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.
Gene Expression Omnibus
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.).
Extended data figures
This file contains oligonucleotides utilized in this study.