Molecular mechanism of neural crest cell differentiation, proliferation and death

小鼠Ncx （Tlx2,Enx,Hox11L1）基因在神经嵴源性组织中表达，调控肠内神经元的神经元细胞死亡。我们采用染色质免疫沉淀法筛选Ncx靶基因。通过筛选，鉴定出了一个新的基因Nczf。Nczf在C端含有一个n端KRAB盒结构域和11个Kruppel C2H2型锌指基序。Nczf基因启动子区包含5个Ncx结合基序的一致序列。用不同量的Ncx表达载体瞬时转染与荧光素酶报告基因融合的5′侧区，结果显示Nczf启动子活性呈剂量依赖性增加。在肠神经元中检测到Nczf mRNA的表达，与Ncx类似。胚胎发育过程中肠神经节中Nczf mRNA的表达量与Ncx的表达量大致相关。Nczf定位于细胞核并作为转录抑制因子发挥作用。核心核苷酸的一致结合序列为（A/T/C）CTTT(A/G)TTNT。在凝胶迁移转移试验中，含有这些序列的探针与融合蛋白结合。在计算机分析中，在内皮素受体B和小眼相关转录因子基因的调控区域发现了这些一致的序列，这些基因与神经嵴发育有关。此外，Nczf在神经母细胞瘤和成纤维细胞系中的过表达导致细胞凋亡。这些结果表明Nczf作为序列特异性转录抑制因子调控神经嵴细胞发育。

The murine Ncx (Tlx2,Enx,Hox11L1) gene is expressed in neural crest derived tissues and regulates neuronal cell death in enteric neurons. We have used chromatin immunoprecipitation method to screen for target genes for Ncx. This screen led to the identification of novel gene termed Nczf. Nczf contains an N-terminal KRAB box domain and 11 Kruppel C2H2 type zinc finger motifs at C terminus. Promoter region of the Nczf gene contains 5 consensus sequences for Ncx binding motifs. Transient transfection assays of the 5'-flanking region fused to the luciferase reporter gene with various amount of Ncx expression vector showed dose dependent increase of the Nczf promoteractivity. Expression of Nczf mRNA was detected in enteric neurons like that of Ncx. The amount of Nczf mRNA roughly correlated with that of the Ncx in enteric ganglia during embryonic development. Nczf localized in the nucleus and functioned as a transcriptional repressor. The consensus binding sequence of core nucleotides contains (A/T/C)CTTT(A/G)TTNT. In a gel mobility shift assay, the probe containing these sequences bound to the fusion protein. In silico analysis, these consensus sequences were found on regulatory regions of the endothelin receptor B and the microphthalmia-associated transcription factor genes, which are involved in neural crest development. Furthermore, overexpression of Nczf incultured neuroblastoma and fibroblast cell lines caused apoptotic cell death. These results suggest that Nczf functions as a sequence specific transcription repressor to regulate neural crest cell development.