RESUMEN
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.
Asunto(s)
G-Cuádruplex , Disostosis Mandibulofacial , Animales , Humanos , ADN/metabolismo , Células HEK293 , Células HeLa , Disostosis Mandibulofacial/genética , Disostosis Mandibulofacial/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
The localisation and quantification of constitutive alkali-labile sites (ALSs) were investigated using a protocol of DNA breakage detection plus fluorescence in situ hybridisation (DBD-FISH) and alkaline single-cell gel electrophoresis (SCGE or comet assay), in spermatozoa of infertile and fertile men. Semen samples from 10 normozoospermic patients undergoing infertility treatment and 10 fertile men were included in this study. ALSs were localised and quantified by DBD-FISH. The region most sensitive to alkali treatment in human spermatozoa was located in the basal region of the head. ALSs were more frequent in spermatozoa of infertile men than in those of fertile men. These results were confirmed by SCGE comet assays. In conclusion, the most intense localisation of hybridisation signals in human spermatozoa, representing the highest density of constitutive ALSs, was not randomly distributed and was predominantly located in the base of the head. Moreover, infertile men presented with an increase in ALS frequency. Further studies are necessary to determine the association between ALS, sperm chromatin organisation and infertility.