The role of the Ep400/Kat5 chromatin remodeling complex in the cranial neural crest and orofacial clefting

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE215109 | GEO | 2023/03/23

REPOSITORIES: GEO