Myc and Dnmt1 impede the pluripotent to totipotent state transition in embryonic stem cells

Fu, Xudong; Wu, Xiumei; Djekidel, Mohamed Nadhir; Zhang, Yi

doi:10.1038/s41556-019-0343-0

Cited by 95 publications

(153 citation statements)

References 56 publications

Supporting

Mentioning

138

Contrasting

Unclassified

Order By: Relevance

“…6c, d ). Unlike previous studies which overexpressed a master regulator, Dux, to induce 2C-like state 27 , we did not observe a significant decrease in Sox2 expression in the intermediate state ( Supplementary Fig. 6c ), highlighting the potential difference between the Dux-induced 2C-transitions and the naturally occurring pluripotent-to-totipotent transition process.…”

Section: Resultscontrasting

confidence: 99%

“…DNA-binding proteins, such as TFs and epigenetic regulators, show rapid gene expression changes during pluripotent-to-2C transition 27, 29 . Our analysis of immediate transcription changes during the pluripotent-to-2C transition identified 26 genes encoding DNA-binding proteins that showed a significant difference at new RNA levels between states ( Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Massively parallel, time-resolved single-cell RNA sequencing with scNT-Seq

et al. 2019

Preprint

View full text Add to dashboard Cite

11Single-cell RNA sequencing offers snapshots of whole transcriptomes but obscures the temporal 12 dynamics of RNA biogenesis and decay. Here we present single-cell new transcript tagging sequencing 13 (scNT-Seq), a method for massively parallel analysis of newly-transcribed and pre-existing RNAs from the 14 same cell. This droplet microfluidics-based method enables high-throughput chemical conversion on 15 barcoded beads, efficiently marking metabolically labeled newly-transcribed RNAs with T-to-C 16 substitutions. By simultaneously measuring new and old transcriptomes, scNT-Seq reveals neuronal 17 subtype-specific gene regulatory networks and time-resolved RNA trajectories in response to brief 18 (minutes) versus sustained (hours) neuronal activation. Integrating scNT-Seq with genetic perturbation 19 reveals that DNA methylcytosine dioxygenases may inhibit stepwise transition from pluripotent embryonic 20 stem cell state to intermediate and totipotent two-cell-embryo-like (2C-like) states by promoting global 21 RNA biogenesis. Furthermore, pulse-chase scNT-Seq enables transcriptome-wide measurements of RNA 22 stability in rare 2C-like cells. Time-resolved single-cell transcriptomic analysis thus opens new lines of 23 inquiry regarding cell-type-specific RNA regulatory mechanisms.25Dynamic changes in RNA levels are regulated by the interplay of RNA transcription, processing, and 26 degradation 1, 2 . Understanding the mechanisms by which the transcriptome is regulated in functionally 27 diverse cell-types within multi-cellular organisms thus requires cell-type-specific measurements of the 28 kinetics of RNA biogenesis and decay. Recent advances in single-cell RNA sequencing (scRNA-Seq) 29 technologies are leading to a more complete understanding of heterogeneity in cell types and states 3 . 30 However, standard scRNA-Seq methods capture a mixture of newly-synthesized ("new") and pre-existing 31 ("old") RNAs without being able to temporally resolve RNA dynamics. 32 Accurately capturing changes in the nascent transcriptome over time at single-cell resolution is of 33 particular interest because measuring newly-transcribed RNAs can reveal immediate regulatory changes 34 in response to developmental, environmental, metabolic, and pathological signals 4 . Commonly used 35 approaches for distinguishing newly-transcribed from pre-existing RNAs of the same population of 36 transcripts rely on metabolic labeling that employs thiol-labeled nucleoside analogs such as 4-thiouridine 37 (4sU) and subsequent biochemical enrichment of metabolically labeled RNAs 2 . Although these methods 38 have yielded unprecedented insights into the regulation of RNA dynamics, they require ample starting 39 material and present challenges for enrichment normalization. Several approaches have recently been 40 developed to chemically convert 4sU into cytidine analogs, yielding uracil-to-cytosine (U-to-C) 41 substitutions that label newly-transcribed RNAs after reverse transcription 5-7 . These chemical nucleotide 42 conversion me...

show abstract

Section: Resultscontrasting

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Massively parallel, time-resolved single-cell RNA sequencing with scNT-Seq

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…Given the large-scale epigenetic and transcriptional changes that occur during the maternal-to-zygotic transition (reviewed in Eckersley-Maslin, Alda-Catalinas & Reik 2018), we hypothesized that maternal epigenetic and transcriptional factors have crucial roles in regulating ZGA and consequently focused our screen on such candidate regulators. Due to the technical limitations of high-throughput screening in early embryos, we used mouse ESC as an in vitro proxy to mimic ZGA (Eckersley-Maslin, Alda-Catalinas & Reik 2018;Fu et al 2019;Yan et al 2019;Eckersley-Maslin et al 2019;Rodriguez-Terrones et al 2018), reasoning that overexpression of ZGA regulators may induce a ZGA-like transcriptional signature which can be captured by scRNA-seq.…”

Section: A Crispr-activation Screen For Zga Regulators At Single-cellmentioning

confidence: 99%

“…Recent studies have shown that a ZGA-like state can be mimicked in mouse ESCs (Macfarlan et al 2012;Zalzman et al 2010;Bošković et al 2014;Ishiuchi et al 2015;Akiyama et al 2015;Eckersley-Maslin et al 2016;Rodriguez-Terrones et al 2018). Consequently, these cells represent an ideal system for in vitro screening and have been previously used to identify regulators of ZGA (Fu et al 2019;Yan et al 2019;Eckersley-Maslin et al 2019;Rodriguez-Terrones et al 2018). While most of these studies probing ZGA regulators in ESCs have focused on repressors (Fu et al 2019;Rodriguez-Terrones et al 2018), positive inducers of ZGA have thus far not been interrogated in a systematic manner.…”

Section: Introductionmentioning

confidence: 99%

“…Consequently, these cells represent an ideal system for in vitro screening and have been previously used to identify regulators of ZGA (Fu et al 2019;Yan et al 2019;Eckersley-Maslin et al 2019;Rodriguez-Terrones et al 2018). While most of these studies probing ZGA regulators in ESCs have focused on repressors (Fu et al 2019;Rodriguez-Terrones et al 2018), positive inducers of ZGA have thus far not been interrogated in a systematic manner. Such regulators are more relevant given the transcriptionally inactive state prior to ZGA and could be identified in ESCs by assessing the transcriptional changes triggered downstream of their overexpression (Eckersley-Maslin et al 2019).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A single-cell transcriptomics CRISPR-activation screen identifies new epigenetic regulators of zygotic genome activation

Alda-Catalinas

Bredikhin

Hernando-Herraez

et al. 2019

Preprint

View full text Add to dashboard Cite

Zygotic genome activation (ZGA) is a crucial developmental milestone that remains poorly understood. This first essential transcriptional event in embryonic development coincides with extensive epigenetic reprogramming processes and is orchestrated, in part, by the interplay of transcriptional and epigenetic regulators. Here, we developed a novel high-throughput screening method that combines pooled CRISPR-activation (CRISPRa) with single-cell transcriptomics to systematically probe candidate regulators of ZGA. We screened 230 epigenetic and transcriptional regulators by upregulating their expression with CRISPRa in mouse embryonic stem cells (ESCs). Through single-cell RNAsequencing (scRNA-seq) of CRISPRa-perturbed cells, we generated approximately 200,000 single-cell transcriptomes, each transduced with a unique short-guide RNA (sgRNA) targeting a specific candidate gene promoter. Using integrative dimensionality reduction of the perturbation scRNA-seq profiles, we characterized molecular signatures of ZGA and uncovered 44 factors that promote a ZGA-like response in ESCs, both in the coding and non-coding transcriptome. Upon upregulation of these factors, including the DNA binding protein Dppa2, the chromatin remodeller Smarca5 and the transcription factor Patz1, ESCs adopt an early embryonic-like state. Supporting their roles as ZGA regulators, Dppa2 and Smarca5 knock-out ESCs lose expression of ZGA genes, however, overexpression of Dppa2 in Smarca5 knock-out ESCs, but not vice versa, rescues ZGA-like expression, suggesting that Smarca5 regulates ZGA upstream and via Dppa2. Together, our single-cell transcriptomic profiling of CRISPRaperturbed cells provides comprehensive system-level insights into the molecular mechanisms that orchestrate ZGA. Highlights• First large-scale screen combining pooled CRISPRa with scRNA-seq. • Multi-omics factor analysis identifies a ZGA-like signature for 44 of the candidate regulators. • Dppa2, Smarca5 and Patz1 were validated as strong inducers of ZGA gene expression. • Smarca5 regulates zygotic genome activation in a Dppa2-dependent manner.

show abstract

Epigenetic reprogramming in the transition from pluripotency to totipotency

Han,

Ma,

Liu

2024

Journal Cellular Physiology

View full text Add to dashboard Cite

Mammalian development commences with the zygote, which can differentiate into both embryonic and extraembryonic tissues, a capability known as totipotency. Only the zygote and embryos around zygotic genome activation (ZGA) (two‐cell embryo stage in mice and eight‐cell embryo in humans) are totipotent cells. Epigenetic modifications undergo extremely extensive changes during the acquisition of totipotency and subsequent development of differentiation. However, the underlying molecular mechanisms remain elusive. Recently, the discovery of mouse two‐cell embryo‐like cells, human eight‐cell embryo‐like cells, extended pluripotent stem cells and totipotent‐like stem cells with extra‐embryonic developmental potential has greatly expanded our understanding of totipotency. Experiments with these in vitro models have led to insights into epigenetic changes in the reprogramming of pluri‐to‐totipotency, which have informed the exploration of preimplantation development. In this review, we highlight the recent findings in understanding the mechanisms of epigenetic remodeling during totipotency capture, including RNA splicing, DNA methylation, chromatin configuration, histone modifications, and nuclear organization.

show abstract

Myc and Dnmt1 impede the pluripotent to totipotent state transition in embryonic stem cells

Cited by 95 publications

References 56 publications

Massively parallel, time-resolved single-cell RNA sequencing with scNT-Seq

Massively parallel, time-resolved single-cell RNA sequencing with scNT-Seq

A single-cell transcriptomics CRISPR-activation screen identifies new epigenetic regulators of zygotic genome activation

Epigenetic reprogramming in the transition from pluripotency to totipotency

Contact Info

Product

Resources

About