and epigenetic modifications exert crucial functions in the transition of ESCs to 2CLCs. However, the roles of RNA modification in the regulation of 2C-like state remain elusive. Using Dux-induced 2CLCs system, we examine N6-methyladenosine (m6A) modification landscape in transcriptome-wide, and find that m6A is dynamically regulated during Dux-driven 2C-like reprogramming. Intriguingly, many of 2C-specific transcripts are highly methylated, including Dux and Zscan4 cluster genes. We further identify the m6A reader protein Ythdf2 as a critical regulator of 2C-like state. Depletion of Ythdf2 facilitates robust expressions of 2C transcripts and the transition of ESCs to 2CLCs. Mechanically, Ythdf2 binds to the Dux/Zscan4 transcripts and promotes their degradation through recruiting the key component of RNA deadenylase complex, Cnot1. Consistent with the phenotype of Ythdf2 deficiency, silencing of Cnot1 induces the 2C gene expressions and the transition of ESCs into the 2C-like state. Collectively, our findings reveal novel insights into the epitranscriptomic regulation of the 2C-like state in mouse ESCs.
Project description:Mouse embryonic stem cells (ESCs) consist of a rare population of heterogeneous cells which express 2-cell-stage-specific transcripts and are referred to as 2-cell-like cells (2CLCs). Accumulating evidence has demonstrated that transcription factors and epigenetic modifications exert crucial functions in the transition of ESCs to 2CLCs. However, the roles of RNA modification in the regulation of 2C-like state remain elusive. Using Dux-induced 2CLCs system, we examine N6-methyladenosine (m6A) modification landscape in transcriptome-wide, and find that m6A is dynamically regulated during Dux-driven 2C-like reprogramming. Intriguingly, many of 2C-specific transcripts are highly methylated, including Dux and Zscan4 cluster genes. We further identify the m6A reader protein Ythdf2 as a critical regulator of 2C-like state. Depletion of Ythdf2 facilitates robust expressions of 2C transcripts and the transition of ESCs to 2CLCs. Mechanically, Ythdf2 binds to the Dux/Zscan4 transcripts and promotes their degradation through recruiting the key component of RNA deadenylase complex, Cnot1. Consistent with the phenotype of Ythdf2 deficiency, silencing of Cnot1 induces the 2C gene expressions and the transition of ESCs into the 2C-like state. Collectively, our findings reveal novel insights into the epitranscriptomic regulation of the 2C-like state in mouse ESCs.
Project description:Mouse embryonic stem cells (ESCs) consist of a rare population of heterogeneous cells which express 2-cell-stage-specific transcripts and are referred to as 2-cell-like cells (2CLCs). Accumulating evidence has demonstrated that transcription factors and epigenetic modifications exert crucial functions in the transition of ESCs to 2CLCs. However, the roles of RNA modification in the regulation of 2C-like state remain elusive. Using Dux-induced 2CLCs system, we examine N6-methyladenosine (m6A) modification landscape in transcriptome-wide, and find that m6A is dynamically regulated during Dux-driven 2C-like reprogramming. Intriguingly, many of 2C-specific transcripts are highly methylated, including Dux and Zscan4 cluster genes. We further identify the m6A reader protein Ythdf2 as a critical regulator of 2C-like state. Depletion of Ythdf2 facilitates robust expressions of 2C transcripts and the transition of ESCs to 2CLCs. Mechanically, Ythdf2 binds to the Dux/Zscan4 transcripts and promotes their degradation through recruiting the key component of RNA deadenylase complex, Cnot1. Consistent with the phenotype of Ythdf2 deficiency, silencing of Cnot1 induces the 2C gene expressions and the transition of ESCs into the 2C-like state. Collectively, our findings reveal novel insights into the epitranscriptomic regulation of the 2C-like state in mouse ESCs.
Project description:Mouse embryonic stem cells (ESCs) consist of a rare population of heterogeneous cells which express 2-cell-stage-specific transcripts and are referred to as 2-cell-like cells (2CLCs). Accumulating evidence has demonstrated that transcription factors and epigenetic modifications exert crucial functions in the transition of ESCs to 2CLCs. However, the roles of RNA modification in the regulation of 2C-like state remain elusive. Using Dux-induced 2CLCs system, we examine N6-methyladenosine (m6A) modification landscape in transcriptome-wide, and find that m6A is dynamically regulated during Dux-driven 2C-like reprogramming. Intriguingly, many of 2C-specific transcripts are highly methylated, including Dux and Zscan4 cluster genes. We further identify the m6A reader protein Ythdf2 as a critical regulator of 2C-like state. Depletion of Ythdf2 facilitates robust expressions of 2C transcripts and the transition of ESCs to 2CLCs. Mechanically, Ythdf2 binds to the Dux/Zscan4 transcripts and promotes their degradation through recruiting the key component of RNA deadenylase complex, Cnot1. Consistent with the phenotype of Ythdf2 deficiency, silencing of Cnot1 induces the 2C gene expressions and the transition of ESCs into the 2C-like state. Collectively, our findings reveal novel insights into the epitranscriptomic regulation of the 2C-like state in mouse ESCs.
Project description:Mouse embryonic stem cells (ESCs) consist of a rare population of heterogeneous cells which express 2-cell-stage-specific transcripts and are referred to as 2-cell-like cells (2CLCs). Accumulating evidence has demonstrated that transcription factors and epigenetic modifications exert crucial functions in the transition of ESCs to 2CLCs. However, the roles of RNA modification in the regulation of 2C-like state remain elusive. Using Dux-induced 2CLCs system, we examine N6-methyladenosine (m6A) modification landscape in transcriptome-wide, and find that m6A is dynamically regulated during Dux-driven 2C-like reprogramming. Intriguingly, many of 2C-specific transcripts are highly methylated, including Dux and Zscan4 cluster genes. We further identify the m6A reader protein Ythdf2 as a critical regulator of 2C-like state. Depletion of Ythdf2 facilitates robust expressions of 2C transcripts and the transition of ESCs to 2CLCs. Mechanically, Ythdf2 binds to the Dux/Zscan4 transcripts and promotes their degradation through recruiting the key component of RNA deadenylase complex, Cnot1. Consistent with the phenotype of Ythdf2 deficiency, silencing of Cnot1 induces the 2C gene expressions and the transition of ESCs into the 2C-like state. Collectively, our findings reveal novel insights into the epitranscriptomic regulation of the 2C-like state in mouse ESCs.
Project description:Mouse embryonic stem cells (ESCs) sporadically transition to a transient totipotent state that resembles blastomeres of the 2-cell embryo stage, which has been proposed to contribute to exceptional genomic stability, one of the key features of mouse ESCs. However, the biological significance of the rare population of 2C-like cells (2CLCs) in ESC cultures remains to be tested. Here, we generated an inducible reporter cell system for specific elimination of 2CLCs from the ESC cultures to disrupt the equilibrium between ESCs and 2CLCs. We show that removing 2CLCs from the ESC cultures leads to dramatic accumulation of DNA damage, genomic mutations and rearrangements, indicating impaired genomic instability. Furthermore, 2CLCs removal results in increased apoptosis and reduced proliferation of mouse ESCs. Together, our data reveal that transition to the privileged 2C-like state is a major component of the intrinsic mechanisms that maintain exceptional genomic stability of mouse ESCs for long-term self-renewal.
Project description:As an evolutionarily conserved master regulator of metabolism, mechanistic target of rapamycin complex 1 (mTORC1) regulates cell states and fates in development, cancer and aging. mTORC1 activity regulation was critical for pluripotent stem cells maintenance and cell fate transitions. Inhibition of mTORC1 induces embryonic stem cells (ESCs) entry into a paused state which reversibly arrests self-renewal leaving pluripotency intact. Hyperactivation of mTORC1 impedes both pluripotency re-establishment and exit of PSCs. As shown that mTORC1 mediates TFE3 nuclear translocation block pluripotency exit, whether similar mechanisms through transcription factor TFE3 are involved in these processes, and the detailed mechanism by which mTORC1-TFE3 regulates critical transcriptional processes for these transitions, remain unclear. In this study, we demonstrate that the nuclear translocation of TFE3, induced by hyperactivation of mTORC1, results in its binding to the nucleosome remodeling and deacetylation (NuRD) complex in both re-establishment and exit of pluripotency. This interaction inhibits the expression of various crucial genes during different fate transitions of PSCs. Our findings uncover a common and key role of TFE3-NuRD association as mediator of mTORC1 to block pluripotent cell fate transitions, with implications for various fields including physiological and pathological diseases.