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

Nature Structural & Molecular Biology volume 30, pages 1105–1118 (2023)Cite this article

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Abstract

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.

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Fig. 1: Dazl as a reporter gene for the 2-cell stage, screen design and generation of the Dazl-mScarlet-HygroR (DASH) reporter cell line.
Fig. 2: A genome-wide CRISPR KO screen for Dazl gene reactivation in the DASH reporter line.
Fig. 3: Four loss-of-function mutants that induce Dazl expression: Mcm3ap KO, Spop KO, Zbtb14 KO and Kdm5c KO.
Fig. 4: Induction of a 2-cell-like signature in the Mcm3ap, Spop, Zbtb14 and Kdm5c KOs.
Fig. 5: Epistasis analyses show that KDM5C removal activates Dazl independently of Dux and Dppa2.
Fig. 6: Mechanistic studies on SPOP and ZBTB14.
Fig. 7: Catalysis-independent role of KDM5C.

Data availability

All sequencing data generated in this study are available in the Gene Expression Omnibus under accession number GSE221710. For Figs. 1 and 4 and Extended Data Fig. 5, published RNA-seq were reanalyzed from GSE33923 (Macfarlan)7; E-MTAB-2684 (Ishiuchi)19; GSE75751 (Eckersley-Maslin)23; GSE71434 (Zhang)66 and GSE66390 (Wu)65. Other online databases used in the study were STRING (https://string-db.org/) in Fig. 2 and GSEA (https://www.gsea-msigdb.org/gsea/index.jsp) in Fig. 4 and Extended Data Fig. 4. Source data are provided with this paper.

Code availability

Custom scripts used for WGBS mapping are available at: https://github.com/FumihitoMiura/Project-2.

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Acknowledgements

We are grateful to the following colleagues for useful advice: A. Bardin, B. Rodgers, C. Francastel, C. Rougeulle, S. Polo, P. Navarro, R. Margueron, G. Velasco, G. Filion, M. Casanova, S. Donakonda, M. Weber, L. Tora, Y. Shinkai, S. Khochbin and T. Bartke. We thank the following colleagues for useful reagents: D. Bourc’his (Institut Curie, Paris) for J1 mESCs, M.-E. Torres Padilla (Helmholtz Zentrum Munich) for tbg4 reporter mESCs, M. Timmers (DKFZ Heidelberg) for KDM5C tagged lines, N. Sakaguchi (Kumamoto University) for a mouse GANP cDNA, H. Niwa and Y. Shinkai (RIKEN Saitama) for piggyBac constructs, J. Sharif and H. Koseki (RIKEN Yokohama) for a FLAG-PCGF6 expression plasmid, J. Hackett (EMBL Rome) for a FLAG-DPPA2 expression plasmid. We thank the Vectorology platform, Epigenetics platform, Microscopy platform and Bioinformatics/Biostatistics Core Facility at the CNRS Epigenetics and Cell Fate Unit (Université Paris Cité), for providing access and technical advice. We thank E. Jeannot at Institut Curie for help with ddPCR. We thank S. Bultmann (LMU, Munich) for help with sgRNA sequencing and MAGeCK analysis. We acknowledge the ImagoSeine core facility of the Institut Jacques Monod, member of the France BioImaging (grant no. ANR-10-INBS-04) and the support of La Ligue contre le Cancer (grant no. R03/75-79). Microfluidic RT–qPCR (Fluidigm) analysis was carried out on the qPCR-HD-Genomic Paris Centre Core Facility and was supported by grants from Région Ile-de-France, grant no. DIMBIO-RVT-INSERM-ADR-P11 21016711. P.-A.D. is supported by Agence Nationale de la Recherche (PRCI INTEGER grant no. ANR-19-CE12-0030-01), LabEx ‘Who Am I?’ (grant no. ANR-11-LABX-0071), Université de Paris IdEx (grant no. ANR-18-IDEX-0001) funded by the French Government through its ‘Investments for the Future’ program, Fondation pour la Recherche Médicale, Fondation ARC (Programme Labellisé grant no. PGA1/RF20180206807). P.-A.D. and M.C.V.G. are supported by Agence Nationale de la Recherche (grant no. PRC REMEDY ANR-21-CE12-0015-03). P.-A.D., A.S. and G.C. were supported by grant RETROMET, no. ANR-16-CE12-0020, from Agence Nationale de la Recherche. J.R.A. and M.V.C.G. were supported by Laboratoire d’excellence Who Am I? (grant no. Labex 11-LABX-0071) Emerging Teams Grant and by the European Research Council (grant no. ERC-StG-2019 DyNAmecs). This research was supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under grant no. JP20am0101103 (support no. 2652). K.Y. was the recipient of a postdoctoral fellowship from Fondation Association pour la Recherche sur le Cancer, and of a subsequent postdoc fellowship from Labex WhoAmI. M.L. thanks the Ligue contre le Cancer for a fourth year PhD fellowship.

Author information

Author notes

  1. Nikhil Gupta

    Present address: Joint AZ CRUK Functional Genomics Centre, The Milner Therapeutics Institute, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge, UK

  2. These authors contributed equally: Nikhil Gupta, Lounis Yakhou.

Authors and Affiliations

  1. Epigenetics and Cell Fate, Université Paris Cité, CNRS, Paris, France

    Nikhil Gupta, Lounis Yakhou, Anaelle Azogui, Laure Ferry, Olivier Kirsh, Sarah Battault, Kosuke Yamaguchi, Marthe Laisné, Cécilia Domrane & Pierre-Antoine Defossez

  2. Institut Jacques Monod, Université Paris Cité, CNRS, Paris, France

    Julien Richard Albert & Maxim V. C. Greenberg

  3. Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Fukuoka, Fukuoka, Japan

    Fumihito Miura & Takashi Ito

  4. Epigenetic Chemical Biology, UMR3523, Institut Pasteur, Université Paris Cité, CNRS, Paris, France

    Frédéric Bonhomme

  5. IRCAN, Université Côte d’Azur, Inserm, CNRS, Nice, France

    Arpita Sarkar & Gael Cristofari

  6. High Throughput qPCR Facility, Institut de Biologie de l’École Normale Supérieure (IBENS), Laboratoire de Physique de l’ENS CNRS UMR8023, PSL Research University, Paris, France

    Marine Delagrange & Bertrand Ducos

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Contributions

N.G. and P.-A.D. conceived the project. N.G., L.Y. and P.-A.D. planned the experiments. N.G., L.Y., L.F., F.B., S.B., K.Y. and A.A. performed experiments and analyzed the data. F.M. performed WGBS. C.D. performed MeDIP. F.B. performed mass spectrometry. M.D. and B.D. performed Fluidigm experiments. O.K. performed WGBS analysis. J.R.A., O.K., L.Y., M.L., A.S., K.Y. and G.C. performed other bioinformatic analyses. N.G., L.Y. and P.-A.D. wrote the manuscript. P.-A.D., T.I. and N.G. supervised the project. M.V.C.G., G.C., T.I. and P-.A.D. acquired funding. All authors reviewed the manuscript. J.R.A, A.A., L.F. and O.K. contributed equally.

Corresponding authors

Correspondence to Nikhil Gupta or Pierre-Antoine Defossez.

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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Carolina Perdigoto and Dimitris Typas were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Generation and validation of the Dazl-mScarlet-Hygromycin (DASH) reporter cell line.

(a) WGBS and RNA-seq in our cellular background (DASH cells) confirm that Dazl promoter is highly methylated and repressed in serum, but hypo-methylated and expressed in 2i. CGI: CpG island. (b) RT-qPCR confirms the upregulation of Dazl in ESCs cultured in 2i (relative to serum condition). (c) Dazl exon 6 was targeted by 2 independent sgRNAs (red arrowhead) to insert the reporter cassette by homologous recombination. The donor construct contains Dazl homology arms flanking genes for the red fluorescent protein mScarlet, and the Hygromycin resistance enzyme (HygroR) separated by 2A self-cleaving peptides (P2A, T2A). (d) DASH ESCs have a single insertion at one of the Dazl alleles, as determined by ddPCR. Left panel: blue droplets are positive for the corresponding amplification; black droplets are negative. About 18,000 droplets were analyzed for each amplification. Right panel: quantitative analysis confirming single insertion of the donor construct. Gapdh served as a control present at 2 copies/cell. (e) RT-qPCR showing the up-regulation of Dazl and mScarlet in DASH ESCs cultured in 2i (relative to serum condition). (f) MeDIP assay showing the relative levels of 5mC at Gapdh and Dazl promoters in DASH ESCs grown in serum or 2i conditions. (g) Treatment of DASH cells with Sodium Acetate, a chemical inductor of 2CLCs, activates mScarlet expression. NT: not treated. SA: Sodium Acetate (40 mM, 48 h). **P < 0.01 (two-tailed t-test). (h) Detection of ZCAN4-positive cells in the SA-treated population (n = 633 cells) versus DASH (n = 812 cells). ****P < 0.0001 (two-tailed Mann-Whitney test). (i) Most ZSCAN4-positive cells are also mScarlet-positive. ****P < 0.0001 (two-tailed Mann-Whitney test). Data from n = 3 replicates are presented as mean values +/- SD for panels b, e and g. Data from n = 4 replicates are presented as mean values +/- SD for panels d and f. Data from n = 278 cells (Negative) and n = 228 cells (Positive) are presented for panel i.

Extended Data Fig. 2 Screen quality controls and validations of selected hits.

(a) FACS analysis (50,000 cells per condition) of mScarlet expression after Hygromycin selection. **P < 0.01 (two-tailed t-test). (b) RT-qPCR: comparison of mScarlet-expressing cells to Hygro49 cells. *P < 0.05, ***P < 0.005 (two-sided Holm-Sidak post-hoc test following ANOVA). (c) Decrease of global DNA methylation in mScarlet+ cells in comparison to Hygro49 cells from the screen, as measured by LUMA. *P < 0.05, **P < 0.01 (two-sided Holm-Sidak post-hoc test following ANOVA). (d) MeDIP assay showing the relative levels of 5mC at Gapdh and Dazl promoters in HygroR and mScarlet+ screen samples. (e) Gene ontology (GO) terms (Uniprot keywords) significantly enriched among the top 40 hits. (f) Expression of ‘naïve’ markers (left panel) and ‘FBS’ markers (right panel) in the KOs, FBS-grown and 2i-grown ESC. The KOs behave like FBS-grown ESC, not like 2i-grown cells. (g) Spontaneous differentiation induced by LIF removal is not impeded in the Mcm3ap, Spop, Zbtb14, and Kdm5c KOs. The Tcf7l1 KO is known to be unable to differentiate and is used as a control. Scale bar: 200 μm. (h) RT-qPCR on pluripotency and differentiation markers after LIF removal in the indicated KOs. Data from n = 2 independent screen replicates are presented as mean values +/− SD for panels a-d. Data from n = 3 independent KO clones are presented as mean values +/− SD for panels f and h.

Extended Data Fig. 3 Genetic analysis of the KO clones, rescue, and WGBS confirmation.

(a) Identification of the mutations found in each of the KO clones. (b) RT-qPCR analysis: genetic rescue of each KO suppresses Dazl mRNA induction. (c) WGBS coverage statistics (n = 3 independent KO clones). In the boxplots, the thick line indicates the median, the box limits indicate the upper and lower quartiles, and the whiskers extend to min and max values (d) Principal Component Analysis (PCA) on the WGBS results. The 4 KOs cluster together, away from serum cells and from 2i cells. (e) Liquid chromatography followed by tandem Mass Spectrometry (LC-MS/MS) confirms the decrease of DNA methylation in Mcm3ap and Spop KOs. *P < 0.05, **P < 0.01, ****P < 0.0001 (two-sided Holm-Sidak post-hoc test following ANOVA). (f) A restriction-enzyme based technique (LUMA) confirms the decrease of DNA methylation in Mcm3ap and Spop KOs. ***P < 0.001, ****P < 0.0001 (two-sided Holm-Sidak post-hoc test following ANOVA). Data from n = 3 independent KO clones are presented as mean values +/- SD for panels b and e-f.

Extended Data Fig. 4 Additional characterizations of the transcriptional 2-cell-like signature in the Mcm3ap, Spop, Zbtb14, and Kdm5c Kos.

(a) RNA-seq statistics: differentially expressed genes (|FC | > 2; FDR < 1%) in each KO condition. (b) Genome browser tracks depicting RNA-seq profiles of 2CLC markers reactivated in the KOs. (c) RT-qPCR analysis: genetic rescue of each KO suppresses 2CLC marker induction. (d) Gene Set Enrichment Analysis (GSEA): the 2CLC signature is enriched in each individual KO. (e) GSEA: metabolic pathways downregulated in 2CLCs38 are also downregulated in the KOs. (f) Increased ZSCAN4-positive staining in the indicated KO populations. Data from n = 812 cells; n = 501 cells; n = 757 cells; n = 576 cells and n = 547 cells are presented for, DASH, Mcm3ap KO, Spop KO, Zbtb14 KO and Kdm5c KO, respectively. ****P < 0.0001 (Dunn’s post-hoc test following Kruskal-Wallis test) (g) Reactivation of an LTR-GFP reporter after siRNA of the indicated factors. Data from n = 3 independent replicates are presented as mean values +/- SD. *P < 0.05, **P < 0.01, ***P < 0.001 (two-sided Holm-Sidak post-hoc test following ANOVA).

Extended Data Fig. 5 Trp53 is not required for the activation of 2CLC markers in the Mcm3ap, Spop, Zbtb14, and Kdm5c Kos.

(a) Experimental scheme for Trp53 depletion. (b) RT-qPCR analysis: Trp53 mRNA is efficiently depleted in all KOs. (c) The p53 protein is efficiently depleted, example of western blot on WT cells. (d) RT-qPCR analysis: the induction of 2CLC markers is Trp53-independent. (e) Expression of the indicated genes in single ES cells sorted according to ZSCAN4 and MERVL expression. Data from n = 3 independent KO clones are presented as mean values +/- SD for panels b and d.

Source data

Extended Data Fig. 6 Limited overlap between ZBTB14 binding and transcriptional response to ZBTB14 loss.

(a) MA-plot on the RNA-seq data from Zbtb14 KO cells relative to WT ES cells. This is the same data as in Fig. 4a. (b) Only a minority of promoters bound by ZBTB14 respond transcriptionally to Zbtb14 KO, and vice versa. (c) and (d) ZBTB14 and ZSCAN4 have coevolved closely. The scores are from CladeOScope, http://cladeoscope.cs.huji.ac.il. A smaller score means tighter co-evolution.

Extended Data Fig. 7 KDM5C binds additional germline/2CLC genes in ESCs.

(a) Genome browser tracks illustrating the binding of KDM5C to the Taf7l and Ddx4 promoters. (b) 85% of the germline genes that are regulated similarly to Dazl are bound by KDM5C in ESCs. (c) 25% of the germline genes that are regulated similarly to Dazl18 are bound and silenced by KDM5C in ESCs. (d) KDM5C expression has no effect on the global level of H3K4me1/2/3. Western blotting was performed in the indicated cellular backgrounds. Significance: hypergeometric test for panels b-c.

Source data

Supplementary information

Reporting Summary

Supplementary Table 1

Table 1: MAGeCK output of the screen. Table 2: Oligonucleotide sequences used in this study. Table 3: WGBS basic sequencing metrics. Table 4: RNA-seq differentially expressed genes (DESeq2 output).

Source data

Source Data Fig. 3

Unprocessed western blots for Fig. 3.

Source Data Fig. 4

Unprocessed western blots for Fig. 4.

Source Data Fig. 6

Unprocessed western blots for Fig. 6.

Source Data Fig. 7

Unprocessed western blots for Fig. 7.

Source Data Extended Data Fig. 5

Unprocessed western blots for Extended Data Fig. 5.

Source Data Extended Data Fig. 7

Unprocessed western blots for Extended Data Fig. 7.

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Gupta, N., Yakhou, L., Albert, J.R. et al. A genome-wide screen reveals new regulators of the 2-cell-like cell state. Nat Struct Mol Biol 30, 1105–1118 (2023). https://doi.org/10.1038/s41594-023-01038-z

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