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Tissue-selective effects of nucleolar stress and rDNA damage in developmental disorders

Nature volume 554, pages 112–117 (2018)Cite this article

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Abstract

Many craniofacial disorders are caused by heterozygous mutations in general regulators of housekeeping cellular functions such as transcription or ribosome biogenesis1,2. Although it is understood that many of these malformations are a consequence of defects in cranial neural crest cells, a cell type that gives rise to most of the facial structures during embryogenesis3,4, the mechanism underlying cell-type selectivity of these defects remains largely unknown. By exploring molecular functions of DDX21, a DEAD-box RNA helicase involved in control of both RNA polymerase (Pol) I- and II-dependent transcriptional arms of ribosome biogenesis5, we uncovered a previously unappreciated mechanism linking nucleolar dysfunction, ribosomal DNA (rDNA) damage, and craniofacial malformations. Here we demonstrate that genetic perturbations associated with Treacher Collins syndrome, a craniofacial disorder caused by heterozygous mutations in components of the Pol I transcriptional machinery or its cofactor TCOF1 (ref. 1), lead to relocalization of DDX21 from the nucleolus to the nucleoplasm, its loss from the chromatin targets, as well as inhibition of rRNA processing and downregulation of ribosomal protein gene transcription. These effects are cell-type-selective, cell-autonomous, and involve activation of p53 tumour-suppressor protein. We further show that cranial neural crest cells are sensitized to p53-mediated apoptosis, but blocking DDX21 loss from the nucleolus and chromatin rescues both the susceptibility to apoptosis and the craniofacial phenotypes associated with Treacher Collins syndrome. This mechanism is not restricted to cranial neural crest cells, as blood formation is also hypersensitive to loss of DDX21 functions. Accordingly, ribosomal gene perturbations associated with Diamond–Blackfan anaemia disrupt DDX21 localization. At the molecular level, we demonstrate that impaired rRNA synthesis elicits a DNA damage response, and that rDNA damage results in tissue-selective and dosage-dependent effects on craniofacial development. Taken together, our findings illustrate how disruption in general regulators that compromise nucleolar homeostasis can result in tissue-selective malformations.

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Figure 1: The functions of DDX21 are linked to rRNA synthesis levels and altered by TCS-associated perturbations.
Figure 2: DDX21 deregulation in TCS is both cell-autonomous and cell-type selective.
Figure 3: Selective sensitivity of cNCCs to p53 activation (act.) and DDX21 levels.
Figure 4: rDNA damage induces DDX21 relocalization and impairs craniofacial development.

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References

  1. Yelick, P. C. & Trainor, P. A. Ribosomopathies: global process, tissue specific defects. Rare Dis. 3, e1025185 (2015)

    Article  Google Scholar 

  2. Berdasco, M. & Esteller, M. Genetic syndromes caused by mutations in epigenetic genes. Hum. Genet. 132, 359–383 (2013)

    Article  CAS  Google Scholar 

  3. Trainor, P. A. Craniofacial birth defects: the role of neural crest cells in the etiology and pathogenesis of Treacher Collins syndrome and the potential for prevention. Am. J. Med. Genet. A 152, 2984–2994 (2010)

    Article  Google Scholar 

  4. Bronner, M. E. & LeDouarin, N. M. Development and evolution of the neural crest: an overview. Dev. Biol. 366, 2–9 (2012)

    Article  CAS  Google Scholar 

  5. Calo, E. et al. RNA helicase DDX21 coordinates transcription and ribosomal RNA processing. Nature 518, 249–253 (2015)

    Article  CAS  ADS  Google Scholar 

  6. Narla, A. & Ebert, B. L. Ribosomopathies: human disorders of ribosome dysfunction. Blood 115, 3196–3205 (2010)

    Article  CAS  Google Scholar 

  7. Kadakia, S., Helman, S. N., Badhey, A. K., Saman, M. & Ducic, Y. Treacher Collins syndrome: the genetics of a craniofacial disease. Int. J. Pediatr. Otorhinolaryngol. 78, 893–898 (2014)

    Article  Google Scholar 

  8. Weiner, A. M. J., Scampoli, N. L. & Calcaterra, N. B. Fishing the molecular bases of Treacher Collins syndrome. PLoS ONE 7, e29574 (2012)

    Article  CAS  ADS  Google Scholar 

  9. Lau, M. C. C. et al. Pathogenesis of POLR1C-dependent type 3 Treacher Collins syndrome revealed by a zebrafish model. Biochim. Biophys. Acta 1862, 1147–1158 (2016)

    Article  CAS  Google Scholar 

  10. Noack Watt, K. E., Achilleos, A., Neben, C. L., Merrill, A. E. & Trainor, P. A. The roles of RNA polymerase I and III subunits Polr1c and Polr1d in craniofacial development and in zebrafish models of Treacher Collins syndrome. PLoS Genet. 12, e1006187 (2016)

    Article  Google Scholar 

  11. Valdez, B. C., Henning, D., Perumal, K. & Busch, H. RNA-unwinding and RNA-folding activities of RNA helicase II/Gu: two activities in separate domains of the same protein. Eur. J. Biochem. 250, 800–807 (1997)

    Article  CAS  Google Scholar 

  12. Dixon, J., Brakebusch, C., Fässler, R. & Dixon, M. J. Increased levels of apoptosis in the prefusion neural folds underlie the craniofacial disorder, Treacher Collins syndrome. Hum. Mol. Genet. 9, 1473–1480 (2000)

    Article  CAS  Google Scholar 

  13. Dixon, J. et al. Tcof1/Treacle is required for neural crest cell formation and proliferation deficiencies that cause craniofacial abnormalities. Proc. Natl Acad. Sci. USA 103, 13403–13408 (2006)

    Article  CAS  ADS  Google Scholar 

  14. Sloan, K. E. et al. The association of late-acting snoRNPs with human pre-ribosomal complexes requires the RNA helicase DDX21. Nucleic Acids Res. 43, 553–564 (2015)

    Article  CAS  Google Scholar 

  15. Gonzales, B. et al. The Treacher Collins syndrome (TCOF1) gene product is involved in pre-rRNA methylation. Hum. Mol. Genet. 14, 2035–2043 (2005)

    Article  CAS  Google Scholar 

  16. Jones, N. C. et al. Prevention of the neurocristopathy Treacher Collins syndrome through inhibition of p53 function. Nat. Med. 14, 125–133 (2008)

    Article  CAS  Google Scholar 

  17. Berkson, R. G. et al. Pilot screening programme for small molecule activators of p53. Int. J. Cancer 115, 701–710 (2005)

    Article  CAS  Google Scholar 

  18. Dolma, S., Lessnick, S. L., Hahn, W. C. & Stockwell, B. R. Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells. Cancer Cell 3, 285–296 (2003)

    Article  CAS  Google Scholar 

  19. Van Nostrand, J. L. et al. Inappropriate p53 activation during development induces features of CHARGE syndrome. Nature 514, 228–232 (2014)

    Article  CAS  ADS  Google Scholar 

  20. Rinon, A . et al. p53 coordinates cranial neural crest cell growth and epithelial-mesenchymal transition/delamination processes. Development 138, 1827–1838 (2011)

    Article  CAS  Google Scholar 

  21. Zhang, Z. et al. Assessment of hematopoietic failure due to Rpl11 deficiency in a zebrafish model of Diamond-Blackfan anemia by deep sequencing. BMC Genomics 14, 896 (2013)

    Article  Google Scholar 

  22. Sakai, D., Dixon, J., Achilleos, A., Dixon, M. & Trainor, P. A. Prevention of Treacher Collins syndrome craniofacial anomalies in mouse models via maternal antioxidant supplementation. Nat. Commun. 7, 10328 (2016)

    Article  CAS  ADS  Google Scholar 

  23. Muscarella, D. E., Ellison, E. L., Ruoff, B. M. & Vogt, V. M. Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum. Mol. Cell. Biol. 10, 3386–3396 (1990)

    Article  CAS  Google Scholar 

  24. Flick, K. E., Jurica, M. S., Monnat, R. J. Jr & Stoddard, B. L. DNA binding and cleavage by the nuclear intron-encoded homing endonuclease I-PpoI. Nature 394, 96–101 (1998)

    Article  CAS  ADS  Google Scholar 

  25. Chailleux, C. et al. Quantifying DNA double-strand breaks induced by site-specific endonucleases in living cells by ligation-mediated purification. Nat. Protocols 9, 517–528 (2014)

    Article  CAS  Google Scholar 

  26. Song, C., Hotz-Wagenblatt, A., Voit, R. & Grummt, I. SIRT7 and the DEAD-box helicase DDX21 cooperate to resolve genomic R loops and safeguard genome stability. Genes Dev. http://dx.doi.org/10.1101/gad.300624.117 (2017)

  27. Buecker, C. et al. Reorganization of enhancer patterns in transition from naive to primed pluripotency. Cell Stem Cell 14, 838–853 (2014)

    Article  CAS  Google Scholar 

  28. Bajpai, R. et al. CHD7 cooperates with PBAF to control multipotent neural crest formation. Nature 463, 958–962 (2010)

    Article  CAS  ADS  Google Scholar 

  29. Prescott, S. L. et al. Enhancer divergence and cis-regulatory evolution in the human and chimp neural crest. Cell 163, 68–83 (2015)

    Article  CAS  Google Scholar 

  30. Hu, S. et al. Effects of cellular origin on differentiation of human induced pluripotent stem cell-derived endothelial cells. JCI Insight 1, e85558 (2016)

    Article  Google Scholar 

  31. Burridge, P. W. et al. Chemically defined generation of human cardiomyocytes. Nat. Methods 11, 855–860 (2014)

    Article  CAS  Google Scholar 

  32. Carey, M. F., Peterson, C. L. & Smale, S. T. Dignam and Roeder nuclear extract preparation. Cold Spring Harb. Protoc. 2009, http://dx.doi.org/10.1101/pdb.prot5330 (2009)

  33. Nieuwkoop, P. D . & Faber, J . (eds) Normal Table of Xenopus laevis (Daudin): A Systematical and Chronological Survey of the Development from the Fertilized Egg Till the End of Metamorphosis (Garland, 1994)

  34. Grier, J. D ., Yan, W. & Lozano, G. Conditional allele of mdm2 which encodes a p53 inhibitor. Genesis 32, 145–147 (2002)

    Article  CAS  Google Scholar 

  35. Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K. & McMahon, A. P. Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr. Biol. 8, 1323–1326 (1998)

    Article  CAS  Google Scholar 

  36. Truett, G. E. et al. Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques 29, 52–54 (2000)

    Article  CAS  Google Scholar 

  37. Flynn, R. A. et al. Dissecting noncoding and pathogen RNA–protein interactomes. RNA 21, 135–143 (2015)

    Article  Google Scholar 

  38. Zarnegar, B. J. et al. irCLIP platform for efficient characterization of protein–RNA interactions. Nat. Methods 13, 489–492 (2016)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Stack and J. Wu for providing the endothelial and cardiomyocyte cells, J. Chen for Xenopus splicing morpholino validations, C. Santoriello and L. Zon for ddx21 zebrafish morpholino, the Swanson Biotechnology Center at the Koch Institute for Integrative Cancer Research, especially E. Vasile for microscopy and A. Amsterdam for zebrafish work, and K. Cimprich and members of the Wysocka, Calo, and Chang laboratories for discussions. This work was supported by the Howard Hughes Medical Institute (J.W.), R01 GM112720 (J.W.), the March of Dimes Birth Defects Foundation (J.W.), March of Dimes Foundation grants 6-FY15-189 and RC35CA197591 (L.D.A.), Ludwig Foundation (J.W.), Stanford Medical Scientist Training Program and T32CA09302 (R.A.F.), the Helen Hay Whitney Foundation (E.C.), EMBO (ALTF 275-2015), the European Commission (LTFCOFUND2013, GA-2013-609409), and the Marie Curie Actions (A.Z.), Jane Coffin Childs Memorial Fund postdoctoral fellowship (M.E.B.), Stanford Graduate Student Fellowship (B.G.) and National Institutes of Health P50-HG007735 and R01-ES023168 (H.Y.C.).

Author information

Authors and Affiliations

  1. Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA

    Eliezer Calo, Fardin Aryan & Jialiang Liang

  2. David H. Koch Institute for Integrative Cancer Research, Cambridge, 02139, Massachusetts, USA

    Eliezer Calo

  3. Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, 94305, California, USA

    Bo Gu, Antoine Zalc, Tomek Swigut & Joanna Wysocka

  4. Department of Radiation Oncology, Division of Radiation and Cancer Biology, Stanford University School of Medicine, Stanford, 94305, California, USA

    Margot E. Bowen & Laura D. Attardi

  5. Department of Chemistry, Stanford University, Stanford, 94305, California, USA

    Ryan A. Flynn

  6. Center for Personal Dynamic Regulomes, Stanford University, 269 Campus Drive, Stanford, 94305, California, USA

    Howard Y. Chang

  7. Department of Genetics, Stanford University School of Medicine, Stanford, 94305, California, USA

    Laura D. Attardi

  8. Department of Developmental Biology, Stanford University School of Medicine, Stanford, 94305, California, USA

    Joanna Wysocka

  9. Howard Hughes Medical Institute, Stanford School of Medicine, Stanford University, Stanford, 94305, California, USA

    Joanna Wysocka

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  1. Eliezer Calo
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Contributions

J.W. supervised the project; E.C. conceived and designed the study; E.C. performed experiments with help from F.A. and J.L.; B.G. performed image analyses. E.C. and R.A.F. analysed ChIP–seq data; R.A.F. analysed the iCLIP data; F.A. and E.C. performed zebrafish experiments; M.E.B. performed mouse embryo dissections and immunostainings; A.Z. performed p53 in situ hybridization; J.L. and E.C. performed DNA damage experiments; T.S., L.D.A., and H.Y.C. provided advice on experimental designs, data analyses, and interpretation of the data; E.C. and J.W. wrote the manuscript with input from all co-authors.

Corresponding author

Correspondence to Joanna Wysocka.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks D. Tollervey and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 DDX21 subnuclear localization is sensitive to perturbations in the rRNA synthesis.

a, Representative immunofluorescence depicting DDX21 localization and 5-ethynyl uridine (EU) incorporation in HeLa cells treated with DMSO or iPol I from n = 3 biologically independent experiments. b, c, siRNA pools were developed against TCOF1 or POLRID and transected into HeLa cells. qPCR was used to determine knockdown efficiency. d, An additional pool of siRNAs targeting TCOF1 3′ UTR was generated and transfected into HeLa cells, followed by immunofluorescence to determine DDX21 localization upon TCOF1 knockdown. Shown are representative images from n = 3 biologically independent experiments. e, f, ChIP–qPCR of DDX21 binding at target gene promoters (e) and the rDNA locus (f) upon knockdown of either TCOF1 (siTCOF1) or POLR1D (siPOLR1D). For b, c, e and f, bars represent the average n = 3 biologically independent experiments; error bars, s.e.m.

Extended Data Figure 2 DDX21 knockdown phenocopies TCS-associated perturbations in X. laevis and zebrafish.

a, Representative immunofluorescence images showing strong nucleolar localization signal for TCOF1 in HeLa cells from n = 3 biologically independent experiments. b, Immunoprecipitation of either GFP-tagged TCOF1 (GFP–TCOF1) or DDX21, followed by western blotting with the indicated antibodies. n = 2 biologically independent experiments. c, Table showing the quantification of injected Xenopus embryos with the indicated morpholinos (MO) or in vitro transcribed mRNAs. d, Efficiency of tcof1 splicing morpholino was determined by PCR. n = 10 injected embryos. e, Representative images of stage 49 Xenopus embryo cartilage stainings with alcian blue. Traces of the mandibular and hyoid streams are shown for clarity. Embryos were collected from n = 3 biologically independent experiments. f, Stage 2 embryos were injected with in vitro transcribed mRNAs encoding wild-type or catalytically defective DDX21. Total mRNA was extracted at stage 31, followed by qPCR to determine the expression levels of injected mRNAs in the anterior part of the embryo (see schematics for details). Bars represent the average of n = 3 independent experiments; error bars, s.e.m. g, Representative images of 5-day-old wild-type (WT), polr1d−/−, and polr1c−/− zebrafish embryo cartilage stained with alcian blue from n = 3 independent matings. h, Table showing the quantification of polr1d−/− and polr1c−/− crosses. i, Table showing quantification of zebrafish embryos injected with the indicated morpholino or combination of morpholino and mRNA. j, Representative images of 5-day-old zebrafish embryo cartilage stained with alcian blue after injection of ddx21 morpholino at the indicated dosages or ddx21 morpholino and in vitro transcribed human DDX21 mRNA. Traces of the ceratohyal, platoquadrate, and Meckel’s cartilage are shown for clarity. n = 3 biologically independent sets of injections.

Extended Data Figure 3 Generation of an in vitro model of TCS.

a, Mouse ES cells were co-transfected with CRISPR–Cas9 and sgRNAs targeting the Tcof1 locus. Targeted mouse ES cells were single-cell cloned and screened for loss-of-function mutations in Tcof1. Clones of the indicated genotypes were selected for this study. b, Overexpression of an exogenous, but stably integrated human GFP-tagged TCOF1 construct in mouse cNCCs rescued DDX21 localization defects as determined by immunofluorescence (quantifications are shown in Fig. 2c). Shown are representative images from n = 3 biologically independent experiments. c, Mouse ES cells were differentiated into embryoid bodies. Embryoid body outgrowth explants were further grown in culture and stained with antibodies for Ddx21. Shown are representative images from n = 4 biologically independent experiments. d, Human H9 ES cells were co-transfected with CRISPR–Cas9 and sgRNAs targeting the TCOF1 locus. Targeted ES cells were cloned and screened for loss-of-function mutations in TCOF1; unlike mouse ES cells, we did not recover homozygous mutant alleles for TCOF1 in human cells. The indicated genotype was selected for this study. e, f, Two different commercially available antibodies raised against TCOF1 were used to confirm the heterozygosity of TCOF1+/− ES cells. Shown are representative western blots from n = 2 biologically independent experiments. g, Representative immunofluorescence images showing DDX21 localization in both wild-type and TCOF1+/− human ES cells and cNCCs from n = 3 biologically independent experiments. h, ChIP–qPCR analysis in human cNCCs sampling DDX21 genomic occupancy at a representative panel of DDX21 target promoters and at the rDNA promoter. i, qPCR analyses of DDX21-regulated Pol I and Pol II transcribed ribosomal genes. For h, i, bars represent the average of n = 3 biologically independent experiments; error bars, s.e.m.

Extended Data Figure 4 Pol I inhibition impairs the ability of DDX21 to associate with the 5′ external transcribed spacer (ETS) and the snoRNAs.

a, DDX21 iCLIP 32P-autoradiogram and western blots from control (DMSO) and Pol I inhibited cells. For Pol I inhibition, we used low levels of actinomycin D (ActD; 50 ng ml−1). Samples were loaded with constant input lysate amounts. b, DDX21 iCLIP reads mapped to the transcribed region of the rDNA. The 5′ external transcribed spacer and the mature portions of the 18S, 5.8S, and 28S rRNAs are highlighted. c, Distribution of ENSEMBL annotated regions for DDX21-bound RNAs in both DMSO and actinomycin D conditions. d, e, Scatter plot analysis of normalized iCLIP reverse transcription stops on individual C/D or H/ACA snoRNAs. iCLIP results are from n = 2 biological replicates.

Extended Data Figure 5 Protein p53 is activated in TCS cNCCs and its mRNA is highly expressed in cNCCs in vivo

. a, b, qPCR analyses of the p53-target gene Cdkn1a in mouse ES cells and cNCCs (a) and human cNCCs (b) of indicated genotypes. Bars represent the average of n = 3 biologically independent experiments; error bars, s.e.m. c, Human cNCCs were treated with NSC146109 for 12 h, followed by western blotting with antibodies raised against p53. Shown is a representative western blot from n = 3 biologically independent experiments. d, Immunofluorescence staining of p53 and DDX21 in sections from the dorsal neural tube of Mdm2fl/fl (control; top) and Wnt1-cre;Mdm2fl/fl (bottom) E9.5 mouse embryos. Dotted lines outline the neural fold. n = 5 animals per genotype. e, Representative picture of whole-mount in situ hybridization of E9.5 embryos with a probe recognizing endogenous p53 mRNA. n = 4 animals. f, g, Representative images of p53 in situ hybridization on tissue sections of the frontonasal prominence and first and second pharyngeal arches of E9.5 mouse embryos. n = 2 independent animals.

Extended Data Figure 6 Hyper-activation of p53 in cNCCs renders pharyngeal arches hypoplastic.

Representative images of wild-type and Wnt1-cre;Mdm2fl/fl E10.5 embryos. Whole-embryo pictures (left) and insets (middle) depicting the location of the first and second pharyngeal arches. Right panel shows traces of first and second pharyngeal arches for clarity. n = 8 animals per genotype.

Extended Data Figure 7 DDX21 overexpression rescues TCS and its function is deregulated by knockdown of other ribosomopathy-associated genes

. a, Representative western blot for DDX21 in different cell types from n = 3 biologically independent experiments. b, FACS analyses to determine the sensitivity of TCOF1+/− cNCC to p53-mediated apoptosis. cNCCs were treated with NSC146109 for 4–6 h (note that this time point is significantly shorter than the one used in Fig. 3f and g). Apoptosis was quantified by FACS of annexin V staining. Bars represent the average of n = 3 independent experiments; error bars, s.e.m. c, Quantification of Xenopus cranofacial development rescue experiments by measuring the length of the hyoid stream upon overexpression of TCOF1, DDX21, or p53 knockdown. Embryos were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P < 0.001, two-sided Wilcoxon–Mann–Whitney test. d, Rescue of TCS-associated craniofacial malformations in Xenopus by injection of the embryos with the indicated in vitro transcribed mRNAs and/or morpholinos (quantification is shown in c). e, Representative immunofluorescence images of mouse Tcof1−/− cNCCs upon overexpression of human GFP-tagged DDX21. n = 3 biologically independent experiments. f, ChIP–qPCR analysis, in mouse cNCCs sampling Ddx21 genomic occupancy, at a representative panel of Ddx21 target promoters and at the rDNA locus. n = 3 biologically independent experiments. Bars represent the average of n = 3 independent experiments; error bars, s.e.m. g, siRNA pools against a subset of ribosomopathy-associated genes were transected into HeLa cells. qPCR was used to determine the efficiency of the knockdowns. Bars represent the average of n = 3 independent experiments; error bars, s.e.m. h, Representative immunofluorescence images showing DDX21 localization changes in HeLa cells transfected with the indicated pools of siRNAs (quantification is on Fig. 3i). n = 3 biologically independent experiments. i, Tables quantifying the number of embryos stained for haemoglobin with o-dianisidine for the indicated genotypes. In the case of rpl1 zebrafish, embryos were collected from three independent matings. For DDX21, three independent batches of embryos were injected and stained.

Extended Data Figure 8 Inhibition of Pol I results in DNA damage in a subset of cells.

a, Representative immunofluorescence images of wild-type and TCOF1+/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b. c, Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d. e, Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f. For a–f, cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P < 0.001, two-sided Wilcoxon–Mann–Whitney test. g, Fraction of DNA-damaged HeLa cells displaying perinucleolar γH2A.X signal after 1 h incubation with iPol I. Cells were collected from n = 3 biologically independent experiments. h–j, Single-cell correlation plots of p53 activation and γH2A.X signal in control and cells expressing either AsiSI or I-PpoI. Cells were collected from n = 4 biologically independent experiments. ρ, Pearson correlation coefficient. P, two-sided Wilcoxon–Mann–Whitney test. k, Single-cell quantification of γH2A.X signal in control and cells expressing either AsiSI or I-PpoI. Cells were collected from n = 4 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. P, two-sided Wilcoxon–Mann–Whitney test.

Source data

Extended Data Figure 9 rDNA damage impairs DDX21 functions and causes craniofacial deformities.

a, b, Time course of phosphorylated DNA-PKcs as measured by auto-phosphorylation of the kinase in Ser2056 (S2056) upon iPol I treatment. Cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. P, two-sided Wilcoxon–Mann–Whitney test. c, Time course of DDX21 exclusion from the nucleolus to the nucleoplasm upon iPol I treatment. Cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. P, two-sided Wilcoxon–Mann–Whitney test. d, Representative immunofluorescence images of HeLa cells transfected with in vitro transcribed I-PpoI and treated or not with inhibitors for ATM (iATM) and DNAPK (iDPK). After treatment, cells were stained with antibodies against DDX21 and γH2A.X. e, Box plot quantifying the number of γH2A.X foci of HeLa cells transfected with I-PpoI and treated or not with iATM and iDPK. Cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P < 0.001, two-sided Wilcoxon–Mann–Whitney test, not significant (NS). f, Western blot showing DNA-PKcs by auto-phosphorylation of S2056 upon knockdown of DDX21. Two different siRNAs were used in this experiment (see Methods for details). n = 3 biologically independent experiments. g, Representative bright-field images of stage 49 Xenopus embryos either uninjected or injected with in vitro transcribed I-PpoI and (h) alcian blue stainings of Xenopus cranial cartilage from embryos injected with the indicated doses of in vitro transcribed I-PpoI. Embryos were collected from n = 4 biologically independent injections.

Source data

Extended Data Figure 10 Model explaining cNCC-type selective effects of nucleolar dysfunction and rDNA damage in TCS.

cNCCs express high levels of p53 mRNA, but during normal development p53 is under post-transcriptional control of its E3 ligase, Mdm2. Upon nucleolar stress and/or rDNA damage, activation of p53 and loss of DDX21 from chromatin result in apoptosis of a subset of cNCCs. This diminishes the population of cNCCs that can be allocated to the lower face, leading to malformations of the developing craniofacial structures. Thus, factors whose perturbations may ultimately induce defects in rRNA synthesis and rDNA damage are likely to be associated with craniofacial malformations.

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Calo, E., Gu, B., Bowen, M. et al. Tissue-selective effects of nucleolar stress and rDNA damage in developmental disorders. Nature 554, 112–117 (2018). https://doi.org/10.1038/nature25449

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