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p53 convergently activates Dux/DUX4 in embryonic stem cells and in facioscapulohumeral muscular dystrophy cell models

Nature Genetics volume 53, pages 1207–1220 (2021)Cite this article

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Abstract

In mammalian embryos, proper zygotic genome activation (ZGA) underlies totipotent development. Double homeobox (DUX)-family factors participate in ZGA, and mouse Dux is required for forming cultured two-cell (2C)-like cells. Remarkably, in mouse embryonic stem cells, Dux is activated by the tumor suppressor p53, and Dux expression promotes differentiation into expanded-fate cell types. Long-read sequencing and assembly of the mouse Dux locus reveals its complex chromatin regulation including putative positive and negative feedback loops. We show that the p53–DUX/DUX4 regulatory axis is conserved in humans. Furthermore, we demonstrate that cells derived from patients with facioscapulohumeral muscular dystrophy (FSHD) activate human DUX4 during p53 signaling via a p53-binding site in a primate-specific subtelomeric long terminal repeat (LTR)10C element. In summary, our work shows that p53 activation convergently evolved to couple p53 to Dux/DUX4 activation in embryonic stem cells, embryos and cells from patients with FSHD, potentially uniting the developmental and disease regulation of DUX-family factors and identifying evidence-based therapeutic opportunities for FSHD.

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Fig. 1: DNA damage induces a 2CLC signature in mESCs and requires Dux.
Fig. 2: 2CLC emergence and Dux expression after doxorubicin treatment require canonical DDR signaling and p53.
Fig. 3: Assembly of the mouse Dux locus reveals chromatin and transcriptional regulatory features.
Fig. 4: p53 accumulates in mouse zygotes after fertilization, and MZ Trp53-KO embryos have lower DUX and DUX-target expression.
Fig. 5: Transient DUX expression in mESCs confers increased expanded-fate potential.
Fig. 6: p53 is required for DUX4 activation after DNA damage in FSHD cells.
Fig. 7: The subtelomeric p53-binding site at the LTR10C is required for full activation of the DUX4 locus, and myoblasts from patients with FSHD are similarly hypersensitive to DNA damage.

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Data availability

All data, cell lines, reagents and unique materials are available upon request. ChIP–seq, ATAC-seq, scRNA-seq and RNA-seq data were deposited under GSE149267 and are detailed in Supplementary Table 4. Source data are provided with this paper.

References

  1. Jukam, D., Shariati, S. A. M. & Skotheim, J. M. Zygotic genome activation in vertebrates. Dev. Cell 42, 316–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Hendrickson, P. G. et al. Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons. Nat. Genet. 49, 925–934 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. De Iaco, A. et al. DUX-family transcription factors regulate zygotic genome activation in placental mammals. Nat. Genet. 49, 941–945 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Iaco, A. D., Verp, S., Offner, S., Grun, D. & Trono, D. DUX is a non-essential synchronizer of zygotic genome activation. Development 147, dev177725 (2020).

    PubMed  Google Scholar 

  5. Vuoristo, S. et al. DUX4 regulates oocyte to embryo transition in human. Preprint at bioRxiv https://doi.org/10.1101/732289 (2019).

  6. Chen, Z. & Zhang, Y. Loss of DUX causes minor defects in zygotic genome activation and is compatible with mouse development. Nat. Genet. 51, 947–951 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Macfarlan, T. S. et al. Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature 487, 57–63 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ishiuchi, T. et al. Early embryonic-like cells are induced by downregulating replication-dependent chromatin assembly. Nat. Struct. Mol. Biol. 22, 662–671 (2015).

    Article  CAS  PubMed  Google Scholar 

  9. Percharde, M. et al. A LINE1–Nucleolin partnership regulates early development and ESC identity. Cell 174, 391–405 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Rodriguez-Terrones, D. et al. A molecular roadmap for the emergence of early-embryonic-like cells in culture. Nat. Genet. 50, 106–119 (2018).

    Article  CAS  PubMed  Google Scholar 

  11. Eckersley-Maslin, M. et al. Dppa2 and Dppa4 directly regulate the Dux-driven zygotic transcriptional program. Genes Dev. 33, 194–208 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hu, Z. et al. Maternal factor NELFA drives a 2C-like state in mouse embryonic stem cells. Nat. Cell Biol. 22, 175–186 (2020).

    Article  CAS  PubMed  Google Scholar 

  13. Dixit, M. et al. DUX4, a candidate gene of facioscapulohumeral muscular dystrophy, encodes a transcriptional activator of PITX1. Proc. Natl Acad. Sci. USA 104, 18157–18162 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Himeda, C. L. & Jones, P. L. The genetics and epigenetics of facioscapulohumeral muscular dystrophy. Annu. Rev. Genomics Hum. Genet. 20, 265–291 (2019).

    Article  CAS  PubMed  Google Scholar 

  15. Lemmers, R. J. L. F. et al. A unifying genetic model for facioscapulohumeral muscular dystrophy. Science 329, 1650–1653 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Blewitt, M. E. et al. SmcHD1, containing a structural-maintenance-of-chromosomes hinge domain, has a critical role in X inactivation. Nat. Genet. 40, 663–669 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. Lemmers, R. J. L. F. et al. Digenic inheritance of an SMCHD1 mutation and an FSHD-permissive D4Z4 allele causes facioscapulohumeral muscular dystrophy type 2. Nat. Genet. 44, 1370–1374 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Shadle, S. C. et al. DUX4-induced dsRNA and MYC mRNA stabilization activate apoptotic pathways in human cell models of facioscapulohumeral dystrophy. PLoS Genet. 13, e1006658 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Campbell, A. E. et al. NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins. eLife 7, e31023 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Atashpaz, S. et al. ATR expands embryonic stem cell fate potential in response to replication stress. eLife 9, e54756 (2020).

  21. Ziegler-Birling, C., Helmrich, A., Tora, L. & Torres-Padilla, M.-E. Distribution of p53 binding protein 1 (53BP1) and phosphorylated H2A.X during mouse preimplantation development in the absence of DNA damage. Int. J. Dev. Biol. 53, 1003–1011 (2009).

    Article  CAS  PubMed  Google Scholar 

  22. Liu, L. et al. Telomere lengthening early in development. Nat. Cell Biol. 9, 1436–1441 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Wossidlo, M. et al. Dynamic link of DNA demethylation, DNA strand breaks and repair in mouse zygotes. EMBO J. 29, 1877–1888 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Srinivasan, R. et al. Zscan4 binds nucleosomal microsatellite DNA and protects mouse two-cell embryos from DNA damage. Sci. Adv. 6, eaaz9115 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Storm, M. P. et al. Zscan4 is regulated by PI3-kinase and DNA-damaging agents and directly interacts with the transcriptional repressors LSD1 and CtBP2 in mouse embryonic stem cells. PLoS ONE 9, e89821 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Britton, S., Coates, J. & Jackson, S. P. A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair. J. Cell Biol. 202, 579–595 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Fischer, M. Census and evaluation of p53 target genes. Oncogene 36, 3943–3956 (2017).

  28. Blackford, A. N. & Jackson, S. P. ATM, ATR, and DNA-PK: the trinity at the heart of the DNA damage response. Mol. Cell 66, 801–817 (2017).

    Article  CAS  PubMed  Google Scholar 

  29. Eckersley-Maslin, M. A. et al. MERVL/Zscan4 network activation results in transient genome-wide DNA demethylation of mESCs. Cell Rep. 17, 179–192 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Eckersley-Maslin, M. A. et al. Epigenetic priming by Dppa2 and 4 in pluripotency facilitates multi-lineage commitment. Nat. Struct. Mol. Biol. 27, 696–705 (2020).

    Article  CAS  PubMed  Google Scholar 

  31. Clapp, J. et al. Evolutionary conservation of a coding function for D4Z4, the tandem DNA repeat mutated in facioscapulohumeral muscular dystrophy. Am. J. Hum. Genet. 81, 264–279 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Rada-Iglesias, A. et al. A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470, 279–283 (2011).

    Article  CAS  PubMed  Google Scholar 

  33. Hu, W., Feng, Z., Teresky, A. K. & Levine, A. J. p53 regulates maternal reproduction through LIF. Nature 450, 721–724 (2007).

    Article  CAS  PubMed  Google Scholar 

  34. Sah, V. P. et al. A subset of p53-deficient embryos exhibit exencephaly. Nat. Genet. 10, 175–180 (1995).

    Article  CAS  PubMed  Google Scholar 

  35. Delbridge, A. R. D. et al. Loss of p53 causes stochastic aberrant X-chromosome inactivation and female-specific neural tube defects. Cell Rep. 27, 442–454 (2019).

    Article  CAS  PubMed  Google Scholar 

  36. Hoi, Y. J. et al. Deficiency of microRNA miR-34a expands cell fate potential in pluripotent stem cells. Science 355, aag1927 (2017).

  37. De Los Angeles, A. et al. Hallmarks of pluripotency. Nature 525, 469–478 (2015).

    Article  PubMed  CAS  Google Scholar 

  38. Behringer, R., Gertsenstein, M., Nagy, K. V. & Nagy, A. Differentiating mouse embryonic stem cells into embryoid bodies by hanging-drop cultures. Cold Spring Harb. Protoc. https://doi.org/10.1101/pdb.prot092429 (2016).

  39. Ibarra-Soria, X. et al. Defining murine organogenesis at single-cell resolution reveals a role for the leukotriene pathway in regulating blood progenitor formation. Nat. Cell Biol. 20, 127–134 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Chan, M. M. et al. Molecular recording of mammalian embryogenesis. Nature 570, 77–82 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. He, H. et al. p53 and p73 regulate apoptosis but not cell-cycle progression in mouse embryonic stem cells upon DNA damage and differentiation. Stem Cell Rep. 7, 1087–1098 (2016).

    Article  CAS  Google Scholar 

  42. Yamanaka, Y., Lanner, F. & Rossant, J. FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst. Development 137, 715–724 (2010).

    Article  CAS  PubMed  Google Scholar 

  43. Niakan, K. K., Schrode, N., Cho, L. T. Y. & Hadjantonakis, A.-K. Derivation of extraembryonic endoderm stem (XEN) cells from mouse embryos and embryonic stem cells. Nat. Protoc. 8, 1028–1041 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Leidenroth, A. et al. Evolution of DUX gene macrosatellites in placental mammals. Chromosoma 121, 489–497 (2012).

    Article  CAS  PubMed  Google Scholar 

  45. Catizone, A. N., Good, C. R., Alexander, K. A., Berger, S. L. & Sammons, M. A. Comparison of genotoxic versus nongenotoxic stabilization of p53 provides insight into parallel stress-responsive transcriptional networks. Cell Cycle 18, 809–823 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Karsli Uzunbas, G., Ahmed, F. & Sammons, M. A. Control of p53-dependent transcription and enhancer activity by the p53 family member p63. J. Biol. Chem. 294, 10720–10736 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Akdemir, K. C. et al. Genome-wide profiling reveals stimulus-specific functions of p53 during differentiation and DNA damage of human embryonic stem cells. Nucleic Acids Res. 42, 205–223 (2014).

    Article  CAS  PubMed  Google Scholar 

  48. Wang, T. et al. Species-specific endogenous retroviruses shape the transcriptional network of the human tumor suppressor protein p53. Proc. Natl Acad. Sci. USA 104, 18613–18618 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Tutton, S. et al. Subtelomeric p53 binding prevents accumulation of DNA damage at human telomeres. EMBO J. 35, 193–207 (2016).

    Article  CAS  PubMed  Google Scholar 

  50. Yeo, N. C. et al. An enhanced CRISPR repressor for targeted mammalian gene regulation. Nat. Methods 15, 611–616 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Hardy, K. Apoptosis in the human embryo. Rev. Reprod. 4, 125–134 (1999).

  52. Butuči, M., Williams, A. B., Wong, M. M., Kramer, B. & Michael, W. M. Zygotic genome activation triggers chromosome damage and checkpoint signaling in C. elegans primordial germ cells. Dev. Cell 34, 85–95 (2015).

    Article  PubMed  CAS  Google Scholar 

  53. Wang, G., Christensen, L. A. & Vasquez, K. M. Z-DNA-forming sequences generate large-scale deletions in mammalian cells. Proc. Natl Acad. Sci. USA 103, 2677–2682 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Zalzman, M. et al. Zscan4 regulates telomere elongation and genomic stability in ES cells. Nature 464, 858–863 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Elde, N. C. et al. Poxviruses deploy genomic accordions to adapt rapidly against host antiviral defenses. Cell 150, 831–841 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Horii, T. et al. p53 suppresses tetraploid development in mice. Sci. Rep. 5, 8907 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Yang, Y. et al. Derivation of pluripotent stem cells with in vivo embryonic and extraembryonic potency. Cell 169, 243–257 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Wu, T. et al. Histone variant H2A.X deposition pattern serves as a functional epigenetic mark for distinguishing the developmental potentials of iPSCs. Cell Stem Cell 15, 281–294 (2014).

    Article  CAS  PubMed  Google Scholar 

  59. Rokavec, M., Li, H., Jiang, L. & Hermeking, H. The p53/miR-34 axis in development and disease. J. Mol. Cell Biol. 6, 214–230 (2014).

    Article  CAS  PubMed  Google Scholar 

  60. Cossec, J.-C. et al. SUMO safeguards somatic and pluripotent cell identities by enforcing distinct chromatin states. Cell Stem Cell 23, 742–757 (2018).

    Article  CAS  PubMed  Google Scholar 

  61. Kahyo, T., Nishida, T. & Yasuda, H. Involvement of PIAS1 in the sumoylation of tumor suppressor p53. Mol. Cell 8, 713–718 (2001).

    Article  CAS  PubMed  Google Scholar 

  62. Association of Ubc9, an E2 ligase for SUMO conjugation, with p53 is regulated by phosphorylation of p53. FEBS Lett. 573, 15–18 (2004).

  63. Liu, J. et al. The RNA m6A reader YTHDC1 silences retrotransposons and guards ES cell identity. Nature 591, 322–326 (2021).

  64. Sasaki-Honda, M. et al. A patient-derived iPSC model revealed oxidative stress increases facioscapulohumeral muscular dystrophy-causative DUX4. Hum. Mol. Genet. 27, 4024–4035 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Le Roux, I., Konge, J., Le Cam, L., Flamant, P. & Tajbakhsh, S. Numb is required to prevent p53-dependent senescence following skeletal muscle injury. Nat. Commun. 6, 8528 (2015).

    Article  PubMed  CAS  Google Scholar 

  66. Dent, P. et al. CHK1 inhibitors in combination chemotherapy. Mol. Interv. 11, 133–140 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank members of the Cairns laboratory and M.B. Chandrasekharan for fruitful discussions. We are grateful to the patients who made this work possible. We thank T. Oliver for the Trp53fl/fl mouse, S.J. Tapscott for FSHD1 and FSHD2 myoblasts, F. Zhang for the px330-Cas9 plasmid, S. Jackson for the pICE-HA-NLS-I-PpoI plasmid, A. Chavez and G. Church for the dCas9-KRAB-MeCP2 plasmid and C. Gersbach for the pcDNA-dCas9-p300(HAT) plasmid. We also thank B. Dalley in the HCI High-Throughput Genomics and Bioinformatic Analysis Shared Resource (NCI grant P30CA042014), the CCTS Stem Cell Facility (National Institutes of Health (NIH), UL1TR002538), J. Marvin and the University of Utah Flow Cytometry Facility (NIH, 1S10RR026802-01; NCI, 5P30CA042014-24) and the University of Utah Cell Imaging Core. Funding for this work supported C.J.W. in part from the Intramural Research Program of the NIH (NIEHS, 1ZIAES102985), the NCI (P30 CA015704-45S6) and W.OP.14-01 from the Prinses Beatrix Spierfonds to S.M.v.d.M., and additional funding was from Wellstone Center from UMass (NICHD, P50HD060848) to R.J.B., the NIH (F30HD098000) to B.D.W., the NICHD (F32HD104442) to S.C.S., the NICHD (F32HD094500) and Lalor Foundation Fellowship 10041116 to E.J.G. and the Howard Hughes Medical Institute and the NICHD (1R01HD095833) to B.R.C. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

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Author notes

  1. These authors contributed equally: Bradley D. Weaver, Christina M. Smith.

Authors and Affiliations

  1. Howard Hughes Medical Institute, Department of Oncological Sciences and Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA

    Edward J. Grow, Bradley D. Weaver, Christina M. Smith, Jingtao Guo, Sean C. Shadle, Peter G. Hendrickson & Bradley R. Cairns

  2. Division of Urology, Department of Surgery, University of Utah School of Medicine, Salt Lake City, UT, USA

    Jingtao Guo

  3. Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA

    Paula Stein & Carmen J. Williams

  4. Department of Neurology, University of Utah School of Medicine, Salt Lake City, UT, USA

    Nicholas E. Johnson & Russell J. Butterfield

  5. Leiden Genome Technology Center, Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands

    Roberta Menafra & Susan L. Kloet

  6. Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands

    Silvère M. van der Maarel

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Contributions

IRB processing, patient consent, patient management and sample selection and processing were overseen by N.E.J. and R.J.B. Experiments and analyses were conducted by E.J.G., B.D.W., C.M.S., J.G., S.C.S., P.G.H., P.S., R.M. and S.L.K. with contributions by C.J.W. and S.M.v.d.M. E.J.G. and B.R.C. designed the study and wrote the manuscript with input from co-authors.

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Correspondence to Bradley R. Cairns.

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Peer review information Nature Genetics thanks Guillermina Lozano and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Related to Fig. 1.

a, Dux expression in aphidicolin or vehicle control treatment of Trp53 WT mESC or Trp53 KO mESC. RT-qPCR, n = 3 biological replicates, * pvalue<0.05, for p53WT p-value = 0.02041, for p53KO p-value= p-value = 0.05449, t-test, one sided. b, Schematic of Dux locus in mm10 genome assembly, the design of the targeting construct, location of gRNAs for Dux KO mESC line generation. Shown below are locations of genotyping primers. c, RT-qPCR analysis Dux KO mESC clones #1 and #2 treated with doxorubicin to induce endogenous Dux expression, which were used for experiments in Fig. 1. Shown is a representative analysis of three independent experiments. d, Design of DUX peptide antigen for antibody creation. e, immunofluorescence with the rabbit polyclonal anti-DUX antibody using mESC with a tetracycline inducible DUX-3xHA transgene. Representative image from 3 independent experiments. Merge: DAPI = blue, DUX = red. Scale bar = 125 micrometers. f, Kinetic analysis of Dux and Zscan4 transcript induction in WT mESC treated with 1uM of doxorubicin for indicated times. Note earlier induction of Dux compared to Zscan4, the RNAseq from Fig1 and Fig2 is using the later time point 18H. FACS/flow cytometry gating scheme to exclude doublets. For Extended Data Fig 1a, the median is shown as a line in the box, and the outline of the box is depicted at the 25th and 75th percentile. The extended whiskers depict Q1-1.5*IQR and Q3 + 1.5*IQR. Outliers points are depicted as dots.

Extended Data Fig. 2 Related to Fig. 2.

a, Western blot analysis and KO deletion allele Sanger sequencing results for two independent Trp53 KO mESC clones (used for Fig. 2). b, RT-qPCR measure of Dux or Zscan4 expression levels in two independent Dppa2/4 dKO mESC clones. Dppa2/4 dKO clone #1 was used for Fig. 2e with p53 rescue experiments. *<0.05 FDR, One-way ANOVA. c, Western blot confirmation of dKO Dppa2/4 mESC clones #1, 2. d, Dux expression in Trp53 WT mESC after control or Trp53 siRNA knockdown, with vehicle or doxorubicin treatment. RT-qPCR, N = 6 biological replicates, *<0.05 FDR, One-way ANOVA. e, Co-overexpression of DPPA2/4 in Trp53 WT mESC does not activate Dux expression. Merge: DAPI = cyan, DPPA2 = yellow, DPPA4 = magenta. Representative image from 3 independent experiments. Scale bar = 125 micrometers. For Extended Data Fig 2b, d, the median is shown as a line in the box, and the outline of the box is depicted at the 25th and 75th percentile. The extended whiskers depict Q1-1.5*IQR and Q3 + 1.5*IQR. Outliers points are depicted as dots.

Source data

Extended Data Fig. 3 Related to Fig. 3.

a, Schematic of long-read (PacBio) sequenced and assembled mouse Dux locus. b, CRISPR-A experiment in Trp53 KO mESC. CRIPSR-A with the Dux promoter-targeted gRNAs strongly activates Dux expression. RT-qPCR, n = 3 biological replicates. c, Mouse Nelfa locus showing enrichment of DUX binding at the 3’ end of the gene at intronic MERVL element. Bottom panel is zoomed (n = 2 biological replicates for each condition). Barplot (middle panel) of doxycycline induced Dux transgenic mESC showing strong induction of Nelfa transcripts (RNA-seq, n = 2 biological replicates, * FDR < 0.05, DESeq2, data reprocessed from Hendrickson, et al. Nature Genetics 2017). Right-most panel: Metagene plot of the 28xDux repeat units showing p53-ChIP-seq and input control and NELFA ChIP-seq and input control (blue and black lines respectively from Hu, et al. Nature Cell Biology, 2020—note lower NELFA ChIP-seq signal compared to the matched-control input). d, Nelfa is transcriptionally induced by doxorubicin treatment, and this requires both p53 and DUX. RNA-seq from this paper, n = 2 biological replicates, * FDR < 0.05 DESeq2. e, Mdm2 and Krt5 are direct p53 targets. p53 ChIP-seq and H3K27ac ChIP-seq (n = 2 biological replicates). RT-qPCR measuring Mdm2 expression in control mESC or ZSCAN4-OE mESC, treated with vehicle control or doxorubicin; n = 5 biological replicates, *p-value <0.05, one-sides t-test. f, Immunofluorescence staining of control mESC (n = 135 cells) or clonal ZSCAN4-OE mESC (n = 487 cells) using phospho-p53 antibodies after doxorubicin treatment, n. *<0.001, one-sided Wilcox test. g, Brightfield image showing decreased cell death after doxorubicin treatment in ZSCAN4-OE mESC compared to control. Image is representative from 3 independent experiments. Scale bar = 125 micrometers. For Extended Data Fig 3e, f, the median is shown as a line in the box, and the outline of the box is depicted at the 25th and 75th percentile. The extended whiskers depict Q1-1.5*IQR and Q3 + 1.5*IQR. Outliers points are depicted as dots.

Extended Data Fig. 4 Related to Fig. 4.

a, Immunofluorescence of pronuclei (PN)-stage zygotes showing nuclear phospho-S15 p53 staining (quantified in Fig. 4a). Scale bar = 40 micrometers. b, Single mouse zygote RT-qPCR measuring Dux expression in PN5 stage zygotes (n = 7 p53 MZ-KO, n = 16 p53 WT), * p-value <0.05, p-value= 0.0409, one-sided t-test. For Extended Data Fig 4b, the median is shown as a line in the box, and the outline of the box is depicted at the 25th and 75th percentile. The extended whiskers depict Q1-1.5*IQR and Q3 + 1.5*IQR. Outliers points are depicted as dots.

Extended Data Fig. 5 Related to Fig. 5.

a, Comparison of two biological replicates for EB scRNAseq (control vs Dux-pulsed) showing high concordance between samples. b, Ibarra-Soria, et al. Nature Cell Biology ‘Defining murine organogenesis at single-cell resolution reveals a role for the leukotriene pathway in regulating blood progenitor formation’ depicting different cell types defined in E8.25 mouse embryos, (data retrieved from https://marionilab.cruk.cam.ac.uk/organogenesis/ February 2020). c, Analysis of Ibarra-Soria, et al. data compared to our EB scRNAseq data with indicated markers identifying cell types (see Fig. 4c table). Each plot shows the marker identified in our Seurat analysis of EB scRNAseq as discriminating between other cell type clusters, and the data shows the distribution of that marker in E8.25 mouse in vivo cell types. For Extended Data Fig 5c, the median is shown as a line in the box, and the outline of the box is depicted at the 25th and 75th percentile. The extended whiskers depict Q1-1.5*IQR and Q3 + 1.5*IQR. Outliers points are depicted as dots.

Extended Data Fig. 6 Related to Fig. 6.

a, FSHD1 iPSC (patient #1, 2, 3) immunostaining for pluripotency markers SOX2 and OCT3/4. Representative image from 3 independent experiments. Merge: DAPI = cyan, SOX2 = yellow, OCT4 = magenta. Scale bar = 125 micrometers. b, Thermo-Fisher Karyostat report for FSHD1 iPSC clones patients #1, 2,3. c, RT-qPCR after vehicle control or doxorubicin treatment measuring DUX4 levels in FSHD1 iPSC patient #1, non-FSHD hESC female ‘LSJ2’, and non-FSHD iPSC ‘WT33’. N = 6 biological replicates, *<0.05 FDR, One-way ANOVA. d, Western blot with N- and C-term SMCHD1 antibodies that the 2 independent clones show in Fig. 6c are KO, isogenically created in FSHD1 patient #1. CRISPR/Cas9 deletion strategy shown on right top, with the Sanger sequencing of KO clones shown on bottom right. e, Genome browser snap-shot of ATAC-seq performed in human embryos showing open chromatin signal at the 4qA LTR10C element. f, Luciferase assay testing directionality of the LTR10C element in FSHD1 patient #1 iPSC (p53 WT or isogenic p53 KO). N = 4 biological replicates. g, RNA-seq analysis from Liu, et al. Nature 2021 showing reactivation of direct p53 targets (Mdm2, Cdkn1a (p21), and Krt5) in Ythdc1 conditional knockout (cKO) mESC ± Dux KO, treated with vehicle or 4-OHT (tamoxifen) to eliminate the YTHDC1 protein. For Extended Data Fig 6c, f, the median is shown as a line in the box, and the outline of the box is depicted at the 25th and 75th percentile. The extended whiskers depict Q1-1.5*IQR and Q3 + 1.5*IQR. Outliers points are depicted as dots.

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Source Data Extended Data Fig. 2

Western blot source data for Extended Data Fig. 2.

Source Data Extended Data Fig. 6

Western blot source data for Extended Data Fig. 6.

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Grow, E.J., Weaver, B.D., Smith, C.M. et al. p53 convergently activates Dux/DUX4 in embryonic stem cells and in facioscapulohumeral muscular dystrophy cell models. Nat Genet 53, 1207–1220 (2021). https://doi.org/10.1038/s41588-021-00893-0

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