The transcription of the main gene associated with Treacher-Collins syndrome (TCOF1) is regulated by G-quadruplexes and cellular nucleic acid binding protein (CNBP).

Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with Kd values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD.

Prevention of Treacher Collins syndrome craniofacial anomalies in mouse models via maternal antioxidant supplementation.

Craniofacial anomalies account for approximately one-third of all birth defects and are a significant cause of infant mortality. Since the majority of the bones, cartilage and connective tissues that comprise the head and face are derived from a multipotent migratory progenitor cell population called the neural crest, craniofacial disorders are typically attributed to defects in neural crest cell development. Treacher Collins syndrome (TCS) is a disorder of craniofacial development and although TCS arises primarily through autosomal dominant mutations in TCOF1, no clear genotype-phenotype correlation has been documented. Here we show that Tcof1 haploinsufficiency results in oxidative stress-induced DNA damage and neuroepithelial cell death. Consistent with this discovery, maternal treatment with antioxidants minimizes cell death in the neuroepithelium and substantially ameliorates or prevents the pathogenesis of craniofacial anomalies in Tcof1(+/-) mice. Thus maternal antioxidant dietary supplementation may provide an avenue for protection against the pathogenesis of TCS and similar neurocristopathies.