Stella modulates transcriptional and endogenous retrovirus programs during maternal-to-zygotic transition

Huang, Yun; Kim, Jong; Vinh, Dang; Lee, Caroline; Penfold, Christopher A.; Ż̷ylicz, J.; Marioni, John C.; Hackett, Jim; Surani, M. Azim

doi:10.7554/elife.22345

Cited by 101 publications

(95 citation statements)

References 66 publications

(123 reference statements)

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“…After Zscan4c, Dppa4 was the strongest identified inducer of ZGA transcription and was therefore selected, along with the closely related Dppa2, for further study. Interestingly, the family member Dppa3 (also known as Stella or Pgc7) was not able to induce the 2C-like state, consistent with a previous report (Huang et al 2017). Dppa2 and Dppa4 physically interact and bind to euchromatin in pluripotent stem cells (Nakamura et al 2011;Klein et al 2018).…”

Section: Discussionsupporting

confidence: 90%

Dppa2 and Dppa4 directly regulate the Dux driven zygotic transcriptional programme

Eckersley-Maslin

Alda-Catalinas

Blotenburg

et al. 2018

Preprint

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The molecular regulation of zygotic genome activation (ZGA) in mammals remains poorly understood. Primed mouse embryonic stem cells contain a rare subset of "2C-like" cells that are epigenetically and transcriptionally similar to the two cell embryo and thus represent an ideal system for studying ZGA transcription regulation. Recently, the transcription factor Dux, expressed exclusively in the minor wave of ZGA, was described to activate many downstream ZGA transcripts. However, it remains unknown what upstream maternal factors initiate ZGA either in a Dux dependent or independent manner. Here we performed a candidate-based overexpression screen, identifying, amongst others, Developmental Pluripotency Associated 2 (Dppa2) and 4 (Dppa4) as positive regulators of 2C-like cells and ZGA transcription. In the germ line, promoter DNA demethylation coincides with upregulation of Dppa2 and Dppa4 which remain expressed until E7.5 when their promoters are remethylated. Furthermore, Dppa2 and Dppa4 are also expressed during iPSC reprogramming at the time 2C-like ZGA transcription transiently peaks. Through a combination of overexpression, knockdown, knockout and rescue experiments, together with transcriptional analyses, we show that Dppa2 and Dppa4 directly regulate the 2C-like cell population and associated transcripts, including Dux and the Zscan4 cluster. Importantly, we tease apart the molecular hierarchy in which the 2C-like transcriptional programme is initiated and stabilised. Dppa2 and Dppa4 require Dux to initiate 2C-like ZGA transcription, suggesting they act upstream by directly regulating Dux. Supporting this, ChIP-seq analysis revealed Dppa2 and Dppa4 bind to the Dux promoter and gene body and drive its expression. Zscan4c is also able to induce 2C-like cells in wild type cells, but, in contrast to Dux, can no longer do so in Dppa2/4 double knockout cells, suggesting it may act to stabilise rather than drive the transcriptional network. Our findings suggest a model in which Dppa2/4 binding to the Dux promoter leads to Dux upregulation and activation of the 2C-like transcriptional programme which is subsequently reinforced by Zscan4c.2

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Section: Discussionsupporting

confidence: 90%

Dppa2 and Dppa4 directly regulate the Dux driven zygotic transcriptional programme

Eckersley-Maslin

Alda-Catalinas

Blotenburg

et al. 2018

Preprint

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“…S1G). The upregulation of ZGA genes upon MERVL LTR activation is consistent with MERVL LTRs acting as functional promoters driving the expression of hundreds of chimeric ZGA transcripts (Macfarlan et al 2012;Huang et al 2017;Franke et al 2017). Similarly, Zscan4c cDNA overexpression has recently been shown to induce the expression of ZGA genes (Eckersley-Maslin et al 2019;Zhang et al 2019).…”

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

confidence: 58%

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

Alda-Catalinas

Bredikhin

Hernando-Herraez

et al. 2019

Preprint

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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.

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“…Such truncation can be undesirable when investigating complex developmental programs, where signalling from adjacent tissues influence cell fate decisions, making it necessary to place branches on consistent timeframes. Finally, in the past, scRNA-seq datasets tended to be of low temporal resolution, albeit with observations in many cells, which reflect a continuum of developmental states Petropoulos et al 2016;Borensztein et al 2017;Huang et al 2017). However, due to decreased costs, scRNA-seq datasets are now routinely generated over finely resolved time series, including the early stages of embryogenesis Petropoulos et al 2016;Borensztein et al 2017;Huang et al 2017;Ibarra-Soria et al 2018;Han et al 2018) and during PGC development Li et al 2017).…”

Section: Introductionmentioning

confidence: 99%

“…Finally, in the past, scRNA-seq datasets tended to be of low temporal resolution, albeit with observations in many cells, which reflect a continuum of developmental states Petropoulos et al 2016;Borensztein et al 2017;Huang et al 2017). However, due to decreased costs, scRNA-seq datasets are now routinely generated over finely resolved time series, including the early stages of embryogenesis Petropoulos et al 2016;Borensztein et al 2017;Huang et al 2017;Ibarra-Soria et al 2018;Han et al 2018) and during PGC development Li et al 2017). While individual populations of cells associated with the different stages still reflect a continuum of states, with some degree of overlap between stages, the existence of a well-defined capture time provides highly informative prior information about the ordering of cells, which most approaches are incapable of utilising.…”

Section: Introductionmentioning

confidence: 99%

Bayesian inference of transcriptional branching identifies regulators of early germ cell development in humans

Penfold

Sybirna

Reid

et al. 2017

Preprint

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During embryonic development, cells undertake a series of fate decisions to form a complete organism comprised of various cell types, epitomising a branching process. A striking example of branching occurs in humans around the time of implantation, when primordial germ cells (PGCs), precursors of sperm and eggs, and somatic lineages are specified. Due to inaccessibility of human embryos at this stage of development, understanding the mechanisms of PGC specification remains difficult. The integrative modelling of single cell transcriptomics data from embryos and appropriate in vitro models should prove to be a useful resource for investigating this system, provided that the cells can be suitably ordered over a developmental axis. Unfortunately, most methods for inferring cell ordering were not designed with structured (time series) data in mind. Although some probabilistic approaches address these limitations by incorporating prior information about the developmental stage (capture time) of the cell, they do not allow the ordering of cells over processes with more than one terminal cell fate. To investigate the mechanisms of PGC specification, we develop a probabilistic pseudotime approach, branch-recombinant Gaussian process latent variable models (B-RGPLVMs), that use an explicit model of transcriptional branching in individual marker genes, allowing the ordering of cells over developmental trajectories with arbitrary numbers of branches. We use first demonstrate the advantage of our approach over existing pseudotime algorithms and subsequently use it to investigate early human development, as primordial germ cells (PGCs) and somatic cells diverge. We identify known master regulators of human PGCs, and predict roles for a variety of signalling pathways, transcription factors, and epigenetic modifiers. By concentrating on the earliest branched signalling events, we identified an antagonistic role for FGF receptor (FGFR) signalling pathway in the acquisition of competence for human PGC fate, and identify putative roles for PRC1 and PRC2 in PGC specification. We experimentally validate our predictions using pharmacological blocking of FGFR or its downstream effectors (MEK, PI3K and JAK), and demonstrate enhanced competency for PGC fate in vitro, whilst small molecule inhibition of the enzymatic component of PRC1/PRC2 reveals reduced capacity of cells to form PGCs in vitro. Thus, B-RGPLVMs represent a powerful and flexible data-driven approach for dissecting the temporal dynamics of cell fate decisions, providing unique insights into the mechanisms of early embryogenesis. Scripts relating to this analysis are available from: https://github.com/cap76/PGCPseudotime

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Stella modulates transcriptional and endogenous retrovirus programs during maternal-to-zygotic transition

Cited by 101 publications

References 66 publications

Dppa2 and Dppa4 directly regulate the Dux driven zygotic transcriptional programme

Dppa2 and Dppa4 directly regulate the Dux driven zygotic transcriptional programme

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

Bayesian inference of transcriptional branching identifies regulators of early germ cell development in humans

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