Project description:The cranial neural crest plays a fundamental role in orofacial development and morphogenesis. As a pluripotent and dynamic cell population, the cranial neural crest is undergoing vast transcriptional alterations throughout embryogenesis and the formation of facial structures. These transcriptional changes are regulated by several transcription factors and remodeling complexes. Previously, we revealed the relevance of the Ep400/Kat5 histone acetyltransferase complex in the cranial neural crest and showed that the knockout of Ep400 causes neural crest-related malformations such as orofacial clefting. Furthermore, a case study identified three patients carrying missense mutations in Kat5 who showed severe mental impairments as well as orofacial clefts.2 The exact molecular causes and mechanisms, however, are still unknown. In this study we selectively knocked out Ep400 or Kat5 in the murine cranial neural crest cell line O9-1 to examine its roles in neural crest biology. To understand the regulatory effects of Ep400 and Kat5, knockout neural crest cells were investigated via bulk RNA sequencing to unravel transcriptomic changes in the affected cells. Bioinformatic analyses hinted at the regulation of major cellular functions such as proliferation, ATP generation and protein synthesis by the Ep400/Kat5 complex. Reduced proliferation was confirmed by crystal violet staining, phospho-histone H3 staining and the determination of mitotic cells with condensed chromatin in vitro. We did not detect increased apoptosis in the knockout cell lines. The energetic profile of the cells was investigated by Seahorse technology. The ATP-rate assay showed a decreased glycolytic activity in Ep400- or Kat5-deficient cells. An O-propargyl-puromycin (OPP) Click-iT assay revealed a significant reduction in protein synthesis. To verify in vivo the discovered in vitro effects, Ep400 and Kat5 were selectively ablated in cranial neural crest using Wnt1-Cre in transgenic mice. The knockout of each of the subunits resulted in severe craniofacial malformations from E12.5 onwards. At E10.5 a significant reduction in neural crest-derived tissue and proliferation rate was evident. The strong defect in orofacial structures of mice lacking Kat5 or Ep400 completely correspond to the milder orofacial malformations in patients carrying heterozygous missense mutations. Our results furthermore argue that changes of metabolism, protein synthesis and proliferation in cranial neural crest cells are responsible for the orofacial defects observed in patients.

Project description:Orofacial clefts are the most common form of congenital craniofacial malformations worldwide. The etiology of these birth defects is multifactorial, involving genetic and environmental factors. In most cases, however, the underlying causes remain unexplained, precluding molecular understanding of disease mechanisms. Here, we integrated genome-wide association data, targeted re-sequencing of case and control cohorts, cell type-specific epigenomic profiling, and genome architecture analyses, to functionally and molecularly dissect a genomic locus associated with an increased risk of non-syndromic orofacial cleft. We found that common and rare risk variants associated with orofacial cleft intersect with a conserved enhancer (e2p24.2) that becomes activated in cranial neural crest cells—the embryonic cell type responsible for sculpting the craniofacial complex. We mapped e2p24.2 long-range interactions to a topologically associated domain harboring MYCN and DDX1 and demonstrated that both MYCN and DDX1 are required for craniofacial development in chicken embryos. We found that e2p24.2 regulates the expression of MYCN, but not DDX1, in cranial neural crest cells. In turn, DDX1 is a target of the MYC family of transcription factors and a component of the tRNA splicing complex. The loss of DDX1 in cranial neural crest cells resulted in the accumulation of unspliced tRNA fragments, depletion of the mature pool of intron-containing tRNAs, and ribosome stalling at codons decoded by these tRNAs. These effects were accompanied by defects in both global protein synthesis and cranial neural crest cell migration. We further showed that the induction of tRNA fragments is sufficient to disrupt craniofacial development. Together, these results uncovered a molecular mechanism in which impaired tRNA splicing affects neural crest and craniofacial development and positioned MYCN, DDX1, and tRNA processing defects as risk factors in the pathogenesis of orofacial clefts.

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Genetic Analysis of Syndromic Orofacial Clefting

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