PDF(Pubmed)
Abstract：
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.
摘要：
反应核糖体生物发生因子1（TCOF1）约占下颌骨骨发育不全（MD）病例的80％。我们以前已经鉴定了人类间充质细胞中TCOF1和CNBP（CCHC型锌指核酸结合蛋白）表达之间的相关性。鉴于CNBP在延髓发育过程中基因调控中的作用，我们探索了CNBP调节TCOF1转录的潜力。TCOF1启动子中CNBP结合位点（CNBP-BS）的计算分析揭示了几个假定的结合位点，其中两个（Hs791和Hs2160）与推定的G-四链体（G4）序列（PQSs）重叠。我们验证了这些测量圆二色性和适当合成寡核苷酸的荧光的PQSs的折叠。体外研究证实纯化的CNBP与靶PQSs(折叠为G4和未折叠)的结合,Kd值在nM范围内。在HeLa细胞中进行的ChIP测定染色质检测了CNBP与TCOF1启动子的结合。HEK293细胞的瞬时转染显示Hs2160克隆上游SV40启动子增加下游萤火虫荧光素酶报告基因的转录。我们还在斑马鱼TCOF1直向同源启动子(nolc1)中检测到了CNBP-BS和PQS(Dr2393)。通过微注射与Dr2393互补的DNA反义寡核苷酸来破坏斑马鱼胚胎中的G4可降低nolc1的转录，并概括了TreacherCollins综合征的颅面异常特征。斑马鱼中cnbp过表达和吗啉代介导的敲低均诱导nolc1转录。这些结果表明，CNBP通过涉及G-四链体折叠/解折叠的机制调节TCOF1的转录表达，这种调节在脊椎动物中很活跃，就像硬骨鱼和人类一样。这些发现可能对理解和治疗MD有影响。
