Tracing the transitions from pluripotency to germ cell fate with CRISPR screening

Hackett, Jim; Huang, Yun; Günesdogan, Ufuk; Gretarsson, Kristjan H.; Kobayashi, Toshihiro; Surani, M. Azim

doi:10.1038/s41467-018-06230-0

Cited by 71 publications

(64 citation statements)

References 55 publications

(61 reference statements)

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“…This was rescued by transgenic Zmym2 expression. This is in agreement with a recently published Cas9 ESC differentiation screen which demonstrated that Zmym2 KO ESCs resist differentiation 25 .…”

Section: Resultssupporting

confidence: 93%

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ZMYM2 inhibits NANOG-mediated reprogramming

Lawrence¹,

Theunissen²,

Lombard³

et al. 2019

Wellcome Open Res

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Background: NANOG is a homeodomain-containing transcription factor which forms one of the hubs in the pluripotency network and plays a key role in the reprogramming of somatic cells and epiblast stem cells to naïve pluripotency. Studies have found that NANOG has many interacting partners and some of these were shown to play a role in its ability to mediate reprogramming. In this study, we set out to analyse the effect of NANOG interactors on the reprogramming process. Methods: Epiblast stem cells and somatic cells were reprogrammed to naïve pluripotency using MEK/ERK inhibitor PD0325901, GSK3β inhibitor CHIR99021 and Leukaemia Inhibitory Factor (together termed 2i Plus LIF). Zmym2 was knocked out using the CRISPR/Cas9 system or overexpressed using the PiggyBac system. Reprogramming was quantified after ZMYM2 deletion or overexpression, in diverse reprogramming systems. In addition, embryonic stem cell self renewal was quantified in differentiation assays after ZMYM2 removal or overexpression. Results: In this work, we identified ZMYM2/ZFP198, which physically associates with NANOG as a key negative regulator of NANOG-mediated reprogramming of both epiblast stem cells and somatic cells. In addition, ZMYM2 impairs the self renewal of embryonic stem cells and its overexpression promotes differentiation. Conclusions: We propose that ZMYM2 curtails NANOG’s actions during the reprogramming of both somatic cells and epiblast stem cells and impedes embryonic stem cell self renewal, promoting differentiation.

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Section: Resultssupporting

confidence: 93%

“…We also observe that Zmym2 promotes embryonic stem cell differentiation. This has also been reported in a Cas9 screen for differentiation-promoting factors 25 . Future work could examine whether the absence of Zmym2 impacts mouse development and elucidate its role in vivo .…”

Section: Discussionsupporting

confidence: 72%

ZMYM2 inhibits NANOG-mediated reprogramming

Lawrence¹,

Theunissen²,

Lombard³

et al. 2019

Wellcome Open Res

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“…So far, CRISPR screen studies on mESC pluripotency or naive and primed state transition is mainly based on genetically labeled fluorescence reporters as readout (Hackett et al, 2018; Li et al, 2018; Seruggia et al, 2019), which is limited by only one or two genes. Here we employed a single-cell RNA-seq combined with CRISPR screening technology (CROP-seq) and used whole cell transcriptome as readout of cell fate changes.…”

Section: Discussionmentioning

confidence: 99%

Linking genotypes with multiple phenotypes in single-cell CRISPR screens

Yang

Zhu

et al. 2019

Preprint

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CRISPR/Cas9 based functional screening coupled with single-cell RNA-seq ("single-cell CRISPR screening") unravels gene regulatory networks and enhancer-gene regulations in a large scale. We propose scMAGeCK, a computational framework to systematically identify genes and non-coding elements associated with multiple expression-based phenotypes in single-cell CRISPR screening. scMAGeCK identified genes and enhancers that modulate the expression of a known proliferation marker, MKI67 (Ki-67), a result that resembles the outcome of proliferation-linked CRISPR screening. We further performed single-cell CRISPR screening on mouse embryonic stem cells (mESC), and identified key genes associated with different pluripotency states. scMAGeCK enabled an unbiased construction of genotypephenotype network, where multiple phenotypes can be regulated by different gene perturbations. Finally, we studied key factors that improve the statistical power of single-cell CRISPR screens, including target gene expression and the number of guide RNAs (gRNAs) per cell. Collectively, scMAGeCK is a novel and effective computational tool to study genotypephenotype relationships at a single-cell level.

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“…ESC lines carrying floxed Dnmt1 alleles 23 and WT ESC were transfected with pPB-as Dazl sensor- Kcnq1ot1 -H2B::GFP ( in silico DNA methylated with M.SssI), pPB- EF1a -H2B::mCherry, pPB- spCas9 -Hygro 54 , and PBase using Lipofectamine 3000. Transfected cells were selected for spCas9 integration in titrated 2i/L using Hygromycin (250 μg/ml) for 5 days, and clonally-derived cell lines were subsequently isolated and expanded.…”

Section: Methodsmentioning

confidence: 99%

Dppa2/4CounteractDe NovoMethylation to Establish a Permissive Epigenome for Development

Hackett

Grétarsson

2020

Preprint

Self Cite

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2Early mammalian development entails genome-wide epigenome remodeling, including DNA 3 methylation erasure and reacquisition, which facilitates developmental competence. To uncover the 4 mechanisms that orchestrate DNA methylation (DNAme) dynamics, we coupled a single-cell 5 ratiometric DNAme reporter with unbiased CRISPR screening in ESC. We identify key genes and 6 regulatory pathways that drive global DNA hypomethylation, and characterise roles for Cop1 and 7 Dusp6. We also identify Dppa2 and Dppa4 as essential safeguards of focal epigenetic states. In their 8 absence, developmental genes and evolutionary-young LINE1 elements, which DPPA2 specifically 9 binds, lose H3K4me3 and gain ectopic de novo DNA methylation in pluripotent cells. Consequently, 65 mediated by parallel mechanisms 8 . Amongst these, repression of the maintenance DNA methylation 66 machinery is central and appears to occur through post-translational regulation of UHRF1 9,10 , at least 67 in part via STELLA activity 11,12 . This is further supported by PRDM14, which suppresses the de novo 68 methylases, and is necessary for DNA hypomethylation in naïve pluripotent cells 13,14 . In parallel, 69 replication-independent DNAme erasure occurs on both the maternal and paternal genomes 1 . 70 Counterintuitively, de novo methylation remains active throughout epigenetic reprogramming but is 71 offset, in part, via TET proteins 15 . These collective mechanisms contribute towards resetting the 72 epigenome, but also present an opportunity for transposable elements (TE), such as LINE1, to mobilise 73 due to epigenetic de-restriction. Such LINE1 activation has been linked with key developmental 74 events 16 , but could also represent a hazard to the genome if left unrestrained 17,18 . Epigenetic 75 (re)programming therefore likely strikes a balance between genome-wide resetting to a competent state 76 for development, and targeted regulation. Nevertheless, a complete understanding of the mechanisms 77 that cross-talk to remodel the epigenome, how they interact to balance focal and global effects, and 78 what the full repertoire of genes involved is lacking. 80Here we have coupled a single-cell ratiometric reporter of cellular DNA methylation status with 81 CRISPR screening to unbiasedly identify the gene networks that underpin DNAme remodeling. In 82 doing so we identify upstream regulators of global DNAme erasure in pluripotent cells. We also identify 83 Dppa2 and Dppa4 as key genes that safeguard against focal de novo DNA methylation and epigenetic 84 silencing at lineage-associated genes by integrating chromatin states, and consequently confer 85 developmental competence. Remarkably, LINE1 elements appear to have exapted this Dppa2/4 86 function to escape epigenetic surveillance and enable competence for precocious activation, potentially 87 highlighting an evolving genomic conflict. 88 89 90 RESULTS 92Single-cell monitoring of DNA demethylation 93 To identify regulators of epigenetic remodeling we exploited the reporter for genomic DNA methy...

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Tracing the transitions from pluripotency to germ cell fate with CRISPR screening

Cited by 71 publications

References 55 publications

ZMYM2 inhibits NANOG-mediated reprogramming

ZMYM2 inhibits NANOG-mediated reprogramming

Linking genotypes with multiple phenotypes in single-cell CRISPR screens

Dppa2/4CounteractDe NovoMethylation to Establish a Permissive Epigenome for Development

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